Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s00216-001-1166-x
E D I TO R I A L
Franois Mathey
Analytical and Bioanalytical Chemistry is the sixth mem- trial research is quite obvious. Naturally, the regulations
ber of the European family of chemical journals. Result- are based upon results obtained from analytical testing.
ing from a collaboration between ten European chemical For this reason, Analytical and Bioanalytical Chemistry
societies and two major commercial publishers, these has a major role to play in bridging the divide between the
journals provide a combination of excellent scientific qual- needs of the chemical industry and the environment. Only
ity, reasonable subscription rates and high commercial ef- a reasonable, scientifically sound approach can provide
ficiency. They were founded seven years ago by several the compromise necessary for innovation and economic
chemical societies in continental Europe with the inten- viability within the European chemical industry whilst
tion of publishing the best chemistry from Europe and the minimising risks to the environment and human health.
rest of the world, while promoting and preserving the de- This exciting and never-ending challenge is one to which
sirable and necessary diversity of the scientific publica- the community of analytical chemists will surely contribute
tions. We are proud of the performance of the European handsomely.
system and are quite sure that Analytical and Bioanalyti-
cal Chemistry will become a major force in the near fu-
ture.
The appearance of Analytical and Bioanalytical Chem- Franois Mathey
istry is quite timely. At present, the European Commis- President of the French Chemi-
sion in Brussels is preparing a directive dealing with new cal Society
safety and handling rules for commercial chemical prod- Professor of Chemistry at cole
Polytechnique
ucts, some of which have been on the market for many
Director of Research of C.N.R.S.
years without any problems. The balance between safety
in its broadest sense and the needs of the market is a deli- Member of the French Academy
of Sciences, Leopoldina Acad-
cate one, and the importance of these new rules for the emy and Academia Europaea.
chemical industry and its partners in academic and indus-
F. Mathey ()
Socit Franaise de Chimie,
250, rue Saint-Jacques, 75005 Paris, France
e-mail: francois.mathey@polytechnique.fr
Anal Bioanal Chem (2002) 372 : 23
DOI 10.1007/s00216-001-1165-y
E D I TO R I A L
Some years ago, in 1997, several European Chemical So- Society (SFC), the German Chemical Society (GDCh)
cieties decided on a very important step: They agreed to and Springer-Verlag. At the moment of writing I know
join forces, to sacrifice their national general chemistry that our Spanish colleagues, also, will very soon become a
journals and to create the commonly owned and edited Eu- member of the owners group and will merge their journal
ropean Journals. This was decades after the Rome Treaty Qumica Analtica. And many others, with no own na-
establishing the European Community (1958), and even tional journal to merge, will be invited to become partners
the more difficult step of bringing about the European of Analytical and Bioanalytical Chemistry. European co-
Union was achieved earlier in 1992 with the Maastricht operation and European ownership have nothing to do
Treaty. So, politics was more courageous than science? with a geographic delimitation: the authorship, readership
Yes and No. Yes, in the sense of supporting a necessary and the visibility of science published in Analytical and
development to overcome too weak and small-scale struc- Bioanalytical Chemistry will be world-wide.
tures. No, if one compares the irreversible and complete During the 20th century the art of synthesis occupied a
merger of the old, traditional journals with the small quanta premier position in chemistry. Analytical chemists in Ger-
of sovereignty transferred by individual nations to the many frequently complained about ever lesser impact of
central European authorities. their field in the university curricula or about the low es-
The concept of natural sciences lies in the search for teem of their work in chemical companies. This was the
truth, objectivity and reproducibility. Thus science should situation yesterday; synthesis may still dominate as long
develop along lines which do not depend on national bor- as mass products and commodities are profitable for clas-
ders and similar barriers. In reality, progress in science has sical chemical industries and craftsmens techniques pre-
unfolded in parallel to societies, sometimes in a national vail in education. But tomorrow added value will largely
direction and sometimes la mode. depend on analytical science. Product liability and quality
Once born and realised, the idea of creating European management, GLP and GMP, accreditation and certifica-
Journals set further steps in motion. The British Royal So- tion all these are terms that demonstrate the leading role
ciety of Chemistry built bridges to the continent with their of the analytical sciences and their great impact on syn-
offer to form the journal Physical Chemistry Chemical thesis and production. Health, safety, and environmental
Physics. From the very beginning it was evident that the care are fields that relate to qualities, quantities, properties
field of analytical chemistry needed a corresponding ini- and effects, which can only be defined, determined and
tiative, although this most important scientific domain is judged with analytical tools. Modern chemical biology is
already weakened by so many specialised journals. At the a striking example of the dominance of analytical science.
worlds largest book fair in October 2000 in Frankfurt the The syntheses were developed and optimised by nature.
idea was presented to publishing colleagues at Springer- Synthetic tools to prepare GMOs are nature-borne. And it
Verlag. It took only a few weeks to develop the outline for is a mere analytical task to decipher the letters, the words
a publishing project in Analytical and Bioanalytical Chem- and the entire script of life. Genomics, proteomics and
istry. Thereafter, the merger of the French journal Analu- forthcoming even more complex omics are chemical
sis and the German journal Fresenius Journal of Analyt- and bioanalytical challenges. The efforts of mankind to
ical Chemistry was agreed on by the French Chemical influence the cellular game, for example, in drug develop-
ment, from combinatorial chemistry to clinical testing
how much of this is synthesis, how dominant is analysis!
H. tom Dieck ()
Gesellschaft Deutscher Chemiker,
Even the world of economics will soon comprehend that
Postfach 900440, 60444 Frankfurt, Germany innovative value creation depends more on the analytical
e-mail: htd@gdch.de sciences than on chemical synthesis.
3
Thus, the prospects for our new journal Analytical and Heindirk tom Dieck is the Man-
Bioanalytical Chemistry, the result of a synthesis from a aging Director of the Gesellschaft
Deutscher Chemiker, Frankfurt,
manifold of components the merged journals, the co-op- Germany, since 1991. He re-
erating societies, the experienced publisher are excel- ceived his Ph.D. in chemistry
lent. (1966) after studies at the Uni-
versities of Gttingen and Mn-
chen. After his Habilitation early
in 1971 he became an Associate
Professor of Organometallic and
Coordination Chemistry at the
Goethe University of Frankfurt/
Main and, in 1977, Full Profes-
sor and Director of the Institute
of Inorganic and Applied Chem-
istry, University of Hamburg.
Anal Bioanal Chem (2002) 372 : 45
DOI 10.1007/s00216-001-1167-9
E D I TO R I A L
Dear Readers
Welcome to the inaugural issue of Analytical and Bioana- Analytical and Bioanalytical Chemistry is published
lytical Chemistry. Three chemical societies, GDCh, SFC twice monthly under the direction of an eminent team of
and SEQA, and Springer-Verlag have joined forces to cre- six internationally renowned Editors: S. Daunert, P. Gar-
ate this new journal, which is the merger of three distin- rigues, G. Gauglitz, K.G. Heumann, K. Jinno and S.A.
guished European journals (Fresenius Journal of Analyt- Wise. You can read their informative biographical por-
ical Chemistry, Analusis and Qumica Analtica). The in- traits on the following pages. Committed to the publication
creasing need to develop fast, reliable and environmen- of high-quality and high-impact analytical research, the
tally friendly analytical procedures or even complete ana- Editors will ensure that especially innovative research pa-
lytical strategies within a very narrow time frame is a task pers are published rapidly within 8 weeks of receipt, as
which requires more than ever before the extended in- Papers in Forefront. Furthermore, it is upon their initia-
terdisciplinary cooperation between experts in the fields. tive that color images necessary to the understanding of a
Therefore, the mission of Analytical and Bioanalytical contribution will now be printed free-of-charge to the au-
Chemistry is: to establish a truly international forum for thor. Moreover, the work of the Editors will be strength-
analytical and bioanalytical science with a pronounced in- ened by a newly formed International Advisory Board. Its
terdisciplinary, and also a practice-oriented character, and members represent 20 nations as well as the most im-
to publish the information fast in print and electronic for- portant fields of analytical and bioanalytical research.
mat. In this first issue we are pleased to present a number of
Consequently, the scope of the merged journal will be special contributions. A feature article describing the chal-
broad, encompassing the entire range of analytical and bio- lenges facing entrepreneurs in bioanalytical chemistry is
analytical research and its application, including topics at followed by Trends 2002, with 17 invited overviews by
the interfaces with the materials sciences, environmental predominantly younger scientists spotlighting current
science, the life sciences, and others. It will embody and trends in diverse areas of research. Four critical reviews
encourage a multidisciplinary approach to solving analyt- then provide in-depth coverage of several topics of high
ical and bioanalytical problems. Analytical and Bioanalyt- analytical and bioanalytical interest, including the chal-
ical Chemistry will give greater coverage to the flourishing lenges of ion mobility spectrometry and the potential of ap-
and vibrant field of bioanalysis than its predecessors, both tasensors. The main part of the issue is devoted to the pre-
in regular issues and in a number of special issues. In fact, sentation of high-quality, peer-reviewed original research
the next issue will be a special issue dedicated to Biosen- papers. One of them is accompanied by a video chro-
sors and Biochips. matogram which can be viewed as supplementary elec-
tronic material under the journals homepage in Springers
LINK service (http://link.springer.de/journals/abc). The
issue closes with Industry news, which describes how new
biochemical methods are revolutionizing work in the in-
dustrial laboratory.
C.E. Dyllick () P. Enders
Analytical and Bioanalytical Chemistry, Springer-Verlag, We invite you to turn the pages of this inaugural issue
Tiergartenstrasse 17, 69121 Heidelberg, Germany and take a look at the breadth and variety of papers pub-
e-mail: abc@springer.de lished in Analytical and Bioanalytical Chemistry.
5
Christina Dyllick is Managing Peter Enders is Senior Execu-
Editor of Analytical and Bioan- tive Editor Chemistry and Food
alytical Chemistry. She has been Science with Springer-Verlag in
involved in the editing and pub- Heidelberg, Germany. He has
lishing of scientific books and been active in publishing since
journals since 1981. She received 1975. He received his education
her Ph.D. in chemistry from ETH, in chemistry, forensic science and
Zrich (1976) and her under- toxicology at the University of
graduate education in chemistry Erlangen (Germany).
from the University of Bristol
(UK).
Anal Bioanal Chem (2002) 372 : 68
DOI 10.1007/s00216-001-1168-8
E D I TO R S
Analytical and Bioanalytical Chemistry is prepared under the leadership of six eminent analytical and bioanalytical sci-
entists. We invite you to meet the Editors of the new journal in the following biographical portraits.
Philippe Garrigues is currently a CNRS Research Director and the Head of the
Environmental and Toxicological Chemistry Laboratory (UMR 5472 CNRS) at the
University of Bordeaux I, France. He received his degree in Chemical Engineering at
the University Louis Pasteur (Strasbourg) in 1978 and his Ph.D. degree in 1985 in
Physical Chemistry at the University of Bordeaux I. He also received a degree in Ma-
rine Ecology and Biology in 1979.
Dr. Garrigues research interests are the analytical aspects (chromatographic and
spectroscopic) related to the detection of organic pollutants and the fate of these com-
pounds in the environment. In particular, he has demonstrated the capability of low
temperature spectrofluorimetry (Shpolskii Effect) for the analysis of aromatic com-
pounds. In cooperation with colleagues in toxicology, he has developed the use of bio-
chemical markers as early warning systems for the evaluation of the ecosystem health
of aquatic ecosystems through the coordination of large research projects (BIOMAR,
BEEP), mainly supported by DG Research (European Commission, Brussels).
Dr. Garrigues has authored about 150 publications. He has been the President of
ISPAC (International Society of Polycyclic Aromatic Compounds; 19971999) and
member of the Board of Directors of SETAC-Europe (Society of Environmental Tox-
icology and Chemistry ; 19961998). He chaired the International Symposia on Poly-
cyclic Aromatic Compounds (ISPAC) (Bordeaux ; 1991 and 1999) ) and was the Sci-
entific chair of the 8th SETAC-Europe Meeting (Bordeaux, 1998). He is also the
French representative in the Division Chemistry in the Environment of the Federa-
tion of the European Chemical Societies (FECS), and is a member of various scien-
tific committees at the national (French Ministries of Research, Environment, Indus-
try ; CNRS) and European level dealing with analytical, toxicological and environ-
mental aspects. He also presently serves as Editor-in-Chief for the journal Polycyclic
Aromatic Compounds and is a member of the editorial boards of various international
journals dealing with analytical and environmental chemistry.
7
Gnter Gauglitz is Professor in the Institute of Physical and Theoretical Chemistry
at the Eberhard - Karls - University, Tuebingen, Germany, since 1987. He received his
Master of Science in 1966, at the State University of Iowa, and his Ph.D. degree in
1972, in Tuebingen, Germany. Since 1992, he is a titular member of the IUPAC com-
mission V.4 in the Analytical Chemistry Division, and has chaired this commission
since 2000. In 2001, he was elected a member of the IUPAC Division of Analytical
Chemistry. Since 1994, Dr. Gauglitz is a Member of the Graduiertenkolleg Analyti-
cal Chemistry at the University of Tuebingen, and acts as chairman for this institution
since 2000.
In 1997, he received the Wallac Award of the Society of Biomolecular Screening for
his research in fluorescent high-throughput screening. He is a member of the board of
the Deutscher Arbeitskreis fr Angewandte Spektroskopie (DASp), and is chairman
for the working group of Chemometrics and Lab Data Processing within the Analyt-
ical Chemistry Division of the Gesellschaft Deutscher Chemiker (GDCh). For the
past two years he has chaired the working group Chemical and Biochemical Sensors.
He is a member of the Editorial Board of the journals Trends in Analytical Chemistry
and Chemometrics and Intelligent laboratory Systems.
At present, there are over 20 Ph.D. and Master students in his research group, work-
ing in the field of UV absorption and fluorescence spectrometry, in the polymeriza-
tion of silicon oligomers, photochemical processes in photochromic systems and
liquid crystals, spectral refractometry in micro volume sizes and in the develop-
ment of various types of optical detection principles for chemical and biochemical
sensing, such as reflectometric interference spectroscopy, surface plasmon resonance,
integrated optical Mach-Zehnder devices, total internal reflexion fluorescence, and
fluorescence resonance energy transfer. He uses spectral ellipsometry to characterize
interphases of polymers and biomembrane surfaces applied to the detection and quan-
tification of molecular and biomolecular interaction, such as, the measurement of
volatile organic compounds in combination with various methods of multivariate data
analysis using chemometrics, and in the field of biosensing. Antibody and antigene
interactions are used in the case of pesticides and estrogens, and recently DNA and
protein interaction have been added, providing methods for high-throughput screen-
ing in the area of drug screening, as well as aiming toward the areas of genomics and
proteomics. The scientific results of his research group have been published in over
185 publications, many book chapters, and 3 monographs and have also been pre-
sented in many lectures at national and international conferences, resulting in many
proceedings and short communications. In addition, more than 10 patents have been
obtained in the fields of actinometry, integrated optics, interferometry, and high-
throughput screening devices.
Stephen A. Wise is the Leader of the Organic Analytical Methods Group within the
Analytical Chemistry Division of the National Institute of Standards and Technology
(NIST), Gaithersburg, Maryland, USA. He received a B.A. in Chemistry from Weber
State University (Ogden, Utah) and a Ph.D. in Analytical Chemistry from Arizona
State University (Tempe, Arizona). Dr. Wise joined NIST (then the National Bureau
of Standards) in 1976 as a research chemist working primarily in the area of chro-
matographic separations. His research interests have focused on: (1) the study of re-
tention and selectivity in reversed-phase liquid chromatography; (2) the development
of chromatographic methods for the determination of organic pollutants (e.g., poly-
cyclic aromatic hydrocarbons, polychlorinated biphenyls, and chlorinated pesticides)
in environmental matrices such as sediment, tissue, and air particulate matter; (3) the
development of Standard Reference Materials for trace organic constituents in envi-
ronmental, clinical, and food matrices; and (4) environmental specimen banking pro-
cedures. He has authored or co-authored over 220 publications in these areas.
Dr. Wise has been associated with Fresenius Journal of Analytical Chemistry since
1991, first as a member of the Advisory Board, and since 1995 as Regional Editor for
North America. He is currently the Topical Editor for Analytical Separation Tech-
niques for Polycyclic Aromatic Compounds. Dr. Wise served as Chairman of the
Division of Analytical Chemistry of the American Chemical Society in 1996 and as
a member of the Editorial Advisory Board for Analytical Chemistry (19962000)
and the Journal of Microcolumn Separations (19912001). Since 1988 he has been an
adjunct faculty member in the Department of Chemistry at Georgetown University
(Washington, DC).
Anal Bioanal Chem (2002) 372 : 911
DOI 10.1007/s00216-001-1143-4
F E AT U R E A RT I C L E
Patricia E. Eisenhardt
As our economy is increasingly dominated by globaliza- 2001: Integration, Jan Leschly, Chairman and CEO of Care
tion, at a time when connectivity is rapidly erasing age-old Capital, LLC recommends that biotech companies think
geographic and cultural boundaries, a remarkable thing is about...going up the value chain themselves, before going
happening in the business world. There beneath the shadow to big pharma [3].
of world-wide corporate conglomerates, cottage industry Often, the same dynamics that affect the business cli-
is resurgent...literally. mate for biotechnology also affect that of bioanalytical
Inc. 500, the 2000 ranking of the fastest-growing pri- chemistry, bringing similar opportunities for company
vate companies in America, reports that 61% of the com- formation. In an interview with the Wall Street Corporate
panies ranked were started in the founders home, as op- Reporter, Peter Kissinger, Chairman and CEO of Bioana-
posed to 39% of companies listed in the Inc. 500 of 1995 lytical Systems, Inc. explains: there is...an ecosystem with
[1]. Many of these companies are high-tech and yet, over regards to drug development where pharmaceutical com-
half had initial start-up capital of less than $20,000 (USD). panies...have numerous partnerships with biotechnology,
The interesting point here is that some of the most vigor- drug discovery, development and specialty chemical com-
ous companies around today are those that start out small panies to whom they will outsource discovery, toxicology,
and independent. Even in the life-sciences sector it is evi- clinical trials (etc.). According to Kissinger, the bottom
dent that there are forces at work that favor success of line is that this outsource approach will be far more pro-
small ventures. Now, this is not necessarily to suggest that ductive than building the infrastructure to do everything
you, the bioanalytical chemist, go set up a laboratory in internally[4]. So it is that big pharma scouts for small spe-
your kitchen, but rather to emphasize that you are blessed cialized shops that might be strung together in a network
with many viable opportunities for bioentrepreneurship. of resources that collectively can make a new drug a suc-
Looking out from the grassy knoll of expansive re- cess in the market.
search parks where pharma giants roam free, this idea of a This climate is one to which the classical bioanalytical
cottage industry might seem incredulous. One has only to chemistry business model of a contract research organiza-
glance at Focus on Fundamentals: The Biotechnology Re- tion is well suited. Traditionally, this is a boutique busi-
port, Ernst & Youngs 15th Annual Review, to see abundant ness, wherein small independent laboratories boast of highly
alliances among pharma and biotech that include record- specialized and diverse capabilities. In recent years, some
breaking deals. In todays global marketplace, cross-bor- have formed alliances and manage to run impressive full
der consolidation is generally accepted as a key to sur- service operations. Industry expert Alex MacDonald of
vival and growth, but inherent in this situation is a fasci- North Caldwell, NJ remarks that, irrespective of size, con-
nating twist. Authors Scott Morrison and Glen Giovan- tract research firms succeed by making investments in
netti explain that ...this global network has allowed com- people, technology, and the information systems to man-
panies from smaller regional markets to grow and com- age it all [5]. Return on these investments tends to take
pete [2]. So, as quickly as more mature life science com- the form of a fee for service or revenue from sales of prod-
panies are consolidating, new ones are emerging and seem ucts such as software, reagents, or instruments originally
to promise to achieve even greater growth and vertical in- made for in-house use. While one can derive a healthy in-
tegration than their forerunners. In European Life Sciences come in this way, the ebb and flow of demand drives many
to search for alternative, stabilizing sources of cash flow.
As one expands ones view of ways in which bioanalyt-
P.E. Eisenhardt () ical chemistry capability could generate revenue, new op-
ChipRx, Inc., Columbus, OH, USA portunities become apparent. For example James Scozzie,
e-mail: eisenhardt@columbus.rr.com President of Ricerca, LLC explains that his firm is giving
10
and often make great personal and financial sacrifices for A founder and Vice President of
the sake of future prosperity. Business start-up is challeng- ChipRx, Inc., Patricia Eisenhardt
facilitates daily operations and
ing, so it is important to build as good a home as possible strategic corporate development.
for it, even if it is only a modest cottage at first. Now, if Her work in the life sciences has
youll excuse me, I must go tend my work, to keep the spanned from bench to hospital
home fires burning. bedside, and from the teachers
desk of a high school classroom
to the halls of a high-tech busi-
ness incubator. Central to Patri-
References cia Eisenhardts career is her de-
sire to enhance the value and ac-
1. Gendron G (2000) (ed) Inc. 22:6465 cessibility of scientific discover-
2. Ernst and Young (2001) Focus on fundamentals: the biotech- ies to society, and thus to im-
nology report, Ernst and Youngs 15th annual review. Ernst and prove quality of life
Young, LLP, New York, NY
3. Ernst and Young (2001) European life sciences 2001: integra-
tion. Ernst and Young, Cambridge, UK
4. OHanlon J (2001) Wall Street Corporate Reporter 6:14
5. Interview notes. Alex MacDonald may be reached at
BeeMac201@aol.com
6. Interview notes. More information is available at http://www.
ricerca.com
7. Interview notes. More information is available at http://www.
labbook.com
8. More information is available at http://www.chiprx.com
9. Young R (2001) Nature Biotechnol 19:BE9-BE10
10. Peters T (2001) Fast Company 44:124140
Anal Bioanal Chem (2002) 372 : 1213
DOI 10.1007/s00216-001-1163-0
TRENDS
Andrew J. de Mello
Miniaturization
In his now celebrated lecture on the 29th December 1959 technologies from conventional to microfluidic (chip-
Richard Feynman pondered the potential of miniaturiza- based) formats. In particular, huge leaps in the efficiency
tion in the physical sciences [1]. His vision, based on and application of separation techniques have been facili-
known technology, examined the limits set by physical tated by miniaturizing column dimensions and creating
principles and proposed a variety of new nano-tools in- monolithic fluidic networks on planar substrates. Indeed,
cluding the concept of atom by atom fabrication. In the a survey of the current literature demonstrates that almost
intervening decades, many of these predictions have be- all separation methods (based on electrophoretic or chro-
come reality; microelectronic systems have shrunk to matographic partitioning mechanisms) have been success-
sizes close to the molecular level, scanning probe micro- fully transferred to micromachined formats [3].
scopes (e.g. STM and AFM) enable us to image and ma- Small volumes of sample and reagent (pLnL) are rep-
nipulate individual atoms, and the molecular machinery resentative of most miniaturized systems. This character-
of living systems is now being more fully understood and istic has clear advantages associated with cost and analyt-
harnessed. ical throughput, but does pose constraints on appropriate
Surprisingly, it is only within the last decade that the detection methods. Consequently, much research has re-
concepts of miniaturization have been seriously applied to cently focused on the development of detection tech-
chemical and biological problems. Of particular focus and niques that are highly sensitive, universal, miniaturized,
interest has been the development and application of lab- and cost-effective. Although the vast majority still utilize
on-a-chip technology. These microscale analytical instru- fluorescent schemes (because of exceptional sensitivity)
ments employ micromachined features (such as channels, other approaches to on-chip detection have been intro-
electrodes, reactors, and filters) and are able to manipulate duced. These include electrochemical and electrochemilu-
fluid samples with high precision and efficiency. Micro- minescent methods, absorption, indirect fluorescence, re-
fluidic chip devices have been used in a wide variety of fractive index variation, plasma, and thermal conductivity
applications including nucleic acid separations, protein methods [4]. Although many novel detection methods
analysis, process control, small-molecule organic synthe- have been reported for on-chip applications, future studies
sis, DNA amplification, immunoassays, DNA sequenc- must focus on improving concentration detection limits
ing, and cell manipulations [2, 3]. In a fundamental sense, and complete integration of the detector with the rest of
chip-based analytical systems have been shown to have the analytical system (Fig. 1).
many advantages over their conventional (larger) ana- Of notable current interest is the use of microfabricated
logues. These include improved efficiency with regard to structures for chemical and biological synthesis. Because
sample size, response times, cost, analytical performance, of the unique environments afforded within microfluidic
process control, integration, throughput, and automation. networks, a variety of synthetic processes can be per-
Much of the pioneering work in microfluidics has fo- formed in continuous flow and batch formats. Increased
cused on the successful transfer of established analytical efficiencies of mixing and separation combined with high
rates of thermal and mass transfer make microreactors
ideal for processing valuable or hazardous reaction com-
ponents and improving reaction selectivities (thus yield-
A.J. de Mello () ing higher quality products). DNA amplification, com-
AstraZeneca/SmithKline Beecham Centre binatorial (and high throughput) chemistry, immuno- and
for Analytical Sciences, Department of Chemistry,
Imperial College of Science, Technology and Medicine,
bioassays, and tissue and cell culture are areas which have
Exhibition Road, South Kensington, London SW7 2AY, UK all recently benefited from advances in microfluidic chip
e-mail: a.demello@ic.ac.uk technology [2, 5, 6].
13
Fig. 1 An integrated analysis system
in which DNA and reagent solutions
are placed on the device and elec-
tronic signals corresponding to ge-
netic information are the primary out-
put. Image taken from Ref. [8]
One of the primary advantages and goals of microfab- beyond those originally defined in the early nineteen-
ricating analytical instruments is the large-scale integra- nineties. The first commercial devices have been intro-
tion of functional components on a single monolithic de- duced to market (see Table 1 in Ref. [3]), and it is almost
vice. Indeed, the ability to extract the required informa- certain that the base technologies defined over the past
tion from a chemical or biological system almost always decade will have a high future profile in the fields of pro-
involves performing a number of distinct analytical oper- teomics, drug discovery, cellomics, clinical diagnostics,
ations. Consequently, a complete device will ideally in- genomics, and chemical production [10].
clude all relevant steps in a complete analytical procedure
(including sample handling, sample pre-treatment, reac-
tion, analytical separation, analyte detection, and product References
isolation). Although, this objective is widely recognized
few studies have yet embraced the concept of integration 1. Feynman RP (1960) Eng Sci 23:22
fully. Of these the focus has primarily centered on the am- 2. Krishnan M, Namasivayam V, Lin R, Pal R, Burns MA (2001)
Curr Opin Biotech 12:92
plification and analysis of DNA fragments [2, 7, 8]. Fu- 3. Jakeway SC, de Mello AJ, Russell EL (2000) Fresenius J Anal
ture work in this area will need to address integration in a Chem 366:525
more general sense and solve problems associated with 4. Schwarz MA, Hauser PC (2001) Lab-on-a-Chip 1:1
materials compatibility and the handling of real-world 5. Mitchell MC, Spikmans V, Manz A, de Mello AJ (2001)
J Chem Soc Perkin Trans 1:514
samples. It is also likely than many future microfluidic 6. Jensen KF (2001) Chem Eng Sci 56:293
devices will be constructed from polymeric materials 7. Woolley AT, Hadley D, Landre P, de Mello AJ, Mathies RA,
(rather than silicon or glass), because of the wide avail- Northrup MA (1996) Anal Chem 68:4081
ability of base materials and highly cost-effective fabrica- 8. Burns MA, Johnson BN, Brahmasandra SN, Hanique K, Web-
ster JR, Krishnan M, Sammarco TS, Man PM, Jones D, Held-
tion methods (such as injection molding and hot-emboss- singer D, Mastrangelo CH, Burke DT (1998) Science 282:484
ing) [9]. 9. Becker H, Gartner C (2000) Electrophoresis 21:12
It is fair to say that the envisaged applications of mi- 10. Sanders GWH, Manz A (2000) Trends Anal Chem 19:364
crofluidic analysis chips have undoubtedly expanded well
Anal Bioanal Chem (2002) 372 : 1415
DOI 10.1007/s00216-001-1156-z
TRENDS
Yoshinobu Baba
Genomics
technology based on microchip and nanotechnology, will 3. Baba Y, Figeys D (2001) Genomics and proteomics for analyt-
be launched and enable us to obtain the huge amounts of ical chemists. John Wiley and Sons, New York
4. Vulkmirovic OG, Tilghman SM (2000) Nature 405:820822
information required for personalized medicine. 5. Service RF (2000) Science 287:19541956
6. Pandey A, Mann M (2000) Nature 405:837846
7. Roses AD (2000) Nature 405:85786
References 8. Evans WE, Relling MV (1999) Science 286:487491
9. Sander C (2000) Science 287:19771978
1. International Human Genome Sequencing Consortium (2001) 10. Heller MH, Guttman A (2001) (eds) Integrated microfabricated
Nature 409:860921 device technologies: advances in genomic, drug discovery, and
2. Venter, JC et al. (2001) Science 291:13041351 clinical diagnostic applications. Marcel Dekker, New York
Anal Bioanal Chem (2002) 372 : 1617
DOI 10.1007/s00216-001-1149-y
TRENDS
Gunther Wittstock
Electrochemical sensors are based either on the measure- microelectrode arrays, with a larger inter-electrode dis-
ment of a potentiometric, impedimetric, capacitive, ampero- tance, from a variety of electrode materials [2, 3]. The
metric, or voltammetric response of a specifically designed commercially available RAM electrodes (random assem-
electrode. This contribution considers transducers based bly of microelectrodes) also fall in this category [4]. An
on amperometric and voltammetric principles. In contrast important application is sensors for anodic stripping analy-
with other detection principles these signals are generated sis of heavy metals which make use of the effective mass
by ongoing electrochemical conversion of the species to be transport during the electrolytic deposition process. De-
detected. If such sensors are fabricated with one dimen- tection in flow systems would be another field in which
sion smaller than the diffusion length of the analyte, their such structures have potential advantages because their
performance might be dramatically improved because of: response to fluctuations in the flow rate is much less sen-
sitive than that of macroscopic electrodes. Parallel ultra-
1. increased mass transport towards the transducer (radial
microelectrode arrays with small inter-electrode distances
diffusion);
behave similarly to conventional macroscopic electrodes
2. reduced double-layer capacity (small electrode area re-
on the typical time-scales used for sensors, because the
sults in lower background current); and
diffusion layers of the individual electrodes overlap. Dra-
3. reduced Ohmic drop (small currents).
matically improved detection limits have, however, been
The term ultramicroelectrode is used to characterize elec- demonstrated with electrode arrays template-synthesized
trodes with such properties. The use of such miniaturized from track-etch membranes [5], because of their extremely
systems in modern electroanalytical chemistry follows three
basic strategies (Fig. 1a):
An assembly of such microelectrodes is operated in par-
allel (array sensors) to exploit the advantageous response
properties of such electrodes while increasing the cur-
rent to a level easily measurable even outside a labora-
tory environment (Fig. 1a).
Two comb-shaped arrangements are interdigitated to en-
able redox recycling between the two combs acting as
anode and cathode (Fig. 1b).
Independent addressable sensors are used to construct
multianalyte sensors (sensor arrays; Fig. 1c).
There is already a rather large body of literature dealing
with the fabrication and characterization [1] of array sen-
sors. Recent reports describe efforts to produce parallel
G. Wittstock ()
Carl von Ossietzky University Oldenburg,
Department of Chemistry (FB9) and Institute of Chemistry Fig. 1 Schematic diagrams of: (a) in-parallel-operated ultramicro-
and Biology of the Marine Environment (ICBM), electrodes (array sensor), (b) an interdigitated electrode array for
PF 2503, 26111 Oldenburg, Germany sensitivity enhancement by redox recycling, and (c) independently
e-mail: gunther.wittstock@uni-oldenburg.de addressable ultramicroelectrodes (sensor arrays)
17
low double-layer capacity. Such electrodes are, further- out for suppression of cross-talk is another fundamental
more, also intensively investigated as microcompartmen- problem. Cross-talk in electrochemical enzymatic sensor
talized electrodes [6]. Such surfaces unite segregated mi- arrays occurs if a product of an enzymatic reaction, e.g.
croscopic domains for different functions, for example sup- H2O2, diffuses to the neighboring array element causing a
port of biomolecular layers and bare regions for efficient signal even though the corresponding analyte is not pre-
electrochemical detection [7]. Sensors based on such prin- sent. Strategies used to avoid this situation include:
ciples can have very short response times.
use of interference-suppressing enzyme layers (e.g., con-
Interdigitated electrode arrays (IDA) consist of two elec-
taining catalase to decompose H2O2) [12];
trodes, one serving as a generator, the other operating as a
electrolysis of the interference-causing compound at
collector at which a reversible or quasi-reversible redox
band electrodes placed between the individual sensors
couple can undergo multiple oxidation reduction cycles.
[13]; or
The different redox forms are transported by diffusion be-
splitting the sample flow into independent parallel
tween the adjacent band electrodes. Theory predicts a dra-
flows before reaching the individual sensors [14].
matic increase of sensitivity as the inter-electrode distance
is reduced and the number of band electrodes per given
area is increased [8]. The integration of biological recog-
References
nition elements adjacent to or between the electrode array
has also been advanced. Redox-active species generated 1. Wittstock G, Grndig B, Strehlitz B, Zimmer K (1998) Elec-
by an label enzyme (e.g. 4-aminophenol formed by alka- troanalysis 10:526531
line phosphatase) are then redox-recycled at the IDA. Ef- 2. Feeney R, Kounaves SP (2000) Electroanalysis 12:677684
3. Suzuki H (2000) Electroanalysis 12:703715
fort is currently being devoted to the construction of ar- 4. Fletcher S, Horn MD (1999) Electrochem Commun 1:502512
rays of IDA for the parallelization of such measurements 5. Menon VP, Martin CR (1995) Anal Chem 67:19201928
[9]. 6. Ratcliff BB, Klancke JW, Koppang MD, Engstrom RC (1996)
Arrays of individually addressable electrodes can be Anal Chem 68:20102014
used to determine different analytes or to provide spatially 7. Nowall W, Dontha N, Kuhr WG (1998) Biosensors Bioelec-
tronics 13:12371244
resolved measurements. Exposing the cross sections of 8. Aoki K, Morita M, Niwa O, Tabei H (1988) J Electroanal
bonding wires of conventional electronic circuits provides Chem 256:269282
convenient access to individually addressable electrode 9. Hintsche R, Albers J, Bernt H, Eder A (2000) Electroanalysis
arrays in explorative research [10]. Although modification 12:660665 (and references cited therein)
10. Matos RC, Augelli MA, Lago CL, Angnes L (2000) Anal Chim
of the individual electrodes with different biological Acta 404:151157
recognition elements would enable the construction of 11. Wittstock G (2001) Fresenius J Anal Chem 370:303315
miniaturized biosensor arrays, the directed immobiliza- 12. Urban GA, Jobst G (1997) In Scheller FW, Schubert F,
tion of functional proteins on individual microscopic re- Fedrowitz J (eds) Frontiers in biosensorics, vol 2. Birkhuser,
Basel, pp 161171
gions is still a challenge. Scanning electrochemical mi- 13. Strike DJ, Hengstenberg A, Quinto M, Kurzawa C, Koudelka-
croscopy (SECM) has become an ideal tool both for pro- Hep M, Schuhmann W (1999) Mikrochim Acta 131:4755
totyping of such structures and for the analysis of differ- 14. Kurita R, Tabei H, Hayashi K, Horiuchi T, Torimitsu K, Niwa
ent aspects of their performance [11]. Optimizing the lay- O (2001) Anal Chim Acta 441:165174
Anal Bioanal Chem (2002) 372 : 1819
DOI 10.1007/s00216-001-1151-4
TRENDS
Joseph J. Dalluge
Abbreviations MALDI-MS Matrix-assisted laser spectra to database sequences allow monitoring of the ex-
desorption ionization mass spectrometry GPC gel pression of any protein from any organism for which the
permeation chromatography SNP single nucleotide corresponding genome has been defined. In addition,
polymorphism MALDI-MS has the capability of identifying posttransla-
tional modifications that alter the mass of a protein. As
such, MALDI-MS is being employed to characterize pro-
teinprotein interactions, cellular compartments, and sig-
Matrix-assisted laser desorption ionization mass spectrom- naling pathways. Recent review articles detailing these
etry (MALDI-MS) [1] is one of two modern mass spec- endeavors are available to the interested reader [2, 3, 4].
trometric techniques (the other being electrospray ioniza- Some of the most compelling applications of MALDI-MS
tion mass spectrometry) that have, over the past decade, involve direct characterization of genes themselves, partic-
become essential tools in the analytical and life sciences ularly single nucleotide polymorphism (SNP) analysis [5].
laboratories. Because the early history of MALDI-MS, SNPs are the most abundant type of variation found in the
discussions of instrumental and experimental design, and human genome. As such, they are becoming popular ge-
proposed ion desorption mechanisms are detailed in netic markers for genome mapping studies, medical diag-
nearly every reference cited herein, these topics will not nostics, and identity testing. MALDI-MS has been demon-
be reviewed. Instead, this article will focus on selected strated as a promising tool for high-throughput screening
scientific endeavors that rely on MALDI-MS as a stan- of SNPs [5, 6], with the potential to become the method of
dard tool for identification and characterization of the choice for direct genetic analysis. In addition, MALDI-MS
wide variety of analytes that are measurable using this has been employed effectively for peptide nucleic acid
technique. While this overview by no means encompasses (PNA) characterization [7]. PNAs are nucleic acid mimics
every area of research in which MALDI-MS is involved, consisting of nucleobases connected via a peptide-like
it should illustrate the versatility of the technique, and the backbone. Because PNAs are resistant to nuclease and pro-
leading role MALDI mass spectrometry plays in the mod- tease digestion, they are being investigated as gene thera-
ern analytical laboratory. peutic drugs, and are employed in a variety of important
MALDI-MS is a proven technology in the emerging hybridization technologies [8].
field of proteomics (functional genomics). With the eluci- Identification of microbes in their natural environment
dation of the human genome complete, efforts are now has great importance in the diagnosis of disease, and char-
shifting toward the large-scale analysis of the function of acterization of biological and environmental hazards. The
genes that is regulation of protein expression. Genome se- use of MALDI-MS for identification of bacteria based on
quence information has provided a comprehensive re- the measurement of cellular proteins has shown great
source for protein identification using data produced by promise [9], but the limitations of microorganism identifi-
MALDI-MS. New methods for direct identification of pro- cation using mass spectrometry are recognized [9, 10].
teins in mixtures using enzymatic proteolysis, MALDI-MS, Future development of improved methodologies and data
and computer algorithms designed to match peptide mass analysis techniques should advance MALDI-MS as a vital
technology in this area of research.
Characterization of synthetic polymer materials has tra-
ditionally been accomplished using chromatographic tech-
J.J. Dalluge ()
Central Research, Cargill, Incorporated,
niques such as gel permeation chromatography (GPC).
P.O. 5702, Minneapolis, MN 554405702, USA Molecular weights of polymers obtained using GPC are
e-mail: joseph_dalluge@cargill.com calculated based on relative comparison with molecular
19
TRENDS
Frank Vanhaecke
ICPMS
Alternative means for the elimination of interferences
The success of ICPmass spectrometry is the result of its strated for certified reference materials of different origin
low limits of detection (LOD), wide linear dynamic range, [2]. Up to 60 elements could be determined in whole blood
multi-element capabilities, surveyable spectra, high sam- after microwave-assisted acid digestion [3]. Increasing the
ple throughput and ease with which it can be used with al- number of elements for which reliable data can be ob-
ternative sample-introduction systems and chromatographic tained obviously leads to enhanced diagnostic capabilities
separation techniques. Very soon after the introduction of and a more profound insight into the consequences of en-
ICPMS, it became clear that non-spectral (matrix-in- vironmental or occupational exposure. For soil digests also,
duced signal suppression or enhancement) and spectral a multitude of spectral interferences complicate analysis
(overlap of the signals from ions with a mass difference with quadrupole-based ICPMS (ICPQMS), whereas ap-
<0.5 u) interferences were its most prominent disadvan- plication of higher mass resolution enables elegant avoid-
tages. Whereas non-spectral interferences could be fairly ance of these problems [4]. The only limitations are that
easily coped with e.g., by means of sample dilution, the for a small minority of analyses the maximum resolution
use of (an) internal standard(s) or application of standard offered by this instrumentation (up to 12,000) is still in-
additions or isotope dilution instead of external calibration sufficient, and there is an inverse relationship between
spectral interferences proved more troublesome. Espe- mass resolution and ion transmission efficiency (signal in-
cially for complex matrices and in the low mass-range tensity).
(80 u), obtaining reliable results or sufficiently low LOD The success of ICPSFMS has, apparently, also trig-
is, therefore, not straightforward. Of course, efforts were gered important developments in ICPQMS instrumenta-
made to cope with this problem and, from the beginning, tion, the dynamic reaction cell (DRC) [5] and, to a lesser
blank subtraction, mathematical correction or, if really nec- extent, the hexapole and octopole collision cells [6] being
essary, matrix/trace separation were used to warrant accu- the most appealing. These devices enable suppression of
racy. Later, also improved aerosol desolvation devices and the signal intensity of unwanted ions by orders of magni-
cool plasma conditions were used. tude as a result of the (selective!) reaction of the latter
The only universal means of overcoming spectral over-
lap was, however, using a double-focusing sector field
mass spectrometer in ICPMS instrumentation, instead of
the more traditional quadrupole filter. Sector field mass
spectrometers offer a much higher mass resolution than
do quadrupole filters, and so ions differing in mass by a
fraction of a mass unit only can still be separated (Fig. 1)
and accurate quantification becomes straightforward [1].
As a result, sector field ICPMS (ICPSFMS) is better
suited for analyzing demanding matrices. Accurate deter-
mination of the first-row transition metals a mass range
especially prone to spectral overlap was, e.g., demon-
with an appropriate gas. New unwanted ions can, unfortu- high mass or chemical resolution. It can, of course, be
nately, also be created in this way. To avoid this, the gases assumed that for ICPSFMS the purchase price (approxi-
used with a hexapole or octopole collision cell are limited mately twice that of an ICPQMS instrument) is often a
to H2 and noble gases (mainly He for thermalization), so major obstacle. Larger numbers of sales will probably be
the number of spectral interferences that can be tackled is achieved if the analytical community is convinced that
relatively limited. With the quadrupole set-up of the DRC, ICPSFMS is sufficiently robust and that instrument op-
on the other hand, the cell can be operated simultaneously as eration is not extremely complicated. The success of
a band-pass mass filter, removing these newly formed ion DRCICPMS which was only introduced at the end of
species from the beam. As a result, in a DRC a large(r) va- the nineties on the other hand, will mainly depend on
riety of gases (e.g., also NH3, CH4 or O2) can be used and the availability of data on the selective ionmolecule re-
the application range becomes virtually unlimited. Chem- actions that have to be used to tackle specific problems
ical resolution in a DRC can thus be used not only to over- and the maintenance of a purchase price that is suffi-
come more traditional spectral overlap (e.g., 40Ca+/40Ar+, ciently different from (i.e. lower than) that of a sector
56Fe+/40Ar16O+ or 75As+/40Ar35Cl+) which can also be field unit.
avoided by using a collision cell [6] but even to resolve The first ICPSFMS instrument equipped with a hexa-
the signals of isobaric isotopes, which would require a mass pole collision cell was introduced very recently. This im-
resolution substantially exceeding the maximum mass res- plies that the analyst has both high mass-resolution and
olution offered by modern ICPSFMS instrumentation. In chemical resolution at his or her disposal. A combination
this context, the possibility for Sr isotopic analysis of ge- of a sector field mass spectrometer and a DRC within the
ological samples without previous Rb/Sr separation has same instrument would, on the other hand, result in the
been demonstrated [7] (isobaric overlap of the signals from most effective and versatile ICPmass spectrometer ever
87Rb+ and 87Sr+ was avoided by selectively converting the produced. For this to become reality, however, some tech-
Sr+ ions into the corresponding SrF+ species by use of nical problems must be overcome, and difficulties related
CH3F). to patents owned by different (competing) manufacturers
To exploit the advantages of DRCICPMS fully, how- can be expected. Even if the latter issue could be resolved,
ever, a collection of gases and considerable operator expe- a marketing study assessing the interest from the analyti-
rience are required. It should, on the other hand, be stressed cal community and the money available for this instrumen-
that the use of NH3 as a single reaction gas enables elim- tation would probably be very useful.
ination of the most abundant interferences and enables
ng L1 LOD for traditionally difficult elements such as K,
Ca, and Fe. Advantages of mass resolution over chemical References
resolution are: 1. the straightforwardness of the former
approach; 2. the direct, visual detection of the presence of 1. Jakubowski N, Moens L, Vanhaecke F (1998) Spectrochim Acta
B 53:17391763
(molecular) ions that would interfere with the determina- 2. Townsend AT (2000) J Anal At Spectrom 15:307317
tion at low resolution and the possibility to estimate their 3. Rodushkin I, Axelsson MD (2000) Sci Total Environ 250:83
importance (Fig. 1); and 3. the simultaneous solution for 100
spectral interferences by different types of interfering ions. 4. Latkoczy C, Prohaska T, Stingeder G, Wenzel WW (2000) Fre-
senius J Anal Chem 368:256262
ICPSFMS instrumentation is, however, significantly 5. Tanner SD, Baranov VI (1999) At Spectrosc 20:4552
more expensive than ICPQMS instrumentation equipped 6. Feldmann I, Jakubowski N, Thomas C, Stuewer D (1999) Frese-
with a DRC; this might explain why the number of DRC- nius J Anal Chem 365:422428
units sold so far is starting to approach the number of 7. Moens LJ, Vanhaecke FF, Bandura DR, Baranov VI, Tanner SD
(2001) J Anal At Spectrom, in press
ICPSFMS instruments used world-wide.
Despite the undeniable advantage, only a minority
(10%) of the ICPMS instruments used worldwide offer
Anal Bioanal Chem (2002) 372 : 22
DOI 10.1007/s00216-001-1152-3
TRENDS
Karen W. Phinney
Chiral separations
The pharmaceutical industry has largely been the driving charged cyclodextrins are the most common chiral selec-
force behind the development of chiral separation tech- tors in CE, but new additives are also being identified [5].
niques. This focus helps to explain the growth in the area Capillary electrochromatography (CEC) has not played a
of preparative scale enantioselective separations in recent major role thus far; however, the lure of high peak effi-
years. A few years ago, preparative scale chromatographic ciency associated with CE coupled with chromatographic
separations would not have been considered a cost effective separation mechanisms continues to fuel research in this
approach to enantiomer production. However, the transition area. Continuous free flow electrophoresis (CFFE) is a re-
from traditional batch processes to simulated moving bed cent addition to the field of chiral separations. This tech-
(SMB) technology has dramatically changed the acceptance nique seeks to exploit the resolving power of electrophore-
of chromatography for large-scale production. Both batch sis for a continuous feed process [6]. As in CE, a chiral
and SMB methods utilize chiral stationary phases (CSPs) to additive is incorporated into the background electrolyte.
achieve the desired separation. Although most systems in- Applications of supercritical fluid chromatography
corporate liquid eluents, the use of gases and supercritical (SFC) to enantioselective separations have also expanded
fluids as eluents has also been reported [1, 2]. because SFC offers an attractive alternative to liquid chro-
Evolution continues to occur in the area of CSPs, pri- matographic techniques. Replacement of traditional liquid
marily for liquid chromatography (LC). The desirable prop- eluents with supercritical fluids commonly results in in-
erties of CSPs can differ significantly depending upon their creased efficiency, simplified method development, and
intended use. On one hand, stationary phases possessing reduced waste generation [7, 8]. In general, the CSPs used
enantioselectivity towards a broad spectrum of compounds for enantioselective separations in SFC are the same as
are advantageous for analytical scale separations. This prop- those used in LC.
erty reduces the number of different columns that must be Additional types of separation procedures, such as
evaluated to achieve the desired separation. Extremely high membrane-based approaches, are gaining attention for enan-
stereoselectivity merely increases analysis time to unde- tioseparations [9]. Clearly, no single technique can address
sirable levels. On the other hand, a stationary phase with a all the current challenges, and the need for high through-
very high level of enantioselectivity towards a single tar- put as well as low cost will likely continue to steer future
get is often preferable for preparative scale separations. developments in chiral separations.
Combinatorial chemistry is increasingly being used to iden-
tify chiral selectors with high levels of enantioselectivity
for a target compound. Suitable chiral selectors are identi- References
fied by screening mixture libraries [3] or individual library
compounds [4] against the target. 1. Francotte ER (2001) J Chromatogr A 906:379397
2. Juza M, Mazzotti M, Morbidelli M (2000) Trends Biotechnol
Capillary electrophoresis continues to grow in popular- 18:108118
ity for enantioselective separations, although reproducibil- 3. Bluhm LH, Wang Y, Li T (2000) Anal Chem 72:52015205
ity and sensitivity remain issues of concern. Neutral and 4. Welch CJ, Bhat G, Protopopova MN (1999) J Comb Chem 1:
364367
5. Chankvetadze B, Blaschke G (2001) J Chromatogr A 906:309
363
K.W. Phinney () 6. Stalcup AM, Sutton RMC, Painuly P, Rodrigo JV, Gratz SR,
Analytical Chemistry Division, Yanes EG (2000) Analyst 125:17191724
Chemical Science and Technology Laboratory, 7. Phinney KW (2000) Anal Chem 72:204A211A
National Institute of Standards and Technology, 8. Terfloth GJ (2001) J Chromatogr A 906:301307
Gaithersburg, MD 20899, USA 9. Maier NM, Franco P, Lindner W (2001) J Chromatogr A 906:
e-mail: karen.phinney@nist.gov 333
Anal Bioanal Chem (2002) 372 : 2324
DOI 10.1007/s00216-001-1146-1
TRENDS
Ulrich Panne
therefore, becoming a routine tool in microcolumn sepa- tion, but can already be seen in several real-world appli-
ration and analysis of single (bio)molecules [8]. A further cations.
impact is observed in fluorescence microscopy, where multi-
photon LIF and fluorescence correlation spectroscopy (FCS)
with femtosecond-lasers have enabled the creation of a new References
user-friendly generation of these lasers [9]. Advances in
1. Imasaka T (1999) Talanta 48:305
near-field microscopy with LIF (and other laser-based 2. Gooijer C, Mank AJG (1999) Anal Chim Acta 400:281
techniques such as Raman spectroscopy) will provide a 3. Panne U, Niessner R (1997) In: Gnzler H, Bahadir AM, Bors-
nano-scale spectrochemical analysis beyond the confocal dorf R, Danzer K, Fresenius W, Galensa R, Huber W, Lin-
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lytiker Taschenbuch, Vol. 16, Springer, Berlin, p 155
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5. Gnther D, Jackson SE, Longerich HP (1999) Spectrochim
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6. Rusak DA, Castle BC, Smith BW, Winefordner JD (1998)
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7. Winefordner JD, Gornushkin IB, Pappas D, Matveev OI, Smith
laser Raman spectroscopy since the mid 1960s, it was not BW (2000) J Anal At Spectrom 15:1161
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10. Rigler R, Elson E (2001) (eds) Fluorescence correlation spec-
Raman microscopy, process analysis with NIR diode lasers, troscopy. Springer, Berlin
and UV resonance Raman at wavelengths below 255 nm 11. McCreery RL (2000) Raman spectroscopy for chemical analy-
are not only currently receiving serious academic atten- sis. John Wiley and Sons, New York
Anal Bioanal Chem (2002) 372 : 2526
DOI 10.1007/s00216-001-1158-x
TRENDS
Hyphenated techniques
TRENDS
Uwe Karst
Few analytical techniques have reached such a high degree the analytes that can be determined with the best sensitiv-
of maturity and applicability for routine analysis as liquid ity in LC/MS. On the other hand, analytes of low polarity
chromatography (LC). From this point of view, it may be are only susceptible to these methods with moderate per-
surprising that there has been such a dynamic develop- formance. One of the major challenges for the future is
ment with respect to modern detection techniques for liq- therefore the development of LC/MS methods and instru-
uid chromatography. On the other hand, due to the tremen- mentation that are capable of analyzing substances with
dous impact of LC separations on such diverse topics as limited polarity at extremely low concentrations.
the purification of pharmaceuticals, clinical chemistry or Very recently, some approaches for expanding the range
environmental speciation analysis, the need for sensitive of applications to less polar compounds have been pub-
and selective detectors becomes increasingly apparent. lished. Bruins et al. [7] have introduced atmospheric pres-
In the 1990s, several detectors for LC were invented sure photoionization (APPI) to liquid chromatography/mass
or greatly improved; these included the evaporative light- spectrometry. The technique allows one to carry out sen-
scattering detector as a universal device [1]. The com- sitive determination of non-polar compounds, e.g., poly-
bination of LC and nuclear magnetic resonance (NMR) cyclic aromatic hydrocarbons that can hardly be detected
spectroscopy allows one to carry out the structural eluci- under ESI or APCI conditions. Blair et al. [8] have inves-
dation of analytes [2]. Laser-induced fluorescence (LIF) tigated pentafluorobenzyl derivatives of biomolecules and
detection of native or labeled analytes has become an im- drugs and have observed dissociative electron capture
portant tool for bioanalysis at very low concentration lev- ionization with excellent sensitivity. Therefore the trans-
els [3]. Despite these and other significant achievements, fer of derivatization techniques based on multihalogenated
the progress in liquid chromatography/mass spectrometry derivatization reagents, which are frequently applied in gas
(LC/MS) is outstanding [4]. chromatography with electron-capture mass spectrometry
Undoubtedly, the development of electrospray ioniza- [9] appears to be possible. For nitroaromatic compounds,
tion (ESI) [5] and atmospheric pressure chemical ioniza- similar electron capture effects are observed and have been
tion (APCI) [6] has triggered a revolution in the field of used for the development of derivatizing agents [10].
detection in liquid chromatography, because LC/MS is Another method of ionizing substances of limited po-
now readily available as a routine tool for pharmaceutical, larity is their electrochemical oxidation at the ESI inter-
biomedical and environmental analysis as well as for com- face [11] or an additional electrochemical cell [12]. Dedi-
binatorial chemistry. In just over a decade, LC/MS has cated derivatizing agents have been developed to be used
completely lost its former reputation of being an expen- with electrochemistry/ESI-MS [11]. Brajter-Toth and co-
sive method of limited sensitivity which could only be ap- workers [13] performed on-line LC/particle beam-MS stud-
plied by specialists. As protonation and deprotonation are ies of the reaction products formed in an electrochemical
the most abundant ionization mechanisms when using ESI cell. Very recently, a LC/electrochemistry/MS system based
and APCI in the positive and negative mode, respectively, on the quantitative post-column oxidation of non-polar
polar or even charged analytes can be readily analyzed. analytes to charged products was realized [14].
For this reason, selected groups of biomolecules are among In the coming years, it can be expected that there will
be an intense development of methods for the LC/MS
analysis of less polar substances. All of the approaches
U. Karst () presented above have strong advantages for selected
University of Twente, Faculty of Chemical Technology,
Chair of Chemical Analysis,
groups of analytes, but only few can be used universally
P.O. Box 217, 7500 AE Enschede, The Netherlands as they may either require particular properties of the an-
e-mail: u.karst@ct.utwente.nl alytes (e.g., electrochemical activity) or a derivatization
28
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techniques will become important complements to ESI and Anal Chem 57:675
6. Covey TR, Lee ED, Henion JD (1986) Anal Chem 58:2453
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9. Giese RW (2000) J Chromatogr A 892:329
10. Hayen H, Jachmann N, Vogel M, Karst U (2001) German
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Anal Bioanal Chem (2002) 372 : 2930
DOI 10.1007/s00216-001-1153-2
TRENDS
Bernd Speiser
Molecular electrochemistry
Electroanalytical chemistry traditionally involves: ments was recently presented [4]. A dendrimeric polymer
with redox-active Ru(tpy)2 groups was adsorbed on an
1. detailed investigation of processes, including electron
electrode, and the diffusion process through the molecules
transfer, at electrodes (molecular electrochemistry), and
(diameter5 nm) was dissected in the manner of an elec-
2. determination of concentrations of redox-active species
trochemical microtome.
by electrochemical means.
Much UME work has recently been devoted to electro-
Common to both sub-fields is the use of the same arsenal analytical experiments in media of low conductivity [5].
of electroanalytical techniques. This overview will mainly This opens the way to electrochemistry in non-polar me-
deal with sub-field 1. Fairly broad coverage of the entire dia or even in the gas phase [2].
field can be found bi-annually elsewhere [1]. Selected The use of electroanalytical techniques in molecular
trends which have emerged recently and are expected to electrochemistry is often accompanied by simulation of
develop into important lines of research and applications experiments. Thus, a physical model of the real processes
will be highlighted here. (transport, electron transfer, chemical reactions, adsorp-
First, the use of ultramicroelectrodes (UME, i.e. elec- tion) is formulated in the form of mathematical (often dif-
trodes with a characteristic dimension <25 m [2], or those ferential) equations and solved [6]. The result predicts the
characterized by dimensions comparable with or smaller experimental outcome on the basis of the physical model,
than the diffusion layer thickness) has influenced electro- and, by comparison with the real experiment, enables qual-
analytical experiments in a variety of ways. Their minute itative testing of the validity of the model and quantitative
size enables investigation of electrochemical processes in evaluation of mechanistic information.
small lateral dimensions. Thus, local redox properties can General application of simulation in the context of cyclic
be probed, and an important development in this respect is voltammetry was boosted a decade ago by DigiSim [7],
the scanning electrochemical microscope (SECM), in the first commercial software based on graphical user in-
which the UME is scanned along a surface. Although the teraction, which enables the formulation of the physical
resolution of the SECM is, by necessity, limited to the di- model in the convenient form of chemical equations. The
mensions of the diffusion layer, and is thus less than that sophistication of numerical solution methods continues to
of other scanning probe techniques, its main advantage is increase [8, 9] and the development of computing facili-
related to the flow of a Faradaic current which results ties and, in particular, the internet has initiated new possi-
from a defined redox process. Consequently, the redox ac- bilities. Artificial intelligence approaches using a knowl-
tivity of the surface can be separated from its topography edge-based system to elucidate reaction mechanisms have
and is imaged, e.g. over biological samples [3]. been described [10]. Compton et al. provide a web-based
Another advantage of the UME was recognized early service for the evaluation of kinetic data [11]. Virtual in-
because of the small iR drop and double-layer charging strumentation for electroanalytical experimentation en-
effects, high scan rate voltammograms (v>1 MV s1) are hances data generation and analysis [12]. Such applica-
available without such artifacts. One of the most striking tions certainly qualify as facets of complex software which
applications of the thin diffusion layers and high temporal has been introduced as a problem solving environment
resolution attainable with such short time-scale experi- in electrochemistry [13]. It is expected that software of
this type will increasingly be developed in the future. In
the opinion of this author, this development could benefit
B. Speiser ()
Institut fr Organische Chemie,
even more from open and co-operative development of
Auf der Morgenstelle 18, 72076 Tbingen, Germany modular software integrating the approaches of different
e-mail: bernd.speiser@uni-tuebingen.de authors.
30
6. Speiser B in Bard AJ, Rubinstein I (1996) (eds) Electroanalyti-
References cal chemistry, vol 19. Marcel Dekker, New York, pp 1108
7. Rudolph M, Reddy DP, Feldberg SW (1994) Anal Chem 66:
1. Anderson JL, Coury LA Jr, Leddy J (2000) Anal Chem 72: 589A600A
44974520 8. Bieniasz LK, Britz D (2001) J Electroanal Chem 503:141152
2. Heinze J (1993) Angew Chem 105:13271349; Angew Chem 9. Harriman K, Gavaghan DJ, Houston P, Sli E (2000) Elec-
Int Ed Engl 32:12681288 trochem Commun 2:150156
3. Hengstenberg A, Blchl A, Dietzel ID, Schuhmann W (2001) 10. Pays MJ, Bos M, van der Linden WE (1993) Anal Chim Acta
Angew Chem 113:942946; Angew Chem Int Ed Engl 40: 283:811829
905908 11. Alden JA, Compton RG (1998) Electroanalysis 10:207209
4. Amatore C, Bouret Y, Maisonhaute E, Goldsmith JI, Abrua 12. Economou AS, Volikakis GJ, Efstathiou CE (1999) J Autom
HD (2001) Chem Eur J 7:22062226 Method Manag Chem 21:3338
5. Myland JC, Oldham KB (2000) Anal Chem 72:39723980 13. Bieniasz LK (1997) Comput Chem 21:112
Anal Bioanal Chem (2002) 372 : 3132
DOI 10.1007/s00216-001-1148-z
TRENDS
Detlef Gnther
The first application of laser-ablation inductively-coupled action is less matrix-dependent if UV lasers are used (im-
plasma mass spectrometry (LAICPMS) by Gray (1985) proved absorption). This initiated studies using all avail-
marked the creation of one of the most versatile direct able UV laser wavelengths (quadrupled 266 nm, quintu-
solid-sampling techniques for both qualitative and quanti- pled 213 nm Nd:YAG and excimer wavelengths such as
tative analysis in the last 16 years [1]. This method prof- 308 nm, 248 nm, 222 nm, 193 nm, and 157 nm) for
ited from early work performed in laser-induced break- LAICPMS. This increased flexibility led to new appli-
down spectroscopy (LIBS) and studies in laser-ablation cations. In addition to progress in laser technology, instru-
inductively-coupled plasma optical-emission spectrome- mental developments in ICPMS, especially higher sensi-
try (LAICPOES) [2, 3]. tivity, increased linear dynamic range, and the greatly im-
From the beginning several keywords, e.g. rapid, proved the speed of data acquisition, are further break-
cheap, easy to use, bulk to micro-scale, trace ele- throughs in laser ablation which enabled simultaneous de-
ment, quasi-non-destructive, no sample preparation, tection of major and trace elements with a single mea-
and quantitative analysis, were applied to LAICPMS. surement. The amount of sample preparation is, however,
The general principle and the advantages of this technique related to the spatial resolution required. For many appli-
have not changed over the last two decades. A laser beam cations determination of minor, trace, and ultra-trace con-
is directed on to the surface of a sample in an air-tight ab- centrations at limits of detection in the low ng g1 range
lation cell. Interaction of the laser beam with the sample (depending on the spatial resolution) by use of matrix-
leads to particles and/or vapour, which are transported by matched and non-matrix-matched reference materials, has
a carrier gas flow into the ICP. The ions produced in the ICP been reported [5, 6]. Unfortunately, until now quantitative
are extracted through the vacuum interface and analysed analysis has required knowledge of the concentration of an
by use of a variety of mass analysers and different detec- internal standard determined by another technique before
tor systems. the analysis. Beside the progress in LAICPMS, ele-
A few years after Grays initial work the most severe mental fractionation [7] became the dominant keyword
drawback of this technique, the non-sample-related varia- with regard to calibration and accuracy. Despite many
tion of the relative signal response during analysis (so-called successful approaches and even quantification without
elemental fractionation) was observed and the lack of ref- standards (possible when all matrix elements can be mea-
erence materials for calibration was seen as a limitation of sured simultaneously during analysis [8]), discussions on
this technique. This led to loss of some of the keywords LAICPMS remain focused on elemental fractionation.
mentioned above, e.g. quantitative analysis. A milestone The source of this phenomenon, which could occur during
report by Jackson et al. in 1992 [4] contained quantitative the ablation process, during transport, or during atomisa-
results for a variety of elements in minerals, obtained by tion and excitation, is still unknown. The number of vari-
use of an in-house-built laser-ablation system (1064 nm ables determining the performance of LAICPMS makes
Nd:YAG laser), with non-matrix matched calibration. it very difficult to evaluate the individual effect of each.
In the early nineties the first quadrupled 266 nm Recent work by Russo et al. [9] has shown that changing
Nd:YAG laser was used to show that lasersample inter- the laser conditions directly affects the analytical charac-
teristics. The transport efficiency is related to the carrier
gas [10] and to the laser wavelength [11] but is less influ-
D. Gnther () enced by the transport system [12].
Eidgenssische Technische Hochschule Zrich,
Laboratorium fr Anorganische Chemie,
The ion source used in LAICPMS was originally de-
ETH Hnggerberg, HCI G113, 8093 Zrich, Switzerland signed for solution nebulisation and not for dry-aerosol
e-mail: guenther@inorg.chem.ethz.ch introduction. Modifications of a set-up used for solution
32
Fig. 1 Limits of detection
of a commercially available
ICPMS for a selection of ele-
ments. Improved expansion
characteristics lead to en-
hanced sensitivity and reduced
background in a modified in-
strument coupled to laser abla-
tion (laser conditions were kept
constant)
References
1. Gray AL (1985) Analyst 110:551
2. Moenke-Blankenburg L (1989) Laser microanalysis. John Wiley
and Sons, Toronto
Fig. 2 Th/U correlation plots for a transient signal acquired by use 3. Moenke L (1993) Spectrochim Acta Rev 15:1
of a 193-nm excimer laser system with different ICPMS instru- 4. Jackson SE, Longerich HP, Dunning GR, Freyer BJ (1992)
ments. The ablation conditions were maintained constant for both Can Min 30:1049
experiments. The slopes of approx. 1 and 2 acquired under opti- 5. Becker JS, Tenzler D (2001) Fresenius J Anal Chem 370:637
mum operating conditions are indicative of ICP plasma-based ele- 6. Durrant SF (1999) J Anal At Spectrom 14:1385
mental fractionation. 7. Fryer BJ, Jackson SE, Longerich HP (1995) Can Min 33:303
8. Leach AM, Hieftje GM (2000) J Anal At Spectrom 15:1121
9. Russo RE, Mao XL, Borisov OV, Liu HC (2000) J Anal At
Spectrom 15:1115
nebulisation can lead to dramatic improvements in the de- 10. Mank A, Mason P (1999) J Anal Atom Spectrom 14:1143
tection capability, as illustrated in Fig. 1. The portion of 10. Eggins SM, Kinsley LPJ, Shelley JMG (1998) Appl Surf Sci-
elemental fractionation occurring in the ionisation source ence 129:278
is, therefore, unknown. The signal response from a 193 nm 11. Gnther D, Heinrich CA (1999) J Anal At Spectrom 14:1369
ArF Excimer laser using two different ICP, shown in 12. Bleiner D, Gnther D (2001) J Anal At Spectrom 16:449
Fig. 2, is indicative of an influence of the ion source on el-
Anal Bioanal Chem (2002) 372 : 3334
DOI 10.1007/s00216-001-1159-9
TRENDS
J. Bettmer
Elemental speciation
Interest in elemental or metal speciation has increased sig- for monitoring irregularities during the analytical process
nificantly in analytical chemistry since the importance of seems to be isotope-dilution mass spectrometry (IDMS),
the determination of individual species was recognised1. well-known as a relatively accurate and precise definitive
The identity of the species present has a decisive effect on method, as demonstrated recently in the determination of
the role and properties of a metal in living organisms and methylmercury [3]. Although degradation of methylmer-
in dead matter. To acquire more knowledge about the ef- cury to elemental mercury could be observed under some
fects and pathways of essential and toxic elements highly conditions, species-specific IDMS enabled accurate analy-
sophisticated, sensitive and selective analytical methods sis of methylmercury.
have been developed. Major target species include organo- During recent years the speciation of biometals com-
metallic compounds (e.g. methylmercury, butyltin), redox plexed with proteins and peptides, etc., has become a very
systems (e.g. Cr(III)/Cr(VI), As(III)/As(V)), and com- important task for analytical chemists [9]. Because com-
plexes of metals and metalloids with biomolecules (e.g. pletely characterised standards which can be used in the
metallothioneins). This paper briefly emphasises two de- same way as well-defined alkyl- or aryl-metal compounds
velopments in analytical chemistry which have started to are rare, this field demands a different approach and, as a
make important contributions to the field of elemental consequence, analysis of these metal-containing biomole-
speciation. cules is more complex. The application of multidimen-
ICPMS (inductively coupled plasmamass spectrom- sional liquid chromatography (LC) or a combination of LC
etry) is well known as a sensitive, element-selective, and and capillary electrophoresis (CE) is essential to guaran-
multi-element detector in trace and ultra-trace speciation tee the purity of the separated compounds entering the de-
analysis [2]. The analytical possibilities of ICPMS are tection system(s). In addition to the element-selective in-
unique compared with other (optical) detectors, partly be- formation about the metalloproteins given by ICPMS as
cause of the ease with which it can be coupled to chro- detector, evaluation of the molecular structure requires
matographic or electrophoretic separation, and partly be- additional mass spectrometric coupling. Electrospray MS
cause it is possible to monitor the isotopes of the element has proven to be an essential tool for further identification
of interest. This additional information enables essential of macromolecules bound to metals [10], and the applica-
investigations on artefact formation and species transfor- tion of more than one detector will significantly improve
mations during sample pre-treatment to be performed us- our knowledge about the nature of metal complexes with
ing isotopically labeled species [3, 4, 5, 6]. One example biomolecules in the near future. These investigations should
in which transformations could influence the quality of re- be performed in an interdisciplinary environment to guar-
sults in speciation analysis is the determination of methyl- antee progressive and effective continuation of our under-
mercury in certified reference materials, a topic about standing the role of metals in our life.
which there is some controversy [7, 8]. A promising tool
1 Definitions
of the terms species, speciation, and speciation analysis References
have been recommended by the IUPAC [1].
1. Templeton DM, Ariese F, Cornelis R, Danielssen LG, Muntau
H, van Leeuwen HP, Lobinski R (2000) Pure Appl Chem 72:
14531470
J. Bettmer () 2. Szpunar J, McSheeny S, Pole K, Vacchina V, Mounicou S,
Johannes Gutenberg-Universitt Mainz, Rodriguez I, Lobinski R (2000) Spectrochim Acta B 55:779
Duesbergweg 1014, 55099 Mainz, Germany 793
e-mail: bettmer@mail.uni-mainz.de 3. Demuth N, Heumann KG (2001) Anal Chem 73:40204027
34
4. Hintelmann H, Falter R, Ilgen G, Evans RD (1997) Fresenius J 8. Bloom NS, Evans RD, Hintelmann H, Wilken RD (1999) Anal
Anal Chem 358:363370 Chem 71:575A
5. Ruiz Encinar J, Monterde Villar MI, Gotor Santamaria V, Gar- 9. Lobinski R, Chassaigne H, Szpunar J (1998) Talanta 46:271
ca Alonso JI, Sanz-Medel A (2001) Anal Chem 73:31743180 289
6. Hill SJ, Pitts LJ, Fisher AS (2000) Trends Anal Chem 19: 10. Chassaigne H, Vacchina V, Lobinski R (2000) Trends Anal
120126 Chem 19:300313
7. Quevauviller P, Horvat M (1999) Anal Chem 71:155A-156A
Anal Bioanal Chem (2002) 372 : 3536
DOI 10.1007/s00216-001-1154-1
TRENDS
TRENDS
Abbreviations MIT Molecular imprinting technology tion technology the gain of using MIPs lies in the ability
MIP molecularly imprinted polymer CEC capillary to perform demanding separations, e.g. enantiomer sepa-
electrochromatography SPE solid phase extraction rations, by using one of the tricky analytes as imprint
molecule in the preparation of the MIP. Bulk polymerised,
ground, sieved and slurry packed MIP HPLC columns have
been used for a broad range of demanding separations [5].
Molecular imprinting technology (MIT) offers a means of Recently, also capillary electrochromatography (CEC), that
preparing materials with cavities that are able to recognise offers improved separation efficiency when compared to
a certain molecule in terms of shape, size and chemical HPLC, has been used successfully in combination with
functionality. In order to obtain a highly selective recog- MIPs [6]. The miniaturised separation channels used in
nition of a certain molecule, this molecule is incorporated this technique offers a diminution in chemical consump-
during the synthesis of the material as a template. When tion both in the preparation of the MIP, and then espe-
the synthesis is completed, the molecule is extracted, thus cially the precious template, and in the analysis. Also, the
leaving behind a three dimensional physical and chemical miniaturised format has forced new MIP formats to be de-
imprint of itself. The first appearance of MIT dates back veloped [7, 8, 9]. However, so far the expectations of the
to the early 1930s [1] and the material used was silica MIP-CEC system in terms of efficiency have not been
gels. In the 1940s the work mainly constituted fundamen- fully achieved. During the past few years much effort has
tal studies on the mechanism of antibody antigen interac- been put into the development of MIP materials for solid
tions using MIT to prove the hypothesis that the antibody phase extraction (SPE) [10]. MIP-SPE has been shown
adapted its selectivity first upon presentation to the anti- superior to ordinary SPE and liquid-liquid extraction in its
gen [2]. This hypothesis of antibody formation was later ability to produce much cleaner extracts [11]. The selec-
proven wrong, and as limitations of the silica material for tivity and the stability of the MIP also offer faster method
MIT were identified, there was a decline in MIT research. development and cost efficiency. Leakage of imprint mol-
In 1972 covalent MIT was introduced by Wulff at al. [3] ecule from the MIP is a problem that is arising from the
and in the early 1980s a successful preparation of a mole- difficulties in removing all of the template used in the
cularly imprinted polymer (MIP) using non-covalent MIT synthesis from the resulting polymer. This problem, that
was presented by Mosbach et al. [4]. These techniques of- can be disastrous when MIP-SPE is used in trace analysis,
fered a means of preparing molecular imprints in organic can be circumvented by the use of an analogue to the im-
matrices (Fig. 1). The limitations of the silica material, i.e. print molecule in the synthesis [11]. Using the template
stability and reproducibility, were avoided and the interest analogue approach the difficulties in finding a pure sam-
in MIT accelerated to yield an almost exponential increase ple of the analyte to be used as template can be circum-
in published papers on MIT during the past few years. vented. Apart from the more commonly used MIP based
The first works with these molecularly imprinted poly- separation techniques mentioned above, MIPs have also
mers concerned chromatographic separations. In separa- been used in thin layer chromatography [12], membrane
separations [13] and bubble fractionation [14]. The anti-
body analogue features of the MIP have introduced it as a
complement to the biological antibodies in different types
P. Spgel L. Schweitz S. Nilsson () of binding assays [10]. Another application of the MIP
Technical Analytical Chemistry,
Center for Chemistry and Chemical Engineering,
that is growing in interest is the use of the MIP as the
Lund University, PO Box 124, SE-221 00 Lund, Sweden recognition element in sensors [15]. Using a general de-
e-mail: Staffan.Nilsson@teknlk.lth.se tection method, e.g. a mass sensitive sensor, the high se-
38
Fig. 1 Schematics of MIP
preparation. The non-covalent
approach: (A) Functional
monomers, cross-linking
monomers, radical initiator and
imprint molecule are mixed in
a proper solvent to allow com-
plexes to form between the
functional monomers and the
imprint molecule. (B) The
functional monomers are
locked in position by the poly-
merisation reaction, and (C) af-
ter having extracted the imprint
molecule (D) the MIP is able
to recognise the imprint mole-
cule. The covalent approach:
(A) Imprint molecules with
substitutions of polymerisable
groups are mixed with cross-
linking monomers and radical
initiator in a proper solvent and
polymerisation is initiated.
(C) After completed polymeri-
sation the covalent bonds be-
tween the imprint molecule
and the MIP can be broken and
(D) the MIP is able to rebind
the imprint molecule utilising
covalent bonds
TRENDS
Valrie Camel
Extraction techniques
Over the past few years, efforts of the analysts focused on samples [4]. Of interest for certain applications is also stir
the development of extraction techniques that allow effi- bar sorptive extraction (SBSE) due to its low cost and
cient extraction along with reduced solvent volumes and simplicity [5]. This technique can be considered as a vari-
time, and a high level of automation. The recent extrac- ant of SPME, as a stir-bar covered with a polymeric phase
tion techniques available, along with the traditional ones, is put into the sample, and partitioning of the solutes en-
are presented below (Fig. 1). ables their extraction. Analysis is further performed after
When extracting liquid samples, the traditional liquid- thermal desorption. However, both techniques are limited
liquid extraction faces several limitations, namely use of an by the nature of the sorbent phase available, thus restrict-
extractant non-miscible with the sample, difficulty in ex- ing their applications. In addition, extractions are rarely
tracting polar and ionic compounds from water, large or- complete due to the low volume of the sorbent phase.
ganic solvent volumes resulting in a diluted extract. To pre- For solid matrices, from which solutes are difficult to
vent these major drawbacks, two major techniques have extract due to the frequently strong interactions with the
merged: solid-phase extraction (SPE) and solid-phase mi- matrix, the traditional techniques (Soxhlet and ultrasonic
croextraction (SPME). In SPE [1, 2], the sample is perco- extractions) now face severe competition due to the de-
lated through a solid phase which retains the solutes of in- velopment of supercritical fluid extraction (SFE), pressur-
terest. They are further eluted with a small solvent vol- ized fluid extraction (PFE) and microwave-assisted ex-
ume. This technique offers the unique advantages of high traction (MAE). As the extraction temperature is a crucial
concentration of the final extract, selectivity, as well as parameter, these recent techniques afford the opportunity
wide choice of the solid phase enabling the extraction of of elevated temperatures. In that way, rapid and efficient
virtually all compounds from aqueous or organic matrices. extractions can be performed due to enhanced solubilities,
In addition several systems are now available (manual mul- higher diffusion coefficients, lower viscosities, as well as
tiple cartridge systems, disks and multiwell plates) with better desorptions of the solutes from the matrix (when
possible automation and coupling to chromatography. The solutes are stable under high temperatures). The interest in
main drawback is the sometimes non-compatibility of the SFE [1, 6], which uses supercritical fluids as the extrac-
final extract solvent with the analytical system, leading to tant (with properties intermediate between those of gas
a solvent evaporation and further dissolution of the residue and liquids), has dramatically decreased in the past five
in an acceptable solvent. This problem is overcome by the years, in spite of its unique elevated selectivity. This is
use of SPME, which is a real non-solvent technique [1, 3]. due to the high dependence of the extraction conditions on
A fiber, covered with a polymeric phase, is put into the the sample, leading to fastidious optimization procedures
sample or its headspace, and the solutes partition between and difficulty in using this technique routinely. The lower
the sample and the phase. Desorption into a gas chromato- interest in SFE is also partly due to the development of
graph injector enables analysis of the solutes to be carried PFE [1, 7], which uses a liquid solvent as the extractant.
out. Recently, SPME coupling with liquid chromatography The liquid state is maintained under elevated temperatures
has appeared, which should increase the use of this tech- upon application of a moderate pressure. Optimization of
nique. In addition, SPME may also be used for gaseous extraction conditions is facilitated as organic solvents rec-
ommended in traditional techniques may be used most of
the time and pressure has a low influence. In addition, de-
V. Camel () veloped systems offer a high level of automation. Yet,
Institut National Agronomique Paris-Grignon, only one extraction at a time can be conducted. On the other
Laboratoire de Chimie Analytique,
16 rue Claude Bernard, 75231 Paris Cedex 05, France hand, MAE [1, 8] affords the opportunity of performing
e-mail: camel@inapg.inra.fr several simultaneous extractions with closed-vessel sys-
40
Fig. 1 Main extraction tech-
niques for gaseous, liquid and
solid samples
TRENDS
Alexander Jung
sulted from the combination of modern optical technology but also dynamic measurement of association and dissoci-
with, for instance, direct immobilization of DNA features ation are performed online. Sub-nanomolar concentra-
within a standard DNA-synthesizer by use of micro-mir- tions could be measured in label-free, flow-through hy-
ror arrays [9]. bridization experiments.
The availability of this technology for simultaneous,
time-resolved detection of hundreds of hybridization
Detection methods events on a DNA chip is an achievable goal in the near fu-
ture.
The complex analytical questions that must be solved by
DNA chips lead to the demand for improvement of the in-
formation depth of a DNA chip experiment. On commer- References
cially available systems array detection is achieved almost
solely by use of fluorescently labeled oligonucleotides. 1. Anderson RC, Su X, Bogdan J, Fenton J (2000) Nucleic Acids
Res 28:e60
Several instruments are available which can be used to de- 2. Freeman WM, Robertson DJ, Vrana KE (2000) Biotechniques
tect hybridization on label-free systems (biosensors), for 29:10421055
example SPR (Biacore), resonant mirror (IAsys), or re- 3. Blohm DH, Guiseppi-Elie A (2001) Curr Opin Biotech 12:41
flectometric interference spectroscopy (RIfS) [10]. Because 47
4. http://www.affymetrix.com
these surface-based detection methods do not require the 5. Beier M, Hoheisel JD (2000) Nucleic Acids Res 28:e11
use of labeled compounds, one can detect directly, in ex- 6. http://www.clondiag.com
tracts, PCR products or other complex matrices. Possible 7. Blanchard AP, Kaiser RJ, Hood LE (1996) Biosens Bioelectron
influence of the interaction through the label, influences 11:687690
8. Beier M, Hoheisel JD (1999) Nucleic Acids Res 27:19701977
of the surface on fluorescence intensity, or any unspecific 9. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F,
quenching effects or photobleaching can also be avoided. Sussman MR, Cerrina F (1999) Nat Biotechnol 17:974978
Label-free measurement enables one to keep hybrid- 10. Sauer M, Brecht A, Chariss A, Stemmler I, Gauglitz G, Bayer
ization times short, because not only endpoint detection E (1999) Anal Chem 71:28502857
Anal Bioanal Chem (2002) 372 : 43
DOI 10.1007/s00216-001-1150-5
TRENDS
John G. Westerfeld
In the early days of high throughput screening (HTS) for waste disposal and handling are safer than other methods.
drug discovery the detection of macromolecules and the Also in this area of detection is electrochemilumines-
ligands that bond to them have generally been limited to a cence, in which certain chemical compounds emit light
small number of techniques, namely absorbance (colori- when electrochemically stimulated, using ruthenium com-
metric), radiolabeling and fluorescence intensity measure- plexed ions as the progenitor.
ments. As the art and science of HTS is evolving, many Other detection methods, which have been around for
more methods for detecting molecules of interest are be- some time, are being re-tooled for applications in HTS.
ing explored. One such method is time resolved fluores- Capillary electrophoresis (CE) is undergoing research for
cence (TRF) and its progeny, homogeneous time resolved adaptation to HTS. Mass spectrometry (MS) and variations
fluorescence (hTRF) and fluorescence resonance energy of MS, such as, matrix-assisted laser desorption ioniza-
transfer (FRET) [1]. These are fluorometric methods that tion-time-of-flight MS (MALDI-TOF-MS) [6] are being
use lanthanide chelates to give a long-term emission sig- investigated as potential tools in protein and peptide mea-
nal (TRF) or closely spaced moieties in order to transfer surements in HTS. Another technology that holds hope
light between a donor and acceptor (FRET) or a combina- for the future is surface plasmon resonance (SPR) [7], like
tion of the two (hTRF). CE and MS, a label free detection method. The principle
Another fluorescent method gaining wide utilization is of SPR is that incident light of a certain wavelength that
fluorescent polarization (FP) [2], in which large and small impinges upon a metal surface to which specific mole-
molecule interactions enhance or diminish polarized fluo- cules, like a DNA fragment, are attached will interact with
rescence signal. Its chief advantage is that it is a homoge- the electrons of the metal and cause a plasmon phenome-
neous assay. One recent survey of HTS research directors non. Any molecules that bond with the attached mole-
indicates that in five years the three most widely used de- cules will shift the wavelength of the light, which can then
tection labels will all be fluorometric [3]. be measured and quantified. This is expected to have great
One more fluorescence technique that is in its incipient promise in DNA screening.
phase toward HTS is fluorescence correlation spectroscopy The future of HTS will change as the detection tech-
(FCS) [4]. The principle is to measure the fluctuations in nologies change and the detection technologies are sure to
fluorescence intensity caused by labeled molecules enter- change as the knowledge of chemistry, physics and biol-
ing and leaving a small volume defined by a sharply focused ogy continues to grow and new detection methods are dis-
laser beam. This evaluation provides information on diffu- covered.
sion times of molecules and aggregates in the sample and
number of molecules corresponding to a certain diffusion
time, which reflects molecular weight and structure. FCS References
can measure a wide range of parameters, such as, protein
1. Hemmil I, Webb S (1997) Drug Discov Today 2:373381
mass, equilibrium constants, and stoichiometry of binding. 2. Prystasy L, Gosselin M, Banks P (2001) J Biomol Screening 6:
Also on the rise as an alternative to radiometric and 141150
fluorometric based methods is the use of chemi/biolumi- 3. Fox S, Sopchak L, Khoury R, Wang H (2001) Drug Discov Dev
nescence detection [5]. Generally the light produced by 4(4)
4. Rigler R, Elson E (2001) (eds) Fluorescence correlation spec-
these reactions is as, or more, sensitive than the former troscopy. Springer, Berlin
methods with a wider dynamic range. With luminescence, 5. Roda A, Pazzali M, Kricka LJ, Stanley PE, (eds) Biolumines-
cence and chemiluminescence: perspective for the 21st Century:
proceedings of the 10th international symposium on biolumines-
J.G. Westerfeld () cence and chemiluminescence (1998) Sept. 48, Bologna, Italy,
Senior Account Specialist, BioWhittaker, Inc., John Wiley and Sons
Walkersville, MD 21793, USA 6. Sinclair B (1999) The Scientist 13:18
e-mail: John.Westerfeld@biowhittaker.com 7. www.biacore.com
Anal Bioanal Chem (2002) 372 : 4448
DOI 10.1007/s00216-001-1189-3
REVIEW
Ciara K. OSullivan
Received: 4 September 2001 / Revised: 19 September 2001 / Accepted: 2 October 2001 / Published online: 13 December 2001
Springer-Verlag 2001
Abstract Aptamers are artificial nucleic acid ligands that from the latin aptus, meaning, to fit [4]. The last decade
can be generated against amino acids, drugs, proteins and has seen the selection of an extensive range of aptamers
other molecules. They are isolated from combinatorial li- with abilities to bind to small molecules, peptides, pro-
braries of synthetic nucleic acid by an iterative process of teins, cells etc. with selectivity, specificity and affinity
adsorption, recovery and reamplification. Aptamers, first equal and often superior to those of antibodies [5, 6, 7, 8,
reported in 1990, are attracting interest in the areas of 9]. Aptamers possess the ability to discriminate targets on
therapeutics and diagnostics and offer themselves as ideal the basis of subtle structural differences such as the pres-
candidates for use as biocomponents in biosensors (ap- ence or absence of a methyl or hydroxy group or the D-
tasensors), possessing many advantages over state of the vs. L-enantiomeric configuration of the target [10]. In par-
art affinity sensors. The properties of aptamers, their ap- allel, the body of literature covering immunosensor devel-
plicability to biosensor technology, current research and opment has exploded over the last twenty years with nu-
future prospects are addressed in this short review. merous generic transduction platforms reported. How-
ever, as outlined in Table 1, antibody generation, particu-
Keywords Aptamers ELONA Aptasensors SELEX larly for use in biosensors, has several fallbacks that are
addressed by aptamers and it can be envisaged that aptasen-
Abbreviations SELEX systematic evolution of ligands sors, using already developed or novel transduction plat-
by exponential enrichment Aptasensors biosensors forms, will be increasingly exploited in the coming years.
utilising aptamers as biocomponent ELISA enzyme
linked immunosorbent assay ELONA enzyme linked
oligonucleotide assay FITC fluoroscein isothiocyanate Aptamer selection The SELEX process
lection/amplification cycle. The efficiency of enrichment the 2 positions of pyrimidine nucleotides with amino/flu-
of high-affinity binders is governed by the stringency of oro groups has been demonstrated to dramatically increase
selection of each round. To this end, negative selection (re- the half life of aptamers in biological fluids with the mod-
moval of aptamers that bind ligand support) and counter ified structures still being recognised as substrates for the
selection (removal of aptamers that bind to structures sim- enzymes utilised in the SELEX process [15, 16].
ilar to that of the target) are employed. The length of in-
cubation time of library with target used is manipulated to
yield aptamers of desired kinetics. Upon achieving affin- Aptamers in analysis
ity saturation, typically after 815 cycles of selection/am-
plification, the enriched library is cloned and sequenced a. ELONA Enzyme linked oligonucleotide assay
and individual sequences investigated for their ability to
bind to the target (e.g. using surface plasmon resonance) As antibodies are used in ELISA, aptamers may be utilised,
[13]. Aptamers can then be truncated as desired, eliminat- but with a much greater degree of flexibility in ELONAs.
ing the fixed primer sequences as well as those identified As depicted in Fig. 2, various formats can be exploited us-
not to be part of the consensus motif (i.e. sequence required ing, for example,
for binding). When the desired sequence has been identi-
aptamers as capture molecules and antibodies as reporter
fied the aptamer can be produced in sizeable quantities by
molecules
chemical synthesis [4].
antibodies as capture molecules and labeled aptamers
against the antibody-analyte immunocomplex as reporter
molecules
Nuclease resistant aptamers
aptamers as capture molecules and labeled aptamers
against the aptamer-analyte complex as reporter molecules
Unmodified oligonucleotides, especially RNA, have a very
short lifetime in biological fluids (typically <10 min) due The use of aptamers thus facilitates the detection of small
to the action of nucleases [14]. Reports have been pub- and large molecular weight analytes in a sandwich-type
lished detailing approaches to make oligonucleotide se- format overcoming the limitations of competitive-type as-
quences resistant to nucleases by modifying the oligonu- says, allowing the development of robust, sensitive assays.
cleotide backbone. However, most of these modifications The use of ELONA was patented by NexStar [17] (now
result in structures not recognised by the enzymes used in Gilead Sciences, Foster City California) in 1997 and a
the SELEX process. Recently however, modification of handful of publications have also appeared [18].
specifically in the presence of TAT-1 protein, derived from Tarragona, Spain for insightful discussions on the potential of ap-
HIV-1 or HIV-2, but not in the presence of RNA binding tasensors. The author acknowledges the financial assistance of Marie
Curie Fellowship program for funding of MCFI-200001246.
proteins and are working towards its incorporation in an
aptasensor.
References
Conclusion and outlook 1. Robertson and Joyce, GF (1990) Nature 344:467468
2. Ellington, AD, and Szostak, JW (1990) Nature 346:818822
3. Tuerk, C and Gold, L (1990) Science 249:505510
This review gives an overall introduction into the selec- 4. Jayasena, S (1999) Clin Chem 45(9):16281650
tion of aptamers and their potential uses in analysis via 5. Famulok, M (1994) J Am Chem Soc 116:16981706
ELONAs and aptasensors, outlining their advantages over 6. Haller, AA and Sarnow, P (1997) Proc Natl Acad Sci 94:8521
the state of the art in affinity based analysis and reviewing 8526
publications in the area. Despite the wealth of literature 7. Gebhardt, K, Shokraei, A, Babane, G, Lindquist, BH (2000)
Biochemistry 39(24):72557265
detailing the use of aptamers in therapeutics, the use of 8. Wilson, DS, Keefe, AD, Szostak, JW (2001) Proc Natl Acad
aptamers in in vitro diagnostics is a field that is still in its Sci 98(7):37503755
infancy with the main focus of literature currently available 9. Kawakami, J, Imanaka, H, Yokota, Y, Sugimoto, N (2001)
being on the detection of the aptamer-target binding event J Inorg Biochem 82(14):197206
10. Geiger, A, Burgstaller, P, von der Eltz, H, Roeder, A, Famulok,
using fluorescence transduction. However, with the advent M. (1996) Nucleic Acids Res 24(6):10291036
of automated platforms for aptamer selection coupled with 11. Burke, D and Gold, L (1997) Nucleic Acids Res 25(10):2020
the advances being made in increasing the stability of ap- 2024
tamers to nucleases, it is anticipated that aptamers will be- 12. Holeman, LA, Robinson, SL, Szostak, JW and Wilson, C (1998)
Folding & Design 3:423431
come increasingly accessible to researchers in the biosen- 13. Jeong, S, Eom, TY, Kim, SJ, Lee, SW, Yu, J (2001) Biochem
sor field. Biophys Res Comm 281(1):237243
The plethora of reported transduction platforms for 14. Kusser, W (2000) Rev Modern Biotech 74:2738
affinity sensing can be readily adapted to the use of ap- 15. Kujau, MJ and Wlfl, S (1997) Nucleic Acids Res 26:1851
tameric biocomponents. In addition, catalytic aptamers 1853
16. Kubik, MF, Bell, C, Fitzwater, T, Watson, SR, Tasset, DM
(aptazymes) have been reported, offering further modes of (1997) J.Immuno 159:259267
binding event detection. Moreover, it can be anticipated 17. Drolet, DW, Jayasena, SD, Gold, L (1998) US Patent 5 789
that the use of immobilisable molecular beacons will be 163
exploited in the biosensor field (to date a handful of pub- 18. Ito, Y, Fujita, S, Kawazoe, N, Imanishi, Y (1998) Anal Chem
70(16):35103512
lications exist), a mode of detection highly suited to ap- 19. Kleinjung, F, Klussmann, S, Erdmann, VA, Scheller, FW,
tasensors facilitating reagentless one step analysis. Frste, JP, Bier, FF (1998) Anal Chem 70:328331
Overall, the potential of aptasensors is immense, and 20. Potyrailo, RA, Conrad, RC, Ellington, AD, Hieftje, GM (1998)
this exciting area is on the brink of exponential growth. Anal Chem 70:34193425
21. Lee, M, Walt, DR (2000) Anal Biochem 282(1):142146
The ability to develop affinity based detection systems of 22. a. Hesselberth, J, Robertson, MP, Jhaveri, S, Ellington, AD
tailor designed characteristics, which can be applied to (2000) Rev Modern Biotech 74:1525 b. Jhaveri, S, Rajendran,
analysis of analytes, unlimited, for example, by size, tox- M, Ellington, AD (2000) Nat Biotechnol 18(12):12931297
icity and matrix effects, offers the field of biosensing the 23. a. Stojanovic, MN, de Prada, P, Landry, DW (2001) J Am
opportunity to explore new and dynamic routes of sensor Chem Soc 123:49284931 b. Stojanovic, MN, de Prada, P,
Landry, DW (2000) J Am Chem Soc 122:1154711548
development and the availability of commercial aptasensors 24. Tyagi, S and Kramer, FR (1996) Nat Biotechnol 14:3038
by the end of the decade can definitively be anticipated. 25. Yamamoto R and Kumar, PKR (2000) Genes to Cells 5:389
396
Acknowledgements The author wishes to thank Jean-Jacques
Toulm of INSERM Unit 386 at the Universit Victor Segalen II,
Bordeaux, France and Ioanis Katakis of Universitat Rovira i Virgili,
Anal Bioanal Chem (2002) 372 : 4965
DOI 10.1007/s00216-001-1191-9
REVIEW
Received: 24 September 2001 / Accepted: 23 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract Completion of the Human Genome Project is Project is driving the rapid development of molecular di-
driving the rapid development of molecular diagnostics in agnostics in the laboratory. Micromachined instrumenta-
the laboratory. To accelerate the penetration of genetic tion can revolutionize the commercialization of these di-
tests and other nucleic acid-based tests into clinical mar- agnostic tests by development of compact, potentially dis-
kets, simple, compact, automatic sample-preparation sys- posable, automated diagnostic systems that could be used
tems for molecular diagnostics must be developed. Micro- to perform molecular diagnostics in a clinical laboratory
electromechanical systems (MEMS) is a promising ap- or a point-of-care setting. Microelectromechanical sys-
proach for the development of automated sample prepara- tems (MEMS) have already permeated the molecular biol-
tion for the clinical laboratory or point-of-care setting. ogy and chemistry arenas and are having a profound im-
This review discusses MEMS-based components that pact on instrument and assay development. The principal
could be applied to the different stages of the sample- advantages associated with a BIOMEMS approach in-
preparation process such as cell separation, nucleic acid clude:
purification, and nucleic acid amplification. Examples of
1. small instrument footprint
functional component integration are given. Issues dis-
2. multiplexing so that several analyses can be completed
cussed include partitioning of functions between the in-
simultaneously
strument and disposable unit, methods of propulsion of
3. microfluidics for fluid manipulation, lessening the need
fluids and particles, vapor and liquid barriers, and sample
for traditional macro pumps
size. Although further evaluation and development are
4. potential for integrating various functional components
needed to provide practical solutions to some of these is-
on to the device
sues, we conclude that MEMS-based components might
5. reduced volume requirements for sample and reagents
contribute to some components in a sample-preparation
6. unsupervised automation
system consisting of modular instruments and disposable
units, but will not provide a generic or a totally integrated In this review we discuss MEMS contributions to sample
solution. preparation in molecular diagnostics. Specifically, we ex-
amine the feasibility of sample preparation in a small, in-
Keywords Sample preparation MEMS Molecular tegrated footprint instrument capable of performing all the
diagnostics aspects of a common DNA assay. Like packaging in the
semiconductor industry, sample preparation is an area that
has been pursued with less vigor than development of the
Introduction assay itself; perhaps it is perceived as somewhat less
glamorous just like packaging in the IC industry. But,
Critical advances in micromachining and genomics over like IC packaging, sample preparation is of overwhelming
the last decade have brought the two fields to the point importance to the end customer.
where synergy can lead to revolutionary changes in bio-
medical diagnostics. Completion of the Human Genome
Elements of sample preparation
for molecular diagnosis
Y. Huang E.L. Mather J.L. Bell M. Madou () Sample types and cell separation
Nanogen Inc., 10398 Pacific Center Court,
San Diego, California, USA 92121 Preparation of biomedical samples for DNA or RNA
e-mail: Mmadou@nanogen.com analysis prior to the detection step tends to be relatively
50
Cell lysis and nucleic acid purification amplification are isothermal, e.g. the strand displacement
amplification (SDA) developed by Becton Dickenson
After isolating the desired cells from the raw sample, the [25] and the transcription-mediated assays, TMA [26] and
cells must be lysed or disrupted to release the nucleic NASBA [27] used by Gen-Probe and Organo-Teknica, re-
acids. For blood, disruption of the nucleated white cells spectively.
can be performed by heating or by the addition of lysing A variety of signal-amplification methods have been
reagents such as detergents. With tissues, one or more en- employed in commercial assays. These methods do not
zymatic steps may also be used. A variety of approaches produce more copies of target DNA, but instead have a
can then be used to purify nucleic acids after the lysis mechanism for amplifying a reporter molecule when the
step. Traditionally DNA and RNA were purified by diges- target is present. Two examples of this type of assay are
tion of proteins with proteinase K in the presence of de- the isothermal enzymatic Invader assay developed by
tergents, extraction with phenolchloroform, and concen- Third Wave [28] and the sequencing by synthesis devel-
tration by precipitation with alcohol in the presence of salt oped by Pyrosequencing AB [29].
[12]. Today DNA and RNA are often purified using As can be seen from the discussion above, the sample
chaotropic reagents such as guanidinium hydrochloride or preparation process generally requires human intervention
guanidinium thiocyanate. This purification procedure either during a step or between steps. In addition to the
takes advantage of the observation that in the presence of cost and the effect on workflow, possibilities for contami-
the chaotropic reagent, DNA and RNA bind to silica and nation and operator error increase. It would, therefore, be
in its absence will be released [13, 14]. Non-nucleic acid desirable to develop a small-footprint instrument with
components of the cell lysates are washed away in the test-specific disposable cartridges that can perform all the
presence of the chaotropic reagent and then the purified sample-preparation steps in an automated and integrated
nucleic acid can be eluted in a small volume of water or fashion.
low-salt buffer.
The efficient lysis of many bacteria, yeast, and spores is
more difficult. Common methods for lysing gram-negative MEMS-based sample-preparation
bacteria include treating the cells with alkaline buffer, with functional components
heat, or with guanidinium thiocyanate [15, 16]. Gram-pos-
itive bacteria are more difficult to lyse efficiently. A vari- By adapting microfabrication techniques in IC to biologi-
ety of enzymes that are specific for gram-positive cell-wall cal applications, a variety of miniaturized devices have
components have often been used to assist in the lysis been developed as sample-preparation functional compo-
process. The enzymatic step is often followed by addition nents. They will be discussed in the following sections ac-
of detergent and then heating [17, 18]. Other approaches cording to their function in the sample-preparation process.
include boiling in the presence of a Chelex-100 (Bio-Rad)
[19] or the use of the bead beater, produced by Biospec
Products, in the presence of 100 L beads and Chelex-100 Cell separation
[20]. Sonication has also been used to lyse bacteria and
fungi [21, 22]. It should be noted that efficient lysis of mi- As discussed above, some applications of sample prepara-
croorganisms is critical for the direct analysis of clinical tion will require cell separation to reduce the complexity
specimens, because for many of these specimens the copy of the original sample. Currently, several techniques for
number of microorganisms is quite low. cell separation have been or could be used on a micro
scale.
One technique, microfiltration, utilizes the size differ-
Amplification ences among cell types. Using conventional computer-
controlled (CNC) machining, different designs and sizes
The last step of sample preparation is amplification of low of filters such as arrays of posts, tortuous channels [30]
copy numbers of target nucleic acids to higher copy num- comb-shaped filters [31] and weir-type filters [32] have
bers that will fall within the limits of the detection tech- been fabricated from glasssilicon mainly for the purpose
nology used. Several amplification methods are in use to- of efficient isolation of white blood cells from whole
day. The most widely used is the enzymatic polymerase blood. Recently Yuen et al. [32] demonstrated that a weir-
chain reaction (PCR) [23]. PCR is usually considered ca- type filter with a 3.5 m gap could isolate white cells with
pable of amplifying as few as 20 copies of a target in any reasonable capture efficiency (7%) of white cells and
given, purified, biological sample to over hundreds of >99.9% elimination of RBC. From the white blood cells,
millions of copies. It generally uses temperatures above a 226-bp region in the human coagulation Factor V gene
92 C to denature the complementary strands of DNA and was successfully amplified in a microchip-based PCR re-
then lower temperatures to enable annealing of primers action. An advantage of microfiltration is that it does not
and enzymatic extension of the primers. Generally 2535 require specific buffer conditions. It does, however, re-
of these cycles are run. Other amplification procedures, quire a size difference between the desirable and undesir-
such as the ligase chain reaction (LCR), requires a similar able cell types. For example, it would not effectively sep-
thermal cycling procedure [24]. Some methods of target arate the different types of white blood cell.
52
Dielectrophoresis (DEP) [33, 34, 35], which utilizes FACS, because of its low cost and potentially easy inte-
the interactions between the intrinsic dielectric properties gration with detectors and optical filters.
of the cells and the applied a.c. electric field, is an active
separation approach. The dielectric property of a cell is a
function of the applied a.c. electric field. Different types Cell disruption and DNA purification
of cell have different responses to the applied electric
field. Therefore, by selecting the appropriate frequency of Many mechanical and non-mechanical methods can be
the a.c. electric field for a sample type one can separate used for cell disruption; we will briefly review both types
different types of cell. A DEP device can be made by fab- of approach and consider their relative merit for integra-
ricating microelectrodes on glass or silicon substrates by tion in microsystems.
photolithography. A mixture of cells can be focused to
different regions of the microelectrodes depending on
their individual dielectric responses to the applied electric Mechanical methods
field. Because of its simplicity and flexibility, DEP has
been widely investigated for chip-based cell separations. In mechanical disruption the cell envelope is physically
Using DEP, a variety of biological cells has been sepa- broken, releasing all intracellular components into the
rated including bacteria [36, 37, 38, 39], cancer cells [40, surrounding medium. Several types of equipment for me-
41, 42, 43], stem cells [44, 45], and leukocyte subpopula- chanical cell disruption are commercially available. In a
tions [46]. Unlike microfiltration, DEP-based separation high-pressure homogenizer, a pump forces a slurry of
is not limited by the size differences between different cell sample through a restricted-orifice valve. High pressure
types. In principle, different types of cell can be separated (up to 150 Mpa) is followed by instant expansion through
with the same electrode design by selecting the appropri- a special exiting nozzle. Cell disruption is accomplished
ate frequency for each cell type. DEP also has the advan- by three different mechanisms in this system: impinge-
tages of flexibility, controllability, and ease of application ment on the valve, high liquid shear in the orifice, and
to automation, and it does not require immuno-labeling. sudden pressure drop upon discharge which causes explo-
Compared with microfiltration, most DEP separations re- sion of the cell.
quire low conductivity separation buffer, which means In a bead mill cells are agitated in suspension with
cells cannot be directly separated from their original sam- small abrasive particles. Cells break because of shear
ple solution, though Mller et al. [43] have reported the forces, grinding between beads, and collisions with beads.
separation of Jurkat cells from red blood cells in phos- This approach does not seem amenable to miniaturization.
phate-buffered saline. To make DEP separation more High-frequency ultrasound is another method widely
adaptable to real samples, more effort needs to be focused used for cell lysis. Ultrasonication devices generate in-
on improving the sensitivity, efficiency, and throughput. tense sonic pressure waves in liquid media. Under the
The Evotec Cytocon [47] is an example of a commercial right conditions, the pressure waves cause formation of
microsystem for cell separation and cell sorting that is microbubbles which grow and collapse violently. The im-
based on dielectrophoresis. plosion generates a shock wave with enough energy to
Both hydrodynamic flow [48, 49, 50] and electroos- break cell membranes. Modern ultrasonic processors use
motic [51] flow have been used to transport and sort cells piezoelectric generators made of lead zirconate titanate
on microchips. Micromachined cytometers that incorpo- crystals. The vibrations are transmitted down a titanium
rate electrokinetic focusing to confine cells spatially have metal horn or probe tuned to make the processor unit res-
been pursued by several groups [49, 52]. Another MEMS onate at 1525 kHz. Disadvantages of this method are that
method for cell concentration is based on ultrasonic stand- ultrasonic disintegrators generate considerable heat dur-
ing-wave technology. Using acoustic waves, cells and ing processing; they also generate free radicals that can
particles have been concentrated from bulk solutions us- react with biomolecules. The advantages of this method
ing either batch systems or flow systems. Coakley et al. are that it is suitable for hard-to-lyse cells and spores. Ul-
reported separation of cells from whole blood using ultra- trasonic lysis is an attractive approach for miniaturization
sonic cell levitation and transportation in standing acoustic of the cell-disruption process [57].
waves [53]. A particularly interesting acoustic MEMS ap- Mechanical disruption methods have several draw-
proach to cell manipulation is the use of flexural plate backs. Because cells are broken completely, all intracellu-
wave devices (FPW) reported by Ballantine et al. [54]. lar materials are released. Therefore, the DNA must be
Compact planar micropumps based on FPW could be separated from a complex mixture of proteins, and mem-
used to transport, mix, concentrate, and even lyse cells brane fragments. The cell debris produced by mechanical
(also see below). lysis often consists of very small fragments, making the
Fu et al. recently sorted Escherichia coli cells express- solution difficult to clarify. Moreover, complete product
ing green fluorescent protein from a background of non- release often requires more than one pass through the
fluorescent E. coli cells by use of a disposable microfabri- membrane disruption device, exacerbating the problem by
cated fluorescence-activated cell sorter (FACS) [55]. further reducing the size of the fragments.
This cell sorter was made using a soft lithograph proce-
dure [56]. FACS offers advantages over conventional
53
Non-mechanical methods for samples that contain low levels of bacteria or hard-to-
lyse spores.
Non-mechanical lysis methods include chemical, enzy- Cells can also be lysed electrically on microelectrode
matic, osmotic, thermal, and electric methods. Chemical arrays after pulsed high-electric-field treatment [38, 60].
methods extract intracellular components from microor- In particular, E. coli lysate obtained by electric lysis has
ganisms by making the outer cell wall permeable with or- been reported to be detected by electric-field-driven hy-
ganic solvents that create channels through the cell mem- bridization on microelectronic chips [38].
brane. Solvents used for this purpose include toluene, ether, Of the different types of non mechanical lysis, the elec-
phenylethyl alcohol, DMSO, benzene, methanol, and chlo- tric and thermal approaches are the most amenable to
roform. As discussed previously, chaotropic agents, deter- miniaturization. The chemical approaches might be ranked
gents, and enzymes have been used for cell lysis, as have according to the amount of chemicals required (the lower
antibiotics, thionins, and chelating agents. One aspect of the amount the more suited the approach to miniaturiza-
the use of these agents is that they must be removed from tion) as well as how critical it is to remove them. A possi-
the sample before subsequent enzyme-mediated amplifi- ble solution when large volumes of lysing solution are re-
cation. This removal step could be a drawback in an quired is to use an external storage bottle and implement a
MEMS device. Another problem is that the amount of flow-through system. Table 1 summarizes some of the ly-
lysing solution required might preclude on-chip storage. sis methods, purification, and recovery steps.
Despite these drawbacks, Tian et al. have built and char-
acterized a miniaturized purification device that makes
use of silica resins and chaotropic agents to purify ge- Amplification
nomic DNA for subsequent PCR amplification [58].
Although cells exposed to slowly varying extracellular Most reports on miniaturized DNA amplification have fo-
osmotic pressure can usually adapt to such changes, cells cused on developing a micro PCR device using IC and
exposed to rapid changes in external osmolarity can be MEMS technologies. The first miniaturized DNA ampli-
mechanically injured. This procedure is typically con- fier reported by Northrup et al. [61] in 1993 had polysili-
ducted by first letting the cells equilibrate internal and ex- con heaters patterned on a bottom Si3N4 membrane and
ternal osmotic pressure in a high sucrose medium, and was sealed from the top by a glass slide. Since then, many
then rapidly diluting away the sucrose. The immediate studies have been performed to enhance the design of the
overpressure of the cytosol is assumed to damage the cell microreaction chambers [62, 63, 64, 65, 66, 67, 68, 69,
membrane. 70]. Efforts have been made to improve temperature con-
Repeated freezing and thawing or elevated temperature trol, enhance temperature ramping rates, and reduce
will also lyse mammalian cells. In some applications the power consumption by exploring different materials such
target DNA can be released by elevated temperatures si- as silicon [71], glass [72], and polyimide [73] and differ-
multaneously with the denaturing step performed in PCR ent methods of heating such as Peltier thermoelectric ele-
[59]. These thermal methods are not, however, suitable ments [63], IR heating with a tungsten lamp [72], and so-
Lysis method
Mechanical High High High Of the mechanical lysis methods reviewed,
ultrasonic is most amenable.
Chaotropic Medium Medium Medium Depends strongly on the volume of reagents involved.
Can be incorporated into a membrane, facilitating removal.
Thermal Low Low Low Easy to miniaturize, but not suitable for all cell types
Electronic Low Low Low Easy to miniaturize
Purification method
Ion exchange Medium Medium High Easy to implement on a miniaturized platform
Beads Medium Medium Medium Somewhat more cumbersome
Molecular weight cut-off Low Low Low Easy to implement on a miniaturized platform
(MWCO) membranes
Recovery method
Silica elution Low Low High If a flow-through system is used miniaturization
of low-salt buffer is easy to accommodate
Beads elution Low Low Medium More cumbersome
Membrane elution Low Low Low Easiest to miniaturize
54
Fig. 2 LabCD for PCR (Tecan). Left: Top view of one area of 3. DNA amplification by strand-displacement amplifica-
the CD. The center of the disc would be in the upper left and the tion (SDA);
outer edge at the bottom. The elements are: (a) sample, (b) NaOH
for lysing cells, (c) tris-HCl from neutralizing the lysate, (d) capil- 4. desalting and histidine-buffer exchange;
lary valves, (e) mixing channels, (f) lysis chamber, (g) tris-HCl 5. denaturation of amplified/desalted amplicon; and
holding chamber, (h) neutralized lysate holding chamber, (i) PCR 6. DNA hybridization assay.
reagents, (j) thermal cycling chamber, and (k) air gap. Fluids
loaded in (a), (b), and (c) are driven at a first RPM into reservoirs At the core of this instrument is a small two-level-stacked
(g) and (f), at which time (g) is heated to 95 C. The RPM is in- microlaboratory that is 7676 mm2 and 2.77 mm thick.
creased and the fluids are driven into (h). The RPM is increased The stacked structure is constructed from a set of lami-
and fluids in (h) and (i) flow into (j). Right: The cross section
shows the disc body (m), air gap (k), sealing layers (n), heat-sink nated flexible substrates with fluidic cutouts, pressure-
(l), thermoelectric (p), PC-board (q), and thermistor (o). Reprinted sensitive adhesive layers, electrode arrays, and two Si
with kind permission from Kluwer Academic Press [91] chips. The completed stacked structure has the DEP chip
in the upper level chamber for dielectrophoretic collection
of bioparticles and the 1600-site chip in the lower level
for performing automated electric-field-driven assays
chambers and channels. These chambers and channels
(Fig. 3).
form the sample and reagent storage reservoirs, fluidic
The stacked structure was integrated into a fluidic sys-
controlling valves, fluidic mixing chambers, and heating
tem to form a prototype instrument (Fig. 4). In addition to
chambers. By rotating the disk at different speeds, cells
the stacked microstructure, the instrument also features
(bacteria or blood) and reagents can be sequentially moved
separate modules for isothermal SDA, desalting, and DNA
to different compartments to accomplish most sample-
denaturation to support DNA hybridization assays in the
preparation steps from cell lysis to DNA purification and
stacked structure. The SDA module is 76298.5 mm2 and
finally to the PCR amplification chamber. A centrifuge
consists of a denaturation chamber (Teflon tubing snaked
platform is an attractive sample-preparation stage because
through a channel in an aluminium heating block) and an
of the wide dynamic range and relative insensitivity to
amplification chamber (a polystyrene micro well in a sec-
fluid composition. It is also believed that the throughput
ond aluminium heating block) (Fig. 4).
could be augmented by placing multiple structures of the
Because electric-field-facilitated DNA hybridization
type shown in Fig. 2 on one plastic disc.
assays are performed in a low-salt environment, a desalt-
One major issue associated with this platform is yet to
ing module was designed to remove salts used in DNA
be resolved. Unless one works with all-dry reagents and
amplification and, simultaneously, to mix the amplified
reconstitutes them upon first use of the disk, the liquids
DNA with histidine buffer used in the DNA assay. The
held in the sample reservoirs will evaporate and distribute
2-stage desalting module (255776 mm3) consists of
as time passes, because there is no vapor barrier to stop
two chambers; salts are removed with ion-exchange resin
them from expanding into the open volume of the disk.
in the first chamber (BioRad) and histidine is exchanged
The fluid gating in the disk only works for fluids, not for
in the second. The amplified DNA is pumped sequentially
vapors.
through a hollow fiber (30,000 molecular weight cutoff,
At Nanogen, a sample-preparation prototype instru-
A/G Technology) coiled in each chamber to enable maxi-
ment was developed to perform both DNA hybridization
mum surface area for diffusion. Finally, the desalted DNA
assays and protein immunoassays from mixed samples
is denatured and pumped back to the stacked structure for
[92]. Sample preparation starts on one Si chip where di-
the DNA hybridization assay and fluorescent detection.
electrophoresis separates and concentrates intact E. coli
One sample-to-answer instrument that is beyond the
and/or protein toxins in the sample. After the dielec-
prototype stage has been developed by Cepheid. The
trophoresis step protein antigens are pumped to a second
GeneXpert instrument was introduced for pathogen detec-
Si chip for immunoassays. For the DNA assay the inte-
tion in 2001 [97]. This instrument is reported to process a
grated protocol consists of six steps:
raw biological sample and provide PCR results for the sam-
1. collection of E. coli by dielectrophoresis; ple in 30 min. Sample preparation occurs in disposable,
2. lysis of the bacteria and denaturation; single-use fluidic cartridges that separate and concentrate
56
Fig. 3 Stacked microlaboratory: (a) Fabrication of the stacked ficiency reaction chambers with integrated heaters and
structure 7676 mm2 square and 2.77 mm thick. The first patterned simple, inexpensive electronics to control the reaction
Kapton layer features a flip-chip bonded CMOS chip (78 mm2
with 4040 electrodes) for DNA hybridization assays or im- temperatures. Four-color detection within the module en-
munoassays. Layers 2, 4, and 6 are double-sided pressure-sensitive ables real-time identification and quantification of four
acrylic adhesive (PSA) layers (0.152 mm thickness per PSA layer), different targets in each reaction chamber. Multiple ICORE
some with fluidic cutouts. The third layer is an optically transpar- modules can be loaded and run at same time in the
ent poly(methyl methacrylate) (PMMA) film (0.178 mm thick) put
over the fluidic cutout to make a first enclosed channel/chamber.
GeneXpert instrument. In contrast with traditional thermal
The next patterned Kapton layer (5) holds the flip-chip-mounted cycling systems in which all samples are subjected to the
dielectrophoretic (DEP) chip (11 cm2 with sixteen large and nine same time/temperature/optical procedure, each I-CORE
small platinum electrodes). Layer 7 is a glass plate (1.27 mm) unit can be operated and controlled independently en-
which encloses a second fluidic chamber. (b) Completed stacked abling the user to run different cycling procedures.
structure showing top and bottom views. Electrode patterns at the
opposite edges of the stacked laboratory enable electrical contact
to an external control circuit; fluidic connections are located on the
bottom of the stacked structure [92] Issues associated with MEMS-based
sample preparation
cells by means of filters. Cells are lysed using ultrasonic Partitioning of functions between instrument
techniques, and DNA or RNA are enriched and purified. and disposable unit
The nucleic acids and reagent mixes for PCR reactions are
delivered into a thin-walled disposable reaction chambers Microfluidic systems comprising nozzles, pumps, chan-
that have an optical window suitable for real-time detec- nels, reservoirs, columns, mixers, valves, etc. can be used
tion of PCR products. This chamber fits into the ICORE for a variety of sample-preparation applications. The ad-
module (ICORE for intelligent, cooling/heating, optical vantages of these devices include lower fabrication costs,
reaction; Fig. 5). This module uses silicon-based, high-ef- enhancement of analytical performance, lower power re-
57
Fig. 4 Nanogens integrated fluidic system for the stacked mi- throughput drug screening, the electronics, the power for
crostructure. For a complete automated assay of the SLTI gene, the propulsion mechanism, heaters, etc., can be integrated
70 L E. coli suspension (109 mL1) is introduced into the fluidic
system through the micro-splitter valve (1) and delivered to the
with the disposable unit or be part of the instrument. In
DEP chip on the stacked structure (2) held in a clamping fixture. high-throughput screening fluid handling is more sophis-
For capturing the E. coli, an ac voltage of 5 kHz, 10 V peak to peak ticated and is typically incorporated into a large, table-top,
is employed (52 min). With the ac voltage on, the chip is washed costly research instrument. In this case, the disposable
with 50 mmol L1 histidine from the storage bottles (3). The ac units for screening are less challenging than those for di-
voltage is then turned off to release collected bacteria and the bac-
teria are mixed with SDA reagents and delivered to the SDA mod- agnostic application, because there is no need to contain
ule for bacterial lysis, DNA denaturation (4) and amplification of sealed fluid or dry stored chemicals on the units. For di-
SLT1 DNA (5). The DNA amplification product is desalted, ex- agnostics the disposable unit should be inexpensive and
changed with buffer (6) and denatured (7) for the DNA hybridiza- should be as uncomplicated as possible perhaps only
tion assay back in the stacked microstructure (8). The hybridized
SLT1 gene is detected with a fluorescence-labeled DNA reporter reagents, plastic substrate, and fluidic conduits. The de-
and viewed from the microscope lens above the stacked structure tection device should be inexpensive, small, preferably
(9) [92] hand-held, and user-friendly. A major challenge for diag-
nostic applications is to determine how to store wet or dry
chemicals in sealed reservoirs within the disposable pack-
quirement, and lower consumption of chemicals than tra- age.
ditional systems [98]. The generic technical challenges in
developing a sample-preparation instrument based on
those MEMS components include the partitioning of the
Choice of fluid and particle-propulsion method
different instrument functions such as power supply,
mechanism for getting the sample into and out of the in-
strument, sample concentration, providing fresh viable A variety of technologies can be used to move small
reagents, and detection. At one extreme one might envi- quantities of fluids or suspended particles in microfluidic
sion a totally integrated option i.e. a micro total analysis channels and networks. Several standard methods have
system (-TAS). In such a monolithic MEMS approach, been used to accomplish fluid transport including syringe
electronics and MEMS elements are co-fabricated within and peristaltic pumping and more sophisticated methods
one single, lengthy sequential silicon process. In a hybrid based on electrochemical bubble generation, acoustics,
MEMS approach, the electronic parts are kept separate magnetics, DC and AC electrokinetics, and centripetal
from the sensor part and both Si and non-Si machining forces. In Table 3 we compare characteristics of a number
processes are used. The device components in such a hy- of MEMS fluid propulsion methods [99]. Only the sim-
brid remain more serviceable, and because the packaging plest and least expensive pumping schemes should be in-
and interconnects determine the final size of the instru- tegrated within the disposable unit. Here we review vari-
ment, hybrids are often not much larger than their coun- ous propulsion techniques with regard to their applicabil-
terparts based on a more integrated Si solution. Hybrids ity to sample preparation.
are usually simpler to design and can be produced more
economically in smaller production volume runs. For
chemical and biosensor based instruments, they often rep- Mechanical pumps
resent the most viable means of implementing microma-
chining technology. The pressure that mechanical pumps must generate to pro-
Partitioning is highly application-specific. Depending pel fluids through capillaries becomes higher the narrower
on the application, e.g. clinical diagnostics or high- the fluidic channels. With piezoelectric (acoustic), elec-
58
Fig. 5 Sample preparation and optical
real-time PCR (Cepheid) in ICORE
cartridge. Adapted from Ref. [97]
tro-osmotic (DC electrokinetic), electro-wetting (DC elec- when the device dimensions are reduced and the resis-
trokinetic), and electro-hydrodynamic-based pumping (AC tance to fluid flow and required driving pressure increase.
electrokinetic), on the other hand, pumping efficiency im- An example of a Si micromachined mechanical pump is
proves with smaller capillary diameter. In other words that from Debiotech for insulin infusion. This belt-worn
most non-mechanical pumping mechanisms scale more drug-delivery pump features a piezoelectric design pio-
favorably in the micro domain. Despite this, almost all neered at the University of Twente [100]. The size of most
modern biotechnology equipment is based on traditional Si micromachined pumps is quite large and because cost
external syringe or peristaltic pumps. Micromachined me- advantages in Si manufacture are realized with small foot-
chanical pumps have not achieved commercial success. print devices only, plastic micro molding technology is
Although integrated micromachined pumps based on two being explored as a viable alternative [101, 102].
one-way valves can achieve precise flow control of the or- Another example of mechanical fluid propulsion is
der of 1 L min1 with fast response, high sensitivity, and embodied in the Johnson and Johnson blister pouch HIV
negligible dead volume, these pumps require complicated test shown in Fig. 6 [103]. An automated mechanical
fabrication processes, generate only modest flow rates and roller, similar to that used in credit card image transfer, is
low pressures, and consume a large amount of chip area used to squeeze a plastic pouch and move the reagents
and a substantial amount of power. As a consequence of through the miniature channels in it. This approach meets
the low pumping pressure, they often become useless the goal for a low-cost diagnostic disposable unit contain-
59
Centripetal force Yes for R&D Density, wetting, Rotary motor Plastics Good From
liquids level contact angle, <1 nL s1 to
and viscosity >100 mL s 1
Pressure (e.g. Yes for Product Generic Mechanical Plastics Good 20500 mL s 1
blister pouch) liquids level roller (Fig.6)
and vapor
Acoustic No R&D Generic 5 to 40 V Piezoelectrics High 20 mL s 1
level (peak to peak)
Electrokinetic Yes for Product pH, ionic strength 10 kV Glass, plastics Low 0.0011 mL s 1
(osmosis) liquids level
Electro-wetting Yes for R&D pH, ionic strength 110 kV Glass Low 0.3 mL s 1
liquids level
Dielectrophoresis No R&D Dielectric property 110 V Glass, silicon Good <100 mL s 1
Acoustic pumping
Electrophoresis and electroosmosis sures can be obtained. Another advantage is that the fluid
propulsion force does not depend on the type of sample.
Electrokinetic pumping in a capillary is a method that These are attractive features for sample preparation.
does not involve moving parts. The fluid is driven and
guided along the capillary by electrokinetic pressure a
combination of electrophoresis and electroosmosis in Vapor and liquid barriers for storage of liquids
which DNA molecules are moved by their charges and
fluid is moved by interaction with capillary wall surfaces One major issue to be resolved with MEMS-based dispos-
under the influence of an electrical potential applied along able units is the incorporation of a fluidic network with
the channel length. Typical electro-osmotic flow veloci- on-board reservoirs containing wet chemicals. If no vapor
ties are of the order of 1 mm s1 in a 1200 V cm1 applied barrier stops them from expanding into the available open
electric field. For example, Jorgenson et al. reported elec- spaces, liquids held in reservoirs on a disposable unit can
tro-osmotic flow of 1.7 mm s1 in free-flow capillary elec- evaporate and distribute slowly throughout the fluidic net-
trophoresis [106]; this is fast enough for most analytical work. For the disposable unit illustrated in Fig. 6, reagents
purposes. The disadvantages of electroosmosis are the of interest are sealed in blister pouches in a plastic lami-
high voltage needed (130 kV power supply), the direct nate and small nips in the plastic channels seal the liquid
electrical-to-fluid contact, and the flow rate sensitivity to and vapor. Upon use, a mechanical roller breaks the liquid
the capillary wall charge, to the ionic strength, and to the and vapor seals. The nips form one-time-use valves only;
pH of the solution. It is, consequently, more difficult to when the plastic nips are ruptured they cannot be closed
make it into a generic propulsion method. For example, again. In most disposable diagnostic applications such
liquids with high ionic strength suffer from excessive one-time-use valves will suffice. Most of the MEMS-
Joule heating and it is therefore difficult or impossible to based diagnostic platforms proposed in the literature do
pump biological samples such as blood and urine. not provide a vapor barrier between channels and reser-
voirs and will not work in a practical diagnostic setting
where shelf lives of six months or longer are desirable. To
Centrifugal pumping circumvent the vapor-valving problem one might consider
dry-storage of reagents. These reagents can be wetted by
Using a rotating disk, centrifugal pumping can provide the sample itself upon use of the diagnostic kit. Depend-
flow rates ranging from less than 10 nL s1 to greater than ing on the response time required and the specific
100 L s1 depending on disk geometry, rotational rate chemistries involved, however, this method might not be
(RPM), and fluid properties [91]. Pumping is relatively in- suitable for all biological applications.
sensitive to chemical properties such as pH, ionic strength,
or chemical composition. Aqueous solutions, solvents (e.g.
DMSO), surfactants, and biological fluids (blood, milk, Sample-size issues
urine) have all been pumped successfully. Fluid gating is
accomplished by using capillary valves in which capil- Sample sizes
lary forces retain fluids at an enlargement in a channel un-
til rotationally induced pressure is sufficient to overcome A major difference between traditional clinical assays and
the capillary pressure at the so-called burst frequency. molecular assays is the amount of target material in a
Compared with electrokinetic pumping, the ability to sample. Chemical analytes are often present at between
move a variety of liquid types and volumes makes the 1014 and 1020 entities mL1. Thus typical clinical chem-
centripetal platform amenable to sample preparation tasks. istry assays can be readily performed with very small sam-
ple volumes, in the picoliter and microliter range. Typical
immunological assays are used to detect antigens present
Vacuum pressure reservoir at between 107 and 1018 species mL1. In contrast DNA
and RNA assays are used to detect far fewer targets
Fluid transport can also be accomplished by employing from less than 1 to 107 species mL1 (Fig. 7). These sen-
pressure difference between the sample, reagent reser- sitivity requirements set fundamental physical limitations
voirs, and other chambers. Valves are required to separate on the minimum quantities of the specimen analyzed.
the evacuated or pressurized chambers from the rest of the Table 4 shows the minimum volumes necessary for
fluid path until fluid motion is required. Opening a valve statistically valid results for several targets assayed by im-
initiates the flow of fluid toward an evacuated chamber or munological assays and for some targets that could be as-
away from a pressurized chamber. The drawbacks of this sayed by nucleic acid techniques. Despite the tremendous
mechanism are that it can only be actuated once, it can be advantage gained by being able to amplify nucleic acids
difficult to implement efficient valving, and reliable man- by factors of >108, when rare cells are sought, there are
ufacture of pressurized or evacuated chambers can be still major issues to be overcome in terms of reducing
challenging. An advantage over the previously described sample volumes to workable levels in microdevices.
methods is that it is a straightforward means of generating
a large fluid-motive force high flow rates and high pres-
61
Fig. 7 Scaling of sample size in mole-
cular diagnostics. Reprinted from [90]
Table 4 Minimum amount of sample need for statistically valid sample with minimum dead volumes is one of the greatest
results assuming near-perfect efficiency of capture and detection challenges in micromachining. Very few machining tech-
Analyte Copies mL1 Type of assay Amount niques can provide the flexibility to cover the dynamic
blood of blood size-range required. Because the sample introduction
needed method can place important constraints on the manufac-
turing process, a few sample-introduction options are con-
Myoglobin ~21012 Immunoassay 25 fL
Troponin I or T ~2109 Immunoassay 25 pL
sidered here. Traditional sample-injection systems such as
Specific antibodies ~1108 Immunoassay 500 pL
flow-injection analysis (FIA) rely on some sort of com-
Single copy gene ~1107 DNA 5 nL mutating valve, either rotary or sliding, actuated manually
Virus such as HIV 5100 RNA 0.510 mL or mechanically. Harrow et al. [107], compared different
Blood infections <110 DNA 6>60 mL commercially available sample-injection systems and
Shoji et al. [108] were among the first to micromachine a
sample injector consisting of two three-way micro valves
Sample-introduction choices and a channel. These valve systems are, however, too
complex and expensive to consider integrating them in a
The exact mechanism for introducing samples into a mi- disposable unit. Perhaps the simplest way to introduce the
croinstrument depends on the sample type. Pretreatment sample into a micro-instrument is by creating wells into
of the sample might be required and relatively bulky sam- which the sample is dropped. For example, most of the
ples, often collected manually, must be introduced into the substrates with electro-osmotic pumps have plastic tubes
microscale channels of the device. Introduction of the glued to them to make sample reservoirs. The Biotrack
62
Fig. 8 (a) SEM photograph of
a fluidic coupler with a 110-m
thick sleeve around the bore to
prevent blocking of capillaries
with adhesive. The silicon is
partially cleaved to show the
sleeve. (b) SEM photograph
of capillaries inserted into the
sleeve couplers. Courtesy Pro-
fessor G. Kovacs, Stanford.
Reprinted from [110]
blood-coagulation device requires that a drop of sample silicon-based MEMS detectors, fluid propulsion systems,
be placed in a depression on the instrument surface [109]. and actuators have been successfully utilized and demon-
The sample is then transferred into an internal chamber by strated in a number of applications, interfacing MEMS
capillary action through a wick. Problems with sample detection devices with sample acquisition remains a major
wells include the large volume that they require, or the challenge in designing practical devices for molecular di-
need to align the sample accurately with the well if the agnostics. The principle advantages of Si-based manufac-
sample well is small. Because the wells are open, one turing methods for making micro-instruments are that they
risks sample contamination or inclusion of air bubbles in offer fabrication of intricate structures with very fine fea-
the sample. The latter are a major problem in microfluidic tures; thus heaters, electroosmosis electrodes, and acoustic
systems, because of the associated surface tension, and streaming pumps can be readily realized. On the other
the addition of a bulky de-bubbler is not a suitable solu- hand, non-traditional microfabrication from plastic mate-
tion in microfluidic systems. Samples can also be injected rials is continually improving and is becoming competi-
directly into the micro instrument or disposable cassette. tive compared with silicon micromachining. If larger
An example of this is the Johnson and Johnson pouch structures are built, those methods could result in lower
HIV test in which an injection septum is provided in the manufacturing cost. Because of a broad range of applica-
disposable pouch (Fig. 6). This reduces the chance of sam- tions and design requirements for each particular assay
ple contamination but again requires exact alignment of and tremendous advancements in microfabrication tech-
the injection needle with the sample-injection port. Manu- nologies, it is difficult to provide general recommenda-
facture of the pouch is, furthermore, complicated by the tions about which technology should be used to fabricate
need for an injection septum. In an alternate approach, the a disposable unit. It is likely that both microfabrication
collection device could be disposable. For example, the approaches would be used in a device. The cost of manu-
diagnostic device could be manufactured in the form of a facturing the disposable unit will dictate the choice of
blood-collection tube (Vacutainer, BectonDickinson), a manufacturing technology and the partitioning of instru-
syringe, a pipette, or a swab. This would, however, con- ment function.
siderably complicate manufacture of the disposable unit
and might limit the design to a specific type.
Interfacing fluidic platforms with the outside world is Conclusions
starting to be addressed by micromachinists. For example,
Gray et al. [110] have demonstrated different types of The wide range of sample types and sizes in molecular di-
minimum dead-space fluidic couplers for standard fused agnostics makes it very unlikely that a universal sample-
silica tubing to MEMS devices. An example is shown in preparation solution can be developed. In general, a sam-
Fig. 8. ple-preparation system should consist of a modular instru-
ment and disposable units. When designing a sample-
preparation system several considerations should be taken
Choice of manufacturing techniques for disposable units into account.
1. Partitioning of functions between the instrument and
Biological samples for molecular diagnostics are often the disposable units are very application specific. For
sampled at volumes that are not always compatible with molecular diagnostics, a modular instrument approach
the microfluidic environment of MEMS devices. Although is appropriate in order to have the capability to handle
63
a small or large number of samples of different sizes MEMS community and will provide major market ad-
and types and to perform different tests on the same or vantages to those who provide efficient solutions to
multiple samples. The design of the disposable unit these problems.
should be simple and the manufacturing cost should be
low. A good balance between traditional machined Acknowledgements. The authors would like to thank Drs Dalibor
Hodko and Jim Zoval for critical reading of the manuscript and
parts and micromachining of selected components helpful discussions.
seems to be the optimal approach.
2. The method for fluid propulsion. The most popular
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Anal Bioanal Chem (2002) 372 : 6673
DOI 10.1007/s00216-001-1195-5
REVIEW
D. C. Collins M. L. Lee
Received: 19 July 2001 / Revised: 11 October 2001 / Accepted: 16 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract Ion mobility spectrometry (IMS) has been used Interest in IMS blossomed in the early 1970s because
for over 30 years as a sensitive detector of organic com- of its analytical versatility, excellent detection limits, suit-
pounds. The following is a brief review of IMS and its ability for real-time monitoring, and low cost. Despite
principles with an emphasis on its usage when coupled to these attractive features, however, interest declined after
mass spectrometry. Since its inception, IMS has been in- 1976 [3]. Loss of interest was associated with universal
terfaced with quadrupole, time-of-flight, and Fourier-trans- disappointment by unmet expectations and possible mis-
form ion cyclotron resonance mass spectrometry. These understandings. IMS was mistakenly compared with mass
hybrid instruments have been employed for the analysis spectrometry (MS), despite its much lower peak resolu-
of a variety of target analytes, including biomolecules, ex- tion and lack of mass information. In addition, chemical
plosives, chemical warfare degradation products, and il- aspects of ion formation at atmospheric pressure were not
licit drugs. fully appreciated or understood. This caused many to con-
clude that IMS was an interesting but not practical or us-
Keywords Ion mobility spectrometry Mass able technology [3, 4, 5, 6].
spectrometry Gas-phase electrophoresis Plasma Between 1980 and the early 1990s, an interest in IMS
chromatography was renewed as a variety of modifications were made to
instrument design, resulting in its suitability for use for a
wide range of applications. During this time, IMS was fur-
Introduction ther employed as a detector for gas chromatography [7, 8]
and supercritical-fluid chromatography [9, 10]. It was also
Ion-mobility spectrometry (IMS) was first introduced in used for the detection of bacteria [11] and military moni-
1970 under the name plasma chromatography. Initially toring in hostile environments [3]. Many attempts were
considered as an ion-separation technique with ion drift also made to incorporate unconventional ionization sour-
times analogous to chromatographic retention times [1, ces, such as laser ionization [12] and a form of electrospray
2], it later became more commonly viewed as a technique ionization (i.e., coronaspray) into its design [13, 14].
for the selective detection of organic compounds [3, 4]. In
IMS, radioactive 63Ni (typically) is used to ionize vapors
of organic compounds through a series of ionmolecule
Principles of IMS
reactions. The ions are then carried under an electric po-
tential through a drift region to an ion collector (i.e. Fara-
The ability to distinguish ions occurs within the drift re-
day plate or mass spectrometer). Ions are selectively de-
gion. In IMS, ions are separated on the basis of their dif-
tected on the basis of their unique drift times traveling
ferent velocities attained when accelerated through a drift
through the drift region.
tube, by a constant electric field, against a counter-flow of
neutral gas. The average velocity of an ion, vd (cm s1), is
determined by the number of collisions it experiences
D.C. Collins within the drift tube with the neutral drift-gas molecules,
Weber State University, Department of Criminal Justice,
Ogden, Utah 844081206, USA
which is directly proportional to the applied electric field
strength, E (V cm1)
M.L. Lee ()
Brigham Young University, vd = KE (1)
Department of Chemistry and Biochemistry,
Provo, Utah 846025700, USA where K is the ion mobility constant in units of cm2 V1 s1
e-mail: milton_lee@byu.edu and is a combined property of both the nature of the ion of
67
interest and the drift gas. Equation 1 is valid only at low only approximate correlation with ion mass. Its unique ad-
field strengths (e.g. <1000 V cm1). At increasing field vantage over mass spectrometry is its ability to operate at
strengths, K is no longer directly proportional to E [15]. atmospheric pressure or near atmospheric pressure. In ad-
K can also be calculated at low electric field strengths by dition, data acquired can be used to calculate structural in-
use of the MasonSchamp equation: formation (e.g. average cross-sectional area) [16, 17, 18].
By interfacing IMS with MS, a somewhat orthogonal com-
K = 3/16 ze/No (2/kT)1/2 (1/D ) (2)
prehensive separation (i.e., structural information) and de-
where z is the integer charge of the ion, e is the charge of tection (i.e., mass information) device can be constructed.
an electron in Coulombs (i.e., 1.6021019 C), No is the
number density of the drift gas (molecules cm1), is the
reduced mass of a colliding iondrift-gas pair [=mM/ IMSMS developments and applications
(m+M)], m is the mass of the ion, M is the mass of a neu-
tral drift molecule, k is Boltzmanns constant, T is the op- Most commercial IMS instruments use Faraday plate de-
erating temperature in Kelvin, and D is the ionneutral tection; that is, ions are detected as they strike a metal plate
average cross-sectional area; D is derived through a se- and induce a current. Faraday plate detection is simple, in-
ries of integrations that average the ionneutral collisions expensive, and can be used for both positive and negative
over all possible scattering angles and energies. For rigid- ions. Unfortunately, such detection does not afford addi-
sphere collisions, integrating analytically yields D=d2, tional qualitative information associated with the separa-
where d is the sum of the ion and drift-gas radii. As can be tion of ions. Because of this, some have chosen to use MS
seen, assuming pressure and temperature are constant, as a means of ion detection and identification.
ze/1/2D is what is actually measured in IMS experiments. The advantages of interfacing IMS with MS were un-
For small atomic ions relative to the drift gas molecules derstood early in the development of IMS, and the cou-
(e.g. N2, He, or air) mobility is largely controlled by re- pling of the two techniques is virtually as old as IMS it-
duced mass whereas for heavy ions is essentially equal self. In early 1971, when IMS was referred to as plasma
to M. The distinguishing property upon which relatively chromatography (PC), the Franklin GNO Corporation de-
large ions (the majority of ions analyzed) are separated is, veloped the first commercial ion mobility spectrome-
therefore, ionic size or D. Thus, ions of identical mass, termass spectrometer (IMSMS) and demonstrated its
but different shapes (i.e., isomers), can be separated [3, 4, use as a detector for the identification and analysis of trace
5, 6]. amounts of oxygenated compounds (i.e., 1-octanol and
Because K, as shown in Eq. (2), is found to be depen- 1-nonanol) [19]. The instrument was called the Alpha II
dent on both temperature and pressure (No is a function of PC/MS. It was a two-gated IMS instrument made from
pressure), the value calculated by use of Eq. (1) is often stacked rings that had been joined to a modified Finnigan
converted to a reduced mobility value, Ko quadrupole mass spectrometer. The IMS section operated
at atmospheric pressure and the quadrupole at approxi-
Ko = K(P/760)(273/T) (3)
mately 105 Torr (1 Torr=133.322 Pa). The interface between
where P is the operating pressure in Torr and T is the op- the two included an ion lens for focusing of ions exiting
erating temperature in Kelvin. This normalizes mobility the aperture of the IMS into the quadrupole mass spec-
values calculated at different temperatures and pressures trometer. The ions were then detected with an electron mul-
for the purpose of comparison. tiplier placed at the end of the quadrupole, as in Fig. 1 [20].
During its early years IMS was periodically compared The instrument was soon used for comparison of IMS
with time-of-flight mass spectrometry (TOFMS), which MS data obtained by 63Ni ionization in nitrogen or pure
was unfortunate because of its low resolving power and air, with mass spectral data obtained by chemical ioniza-
opening and closing in such a fashion that only ions of a trometermass spectrometer. Several models were ulti-
desired drift time could pass into the mass spectrometer. mately produced; in all of these the basic operation of the
The mass spectrometer would then be tuned to a previ- new instrument remained the same as that of its predeces-
ously assumed mass-to-charge ratio (m/z) for the purpose sor. To acquire both IMS and MS data the instrument was
of identification. In this manner, direct correlation could gated for a certain drift time and the mass spectrometer
be made between a given IMS peak and its corresponding was tuned to a specific m/z value for the analyte of inter-
mass (or masses, for more than one compound of distinct est. The IMSMS instrument was primarily regarded (and
m/z defining a given IMS peak). Figure 2 shows data col- used) as a selective detector.
lected using both the total- and selected-ion-monitoring Most of the limited IMSMS work in the 1980s was,
modes of the mass spectrometer [20]. as in the 1970s, performed using the commercially avail-
In 1975 Franklin GNO Corporation halted its manufac- able instrument supplied through PCP. Work with this in-
turing operation. Shortly after, however, two former foun- strument in the 1980s included the identification and char-
ders, M. J. Cohen and R. F. Wernlund, formed a new com- acterization of illicit and prescription drugs. It was thought
pany, PCP (West Palm Beach, FL, USA) to continue the that an IMSMS system could be used to aid rapid identi-
technology under the patent license of Franklin GNO. Un-
fortunately, as mentioned previously, interest in ion mobil-
ity spectrometry began to wane in the late 1970s and
through the mid-1980s. The 1980s brought a better under-
standing of IMS, however, and this, in turn, sparked a re-
newal of interest. At this time, many felt IMS was more a
spectrometric than chromatographic process. This new
consensus fostered a name change of the process from
plasma chromatography to ion mobility spectrometry. PCP
began manufacturing an early version of the Alpha II
PC/MS as the Phemto-Chem MMS-160 ion mobility spec-
fication of drug residues on the hands of patients admitted drift times associated with a nested flight time. Sample
to the hospital with a drug overdose [28, 29] (Fig. 3). The ionization was achieved by electrospray ionization. With
instrument was also used to identify structurally different the information acquired, a contour plot of flight time
ions with the same m/z values [30]. A modified version of against drift time could be generated (Fig. 6). Clemmer
the instrument was used by Lubman and coworkers, after also performed high-resolution IMS studies. An ion trap
laser desorption of solid samples, including 63Ni ioniza- and octapole collision cell were incorporated into the de-
tion in the normal fashion. They used this method to de- sign to increase the duty cycle and obtain fragment infor-
tect explosives (i.e., trinitrotoluene, cyclotetramethylene- mation, respectively.
tetranitramine, and cyclotrimethylene-trinitramine). In ad- Recently, Hill et al. designed and constructed an in-
dition, throughout their studies, 63Ni ionization was com- house electrospray IMS instrument, and interfaced it with
pared with laser ionization (Fig. 4) [12, 31]. a C50-Q (ABB Extrel, Pittsburgh, PA, USA) quadrupole
In the 1990s, work continued in the field of IMSMS, mass spectrometer. The instrument was used successfully
but with more of a departure from the use of the commer- for the study of isomeric peptides, illicit drugs, chemical
cially available design offered through PCP. Guevremont warfare degradation products, explosives, and IMS selec-
and coworkers purchased an IMSMS instrument from tivity [46, 47, 48, 49, 50, 51] (Fig. 7). High IMS resolution
PCP and modified it to accommodate a time-of-flight mass values were also reported and associated with the very
spectrometer to study ions formed during the initial stages small diameter (i.e., 40 m) entrance orifice into the mass
of electrospray ionization (Fig. 5). A LeCroy digital oscil- spectrometer, which enabled the sampling of ions from
loscope (Model 9350, Chestnut Ridge, NY, USA) was used the most homogenous region of the electric field within
for data collection and processing [32]. TOFMS enabled the drift tube [52]. Modes of operation were equivalent to
complete mass spectra of individual peaks separated in those offered by the commercial IMSMS instrument
the IMS instrument to be obtained. This eliminated the te- available through PCP.
dious task of having to tune the mass spectrometer to the Russell et al. and Ionwerks (Houston, TX, USA) recent-
assumed m/z value of an IMS peak. ly collaborated in the development of an IMSTOFMS in-
In 1997, Clemmer et al. designed an IMSMS instru- strument with a high-pressure matrix-assisted laser des-
ment with a quadrupole mass spectrometer for characteri- orption ionization source for sample ionization. The in-
zation of oligosaccharides and proteins [33, 34, 35, 36]. strument was used for the analysis of peptide mixtures
Later, they constructed an IMSMS instrument with a (Fig. 8) [53]. Data were collected and processed in an ion-
time-of-flight mass spectrometer to study biomolecules, counting mode by means of an Ionwerks time-to-digital
incorporating a unique time-to-digital converter data sys- converter (Model TDCX4). They also designed a Fourier-
tem linked to, and operated by, a Pentium computer [37, transform ion cyclotron resonance mass spectrometerion
38, 39, 40, 41, 42, 43, 44, 45]. Data were presented as mobility spectrometer with TOFMS detection [54].
71
REVIEW
Received: 7 June 2001 / Revised: 10 September 2001 / Accepted: 20 September 2001 / Published online: 13 December 2001
Springer-Verlag 2001
Abstract Because of their high toxicity and widespread the total amount of metal in the sample, and they must be
distribution, the reliable selective quantification of alkyl combined with a separation technique before individual
and aryl species containing mercury, tin, or lead has been species can be quantified [2, 3]. Speciation analysis, there-
one of the goals of speciation analysis in recent years. fore, normally relies on hyphenated techniques.
Since becoming commercially available, GCMIPAED In this review we concentrate on what, with the excep-
has been one of the most-used tools in this work. In this tion of GCMS, has probably been the most successful
paper, the value and limitations of GCMIPAED for the commercially marketed GC-based hyphenated technique,
speciation of Hg, Sn, and Pb compounds in environmental the combination of GC with detection of atomic emission
samples are reviewed and compared with the analytical stimulated by use of a microwave-induced helium plasma
characteristics of other hyphenated GC-based techniques. (GCMIPAED).
Because quantification of Hg, Sn, and Pb species by GC Because microwave-induced helium plasmas (MIP)
techniques normally requires complex sample preparation are formed at lower temperatures than inductively cou-
involving several steps, the effect of sample-preparation pled argon plasmas (ICP), liquid samples cannot be intro-
methods on the accuracy and precision of the results is duced into them, because they are extinguished by even
discussed. Finally, we describe the current status of a small amounts of organic vapor. When MIPAES is com-
rapid, low-cost GCMIPPED system specifically de- bined with GC it is, therefore, necessary to vent the GC
signed for routine quantification of Hg, Sn, and Pb species solvent front before it arrives at the discharge tube. When
in environmental control laboratories. this problem had been solved, GCMIPAED proved to
have several advantages over GCICPAED. The most
Keywords Speciation Mercury Tin Lead Gas important are, perhaps, the higher electron temperature
chromatographymicrowave-induced plasmaatomic and ionization energy of MIP, which enable quantification
emission not only of metals but also of semi-metals, and even of
other organic compounds containing elements with high
ionization potentials (fluorine, chlorine, etc.) [4, 5, 6].
Introduction Other advantages of MIP are the small dead volume of the
discharge tube, the compatibility of the plasma with the
Speciation analysis is the separation and quantification of low carrier gas flows used in capillary GC columns, and
identifiable chemical species containing a particular ele- the consumption of less gas than ICP. AES detection of el-
ment [1]. Its aim is to determine the distribution of the ements excited by an MIP is, furthermore, both very se-
element among the different chemical species in a sample. lective and often (e.g. for selenium and for most elements
Few of the instrumental methods available for elemental with atomic masses lower than 40) less subject to interfer-
analysis have sufficient selectivity for direct quantifica- ence than the more sensitive ICPMS system [3, 7]. There
tion of individual metal-containing species. Spectroscopic is no doubt that these properties that were responsible for
techniques are very sensitive but usually determine only the worldwide commercial success of the GCMIPAED
system marketed by HewlettPackard at the end of the
nineteen-eighties.
One of the most important environmental applications
I.R. Pereiro () A.C. Daz of GCMIPAED is the quantification of alkylmetal species
Dpto. Qumica Analtica, Nutricin y Bromatologa,
Facultad de Qumica, Universidad de Santiago de Compostela,
of low molecular-weight. These species are highly toxic at
Santiago 15782, Spain low concentrations and are easily bioaccumulated; some
e-mail: qnisaac@usc.es are regarded as endocrine system disrupters. In environ-
75
Table 1 Summary of GCAES methods for the determination of inorganic mercury and organomercury species in various sample
matrices
Species; sample Sample preparation LOD CRM used Determination Date Ref.
matrix technique
MeHg; fish Alkaline digestion 0.4 pg MeHg as CRM 464, GCMIPAED 2000 [38]
tissue (KOHMeOH)+derivatization Hg DORM-2
(HClCH 2Cl2NaBPh 4)
MeHg, EtHg; (1) Ethylation (NaBEt4) (1) 0.04 pg GCMIPAED 2000 [24]
standard aqueous MeHg as Hg
solutions (2) Phenylation (NaBPh4) (2) 0.02 pg GCAFS
MeHg as Hg
(2) 0.03 pg GCMS
EtHg as Hg
MeHg, Hg2+; (1) Derivatization (Grignard reagent) 0.1 ng g1 IAEA 356, GCMIPAED 2000 [31]
soil, sediment (2) CH 2Cl2 extraction+ethylation MeHg CRM 580,
(NaBEt4)+Tenax collection PACS-1
MeHg, EtHg, Derivatization (Grignard reagent) 0.2 pg MeHg DORM-2, GCrf-HCGDAED 2000 [23]
Hg2+; dogfish and EtH, 0.3 pg DOLT-2
tissue Hg2+
MeHg; tuna fish, MAE (HAc)+phenylation (NaBPh4) 0.04 pg MeHg CRM 463, GCMIPAED 2000 [43]
cockle, mussel, as Hg CRM 464,
dogfish DORM-2
MeHg, Me2Hg, (1) Ethylation (NaBEt4) <1 ng L1 PTIGCMIPAED 1999 [49]
Hg2+; water, soil, (2) Hydride generation
sediment (NaBH 4)+preconcentration on
Chromosorb and thermal desorption
Me2Hg, Et2Hg, (1) Direct SPME (1) 144 pg mL1 GCMIPAED 1999 [34]
Ph2Hg; water, Me2Hg as Hg,
soil, sediment 30 pg mL 1
Et2Hg as Hg
(2) HS-SPME (2) 30 pg mL 1
Me2Hg as Hg,
25 pg mL 1
Et2Hg as Hg
MeHg, EtHg, Acid hydrolysis 1.3 ng mL 1 DORM-2, GCrf-GDAED, 1998 [22]
Hg2+; fish tissue (HCl)+DDTC+derivatization MeHg and DOLT-2 GCdc-GDAED
(Grignard reagent) EtHg, 3.0 ng
mL 1 Hg2+
MeHg, Me2Hg, Derivatization (Grignard reagent) (1) 0.5 (2) (1) GCFAPES 1998 [21]
Hg2+; natural gas 2.1 pg Me 2Hg
condensate (1) 0.9 (2) (2) GCMIPAED
4.1 pg MeHg
(1) 1.7 (2)
4.1 pg Hg2+
MeHg; grain, Acid hydrolysis (HCl)+Celite 545 0.24 pg as Hg GCAED 1998 [54]
cereal products, column+CH 2Cl2 elution+Kuderna
fruit, vegetables Danish concentrator+stannic
chlorideMeOH
MeHg; fish Alkaline digestion 5 pg MeHg PTIGCMIPAED 1998 [39]
(KOHMeOH)+ethylation
(NaBEt4)+Carbotrap column
MeHg; fish MAE (TMAH)+(1) ethylation (1) 3 pg g1 CRM 464 PTIGCMIPAED 1997 [41]
(NaBEt4) MeHg
MAE (TMAH)+(2) hydride (2) 12.5 pg g 1
generation (NaBH 4) MeHg
MeHg, Hg2+; TMAH digestion+ethylation 7 pg MeHg, DOLT-2, PTIGCFAPES 1997 [44]
fish tissues (NaBEt4)+Tenax-TA column+thermal 1 pg Hg2+ TORT-2,
desorption DORM-2
76
Table 1 (continued)
Species; sample Sample preparation LOD CRM used Determination Date Ref.
matrix technique
Table 1 (continued)
Species; sample Sample preparation LOD CRM used Determination Date Ref.
matrix technique
MeHg, Me2Hg; Acid hydrolysis (HBrHI)+benzene 2 pg Me2Hg, GCECD, GC-MIPa 1975 [28]
water, fish 0.5 pg MeHg
Organic Hg; Acid hydrolysis 0.1 ng MeHgCl GC-MIP a 1971 [27]
salmon (HCl)+HgCl2+Benzene+clean-up
with thiosulphate
a
Non-commercial microwave-induced plasma
mental samples they are normally present in polar species, pressure MIP, hydrogen should be used as scavenger gas
making their conversion to compounds that are less polar [14].) Table 1 lists studies of the application of GC
and thermally stable a pre-requisite for gas chromato- MIPAED, and of some other GC methods, to the deter-
graphic separation [7]. CGMIPAED is one of the best mination of Hg species in a variety of sample matrices.
and most frequently used techniques for quantification of Note that in each example the table specifies the certified
the resulting derivatives. reference materials (CRM) used; in species analysis, as in
This paper reviews the application of CGMIPAED other areas of analytical chemistry, the use of CRM is es-
to the speciation of alkyl species containing mercury, tin, sential for good quality control and traceability [15, 16].
and lead in environmental samples mainly water, sedi- Although packed columns are sometimes useful, capil-
ments, and biological tissues. The scope and limitations of lary columns are generally essential for complex environ-
the technique are discussed, and its analytical perfor- mental samples [17]. Because of its flexibility, ease of
mance is compared with that of other hyphenated tech- use, inertness, and ability to retain a variety of coatings,
niques used in species analysis, including GCAED sys- fused silica is the preferred material for capillary columns,
tems with other plasma sources. Particular attention is its inertness being especially important in the determina-
given to sample preparation procedures and derivatization tion of chemically active compounds such as organomer-
methods, because of their great influence on the precision cury compounds [14].
and accuracy of the analytical results.
ing in particular, the LOD for alkylmercury compounds Sample pretreatment for mercury speciation
in the atmosphere was 20 times lower with the axial with GCMIPAED
method.
After an acid-hydrolysis extraction procedure, CGMIP Extraction
AED enables accurate quantification of MeHg in unclean
marine samples and samples with a high fat content (e.g. The first extraction method developed specifically for
mussels), which pose serious resolution problems for GC quantification of mercury species involved the leaching of
ECD systems [20]. Hg compounds from the sample using hydrochloric acid
Frech et al. compared MIPAED and furnace-atomiza- [25, 26]. Since the nineteen-seventies this method has
tion plasma-excitation spectrometry (FAPES) as detection been used to prepare fish samples for the determination of
systems for use with high-resolution GC [21]. The test an- several organic mercury salts by GCMIPAED [27, 28]
alytes were the mercury species in a natural gas conden- (Talmis paper [28] describes a procedure that makes the
sate, which had been derivatized by use of butylmagne- clean-up step of the West method unnecessary.) The to-
sium chloride in tetrahydrofuran. Better LOD were achieved tal analysis time for MeHg in fish is less than 15 min, and
with FAPES, and MIPAED measurement of Me2Hg was, more than 30 benzene extracts can be analyzed per hour.
in fact, impossible, because the plasma could not with- As mentioned above, a modified acid hydrolysis proce-
stand loading with hydrocarbon solvents. Although base- dure has also been applied to marine samples before quan-
line disturbances occurred with both sources during sam- tification of MeHg by CGMIPAED [20].
ple elution, in FAPES they resulted from changes in back- Supercritical-fluid extraction (SFE) has been used to
ground emission, a problem that could be ameliorated by isolate MeHg from sediments before quantification by
means of a suitable background correction system, CGMIPAED [29]. SFE is fast and reliable, and is more
whereas in MIPAED they were because the plasma lost sparing of solvents, labor and chemicals than other cur-
excitation capability, a problem little can be done to solve. rently used methods such as steam distillation (the refer-
Recently, the analytical potential of capillary GC with ence method in the study given in Ref. [29]).
radiofrequency (rf) and direct current (dc) glow dis- Acid leaching procedures have been applied to both
charges (GD) and AED has been investigated for quantifi- marine tissues and sediments [30, 31]. Interfering sulfur-
cation of low levels of MeHg, ethylmercury (EtHg) and containing species can be removed during sample extrac-
inorganic mercury (Hg2+) in fish tissues [22]. Both tion with copper powder, the extracts subsequently being
dc-GDAED and rf-GDAED performed at a level similar themselves extracted with toluene after acidification with
to that of more common AED methods. GCGDAED potassium bromide solution [30, 32]. High molecular-
enabled accurate determination of the Hg species without weight pigments and lipids can be removed from the ex-
the need to resort to standard addition techniques. When a tracts by preparative gel-permeation chromatography
hollow-cathode was used to increase emission intensities, (GPC) before GC analysis [30].
optimization of discharge conditions (pressure, He flow Few studies have used solid-phase microextraction
rate, rf power) afforded LOD that were 510 times better (SPME) to prepare samples for quantification of Hg species
than those obtained by use of flat-cathode GDAED and by GCMIPAED [33, 34]. SPME extracts volatile or
were also lower than those obtained with MIPAED [23]. semivolatile organic compounds directly from an aqueous
The hollow-cathode GD-AED system had a low construc- or gaseous sample passed through a capillary or over a
tion cost and consumed 1020 times less He than MIP fused-silica fiber coated with an appropriate stationary
AED. phase. In combination with headspace sampling (HS),
The performance indices of GC in combination with SPME has proved suitable for extraction of Me2Hg and
MIPAES, mass spectrometry (MS), or atomic fluorescence diethylmercury (Et2Hg) in the analysis of aqueous and
spectrometry (AFS) have been evaluated for quantifica- soil samples. It is also possible to use direct SPME for
tion of MeHg and EtHg after aqueous derivatization with less volatile organomercury compounds in aqueous sam-
sodium tetraethylborate and sodium tetraphenylborate ples [34].
[24]. Both GCAFS and GCMIPAED had broad linear
ranges, although the AFS sensitivity setting had to be ad-
justed for each particular sample on the basis of its Hg Derivatization
concentration. Phenylation seemed to be the better deriva-
tization method, because it distinguished better between The derivatization of analytes before their determination
EtHg and Hg2+ and was less expensive than ethylation. It by GC with low-pressure or atmospheric-pressure MIP
was concluded that although GCMS is, perhaps, the AED avoids capillary column passivation problems even
most important technique for identification and confirma- if no prior extraction stage is used [35]. Direct ethylation
tion, it is unlikely to provide the sensitivity required for or phenylation in an aqueous medium, and butylation with
determination of Hg in most environmental samples. a Grignard reagent, have been compared with regard to
their improvement of the multi-element determination of
several organomethylates in biological tissue [36]. The
addition of traces of a mixture of oxygen and hydrogen to
the plasma improves the shape of the chromatogram peaks
79
by removing carbon deposits and preventing the forma- Combination of MAE with a suitable derivatization pro-
tion of refractory oxides. For samples for which there is cedure avoids the need for previous column treatment
no information about the species present, derivatization with inorganic salts before GCMIPAED, and also re-
by at least two procedures is recommended. duces solvent volume and analysis time.
PTI has also been used in other studies of the applica-
tion of GCMIPAED [41] or GCFAPES [44] to quan-
Extraction and derivatization tification of MeHg and Hg2+ in biological tissues. In the
latter technique samples were solubilized with TMAH
Fish and marine samples. Combining derivatization with and the ionic species were purged from aqueous solution
extraction improves the chromatographic characteristics after ethylation; the species were then preconcentrated on
of the species to be separated. For MeHg and EtHg in fish, Tenax-TA and thermally desorbed on to an isothermal GC
samples can be extracted with saturated sodium chloride column.
solution and HCl; after the extracts have been shaken vig-
orously with sodium diethyldithiocarbamate (DDTC) and Water samples. In the quantification of Hg compounds in
toluene, the toluene phase can be derivatized with butyl- water samples, low concentration is without doubt the
magnesium chloride in tetrahydrofuran (THF) [37]. Col- most serious problem; occasionally very large sample vol-
umn efficiency, detection limits, and resolution of MeHg umes must be processed. The method developed by Emte-
and EtHg are better in capillary GCMIPAED than borg et al. [45] for simultaneous determination of mercury
packed column GCECD. species in natural waters involves large sample volumes,
Alkaline digestion with KOHmethanol is an obvious preconcentration on a dithiocarbamate (DTC) resin-loaded
alternative to acid treatment before extraction of Hg com- microcolumn, extraction of the eluted species into toluene,
pounds with dichloromethane then phenylation [38] or and derivatization with a Grignard reagent; these deriva-
ethylation [39]. When used to isolate MeHg from fish tives are separated and quantified by GCMIPAED. One
samples, alkaline digestion has been found to eliminate disadvantage of the method is that the preconcentration
interferences with the derivatization reaction before GC step fails in the presence of high concentrations of humic
MIPAED determination [38, 39]. International intercali- substances. To solve this problem a batch method has
bration studies of the simultaneous determination of been developed [46] Hg species enrichment is per-
MeHg and Hg2+ from reference and candidate reference formed by adding purified DTC resin directly to the water
materials by CGMIPAED after acid leaching or alka- sample. The accuracy of this method has been assessed, in
line digestion and subsequent derivatization revealed very part, by means of interlaboratory comparison with results
large differences between the extraction efficiencies of from GCAFS, and the ability of the columns to retain
different sample work-up procedures. Artifact formation, alkyl-Hg and Hg2+ compounds has been studied [47]. In a
detector selectivity, chromatographic performance, and later variant, large volumes of the sample are injected in a
the stability of Hg compounds are discussed on the basis packed-column GC system connected to a capillary GC
of their results [40]. system coupled to a MIPAED device [48]. In this way
Microwave-assisted extraction (MAE) is a well ac- the Hg species are focused before separation in the ana-
cepted technique in elemental trace analysis and has also lytical column; this minimizes the risk of the plasma be-
been used for quantification of organometallic compounds. ing extinguished by excess solvent. This variant has been
Under the usual conditions (high applied energy, high applied to the determination of Hg species in river water.
pressures, and high temperatures) most compounds are In another study [49] Hg species were either ethylated
destroyed and all speciation information is lost [41], but or were converted to hydride with NaBH4, stripped from
when performed under milder conditions in a closed sys- solution with He, pre-concentrated on Chromosorb at
tem with sophisticated pressure and temperature control it room temperature or 160 C, released from the adsorbent
achieves successful extraction of organomercury com- by thermal desorption, and quantified by PTIGCMIP
pounds [42]. Alternatively, an open system using focused AED. Both these procedures were designed for analysis
microwaves has also been successfully applied to the di- of soil and sediments and water.
gestion of fish samples before Hg species analysis by Because of problems associated with the production of
GCMIPAED [41]. Digestion of fish tissues with tetra- natural materials and the stability of Hg compounds in
methylammonium hydroxide (TMAH) is quick and sim- aqueous media, very few reliable reference materials are
ple, and losses of volatile analytes as a result of heating by available for the quantification of mercury compounds in
the microwave field are reasonably low and reproducible. natural waters [50, 51]. Because of this the possibility of
Two different derivatization and injection procedures storage on a solid support has been studied [52]. The sta-
have been examined (ethylation, extraction into hexane, bility of Hg species from natural waters immobilized on
and injection with a cooled injection system; and hydride sulfhydryl cotton fibers (SCF) was examined by elution
generation with sodium tetrahydroborate then purge-and- into HCl solution, derivatization with NaBPh4, and deter-
trap injection (PTI)). mination by GCMIPAED [53].
For determination of MeHg with the closed-vessel sys-
tem the extraction and derivatization conditions have been Gaseous samples. Hg species present in natural gas con-
optimized by use of a factorial experimental design [43]. densate have been extracted for determination by GC
80
MIPAED using an on-line amalgamation trap [33 ]. The by degradation of TBT. Triphenyltin (TPhT), although
Hg compounds can be collected from the column eluate also used as an antifouling agent in paints, is mainly em-
within a specific time window and subsequently passed to ployed as a fungicide and biocide in agriculture [58]. Nat-
the plasma as Hg vapor in a flow of pure helium. Use of ural degradation of TPhT to diphenyltin (DPhT) and mono-
the amalgamation trap improves the determination of phenyltin (MPhT) is well documented. Nowadays, use of
mercury species in two ways. First, by separating analyte all these compounds (especially TBT) is restricted, but
material from the matrix before detection it enables the in- they are still present in marine environments (mainly in
troduction of samples containing large amounts of carbon sediments, from which they are slowly released into the
compounds without any need to use split injectors or di- aquatic environment). Naturally occurring organotin com-
luted or smaller sample masses to preserve chromato- pounds are mainly biomethylated species generated from
graphic resolution and detector efficiency. Second, use of inorganic tin (Sn4+) in sediments and estuarine environ-
the trap enables the addition of standards directly after the ments. Natural methylation of butyltin compounds has
gas chromatograph by injecting samples of air saturated also been reported [59].
with mercury vapor; this procedure enables independent The toxicity of organotin compounds depends on the
calibration for standards separated by the chromatograph. number and kind of organic groups bound to the Sn atom.
Results obtained by use of the trap were compared with Species analysis is therefore essential for understanding
data obtained by HS-SPME and liquid sample injection the biological effects and environmental impact of these
followed by direct measurement of the column eluate. compounds. Accurate identification and quantification of
SPME might be a simple and useful alternative method tin species in environmental samples needs reliable ana-
for the determination of derivatized polar Hg species [33]. lytical methods based on the combination of adequate
sample preparation procedures with selective, sensitive
Soil samples. In addition to the method mentioned above analytical techniques. Most viable methods are based on a
in the section Water samples [49], extraction with di- separation step followed by quantification, by use of an
chloromethane then in situ ethylation and collection of the appropriate detector. Gas chromatographic separation is
species on Tenax has been used for determination of more widely used than HPLC because of the higher re-
MeHg in forest soils by GCMIPAED [31]. solving power of GC columns, the smaller requirement for
hazardous solvents, and the availability of detectors that
Food samples. Historically, Hg in food products other are more sensitive and more selective. Among these tech-
than seafood was not subjected to species analysis be- niques, GCMIPAED has been one of the most widely
cause of the lack of adequate methodology. The high used in the past 10 years.
specificity and selectivity of GCMIPAED, and the min-
imum analyte isolation required by this method (irrelevant
matrix components being transparent to the detector) Experimental conditions and analytical features
have, however, enabled its use for the determination of of GCMIPAED
MeHg in thirty-two samples of grains, cereal products,
fruits, and vegetables [54]. After acid hydrolysis with HCl Optimization of MIPAED conditions for the analysis of
the resulting chlorinated species were eluted from a Celite organotin compounds was first performed by Lobinski et
545 sample homogenate column with dichloromethane, al. [60], and later by several others (Table 2). Although
the eluate was treated with stannic chloride, and the ana- there are small differences among the measurement con-
lyte was isolated from co-extracted materials by use of a ditions used in these studies, there is general agreement
wide-bore capillary column for GCMIPAED. that a helium make-up flow rate higher than 200 mL min1
is necessary to prevent the interaction of tin compounds
with the quartz discharge tube and to achieve maximum
Speciation of tin compounds sensitivity. The presence of oxygen in the helium plasma
was also found to be necessary to prevent deposition of
Organotin compounds found in environmental samples carbon on the walls of the discharge tube, even though
(water, sediments, particulate matter and living organ- oxygen leads to the formation of refractory tin oxides and
isms) have both anthropogenic and natural origins. The a consequent decrease in the emission signal. The nega-
most important anthropogenic compounds are butyl- and tive effect of oxygen can be compensated by addition of
phenyltin species. Tributyltin (TBT), used mainly as an hydrogen to the plasma gas; hydrogen probably produces
antifouling compound in paints, is highly toxic to both very volatile hydrides that are easily atomized and ex-
target and non-target marine organisms, and has recently cited, thereby reducing the detection limit of the tech-
been included in the list of endocrine system disrupters nique [61, 62].
[55]. Dialkyltins with butyl and octyl groups are used as Although some authors claim absolute detection limits
polymer stabilizers in, for example, PVC pipes, from <0.2 pg of tin [60, 63, 64, 65], most authors have reported
which they can be released into water [56], or even into values between 0.5 and 5 pg. Slight variations in the he-
air, in fires or during processing of plastic materials at lium make-up flow and reagent gas pressure are probably
high temperature [57]. Dibutyltin (DBT) and mono- largely responsible for these small differences. With re-
butyltin (MBT) can also be generated in the environment gard to the effect of the emission line selected, Tutschku
81
et al. [62] obtained lower detection limits at 326.23 nm With regard to the linearity of response, several papers
than with the more usual 270.651 and 303.419 nm lines. have reported GCMIPAED calibration curves that are
Table 2 summarizes MIPAED conditions and detection linear over the range 10 to 2500 ng tin mL1, with corre-
limits for organotin compounds. lation coefficients >0.999 [68, 69]. The detection limits
Table 3 lists the detection limits achieved by GC-based noted above and the elemental selectivity of CGMIP
systems other than GCMIPAED for quantification of AED have made this technique the workhorse for quan-
organotin compounds. Those achieved by GCMIPAED tification of organotin compounds in environmental sam-
are similar to those of the best GC-FPD instruments and ples.
of GCMS systems with a quadrupole mass analyzer op- In the discussion below the possibilities and limitations
erating in SIM mode (ion-trap mass analyzers are a valu- of GCMIPAED for the speciation of organotin com-
able alternative to high-sensitivity scanning, and have de- pounds in a variety of different matrices are discussed.
tection limits similar to those of quadrupole instruments Some comments on sample-preparation and derivatization
in SIM mode) [66]. In GCMIPAED, however, all strategies are also made.
organotin compounds are quantified under identical con-
ditions, whereas with quadrupole SIM MS detection sev-
eral different masses must be monitored [67]. The combi- Water samples
nation of gas chromatography with AAS is less sensitive
for tin compounds than GCMIPAED, because of the The levels of organotin compounds in polluted waters
high atomization temperatures of tin and the possible for- (rivers, estuaries, harbors, shipyard areas, and tap water in
mation of refractory oxides [63]. Detection limits one or contact with certain polymeric materials) are below the
two orders of magnitude lower than those of CGMIP detection limits of GCMIPAED or any other gas chro-
AED have only been achieved with customer developed matography-based technique used in organotin species
combinations of gas chromatography with ICPMS detec- analysis. A concentration step is therefore necessary be-
tion. fore analytical determination. In general, concentration
strategies are very similar for all gas chromatographic
Table 3 Absolute detection limits (S/N=3) for organotin com- techniques used in the speciation of tin, Table 4.
pounds achieved by gas chromatography systems other than Classical approaches are based on the extraction of
GCMIPAED organotin compounds (mainly butyl and phenyl com-
pounds) using a complexing agent (tropolone or sodium
System Instrumental detection Ref.
limits (pg tin) diethyldithiocarbamate) in combination with a volatile or-
ganic solvent, followed by derivatization with a Grignard
GC-FPD 3 [148] reagent [60, 67]. A faster option is the use of NaBEt4, and
0.2 [149] more recently NaBPr4, as derivatization agent (the latter
24 [150] enables the investigation of ethyltin compounds in water
0.20.5 [151] samples) [61, 64, 70]; in both reactions alkyl derivatives
0.318 [152] are formed in the aqueous medium and extracted simulta-
GC-QF AAS 3371 [63] neously into an organic solvent. NaBEt4 has also been
10100 [153] used to derivatize polar organotin species previously re-
GCMS 110 [66, 67] tained on a solid extraction cartridge [65].
1.58 [154] Methyltin compounds can be ethylated in aqueous
samples using NaBEt4, and simultaneously extracted and
GCICPMS 0.050 [155]
concentrated with a purge-and-trap device; these injectors
0.0150.034 [156]
can use cryogenically cooled capillary traps [71] or solid
0.0520.17 [112]
adsorbents operating at room temperature [49]. Polar butyl-
0.050.08 [157]
tin species can be converted by treatment with NaBH4 into
82
Table 4 Summary of GCMIPAED procedures for the quantification of organotin compounds in water samples
Compounds Sample Extraction technique Derivatization Detection Ref.
volume reagent limits
(mL) (ng L1)
Butyl and phenyltin 50 LLE (hexane) NaBEt4 0.1b [64]
Butyl and phenyltin 50 SPE, C18a NaBEt4 0.1b [65]
Butyl, methyl and diphenyltin 100250 LLE (hexanetropolone) EtMgBr 1015 [147]
Butyl and methyltin 1500 LLE (pentaneNaDDTC) PeMgBr [60]
Butyl and phenyltin 100 LLE (hexane) NaBEt4 1733 [61]
Butyl and phenyltin 100 LLE (hexane) NaBPr4 312 [70]
Methyltin 10 P&T NaBEt4 0.15 [71]
Butyltin 4 Direct SPME (PDMS fibers) NaBEt4 ca. 20 [73]
aDerivatization of polar compounds on the stationary phase of the bCalculated for an injection volume of 25 L, using a PTV injector
adsorbent cartridge Not available
volatile compounds that can be purged from water sam- the analysis of butyltin compounds at low concentrations
ples, an option that has normally involved the use of a when using HS SPME with GCMIPAED [82]. Apart
packed column coupled to a QF-AAS instrument [72]; as from problems described by the authors in relation to the
far as we are aware, analysis of butyltin compounds as hy- SPME fiber surface, it is also possible that blank signals
drides by GCMIPAED has not been reported in the lit- are also responsible for this lack of repeatability when
erature. organotin compounds are analyzed at concentrations be-
The possibility of direct derivatization in aqueous sam- low the g L1 level.
ples enables the use of solid-phase microextraction
(SPME) as a concentration technique [73, 74, 75, 76].
Non-polar fibers are normally used (mainly with polydi- Solid matrices
methylsiloxane as stationary phase). The sampling step
can be performed directly in the aqueous phase or in the Sample preparation procedures for determination of organo-
headspace over the sample. The latter procedure enables tin compounds in solid matrices by gas chromatography
faster equilibration between sample and fiber for volatile comprise several steps, the precise number of which de-
and semivolatile compounds, and also more selective ex- pends on the derivatization technique (Grignard reaction,
traction. Although methyl- and butyltin species are nor- direct alkylation with sodium tetraalkylborates in aqueous
mally sampled at room temperature [77, 78], for less solution, or hydride generation) [83] and on whether a
volatile species such as phenyltin compounds better sensi- clean-up step is needed, which in turn depends on the de-
tivity is achieved at high temperature [79]. rivatization technique (alkylborates normally lead to
Despite the high concentration factors achieved with cleaner extracts than Grignard reactions, which require
these techniques, and their low or at best medium selec- the use of complexing reagents such as tropolone [84]),
tivity, GCMIPAED suffers from no interferences at tin on the kind of sample, and above all on the selectivity of
emission lines (271, 303 and 326 nm). Some problems the GC detector. For GCMIPAED systems, concentra-
with blanks have, however, been reported. Szpunar et al. tion of the final extracts from solid matrices is not nor-
[80], who used Grignard derivatization with pentylmagne- mally necessary, because instrument sensitivity matches
sium bromide, observed organotin signals in blanks when target detection limits for environmental samples (approx.
several microliters of the organic phase were introduced 1 ng g1) [85].
into the chromatography column by use of a PTV injector.
Blanks with organotin signals have also been detected
when NaBEt4 was used as derivatization agent; possible Sediment samples
sources of this contamination are plastic holders, the
polypropylene body of SPE cartridges [65], and reagents Because organotin compounds are not involved in miner-
used in sample preparation, including the NaBEt4 [81]. alogical processes and bind only to the surface of sedi-
Szpunar et al. also reported that the use of sodium di- ments, complete dissolution of the matrix is not consid-
ethyldithiocarbamate (NaDDTC) as a complexing agent ered necessary. Acid leaching combined with mechanical
and then Grignard derivatization of the organic extract led shaking [86] or sonication [77], and microwave-assisted
to noisy baselines, probably because of reaction of the leaching [87, 88], are, therefore, the basic approaches used
Grignard reagent with decomposition products of NaDDTC to release organotin compounds from sediments. Leach-
[80]. Stb et al. [67] using GCMS have identified MBT ing can be achieved by use of dilute acids with complex-
and DBT, at levels of 512 ng L1, in blank chromato- ing agents (tropolone or sodium diethyldithiocarbamate)
grams corresponding to the analysis of water. Finally, dissolved in an organic solvent [84, 89], or using only
Rosenberg et al. have reported variations of 3040% in aqueous or methanolic solutions of acetic or hydrochloric
83
acids [88]; normally the former choice is followed by Some authors have found that GCMIPAED chro-
Grignard derivatization and the latter by direct alkylation matograms for some sediment samples show not only
with NaBEt4 or NaBPr4. Methods based on hydride gen- methyl-, butyl-, and phenyltin compounds but also peaks
eration have been shown to be poorly efficient for extracts corresponding to minor organotin species [96]. Identifica-
with high sulfur, hydrocarbon, or inorganic metal content, tion of these species is the first step towards understand-
and are therefore not advisable for sediment analysis [72]. ing their origin and environmental behavior. GCMS is
The element selectivity of GCMIPAED makes it un- currently the only hyphenated technique capable of pro-
necessary to perform the time-consuming desulfuration pro- viding information about their structures. Because the de-
cedures described in the literature for the determination of tection limits of quadrupole mass analyzers, working in
organotin species in sediments by GC-FPD [90, 91]. In gen- scan mode, are still far above those achieved with MIP
eral, no clean-up procedures are necessary with GCMIP AED, the use of ion-trap MS instruments is advisable
AED, although Ceulemans et al. [89] have recommended [66].
that if tropolone is used as complexing reagent the final or-
ganic extract should be passed over basic alumina, or ex-
cess tropolone can contaminate the head of the column. Biological tissues
The analytical characteristics (sensitivity and selectiv-
ity) of GCMIPAED are, in principle, good enough to Speciation of tin compounds in fish and other seafood tis-
solve the detection step in the analysis of tin compounds sue has been studied for the past 15 years, since their neg-
in sediments. Problems mainly arise in sample prepara- ative effects on the reproduction of marine organisms
tion [92] and in the limited number of reference materials such as oysters and other mollusks were discovered [97,
(currently only reference sediment material PACS-2, with 98]. Nowadays, two reference materials are available
certified concentrations of MBT, DBT, and TBT, is com- NIES-11 (fish tissue from the National Institute for Envi-
mercially available). As a consequence, validation of ana- ronment Studies, Japan) and BCR-477 (mussel tissue pre-
lytical procedures for phenyltin species is a difficult task. pared by the former BCR). The concentration of TBT in
Some of the problems related with the analysis of organo- the former is certified and an approximate level is also in-
tin compounds (mainly MBT and phenyltin species) in dicated for TPhT; the latter contains MBT, DBT, TBT,
sediments are well known and clearly described in the lit- MPhT, and TPhT, at least, although only the concentra-
erature. Few sample extraction procedures for MBT claim tions of MBT, DBT, and TBT are certified.
quantitative yields, and recoveries are sometimes esti- As for sediments, sample preparation is usually per-
mated by use of spiked samples [84, 93]. In real polluted formed in several steps, the first of which is sample di-
sediments interaction of MBT with the matrix, which is gestion. Because organotin compounds can be incorpo-
directly related to sample sulfur and organic carbon con- rated in tissues, complete sample decomposition is a pre-
tent [84], is probably even stronger than in the spiked requisite for quantitative extraction. Acid solutions (mainly
samples, and the extraction yield accordingly lower. Be- acetic acid and dilute hydrochloric acid), modified super-
cause of its structure and polarity, similar behavior is ex- critical CO2, and mixtures of lipase and protease have all
pected of MPhT. Another problem is encountered in the been used to release organotin compounds from biologi-
extraction of phenyltin compounds these species are cal tissues, but highest yields seem to be achieved with
partially degraded by some leaching procedures designed aqueous TMAH solutions [99, 100, 101, 102, 103]. The
for butyltin compounds. Degradation is of special signifi- second step is derivatization and extraction of the organo-
cance when acid conditions are combined with high tem- tin derivatives into an organic solvent (except for hydride-
peratures. Despite their lability during sample prepara- generation techniques, when cryogenic purge-and-trap de-
tion, however, surveys of phenyltin compounds, espe- vices are used). Fast, integrated sample-preparation pro-
cially TPhT, in sediments are of environmental interest, cedures have been described in which tissue digestion,
because of their toxicity and their persistence under the derivatization (ethylation), and extraction of the ethylated
anaerobic conditions obtaining in sediments [67]. derivatives into an organic solvent occur simultaneously
Both problems (non-quantitative extraction and degra- in only 3 min under the action of a microwave field [83].
dation) could be compensated by use of isotope-labeled The problems encountered in the sample preparation
organotin compounds as internal standards. If the label is step are similar to those reported for sediment samples:
introduced in the tin atom [94] the excellent sensitivity sub-quantitative recovery and the degradation of phenyltin
and multielemental capabilities of ICPMS might justify compounds [101, 104]. TPhT seems to be more stable at
marketing an interface between GC and ICPMS as a basic pH [79, 99] than in acid media [101], however,
complementary technique to GCMIPAED for organo- which is another reason for preferring solubilization with
tin speciation (and for volatile organometallic speciation TMAH to acid leaching.
in general) [3]. A similar effect could be achieved with Irrespective of the digestion and derivatization agents
13C or deuterated organotin standards; with these, deter- used, the final extracts from biological samples are col-
mination can be achieved by use of conventional quadru- ored solutions containing large amounts of lipids. Clean-
pole GCMS [95], a technique available in most analyti- up of the organic extract containing the derivatized organo-
cal chemistry laboratories which is sufficiently sensitive tin species has therefore been recommended, to prevent
for the analysis of organotin compounds in solid matrices. column deterioration and to eliminate baseline drift with
84
Fig. 1 GCMIPAED chromato-
grams obtained from a mussel-tissue
extract, with clean-up over alumina
(solid line) and without clean-up
(dotted line). Extraction was per-
formed as described elsewhere [99].
1. TPT (IS), 2. MPhT, 3. TPhT.
A. 248 nm carbon emission line.
B. 271 nm tin emission line
increasing oven temperature when relatively poorly selec- still used in some countries as anti-knocking additives in
tive detectors such as scan-mode MS or FPD are used. Po- gasoline [107], can penetrate the skin and biological
lar adsorbents such as silica [100], alumina [80, 99] and membranes and are readily absorbed via the lungs. The
Florisil [105, 106] are used for this purpose. Szpunar et al. toxicity of organolead compounds depends on the organic
have shown that with CGMIPAED a stable, flat base- groups bound to the lead atom methylated species are
line is obtained even without clean-up, although they also less toxic than the corresponding ethylated compounds,
noted that the response to ethylated TPhT depended on but are more stable and volatile [108].
whether or not clean-up had been performed [80]. In our TAL enter the atmosphere via vehicle exhaust pipes
laboratory we have had the same problem with the deter- and as a result of accidental spillage. There they are de-
mination of TPhT in biological materials; the 248 nm car- composed to inorganic lead (Pb2+) via tri- and dialkyl
bon channel, monitored simultaneously with the 271 nm compounds. Photolysis and radical reactions are responsi-
tin line, shows a large, broad signal at the retention time ble for the fast decomposition of Me4Pb and Et4Pb
of TPhT (Fig. 1). The species responsible for this non-spe- (t1/2=41 and 8 h, respectively). Ionic organolead species
cific signal (probably lipids) might temporarily modify (Me3Pb+, Et3Pb+, Et2Pb2+, and Me2Pb2+) are less toxic
the internal wall of the quartz discharge tube, causing loss than tetraalkylated species but have longer half-lives, en-
of sensitivity in tin emission signals. abling them to be transported considerable distances from
their anthropogenic sources [109].
The combination of gas chromatography with spectro-
Speciation of lead compounds scopic techniques (AAS, MIPAED, MS or ICPMS) is
common in the speciation of lead compounds. As for tin,
Organolead compounds are ubiquitous pollutants in air, the separation of lead compounds by gas chromatography
atmospheric aerosols, water, and sediments. Tetraalkyllead requires prior derivatization of ionic alkylated species to
species (TAL; mainly Et4Pb, Me4Pb, and MenEt4nPb), thermally stable volatile compounds. This reaction can be
85
performed with Grignard reagents after chelation of these Analytical features of GCMIPAED
species and extraction into an organic solvent such as
hexane or pentane. Another option is to use borate reagents. Detection limits obtained in the speciation of lead com-
Although sodium tetraethylborate is only useful for meth- pounds by use of a variety of different GC-based hyphen-
ylated lead compounds (both inorganic and ethylated lead ated techniques are compared in Table 5. The detection
species yield Et4Pb [110]), Pawliszyn et al. have recent- limits of GCMIPAED are similar to those reported for
ly overcome this problem by using deuterium-labeled GCICPMS, and between two and three orders of mag-
NaBEt4 [111]; because the isotope-labeled ethyl group is not nitude better than those obtained using GCMS systems
present in environmental samples, it can be used to distin- or custom-made combinations of GC with AAS.
guish between ethylated inorganic lead and native ethyl- The GCMIPAED system enables measurement of
lead species. Lower-cost alternative derivatization reagents lead emission at 261.418 nm and 405.783 nm. The latter
are sodium tetrapropylborate [10, 112] and tetrabutylam- line is preferred because of its higher sensitivity, which is
monium tetrabutylborate [113, 114], both of which enable maximum with a helium make-up flow of approximately
simultaneous determination of most of the ionic alkyllead 300330 mL min1 [114, 116, 117, 118]. Oxygen and hy-
(IAL) species commonly found in environmental samples drogen are also necessary as reagent gases; oxygen pre-
(Me3Pb+, Et3Pb+, and MenEtmPb(4nm)+) [115]. vents carbon from being deposited on the walls of the dis-
charge tube (oxygen pressure is normally adjusted to 138
kPa (20 psig)) and, as for tin, the presence of hydrogen
improves sensitivity (pressures of 550650 kPa are re-
ported as optimum [116, 119, 120]).
With the 405.783 nm emission line, good linearity has
Table 5 Absolute detection limits (S/N=3) for organolead com- been obtained for injected amounts of organolead species
pounds achieved by GC-based hyphenated techniques
between 0.1100 pg, as Pb. If very high levels of organo-
Hyphenated Absolute detection limits Ref. lead species reach the plasma, lead can condense on the
technique (pg as lead) wall of the discharge tube, causing peak tailing and re-
GCMIPAED 0.031 [107, 116] duced sensitivity [116]. Because more than 99.9% of the
0.01 [123] lead in most samples is Pb2+ [121], and part of this inor-
0.040.08 [114] ganic lead can be derivatized and extracted with the native
organolead species, it is advisable to vent the chromato-
GCICPMS 0.010.016 [122]
graphic peak corresponding to native Pb2+, to prevent con-
0.7 [158]
tamination of the discharge tube [120].
0.040.09 [112]
GCAAS 4095 (Flame) [125]
3045 (Quartz furnace) [47] Water and other aqueous samples
12 (Quartz furnace) [159]
Because of their short half-lives and low solubility in wa-
GCMS 2 [110] ter, the presence of TAL compounds in aqueous samples
4 [130]
is not common [107]. The levels of IAL species in rain,
78 [131]
surface, and tap water (mainly dimethyl-, trimethyl-, di-
ethyl-, and triethyllead) are at most a few ng L1 [47, 117, ports of transalkylation reactions; Pb2+ is, nevertheless,
118, 121, 122]. The concentration of organolead com- usually masked with EDTA to reduce consumption of bo-
pounds in polar snow is even lower, although it is indica- rate [110, 114]. The presence of EDTA does not affect the
tive of the atmospheric distribution of these pollutants and derivatization of IAL because only inorganic lead forms
of the world production of leaded petrol [120, 123, 124]. stable chelates [113].
Because of these low concentrations, the first step in the Solid-phase procedures can be used as an alternative to
quantification of organolead compounds in aqueous sam- liquidliquid extraction for the quantification of lead
ples is enrichment. Table 6 lists reported sample prepara- species in water samples [47, 115, 125]; adsorbents con-
tion procedures. The earliest concentration techniques taining dithizone or dithiocarbamate groups are usually
were based on liquidliquid extraction of IAL species used. Purge-and-trap and SPME are also alternatives that
with hexane or pentane after complexation with sodium can be used after in-situ derivatization with borate
diethyldithiocarbamate at pH 89; this was followed by reagents [74, 111, 112, 121, 126, 127]; both techniques re-
derivatization with Grignard reagents such as propyl- or quire smaller volumes of sample than LLE (Table 6) and
butylmagnesium chloride [117, 120, 122, 123, 124]. Si- airborne contamination is also avoided, because samples
multaneous alkylation and extraction of IAL into organic are processed in closed vessels; contamination problems
solvents can be performed by use of NaBEt4 [110, 111], arising from derivatization reagents cannot, however, be
NaBPr4 [10, 112] or Bu4NBBu4 [113, 114], for all of circumvented by use of these approaches.
which the highest yield is achieved at pH 4. Irrespective
of whether a Grignard reagent or a borate is used, inor-
ganic lead present in the sample is co-extracted and de- Air samples
rivatized; to prevent large quantities of alkylated inor-
ganic lead from contaminating the MIP discharge tube (or Determination of IAL and TAL compounds in the atmo-
the ion source of GCMS systems), inorganic lead is nor- sphere, in which they are found at levels below 1 ng m3,
mally masked with EDTA before extraction of IAL com- requires the concentration of large volumes of air. Filters,
pounds. solid adsorbents such as Tenax, Porapak Q, and Amber-
With initial sample volumes of 100200 mL and the lites, water-filled gas bubblers, and cryogenic traps have
injection of 2550 L of the final organic extract into the all been used to extract organolead compounds from at-
chromatographic column, GCMIPAED achieves detec- mospheric aerosol and gas phases [108, 109, 128, 129].
tion limits of the order of 0.1 ng L1 (Table 6). At these Because of the possibility of decomposition of TAL com-
low concentrations, however, the accuracy and precision pounds in the presence of ozone, a layer of ferrous sul-
of the analysis can easily be disturbed by blank signals phate is normally placed before to the trap [129].
and the presence of artifacts. In fact, organolead com-
pounds are very often detected in blanks because of con-
tamination and transalkylation reactions. Steps can be Solid samples
taken to prevent this:
In addition to water and air, organolead compounds have
For samples with concentrations below 0.1 pg g1, sam-
been found in soil, sediments, road dust, grass, and tree
ple preparation should be performed in clean cabinets
leaves. Wet atmospheric deposition, direct contamination
to avoid contamination with airborne IAL [123].
(in the vicinity of emission sources) and absorption from
Concentrated standards should be prepared and stored
contaminated waters through plant roots are the mecha-
in a different room from the sample [107].
nisms responsible for the presence of alkyllead species in
Impurities in reagents (buffer solutions and complexing
these samples. The excellent sensitivity and selectivity of
and masking agents) can be removed by pre-extraction
GCMIPAED for lead compounds enables quantifica-
with hexane [116, 117, 119, 123].
tion of levels in these samples without any need for high
Derivatization reagents are another possible source of
concentration factors or extensive clean-up procedures.
problems. Purification of Grignard reagents is very dif-
Because organolead species are labile, however, care must
ficult because of their low stability. PrMgCl is normally
be taken to extract them without modifying their chemical
preferred to BuMgCl and PeMgCl for quantification of
structure.
organolead compounds by GCMIPAED because it
The extraction of lead compounds from sediments and
causes less baseline disturbance [120].
particulate matter can be performed under mild conditions
If Grignard derivatization is used, Pb2+ must be masked by use of organic solvents in the presence of complexing
before extraction of organolead compounds because oth- agents [107, 130], supercritical CO2 modified with
erwise it will react with the Grignard reagent, giving not methanol [131], buffer solutions of pH 4, or direct disso-
only the main product (e.g. Pr4Pb, if PrMgCl is used) but lution in water containing NaCl [121]. Most of these pro-
also minor compounds such as MePr3Pb and EtPr3Pb, cedures give satisfactory yields for Me3Pb+ and Et3Pb+,
which are formed by transalkylation reactions [80]. For but not for dialkyllead species. Validation of the extrac-
the alkylborate derivatization reagents NaBEt4, NaBPr4, tion procedure is, moreover, only possible for Me3Pb+, for
and Bu4NBBu4, which also react quantitatively with both which there is a road dust reference material (BCR 605,
IAL and inorganic lead [111, 112, 121], we know of no re- containing 7.91.2 g kg1 of Me3Pb+).
87
With regard to biological samples, TMAH has been ously found to be optimum for tin, and detection limits
used as a tissue solubilizer for the speciation of lead com- lower than 1 ng L1 of metal were achieved for all com-
pounds in grass and tree leaves. The digestion step takes pounds with a sample intake of 10 mL. Schubert et al.
several hours, and unless performed at low temperature [70] have described the use of NaBEt4 and NaBPr4, then
affords very low yields of dialkyllead compounds, pre- liquidliquid extraction with hexane, for the simultaneous
sumably because of their transformation into inorganic determination of organotin (butyl and phenyl compounds)
lead [132]. Enzymatic hydrolysis of biological tissues has and organolead (Et3Pb+ and Me3Pb+) in water samples;
also been used samples are treated with a mixture of li- the two groups of compounds were analyzed at 270.651
pase and protease at 37 C for 2448 h [107]. SPME has and 261.418 nm, and the 247.857 nm carbon line was also
been described as a promising technique for the analysis monitored to obtain information about possible interfer-
of Pb2+ and organolead compounds in urine and blood ences from organic compounds. Finally, Minganti et al.
after precipitation of erythrocytes (from blood), samples [36] have shown that lead, tin, and mercury speciation in
are diluted with water and adjusted to pH 4, after which water and biological tissues is possible after extraction
lead compounds are derivatized in-situ and adsorbed on to and simultaneous derivatization with n-pentylmagnesium
a 100 m polydimethylsiloxane-coated fused-silica SPME bromide.
fiber [126, 133].
cryogenic purge-and-trap injector [138, 139]. These pro- dards and MS detection. We regard this as the main ad-
cedures have been validated by analysis of reference ma- vantage of GCICPMS over GCMIPAED and other
terials (sediments, biological tissues, urine, and petrol) atomic emission detection systems for quantification of
with certified levels of tin, lead, and mercury compounds. mercury, tin, and lead species.
Other papers have reported the possibility of using iso-
thermal columns as short as 522 cm combined with Acknowledgements Financial support from the Spanish DGICT
ICPMS for analysis of mercury and tin species [140, (project REN 20000984HIP) is acknowledged.
141].
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Anal Bioanal Chem (2002) 372 : 91100
DOI 10.1007/s00216-001-1197-3
T E C H N I C A L R E P O RT
C. Hnel G. Gauglitz
Received: 22 August 2001 / Revised: 5 October 2001 / Accepted: 25 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
detection method used will be described in a little bit more for certain incident angles. The angles of excitation are sen-
detail, because it is not as well known as the other princi- sitive to changes in refractive index [17].
ples. IAsys plus operates with a special cuvette system which
The detection principle of the BIAcore instrument be- contains sample container and sensing layer. To avoid dif-
longs, as do the RM techniques, to the refractometric tech- fusion limitation and ensure rapid sample mixing it includes
niques. Beyond the critical angle, light will be totally in- a microstirrer. Its sample consumption is about 150 L/
ternally reflected at interfaces with different refractive in- measurement. Also, dirty samples can be applied without
dices. When this occurs, an electromagnetic field an plugging the chamber. The cuvette includes two chambers,
evanescent wave passes from the interface to the lower so one could be used as a reference cell.
refractive index medium, where it decays exponentially. A disadvantage could be the user-dependent sample
The resonance angle at which the evanescent field is handling in case of the lack of an autosampler. The sam-
highly enhanced is extremely sensitive to the refractive ple has to be injected manually into the cuvette.
index at the sensor surface. Considering the available surfaces, the same sensing
Surface plasmons are excited at the surface of a thin layers as the BIAcore instrument supplies are provided
metal layer (Au in the case of BIAcore) while the evanes- and recommended.
cent wave interacts with free oscillating electrons. Energy Both evanescent field methods are strongly affected by
from the polarized incident light is lost to the metal film at slight changes in temperature, so a highly sophisticated
a well-defined angle of incidence depending on the refrac- instrumentation for referencing and temperature stabilisa-
tive index of the medium beyond the chip surface [14]. tion is required.
Considering the fluid handling, BIAcore 2000 has a so- RIfS, as a very simple and successful approach, sup-
phisticated, automated microfluidic system which is an plies a very specific method giving cheap, robust and reli-
integral part of the biosensor. It has a very low dead vol- able sensor elements. This reflectometric technique deter-
ume and rapidly takes the sample to the sensing surface, mines the apparent optical thickness (nd) of a thin layer
a constant and highly reproducible flow driven by highly by measurements of interference of white light caused by
precise syringe pumps. It allows a consumption of sample partial reflection at interfaces with a distance of approx-
between 30 and 80 L/cycle by a microfluidic cartridge imately 1 m. Any change in optical thickness causes a
system. Up to four channels can be used, so one of the shift in the resulting interference pattern [18].
channels is mostly utilized as an in-line reference. Sam- In detail, a thin transparent film passed by a light beam
ples containing macromolecular aggregates or entire cells will cause at each of the interfaces a multitude of reflected
could cause problems by plugging up the microchannels. beams. A light beam passing a weakly reflecting thin film
BIAcore is provided with a variety of surfaces for dif- will be reflected in part at each of the interfaces. As the
ferent applications. The most recommended and used sur- two reflected beams travel different optical paths, a phase
face for the commercial systems is the carboxymethylated difference is introduced. If the film thickness is small (a
dextran layer. It is a hydrophilic, three-dimensional, high few micrometers) the difference in optical paths is minute
surface area matrix, bearing charged derivatizable carboxy- and even for short, coherent (white) light sources interfer-
late groups. It is used for most of the kinetic measurements ence effects can be observed. These lead to a modulation
and evaluation of rate constants described in the literature of reflected light intensity due to constructive and de-
[15, 16]. These binding sites within the three-dimensional structive interference, as shown in Fig. 1.
layers provide different accessibility for ligands and there- The use of a simultaneous spectrometer allows fast
fore no homogeneously distributed binding kinetics. acquisition of the reflectance spectra. Any change in opti-
RM, represented by IAsys plus, uses a dielectric cou- cal thickness will lead to a change in the interference
pling layer of high refractive index on an integrated prism spectrum. A resolution of optical thickness (nd) of about
as a waveguide. It is separated from the prism by a low in- 0.5 pm has been achieved [18]. Drift measured with the
dex layer, which allows a coupling into the resonant layer set-up normally ranges smaller than 10 pm/h.
In comparison to the described refractometric methods, 5. Microcal Origin, version 5.0, Northampton, UK, was used to
the reflectometric detection method selectively identifies evaluate kinetic data.
physically attached material on a surface [19] with no need
of referencing or temperature control unit. Materials
The liquid handling for the RIfS system, providing a
single flow through cell, suffices because there is no ne- Chemicals and biochemicals were either from Sigma (Deisenho-
fen, Germany) or Fluka (Neu-Ulm, Germany). The TI (MW 393 g/
cessity of signal referencing. The continuous flow is gained mol) was kindly put at our disposal by Dr. Friedrich, BASF, Lud-
by a robust fluidic system with either syringe pumps or wigshafen, Germany. Polyethyleneglycol (MW 2000 Da) was from
peristaltic pumps, which means a sample consumption of Rapp Polymere, Tbingen.
200 L and 500 L respectively. Agglomerated samples GA (Glutaric anhydride) was used to synthesize diamino-poly-
ethyleneglycol (DA-PEG)-GA surfaces by carboxylation of DA-
are not supposed to cause any problems by plugging up PEG-modified surfaces. Phosphate buffered saline (PBS) consist-
the cell. A disadvantage could be the dead volume of 20 L ed of 150 mM sodium chloride and 10 mM potassium phosphate at
causing dispersion of the sample plug. pH 7.4. Ultrapure water (18.2 MOhmcm, SG, Barsbttel, Ger-
Also, there are suitable sensing layers for various ap- many) was used throughout this study.
Regeneration buffer used with BIAcore was TMM (2%Triton,
plications. For kinetic measurements a plain, two dimen- MgCl2 : Maleate:Tris, 1:1:1, 1 M NaCl), pH=6.8. Sodium acetate
sional sensing layer is recommended, which shows homo- buffer (NaAc) consisted of 10 mM sodium acetate in ultrapure wa-
geneous accessibility of binding sites and for that reason a ter at pH 4.6. For the activating solution 100 mM N-hydroxysuc-
homogeneous kinetic behavior over the whole detection cinimide (NHS) (MW 115.1 g/mol) was diluted in ultrapure water
area [20]. and 400 mM N-ethyl-N-dimethylaminopropylcarbodiimide hydro-
chloride (EDC) (MW 191.7 g/mol) was diluted in ultrapure water
The data obtained with BIA can be used to derive un- also. For activating surfaces both solutions were freshly mixed,
ambiguous answers to many qualitative questions; the 1:1. Blocking solution was ethanolamine in water 1 M, pH 8.0.
quantitative kinetic analysis is often difficult [21]. In some
cases there were multiphasic association and dissociation
Surface chemistry for RIfS transducers
sections found. The deviations were attributed to hetero-
geneity of the binding sites [22, 23]. So the sensing layer Pre-treatment of surfaces. All transducer chips (1.21.2 cm2) were
of the transducer plays an important role. cleaned and modified with a reactive silane derivative by the same
In this study the interaction of thrombine and a throm- procedure. Chips were first cleaned in NaOH in an ultrasonic bath
for 2 min, then washed with tap water. The surface was mechani-
bine inhibitor (TI) is observed. TI was immobilized to the cally cleaned and dried with Kim Wipes. Freshly prepared Piranha
surface and the binding of thrombine was monitored re- solution (concentrated H2SO4 and H2O2 (30%), mixed 7+3) in an
solved in time. Comparing general parameters such as ultrasonic bath for 15 min was used as the final cleaning step and
noise and drift and the evaluation of kinetic rate constants to ensure a surface rich in reactive silanol groups (security advice:
freshly prepared Piranha solution is extremely hot and aggressive.
should prove that determination of kinetic rate constants Use a fume cupboard and appropriate security means). Afterwards,
obtained from the RIfS set-up is comparable with those transducers were washed thoroughly with ultrapure water and dried
received from SPR and RM instruments. in a nitrogen stream. To generate a surface provided with amino-
groups for coupling, the transducers were covered first with 10 L
3-glycidyloxypropyl-trimethoxysilane (GOPTS) for 1 h, followed
by cleaning with dry acetone and drying in a nitrogen stream, and
Material and methods immediately after this pre-treatment 30 L of a solution of DA-
Devices PEG in dimethylsulfoxide (4 mg/1 mL) was placed on each trans-
ducer and melted at 70 C overnight. Afterwards the transducers
For the SPR-based measurements a BIAcore 2000 device with au- were rinsed thoroughly with ultrapure water and dried. For con-
tosampler of the company Biacore (Uppsala, Sweden) was used; verting amino functions into carboxylate groups, 10 L of a GA/
Sensorchips CM5 (carboxylate dextran surface) were also deliv- N,N-dimethylformamide (DMF) solution (2 mg/1 L) were put
ered from there. onto each transducer and than a second transducer was used to
The RM technique was performed by IAsys plus of the com- cover (sandwich technique). The reaction needed 6 h within a
pany Labsystems (Affinity Sensors division). Carboxylate dextran DMF saturated chamber. Then the transducers were washed with
(CMD)-modified cuvettes have been supplied from there. DMF and ultrapure water and were dried.
For the RIfS set-up components from several companies were
used: Immobilization of TI on the carboxylic surfaces on the RIfS trans-
ducer. On the carboxymethylated surfaces, 10 L of a TI/DMF/di-
1. Flow injection analysis device ASIA from Ismatec, Wertheim. isopropylcarbodiimide solution (1 mg/10 L/1.5 L) were placed
2. Diode array spectrophotometer SPEKOL, Analytik Jena, Ger- and sandwiched with a second transducer. After 6 h in a DMF sat-
many, modified according to [10] with a halogen reflector lamp urated chamber the transducers were rinsed with DMF and ultra-
from Welch Allyn, New York, USA, and a polymer fibre of pure water and dried.
PMMA 1 mm diameter from Ratioplast, Optoelectronics, Lhne,
Germany. Characterization of immobilized TI with RIfS. Transducers were
3. Optical transducers consisting of float glass coated with an in- characterized for maximum specific binding of thrombine by a
terference layer system (10 nm Ta2O5/330 nm SiO2), obtained sample concentration of 50 g/ml and reached a maximum signal
from Schott, Mainz, Germany. The term transducer provides of 500 pm, which is very low. A low capacity of binding sites is an
the additional information, that a physical property of the used advantage to kinetic measurements, because measuring near the
surface, here the interference-layer, is used to obtain the physi- limit of capacity reduces the probability of rebinding. Determina-
cal measurement parameter. Therefore the word chip is not tion of dissociation rate constants is more precise.
used in this context; nevertheless it could be used as a synonym. For non-specific binding of another protein (below 10 pm),
4. The flow cell has the following dimensions 410.05 mm3, ovalbumin from chicken egg was injected. For proof of the stabil-
< 200 nL. ity of the immobilized TI layer,l several regenerations were carried
94
out. RIfS as a label-free optical detection technique allows the microlitres of TI solution (1 mg/ml in acetate buffer) was added for
monitoring of real time changes in the optical thickness (physical 5 min and washed with PBS buffer. Subsequently the surface was
thickness drefractive index n) of a thin transparent interference blocked by injection of ethanolamine for about 3 min. Then the cu-
layer coated to a glass carrier. The interference layer was exposed vette was rinsed twice with NaCl (1 M in utrapure water) and in
to the sample, while it was illuminated with white light through the between with PBS.
glass carrier from the back side. Interference effects lead to a char-
acteristic spectral modulation of reflected intensity, which was
recorded by a diode array spectrometer. Changes in the reflectance Measurement protocols
pattern allow on-line detection of binding events [24, 25, 26], with
a resolution down to some picograms per millimetres squared [10, RIfS measurement protocol. For binding measurements of throm-
27]. The set-up used for RIfS-measurements consisted of a 20 W bine to immobilized TI several sample concentrations were in-
tungsten halogen reflector lamp (Welch Allyn, New York), a bi- jected into the flow cell. During all these measurements an au-
furcated HCP silica fibre coupler (600 m fibre diameter) and a tosampler was used and the measurements were carried out by pro-
diode array spectrometer MMS Spekol (Analytik Jena, Jena, Ger- gram. After a 120 s baseline measurement with PBS buffer the
many). The transducer chips were mounted to a flow cell (41 sample was injected for 180 s at a flow rate of 100 L/min. After
0.05 mm3, <200 nL) and matched to the fibre with glycerol. A FIA that association period, buffer was injected for a dissociation pe-
system (ASIA, Ismatec, Zrich, Switzerland) was used for sample riod of 150 s. Afterwards the surface was regenerated by a 250 s
handling. pulse of low pH (0.01 M HCl, 30 L/min) and washed with buffer
The maximum specific binding capacity of transducers was for 120 s.
characterized with solutions of 50 g/mL of thrombine in PBS:
1 mL of sample solution was loaded into the sample loop (0.8 mm Measurement protocol for SPR. During measurements with BIAcore,
inner diameter, 800 L) of the liquid handling system and injected the autosampler was used and a flow rate of 10 L was steadily
with an initial flow rate of 240 L/min (10 s) followed by a flow run. After 120 s of buffer pre-run the sample was injected for 180 s.
rate of 100 L/min (200 s). Subsequently the flow cell was rinsed Subsequently the surface was washed with running buffer for 270 s
with PBS at 240 L/min (125 s). For the regeneration of the trans- and after that dissociation period the thrombine was removed off
ducer surface it was rinsed with a pulse of low pH (0.01 M HCl, the chip by a 180 s pulse of TMM solution. Afterwards the surface
250 s, 100 L/min). Non-specific interactions were checked using was rinsed for 100 s with buffer. Spot 1 was used as reference and
the same injection protocol with 100 mg/mL of ovalbumin. spot 2 as measuring spot.
During all steps of the incubation the reflectance spectra were
recorded continuously and averaged over time intervals of 5 s. Measurement protocol using RM technique. Measurements with
From the resulting interference spectra the apparent change in op- IAsys plus were carried out without the use of an autosampler. All
tical thickness was calculated on-line. For binding experiments the injections were done by hand using an Eppendorf pipette. The in-
signal shift from the pre-run baseline to the signal after binding strument gave the ability to suck dry the cuvette. Every exchange
and rinsing was determined. For proteins a change in apparent op- of liquid was done three times in order to ensure a complete re-
tical thickness of 1 pm corresponds to a surface loading of about moval of the previous liquid.
1.6 pg/mm2, which is calibrated by measurements of the specific After 2 min pre-run with buffer the sample was injected and
interaction between immobilized biotin and 125I-labelled strepta- stayed in the cuvette for 5 min. Afterwards it was washed with
vidin. A publication regarding this is still in preparation. PBS buffer for 3 min and thrombine removed by low pH (0.01 M
HCl) for 2 min. After that the surface was washed with buffer for
Immobilization of TI on BIAcore chips. Immobilization of TI on 2 min. Every step was carried out in both cells in parallel.
BIAcore chip CM5 was carried out on-line. The chip has a carbox-
ylate-modified surface as described above. TI (2.1 mg) was dis-
solved in 100 L NaAc buffer. After activation of the surface by a
200 L pulse of EDC/NHS (1:1), the injection block was washed Results and discussion
with running buffer. TI solution (80 L) was injected at a flow rate
of 10 L/min. Subsequently the surface was blocked by a 70 L Biomolecular interaction between immobilized TI and
pulse of ethanolamine solution. After this treatment the chip was
regenerated with 20 L of 1 M NaCl solution. thrombine using different techniques will be compared.
The chip was given the ability to immobilize targets on four Kinetic data achieved from measurements with RIfS set-
different spots. Every spot can be accessed individually during up, SPR (using BIAcore 2000) and RM (using IAsys plus)
immobilization. During measurements any desired spot can be will be evaluated and compared.
switched in a row and used in parallel. So the first spot was chosen
for the reference measurement and the second one used for immo-
bilisation of the considered compound. So every sample hit the ref-
erence spot first and afterwards the measuring point in order not to Short description of the methods used
reduce the concentration of the sample before measuring.
RIfS is one of the reflectometric techniques that have
Immobilization of TI in IAsys cuvettes. Every cuvette consists of been applied to direct immunosensing. The basic effect is
two measuring chambers. One was used as reference cell and the
other as measuring cell. For referencing the signal in both cells the white light interference at thin transparent films. The prin-
same liquid was injected but only in the measuring cell TI was im- ciple is extensively discussed elsewhere [11, 24].
mobilized. SPR is also a direct optical method that is used in de-
For measurements of thrombine kinetics with RM a CMD cu- tection of solid phase interactions. The basis of this tech-
vette was used. In one of the two cuvette cells the immobilization
was accomplished. The other was used as reference cell. In order nique is reflection of incident radiation at an interface be-
to change the liquid in the cell completely every injection was car- tween media with different refractive indices. Beyond a
ried out three times. The liquid (always 50 L) was handled with a critical angle the radiation will not pass through the inter-
pipette and the cuvette was sucked dry automatically. Stirrer speed face but will be totally reflected, the light is guided. An
used was always 100 times /min and the immobilization was car-
ried out on-line.
evanescent field exists close to the interface in the me-
After activating with EDC/NHS within 7 min the surface was dium of lower refractive index and/or the transmittance.
washed with PBS and afterwards changed to acetate buffer. Ten The electric field of the reflected radiation depends on the
95
refractive index, so any changes in these properties influ- index. It is separated from the prism by a low index layer
ence the evanescent field. thin enough that light may couple into the resonant layer
Surface plasmon waves are concerted oscillations of via evanescent field. Efficient coupling occurs only for
electrons at a surface of a guiding material. The impact of certain incident angles where phase matching between the
incident photons (only the parallel polarized part of the ra- incident beam and the resonant modes of the high index
diation, TM mode) can excite such surface plasmons to layer is achieved. At the resonance point, light couples
oscillate in resonance with the frequency of the radiation. into the high index layer and propagates some distance
In general, a prism is used to excite plasmons in a sec- along the sensing layer interface before coupling back into
ond interface (a metal like Au, Ag) which is coated on the the prism. Resonance happens for TE and TM polariza-
prism. The metal is used as a guiding material, incident tion and is observable as fine structure in reflected light.
radiation penetrates this metal layer and excites the plas- The angle of excitation is sensitive to changes at the sens-
mons at the interface to the analyte. These plasmons ex- ing layer caused by changes of refractive index during in-
hibit resonance in the visible range. Under conditions of teractions at the surface. Light is reflected at any incident
resonance, the intensity of reflected light is reduced like angle, but a shift in the phase of reflected light during res-
an absorption peak at the scale of the angle of incidence onance can be detected.
or wavelength of the exciting radiation.
So this technique gives a highly sensitive probe for de-
tecting changes in refractive index while binding to the Liquid handling
surface. But it needs a highly precise temperature control-
ling system in order to detect only changes in refractive In flow-through systems, time resolution of the detection
index caused from binding, not from media. is limited by sample handling, for example by the time
The third method used in this study is a RM technique, which is necessary for exchange of solution in the flow
which resembles the SPR sensor in its construction [17]. cavity. In this study there were only two flow-through sys-
Light is totally reflected from the sensing surface by tems used for sample handling.
means of a prism. At the sensing surface, the metal layer In order to evaluate data for determination of kinetic
is replaced by a dielectric resonant layer of high refractive rate constants it is necessary to know the region of maxi-
mum sample concentration. Concentration profiles were Table 1 Comparison of general parameters such as noise and drift
measured with the RIfS system to register the peak of the derived from baseline measurements with reflectometric interfer-
ence spectroscopy (RIfS), BIAcore 2000 and IAsys plus. Data were
sample during the flow injection and specify the distribu- taken during investigations of interaction between thrombine and
tion. To this purpose, the reflected intensity was moni- immobilized thrombine inhibitor (TI). For a better comparison the
tored while dye solutions were injected under the same results of the signal calibration by using radioactive labelled com-
conditions as the thrombine samples. Due to the different pounds is shown. Signal calibration: 1 ng protein/mm2=1600 pm
optical thickness (RIfS), 163 arcsec (IAsys), 1000 RU (BIAcore)
diffusion dyes with different molecular weights used,
namely indigocarmin (MW 466 g/mol), dextranblue (MW Instru- rms-noise Limit of detec- Drift Drift
1 million g/mol) and Cy5-labelled antibody (150,000 g/ ment tion (3rms)/ (in units of
mol). Thrombine has a molecular weight of 39,000 g/mol. sensitivity concentration)
Concentration profiles are given in Fig. 2. Maximum con- RIfS 0.80 pm 1.50 pg/mm2 10 pm/h 6 pg/h mm2
centration was set to 100% in this plot. BIAcore 0.26 RU 0.78 pg/mm2 6 RU/h 6 pg/h mm2
Using this plot, the interesting evaluable region can be IAsys 0.40 arcsec 7.36 pg/mm2 4 arcsec/h 24.5 pg/h mm2
defined. Maximum concentration is reached at nearly the
same time for each dye. Within 10 s up to 90% and within
20 s maximum concentration is reached using peristaltic signal after subtraction of linear drift. It was taken from
pumps. Removing the sample from the flow chamber 5 min pre-run baseline measurements with every instru-
strongly depends on the molecular weight of the dissolved ment using the surfaces with the immobilized TI.
dye. When the sample plug is directly flanked by carrier Another limitation is the drift, which should not exceed
liquid, diffusion takes place. Smaller molecules diffuse the signal. Detection of slow interactions is limited. The
faster than bigger molecules. Fast radial diffusion inhibits drift is obtained from linear regression of this baseline
dispersion of the sample plug caused by axial convection, runs. Results are shown in Table 1.
which means samples of small molecules do not mix with Comparable data resulting from signal noise are the
the carrier stream as fast as larger molecules [28]. LODs received from each instrument (3rms/sensitivity).
BIAcore 2000 has a very low dead volume and ensures The lowest values were obtained from BIAcore 2000 in
a highly precise exchange of sample in the flow cavity by this study. LOD of RIfS measurements is twice as high as
using a micro-flow cartridge. Because of successively fill- BIAcore data, and noise data achieved with IAsys plus in
ing the chamber, the different spots are reached one after this measurements gives an LOD more than 9 times
the other. The marginal resulting offset can be nearly higher than the BIAcore data. All achieved values are
eliminated by software. within one order of magnitude. Binding of proteins where
IAsys plus uses stirred liquid in a cuvette with two masses are in a range of several kilo Daltons can be easily
cells. Limitations in this case could be not having a total resolved and monitored.
change of liquid while pipetting into the cuvettes, not fill-
ing both chambers simultaneously and evaporation during
long lasting equilibrium measurements because there is Evaluation of rate constants
no lid to cover the chambers.
By changing liquid three times, total exchange of liquid For the evaluation of rate constants several concentrations
is ensured. Nevertheless, a slight offset between reference of thrombine binding to the TI-modified surface were
and measuring chamber caused by a time shift is unavoid- monitored. Each sample concentration was measured three
able; a correction by software is possible. Temperature con- times. For SPR and RM, referencing of the signal was nec-
trol and short measurement times keep evaporation low. essary. Signals obtained from the reference cell were sub-
The comparison of the fluid handling systems shows tracted from that received from the measuring cell. RIfS
that RIfS and IAsys have to manage without a sophisti- measurements were carried out without referencing. Bind-
cated fluidic system. This could be a disadvantage in the ing curves for RIfS, referenced signals obtained with
reproducibility of data and should be considered through- BIAcore and also referenced binding curves for IAsys are
out evaluation. presented in Fig. 3.
The performance of a method first of all depends on gen- Considering the binding curves, the reproducibility of the
eral parameters such as noise or drift. So first of all, noise binding of thrombine with a certain concentration can be
or drift obtained during the measurements should be com- estimated. Triple experiments have been carried out and
pared. For determination of LOD, signal calibration has to the standard deviation of the kobs values, which has been
be known. Measurements with radioactive labelled pro- percentally referred to the kobs values has been evaluated.
teins give the signal level for each method [29, 30] as The average of the percental standard deviation is shown
shown in Table 1. in Table 2.
The noise of the signal was derived from linear regres- Data show that RIfS provides the lowest standard devi-
sion of recorded baselines as standard deviations of the ations throughout the measurement. Despite the use of
97
Table 2 Comparison of the average of the percental standard de-
viationof kobs measured with RIfS, BIAcore 2000 and IAsys plus
Instrument Mean value of the standard deviation
of kobs in percent referred to kobs [%]
RIfS 8
BIAcore 2000 15
IAsys plus 15
for steady and reproducible measurements, it would be 11. Brecht A, Gauglitz G, Polster J (1993) Biosens Bioelectron
favourable to evaluate temperature dependency of reac- 8:387392
12. Piehler J, Schreiber G (2001) Anal Biochem 289:173186
tions at the surface. 13. Piehler J, Brecht A, Gauglitz G (1996) Anal Chem 68, 139143
Further, a miniaturization of the device is possible. In- 14. Brigham-Burke M, OShannessy DJ, Smith (1993) Chromato-
stead of using white light providing the whole spectra for graphia 35:459
the interference measurement, only four LEDs can be 15. Abraham R, Buxbaum S, Link J, Smith R, Venti C, Darsley M
(1995) J Immunol Methods 183:119125
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with photodiodes instead of an diode array spectrometer, ods 183:5163
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Anal Bioanal Chem (2002) 372 : 101108
DOI 10.1007/s00216-001-1198-2
O R I G I N A L PA P E R
K. W. Phinney L. C. Sander
Received: 20 August 2001 / Revised: 8 October 2001 / Accepted: 16 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract The applicability of a new Standard Reference Chromatographic separations of enantiomers on CSPs
Material (SRM) for the evaluation of chiral stationary depend upon the formation of transient diastereomeric
phase (CSP) performance was demonstrated by utilizing complexes between the enantiomers and a chiral selector
the SRM to characterize the chromatographic behavior of incorporated into the column packing material. Separation
eight commercially available CSPs in liquid and super- of the enantiomers occurs when one enantiomer forms a
critical fluid chromatography. The SRM consists of five more stable complex and is retained longer than the other
ethanolic solutions, each containing one chiral compound. enantiomer [4]. CSPs are often classified by the nature of
These test mixtures can be used to assess changes in col- the chiral selector or by the type of interactions responsi-
umn performance over time and to evaluate lot-to-lot vari- ble for chiral recognition. Common chiral selectors in-
ability in column manufacturing. The SRM was also used clude macrocyclic compounds, proteins, modified amino
to probe the effect of various parameters on column per- acids and derivatized polysaccharides [5].
formance. Evolution of CSPs has continued to occur as new CSPs
are prepared and commercialized. Chiral recognition stud-
Keywords Chiral stationary phase Enantiomers ies have guided the development of modified versions of
Standard reference material early CSPs and the second generation versions of some
CSPs also offer improved stability [6, 7]. Many of the
CSPs initially developed for LC have now been employed
Introduction in supercritical fluid chromatography (SFC), where the
traditional liquid mobile phase is commonly replaced by a
Chiral stationary phases (CSPs) for liquid chromatogra- carbon dioxide-based eluent [8].
phy (LC) have had a tremendous impact on enantioselec- The validity of chiral chromatographic results is con-
tive separations. Within the past two decades, the com- tingent upon the performance of the CSP. Once a suitable
mercialization of CSPs for LC has been accompanied by a CSP has been identified for the desired separation, enan-
dramatic increase in the number of successful enantiose- tioselectivity is typically optimized through changes in
lective separations, and many chiral compounds can now mobile phase composition, temperature, and flow rate [9,
be resolved on more than one CSP [1]. The predominant 10]. Inappropriate mobile phases, adsorption of sample
application of CSPs has been to the separation of drug components, and leaching of bonded selectors can have
enantiomers, but the importance of stereochemical analy- deleterious effects on column behavior. Changes in col-
sis has been established in additional areas such as geo- umn performance often occur gradually and may not be
chemistry and environmental science [2, 3]. noticeable until the column fails to produce the desired
separation. Lot-to-lot variability in columns from the
same manufacturer may also have some bearing on the
suitability of a CSP for a particular analysis [11].
We have developed a Standard Reference Material
Contribution of the National Institute of Standards (SRM) for the evaluation of CSP performance in LC and
and Technology. Not subject to copyright
SFC. The SRM consists of five ethanol solutions, each
K.W. Phinney () L.C. Sander containing one racemic or enantiomerically enriched (non-
Analytical Chemistry Division, racemic) compound. The five test compounds are indap-
Chemical Science and Technology Laboratory, amide, ketoprofen, N-carbobenzyloxy-phenylalanine (N-CBZ-
National Institute of Standards and Technology,
Gaithersburg, MD 20899, USA phenylalanine), propranolol, and warfarin. The structures
e-mail: karen.phinney@nist.gov of the test compounds are shown in Fig. 1, and the com-
102
Experimental
Certain commercial equipment, instruments, or materials are iden-
tified in this report to specify adequately the experimental proce-
dure. Such identification does not imply recommendation or en-
dorsement by the National Institute of Standards and Technology,
nor does it imply that the materials or equipment identified are
necessarily the best available for the purpose.
Reagents
The CSPs used in this study are listed in Table 2. These columns
were selected as a broad sample of dissimilar CSPs. Columns were
obtained from the following sources: Chiralcel OD and Chiralpak
AD (Chiral Technologies Inc., Exton, PA, USA), Chirex 3005 and
Chirex 3022 (Phenomenex, Torrance, CA, USA), Chirobiotic T,
Chirobiotic V, Cyclobond I 2000, and Cyclobond I 2000 RSP (Ad-
vanced Separation Technologies, Inc., Whippany, NJ, USA). All
columns were 4.6 mm25.0 cm. The same columns were used for
both LC and SFC. Columns were flushed with ethanol prior to
switching from one chromatographic technique (LC or SFC) to an-
other.
Chromatographic conditions
Fig. 1 Structures of components of SRM 877. The chiral center of
each compound is indicated by an asterisk Liquid chromatographic separations were performed at a tempera-
ture of 25 C and a flow rate of 1.0 mL/min. The injection volume
was 20 L. A solvent exchange to dissolve the test compound in
Table 1 Components and enantiomeric compositions of SRM 877
the mobile phase was necessary to avoid peak distortion with cer-
solutions
tain mobile phases. Supercritical fluid chromatography was per-
Compound Enantiomeric Major formed at a temperature of 30 C, a flow rate of 2.0 mL/min, and a
ratioa,b enantiomer pressure of 15 MPa. The injection volume was 5 L. Methanol
with 0.5% (v/v) isopropylamine was used as the modifier for all
Indapamide 1.0 N/Ac SFC separations. Detection in LC and SFC was performed by mea-
Ketoprofen 2.3 R surement of UV absorbance at 254 nm.
N-CBZ-phenylalanine 4.0 L
Propranolol hydrochloride 2.3 S
Warfarin 1.0 N/A Results and discussion
aData are provided for information purposes only and are not to be
A number of factors influence the performance of CSPs.
used for quantification
bEnantiomeric ratio (er)=mass E /mass E , where E is the major
1 2 1
Although the nature of the chiral selector has the greatest
enantiomer influence on the enantioselectivity of the CSP, other fac-
cNot applicable. The racemate was used
tors such as mobile phase composition, temperature, and
column age also have a bearing on column behavior. Suc-
cessful enantioselective separations are often achieved
position of the solutions is shown in Table 1. Potential ap- within a narrow range of mobile phase compositions, and
plications of this SRM include use as a control material CSPs are typically more sensitive to slight changes in mo-
for monitoring column performance, comparisons of bile phase composition than their nonchiral counterparts
columns having similar chiral selectors, and use in quality [11]. Temperature is an important parameter for chiral sep-
control for column manufacturing. We assessed the capa- arations in both LC and SFC because changes in tempera-
bilities of this SRM for testing column performance by ture can result in reversal of elution order [12]. In general,
using the test mixtures to evaluate the enantioselectivity enantioselectivity decreases with increasing temperature.
of eight commercial CSPs in LC and SFC. The performance of all CSPs degrades over time, but the
speed of this process varies. These changes may not be ap-
103
Table 3 Suggested mobile phase compositions for liquid chro- Table 4 Suggested mobile phase compositions for supercritical
matographic separations of SRM 877 fluid chromatographic separations of SRM 877
Table 6 Supercritical fluid chromatographic data for selected commercial chiral stationary phases
Compound Chiralcel Chiralpak Chirex Chirex Chirobiotic Chirobiotic Cyclobond Cyclobond
OD AD 3005 3022 T V I 2000 I 2000 RSP
Indapamide
mobile phase EE CC CC DD CC CC CC CC
K(1)a 3.16 12.14 14.15 11.56 19.16 22.59 9.31 18.08
1.35 1.00 1.00 1.05 1.03 1.04 1.00 1.00
Rs 5.55 0.00 0.00 1.40 1.08 1.12 0.00 0.00
Ketoprofen
mobile phase BB AA CC CC CC CC CC CC
K(1) 4.28 11.83 9.89 7.67 8.50 10.95 7.73 6.10
1.00 1.11 1.08 1.00 1.00 1.00 1.00 1.00
Rs 0.00 3.17 1.78 0.00 0.00 0.00 0.00 0.00
Phenylalanine
mobile phase BB BB DD DD CC CC CC CC
K(1) 8.38 5.50 15.91 12.62 18.69 19.20 19.00 16.26
1.10 1.11 1.00 1.00 1.04 1.00 1.00 1.00
Rs 2.18 2.66 0.00 0.00 1.33 0.00 0.00 0.00
Propranolol
mobile phase DD BB CC CC CC CC CC CC
K(1) 4.36 4.07 11.94 8.87 25.50 23.80 7.66 9.45
1.70 1.30 1.00 1.00 1.09 1.07 1.00 1.00
Rs 12.41 7.19 0.00 0.00 3.51 2.45 0.00 0.00
Warfarin
mobile phase DD DD CC CC CC CC CC CC
K(1) 2.80 2.12 9.95 12.14 9.93 10.64 7.54 8.31
1.72 1.94 1.07 1.00 1.00 1.02 1.00 1.00
Rs 5.64 14.72 1.33 0.00 0.00 0.73 0.00 0.00
aretention factor of the first eluting enantiomer
105
same number of test compounds was resolved on the two mobile phase was used for both columns, and therefore,
CSPs in SFC, although the compounds resolved on the the variation shown likely did not arise from changes in
Chiralpak AD CSP were not the same in LC and SFC. mobile phase composition.
Separations of indapamide enantiomers by LC on the
polysaccharide-based CSPs, as well as two other CSPs,
are shown in Fig. 2. In some cases, a reversal of elution Macrocyclic antibiotic-based CSPs
order was observed in the transition from LC to SFC.
Figure 3 illustrates the reversal of elution order that oc-
curred for ketoprofen enantiomers when a liquid eluent The Chirobiotic T and Chirobiotic V CSPs utilize slightly
in LC was replaced with a carbon dioxide-based eluent different macrocycles as the chiral selector. Enantioselec-
in SFC. tive separations of four of the five test mixtures were ob-
served on the Chirobiotic T CSP, but the low resolution
value for ketoprofen (Rs=0.41) makes this a poor choice
for column evaluations. In addition, Rs could not be reli-
Brush-type CSPs ably determined for N-CBZ-phenylalanine. All five of the
test compounds were separated to some extent on the Chi-
The two brush-type (Pirkle-type) CSPs, Chirex 3005 and robiotic V stationary phase, but the minimal degree of res-
Chirex 3022, incorporate modified amino acids as the chi- olution obtained for some of the compounds makes them
ral selector [18]. Partial separation (Rs=1.0) of ketoprofen poor candidates for column monitoring. Only propranolol
enantiomers was observed on the Chirex 3005 CSP in LC. and warfarin were well resolved in LC. Separations of
Enantioselective separations for three of the test com- propranolol enantiomers on the Chirobiotic T and V
pounds were obtained on the Chirex 3022 CSP in LC, but CSPs, as well as two other CSPs, are illustrated in Fig. 5.
resolution was less than 1.5 in each case. In contrast, both In SFC, three of the test compounds were enantioresolved
ketoprofen and warfarin were resolved on the Chirex on each CSP. Although the enantiomers of indapamide
3005 CSP in SFC. Only indapamide was enantioresolved were not separated on the Chirobiotic T CSP in LC, a par-
on the Chirex 3022 CSP in SFC. tial separation was observed in SFC, as shown in Fig. 6.
SRM 877 was used to evaluate the differences between
two brush-type columns with the same chiral selector and
obtained from the same vendor. One column was pur-
chased specifically for this study. The second column had
been used for six months with various mobile phases. Fig-
ure 4 shows the separation of indapamide enantiomers on
two Chirex 3022 CSPs. As shown in the figure, slightly
better separation factor and resolution were observed on
the column that had been used for six months. The same
Fig. 4 Separation of indapamide enantiomers by LC on a new col- Fig. 5 Separations of propranolol enantiomers on commercial chi-
umn (A), and a column that had been used for six months (B) ral stationary phases by liquid chromatography
107
Table 7 Effect of temperature on the separation of propranolol Fig. 7 Separations of warfarin enantiomers on commercial chiral
enantiomers stationary phases by liquid chromatography
O R I G I N A L PA P E R
Received: 27 July 2001 / Accepted: 20 September 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract A comparison between a laser-induced break- times the term round robin study is used [3]. Finally, an
down spectrometry-partial least squares (LIBS-PLS) meth- interlaboratory study is used for the determination of the
od and methods based on some well-known techniques, reference value of a reference material or a certified refer-
such as induced-coupled plasma-atomic emission spec- ence material in which several independent methods known
trometry (ICP-AES), flame atomic absorption spectrome- for their accuracy are selected [4]. An issue of the Frese-
try (FAAS), and atomic scanning microscopy (ASM) is pre- nius J. Anal. Chem. devoted to reference materials in both
sented in order to both validate the content of gold and sil- biological and environmental fields has recently been
ver in alloys to be used as solid standards and develop an published [5].
alternative to the established methods for the hallmark of Most of the analytical methods used in the systematic
gold and silver in jewelry pieces. 17 alloys with gold con- analysis of solid samples are applied after wet digestion or
centrations ranging from 100 to 50% and 8 alloys with sil- leaching and thus standard solutions commercially avail-
ver concentrations between 100 and 80% and variable con- able can be used for calibration. These wet treatments
centrations of other metals usually present in jewels were consist of more or less complex steps which are generally
used as solid standards in LIBS in order to develop a meth- critical parts of the overall analytical process because they
od as general as possible. The results obtained in the anal- are responsible for the most significant errors. This is par-
ysis of some alloys (9 for gold and 7 for silver) show that ticularly true for solid samples, which have to be gener-
the proposed method is comparable with the official one. ally brought into either complete dissolution or leaching
in order to fulfill the requirements for sample introduction
Keywords LIBS Jewelry Hallmark Gold Silver of the majority of the spectroscopic instruments of general
use in routine laboratories [6]. Methods for direct solid
sample analysis are the most desirable for saving both
Introduction reagents and time.
Laser-induced breakdown spectrometry (LIBS) inte-
A number of interlaboratory studies have been developed grates ablation of solid samples and excitation of the re-
in Europe, particularly in the last decade. Most of them sulting aerosol through plasma formation. Thus, the meth-
have been fostered in the light of the necessities creat- ods based on this technique are drastically simplified and
ed by EU Directives. The different kinds of interlabora- shortened as regards to methods involving sample disso-
tory experiments depend on the aim for which they are lution. Examples of satisfactory applications of LIBS are
planned. Thus, when the performance of a single method the determination of copper in aluminium samples [7], the
has to be tested the study is called collaborative trial or simultaneous quantification of Al, Cu, Fe, Pb and Sn in
method performance study [1]; the comparison of differ- zinc-based alloys [8] or the determination of aluminium in
ent laboratories that perform comparable analyses with Zn alloys [9]. The most remarkable aspects of LIBS are
their own individual methods is often called proficiency rapidity (analysis time in the order of seconds), no appar-
testing or laboratory performance study [2], though some- ent sample destruction (only a small crater of micron-di-
ameter), in addition to the selectivity, sensitivity and pre-
cision characteristic of laser-based atomic techniques.
Nevertheless, LIBS shares with other techniques for direct
A. Jurado-Lpez M.D. Luque de Castro () measurements in solids the lack of standards to run cali-
Analytical Chemistry Division, Annex C-3,
Campus of Rabanales, University of Cordoba,
bration properly.
14071 Crdoba, Spain The official methods for the hallmark of gold and sil-
e-mail: qa1lucam@uco.es ver pieces are absolute methods (copelation and potentio-
110
metric titration, respectively). These methods involve se- The special characteristics of this validation study
rious drawbacks, such as: (namely, 9 different materials for gold and 7 different ma-
terials for silver with different contents) made also man-
a) the samples must be completely destroyed,
datory special statistical treatments of the data, different
b) the analytical procedures are tedious and time-con-
from those used for conventional interlaboratory studies.
suming, involving multiple steps of high safety risk for
the operators, and
c) the energy costs are high.
Materials and methods
Therefore, the development of reliable analytical methods
which, taking advantage of new analytical techniques, en- Apparatus and instruments
ables the drawbacks of the conventional methods to be The experimental setup for carrying out the laser-induced break-
circumvented should be promoted [10]. down spectroscopic method is depicted in Fig. 1. A pulsed Nd:YAG
One of the present research guidelines of the authors laser (Continuum, Model Minilite II) was used as excitation source,
team is focused on the development of methods for help- operating at the second harmonic, wavelength 532 nm, with a laser
energy of 1.73 mJ with a 1 Hz repetition frequency. The pulse en-
ing jewelry trade, one of the most significant industrial ar- ergy was measured with a pyroelectric joulimeter (Gentec, Model
eas in Crdoba. One of the main authors aims is to de- ED-200L, nominal sensitivity of 9.86 VJ1) coupled to a digital
velop alternative methods which can be promoted for sub- real-time oscilloscope (Tektronix, Model TDS 380). Owing to the
stituting those officially established for the hallmark of laser module does not permit the change of the beam energy, it was
modified by dispersing the beam (ca. 3 mm size at the output) us-
gold and silver jewelry pieces. With this aim, we planned ing a 33 mm focal length plano-convex lens (Oriel, 36 mm outside
to apply LIBS for establishing not only the content in gold diameter, 236 mm aperture range). The beam was guided towards
or silver in the target piece but also the content of other the sample with a flat aluminised mirror (Oriel, 5050 mm, coated
components of the alloys used for making the jewel. One with aluminium and overcoated with MgFa) and focused at normal
of the main shortcomings to be circumvented was the lack incidence on the target by a 135 mm focal length plano-convex lens
(Melles-Griot, 25 mm in diameter). The radiation emitted by the
of the appropriate standards for running the calibration plasma was collected and transmitted by a fiber optic to a Czemy-
curve. A series of nine Au and seven Ag alloy potential Turner l/8 m MS125 spectrograph (Oriel, Model 77400), equipped
standards were prepared with contents in gold and silver with an entrance slit of 25 m (Oriel, Model 77220) and a grating
ranging between 50100% and 79l00%, respectively. A of 2400 grooves mm1 (Oriel, Model 77420). The detector was a
charge-coupled device (Andor-Oriel, Instaspec IV Model 78430-V),
sui generis interlaboratory validation was planned with which consists of 1024128 elements and a total photoactive area
the dual objective of assessing the metal concentrations in of 26 m2, connected via a 16-bit ISA card to a Pentium II micro-
the standards and comparing the results obtained using processor computer. A multiple I/O box (Oriel, 10140) allowed
well-established methods such as the official ones and the connection between the Instaspec card and the laser power sup-
ply in order to synchronize the Q-switch with data acquisition, The
others based on ICP-AES, FAAS, etc. with those provided sample was placed in a manual X-Y-Z translation stage (Oriel,
by LIBS. For making this, the alloys were analysed by Model 16921), which made possible sample manipulation as re-
five laboratories using different techniques. quired. A 13.5 mm longitudinal movement could be achieved for
Table 3 Content of silver of the target alloys obtained by ICP-AES, ASM and FAAS (values in brackets correspond to the relative er-
ror in percentage)
Pieces Nominal ICP-AES (1) ASM (2) FAAS (2) ICP-AES (2) ICP-AES(3) FAAS
value
1 99.68 99.98 (0.300) 99.92 (0.241) 100.70 (1.023) 102.23 (2.558) 95.99 (3.702) 100.86 (1.214)
2 97.70 98.44 (0.757) 97.95 (0.256) 99.73 (2.078) 99.94 (2.293) 98.21 (0.583) 96.81 (0.911)
3 95.61 96.55 (0.920) 96.31 (0.669) 98.79 (3.261) 99.16 (3.648) 98.69 (3.157) 93 35 (2.425)
4 92.11 93.40 (0.744) 92.70 (0.011) 93.28 (0.615) 95.17 (2.653) 93.59 (0.949) 91.16 (1.672)
5 89.63 90.26 (0.703) 89.46 (0.190) 91.61 (2.209) 94.57 (5.512) 95.55 (6.605) 88.72 (1.015)
6 84.66 85.40 (0.874) 83.49 (1.382) 84.79 (0.154) 83.43 (1.453) 84.61 (0.059) 82.03 (3.107)
7 79.67 19.77 (0.125) 79.27 (0.502) 80.59 (1.155) 79.45 (0.276) 78.77 (1.130) 77.08 (3.251)
Number in brackets after the technique acronym indicates the laboratory where the analysis were developed. The absence of number in-
dicates the authors laboratory.
113
Comparison of methods
In addition to the analyses by the reference and authors
laboratories, where the target alloys were analysed both by
LIBS and FAAS, they were delivered to three other labo-
ratories in order to analyse them by other well-established
techniques for metals determination for their subsequent
comparison with the proposed LIBS method, taking as
true values those provided by the official method devel-
oped by the Spanish assay office. These laboratories were:
Istituto Superiore di Sanit, Rome (Italy), where the tech-
nique used for the analysis of both metals was ICP-AES
(labelled as 1); Analytical Chemistry Division, University
of Zaragoza (Spain), where the gold samples were anal-
ysed by ASM (Analytical Scanning Microscopy), while
the silver ones were analysed by ASM and also by FAAS
and ICP-AES (labelled as 2); Analytical Chemistry Divi-
sion, University of Malaga (Spain) where ICP-AES was Fig. 2 Plot of the slope and its standard deviation calculated from
used (labelled as 3). The results obtained for gold and sil- the application of the minimum squares regression method to the
comparison between the results obtained for gold with each tech-
ver are shown in Tables 2 and 3, respectively. Each result nique and copelation. Number in brackets indicates the laboratory
is the mean of four replicates, except in the case of the of- where the analysis was made; the absence of number indicates the
ficial method, which made ten replicates. authors laboratory (for details, see text)
The comparison of the results provided by the instru-
mental methods and those provided by the reference one
was done using a two-tailed t test to compare the means of The statistical parameters are shown in Table 4. The cal-
related (paired) values obtained from each method and the culated t-values were compared with the theoretical one at
official one in order to evaluate if the methods yield simi- a=0.05 and 8 degrees of freedom for gold (t=2.306) and
lar results at the 95% confidence level. The expressions 6 degrees of freedom for silver (t=2.447). As can be seen,
used were: ICP-AES and LIBS are the methods that yield values
1/2 comparable to those obtained from the official method for
d 2
d
2
/n
di i i gold, while for silver the ASM method can also be in-
d = sd =
cluded. Then, the results provided by every method were
n n1
plotted versus those obtained by the official one and the
d minimum squares regression method was applied for test-
t= ing linearity between them. Taking into account the corre-
sd / (n)1/2
lation coefficient (because the absolute character of the
official method does not provide calibration curve, so no
slope is available for comparison), all the instrumental
Table 4 Statistical parameters obtained from the application of methods as well as LIBS had an acceptable linearity with
the two-tailed t-test to the comparison of the values provided by the official one. Then, each slope and its standard devia-
the official method and the instrumental ones tion were plotted to compare the instrumental methods
Instrumental d sd t among themselves. The results for gold are shown in Fig. 2.
method As can be seen, ICP-AES (1) and LIBS are the methods
that provide the more accurate results, while ASM has the
Gold ICP-AES (1) 0.362 1.212 0.897
minor standard deviation, but the slope has the higher dif-
ASM (2) 2.528 1.553 4.883
ference from 1. ICP-AES (3) has nearly the same slope as
ICP-AES (3) 1.539 2.250 2.052
ICP-AES (1), but the former has a higher standard devia-
FAAS 2.621 1.802 4.363
tion. The same was made for silver, as can be seen in Fig. 3,
LIBS 0.056 1.047 0.159
where the results obtained are similar to those provided
Silver ICP-AES (1) 0.583 0.278 5.543 from gold: the most accurate methods are those based on
ASM (2) 0.089 0.582 0.403 ICP-AES and LIBS, while the smaller standard deviations
FAAS (2) 1.396 1.030 3.580 correspond to ICP-AES (1) and ASM. According to Figs.
ICP-AES (2) 2.033 2.113 2.545 2 and 3, the proposed LIBS method provides values com-
ICP-AES (3) 0.821 3.027 0.718 parable to those obtained by the conventional techniques,
FAAS 1.387 1.351 2.716 being one of the more accurate and precise for both gold
LIBS 0.289 0.962 0.794
and silver.
Number in brackets after the technique acronym indicates the lab-
oratory where the analysis were developed. The absence of num-
ber indicates the authors laboratory.
114
O R I G I N A L PA P E R
F. Ehrentreich
Received: 31 May 2001 / Revised: 7 September 2001 / Accepted: 11 September 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract The wavelet transform has been established The wavelet transform (WT) is a mathematical trans-
with the Fourier transform as a data-processing method in formation for hierarchically decomposing functions. It led
analytical chemistry. The main fields of application in an- to a description of a function in terms of a coarse overall
alytical chemistry are related to denoising, compression, shape and details of a graded sequence.
variable reduction, and signal suppression. Analytical ap- In continuous wavelet analysis the wavelet coefficients
plications were selected showing prospects and limita- c are related with the signal according to:
tions of the wavelet transform. An important aspect con-
1 x q
sists in showing the advantage of wavelet transform over c ( p, q) = y (t) dt (1)
Fourier transform with respect to dual localization of a p p
R
signal in both the original and the transformed domain en-
abling principal new application fields in comparison with where x and y describe the original signal, p characterizes
Fourier transform. the scale and resolution and q characterizes the transla-
tion.
Keywords Wavelet transform Wavelet packet Because we deal with discrete data vectors or matrices
transform Raman spectroscopy Spike recognition we make use of the discrete wavelet transform. In discrete
Infra-red spectroscopy Spectra comparison wavelet transform (DWT), p and q take discrete values
according to: p = 2 j , q = k2 j , j N , k Z , leading to:
C (a, b) = C ( j, k) = y (t) j,k (t) (2)
Introduction tZ
A real breakthrough regarding applications of the wavelet
Concerning wavelet transform methods diverse applica- transform in the applied sciences has only been achieved
tion areas have been established in the information sci- after the work of Daubechies [1, 13], Mallat [14, 15] and
ences [1, 2, 3, 4, 5, 6, 7, 8], i.e. denoising, data compress- others. The first one includes suitable filter selection for
ing, detection of discontinuities, edge detection, suppress- decomposition and reconstruction to solve the problems
ing of signals, locating and suppressing outlying values, of aliasing. The progress achieved by Mallat is due to the
detection of missing data, detection of pure frequencies, development of efficient algorithms for performing the
long-term evolution or self-similarity. Some special appli- discrete wavelet transform (DWT) as pyramidal algo-
cations, such as fast multiplication of large matrices also rithms.
exist. The applications are not restricted to the one-di- By wavelet analysis only the approximation coeffi-
mensional case, but wavelet transform methods are suc- cients, will be decomposed at each level j1 yielding new
cessfully applied for two-dimensional image analysis. In- approximation coefficients aj, that represent the low fre-
troductions and overviews from a chemometric point of quencies or coarse shape of aj1, and detail coefficients dj
view have also been published [9, 10, 11, 12]. that correspond to the high-frequency constituent of the
preceding approximation. The original data might be con-
The main part of this work was performed at the TU Dresden, sidered as the approximation at level 0 by the a0 coeffi-
Institute of Analytical Chemistry, working on the project cients. Inversely, during wavelet synthesis, approximation
Eh 121/21 funded by the Deutsche Forschungsgemeinschaft. and detail coefficients from level j+1 will reconstruct the
approximation coefficients at the next finer level j. The
F. Ehrentreich ()
Universitt zu Kln, Institut fr Biochemie,
wavelet packet transform (WPT) is a generalization of the
Zlpicher Str. 47, 50674 Kln, Germany WT [16, 17] because it leads to a complete decomposition
e-mail: f.ehrentreich@uni-koeln.de tree of the original data. It can be best explained as in Fig. 1.
116
Fig. 1 Schematic representation of the decomposition tree of the olding shrinks them towards zero avoiding a gap between
wavelet packet transform (WPT) up to level 4. The solid thick zero and the chosen threshold. Avoiding the discontinuity
lines correspond to the sequence of approximation coefficients and
the dotted thick lines to the appropriate detail coefficients of the is desirable for further data processing but does not make
wavelet transform much sense for pure compression purposes. The appropri-
ate formulas are given in Fig. 2. Pioneering work was
done by Donoho [31, 32, 33] making standard wavelet
A schematic wavelet decomposition tree is drawn from transform procedures for denoising data available to the
level 1 to level 4. The thick solid lines correspond to the scientific community.
decomposed approximation coefficients of the WT and Soft thresholding (path I in Fig. 2) of wavelet detail co-
the thick dotted lines to the details coefficients of the WT. efficients will generally be applied for data denoising. Due
The other decomposition nodes are specific for the wavelet to the fact that denoising is a procedure preceding further
packet transform. data processing methods, the wavelet coefficients are of-
ten not in the focus by itself but the reconstructed signal
after removal of the noisy components.
Applications in analytical chemistry Compression (path II in Fig. 2) is performed very sim-
ilar to denoising. Wavelet analysis does not lead to storage
Most of the applications in analytical chemistry belong to compression by itself. During processing, the coefficients
a common processing scheme, i.e. they obey the sequence: below the threshold are set to zero. Underlying a common
decomposition processing reconstruction. The wavelet data format for all coefficients, the zero coefficients need
transform enriches the palette of procedures comprising as much storage space as the other ones. However, wavelet
methods as principal component analysis and Fourier trans- decomposition with hard thresholding is an excellent pre-
formation. processing step for compression methods such as run length
Despite the diverse analytical disciplines, two main encoding or Huffman coding (a concise introduction is
fields of wavelet applications in analytical chemistry may given elsewhere [34]) because the procedure produces
be extracted that can be categorized to the above-men- large contiguous ranges of zeros. These processed data are
tioned general processing scheme. The first covers appli- subsequently compressed efficiently. Temporarily the
cations for improvement of signal-to-noise ratios [18, 19, compressed intermediate is of interest for storage. Be-
20, 21, 22, 23, 24, 25] and the second concerns data com- cause storage is not performed on his own the reconstruc-
pression [26, 27, 28, 29, 30]. Often, appropriate routines tion of the data is the final point.
are embedded in larger procedures as feature extraction With the software used, both methods have in common
[26], peak recognition [21], calibration [27, 28] or spec- that they do not lead to compression of data in the computer
tral library search [30]. memory after thresholding. Admittedly, it could be possi-
In Fig. 2 a principal scheme is drawn that is applicable ble to gain computer memory by using appropriate data
for wavelet denoising as well as for compression. The structures, e.g. by pointing only to non-zero coefficients.
branches leading to soft thresholding (I) and hard thresh- An even more radical data reduction could be achieved
olding (II) are well investigated by mathematicians. If the by crude wavelet analysis, removing all the detail coeffi-
absolute values of detail coefficients |dj,k| are smaller than cients and keeping only the approximation coefficients
a chosen threshold thrn those values are set to zero in both (path III in Fig. 2). The dotted line in Fig. 2 should indi-
cases, soft and hard thresholding. Soft and hard threshold- cate that also in that case a reconstruction is possible.
ing differ with respect to processing detail coefficients However, in general this strategy should be performed
whose absolute values equal or exceed the threshold. with care because substantial data distortions may result.
Hard thresholding does not change them, but soft thresh- Further, in contrast with the theory developed by Donoho
117
Fig. 2 Scheme of the sequence wave-
let decomposition processing re-
construction of signals y or approxi-
mation coefficients aj1. Three types
of processing wavelet detail coeffi-
cients are important: soft threshold-
ing, hard thresholding and crude pro-
cessing
et al. for thresholding wavelet coefficients [31, 32, 33] better than those obtained by ordinary resolution decreas-
crude processing is not founded on statistical theory. ing. At least, the advice should be given, to compare the
Hence, it should only be performed with deep knowledge results with those of ordinary resolution decreasing.
of the subject, and analytical expertise should guide the Besides these main streams, other applications such as
crude wavelet coefficient processing. Moreover, the ques- recognition of signal discontinuities [36] or contrast en-
tion arises, how the distortion of signals is comparable hancement of edges in image processing [37] have been
with that of simple resolution reduction. reported.
In Figs 3 and 4 resolution-dependent infrared spectra That application class shows a principal advantage of
of 4,8-dimethyl-3,7-nonadienoic acid methyl ester and the WT over Fourier transform (FT) and should be ex-
N-phenyl-2,3-naphthalenedicarboximide are drawn; the plained by Fig. 5. In Figs 5ae, a sine function is drawn
experimental details are given elsewhere [35]. The upper overlaid by a distortion (small spike). In Figs 5fq, results
spectra are shown with the initial resolution of 2 cm1. In of a Sym8 wavelet transform are represented. In Figs
the left column an ordinary resolution reduction was per- 5fh, the approximation coefficients are represented and
formed (4, 8, 16, 32 cm1). At the right side successive in Figs 5ik, the detail coefficients are represented. In Figs
wavelet transformations from level 1 to level 4 based on 5ln, the reconstruction of the approximations is shown
the Symlet basis with 8 vanishing moments (Sym8) are and in Figs 5oq, the reconstruction of the details is
shown corresponding in resolution to the ordinary resolu- shown. Fig. 5r represents the imaginary part of the Fourier
tion reduction. Concerning the preservation of important coefficients. For comparison in Fig. 5s, the undisturbed
spectral features, the spectra are comparable up to level 3 sine is drawn and Fig. 5t represents the imaginary part of
in both examples. At level 4 considerable signal distor- the Fourier coefficients belonging to the pure sine. The
tions are observed. In Fig. 3 the ordinary resolution reduc- favorable property for signal and image processing con-
tion performs better and in Fig. 4 the Sym8 decomposition sists in the fact that a temporary instant is localized over
lead to a better preservation of the spectral contours. the different wavelet scales. The distortion of the sine is
Because the behavior is not known in advance, it clearly recognized not only in the original domain but also
seems advisable to perform even the preliminary data pro- in the frequency analogous scale representation of the
cessing at least with a resolution of 16 cm1. However, at wavelet transform. That is a clear advantage with respect
this level the preservation of significant signal features to Fourier transform where the disruption is localized only
obtained by crude wavelet processing is not substantially in the original domain.
118
Fig. 3 Infrared spectrum of 4,8-di-
methyl-3,7-nonadienoic acid methyl
ester in the range 4002446 cm1. Left
column conventional resolution re-
duction, right column wavelet ap-
proximation coefficients relying on
the Symlet basis with eight vanishing
moments
Fig. 6 Localization preservation in both original and wavelet do- are represented the wavelet transformations from level 1
mains shown for the Raman spectrum of a graphite sample. The to level 4. The columns from left to right represent wavelet
wavelet transform was performed up to level 4 for the Symlet ba-
sis with 8 vanishing moments. From left to right: wavelet approx-
approximation coefficients, wavelet detail coefficients,
imation coefficients, wavelet details coefficients, reconstructed ap- and reconstruction of the approximations and reconstruc-
proximations, and reconstructed details tion of the details. It could be shown [38] that the level 1
details are suitable for discrimination between signal com-
ponents and disturbing spikes. The property of projection
Figs 6 and 7 demonstrate the application of the local- between different wavelet representations enables the lo-
ization principle to Raman spectra of a graphite sample; calization of the spiky components by the wavelet trans-
the experimental details are given elsewhere [38]. form and performing the spike removal afterwards in the
In the upper row of Fig. 6, the initial Raman spectra of original domain by conventional methods as interpolation
a graphite sample are represented for comparison. Below or regression.
120
riched the arsenal of chemometrics. The most important 13. Daubechies I (1988) Comm Pure Appl Math 41:909996
applications rely on wavelet detail coefficient suppression 14. Mallat SG (1989) IEEE Trans Acoustics Speech Signal Proc
37:20912110
in the wavelet domain. However, care should be taken 15. Mallat SG (1989) IEEE Trans Acoustics Speech Signal Proc
when leaving the path founded by theoretical considera- 37:20912110
tions as in the case of crude processing of wavelet detail 16. Wickerhauser MV (1991) INRIA lectures on wavelet packet al-
coefficients. In that case, strong analytical motivation gorithms Proceedings ondelettes et paquets dondes. Rocquen-
court, France, pp 3199
should guide that process and limitations of the method 17. Walczak B, van den Bogaert B, Massart DL (1996) Anal Chem
should be considered. 68:17421747
Another category of applications makes use of the lo- 18. Barclay VJ, Bonner RF, Hamilton I P (1997) Anal Chem
calization property of the wavelet transform. That proce- 69:7890
dure does not follow the classical three-step scheme: de- 19. Woodward AM, Alsberg BK, Kell DB (1998) Chemom Intell
Lab Syst 40:101107
composition processing reconstruction, but according 20. Mittermayr CR, Rosenberg E, Grasserbauer M (1997) Anal
to decomposition analysis reconstruction processing. Commun 34:7375
The selective recognition of instants in the transformed 21. Ehrentreich F, Nikolov S, Wolkenstein M, Hutter H (1998)
domain clearly goes beyond the processing abilities Mikrochim Acta 128:241250
22. Nikolov SG, Hutter H, Grasserbauer M (1996) Chem Int Lab
served by the Fourier transform. Syst 34:263273
23. Cai C, de Harrington P (1998) J Chem Inf Comput Sci 6:
Acknowledgements The support of the company Chemical Con- 11611170
cepts, Weinheim, FRG, for making the SpecInfo infrared spectral 24. Walczak B, Massart DL (1997) Chemom Intell Lab Syst 36:
database available to the author is grateful acknowledged. 8194
25. Depczynski U, Jetter K, Molt K, Niemoller A (1999) Chemom
Intell Lab Syst 49:151161
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122
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Anal Bioanal Chem (2002) 372 : 122127
DOI 10.1007/s00216-001-1132-7
O R I G I N A L PA P E R
Received: 24 June 2001 / Revised: 30 August 2001 / Accepted: 8 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract Evaporation constants of acoustically levitated range of 0.12.5 mm and they are suspended in a gaseous
drops from the homologue series of n-alkanes and 1-alka- environment by a stationary ultrasonic field of approxi-
nols in ambient air have been evaluated by size and tem- mately 155 dB. Acoustic levitation avoids sample conta-
perature measurements. The size of the pure liquid drops, mination and sorption processes by container walls, but
within a diameter range of 0.1 to 2.5 mm, was monitored suffers from evaporation and loss of solvents. To balance
using a CCD camera, while temperature measurements evaporation and condensation on levitated drops during
were carried out by IR thermography. During drop evapo- the experiments, techniques of contact-less droplet size
ration, water from a humid environment with a relative monitoring and solvent and reagent supply have been de-
humidity between 5 and 80% was condensed on the drop veloped [2]. For a quantitative analysis of evaporation pro-
surface and in the case of n-pentane, the condensed water cesses a general understanding of the properties of the drop
froze as a result of the evaporative cooling. and gaseous environments and the technique of acoustic
levitation is needed. Therefore, we have performed evap-
Keywords Drop evaporation Acoustic levitation oration experiments under different environmental condi-
Infrared (IR) thermography Drop freezing tions and with different kinds of acoustically levitated
drops, i.e. distilled water and the homologues of alkanes
(from n-pentane to n-decane) and alkanols (from meth-
Introduction anol to 1-hexanol).
Fig. 5 Drop size and surface temperature of n-alkane and 1-alkanol drops evaporated in ambient air at 21 C and 38% relative humidity,
plotted as a function of time
126
A series of size and temperature measurements for surface decreased linearly as reported in the case of 1-bu-
alkanes from n-pentane to n-nonane is shown in Figs. 5a tanol in the literature [16], and the surface temperature was
to e. The temperature and relative humidity of the envi- nearly constant during the evaporation process. 1-Butanol
ronment during these experiments was 21 C and 38%, re- and 1-pentanol were only partly miscible, while 1-propa-
spectively. The surface of the suspended drops mostly de- nol, ethanol and methanol were miscible with water. Be-
creased linearly with the evaporation time except at the cause of the condensed water, the surface and temperature
end of the measurements when the solvent was evapo- evolution changed in the case of miscible compared to non-
rated. Here, a residual droplet consisting of impurities or miscible solutions. The surface decrease changed slowly
condensed water sometimes remains. The water is con- from that of a pure alkanol drop, to that of a pure water
densed from the humid environment onto the cold surface drop, and the temperature ran continuously from the typi-
of the evaporating drop. In case of n-pentane (see Fig. 5a), cal evaporation temperature of a pure alkanol to that of a
where the surface temperature was close to 20 C, one pure water drop. Moreover, in comparison with alkane
could clearly observe this effect. Furthermore, as a result drops, generally higher amounts of condensed water gath-
of the very low temperature of evaporating n-pentane ered in alkanol drops under the same conditions (size and
drops, ice particle formation from the condensed water surface temperature of the drop and temperature and rela-
could be observed inside the suspended drop. In the ex- tive humidity of the environment). An example is illus-
ample of Fig. 5a, pure n-pentane evaporated into the hu- trated by n-heptane and ethanol (see Figs. 5c and g): only
mid environment. After approximately 33 s, all of the in the case of ethanol drops could water condensation be
pentane had evaporated and an ice particle formed for a observed while surface temperature of the evaporating
few seconds; the ice particle then melted and a water drop drop and temperature and relative humidity of the envi-
remained. A step (between 34 and 38 s in the given ex- ronment were similar.
ample) occurred in the time evolutions of drop surface
and temperature, corresponding to the phase change of wa-
ter. For the temperature calculation of the ice particle from Conclusions
IR thermography images we assumed an emission coeffi-
cient of 0.85 [13]. Condensation of water during evapora- Evaporation of acoustically levitated drops of pure water,
tion processes is also remarkable in the case of n-hexane n-alkanes and 1-alkanols in an environment of ambient
(see Fig. 5b), where a small drop of condensed water re- air, temperature and pressure have been studied. The gen-
mained at the end of the n-hexane evaporation. For the eral and quantitative understanding of this process is nec-
higher homologues n-heptane, n-octane and n-nonane, con- essary for the application of acoustic levitation techniques
densation of water was neglected because of the higher for sample pre-treatment and for the combination with an-
surface temperature of the evaporating drops and the in- alytical techniques. It has been shown that the evaporation
solubility of water in alkanes. The condensed water with- constant K can be calculated from both contact-less size
in a suspended alkane drop in the case of n-pentane and or temperature measurements. The method has been vali-
n-hexane formed a separate droplet-shaped phase which dated by using water drops and the evaluated values of K
was completely surrounded by the alkane phase. Phase for alkane and alkanol drops are in agreement. Therefore,
separation of non-miscible solvents in acoustically levi- the method can also be used to determine binary diffusion
tated drops could be visualised by video images and has coefficients from the evaluated evaporation constant K.
also been reported in the literature [15] in the case of Future work will focus on the evaporation processes of bi-
hexane and water. In that way, the enclosed water did not nary liquid drops. As seen in the case of condensed water
essentially change drop surface and drop temperature evo- in alkane and alkanol drops, different effects of surface
lution during the evaporation process. and mixing processes have to be taken into account.
The surface temperature of the alkane drops during Evaporation rates can be changed by covering the drop
their evaporation was nearly constant within a range of surfaces with monomolecular films [17].
1 C. The suspended drop was cooling down from ambient
temperature to its typical evaporation temperature at the Acknowledgment The authors thank Prof. Dr. H. K. Cammenga
for helpful discussions.
beginning of the measurements only. At the end, when all
alkane had evaporated, a jump in the temperature evolu-
tion occurred.
References
Evaporation of 1-alkanol drops 1. Welter E, Neidhart B (1997) Fresenius J Anal Chem 357:345
350
2. Eberhardt R, Neidhart B (1999) Fresenius J Anal Chem 365:
The evaporation of suspended alkanol drops was per- 475479
formed under the same conditions (ambient air at 21 C 3. Rohling O, Neidhart B (2000) Fresenius J Anal Chem 368:
and with a relative humidity of 38%). The experimental 125129
4. Lierke EG (1996) Acoustica 82:220237
results are shown in Figs. 5f to j. Size and temperature 5. Tuckermann R, Bauerecker S, Neidhart B (2001) Phys Unserer
evolution of 1-butanol and 1-pentanol drops during their Zeit 32:6975
evaporation were similar to those of the alkanes. Drop 6. Fuchs N (1934) Phys Z Sowjetunion 6:224 ff
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58:18 ff Apperatur fr die Probenvorbereitung in der Mikro- und Spuren-
10. Reid RC, Prausnitz JM, Sherwood TK (1987) The Properties of analyse. Dissertation, Universitt Marburg
Gases and Liquids. McGraw-Hill, New York 16. Erbil HY, Dogan M (2000) Langmuir 16:92679273
11. Daidzic N (1995) Nichtlineare Tropfenschwingungen und -hy- 17. Stosch R, Cammenga HK (2000) J Colloid Interface Sci 230:
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Anal Bioanal Chem (2002) 372 : 128135
DOI 10.1007/s00216-001-1112-y
O R I G I N A L PA P E R
Received: 14 June 2001 / Revised: 13 August 2001 / Accepted: 24 August 2001 / Published online: 14 December 2001
Springer-Verlag 2001
Abstract Experimental studies and computer simulations plasmas are formed when N2, O2, air, H2, or He are added
were conducted to identify plasma operating conditions to one or more gas flows of the ICP torch. Almost all
and to explore and contrast the excitation conditions of commercial ICP instruments are sustained in Ar, rather
Ar, Ar-O2, and Ar-He inductively coupled plasmas (ICPs) than less expensive molecular gases such as air, N2, and
for the introduction of microliter volumes of sample solu- O2, mainly because the Ar ICP is formed more easily at
tions with a direct injection high efficiency nebulizer reasonable power levels (12 kW) and generally provides
(DIHEN). The best MgII 280.270 nm/MgI 285.213 nm ratio better detection limits for elements possessing spectral
(6.6) measured with Ar ICP atomic emission spectrome- lines below 350 nm [3, 4]. However, mixed-gas plasmas
try for the DIHEN (RF power = 1500 W; nebulizer gas offer a superior ability to decompose refractory particles,
flow rate = 0.12 L min1) was less than the ratio (8.2) ac- increased tolerance to higher solvent and analyte loads,
quired on the same instrument for conventional nebuliza- and less structured background spectra above 350 nm,
tion (1500 W and 0.6 L min1). Addition of small amounts leading to improved analytical performance in certain ap-
of O2 or He (5%) to the outer gas flow improved excita- plications [3, 5, 6, 7]. These characteristics allow for the
tion conditions in the ICP, that is, a more robust condition analysis of a wider range of samples than are possible us-
(a MgII/MgI ratio of up to 8.9) could be obtained by using ing Ar alone. For example, introduction of O2 or He to the
the DIHEN with Ar-O2 and Ar-He mixed-gas plasmas, outer gas flow of the Ar ICP is effective in suppressing
thereby minimizing some potential spectroscopic and ma- the background caused by organic solvents [8, 9] and in
trix interferences, in comparison to Ar ICPAES. improving the evaporation efficiency of slurries [10, 11]
when conventional nebulizer-spray chamber arrangements
Keywords Direct injection high efficiency nebulizer are used. However, except for two recent conference pre-
DIHEN Mixed-gas inductively coupled plasmas sentations [12, 13], no report is available on studies of
Atomic emission spectrometry Matrix interferences mixed-gas plasmas for the direct injection of the sample.
Computer simulation The principal aim of this research is to explore the ex-
citation conditions of the mixed-gas ICPs for the direct in-
troduction of test solutions using the DIHEN. The DIHEN
Introduction is a low consumption micronebulizer (1100 L min1)
for ICP mass spectrometry (ICPMS) [14, 15, 16, 17, 18,
Argon inductively coupled plasmas (Ar ICPs) are com- 19, 20, 21, 22, 23, 24, 25] that operates at very low nebu-
monly used as desolvation, vaporization, atomization, ex- lizer gas flow rates (ca 0.2 L min1) compared to conven-
citation, ionization sources in analytical atomic emission tional pneumatic nebulizer-spray chamber arrangements
and mass spectrometry [1, 2]. Argon-supported ICP dis- (ca 1.0 L min1). Initial studies describing the use of the
charges provide low detection limits, wide concentration DIHEN for ICP atomic emission spectrometry (ICPAES)
dynamic ranges, and minimal matrix effects. Mixed-gas were presented by Montaser and coworkers [12, 13] and
Todol and Mermet [26, 27]. The latter work, however,
was limited to Ar ICPAES measurements at a maximum
J.R. Chirinos K. Kahen S.-A.E. OBrien A. Montaser () RF power of 1300 W. In contrast, our current and previ-
Department of Chemistry, The George Washington University,
Washington, DC 20052, USA ous accounts [12, 13] consider the efficacy of the DIHEN
e-mail: montaser@gwu.edu for atomic emission studies of the Ar ICP and the mixed-
J.R. Chirinos
gas plasmas operated at 1500 W. Such a study is signifi-
On leave from Centro de Qumica Analtica, cant because plasma-related matrix effects are usually
Universidad Central de Venezuela, Caracas 1041a, Venezuela more serious for direct liquid introduction [27] since the
129
mass of sample (and solvent) presented to the plasma is dynamic equilibrium (LTE), (2) a steady state and laminar flow
greater and usually has larger droplets than the aerosol in- prevails in the ICP torch, (3) the plasma is axially symmetric and
optically thin, (4) viscous dissipation and displacement currents
jected in conventional nebulization. It has been reported are negligible, and (5) flow, electromagnetic, temperature, and
that only 45% of the aerosol produced by the DIHEN are concentration fields are two-dimensional.
composed of droplets less than 10 m [19]. Because the
DIHEN is delicate and relatively expensive, our study
Instrumentation and operating conditions
was initially guided by computer simulation of Ar, Ar-O2,
and Ar-He plasmas to locate a thermally safe region to The instrumentation and operating conditions are shown in Table 1.
place the DIHEN within the torch. To explore the excita- A free-running generator (Elan 5000 ICPMS generator), modified
tion condition or robustness of the mixed-gas ICP versus in our laboratory [30], was used for all ICPAES studies. The outer
the Ar ICP, the ratio of MgII/MgI was also measured for gas flow consisted of either Ar or Ar-O2 and Ar-He mixtures. Ar-
gon was used in the intermediate and nebulizer gas flows.
the DIHEN with the plasma under the end-on viewing To estimate the robustness of the ICP [31], the line intensity
condition and compared to results using conventional ratio MgII 280.270 nm/MgI 285.213 nm was measured using a
nebulization. Finally, the effects of acid concentration and 5 g mL1 solution of Mg prepared by diluting a 1000 g mL1
easily ionized elements on the MgII/MgI ratios were ex- stock solution (Spex Certiprep Inc., Metuchen, NJ) in distilled,
deionized water (18 M cm). The effect of 2 M HNO3 (Optima
amined for the DIHEN and contrasted to previous work grade, Fisher Scientific, Pittsburgh, PA) and 0.1% and 1% NaCl
[26, 27]and our findings for conventional nebulization us- (ACS grade, Fisher Scientific) on plasma robustness was also in-
ing the Ar ICP and the Ar-O2 plasma [12, 13]. vestigated.
Version 2.0 of the High Frequency Induction (HiFI) plasma code In general, ignition of the Ar plasma for the DIHEN-
[28] was used to calculate properties of Ar and mixed-gas plasmas ICPAES measurement is straightforward and similar to
as a function of the operating conditions listed in Table 1. The
code predicts plasma temperature, gas velocity, power input, gas conditions used for conventional nebulization, that is, the
enthalpy, and magnetic flux density [5, 28, 29]. Only the result for plasma is ignited at outer, intermediate, and nebulizer gas
plasma temperature was used in this work as an initial guide for lo- flows of 15, 1.2, and 0 L min1, respectively. The Ar
cating a thermally safe region within the ICP torch to place the ICPAES operating conditions under the end-on viewing
DIHEN, that is, to prevent excessive heating of the DIHEN nozzle
due to the changes in the plasma operating conditions. The results
mode are generally comparable to those used for Ar
of this code must be treated as estimates because the model as- ICPMS [14, 15, 16, 17, 18, 19, 20, 21], that is, the RF
sumes the following: (1) the plasma is in a state of local thermo- power, nebulizer gas flow rate, and solution uptake rate
Fig. 3 Simulated plasma temperature (K) as a function of the dis- Fig. 4 Simulated plasma temperature (K) as a function of the dis-
tance from the end of the torch intermediate tube for Ar-He ICPs. tance from the end of the torch intermediate tube for Ar-O2 ICP
RF power, nebulizer gas flow and intermediate gas flows are with 10% O2 in the outer gas flow: (A) intermediate gas flow=
1500 W, 0.15 L min1 and 1.2 L min1, respectively 1.2 L min1 at different nebulizer gas flows and, (B) nebulizer gas
flow=0.15 L min1 at different intermediate gas flows
above the intermediate tube, respectively. The wide tem- Simulation of the axial temperature profiles
perature variations at the base of the plasma collectively at different nebulizer and intermediate gas flow rates
suggested that the DIHEN nozzle should be positioned at
larger distances below the intermediate tube when the per- The predicted axial temperature profiles of the plasma at
centage of O2 in the outer gas is extensively altered; oth- different nebulizer gas flow rates are shown in Fig. 4A for
erwise, the DIHEN nozzle would melt. Accordingly, the 10% O2 in the outer tube. The plasma temperature in-
DIHEN nozzle was placed 3 mm below the intermediate creases significantly close to the intermediate tube when
tube for Ar-O2 ICP as compared to 2 mm for Ar ICP. One the nebulizer gas flow is reduced or stopped. These results
must note, however, that the temperature rise predicted at are significant for DIHEN operation, considering that the
the base of the mixed-gas plasma is useful analytically be- DIHEN is typically operated at comparatively low nebu-
cause it enhances droplet desolvation and diminishes sol- lizer gas flows (0.10.2 L min1). A nebulizer gas flow
vent effects. Indeed, the cited issues are difficult to attack rate of 0.15 L min1 was chosen for experimental work. At
when the sample aerosol is directly injected into the lower nebulizer gas flows, either the DIHEN nozzle
plasma, especially for the large droplets. would gradually clog due to excessive heating or the
Axial temperature profiles are shown in Fig. 3 for Ar aerosol would not penetrate the axial channel, resulting in
and Ar-He plasmas. At large distances from the nebulizer plasma instabilities.
(4060 mm), the plasma temperature is reduced with the The effect of the intermediate gas flow rate on axial
introduction of He. In contrast to the results obtained with temperature distributions of the plasma is shown in Fig. 4B
Ar-O2 plasmas, the predicted axial channel temperature for 10% O2 in the outer tube. The simulated profiles are
close to the intermediate tube (Fig. 3B) does not signifi- obtained at intermediate gas flow rates of 0.5, 1.2, and
cantly increase as more He replaces Ar in the outer gas, 2.0 L min1. The plasma temperature increases signifi-
suggesting that the DIHEN nozzle could be safely posi- cantly close to the intermediate tube when the intermedi-
tioned 2 mm below the intermediate tube. Based on the ate gas is reduced from 2 to 0.5 L min1. For example, at
predicted temperatures, the spatial structures of the Ar-He an intermediate gas flow of 2.0 L min1, the predicted
ICPs and the Ar ICP are nearly identical, hence the MgII/ temperatures are 350, 414, and 1695 K at 2.5, 4.2, and
MgI values of Ar-He ICPs should be less (see below) than 6.5 mm above the intermediate tube, respectively. At
those measured in Ar-O2 plasmas. 0.5 L min1, the estimated temperatures are 350, 672, and
3875 K at 2.5, 4.2, and 6.5 mm above the intermediate
tube, respectively. These results collectively illustrate that
the intermediate gas flow plays, compared to conven-
133
Plots of MgII/MgII ratios for Ar, ArO2, and Ar-He ICPs Introduction of an inorganic acid, such as HNO3, or NaCl
are shown in Fig. 5 as a function of nebulizer gas flow rate is known to suppress or enhance analytical signal in
for the DIHEN. The ratios are the mean of three measure- ICPAES, but the effects can be minimized for conven-
ments on different days and the precision is within 5%. tional nebulization by using robust plasma conditions,
The Ar-O2 ICPs exhibit higher ratios compared to Ar ICP that is, reduced nebulizer gas flow and high RF power
(Fig. 5A). For example, the MgII/MgI ratio is approxi- [35, 37, 38, 39, 47]. Table 3 includes MgII/MgI ratios for
mately 8.5 and 6 for Ar-O2 ICP and Ar ICP, respectively, the introduction of HNO3 solutions by the DIHEN and
at a nebulizer gas flow of 0.15 L min1. No significant dif- nebulizer-spray chamber arrangement operating under op-
ference was observed for mixed-gas plasmas having 2.5, timal operating conditions. Again, the ratios are the mean
5, and 10% v/v of O2 in the outer gas flow. Fig. 5B shows of five measurements on different days and the precision
similar results for Ar-He plasmas, that is, Ar-He plasmas is within 5%. For the Ar ICP, the MgII/MgI ratios (Table 3)
exhibit higher MgII/MgI ratios compared to the Ar-ICP, measured with the DIHEN decrease only slightly (6.66.2
but the ratios are slightly lower than those obtained with or 6%) as the HNO3 concentration is increased from 0 to
Ar-O2 ICP. 2 M. These data suggest that DIHEN-ICPAES is rela-
The above results jointly suggest that mixed-gas plas- tively robust when Ar ICP is used at 1500 W. Todol and
mas improve the robustness of the ICP when a relatively Mermet also reported no matrix effect by nitric acid for a
small amount of the foreign gas is introduced (ca 2.5%). 1300 W Ar ICP, but a lower solution uptake rate (30
For higher percentages of foreign gas, no significant im- 40 L min1) had to be used [26, 27]. Improved MgII/MgI
provement in the MgII/MgI ratios is realized, but the hot ratios are also noted for Ar-O2 ICP (Table 3), however the
zone of the plasma approaches the tip of the DIHEN noz- level of suppression (6%) remained unchanged when the
134
DIHEN was used. In contrast, with conventional nebu- MgII/MgI greater than 6) for some Ar ICPAES measure-
lization, acid effects are more pronounced with Ar-O2 ICP ments at high RF power (ca 1500 W) and low nebulizer
compared to the Ar ICP, i.e., the mixed-gas plasma is less gas flow (ca 0.15 L min1), however, the ratios were less
robust than the Ar ICP. The main reason for this change in than those noted for a conventional nebulizer-spray cham-
plasma robustness is unclear to us. Obviously, the pres- ber arrangement. Slightly larger MgII/MgI ratios could be
ence of the spray chamber makes interpretation of the re- obtained using Ar-O2 and Ar-He mixed-gas plasmas with
sult more difficult. the DIHEN for the addition of the foreign gas to the outer
Table 3 also includes MgII/MgI ratios in the presence gas flow; nevertheless, the level of suppression (6%) for
of 0.1% NaCl introduced by the DIHEN and the conven- the introduction of a solution of 2 M HNO3 remained un-
tional nebulizer-spray chamber for Ar ICP and Ar-O2 ICP. changed. With conventional nebulization, acid effects were
Solutions containing 1% Na were not used with the more pronounced with Ar-O2 ICP compared to the Ar ICP.
DIHEN because the nebulizer is prone to clogging. The In general, results of these studies at 1500 W on mixed-
data in Table 3 suggest that Na suppresses MgII/MgI gas ICPAES with the DIHEN agree with the work of
more severely than HNO3, both for the conventional neb- Todol and Mermet who operated a 1300 W Ar ICP [26,
ulization system and the DIHEN. Importantly, the use of 27]. Clearly, better MgII/MgI ratios were obtained be-
the Ar-O2 ICP was not very effective in reducing Na ma- cause the ICP was operated at 1500 W. It was also demon-
trix effects at 1500 W. In contrast, the Na matrix effect strated that the plasma required RF power above 1500 W
was negligible at 1300 W and at a solution flow rate of in the presence of the DIHEN to run under robust condi-
nearly 80 L min1 in Ar ICPAES for micronebulization tions, but only 1300 W was necessary for a pneumatic
with a high efficiency nebulizer-cyclonic spray chamber nebulizer-spray chamber arrangement. Todol and Mer-
[26, 27]. Accordingly, to eliminate the Na matrix effect in met also concluded that the acid effects are eliminated for
ICPAES, the DIHEN must present a smaller amount of the DIHEN, but only at a solution uptake rate of 3040 L
sample to the plasma at RF power level greater than 1500 W. min1 [27]. Operation at 1500 W, as demonstrated in this
This conclusion is in line with the work of Todol and work, allows introduction of 80 L min1 with the DIHEN
Mermet [26, 27]. with nearly no HNO3 effect. At 1300 W, Todol and Mer-
The above data on MgII/MgI ratios also brings some met reported minimal Na matrix effect with the DIHEN,
uncertainty to the application of this ratio as an indicator but only for spectral lines possessing low Esum [27]. For
of plasma robustness [31]. At least for direct injection, it lines having high Esum values, as shown in this work, the
is prudent to conduct further experiments to validate the Na matrix effect could not be remedied at 1500 W or
usage of MgII/MgI as an indicator of plasma robustness. through the use of mixed-gas plasma. To create a robust
plasma condition for direct injection, one must reduce the
amount of aerosol and decrease the droplet size intro-
Conclusions duced into the ICP to the level prevalent in conventional
pneumatic nebulization (2040 mg min1), increase the
This paper describes ways to overcome the limitations of RF power beyond the level required for conventional neb-
the DIHEN. Computer simulation was applied to predict ulization, and enhance plasmasample interaction by op-
the operating conditions of mixed-gas ICPs using the erating a mixed-gas plasma.
DIHEN. The simulated temperature profiles show that It would be interesting to introduce a foreign gas to the
Ar-O2 and Ar-He plasmas provide better excitation condi- nebulizer gas flow as no report is currently available on
tions than the Ar ICP but may create a hot zone close to the efficiency of the DIHEN with a large proportion of
the DIHEN nozzle. For mixed-gas plasmas, the DIHEN such a gas. In this case, the axial channel would be wider
should be positioned farther below the torch intermediate [3], but an increase in thermal conductivity may result in
tube compared to its typical location in Ar ICPs. In gen- the central channel. Work in this area is in progress in our
eral, the DIHEN provides robust conditions (values of laboratory.
135
Acknowledgements This research was sponsored by grants from 21. Minnich MG, Montaser A (2000) Appl Spectrosc 54:1261
the US Department of Energy (DE-FG0293ER14320), the Na- 22. McLean JA, Minnich MG, Su J, Lai W, Montaser A (2000)
tional Science Foundation (CHE-9505726 and CHE-9512441), Anal Chem 72:4796
and JE Meinhard Associates, Inc. The authors are grateful to John 23. Minnich MG, McLean JA, Montaser A (2001) Spectrochim
A. McLean and Craig M. Benson for constructive discussions and Acta, Part B 56:1113
assistance and to Mr. Bill Rutkowski for excellent machine shop 24. Acon BW, McLean JA, Montaser A (2001) J Anal At Spectrom
service. 16:852
25. Westphal CS, McLean JA, Acon BW, Allen LA, Montaser A
(2001) Anal Chem (submitted)
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Anal Bioanal Chem (2002) 372 : 136140
DOI 10.1007/s00216-001-1136-3
O R I G I N A L PA P E R
Received: 2 August 2001 / Revised: 19 September 2001 / Accepted: 20 September 2001 / Published online: 12 December 2001
Springer-Verlag 2001
[8, 9]. However, some remarkable differences exist. These should be inert (no reaction with liquids or flame gases) and inex-
are the direct heating of the TS capillary with the flame pensive (for instance nitrogen or technical argon). At the outlet of
the pressure reduction valve a special stainless steel connecting
gases instead of a temperature-controlled electrical heat- piece (commercially available, Drger AG, 47807 Krefeld, Ger-
ing, the much higher temperature (up to 900 C), the short many) was used for a close connection between the reduction
length of the heated area (about 1 cm) with a strong tem- valve and the PEEK capillary (o.d. 1/16, i.d. 0.5 mm) going to the
perature gradient and a larger inner diameter of the capil- sample injection valve.
For sample introduction a six-port sample injection valve
lary. Another remarkable difference is that high pressure (Knauer, Berlin, Germany) with various sample loops (0.350 L)
is not needed for the transport of the liquids, which allows were applied. The sample loops were made using PEEK tubing
the use of peristaltic pumps instead of HPLC pumps [7]. with various inner diameters. The smallest sample loop (0.3 L)
In this paper we show that it is possible to extend the ap- was prepared from the smallest inner diameter PEEK capillary
plicability of the TS-FF-AAS method for determination of available on the market (64 m).
A flexible fused silica capillary (Polymicro Technologies,
elemental traces in submicroliter volumes of samples us- Phoenix, USA) was tightened into the outlet of the sample intro-
ing a fused silica capillary as an easily available, effective duction valve. Since the outer diameter of the fused silica capillary
thermospray vaporizer and a standard gas cylinder as a (365 m) was much smaller than that of the standard capillaries
gas pressure pump for the transport of the sample. (1/16) for these valves, it was screwed with the aid of a short
piece of standard PEEK capillary of 500 m inner diameter
(sheath piece) into the valve. The other end (outlet) of the fused
silica capillary was directed into the flame furnace through the in-
Experimental troduction hole. The length of the fused silica capillary was about
40 cm. In the experiments capillaries with inner diameters of 25,
Spectrometer 50, 75, 100 and 200 m were used. The capillary was fixed with an
adjustable holder in front of the flame furnace.
A Philips model PU9200X atomic absorption spectrometer with The outlet end of the capillary can be considered as a thermo-
deuterium lamp background correction was used. Operating pa- spray vaporizer. The last few millimeters of the capillary are
rameters (wavelength, slit, current for the hollow cathode lamp) strongly heated due to the proximity of the glowing flame furnace.
for the spectrometer were as recommended by the manufacturer. In A tight (hermetically sealed) connection between the end of the
all experiments an acetylene/air flame was used. The original spec- capillary and the hole in the flame furnace is not needed, because
trometer had no suitable data/signal output to display transient sig- the vapor exits from the outlet of the capillary at high speed into
nals, but with a separate personal computer and self-written soft- the tube furnace. The areas of both ends of the flame-heated tube
ware, all signals could be visualized, recorded and stored. The are much larger than the very small free remaining area of the sam-
computer was interfaced to the analog output of the spectrometer ple introduction hole. Therefore, the vapor escaping from the fur-
via an analog/digital converter unit. The software also permitted a nace will only exit at the ends of the tube and not through the in-
transfer of raw data to other computer programs, for example to a troduction hole. The end of the capillary can be easily put in and
chromatography software package (Eurochrom 2000, version 1.2, out of the flame furnace through the introduction hole. The fused
Knauer, Berlin, Germany) for integration of transient signals and silica capillary would melt and curve if it came into direct contact
to a spreadsheet program (Excel, Microsoft) for better displays of with the flame gases, therefore, the flame-heated end of the capil-
the original signals. lary was protected by means of a short piece (4 cm) of stainless
steel capillary (o.d. 1/8, i.d. 1.6 mm) welded into the introduction
hole of the furnace.
TS-FF-AAS system The arrangement of the flame furnace on the burner head of the
spectrometer, the geometry (10/7 mm o.d./i.d.) and the material
The schematic setup of the micro TS-FF-AAS system is shown in (super alloy, ASTM B 167) of the furnace was the same as in Refs.
Fig. 1. The arrangement of the system used in this work was simi- [6, 7]. There was a hole of 3.2 mm centered in the flame furnace
lar to one already described [7]. There are two essential differ- for the introduction of the stainless steel protection tube and four
ences, the type of the pump and the TS capillary vaporizer. While additional holes of 3 mm at the bottom side for ensuring the flame
in the earlier work a peristaltic pump transported the sample, in the gases enter the furnace. Small metal plates were screwed to both
present system a nitrogen or argon gas cylinder was used as a sim- ends of the burner head, in which two ceramic pins of 3 cm length
ple, pulsation-free device capable of forwarding small liquid sam- are fixed. The 10 cm flame-heated tube length was laid on these
ple plugs through a capillary with a small inner diameter into the pins and could be put into/out of the flame easily.
flame furnace. From the gas cylinder 0.12.5 MPa pressure gas
could be obtained via a standard pressure reduction valve. From
the point of view of our work the type of gas is not important. It Thermospray aerosol generation
In all papers about TS sample introduction a electrically heated
capillary is used [8, 9], while in the TS-FF system the capillary is
heated only at one end by the acetylene/air flame over a length of
a few millimeters [7]. In our present work capillaries made from
synthetic fused silica with 25200 m inner diameter were used.
The flow in a capillary can be described by the Hagen-Poiseuille
law. In Fig. 2 the calculated effect of the inner diameter of the cap-
illary on the pressure to be applied and the flow rate of water at
20 C (=0.01 Poise) are shown. For these calculations a liquid
plug of 10 cm length in the capillaries was chosen. A liquid plug of
this length is equal to a volume of 0.19 L, 0.44 L, 0.78 L and
3.14 L in capillaries of 50 m, 75 m, 100 m and 200 m inner
diameter, respectively. According to this diagram, a pressure of
0.05 to 1 MPa and a capillary of 100 m inner diameter result in a
flow rate between 0.08 and 1.6 mL/min, which is a useful range for
Fig. 1 Schematic setup of the micro TS-FF-AAS system for the TS-FF-AAS. Figure 3 shows the effect of various inner diameters
analysis of submicroliter samples of the capillary on the signal height. The highest signals were ob-
138
Fig. 2 Influence of the flow rate and of the inner diameter of the
capillary on the pressure (calculated values according to Hagen- Fig. 4 Signals of 0.3 L samples of 10 g/mL Pb obtained by
Poiseuille law, the considered length of the liquid segment means of TS capillary with 100 m i.d. applying different pres-
amounts to 10 cm) sures
Table 1 contains the limits of detection (3 s values) ob- The described micro TS-FF method is mainly advanta-
tained from 25 measurements of a blank solution. The geous for the analysis of sample volumes smaller than
LOD values for TS-FF-AAS were determined for three 10 L. After the optimization of the operational param-
small sample volumes (0.3, 1 and 10 L) while in con- eters (capillary of 100 m i.d., 1 MPa pressure), samples
O R I G I N A L PA P E R
Received: 28 August 2001 / Revised: 4 October 2001 / Accepted: 24 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract We describe the development of, and analytical of these substances. The substances are pooled in sub-
conditions used for, parallel affinity assay for thrombin stance-libraries which often contain 10 to 300 thousand,
inhibitors adapted to the first label-free optical screening mostly low-molecular-weight, compounds of high struc-
HTS detection set-up fully integrable into a screening tural diversity [1].
platform. To achieve compatibility with pharmaceutical One of the targets is the intensively investigated en-
libraries, an HTS-transducer was realized by gluing the zyme thrombin. Thrombin is a serine protease with a mol-
bottomless scaffolds of 96- and 384-well plastic micro- ecular weight of 34 kDa ( 40 kDa in cows) which consists
plates on to transducer slides. The transducer are coated of two subunits. It catalyses the splitting of fibrinogen to
with a dextran, to ensure biocompatibility and functional- active fibrin and is the main component in the last step of
ity, and a known thrombin inhibitor was attached cova- the blood-coagulation cascade. Exact regulation of the
lently to it. By adapting reflectometric interference spec- blood coagulation is of special importance and determines
troscopy for simultaneous reading of the whole transducer the balance between bleeding and thromboses. This
plate we were able to detect the binding of thrombin in all makes thrombin an important target in pharmaceutical
the wells of the microplates on-line, in parallel, and time drug screening and much effort is being devoted to find-
resolved. By using an inhibition assay, the screening of ing effective thrombin inhibitors.
384 substances for thrombin activity can be performed Whereas nowadays the number of interesting targets is
within an assay time of less than 15 min. We also show numbered in hundreds only, the human genome project
that the data quality is high enough for parallel quantifica- will lead consistently to the discovery of new targets
tion of the IC 50 values of the library substances. which, although not attributed to a discrete function, are
of great importance for the pharmaceutical industry. The
Keywords High throughput screening Label-free main targets actually tested are enzymes in the classes ki-
detection Thrombin Reflectometric interference nases [2] or proteases [3] and membrane-bound or nucleo-
spectroscopy Assay development plasmatic receptors [4]. Screening of these targets is real-
ized either by functional assays or by binding assays.
Functional assays correlate the function of the target di-
Introduction rectly towards the concentration of the test substance.
This function can be as complex as the modulation of
In recent years pharmaceutical research has been searched gene expression or as simple as the activity of an enzyme.
for new ways to discover future drugs and agrochemical Binding or affinity assays determine the biomolecular in-
products and has developed high-throughput screening teraction of two substances. These assays are less com-
(HTS). The objective of this field of research is to screen plex and easier to perform but the target function cannot
enormous numbers of compounds for activity against a be predicted. On the other side, binding assays supply ad-
target of interest without the need for knowledge about ditional information if a competitive assay is used. For
the structure of the target or the mechanism of the action binding assays the availability of very pure targets, e.g.
chromatographically purified, is necessary.
Among the physical methods used for HTS experi-
ments, imaging and scanning optical detection systems
are particularly widely used. The most important strate-
O. Birkert () G. Gauglitz
Institute of Physical Chemistry, University of Tbingen,
gies are fluorescence polarisation spectroscopy [5, 6],
Auf der Morgenstelle 8, 72076 Tbingen, Germany fluorescence correlation spectroscopy [7], and fluores-
e-mail: oliver.birkert@ipc.uni-tuebingen.de cence resonant energy transfer [8].
142
Experimental
Equipment and reagents
AMD was covalently attached to glass transducers by use of a The wells of the microplates were filled with solid glutaric acid an-
modification of the procedure of Piehler et al. [14]. The interfer- hydride and the maximum amount of water was also added. The
ence transducers (73 mm108 mm) were cleaned in freshly pre- plates are covered and shaken gently for 150 min. The wells were
pared, hot H2SO4H2O2, 6:4, rinsed with water and dried in a ni- rinsed thoroughly with water. A freshly prepared aqueous solution
trogen stream. The activated transducers were silanised with glyci- of EDC (9.6 mg mL1, 0.05 mol L1) and NHS (5.9 mg mL1,
doxypropyltrimethoxysilane (400 L) for 1 h. AMD (2.5 g) was 0.05 mol L1) was pipetted into the microplate wells. The plates
dissolved in water (5 mL) and the solution was dropped on the pre- were covered and shaken gently for 20 min. The plates were
treated transducers to furnish a uniform coating. Two such trans- washed with water and immediately filled with 70 L (96-well
ducers were arranged in a sandwich. After a reaction time of 14 h plate) or 30 L (384-well plate) of an aqueous inhibitor solution
in a saturated-water atmosphere the sandwich was separated and (1.5 mg mL1; 3.8 mmol L1). The covered plates were shaken for
both transducers were rinsed with water. After drying the plates in 14 h, washed with water and dried in a nitrogen stream. The mod-
143
ified plates were stored at room temperature. The plates were sta- Changes in the refractive index
ble for months.
To examine the effect of changes in the refractive index, water and
glycerine solution (10% in water) were alternately measured in a
Detection method 96-well microplate. Liquid handling was performed by means of
an automated pipettor control to ensure the plate was repeatedly
Parallel reflectometric interference spectroscopy (RIfS) enables filled with both liquids for 600 s alternately. In this and all further
the optical on-line detection of specific biomolecular interaction in experiments data accumulated when the plates contained no liquid
special 96- and 384-well RIfS microplates without the need for la- were not used for evaluation.
belling techniques. A schematic diagram of the detection and Protein interaction
pipetting unit is shown in Fig. 1. Basic information about single-
channel RIfS is available elsewhere [15, 16]. Protein adsorption was measured with the same pipetting program
The parallel RIfS is based on the spectral distribution of re- for 96- and 384-well microplates, with the exception of the sample
flectance from thin transparent layers on a glass substrate. The volume. In the following, sample volumes are given for measure-
beams reflected at both sides of an interference layer (330 nm ments in 96-well plates, volumes for 384-well plates are given in
thickness) coated with a biopolymer adlayer lead to a interference parentheses.
pattern from which the optical thickness, nd, of this thin layer may Before starting a measurement the plate was filled with PBS
be calculated. Changes in the optical thickness, nd, of the layer for at least 10 min to prevent swelling effects within the data-
caused either by changes in the reflective index or in the physical recording time. The plates were depleted and data recording and
thickness of the adlayer, e.g. because of specific binding to this the pipetting program were started simultaneously. First the RIfS
surface, leads to a shift of the interference pattern which is evalu- microplate was filled with 70 L (60 L) PBS and the baseline
ated in real-time. was recorded for 300 s. The PBS was then removed and 70 L
The new parallel screening system was realized by a simulta- (40 L) protein sample was pipetted from one of the storage mi-
neous illumination of the whole RIfS microplate glass bottom with croplates into the RIfS microplate. After a binding time of at least
a halogen-light source. As a certain spectral resolution of the re- 900 s, the sample was replaced by 80 L (60 L) hydrochloric acid
flected light is necessary for signal evaluation, a monochromator to regenerate the plate. This enables re-use of the same plate for
system was placed directly behind the light source. The mono- more than 100 screening runs.
chromator consists of a filter wheel which enables the subsequent
passage of monochromatic light of seven different wavelengths Screening experiments and evaluation of IC 50 value
(516.0, 543.5, 572.3, 606.8, 643.3, 685.9, and 707.0 nm). A total
turn of the filter wheel takes 18 s and determines the time resolu- For the binding inhibition test 96 samples of 100 mL PBS buffer
tion of the measurement. Diffusion and binding of protein to the solution containing thrombin (6 units mL1) and chicken ovalbu-
transducer surface normally occurs much more slowly. If the num- min (500 g mL1) were pre-incubated with the inhibitors in a
ber of filters can be reduced to three; this leads to better time reso- standard polystyrene microplate. Ovalbumin was added in large
lution and increased signal noise. excess to prevent adsorption of thrombin within the pipettes and
The signal was recorded by means of a 12-bit CCD camera, microplates. These pre-incubated mixtures were measured with the
EEV type CCD0520, which traps the reflected light with the in- RIfS arrangement as described above.
terference information. The beam illuminates half of the CCD As the binding event is controlled by diffusion of antibodies to
chip. Simultaneously a reference beam was detected with the other the surface, the calibrated signal is proportional to the concentra-
half of the chip. This reference beam enables correction for light tion of free antibody in the solution at each time. After the binding
inhomogeneities caused by temporal intensity fluctuations of the event the sensor was regenerated by pipetting hydrochloric acid
light source and by spatial differences in illumination intensity. (pH 1.7) into the wells to prepare it for the next cycle.
An automatic well mask generation program assigns the pixels For Screening experiments high, standardized inhibitor con-
on the CCD chip to the wells of the microplate and accumulates centrations (60 ng mL1, 1.5107 mol L1) were used beside sam-
the signals corresponding to one well. Because the well mask gen- ples without added inhibitor.
eration tool is very flexible, masks can be created for standard 96-, For the determination of the IC 50 value, inhibitor concentra-
384-, and 1536-well microplates and for individual plate formats. tion was varied between 4109 mol L1 and 1107 mol L1. Eval-
From these data the software calculates the average change in uation occurs by plotting the calibrated signal after a binding time
optical thickness for each well and each measurement point in real of 13 min against the inhibitor concentration.
time. The data are saved in common file types. Signals from the
larger wells are calculated from a larger number of CCD camera
pixels; this results in an reduced noise. The root-mean-square
noise (RMS) is proportional to N0.5, where N is the number of il- Results and discussion
luminated CCD camera pixels.
Liquid handling was achieved by means of a modified pipet- Signal drift and noise
ting robot IGEL 250/96 from CyBio (Jena/Germany). Samples can
be transferred between four standard microplates and one RIfS mi-
croplate. The robot was modified in such a way that liquid han- As this is a new arrangement for parallel screening, fun-
dling was possible without lifting the RIfS microplate from the de- damental data analysis was performed to specify the sig-
tection position. This enables continuous data recording totally in- nal noise and the temporal signal drift. Both examinations
dependent of the pipetting steps. were conduct using plates with and without aminodextran
coating. Measurements are performed with dry plates and
with plates filled with water. Aminodextran is a hydrogel
sults are shown in Table 1. The quality of the data was not
affected by the biopolymer adlayer in air or in an aqueous
environment. Both drift and root-mean-square noise
(RMS) were at such a low level that they do not limit de-
tection of biomolecule adsorption.
Fig. 2 Signals arising from: (a) changes in refractive index, (b) non- Specific and non-specific binding of protein
specific adsorption of calf serum, and (c) specific binding of
thrombin, with subsequent surface regeneration. One of 96 regis-
tered curves is given as an example Non-specific binding was examined with calve serum,
bovine albumin, and chicken ovalbumin. Because of the
shield effect of the aminodextran coating, non-specific
with the property of swelling in aqueous solution. To ob- binding of bovine albumin and ovalbumin could be suc-
tain results unaffected by the swelling processes the plates cessfully prevented. Even at high protein concentrations
were equilibrated with water for at least 20 min before the of 1 mg mL1 non-specific binding signal vanishes in the
start of liquid measurements. For both types of plate re- baseline noise. Only with calve serum at very high con-
Fig. 4 Normalized signals for glycerine solution (a) and for the For the specific binding of thrombin, the plates were
specific binding of thrombin in a 96-well microplate (b) and in a covalently modified with a thrombin inhibitor. Thrombin
384-well plate (c)
concentration was 6 units mL1. A single binding curve
for a 96-well plate is shown in Fig. 2c. The binding of
thrombin could be detected with sufficient time resolution
centrations (1:10 diluted in water) non-specific binding and the surface was completely regenerated with hydro-
was detected. One binding curve for a 96-well plate is chloric acid. The sensor surface was not affected by the
shown in Fig. 2b. The high protein concentration results a regeneration solution and stayed stable for more than
rapid coverage of the surface by protein absorption. Such 80 measurement cycles. Because the detected signals rep-
non-specific interaction often is characterized by irre- resent changes in the optical thickness, they are indepen-
versible binding. We found, in fact, that the baseline sig- dent of the size of the microplate wells and signals from
nal in Fig. 2b could not be reached again. This showed 96-well plates can be compared with those from 384-well
that the non-specific interaction of calve serum could not plates without further conversion. Calibrated binding
be regenerated under acidic conditions under which spe- curves for both types of plate are shown in Figs 4b and 4c.
cific thrombin binding could be regenerated. Each well was filled with a thrombin solution of 6 units
mL1. With few exceptions the signals are very homoge- binding signal or the calibrated binding signal, as is ap-
neous for both types of plate and enable well to well com- parent from Fig. 5. Depending on the criteria for hit iden-
parison. The system is, therefore, suitable for binding-in- tification 86 (50% criteria) or 87 (86% criteria) of the
hibition high-throughput screening assays. 88 inhibitor samples were identified on the 384-well
The better results from the 96 plates are a consequence plate. As mentioned above, false negative hits are most
of the lower signal noise, because of the greater well area. problematic in drug screening. The higher number of false
Signal derivation results mainly from the manual surface negative hits should, therefore, be accepted and the more
modification chemistry and might be prevented by an au- generous 60% criterion should be used so that as few hits
tomated preparation process; this is recommended when as possible are missed.
many plates are needed for screening of whole substance In addition to the experiments with the thrombstop-
libraries. analogous thrombin inhibitors, three other inhibitors of
unknown structure were examined; the results confirmed
the high assay stability. Screening was also performed
Screening experiments with 5% DMSO added to the samples. No influence of
DMSO was detected. Because library substances are nor-
To show the suitability of the parallel RIfS system throm- mally stored in DMSO, stability of the assay against
bin binding was examined for an unknown number of DMSO is essential even when a stabilisation of the assay
samples spiked with thrombin inhibitors. The results of compounds is not necessary.
this screening are shown in Fig. 5. For the 384-well plate
signals for every fourth microplate position are shown.
Samples without inhibitor give binding signals of more Evaluation of IC 50 value
than 0.3 nm whereas samples with inhibitor added re-
In addition to the determination of hit compounds, quanti-
sulted in signals <0.2 nm. For most of the positions, the
tative results such as IC50 value or binding constants of
inhibited samples could be re-found even without a cali-
drug candidates are of interest. The stability and reliabil-
bration step. The few curves which resulted in signals
ity of binding signals with the parallel RIfS-screening sys-
which could not be attributed with certainty could be as-
tem enable such examination. A binding inhibition test
signed after performing the standard calibration presented
was performed to determine the IC50 value of the thromb-
above. These results are shown in Fig. 5 as bar chart. The
stop-analogous thrombin inhibitor (Fig. 6). In this assay
samples with additional thrombin inhibitor are plotted in
black. For samples without inhibitors binding signals of
100% were expected whereas inhibited thrombin samples
should result in obviously distinguishable smaller signals.
For both plate types, nearly all of the pure thrombin sam-
ples gave binding signals of 80120% whereas the sam-
ples incubated with inhibitors mostly gave signals of
<40%. Re-finding rates are given in Table 2. To identify a
hit, two criteria of 40% and 50% signal inhibition were
stipulated. Except for two false-positive predictions, all
samples are classified correctly on the 96-well plate in
particular all the inhibitor samples were identified as hits,
which means that no false negative hits were found. This
is of primary importance, because each false negative hit
leads to the loss of a potential drug candidate. False posi-
tive hits in the 96-well plates occurred in wells in which
surface damage as a result of the manual surface coating
had previously been detected. In summary, the 96-well
plate gave absolutely stable and reliable results for the in-
hibitor screening. Fig. 6 Determination of the IC 50 value of a thrombin inhibitor by
The smaller well-size of the 384-well plates resulted in simultaneous detection of 96 different samples within less than
a higher signal noise but did not affect either the absolute 30 min. Inset, the structure of the thrombstop analogue inhibitor
147
the IC 50 value represents the concentration at which half Acknowledgements Part of this work was funded by BMBF pro-
of the original binding signal was obtained. Thrombin ject Librarian II/0310838. Oliver Birkert was supported by the
DFG-Graduiertenkolleg Quantitative Analyse und Charakterisie-
samples (20 g mL1) were incubated with inhibitor at rung pharmazeutischer und biochemisch relevanter Substanzen at
concentrations of 2109 mol L1 to 2107 mol L1. Each the University of Tbingen and by the Fonds der Chemischen In-
sample concentration was detected eightfold and simulta- dustrie.
neously in one 96 RIfS microplate. After data calibration,
an IC 50 value of 50 nmol L1 could be determined from
the binding inhibition curve for this thrombin concentra- References
tion (Fig. 6).
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Molec Design 12:441449
2. Hardcastle A, Boxall C, Newbatt Y, Rowlands MG, Turlais F,
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3. Zhang R, Beyer BM, Durkin J, Ingram R, Njoroge FG, Wind-
sor WT, Malcolm BA (1999) Anal Biochem 270:268275
We have reported the development of a new binding assay 4. Mellentin-Michelotti J, Evangelista LT, Swartzman EE, Mi-
to screen for thrombin inhibitors with HTS methods. We raglia SJ, Werner WE, Yuan P-M (1999) Anal Biochem 272:
used a new, parallel, label-free detection method based on 182190
the RIfS technique. With label-free optical methods it is 5. Seethala R, Menzel R (1999) Anal Biochem 253:210218
6. Lynch BA, Loiacono KA, Tiong CL, Adams SE (1997) Anal
not necessary to wait for equilibrium binding to determine Biochem 247:7782
the binding characteristic [17]. This enables shorter assay 7. Brock R, Vamosi G, Vereb G, Jovin TM (1999) Proc Nat Acad
times. Although we used binding times of 900 s, notice- Sci USA 96:1012310128
able and evaluable signals are available after <250 s. 8. Grahn S, Kurth T, Ullmann D, Jakubke H-D (1999) Biochim
The quality of screening data for 96- and 384-well Biophys Acta 1431:329337
9. Haake H, Schtz A, Gauglitz G (2000) Fresenius J Anal Chem
plates are excellent and promise successful further paral- 366:576585
lelisation on denser plates, e.g. 1536-well plates. The 10. Myszka DG, Rich LR (2000) Pharm Sci Technol Today 9:310
imaging system based on a CCD camera is not restricted 317
to a certain plate density and enables much denser plates 11. Rothmund M, Schutz A, Brecht A, Gauglitz G, Berthel G, Graefe
D (1997) Fresenius J Anal Chem 359:1522
with marginal changes in the evaluation software. Be- 12. Piehler J, Brecht A, Hehl K, Gauglitz G (1999) Colloids Sur-
cause the signal does not depend on the well area and the faces B 13:325336
noise can be reduced by use of a high pixel-to-well ratio 13. Klingler J, Friedrich T (1997) Biophys J 73:21952200
in the CCD camera, even a chip-based arrangement can 14. Piehler J, Brecht A, Gauglitz G (1996) Biosens Bioelectron
11:579590
be realised. The throughput of the system presented is 15. Gauglitz G, Brecht A, Kraus G, Nahm W (1993) Sens Actua-
high enough to fulfill the specification of HTS and even tors B 11:2127
ultra-HTS which demands screening of 100,000 samples 16. Schmitt H-M, Brecht A, Piehler J, Gauglitz G (1997) Biosens.
per day [18]. Bioelectron. 12:809816
Limitations of this new approach are the low sensitiv- 17. Edwards DA (1999) J Appl Math 63:89112
18. Koltermann A, Kettling U, Bieschke J, Winkler T (1998) Proc
ity and therefore the restriction on sample molecules with Natl Acad Sci USA 95: 14211426
a high molecular mass, e.g. enzymes or receptors. Small
molecules, which are of main interest in drug discovery,
can be examined with an indirect binding-inhibition test.
Anal Bioanal Chem (2002) 372 : 148154
DOI 10.1007/s00216-001-1190-x
O R I G I N A L PA P E R
R. M. Lzaro J. J. Garca A. Pi
Received: 3 September 2001 / Revised: 19 October 2001 / Accepted: 23 October 2001 / Published online: 13 December 2001
Springer-Verlag 2001
Abstract Normal values of the parameters of insulin re- Abbreviations N-AA High-affinity receptor number
ceptors in erythrocytes were provided to make a control per erythrocyte Ka-AA affinity constant of high-affinity
group throughout gestation, 24 h postpartum and six weeks receptors %Bo maximum specific insulin binding
after delivery with the aim of comparing them with other
parameters with insulin receptor-related pathologies. Thus,
one of the purposes of this study was the update of a Introduction
method to calculate the parameters of insulin receptors in
erythrocytes, carrying out several modifications that im- Human pregnancy is accompanied by profound changes
proved the assay: during binding studies incubation was in carbohydrate and lipid metabolism [1, 2, 3, 4]. Although
in continuous rotation shaking, and increasing maximal plasma insulin levels rise during pregnancy [2], glucose
insulin binding. Other modifications were the increase in tolerance is reduced [5], which suggests a loss in the re-
the concentration of insulin 125I to 1 ng/mL, and the max- sponsiveness of insulin-sensitive tissues [6]. The mecha-
imal concentration of unlabeled insulin, 100 ng/mL. Eryth- nism that mediates this altered sensibility would be located
rocyte age was considered by the intracellular creatine at the insulin binding level to its receptors, beginning with
content providing control values and allowing the normal- receptor activation through ligand binding, which facili-
ization of the parameters of insulin receptor during gesta- tates receptor dimerization and autophosphorylation of
tion. Data obtained in this study indicated that changes at specific tyrosine residues in the cytoplasmic portion. The
receptor binding level may be also considered to explain phosphotyrosine residues either enhance receptor catalytic
insulin resistance: week 28 showed maximal insulin secre- activity or provide docking sites for downstream signaling
tion (16.701.44 U/mL) whereas plasma glucose concen- proteins [6, 7, 8, 9, 10]. Although skeletal muscle, liver
trations remained almost constant (91.142.37 mg/100 mL) and adipose tissue are the major target tissues for studies
with respect to the 1st and 2nd trimester of pregnancy on insulin receptor binding, ethical reasons limit the avail-
(89.731.38 and 91.712.10 mg/100 mL respectively); ability of cells from these tissues, particularly for longitu-
insulin reached the point of maximal resistance, which is dinal studies in pregnant women. Therefore, most studies
explained by a decrease of maximum specific insulin bind- were carried out on erythrocytes and monocytes. Both un-
ing, %Bo (6.320.51) at minimal values due to a decrease changed [11, 12, 13, 14] and increased [15] insulin bind-
of high-affinity receptor number per erythrocyte, N-AA ing to erythrocytes from normal pregnancies have been
(162) at minimal values. Moreover, the negative correla- reported in agreement [11, 13] or in conflict [16] with re-
tion between progesterone (31.20.2 ng/mL) and Ka-AA sults on monocytes. Moreover, monocytes need more blood
(r=0.71) could possibly be related to this maximal resis- volume for binding measurements, representing only a very
tance. small fraction of the whole blood cells. Finally, insulin re-
ceptors in erythrocytes exhibit many of the characteristics
Keywords Insulin binding to erythrocytes Insulin of other tissues but the reported results on insulin binding
receptor in pregnancy Insulin resistance in pregnancy to erythrocytes in pregnancy were controversial, due to the
different time points during pregnancy at which data were
collected, rendering the comparison of the various results
difficult. Furthermore, only a limited number of studies
R.M. Lzaro () J.J. Garca A. Pi have been performed longitudinally throughout gestation,
Departament of Pharmacology and Physiology,
University of Zaragoza, Domingo Miral, only one or two, and they did not allow reliable identifi-
s/n 50002, Zaragoza, Spain cation of the changes in insulin binding in erythrocytes
e-mail: rosamlg@yahoo.com throughout pregnancy. Therefore, the course of the serial
149
changes in parameters related to insulin binding to eryth- 10 mmol/L CaCl2, 50 mmol/L NaCl, 5 mmol/L KCl, 1 g/L bovine
rocytes has not yet been established. albumin. After centrifuging the cell suspension (400 g, 10 min,
4 C) the buffer was aspirated (with leukocytes) and the cell pellet
This study was designed to obtain a control group data resuspended in buffer G, adjusting the erythrocyte concentration to
of the parameters of insulin binding to its receptors in eryth- 4.4109 cells/mL (estimated in a STKS and DAX 72 of Technicon
rocytes during normal pregnancy. and Cell Coulter System 8000 of Baker Instruments). Leukocyte
contamination: 525.6SD 205.5
third trimesters (Fig. 3). Following oral glucose ingestion, Gestational hormones
plasma insulin increased significantly (p<0.05) [40], in the
third trimester with respect to the first one, 68.157.73 U/ Gestational hormones, 17--estradiol, progesterone, pro-
mL to 85.096.22 U/mL while glucose concentration did lactin and testosterone were studied with the aim of com-
not vary significantly, from 135.16.08 to 144.26.17 mg/ pleting the results of the control group of pregnant women,
100mL (p=0.29). Fasting plasma glucagon levels (Fig. 4) correlating them with insulin receptor parameters and then
increased from the 12th to the 20th week of gestation obtain some explanation about the resistance of insulin
(151.221.8 to 196.127.5 pg/mL) with statistical signifi- action at receptor level. Fasting concentrations of 17--
cance (p<0.05); it then decreased until week 28th (166.0 estradiol, prolactin and testosterone increased progres-
20.0 pg/mL) and increased progressively until it reached sively (Fig. 5) from the 12th to the 36th week of gestation,
the 36th week (172.624.8 and 20522.46 pg/mL to the whereas progesterone levels kept elevated and almost con-
32nd and 36th week respectively, p<0.05). This parameter stant throughout gestation, reaching the maximal value at
decreased in the postpartum (160.617.1 pg/mL) and in- week 20.
creased in the puerperium, reaching the highest values of
the study (272.839.9 pg/mL). In the same figure, fasting
plasma cortisol levels increased progressively from the 12th Creatine content in erythrocytes
to the 28th week of gestation (24.331.02, 33.821.54
and 41.021.45 g/mL corresponding to the 12th, 20th and Creatine concentration of erythrocytes increased progres-
28th week respectively, p<0.05) and decreased progres- sively (Fig. 6) from the 12th to the 36th week of gestation,
sively during the third trimester, postpartum and puerperium without changes in the postpartum, and decreased in the
(39.41.15, 35.411.52, 29.61.72 and 20.461.04 g/mL puerperium. The values obtained provided a control group
corresponding to the 32nd, 36th week, postpartum and to correct the values of the insulin receptor parameters de-
puerperium respectively). pending on erythrocytes age, using the formula proposed
by Muggeo [41].
152
rocytes decrease with cell aging [41, 54, 55, 56], so in this Moreover, at this point of the study, there were high val-
study, creatine concentration in erythrocytes was obtained ues of progesterone and the highest values of cortisol,
in all blood samples, with the aim of providing a control both well-known diabetogenic hormones.
group of this parameter during gestation, to correct the These modifications described with regard to Gambhir
values of the insulin receptor parameters depending on et al.s method have improved the development of the
erythrocytes age. technique and have facilitated the analysis of such a large
A critical point in this study was the method employed number of blood samples with minimum blood taking.
to determine insulin receptor parameters because it was
originally of Gambhir et al. [18], although several varia- Acknowledgements This work was supported by a fellowship re-
tions have been made. Buffer G was made according to ceived by Rosa Ma Lzaro from the Government of Aragn (Ref-
erence: BCM2590) and carried out at the Hormones Laboratory of
Gambhir et al. [18], but the solution with albumin was eas- the Hospital Clnico Universitario Lozano Blesa of Zaragoza.
ily contaminated, so it was of interest to prepare 500 mL
every 3 or 4 days without albumin, keeping it refrigerated,
extracting 100 mL and adding the albumin at the moment References
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Anal Bioanal Chem (2002) 372 : 155163
DOI 10.1007/s00216-001-1164-z
O R I G I N A L PA P E R
Abstract In a new approach to the characterization and A frequently used analytical method for the detection
quantification of metallothionein isoforms an on-line iso- of metal complexes in biological matrices is the coupling
tope-dilution method in combination with the coupling of of a high-resolution separation technique with an induc-
capillary electrophoresis (CE) to an inductively coupled tively coupled plasmamass spectrometer (ICPMS) as
plasmasector field mass spectrometer (ICPSFMS) is re- element-specific detector. High-performance liquid chro-
ported. Metallothionein (MT) isoforms are separated by matography (HPLC) is widely used as the separation tech-
CE and the elements Cu, Zn, Cd, and S are detected si- nique [2] but the application of capillary electrophoresis
multaneously by use of ICPSFMS in the medium resolu- (CE) to biomolecules is also attracting more interest [3].
tion mode. On-line isotope dilution is performed by con- The separation efficiency of CE is high, the sample re-
tinuous introduction of an isotopically enriched, species- quirement small, and the mechanism of separation is dif-
unspecific spike solution after the separation step. MT from ferent from that of HPLC. CE therefore complements
rabbit liver and a further purified MT-1 isoform were quan- HPLC as a separation technique for biomolecules. The
tified by determination of sulfur, and the stoichiometric coupling of CE to ICPMS results in additional element-
compositions of the metalloprotein complexes are charac- specific information about the metal complexes. Majidi
terized by determination of their sulfur-to-metal ratios. [4] recently reviewed progress in the development of
CEICPMS interfaces. The interface introduced by
Keywords Inductively coupled plasma mass Schaumlffel and Prange in 1999 [5, 6] has been com-
spectrometry Capillary electrophoresis Isotope dilution mercialized. The design solves the problem of detrimental
Metallothionein quantification effects of suction by the nebulizer on the CE-separation
and thus the migration times of the analytes are both sta-
ble and reproducible. Work with CEICPMS can, there-
Introduction fore, now focus on optimization of the CE separations and
on application to real samples [7]. Recent publications have
In the field of bio-inorganic analytical chemistry the dealt with the application of CEICPMS to biomolecules,
analysis of metal compounds in living organisms to en- especially to the characterization of metallothioneins [8, 9].
able better understanding of their biochemical behavior Metallothioneins (MT) are cysteine-rich proteins of
has become an area of growing interest. In biological sys- low molecular mass (67 kD) that occur in animal and hu-
tems metals are usually coordinated to bioligands with man cells such as the liver, kidney or brain. They have a
high complexity, e.g. proteins or peptides [1]. variety of physiological functions, e.g. transport, storage,
and detoxification of metals or protection from metal tox-
icity. MT are involved in the metabolism of essential met-
als or act as free-radical scavengers. Four MT isoforms
D. Schaumlffel A. Prange () are known and several subisoforms have been discussed.
GKSS Research Center,
Max-Planck-Strae, 21502 Geesthacht, Germany The amino acid chain includes 21 covalently bonded sul-
e-mail: andreas.prange@gkss.de fur atoms: 20 are bound to the cysteine and one to the
G. Marx K.G. Heumann
N-terminal methionine [10]. The twenty sulfhydryl
Institute of Inorganic Chemistry and Analytical Chemistry, groups of the cysteines can coordinate seven bivalent ions
Johannes Gutenberg-University Mainz, 55099 Mainz, Germany such as Zn2+ or Cd2+, or twelve monovalent ions, e.g. Cu+
P. Brtter
[11].
Hahn-Meitner-Institute, There is still a lack of analytical methods for the quan-
Glienicker Strae 100, 14109 Berlin, Germany tification of MT with high precision, accuracy, and iso-
156
Stock solutions of the enriched isotopes 34S, 65Cu, 68Zn, and was realized as an important precondition for IDMS analysis. The
116Cd were provided courtesy of the group of Professor Heumann concentrations of the enriched isotopes were optimized to obtain
(Johannes Gutenberg-University Mainz, Germany). The isotopic isotope ratios in the range 0.12 in the isotope-diluted samples, to
composition of these spike solutions was 34S=85.9%, 65Cu=99.7%, keep the statistical error of the IDMS result low [21].
68Zn=91.0%, and 116Cd=83.4%. The species-unspecific spike solu-
optimum isotope ratio for 32S/34S in the range of 0.12 with two calibrations of the mass flow of the spike solu-
was achieved over the whole electropherogram (Fig. 3b). tion. The average amount of sulfur was 0.460.05 ng. This
Because of the 32S background values of the system, the could be used to calculate the absolute quantity of rabbit
measured isotope ratio for 32S/34S at the base line in Fig. 3b liver MT-1; this was 4.69 ng, assuming an average molec-
differed from the theoretical isotope ratio given by the ular mass of 6870 D for MT (obtained from Ref. [8]). This
isotopic composition of the spike solution. resulted in an MT-1 concentration of 2.13 mg mL1 for an
Simultaneously, the metal isotopes 63Cu, 65Cu, 64Zn, injection volume of 2.2 nL.
68Zn, 114Cd and 116Cd were monitored during the elec- The amounts of the metals in MT-1 were quantified by
trophoresis of MT-1. Figure 4 shows the isotope ratios for the same procedure, and found to be 0.430.07 ng Cd and
the metals. At the MT-1 peak mainly Cd and Zn were de- 0.040.007 ng Zn. Thus the stoichiometric composition
tected; peaks 5 and 6 in the Cu electropherogram (Fig. 4c) of the MT-1 complex could be characterized by calculat-
were before and behind the MT-1 peak. ing the molar ratio of sulfur to metal, normalized to 21 sul-
By applying Eq. (2) the isotope ratio electropherogram fur atoms per MT molecule. This resulted in the rounded
of MT-1 for S, Cu, Zn and Cd was converted into a mass- ratios S:Cd:Zn=21:6:1 for MT-1. As a consequence it
flow electropherogram (Fig. 5), where the Cu mass flow could be stated that the rabbit liver MT-1 complex inves-
was very low. Integration of the MT-1 peak in the sulfur tigated in this preparation could be characterized as
mass flow electropherogram resulted in the amount of sul- Cd6Zn1-MT-1.
fur. Three independent measurements were performed
160
Analytical characteristics of the MT-1 determination deviation (RSD) of the peak area of MT-1 measured with-
out isotope dilution. In an independent experiment using
From the measurements described above, the limits of de- the same CEICPMS system the precision of the peak
tection could be estimated as 6.8 g mL1 for sulfur and area was determined as 9.9% [7]. The comparison indi-
72.6 g mL1 for MT-1, in accordance with the 3 crite- cates that the main source of error in the reproducibility
rion. Note that 95% of the transmission is lost in lies in the electrophoresis, including sample injection and
ICPSFMS when operating in the medium resolution not in the isotope-dilution technique, where more precise
mode; this strongly influences detection limits. results are expected [21]. To improve the precision of the
The precision of the MT-1 quantification was 11.3%. peak area an internal standard will be used in future in-
This value must be compared with the relative standard vestigations. The application of HPLC as an alternative
161
Fig. 5 Mass-flow electrophe-
rograms of S, Cd, Zn, and Cu
for MT-1 (offset Cd: 0.015,
Zn: 0.0075, Cu: 0.001 for better
view)
separation technique might also result in better precision, trast, the percentage of Cu, Zn, and Cd in MT-1 measured
but in comparison with HPLC capillary electrophoresis by CEICPIDMS matched the TXRF results and also the
has better separation efficiency for MT isoforms even values specified by SigmaAldrich. Although the preci-
subisoforms can be separated. sion of the TXRF measurement was only approximately
30% for these determinations, the results suggested that
the total element contents were indeed represented by the
Mass balance of the MT-1 determination peaks in the mass flow electropherogram.
Conclusions
Fig. 6 Electropherograms of S, Cd, Cu, and Zn of a rabbit liver The coupling of capillary electrophoresis with ICPMS is
MT preparation; given in moles (offset Cd: 0.6, Cu: 0.11 for better suitable for application of an on-line isotope dilution tech-
view)
nique. This enables quantification of elements in unknown
species after CE separation, which can be used to investi-
peak heights instead of the peak areas has the advantage gate metallothionein isoforms. Because of the known
that even peaks that are not fully resolved, such as peaks number of 21 sulfur atoms per MT molecule, the sulfur
4 and 5 in Fig. 6, can be evaluated. isotope-dilution technique is a promising approach to the
The electropherogram in Fig. 6 shows a complex mix- quantification of MT isoforms after CE separation. Addi-
ture of different MT isoforms. Peak 4 and 5 were identi- tional measurements of the metals Cu, Zn, and Cd then
fied as MT-1 and peak 6 as MT-2 by comparison with the determination of the sulfur-to-metal ratio in MT, enables
electropherograms of purified MT-1 and MT-2 prepara- characterization of the stoichiometric composition of the
tions [7]. Peak 5 in Fig. 6 (MT-1) corresponds to peak 3 in metalloprotein complexes.
Fig. 5 and peak 6 in Fig. 6 (MT-2) to peak 4 in Fig. 5. MT-1 The quantification method presented is limited to pro-
and MT-2 in Fig. 6 have longer migration times than in teins such as MT, where the sulfur stoichiometry is known.
Fig. 5. The migration times are shifted in Fig. 6 because of For unknown compounds additional structural informa-
the larger amount of matrix present in the MT preparation tion, which can be obtained, e.g., from electrospray tan-
in comparison with the further purified MT-1 preparation. dem mass spectrometry (ESIMSMS), is necessary.
The MT isoform belonging to peak 3 has not yet been Further investigations will be performed to improve
identified. Further investigations with molecule-specific the sensitivity of sulfur measurements by the use of an
detectors such as electrospray MSMS will therefore be octapole reaction system (ORS) in combination with an
necessary to identify this peak. Peaks 1 and 2 are probably ICPquadrupole MS and the use of more highly enriched
34S and 33S spikes. Details of the application of the
degradation products, because their intensity increases
with sample age. The quantity of sulfur of the species was method to MT determination in animal and human tissues
obtained from the mass-flow electropherogram; the re- will be published separately in the near future.
sults were 8.40.6 ng and 3.80.3 mg mL1 for MT-1 and
5.50.4 ng and 2.50.2 mg mL1 for MT-2. The sum of
163
Acknowledgement The authors thank Dipl. Ing. Burkhard Erbslh 16. Kgi JHR, Himmelhoch SR, Whanger PD, Bethune JL, Vallee
for performing the TXRF measurements. BL (1974) J Biol Chem 249:3537
17. Nordberg GF, Nordberg M, Piscator M, Vesterberg O (1972)
Biochem J 126:491
18. Minami T, Tohno Y, Okazaki Y, Kubo K, Otaki N, Kimura M
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7. Prange A, Schaumlffel D, Brtter P, Richarz A-N, Wolf C 9:13511355
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9. Taylor KA, Sharp BL, Lewis DJ, Crews HM (1998) J Anal At Garca Alonso JI, Sanz-Medel A (1999) J Anal At Spectrom
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11. Stillman MJ (1995) Coord Chem Rev 144:461511 Mainz
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13. Eaton DL, Toal BF (1982) Toxicol Appl Pharmacol 66:134 At Spectrom 13: 10011008
142 28. Main MV, Fritz JS (1989) Anal Chem 61:12721275
14. Vandermallie RJ, Garvey JS (1979) J Biol Chem 254:8416 29. Timerbaev AR, Semenova OP, Bonn GK, Fritz S (1994) Anal
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Anal Bioanal Chem (2002) 372 : 164168
DOI 10.1007/s00216-001-1126-5
O R I G I N A L PA P E R
Abstract Miniaturized fiber-in-tube solid-phase extrac- troscopy (FTIR), mass spectroscopy (MS) and inductively
tion (fiber-in-tube SPE) has been developed as a solvent- coupled plasma spectroscopy (ICP), was developed and
less sample preconcentration technique for microcolumn specific detection with high sensitivities was accom-
liquid-phase separation methods. Short capillaries packed plished by these hyphenated systems. The combination of
with polymer filaments were employed as the extraction a specially designed stationary phase and a highly sensi-
tube and the preconcentration power for phthalates in tive/selective detector made it possible to analyze com-
aqueous solutions was studied. On the basis of the suc- plex sample mixtures.
cessful on-line coupling of this preconcentration method In contrast with many successful applications of mi-
with liquid chromatography (LC), a more miniaturized cro-scale liquid-phase separation techniques with high-
extraction cartridge, which is installed in the rotor of the performance separation and detection, however, the re-
micro-injector, has been developed. With a modified com- ports for this miniaturized sample preparation method,
mercially available valve, on-line coupling of this sample which was specially designed for these separation systems
preconcentration method to capillary electrochromatogra- [3, 4, 5], have been somewhat limited because of the dif-
phy (CEC) was also investigated. ficulties associated with on-line coupling. On the basis of
the previous results from the solid-phase microextraction
Keywords Fiber-in-tube solid-phase extraction (SPME) technique of Pawliszyn et al. [6, 7, 8, 9, 10, 11,
Miniaturization Sample preparation Microcolumn 12, 13, 14] and Jinno et al. [15, 16, 17, 18, 19, 20, 21, 22]
liquid chromatography Capillary a novel miniaturized sample preparation method [23] was
electrochromatography recently developed. In the newly developed method fiber-
in-tube SPE, extraction has been carried out in short cap-
illaries packed with the filaments of synthetic polymer as
Introduction the extraction medium. The results have shown that the
effective contact of the sample solution with several hun-
The development of miniaturized analytical systems has dreds of the fine fibrous extraction media in the extraction
been one of the most important projects in analytical tube could provide miniaturization in this micro-scale
chemistry because it promises a solution to recent require- sample preconcentration device. Down-sizing of the ex-
ments and rapid analysis with low operation cost, but also traction device will also permit direct coupling of the ex-
without environmental pollution. As a typical example of traction process to microcolumn separation methods with-
the miniaturization of separation systems, microcolumn out any disadvantages such as over-load injection and
liquid chromatography (micro-LC) and related techniques poor resolution during separation.
have been widely investigated [1, 2]. Taking the advan- As an extension of the previous work [23], miniatur-
tage of the low volumetric flow-rate of the mobile phase, ized fiber-in-tube SPE has been developed as a solvent-
on-line coupling of micro-LC to various spectroscopic de- less sample-preconcentration technique for microcolumn
tection systems, such as Fourier transform infrared spec- liquid-phase separations, such as micro-LC and capillary
electrochromatography (CEC). For preparation of the ex-
traction tube a short capillary of polyetheretherketone
(PEEK) or polytetrafluoroethylene (PTFE) was packed
Y. Saito M. Imaizumi T. Takeichi K. Jinno () longitudinally with fine polymer filaments having both
School of Materials Science,
Toyohashi University of Technology, solvent resistance and mechanical strength. The extraction
Toyohashi 4418580, Japan cartridge of fiber-in-tube SPE was incorporated in an in-
e-mail: jinno@chrom.tutms.tut.ac.jp jection valve and used as the sample loop itself. Desorp-
165
Experimental
Reagents and materials
Zylon fiber (HM type, Fig. 1) was obtained from Toyobo, Ohtsu,
Japan. All solvents were of analytical grade and obtained from
Kishida Chemical, Osaka, Japan. Dimethylphthalate (DMP), di-
ethylphthalate (DEP), di-n-propylphthalate (DPP), di-n-butylph-
thalate (DBP), and aromatic hydrocarbons were purchased from
Tokyo Chemical Industries, Tokyo, Japan. All PEEK and PTFE
tubing, and fused-silica capillaries were obtained from GL Sci-
ences, Tokyo, Japan. Water was purified by use of a Milli-Q Wa-
ter Purification System (Millipore, Tokyo, Japan). Fig. 2 Miniaturized fiber-in-tube SPEmicro-LC system. The ex-
traction was carried out in the extraction tube between the switch-
ing valve (model 7000, Rheodyne, Cotati, CA, USA) and the in-
Preparation of the extraction tube for fiber-in-tube SPE jection valve (model 7520, Rheodyne)
Fig. 4 On-line coupling of miniaturized fiber-in-tube SPE and and time for the extraction and desorption, were deter-
CEC separation system mined in preliminary experiments. For the fiber-packed
tube of 0.25 mm i.d.24 mm length (Table 1), the extrac-
from the valve to the cathodic vial was 200 mm) and the window
tion flow-rate was 16 L min1 and desorption was per-
for on-column detection was made just after the mid-frit. A pre- formed at a flow-rate of 2 L min1 for 4 min. The injec-
column capillary of 0.25 mm i.d.150 mm length was connected to tion volume was 1 L, and the mobile phase was pumped
the valve port on the other side, to supply the mobile phase from at 4 L min1 for the micro-LC analysis. The preconcen-
anodic vial during the CEC separation. A PTFE tube from a sy- tration factor of this method was calculated by injecting
ringe pump (Microfeeder MF-2, Azumadenki Kogyo, Tokyo,
Japan), to deliver the sample solution during the extraction the standard solution in a conventional way, and was esti-
process, and a short fused-silica capillary (0.030 mm i.d.50 mm mated as about 101 for DBP with the extraction efficiency
length), for waste, were connected to the other two ports. of 21%. The volume of solvent needed for each analysis
was about 60 L.
Data processing No desorption solvent was needed for the extraction
device shown in Fig. 3. The extraction cartridge was in-
For data acquisition and processing, Borwin chromatography data- corporated in the rotor of the micro-injection valve. The
handling software (Jasco) running on a personal computer was cartridge was prepared with a PEEK tube of 0.50 mm
used. All measurements were carried out at least in triplicate and i.d.5.0 mm. Two pieces of blank PEEK tubes, 0.25 mm
the relative standard deviations (RSDs) were less than 4% for all
runs. i.d.1.0 mm length, were employed to retain the fiber-
packed cartridge in the rotor. Furthermore, the modifica-
tion was made with a small piece of PEEK tubing to re-
Results and discussion duce the void volume of the remaining flow-pass in the
valve. During the extraction a certain volume of sample
First, the system shown in Fig. 2 was employed for the solution was pumped by a syringe at the appropriate flow-
analysis of phthalates. The extraction capillary was placed rate. The analytes of interest were adsorbed onto the sur-
between the switching valve and the injection valve, and face of the filaments packed in the extraction tube.
the extraction was carried out as follows. First, an aque- Changing the position of the injector the desorption was
ous sample solution was delivered to the extraction tube carried out by the flow of the mobile phase. As shown in
by means of a syringe pump, and the analyte was ad- Table 1, the total number of packed-filaments was about
sorbed onto the fibers. Next, a certain amount of the des- 1500, and the void volume of the fiber-packed injection
orption solvent was also pumped after changing the valve was about 0.31 L. Figure 5 shows the typical chro-
switching valve. At the same time the injector was matogram obtained with an extraction cartridge of 5-mm
switched to fill the sample loop with the effluent from the length. By this in-valve configuration, the further
extraction tube. The injection was made just after sample miniaturization was accomplished as well as the solvent-
loading. All operational parameters, such as the flow-rate less sample preparation method for micro-LC separation.
167
Table 2 Preconcentration factor and extraction efficiency for di-
n-alkylphthalates [Ph(COOR)2] with fiber-in-tube SPECEC sys-
tem
Preconcentration Extraction
factor efficiency (%)
R=CH3 31.5 11.5
R=C2H5 38.8 14.2
R=n-C3H7 56.5 20.6
R=n-C4H9 80.2 29.4
The total volume of the solvent required, including the relationships between the polarity of the solutes and pre-
volume of the mobile phase, was reduced to about 40 L concentration factor has been carried out. The plot in
for each analysis in this system. Fig. 7 clearly indicates the linear relationship between log
On the basis of successful direct coupling of the fiber- P (where P is the 1-octanol/water partition coefficient)
in-tube SPE to micro-LC separation, the application of [24] and the preconcentration factor, with a correlation
this extraction technique to sample preparation for CEC coefficient (r) of 0.990. The results are in good agreement
analysis was also investigated with the system shown in with our previous results from use of the fibrous packing
Fig. 4. As described in the Experimental section, the fiber- materials as the stationary phase in LC [25, 26] and CEC
packed cartridge was made with a 5-mm piece of PTFE [27, 28, 29], in which the elution order from the fiber-
tubing (0.25 mm i.d.), and the tubing was incorporated packed column is the same as for conventional reversed-
into the rotor of a small 2-way valve. Figure 6 depicts a phase packings.
typical chromatogram for the analysis of phthalate mix-
tures with fiber-in-tube SPECEC system. Although neg-
ative baseline drift, probably due to the injection of a wa- Conclusions
ter plug, was observed, a good separation was obtained.
No solvent was needed for the sample preparation, and A novel miniaturized sample-preparation method has
the total usage of organic solvent (as the mobile phase been developed with a short capillary and fine fibrous
component) for each analysis was about 2.5 L. The pre- packing materials as extraction media. Incorporating the
concentration factor was calculated based on comparison extraction cartridge into the injection valve, a solventless
of the peak areas with fiber-in-tube SPE preconcentration sample preparation technique was realized for the analysis
and that without the preconcentration procedure. In the of water samples by microcolumn liquid-phase separation
latter case, the injection was made by pumping the stan- methods. The results also show the possibility of new fi-
dard sample prepared as the methanol solution, because in brous extraction media having different chemical struc-
the preliminary experiments no preconcentration effect tures and/or coated with other materials, which are spe-
was observed with the methanol as the sample solvent. cially designed with the concept of molecular shape
Table 2 shows the calculated preconcentration factor and recognition [30].
the extraction efficiency. The results demonstrated that The applications of polymer-coated fibrous materials,
good preconcentration was obtained only with a short not only as the sample-preparation media but as the sta-
fiber-packed tubing, and a better preconcentration effect tionary phase materials for various chromatographic sepa-
was also observed for less-polar solutes. Analysis of the ration techniques, such as GC or supercritical-fluid chro-
168
matography (SFC), are currently being investigated in our 13. Kataoka H, Narimatsu S, Lord HL, Pawliszyn J (1999) Anal
laboratory along with the miniaturization of these tech- Chem 71:42374244
14. Kataoka H, Pawliszyn J (1999) Chromatographia 50:532538
niques. 15. Jinno K, Muramatsu T, Saito Y, Kiso Y, Magdic S, Pawliszyn
J (1996) J Chromatogr A 754:137144
Acknowledgements A part of this research was financially sup- 16. Jinno K, Han Y, Sawada H, Taniguchi M (1997) Chro-
ported by Grant-in-Aids for Scientific Basic Research (C (2), No. matographia 46:309314
12640586) and for Young Scientists (A, No. 13740421) from 17. Jinno K, Taniguchi M, Hayashida M (1998) J Pharm Biomed
Japan Society for the Promotion of Sciences. The authors would Anal 17:10811091
like to thank Toyobo Co. Ltd. for the donation of Zylon fiber. YS 18. Jinno K, Sawada H, Han Y (1998) Biomed Chromatogr
also thanks Mr Yutaka Ohtsu (Basic Research Center, Shiseido, 12:126127
Yokohama, Japan) and Dr Norikazu Nagae (Nomura Chemical, 19. Jinno K, Kawazoe M, Hayashida M (2000) Chromatographia
Seto, Japan) for providing stationary phases. 52:309313
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mance liquid chromatography. VCH, New York, NY, USA
(2000) Fresenius J Anal Chem 368:641643
2. Yang FJ (1989) (ed) Microbore column chromatography,
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hydrophobic, electronic, and steric constants. ACS Profes-
York, NY, USA
sional Reference Book, American Chemical Society, Washing-
3. Yang Q, Tomlinson AD, Naylor S (1999) Anal Chem 71:
ton, DC, USA
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25. Kiso Y, Jinno K, Nagoshi T (1986) J High Resol Chromatogr
4. Whang CW, Pawliszyn J (1998) Anal Commun 35:353356
Chromatogr Commun 9:763764
5. Fritz JS, Masso JJ (2001) J Chromatogr A 909:7985
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6. Chen J, Pawliszyn J (1995) Anal Chem 67:25302533
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27. Jinno K, Wu J, Sawada H, Kiso Y (1998) J High Resol Chro-
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8. Eisert R, Pawliszyn J (1997) Anal Chem 69:31403147
28. Jinno K, Watanabe H, Kiso Y (2001) J Biochem Biophys
9. Lord HL, Pawliszyn J (1998) LCGC 16:S41S46
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Anal Bioanal Chem (2002) 372 : 169173
DOI 10.1007/s00216-001-1162-1
O R I G I N A L PA P E R
Received: 20 August 2001 / Revised: 2 October 2001 / Accepted: 15 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract Solid-phase extraction has become one of the opment of reliable, practical, and sensitive methods for
most commonly used techniques for preconcentration of their monitoring and analysis in air and natural water.
analytes from environmental samples. In the standard use The most sensitive methods yet developed for the de-
of solid sorbent phases the extracted pollutants are subse- tection and determination of volatile organic pollutants
quently eluted with a suitable organic solvent before chro- usually include an extraction and concentration step, usu-
matographic analysis. An alternative to this procedure is ally on a solid phase, followed, after desorption or elution,
analysis of the adsorbed and concentrated pollutants by by chromatographic analysis. All these are laboratory meth-
direct application of a spectroscopic method (fluorimetry ods and require sophisticated instruments and complex
or absorptiometry) to the phase. Although this method can- operations and procedures if reliable results are to be ob-
not be expected to give results as precise as those given by tained, and it is usually difficult to adapt these methods
chromatographic methods, it might have valuable applica- for on-site pollution control. Although liquidsolid ex-
tions, particularly for on site pollution monitoring. This traction has benefited from important progress made in
paper reports an evaluation of the capability of the method the development of new solid adsorbents [1, 2], the proce-
for the spectrophotometric detection of BTX (benzene, dure entails an elution step with an organic solvent, the
toluene, xylenes) in aqueous media and in contaminated use of which is increasingly subject to legislative restric-
atmospheres, with polydimethylsiloxane (PDMS) as sor- tion.
bent. The tests performed, with the estimated detection Besides these conventional methods, several systems
limits, indicate that the method is relatively simple and and instruments have been proposed for on site monitor-
easy to operate and sensitive enough for application to the ing of atmospheric VOC pollutants [3, 4, 5, 6] or for wa-
monitoring of pollution both in water and in air in an in- ter monitoring [7, 8]; most of these systems require com-
dustrial ambient atmosphere. plex and heavy instrumentation, however, and although
they seem suitable for some specific applications, their sen-
Keywords Solid sorbents BTX Pollutants sitivity or selectivity do not always meet the requirements
Absorptiometry Fluorimetry imposed by legislation.
As an alternative approach, during the last ten years
there have been attempts to evaluate methods based on the
Introduction detection and/or analysis of hydrophobic organic pollu-
tants from aqueous media by application of a spectro-
Because of the widespread environmental occurrence of scopic method (absorptiometry or fluorimetry) directly on
volatile organic compounds (VOC), and particularly ben- a solid sorbent phase after extraction [9, 10, 11, 12, 13,
zenic-type compounds such as benzene, toluene, and 14, 15, 16, 17, 18] thus avoiding use of organic solvents
xylenes (BTX), there is continuous interest in the devel- for elution and chromatography. Although this method
cannot be expected to give results as precise as those ob-
tained by use of chromatographic methods, it has some
advantages it is, for example, simple to operate and suit-
M. Lamotte () P.F. de Violet P. Garrigues able for on site pollution monitoring.
Laboratoire de Physico-Toxico-Chimie, UMR CNRS 5472, Whatever the type of sorbent used, the analytical
Universit de Bordeaux 1, process comprises two steps:
351 Cours de la Libration, 33405 Talence, France
e-mail: m.lamotte@lptc.u-bordeaux.fr the adsorption and concentration step in which the hy-
M. Hardy drophobic pollutants are selectively adsorbed and con-
Supelco SigmaAldrich, 38298 Saint Quentin Fallavier, France centrated on/or in the solid phase.
170
Experimental
Results and discussion
Preparation of the PDMS phase
BTX detection in water
The PDMS phase (OV1 type from Supelco) was shaped into paral-
lelepiped blocks in a brass mold at reduced temperature (50 C).
Because of the size of the spectrophotometer analyzing beam and
The absorption kinetics of benzene at 100 mg m3 in wa-
because the analyte molecules are concentrated in a very thin depth ter, obtained by following the dependence of absorbance
from the surface [16], the size of the sorbent block was set at by the PDMS block on contact time, is shown in Fig. 1.
6 mm10 mm for the effective absorbing face and 2 mm for the The absorbance increases rapidly initially and then increases
thickness of the optical path. continuously but linearly after approximately 60 min for
The same PDMS block could be used several times by remov-
ing the adsorbed volatile analytes by means of a dry nitrogen benzene and after shorter delays for the other derivatives.
stream at room temperature. The use of a hot nitrogen stream at As a compromise between measurement time and signal
130 C, as applied by Wittkamp et al. [15], was avoided to mini- intensity, a contact time of 60 min was chosen for all mea-
mize any possible modification of the characteristics of the poly- surements.
mer upon heating.
Calibration curves for benzene and toluene are shown
in Fig. 2. Similar good linearity for the dependence of ab-
Procedure for BTX analysis in water sorbance on concentration was also obtained for p- and
o-xylenes; determination coefficients were >0.98. Figure 3
For analysis of BTX in water, test solutions (200 mL) were pre- illustrates the absorbance amplification effect occurring as
pared in a 250-mL stainless-steel container by adding 10 L of a
concentrated stock solution to water (Milli-Q Millipore). The stock a result of the extraction and concentration of the analyte
solutions were prepared in acetonitrile at suitable concentrations. molecules from the dilute water solution on to the adsor-
The PDMS attached to its holder was then immersed in the spiked bent block. A photometric enhancement factor (PEF) of ap-
aqueous solutions for a fixed period of time. After adsorption, the
PDMS block holder was removed and immediately placed in a reg-
ular fused silica cell (1 cm1 cm) in the presence of pure water
(Milli-Q water from Millipore) for absorbance measurement. Water,
instead of the normal atmosphere, was used to surround the adsor-
bent in the measurement cell for two reasons:
1. it prevents desorption of the adsorbed volatile analytes; and
2. because the refractive index of the PDMS used (n1.37) is
closer to that of water than that of air, it greatly improves the
optical transparency of the solid phase.
Fig. 2 Calibration curves showing the dependence of absorbance on at a concentration equal to approximately one third of the
the PDMS sorbent block on the concentration in water, for benzene estimated LOD.
(a) and toluene (b)
Depending on the optical quality of the phase, in prac-
tice the weakest absorbance of a spectrum that could be
proximately 80 was found for benzene. Larger PEF val- measured above background varied from 3 to 5104 ab-
ues, approximately 200 and 300, respectively, were found sorbance units. The limits of detection deduced from these
for toluene and p- and o-xylenes, in agreement with pre- values are indicated in Table 1.
vious results obtained by Wittkamp et al. [15] with a sim- The LOD estimated in this work are lower than those
ilar phase but different experimental conditions. reported previously. Our LOD for benzene is, neverthe-
Limits of detection were determined from the slopes of less, slightly higher than the concentration in tap water
the calibration curves and the lowest absorbance signal tolerated by law. It is, however, not very far from the con-
which could be detected. This signal is usually estimated centration limits in industrial effluents in Europe and very
as three times the mean peak-to-peak noise signal. Because close to the benzene concentration found in Perrier water
of the degradation of the optical properties of the PDMS in 1990 this was accidentally contaminated at levels rang-
block during the adsorption process, the LOD was, in ing from 12 to 20 mg m3 (1220 ppb).
practice, limited by the increase in the background signal For the other compounds, the estimated LOD are much
towards the long wavelength side of the spectrum, which lower than the recommended concentration limits and the
makes it difficult to measure the contribution to the total method seems to be suitable for monitoring these pollu-
absorbance of a weak spectrum. This is illustrated in Fig. 4 tants in most waters, including tap water.
where the absorption spectrum of benzene is reproduced
Table 1 Limits of detection (LOD), in mg m3 (ppb), estimated for monitoring of BTX in water by absorption on an OV1-type PDMS
block (thickness 2 mm) after contact for 60 min, and comparison with other data
BTX LOD LOD, other work Tolerated maximum Concentration limits Maximum values found
(mg m3) (mg m3) concentrations in tap water in cokeries (Europe) in tap water (USA,
(mg m3) (mg m3) Europe) (mg m3)
Benzene 1520 >50a 97b 5 (EPA), 1 (EEC), 10 (OMS) 332 0.3500
Toluene 46 >50a 10b 1000 (EPA), 500 (OMS) 375 14500
o-Xylene 24 >50a 7.8b 500 (OMS) 425440
p-Xylene 24 >50a 5.5b 500 (OMS) 425440
aUVspectroscopy by dynamic derivation of the spectra (from Ref. [19])
bUV spectroscopy on PDMS (from Ref. [15])
EPA: Environmental Protection Agency; OMS: Organisation Mondiale de la Sant; EEC: Economic European Community
was observed after 20 min of warming up when the cat- pected from use of specifically prepared PDMS with suit-
alytic system has attained its working temperature. able conditioning and shaping for optical application.
Conclusions References
The results presented above show that a direct spectro- 1. Hennion M.-C (1999) J Chromatogr A 856:354
2. Lika I (2000) J Chromatogr A 885:316
scopic detection on PDMS might constitute a method for 3. Mortgat B (1997) Environnement & Technique No 168, 2022
the detection of benzenic contaminants in water and pol- 4. Di Benedetto D, Breuil P, Marchand JC, Rousson D (1999)
luted atmospheres. Actes du 9ime Congrs International de Mtrologie. pp 245
The evaluation tests and the estimated detection limits 248
5. Mays P (1999) Environnement & Technique No 187, 4145
obtained for benzene, toluene and xylenes indicate that 6. Aragon P, Atienza J, Climent MD (2000) Crit Rev Anal Chem
the method is relatively simple and easy to operate and sen- 30:121151
sitive enough for application to pollution monitoring in 7. Stuetz R (2001) Int Lab 31:1820
water and in industrial air. 8. Thomas O, Theralaz F, Cerd V, Constant D, Quevauvillier Ph
(1997) Trends Anal Chem 16:419424
In this work the applicability of the method has essen- 9. Carr JW, Harris JM (1988) Anal Chem 60:698702
tially been evaluated on the basis of the limits of detection 10. Vilchez JL, del Olmo M, Avidad R, Capitn-Vallvey LF
which could be attained. The precision and repeatability (1994) Analyst 119:12111214
of the method for quantitative analysis have not been pre- 11. Pozomiek EJ (1991) Anal Lett 24:19131921
cisely determined. These characteristics are highly depen- 12. Eastwood D, Dominguez ME, Lidberg RL, Poziomek EJ (1994)
Analusis 22:305310
dent on the optical properties of the solid phase which in 13. Arruda AF, Campiglia AD (1999) Anal Chim Acta 386:271
turn are sensitive to chemical homogeneity, mechanical 280
stability with time, and optical transparency. The PDMS 14. Wittkamp BL, Tilotta DC (1995) Anal Chem 67:600605
material used in this work is specifically designed for chro- 15. Wittkamp BL, Hawthorne SB (1997) Anal Chem 69:11971203
16. Fornier de Violet P, Garrigues P, Tyteca M, Hardy M (1998)
matography, and not for spectrometric purposes. Accord- Forum Labo 98, mars 1998, CNIT PARIS
ingly it is not totally suitable for the application described 17. Algarra M, Lamotte M, Fornier de Violet Ph, Hernandez M,
in this work, particularly because of the instability of its Garrigues Ph (2000) Polycyclic Arom Compounds 19:241251
transparency. Despite this, calibration curves with deter- 18. Algarra M, Radin C, Fornier de Violet Ph, Lamotte M, Garrigues
Ph, Hardy M, Gillard R (2000) J Fluoresc 10:355359
mination coefficients >0.98 were always obtained, indi- 19. Vogt F, Tacke M, Bohmer W (1999) Spectrosc Eur 11:1218
cating that the method can reasonably be expected to be 20. Schwarzenbach RP, Geshwend PM, Imboden DM (1993) Envi-
sufficiently precise for quantitative evaluation. A substan- ronmental organic chemistry. Wiley and Sons, New York, p 619
tial reduction of the limits of detection and an improve- 21. Chiou RP, Schmedding DW (1982) Environ Sci Technol 16:
ment in the precision of the measurement would be ex- 410
Anal Bioanal Chem (2002) 372 : 174180
DOI 10.1007/s00216-001-1169-7
O R I G I N A L PA P E R
Received: 14 September 2001 / Revised: 22 October 2001 / Accepted: 24 October 2001 / Published online: 13 December 2001
Springer-Verlag 2001
Abstract A sensing system for nickel based on the nickel this metal enters the environment it becomes a potential
binding protein (NBP) from Escherichia coli is shown to health hazard to man and animals. Nickel is known to be
be feasible. The versatility of NBP was demonstrated by its toxic to both prokaryotic and eukaryotic organisms [1, 2].
use in three different assay formats. When the NBP binds Nickel particles present in the air can settle in the ground
nickel, it undergoes a conformational change that can be or be removed from the air by rain. As a result, a signifi-
used as the basis for an optical sensing system for nickel. cant amount of the metal can find its way into ground or
The NBP gene was overexpressed in E. coli and the pro- surface waters. Nickel toxicity can result in adverse health
tein purified in a single step using perfusion anion-ex- effects ranging from allergic dermatitis to lung and nasal
change chromatography. A unique cysteine residue at po- sinus cancers. Because of the hazard of environmental
sition 15 in the NBP was labeled with the fluorophore, nickel, the Environmental Protection Agency (EPA) has es-
N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3- tablished that children should not ingest drinking water con-
carboxamide (MDCC). In a spectrofluorimetric assay, there taining nickel at concentrations of more than 0.04 mg L1
was a maximum of 65% quenching of the fluorescence (6.8107 mol L1) [3].
signal produced by NBPMDCC in the presence of Currently, there are several analytical methods for the
nickel. A response curve for nickel using NBPMDCC detection of nickel. The Department of Natural Resources
revealed a detection limit of 8108 mol L1. NBPMDCC reports detection limits for nickel to be 1.7107 mol L1
was also used to develop assays in microtiter plate and with ICP and 3.4108 mol L1 using ICPMS [4]. These
fiber optic bundle formats. Detection limits for nickel us- traditional methods, including ICPAE spectrometry [5],
ing these formats were also in the submicromolar range. are sensitive enough to detect the metal at levels where it
Selectivity studies conducted with other divalent metals, is toxic to man; however, for field studies, where on-site
including copper, cobalt, iron, cadmium, and manganese, measurements are necessary, these methods are not prac-
showed that fluorescence quenching for cobalt was simi- tical. Determination of nickel via differential-pulse ad-
lar in magnitude but with a detection limit more than sorptive stripping voltammetry at a hanging mercury drop
10-fold higher than for nickel. The quenching responses electrode is a new technique that is sensitive to mol L1
were lower for the other metals, with detection limits at concentrations of the metal [6], but for purposes of
least 10 to 100 times higher than for nickel. These results screening for concentrations of nickel at which it is toxic
suggest that fluorescently labeled NBP is potentially use- to man (7107 mol L1), this method might not be a fea-
ful in the development of a sensing system for nickel. sible option. In designing a sensing system for nickel,
high sensitivity, rapid results, and adaptability of the sys-
Keywords Fluorescence-based sensor Environmental tem to miniaturization and use on-site are of critical im-
monitoring Nickel-binding protein portance.
Nickel is an important nutrient for Escherichia coli
Introduction (E. coli) and other microorganisms. It is an essential com-
ponent of a number of microbial enzymes, which make
Nickel is important in many industrial applications, e.g. in possible such reactions as ureolysis, methane biogenesis,
the making of stainless steel and other metal alloys. When acetogenesis, and hydrogen metabolism [7]. Because of
this, the bacterium has developed a sensitive recognition
and transport system for nickel that is responsible for
L.L.E. Salins E.S. Goldsmith C.M. Ensor S. Daunert () monitoring and regulating the concentration of nickel
Departments of Chemistry and Pharmaceutical Sciences,
University of Kentucky, Lexington, KY 405060055, USA within the cell. The protein components of the nickel-
e-mail: daunert@pop.uky.edu transport system in E. coli are encoded by the nik operon.
175
The amino acid sequences of the five overlapping nik sensitive and can potentially be miniaturized and used for
gene products are homologous to the proteins of peptide- on-site testing of water samples.
binding protein-transport systems in other Gram-negative
bacteria [8]. These five proteins together constitute a sys-
tem that not only enables the transport of nickel atoms be- Experimental
tween the periplasm and the cytoplasm but also makes
nickel chemotaxis possible. Bacterial strains and plasmids
The nikA gene codes for the periplasmic nickel-bind- Plasmid p8602, containing the nikA gene, and E. coli strain B834
ing protein (NBP), the nickel-binding component of the were provided by Dr Long-Fei Wu, at the Laboratoire de Chimie
system. The NBP of E. coli consists of 524 amino acids. Bacterienne, Marseilles, France.
The first 22 amino acids, which make up the signal pep-
tide necessary for transport into the periplasmic space, are Materials
cleaved off when the protein is transported from the cyto-
Luria-Bertani (LB) medium was purchased from GibcoBRL
plasm to the periplasm [9]. The molecular weight of the (Gaithersburg, MD, USA). Tris buffer ([tris(hydroxymethyl)ami-
mature protein is 56 kDa [8]. Quenching of intrinsic tryp- nomethane]) was obtained from VWR Scientific (S. Plainfield, NJ,
tophan fluorescence and equilibrium dialysis reveal that USA). Ampicillin and bovine serum albumin (BSA) were bought
NBP binds a single nickel ion per molecule of protein from Sigma (St Louis, MO, USA). Ethylenediaminetetraacetic
acid (EDTA), dithiothreitol (DTT), and all organic and inorganic
with a dissociation constant (Kd) <0.1 mol L1. This Kd salts were obtained from either Fisher Scientific (Fair Lawn, NJ,
value reflects high specificity for nickel, because other di- USA), VWR Scientific (S. Plainfield, NJ, USA), or Sigma. NBP
valent metal ions (Co2+, Cu2+, Fe2+) bind at least a factor expression was induced with isopropyl--D-thiogalactoside (IPTG),
ten less tightly [10]. A preliminary X-ray diffraction study purchased from GibcoBRL (Gaithersburg, MD, USA). The micro
of NBP achieved a maximum resolution of 3.0 [9]. Al- bicinchoninic acid (BCA) protein assay reagent kit from Pierce
(Rockford, IL, USA) was used to determine the concentration of
though the detailed crystal structure of NBP is not cur- purified NBP. The fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(di-
rently available, the general structure of periplasmic bind- ethylamino)coumarin-3-carboxamide (MDCC), which was conju-
ing proteins is known to consist of two similarly folded gated to NBP, was synthesized in our laboratory by the method of
globular regions, each made up of a -pleated sheet with Corrie [18]. Size-exclusion chromatography on a Sephadex G-25
column (Sigma) was used to separate conjugated protein from
two or three -helices lining each side [9, 11, 12, 13]. excess fluorophore. Fluoronunc Maxisorp 96-well white microtiter
Three short peptide segments in the protein forming a plates were purchased from Fisher Scientific (Norcross, GA,
cleft between the globular domains connect the two do- USA).
mains. The binding site for the nickel lies within this cleft
[14, 15, 16]. The protein undergoes a conformational Apparatus
change when it binds nickel, with the globular domains
approaching one another and reducing the size of the Bacterial colonies were grown on agar plates at 37 C in a Fisher
Scientific Incubator. Cell cultures were grown in an Orbital Shaker
groove around the ligand in the center, excluding mole- from Forma Scientific (Marietta, OH, USA) and pelleted by use of
cules of water or other solvent from the binding site [17]. a Beckman (Palo Alto, CA, USA) J2-MI Centrifuge. Unpurified
This conformational change might be expected to cause protein fractions were filtered with a 0.2 m filter from Nalgene
an observable change in the fluorescence intensity of a (Rochester, NY, USA).
The BioCAD Sprint Perfusion Chromatography System from
fluorophore conjugated to the protein at the appropriate PerSeptive Biosystems (Cambridge, MA, USA) was used for pro-
site. This change in fluorescence could be directly corre- tein purification. Liquid protein samples were lyophilized using
lated with the concentration of nickel present in the sys- the VirTis Bench Top 3 Freeze Dryer (Gardiner, NY, USA). Pro-
tem, enabling the development of a sensitive assay for teins were dialyzed against the proper buffer using a 1214,000
MWCO Spectra/POR molecular porous membrane from Spectrum
nickel. Medical Industries (Los Angeles, CA, USA). Protein purity was
There is only one cysteine residue in NBP, located at verified by sodium dodecyl sulfate polyacrylamide gel electro-
position fifteen. This seems to be a potentially good site phoresis (SDSPAGE) using the PhastSystem from Pharmacia
for conjugation with a sulfhydro-specific fluorophore such Biotech (Uppsala, Sweden). Protein absorbances in the BCA assay
as N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin- were determined with a diode array spectrophotometer (model
8453) from HewlettPackard (Palo Alto, CA, USA). Fluorescence
3-carboxamide (MDCC). In this study NBP was conju- studies were performed on a Spex Industries (Edison, NJ, USA)
gated with MDCC, and steady-state fluorescence experi- Fluorolog-2 spectrofluorimeter, equipped with a 450-Watt xenon
ments were conducted to verify there was a difference be- arc lamp. A Cytofluor series 4000 fluorescence multi-plate reader
tween the fluorescence signals emitted by the labeled pro- from Applied Biosystems (Foster City, CA, USA) was used for the
microtiter plate assay.
tein in the absence and presence of nickel. Selectivity A bifurcated fiber-optic bundle and a dialysis membrane from
studies were performed to evaluate the response charac- Spectra/Por3, MW cut-off 3500 from Spectrum Medical Industries
teristics of NBP to other divalent metals that might be po- (Los Angeles, CA, USA) were used.
tential ligands. The development of a fiber-optic sensor
for nickel was also investigated. A sensor of this kind is a Optical probe, and type of dialysis membrane used
useful tool in environmental analysis, because it could fa-
cilitate remote sensing of the metal in areas not easily ac- Expression of nickel-binding protein (NBP)
cessible for testing. The work presented discusses the de- To determine the time course of NBP production in E. coli, a 5-mL
velopment of a novel fluorescence-based system that is LB-ampicillin (100 g mL1) culture of E. coli B834 containing
176
p8602 was grown overnight in a shaker at 37 C and 250 rpm. The 25 mmol L1 MDCC were added, drop by drop, to the NBP solu-
next day LB-ampicillin (100 g mL1, 50 mL) were inoculated tion to ensure that the final MDCC concentration would be
with the overnight culture and incubated in a shaker at 37 C and 65 mol L1 (five times that of the NBP). The reaction was left to
250 rpm. When the absorbance at 600 nm reached 0.6, IPTG (final proceed for 4 h at room temperature, after which time the sample
concentration of 0.2 mmol L1) was added to the culture. Samples was loaded onto a Sephadex G-25 size-exclusion column for sepa-
(2 mL) were taken 0, 1, 2, 3, 4, and 20.5 h after addition of IPTG. ration of the conjugated protein from excess fluorophore. The con-
Cells from 1 mL of each sample were pelleted in a microcentrifuge jugate was eluted with 10 mmol L1 Tris-HCl, pH 8.0. Fractions
tube. The supernatant was poured off and the pellet resuspended in containing the labeled protein were pooled, lyophilized, dissolved
the drop of medium remaining in the tube. Chloroform (10 L) in 3 mL deionized water, and dialyzed twice against 10 mmol L1
was added to the resuspended cells, which were then vortex-mixed Tris-HCl, pH 8.0, in the dark at 4 C. The labeled protein was
briefly and left at room temperature for 15 min. TE buffer stored at 4 C in the dark to keep the protein from degrading and
(10 mmol L1 Tris-HCl, pH 7.0, 1 mmol L1 EDTA; 100 L) was the fluorophore from photobleaching. The NBPMDCC concen-
then added to the cells and the contents were mixed, and centrifuged tration was determined by BCA analysis to be 11.5 mol L1.
at high speed for 5 min. The upper periplasmic phase was transferred
to a clean microcentrifuge tube. A volume of 20 L of each frac-
tion was run on a 12.5% SDSPAGE, which was then stained with Fluorescence studies of NBPMDCC with nickel
coomassie brilliant blue R-250 to verify the presence of the NBP.
For purification of NBP a single isolated colony of E. coli Standard fluorimetric conditions
B834 containing p8602 was cultivated overnight in 3 mL LB-
ampicillin (100 g mL1) at 37 C and 250 rpm. This culture was The fluorescence spectra of NBPMDCC were obtained by use of
used to inoculate 500 mL LB-ampicillin (100 g mL1) which was a Fluorolog-2 spectrofluorimeter (Spex Industries, Edison NJ,
incubated at 37 C and 250 rpm until the absorbance at 600 nm was USA) equipped with a 450-W xenon arc lamp. The monochroma-
0.6. At that point, IPTG was added to 0.2 mmol L1 final concen- tor slit widths were set at 2 mm, and an excitation wavelength of
tration. The culture was then left to grow for a further 3 h. The 425 nm and an emission monitoring wavelength of 469 nm were
cells were pelleted by centrifugation at 3000 rpm and 4 C for used unless otherwise noted. Different dilutions of NBPMDCC
30 min. The isolated cells were then stored at 4 C. were prepared and read in quartz cuvettes using a final reaction
volume of 1.5 mL. Spectrophotometric readings were taken at
room temperature (RT). From the dilution curve (data not shown)
Purification of NBP an NBPMDCC concentration of 2.3108 mol L1 in 10 mmol L1
Tris-HCl, pH 8.0, was selected and used unless otherwise noted.
Periplasmic NBP was released from cells by the chloroform method This concentration was selected because the signal obtained was
of Ames et al [19]. The cells were resuspended in 5 mL chloroform well above the background signal.
and kept at room temperature for 15 min. This solution was com-
bined with 50 mL of 40 mmol L1 Tris-HCl, 5 mmol L1 EDTA,
0.5 mmol L1 phenylmethylsulfonylfluoride (PMSF), pH 7.0. The Steady-state study of NBPMDCC binding with nickel
solution was centrifuged at 9000 rpm and 4 C for 15 min. The su- in the presence and absence of bovine serum albumin (BSA)
pernatant was saved and lyophilized overnight. The powder was
dissolved in 1 mL deionized water and dialyzed against three A preliminary steady-state study of NBPMDCC was conducted to
changes of 500 mL 10 mmol L1 Tris-HCl, pH 8.0 (Buffer A) over obtain information about whether the presence of BSA would pre-
12 h. The crude sample was centrifuged twice, each time for vent adsorption of the labeled protein by the walls of the cuvette.
15 min, at 9000 rpm and 4 C, and filtered through a 0.2 m mem- The original 11.5 mol L1 stock of conjugated protein was diluted
brane filter. The protein was purified by HQ (quarternized poly- 100-fold in 10 mmol L1 Tris-HCl, pH 8.0, and divided into two
ethyleneimine) perfusion chromatography. The column (2 mL bed samples. One did not contain any BSA whereas 0.01% BSA was
volume) was equilibrated with Buffer A. The flow rate was set at included in the other to prevent adsorption of the protein by the
8 mL min1. The periplasmic extract (1 mL) was injected onto the walls of the cuvette. The fluorescence spectra of both nickel-free
column and the column washed with 10 column volumes of Buffer and nickel-bound (1.6104 mol L1 NiCl2, final concentration)
A. The protein was eluted with a salt gradient from 0 to 100% NBPMDCC were determined by use of the spectrofluorimeter af-
NaCl in Buffer A over 50 column volumes. Protein elution was ter being left to incubate on a 400 rpm shaker for 10 min at 4 C.
monitored by UV absorbance at 280 nm. Fractions containing the
purified protein were pooled and dialyzed against three changes of
500 mL 10 mmol L1 Tris-HCl, pH 8.0 over 16 h. The samples Incubation-time study of the labeled protein with nickel
were loaded on a 12.5% polyacrylamide gel that was then devel-
oped by silver staining. The SDSPAGE was used to determine To determine the effect of incubation time on the change in fluo-
the presence (band appearing at a molecular weight of 56 kDa) and rescence response of NBPMDCC to nickel, the conjugated pro-
purity of the NBP. The purified NBP was concentrated by tein was incubated with nickel for different lengths of time before
lyophilization. The product was dissolved in 3 mL distilled deion- determining fluorescence. The conjugate was incubated with
ized water and dialyzed against four changes of 10 mmol L1 Tris- 25 L 0.01 mol L1 NiCl2 in triplicate on a 400 rpm shaker at 4 C
HCl, pH 8.0. The protein concentration was determined by the for different periods of time. A volume of 25 L 10 mmol L1 Ni
BCA assay using bovine serum albumin as a standard and the en- was used in a total volume of 1.5 mL, which is equivalent to
hanced test tube protocol with a working protein concentration 1.6104 mol L1 Ni. This amount of Ni is sufficient to yield mea-
range of 5250 g mL1 according to the suppliers instructions. surable quenching in the signal (~35%) and hence was selected for
A BCA assay revealed the concentration of NBP to be 13 mol L1. the study. Average fluorescence quenching values were plotted
against the incubation times in min.
Conjugation of NBP with MDCC
Purified NBP (1 mL) was dialyzed against 10 mmol L1 Tris-HCl, Response curve of NBPMDCC to nickel
pH 8.0, containing 10 mmol L1 DTT to eliminate disulfide link-
ages and free up the sulfhydryl group for reaction with the fluo- Samples (25 L) of different concentrations of NiCl2 were added
rophore. The sample was then dialyzed against three changes of to NBPMDCC in triplicate samples and then incubated on a
500 mL 10 mmol L1 Tris-HCl, pH 8.0 over 6 h to remove any ex- 400 rpm shaker at room temperature for 15 min, and analyzed in
cess DTT (to prevent conjugation of that molecule with the fluo- the fluorimeter. Average fluorescence quenching was plotted in re-
rophore, MDCC). In the dark at room temperature, 2.8 L of lation to the log of the concentration of nickel in the sample.
177
Selectivity studies of NBPMDCC with other divalent metals 0h 1h 2h 3h 4h 20.5 h Marker
Copper (II), cobalt, iron (II), cadmium, and manganese used were
in the chloride form, because chlorine is common in many organ-
isms and biosystems. Samples (25 L) containing different con- 60 kDa
centrations of each metal were added to NBPMDCC and incu- 50 kDa
bated on a 400 rpm shaker at RT for 15 min and analyzed in the 40 kDa
fluorimeter. Average fluorescence quenching was plotted in rela-
tion to the log of the metal concentration in the sample. 30 kDa
Samples (25 L) of 0.01 mol L1 and 0.1 mol L1 NiCl2 were added
to 1.5 mL samples of MDCC in 10 mmol L1 Tris-HCl, pH 8. Sam-
ples were analyzed in the fluorimeter as described above, except
that the emission wavelength was 477 nm.
Fluorescence assay on a multi-well plate reader Fig. 1 Time study of the induction of nikA with IPTG. The lanes
of SDSPAGE correspond to induction times of 0, 1, 2, 3, 4, and
A CytoFluor series 4000 fluorescence multi-well plate reader from 20.5 h, respectively. The final lane contains protein molecular-
PerSeptive Biosystems (Framingham, MA, USA) was used with a weight markers
96-well format and sample volumes of 200 L. A 420 nm excita-
tion filter with a band-pass of 50 nm and an emission filter of
460 nm with a band-pass of 40 nm were used to obtain fluores- 1 2 3
cence data. NBPMDCC was incubated with different concentra-
tions of nickel for 15 min at RT.
Results
Time course of NBP production in E. coli Fig. 2 SDSPAGE with silver staining verifying the purity of
NBP. Lane 1, protein marker; lane 2, crude periplasmic extract;
lane 3, purified protein
There is considerable production of the 56 kDa NBP even
1 h after induction, with maximum production at approxi-
mately 3 h (Fig. 1). The 4 h time point showed no greater Steady-state study of NBPMDCC binding
quantity of NBP production than the 3 h time point. At with nickel in the presence and absence
20.5 h, little NBP is present. Therefore a 3 h induction of bovine serum albumin (BSA)
was used for large-scale purification of NBP.
A steady-state study using non-conjugated MDCC indi-
cated no change in fluorescence signal with exposure to
Purification of NBP nickel, demonstrating that nickel by itself does not quench
the fluorescence of MDCC. The results of the incubation
NBP was purified from a periplasmic extract in a single time study of NBPMDCC indicated significantly less
step using perfusion chromatography with a quarternized fluorescence quenching when BSA was included in the
polyethyleneimine (HQ) anion-exchange column. The re- assay. BSA contains a cysteine residue with a sulfhydryl
sulting protein was >95% pure, as demonstrated by group which is negatively charged at pH 8 and which is
SDSPAGE with silver staining (Fig. 2). known to sequester cations. BSA can reduce quenching
178
Stability study
most sensitive systems yet developed for nickel. The rea- protein. A detection limit for nickel of 8108 mol L1 was
son for such significant quenching might be the theoreti- obtained using this method. The system was used to mea-
cal location of the conjugated fluorophore. It is postulated sure nickel in different ways in a conventional static
that when NBP is in the ligand-free form, MDCC might spectrofluorimeter, in a microtiter plate format, and in a
be buried within a hydrophobic pocket of the protein fiber-optic bundle format. The aim of this study was to
structure. When, however, the globular domains of the demonstrate that the system developed was versatile and
protein engulf nickel, the fluorophore should be exposed could be employed for in-situ measurements. The opti-
to solvent molecules, resulting in a weaker signal, owing mization of the system for in-situ and remote sensing is a
to quenching. Previous studies conducted by us with the part of future studies in our laboratory that involve minia-
phosphate binding protein (PBP) revealed such a mecha- turization of the set-up and the sensing system.
nism of action [21]. Given the similar structures and func-
tion of NBP and PBP we hypothesize that the mechanism Acknowledgements We would like to thank the National Aero-
of action should be similar. To elucidate the exact mecha- nautics and Space Administration for funding this research, and
nism by which the change in conformation of NBP affects the Kentucky Research Challenge Trust Fund for a fellowship pro-
the fluorescence emission of the fluorophore, time-re- vided to L.S. S.D. is a Cottrell Scholar and a Lilly Faculty
Awardee.
solved anisotropy measurements should be obtained.
When NBPMDCC was used in a fiber optic biosensor
apparatus, maximum quenching of the fluorescence signal
was only 35%, compared with the 65% quenching ob- References
served in solution studies with the spectrofluorimeter. The
1. Babich H, Stotzky G (1983) Adv Appl Microbiol 29:195265
detection limit for nickel employing this fiber optic set-up 2. Brown SS, Sunderman FWJ (1988) Nickel toxicology. Acade-
was determined to be 1106 mol L1 (calculated as S/N = 3). mic Press, London, pp 14
Although this is slightly above the level considered toxic 3. US Department of Health and Human Services (1996) Toxico-
to humans, this sensor could still be used in remote loca- logical profile for nickel. US Department of Health and Human
Services, Public Health Service, Atlanta, GA
tions to screen waters that have the potential of being con- 4. Environmental Protection Agency (1983) Methods for the
taminated with toxic levels of nickel. Further optimization analysis of water and wastes. Environmental Protection Agency,
of the sensor is being performed, by varying different ex- Washington DC
perimental conditions, to increase its sensitivity. 5. Molinero AL, Castillo JR (1998) Anal Lett 31:903911
Of the metals tested, only for cobalt was fluorescence 6. Mrzljak RI, Bond AM, Cardwell TJ, Cattrall RW, Knight RW,
Newman OMG, Champion BR (1994) Analyst 119:1057
quenching similar in magnitude to that for nickel, al- 7. Hausinger RP (1987) Microbiol Rev 51:2242
though with a detection limit more than ten times higher 8. Navarro C, Wu L-F, Mandrand-Berthelot M-A (1993) Mol
than that for nickel. Studies of intrinsic tryptophan quench- Microbiol 9:11811191
ing in NBP upon binding to a ligand also demonstrated a 9. Charon M-H, Wu L-F, Piras C, de Pina K, Mandrand-Berthelot
M-A, Fontecilla-Camps JCJ (1994) Mol Biol 243:353355
strong response to cobalt [22]. The remaining metals all 10. de Pina K, Navarro C, McWalter L, Boxer DH, Price NC,
showed less maximum quenching than nickel and detec- Kelly SM, Mandrand-Berthelot M-A, Wu L-F (1995) Eur J
tion limits were more than 10 to 100 times higher than for Biochem 227:857865
nickel. The 10-fold or tighter binding of nickel compared 11. Hirshberg M, Henrick K, Haire LL, Vasisht N, Brune M,
Corrie JET, Webb MR (1998) Biochemistry 37:1038110385
with cobalt or copper means that the concentrations of 12. Brune M, Hunter JL, Howell SA, Martin SR, Hazlett TL,
cobalt and copper in a test solution must be equal to or Corrie JET, Webb MR (1998) Biochemistry 37:1037010380
less than that of nickel for the measurement to give a reli- 13. Hsiao C-D, Sun Y-J, Rose J, Wang B-C (1996) J Mol Biol 262:
able measurement for nickel. Even though the specificity 225242
of NBPMDCC for nickel is somewhat less than desired, 14. Nickitenko AV, Trakhanov S, Quiocho FA (1995) Biochem-
istry 34:1658516595
this molecule might be useful in sensitive sensing systems 15. Mowbray S L, Cole LB (1992) J Mol Biol 225:155157
used for prescreening for nickel. It might also find utility 16. Quiocho FA (1990) Phil Trans Roy Soc Ser B 326:341351
in biosensors for areas where nickel contamination has 17. Spurlino JC, Lu GY, Quiocho FA (1991) J Biol Chem 266:
been previously found and it is desirable to monitor regu- 52025219
18. Adams MD, Oxender DL (1989) J Biol Chem 264:15739
larly for the presence of this toxic metal. 15742
19. Corrie JET (1990) J Chem Soc Perkin Trans 1 21522152
20. Ames GF-L, Prody C, Kustu S (1984) J Bacteriol 160:1181
Conclusions 1183
21. Freeman MK, Bachas LG (1990) Anal Chim Acta 241:119
125
In conclusion, a sensitive method for the detection of nickel 22. Lundgren JS, Salins LLE, Kaneva I, Daunert S (1999) Anal
has been developed on the basis of bacterial nickel-binding Chem 71:589595
Anal Bioanal Chem (2002) 372 : 181186
DOI 10.1007/s00216-001-1199-1
O R I G I N A L PA P E R
Abstract In capillary electrophoresis, it is commonly shapes and detection limits of 0.8 M and 0.6 M for ni-
considered that even a moderately high ionic concentra- trate and bromide, respectively. The beneficial effects of
tion in the background electrolyte (BGE) leads to high the non-ionic surfactant on the separation were attributed
currents, resulting in Joule heating and serious peak dis- largely to suppression of the electro-osmotic flow. On the
tortion. As a new approach to overcome this problem, other hand, the zwitterionic surfactant was found to be ca-
zwitterionic (Zwittergent-314) and/or non-ionic (Tween pable of the incorporation of some anions in accordance
20) surfactants have been added to BGEs containing high with the behaviour of these same surfactants in electrosta-
salt concentrations (e.g. 0.3 M NaCl) and have been tic ion chromatography. This incorporation resulted in a
shown to result in acceptable separation currents (<200 A). decreased conductivity of the BGE and also a change in
In turn, these BGEs could be applied to the separation the separation selectivity of the system.
of samples containing high salt concentrations (such as
undiluted seawater) without the occurrence of any sig- Keywords Bromide Nitrate Seawater Capillary
nificant peak broadening due to electrodispersion of the electrophoresis Surfactants
sample. For example, a BGE comprising 10 mM Zwitter-
gent-314, 50 mM Tween 20, 0.3 M NaCl and 5 mM
phosphate (ph 7) could be used for the determination of Introduction
UV-absorbing anions in seawater, giving good peak
Capillary electrophoresis (CE) offers several advantages
over high performance liquid chromatography (HPLC) in
that it is simple, gives high efficiency separations, uses a
M. Mori () K. Tanaka small volume of electrolyte, and usually requires only a
National Institute of Advanced Industrial Science
and Technology at Sero, Sero 489-0884, Japan short analysis time [1]. Most CE studies have been per-
e-mail: masanobu-mori@aist.go.jp formed on samples containing a relatively low total ion
H. Tsue S. Tanaka
concentration, with high salt samples, such as seawater,
Division of Material Science, receiving little attention. The main reason for this is that
Graduate School of Environmental Earth Science, these highly conducting samples cause electrodispersion
Hokkaido University, Sapporo 060-0810, Japan and broadening of the analyte peaks. Therefore, direct in-
W. Hu jections of such samples are rarely used without some pre-
Department of Chemistry, Faculty of Science, treatment such as dilution [2] or removal of salt in the
Hokkaido University, Sapporo 060-0810, Japan sample [3, 4]. High salt sample matrices also usually re-
P.R. Haddad sult in irreproducible injections and migration times [5].
Australian Center for Research on Separation Science, One method to circumvent these problems has been to
School of Chemistry, University of Tasmania, raise the ionic strength of the background electrolyte
GPO Box 252-75, Hobart 7001, Australia
(BGE) such that electrostacking of the sample is restored
J.S. Fritz [6]. However, the addition of high salt concentration to
Department of Chemistry and Ames Laboratory, the BGE causes Joule heating and an increase in current,
Iowa State University, Ames, IA 50011, USA
leading to peak distortion and unstable baselines. Joule
Former address: heating can be minimised by lowering the applied volt-
M. Mori
Division of Material Science,
age, increasing the capillary length, or reducing the inner
Graduate School of Environmental Earth Science, diameter of the capillary, but these approaches usually
Hokkaido University, Sapporo 060-0810, Japan lead to relatively long analysis times. It is therefore of in-
182
Fig. 2 Changes in current with increasing concentration of several Fig. 3 Changes in currents with increasing concentration of NaCl
surfactants added to 5 mM phosphate buffer at pH 7. These plots added to BGEs containing 100 mM of various surfactants and
were the average of currents measured over a 10 min period. 5 mM phosphate buffer (pH 7). No surfactant (white triangle),
Cetyltrimethylammonium chloride (CTAC) (white square), sodium CTAC (white square), SDS (black square), Zwittergent-314
dodecylsulfate (SDS) (black square), Zwittergent-314 (black cir- (black circle), Brij 35 (black diamond) and Tween 20 (white cir-
cle), Brij 35 (black diamond) and Tween 20 (white circle). Capil- cle). Other experimental conditions were as described in Fig. 2
lary length, 41 cm (32.5 cm to detector); voltage, 20 kV; tempera-
ture, 25 C
O R I G I N A L PA P E R
Received: 28 May 2001 / Revised: 6 August 2001 / Accepted: 20 September 2001 / Published online: 13 December 2001
Springer-Verlag 2001
Abstract Methods for determination of both the total and Abbreviations GFAAS Graphite furnace atomic
extractable content of Cr and Co in soil samples were in- absorption spectrometry ETAAS electrothermal atomic
vestigated. For the total content of metal, ultrasonic slurry absorption spectrometry CRM certified reference
sampling graphite furnace atomic absorption spectrome- material EDTA ethylenediaminetetraacetic acid
try was used and compared with conventional analyses af- DTPA diethylenetriaminepentaacetic acid
ter microwave digestion. The influence of grinding, leach-
ing and homogeneity for slurry sampling was also exam-
ined. The concentration of the elements in the analyzed Introduction
materials were in the range: 50 g g1 0.4% for Cr and
814 g g1 for Co. Relative standard deviations (slurry In the present work we focus both on methods for deter-
sampling) were in the range 3%12% for Cr and 0.3% mination of total content of metal as well as on methods to
6% for Co determinations. The detection limit and char- determine the fraction available for plant uptake, i.e. the
acteristic mass (peak-area measurements) for Co were bioavailable part of the element. For the determination
0.14 g g1 and 12.6 pg, respectively. For Cr less sensitive of total Cr and Co in soil, samples were analyzed both as
wavelengths were used. Excellent agreement with certi- slurries and after digestions using different digestion pro-
fied reference material was found for total Cr and Co us- cedures. To obtain accurate results for decomposed soil
ing slurry sampling. samples a total digestion is normally required, which usu-
EDTA and acetic acid extractions were performed, us- ally includes use of hydrofluoric acid in addition to nitric
ing protocols given by the Measurement and Testing Pro- acid. For slurry sampling, other parameters are of impor-
gramme of the European Commission. The percentages tance for the accuracy and precision. The effect of some
extracted for the different soil samples were 0.31.0 for of these parameters, i.e. grinding, homogeneity and per-
Cr and 2.524 for Co. To validate the accuracy of the ex- centage extracted into aqueous phase of the slurry was ex-
tractable Cr, CRM 483 Sewage sludge amended soil was amined and discussed. Slurry sampling graphite furnace
analyzed. The values found were 37% and 32% higher than atomic absorption spectrometry (GFAAS) has previously
the certified value for EDTA and acetic acid extractable been developed and used for determination of the total
Cr, respectively. The precision for extractable concentra- content of various metals in soils and sediments, includ-
tion of Cr and Co was about 6% or less. External calibra- ing some work on Co [1, 2, 3] and Cr [1, 4, 5, 6, 7, 8].
tion with aqueous standards, matched to contain the same Several extraction agents and a variety of procedures
reagents as the samples, was employed. have so far been used to determine the bioavailable frac-
tion of metal in soils, the extraction agents include acids,
Keywords Chromium Cobalt EDTA/acedic acid chelating agents and buffered or unbuffered salt solutions.
extraction Spectrometry, atomic absorption Graphite Rauret [9] summarizes, in a recent paper, state-of-the-art
furnace extraction procedures used for heavy metal determination
in contaminated soil and sediments. For Cr and Co, ethyl-
enediaminetetraacetic acid (EDTA) [10, 11, 12] or acetic
acid [10, 11, 13, 14] have most often been used as extrac-
tion agents.
To judge which extraction agent and procedure that most
B. Lilleengen G. Wibetoe ()
Department of Chemistry, University of Oslo, likely will give the best estimate of the fraction available
P.O. Box 1033, 0315 Oslo, Norway for plant uptake is difficult and not a topic for the present
e-mail: grethe.wibetoe@kjemi.uio.no work. Moreover, at present there is no consensus about a
188
unique method that always gives the best estimate for the Table 1 Furnace programs used for the determination of Cr and
bioavailable fraction because the availability of metal to Co
plants depends on a large number of properties concern- Cr Co
ing the soil, plant and climate.
Fortunately, the problem with the different extraction Wavelengths (nm) 357.9, 429.0a, 425.4a 240.7
agents and procedures used has been dealt with by the Spectral bandpass (nm) 0.7 0.2
Measurement and Testing Programme of the European Injection volume (L) 10 20
Commission, which recently launched a project aiming at Step 1: Drying
harmonizing measurements for extractable trace metal Temperature (C) 90 90
content in soil. Protocols for determination of extractable Ramp time (s) 1 1
trace elements in soils have been described, and reference Hold time (s) 5 5
materials certified for their EDTA, diethylenetriaminepen- Step 2: Drying
taacetic acid (DTPA) and acetic acid extractable trace ele- Temperature (C) 110 110
ments have been prepared [15, 16, 17, 18]. Published re- Ramp time (s) 20 20
sults for the use of these procedures and CRMs are re- Hold time (s) 10 20
quested to see what to expect regarding the accuracy of Step 3: Pyrolysis
these operationally defined fractions. Temperature (C) 1200 1200 (1100b)
In the present work the protocols for EDTA and acetic Ramp time (s) 10 10
acid extraction were used to determine the concentration Hold time (s) 10 10
of metal to estimate the bioavailable part of Cr and Co in Step 4: Atomizationc
agricultural soil and sludge amended soil. The results for Temperature (C) 2300 2400 (1900b)
Cr were validated by analyzing CRM 483 Sewage sludge Ramp time (s) 0 0
amended soil that is certified for EDTA and acetic acid Hold time (s) 3 3 (2b)
extractable part for some trace elements including Cr (but Step 5: Clean out
not Co). Temperature (C) 2650 2650
Except for papers in connection with the development Ramp time (s) 1 1
of the CRM, no results for Cr and Co in CRM 483 Sewage Hold time (s) 4 3
sludge has, to the authors best knowledge, been published
When slurry sampling was used, the slurries were homogenized by
so far. automatic agitation for 30 s just before injection.
aLess sensitive lines used for total content of Cr.
bUsed for analysis of EDTA and acetic acid extracts.
cWall atomization; peak-area measurements; gas stop.
Experimental
Apparatus
Determinations were performed using a PE 5000 Zeeman atomic Reagents and samples
absorption spectrometer with an HGA 400 graphite furnace, an
AS40 autosampler with 0.5 mm (i.d.) tubing and Intensitron hol- Water (>15 M), purified by Elix S water purification system
low cathode lamps (all from Perkin-Elmer, Norwalk CT, USA). from Millipore (Bedford, USA), was used throughout. All reagents
An ultrasonic processor, VT 50 T Vibra cell (Sonics and Materials, were of analytical grade. The acids used were 65% nitric acid, 48%
Danbury, CT, USA) with a 3 mm (o.d.) titanium probe was incor- hydrofluoric acid, 37% hydrochloric acid and 100% acetic acid, all
porated into the autosampler to provide automatic homogenizing of pro analysis quality from Merck (Darmstadt, Germany) plus
of the slurry. Zeeman background corrector was used, and the an- 30% hydrogen peroxide from Fluka (Buchs, Switzerland). Element
alytical signal corrected for background and the background signal stock standard solutions of 1000 mg L1 concentration (Teknolab,
were recorded with the Perkin-Elmer 3600 data system and Drbak, Norway) were used to prepare calibration standards. Other
Anadex DP 9500 B printer. Pyrolytically coated graphite tubes chemicals used were EDTA, free acid (Fluka, Buchs, Switzerland),
without platform (Applied Optics AB, Mlnlycke, Sweden) were about 25% ammonia solution (AnalaR sp. gr.) from BDH Labo-
used. Electrothermal programs used are shown in Table 1. The vials ratory Supplies (Poole, England) and Triton X-100 (Sigma, St.
used in the autosampler were commercial 2 mL polystyrene vials Louis, USA).
(Sarstedt, Ski, Norway) and in some cases in-house-made 15 mL The following reference materials were used: CRM 07411 Chi-
borosilicate vials. For grinding of samples a MM 200 Mixer Mill nese Soil (Standard Materials of Soils Component, Harbin, China)
(Retsch, Haan, Germany) was used. Grinding cups (10 mL) and and CRM 483 Sewage sludge amended soil (IRMM, Geel, Bel-
grinding beads (12 mm diameter, one in each cup) were made of gium).
zirconia. Two real soil samples were also analyzed; an agriculture soil
Particle size measurements were made using a Coulter Multi- (soil-1) and a sludge amended soil (soil-2), both collected in Nor-
sizer II (Coulter Corporations, Miami, FL, USA) with Sampling way.
Stand IIA and an orifice tube with 70 m aperture diameter, cov-
ering the particle diameter range 1.442 m. For other details of
particle measurements see reference [19]. Procedures
Microwave-assisted decompositions were performed in 100 mL
PFA Teflon vessels using ETHOS 1600 Advanced Microwave All samples and reference materials were dried to constant weight
Station from Milestone (Sorisole, Italy) with an HPR-1000/10 S (110 C) and real soil samples were sieved, using a 2 mm diameter
rotor. An in-house-made end-over-end shaker (360o rotation, four sieve.
samples capacity) and a Jouan B4i centrifuge (Nantes, France) For the digestion of samples, 0.25 g aliquots of the material
were used for mixing and separation of suspensions, respectively. were accurately weighed into the decomposition vessels. Two dif-
189
ferent mixtures of concentrated acids were used: (1) 5.0 mL nitric The extracts were immediately filtered or centrifuged. A 0.45 m
acid plus 1.0 mL hydrogen peroxide and (2) 4.0 mL nitric acid, filter was used for filtering. Centrifuging was performed for 5 min
1.0 mL hydrogen peroxide plus 2.0 mL hydrofluoric acid. The sam- at 4000 rpm. For more details of the procedure see reference [17].
ples were digested in closed vessels (max. pressure set to 30 bar, In the final analyses, 0.2 g and 1.0 g sample were used for CRM
i.e. 300 kPa) in the microwave oven (program: linear increase and real samples, respectively.
from ambient temperature to 150 C for 20 min and then 23 min at After extraction (in some cases) the supernatant was washed
210 C). After digestion and cooling, all samples were diluted to with small portions of the extraction agent, dried at 110 C and an-
100 mL. For determination of Cr further dilution was required. alyzed by slurry sampling to determine the non-bioavailable frac-
For preparation of slurries, the samples and reference material tion of Co.
CRM 483 were ground in the mixer mill for a selected time with External calibration with aqueous standards was used for
the amplitude set at maximum. For studying the effect of parti- quantitative analyses. The calibration graphs covered the range
cle size, different grinding times were used. For studying the effect 10150 g L1 for Cr and 1040 g L1 for Co. The aqueous stan-
of acid on the percentage extractable into the aqueous part of the dards and blanks used for slurry sampling, decomposed samples
slurries, samples ground for 30 min were used. For the final analy- and extracts (EDTA and acetic acid) contained the same concen-
ses, the samples were, with some exceptions, ground for 10 min: tration of acids and reagents as the samples analyzed. The graphite
CRM 07411 (particle diameter <42 m) was analyzed as received furnace programs used for all analyses are given in Table 1. The
and soil-1 was ground for 60 min. Slurries were prepared by optimized furnace programs are based on pyrolyses and atomiza-
weighing 24 mg material (0.01 mg accuracy) into the vials of the tion curves for standard solutions and slurries of the different ma-
autosampler before diluent (0.22 M HNO3) was added. For CRM terials. Because of the relatively high content of Cr in the samples,
483 the diluent also contained 0.005% (v/v) Triton X-100. The the less sensitive wavelengths 429.0 nm and 425.4 nm were used
slurry concentration and diluents used for final analysis are given for the determination of total Cr.
in Table 2.
For particle size measurements, the slurries were made in the
same way as for analyses (see Table 2). After ultrasonic agitation Results and discussions
for 20 s (30% full effect), 200 L of the suspension was transferred
to the beaker of the Coulter Multisizer, containing 200 mL Isoton
II electrolyte solution, for particle measurements. For more infor- Determination of total content by slurry sampling
mation on the particle size measurements see Refs. [19, 20].
For the determination of the percentage of metals extracted into Effect of acid concentration on leaching, accuracy and
the liquid phase of the slurry, the elements were first determined in
the slurry and then in the supernatant after centrifuging. precision. Nitric acid of various concentrations is nor-
For determination of the EDTA and acetic acid extractable con- mally chosen as a diluent for slurry sampling GFAAS. In
tent of Cr and Co, the preparation of reagents and extraction was some cases, hydrofluoric acid has been used as diluent
performed (with some minor exception) as described in the proto- with the intention of mobilizing a larger fraction of ana-
col given by the European Commission for CRM 483 [17]. The ex-
traction procedures were also performed with 5 and 25 times lyte into the liquid phase, and, thus improving the accu-
downscaling. The extraction procedures were as follows (param- racy and precision (see, e.g. Ref. [3]). However, strong
eters for 25 times downscaling are given in parentheses): acid is not always needed for a large fraction to be ex-
1. EDTA extraction: 5 g (0.2 g) of soil sample was transferred to tracted; in some cases nearly quantitative extraction is
an 250 mL (10 mL) extraction bottle to which 50 mL (2 mL) of found using diluted nitric acid or even pure water (e.g.
0.05 mol L1 EDTA was added. The obtained mixture was Refs. [20, 21]). On the other hand, in the slurry analysis a
shaken on an end-over-end shaker operating at 3010 rpm for high degree of extraction is usually not a condition to ob-
1 h in a room at 202 C.
2. Acetic acid extraction: 5 g (0.2 g) of soil sample was transferred tain reliable results; good accuracy and precision have
to a 250 mL (10 mL) extraction bottle to which 200 mL (8 mL) been obtained with most of the analyte retained in the
of 0.43 mol L1 acetic acid was added. The obtained mixture solid phase of the slurry [22]. Thus, more work is needed
was shaken on an end-over-end shaker operating at 3010 rpm to learn more about these relations for different samples
for 16 h in a room at 202 C.
and analytes.
In the present work the effect of various concentrations
of nitric acid on the percentage extracted and the accuracy
Table 2 Slurry concentration used for analyses
and precision of the final result was examined for deter-
mination of Cr and Co in the various soils. As can be seen
Sample Slurry concentration in Fig. 1, the percentage extracted increases with nitric
acid concentration specially in the range from 01.1 M
Cr Co
(mg mL1) (mg mL1) acid. For Cr the maximum extracted was in all cases less
than 24% for CRM 07411 and soil-1, but was nearly
CRM 07411 1.32.7 1.32.7 quantitative for CRM 483 when 2.2 M nitric acid was
CRM 483 0.0150.03b 1.32.7 used as diluent. The reason for the high percentage leak-
Soil-1 1.32.7 1.32.7 age for CRM 483 Sewage sludge amended soil, found in
Soil-2 0.030.05 1.32.7 the present work, could be that the added sludge contains
The volume of diluent (0.22 M HNO3a) used was 1.5 mL except more soluble forms of Cr than normally found in soils.
for the determination of Cr in CRM 483 and soil-2, where 10 mL Data from previous work for extraction of Cr from soil
diluent was used. and sediments, in connection with slurry sampling, are
aThe CRM 483 also contained 0.005% (v/v) Triton X-100.
bThe slurry was prepared in two steps: 1.53 mg of sample was
found in the literature. Klemm and Bombach [5] found
transferred to the vial and 10 mL diluent added. After homoge- 26% leakage from diluted nitric acid (pH 2) for polluted
nization by ultrasonic agitation, 1.0 mL of this slurry was trans- river sediments; Dobrowolski [6] found 14%15% using
ferred to a second 15 mL vial and 9.0 mL added diluent. 0.05% nitric acid and 24%47% leakage using 5% nitric
190
Fig. 3 Concentration of Cr in ten sample replicates (three to six the number of replicates, the average content of Cr is not
measurements of each slurry) for: a CRM 07411, b CRM 483, significantly lowered. In routine analyses, when only few
c soil-1 (ground for 10 min), and d soil-1 (ground for 60 min). replicates are taken for analyses outliers may not be expe-
Each bar represents a single replicate. Median particle diameter
was 7.9 m for CRM 07411, 8.6 m for CRM 483, 10.7 and rienced even when the materials contain nuggets, but when
5.5 m for soil-1, ground for 10 min and 60 min, respectively. For they occasionally appear they may contribute significantly
CRM 07411, the certified value for Cr (mean value one standard to the result. Thus, when nuggets are expected, several
deviation) is shown (horizontal lines) replicates should be taken. Kurfrst [24] has published a
paper dealing with the statistical treatment of data from
electrothermal atomic absorption spectrometry (ETAAS)
CRM 483, no outliers were experienced, in spite of the analyses of heterogeneous solid samples.
fact that the number of particles injected for this sample Another way to deal with the problem is to reduce
was about two orders of magnitude less than for the other the effect of nuggets by increasing the amount of sample
two; i.e. the particle size distributions were similar for the or decreasing the particle size by further grinding. In the
three samples but the slurries injected were 100 times more present case, the latter was most practical and soil-1 was
dilute for CRM 483 (see Table 2). Cr in CRM 483 Sewage ground for 60 min before analyses. As seen in Fig. 3d and
sludge amended soil, which contains much higher con- Table 3, the outliers disappeared, and better precision and
centration of Cr than the other samples, is probably more higher concentration were obtained for Cr in soil-1 when
evenly distributed than in the other two samples. The quan- the mean particle diameter was reduced from 10.7 m to
titative results for the determination of Cr with and with- 5.5 m.
out the outliers are shown in Table 3. The precision is No problem with heterogeneity was experienced for the
greatly improved when the outliers are omitted, especially Co determination. No outliers were seen and good preci-
for soil-1, but because the outliers are few compared to sion was obtained for all the samples. The homogeneity
192
for Co in CRM 07411 (analyzed as received) is shown in less sensitive wavelength, 0.4% Cr was determined with
Fig. 4. good precision (5% RSD).
extraction protocol was used for two CRMs and two real extractions were performed. It could be of interest to de-
samples, while the acetic acid extraction was used only termine also this fraction, which could represent the non-
for CRM 483 Sewage sludge amended soil. The latter bioavailable content of the metal. Another reason to de-
CRM is certified both for the EDTA and acetic acid ex- termine this remainder was part of the validation of the
tractable content of Cd, Cr, Cu, Ni, Pb and Zn. The ex- method, to see if the amount extracted plus non-extracted
tractable fractions are operationally defined and it is, added up to the total amount. As Fig. 8 shows, the results
therefore, of outermost importance to follow the given for extractable and non-extractable added up to the same
procedure exactly, because small changes may effect the concentration as the total content, determined by slurry
final results. It is also important that the protocols for op- sampling, both for EDTA and acetic acid extraction.
erationally defined fractions are practical, rugged and eco-
nomical. For this purpose, we looked at the possibility to
do a few changes in the protocol without effecting the re-
sults; the possibilities to use centrifuging instead of the
recommended filtering and to scale down the procedure
were examined.
Our experience is that it is more practical to centrifuge
the soil mixture instead of filtering. Centrifuging is quicker
and the risk of contamination is less, especially when the
samples are centrifuged in the same bottles as used for the
extraction (as used in the present case). The results are
given in Table 4. No significant difference was found, and
centrifuging could preferably be used instead of filtering.
According to the given protocol, 5 g of sample should
be used for extractions. As the CRMs are very expensive,
it is advantageous to reduce analyzed sample amounts.
The procedures were, therefore, scaled down 25 times. The
results for the extractable part of Cr and Co in CRM 483,
using 5 g and 0.2 g sample, shown in Fig. 7, are not sig-
nificantly different (t-test, 95% confidence level).
The quantitative results for extractable (concentration
and percentage of total) and total metals are given in
Table 5 and Table 6. The percentage extractable Cr was
small for all the materials, only 0.3%1.0% of the total.
The percentage extractable Co was higher, 2.5%6.9% for
three of the samples, while for CRM 483 Sewage sludge
amended soil, 21.5% and 24.4% were extracted using
EDTA and acetic acid, respectively. The result for the ex-
tractable concentration of Cr in CRM 483 was higher than
the certified values both for EDTA and acetic acid extrac-
tion, i.e. 37% and 32% higher, respectively. The same de-
gree of accuracy should not be expected for the opera-
tionally defined fractions of metal compared to the total
content, as only small differences in the performance of
the procedure, between laboratories, are inevitable.
O R I G I N A L PA P E R
Received: 20 August 2001 / Revised: 9 October 2001 / Accepted: 11 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract A multi-residue analysis procedure using micro- tivity. Industrial activity and combustion of fossil fuels are
wave-assisted extraction and pre-purification has been de- the main sources of PAH in the environment. PCB were
veloped for the combined analysis of polycyclic aromatic widely used in the electrical industry and as additives in a
hydrocarbons (PAH), polychlorobiphenyls (PCB), and variety of materials such as plastics, inks, and carbon
organochlorine pesticides (OCP) in marine sediments. This copy paper. OCP were used as insecticides, although most
procedure has been validated with certified marine sedi- have been withdrawn because of their environmental per-
ment. Several surrogate standards have been employed sistence. Atmospheric or fluvial processes can transport
and the use of octachloronaphthalene (OCN) as a surro- these contaminants into the marine environment, where they
gate standard for organochlorine determination in this ma- can be adsorbed on to particulate matter and sediment.
trix is discussed. The recoveries of all compounds were To evaluate the impact of pollution on living organ-
high (>70%) and the relative standard deviations are of isms, a variety of biomarkers [1, 2] has been developed for
the same order as the certified values. Different analytical rapid screening of exposure to environmental contaminants.
problems are discussed, including DDT degradation in These techniques analyse the enzyme activity (ethylre-
gas chromatography and laboratory PCB background lev- sorufine-O-deethylase, EROD, acetylcholinesterase AChE)
els. Quantification problems encountered for two pesticides induced by the mechanism of metabolism of xenobiotics.
(cis-chlordane and trans-nonachlor) were attributed to PAH Such activity is not, however, compound-specific and syn-
interference in the GCECD chromatogram. ergetic or antagonistic effects can be observed. For exam-
ple, planar compounds such as PAH and certain PCB [3]
Keywords Aromatic compounds Organochlorine both induce EROD activity. To validate the use of biomark-
compounds Multi-residue analysis ers in environmental evaluation detailed chemical analy-
sis should be performed and there is, therefore, a need for
a single analytical procedure which enables the determi-
Introduction nation of several classes of contaminants.
Several environmental monitoring programmes (Na-
Polycyclic aromatic hydrocarbons (PAH), polychlorinated tional Status and Trends Program [4], Reseau National
biphenyls (PCB), and organochlorine pesticides (OCP) dObservation [5]) specify the routine analysis of marine
such as -hexachlorocyclohexane (HCH) and DDT, are sediments and many environmental studies evaluate a large
three classes of persistent and ubiquitous organic contam- number of samples. To satisfy the demand for increased
inants. These compounds have similar physicochemical productivity and faster analysis of these contaminants an
properties such as hydrophobicity, low degradability, and analytical procedure has been developed in this work which
high affinity for lipids. Because of these properties, these enables the determination of the three classes from the
contaminants are readily accumulated by living organisms. same purified extract of sediment.
They are often found together in environmental samples, Quantitative analysis of these compounds remains a
because they are mostly generated by anthropogenic ac- challenging task, because they often occur at very low
concentrations (ng g1) compared with endogenous mate-
rial, and several purification steps may be necessary to
S. Thompson H. Budzinski K. LeMenach M. Letellier obtain a chromatogram free from interference. Gas chro-
P. Garrigues () matographic separation of all of the PCB is currently im-
Laboratory of Environmental and Toxicological Chemistry,
UMR 5472 CNRS, University of Bordeaux, possible. As Larsen [6] pointed out, no single column is
33405 Talence Cedex, France capable of separating even the seven priority PCB (28, 52,
e-mail: p.garrigues@lptc.u-bordeaux.fr 101, 118, 138, 153, and 180). Indeed, several congeners can
197
which resulted in a large peak (Fig. 3). OCN cannot be Fig. 4 GCECD Chromatograms of 4,4-DDT d8 under different
used as an surrogate standard for this matrix because its chromatographic conditions. (a) Injector temperature=280 C; col-
umn head pressure=140 kPa. (b) Injector temperature=250 C; col-
use will result in systematic overestimation of the concen- umn head pressure=140 kPa. (c) Injector temperature=250 C; col-
trations of the analytes (area of the standard too high). umn head pressure=190 kPa
Optimisation of gas chromatographic injector conditions pound spends less time in the hot glass insert of the injec-
for the analysis of DDT tor and is rapidly deposited in the cooler (oven at 60 C)
column head. Under these conditions (injector tempera-
DDT is a thermolabile compound that can be degraded ture=250 C, and column head pressure=190 kPa) the
during gas chromatographic analysis [17, 18]. For example, degradation is minimal, 4,4-DDT-d8 represents 96% of
the analysis of 4,4-DDT-d8, the surrogate standard used the total area. For this sample-preparation procedure the
for the quantification of chlorinated pesticides under the degradation of DDT can be determined, because the
conditions used for the analysis of PCB leads to the de- deuterated standard will also be degraded and will lead to
tection of three peaks (Fig. 4). These peaks have retention the corresponding deuterated degradation product peaks,
times corresponding to 4,4-DDE-d8, 4,4-DDD-d8 and which are not present in the environmental sample.
4,4-DDT-d8. The DDT compound represents only 60% of
the total area of the three peaks. The analysis was per-
formed at different injector temperatures to determine the PAH interference in GCECD chromatograms
optimum conditions for the GC analysis of 4,4-DDT-d8.
Reduction of the injector temperature to 250 C increases The ECD is a highly sensitive detector that responds to
the area of the 4,4-DDT-d8 peak to 67%. The gas flow electronegative compounds; the signal is proportional to the
through the injector can be increased so that the com- electron affinity of the analyte. the signal from organo-
201
Fig. 5 GCECD Chromatogram of SRM 2260 PAH in Toluene for two pesticides, cis-chlordane and trans-nonachlor, in
GCECD analysis. These two chlorinated pesticides can
co-elute with pyrene and deuterated pyrene. Other authors
chlorine compounds such as PCB and the chlorinated pes- have reported the interference of pyrene with chlorinated
ticides depends on the degree of chlorination. On a nor- pesticides in gas chromatographic analysis [30]. Although
malised scale, hydrocarbons have a relative sensitivity of PAH have low response factors compared with organo-
1, monochlorinated compounds 100 and polychlorinated chlorine compounds, because the PAH can be present at
compounds 106 [26]. The electron affinities of condensed concentrations 100 times greater, their signal can be suffi-
aromatic compounds are, however, much greater than cient to create interference.
those of aliphatic hydrocarbons, and the ECD has been
proposed as a selective detector for the PAH [27, 28], be-
cause there are large differences between the electron affini- Procedural blanks
ties of the different isomers. For example, benzo[a]pyrene
has a relative response factor more than twice that of Procedural blanks are a standard procedure in analytical
benzo[e]pyrene. Indeed, this electron-accepting property chemistry and are a necessary step for exact quantifica-
is thought to be responsible for their toxic behaviour [29]. tion. The procedural blanks performed during these vali-
A GCECD chromatogram obtained from an SRM dation experiments revealed a background level of PCB.
2260 standard solution of PAH is presented in Fig. 5. The There were no significant peaks in the pesticide and PAH
average individual compound concentration in this solu- fraction. The results obtained are shown in Table 2 in ng/
tion is 50 ng L1. The peaks of the PAH are not very in- sample and the overall standard deviation is low (<5%).
tense, but the areas are significant compared with those of The chromatographic profile of these analyses is also con-
the peaks resulting from low concentrations of organo- stant. There are few tri-chlorinated congeners that are typ-
chlorine compounds. Comparison of the retention times of ical of an air source of PCB. The distribution is dominated
the standards of the three classes of contaminant reveals by tetra- to hexachlorinated PCB, which suggests an indoor
that several co-elutions are possible. Because the HPLC source such as sealant [15] or capacitors [13]. PCB were
step in the sample preparation separates the PCB from the used in building material (insulators, electrical transformer
PAH, however, only the pesticides that remain in Fraction fluids) until the 1970s and, as other authors have demon-
2 are affected. Quantification problems were encountered strated [14], it is probable that the air of the laboratory is
202
Table 2 Results (ng/sample) obtained for procedural blanks of Chemistry) the construction of which dates from approxi-
PCB mately 1990. No research has been conducted on PCB in
PCB Laboratory RSD Chemistry school this building. All the material (glassware, silica gel, sodium
sulfate) was prepared in our laboratory but was washed
8 0.27 29.6 0.36 with dichloromethane just before use in the Chemistry
18 0.29 3.4 0.2 School. The results are shown in Table 2, the level of PCB
28 0.53 64.2 0.0 is approximately a third of that in laboratory in the Depart-
44 1.31 29.0 0.39 ment of Chemistry (11 ng/sample compared with 28 ng/
52 2.62 18.7 0.0 sample). Because the glassware and material was prepared
66 4.27 3.3 1.47
in the contaminated laboratory, it is not surprising traces
87 1.66 13.3 0.66
of PCB were found, but the difference is significant. The
101 3.41 20.8 1.51
chromatographic profile is different, however. Although
105 1.36 15.4 0.58
the penta- and hexachlorobiphenyls still dominate, the tri-
118 3.42 6.1 1.85
and tetra-chlorinated congeners were absent. This could
128 0.47 27.6 0.24
138 2.37 26.2 1.54
be explained by the stronger adsorption of heavier PCB
153 1.98 25.3 1.72
congeners (penta- and hexachlorobiphenyls) by glassware
170 0.16 18.7 0.07 compared with the lighter PCB.
180 0.29 33.3 0.25 These results demonstrate that PCB persist in indoor
187 0.15 20.0 0.09 environments even after about 15 years since they were
banned. Indoor sources seem to be the cause of the high lev-
Total 24.56 6.8 11.09 els of PCB, as shown by the distribution of congeners. This
contamination particularly affects buildings that date from
before the PCB ban (19301980), i.e. most public buildings.
contaminated. The laboratory is situated in the Department
of Chemistry building that was constructed in the early
1960s, the period during which PCB were extensively em- Validation of the analytical procedure
ployed. Procedural blanks were performed in other laborato-
ries in the same building, where no PCB research had been The reference material, SRM 1941.a Organics in Marine
performed. The results showed that background levels were Sediment was analysed to determine the precision of the
the same, with the same chromatographic profile, thus the procedure. Three separate extractions were performed ac-
contamination was not caused by the manipulation of PCB. cording to the sample-preparation procedure (Fig. 1), en-
It was suggested by Wallace et al. [12] that contamina- abling calculation of the standard deviation of the mea-
tion of procedural blanks occurs during clean-up and treat- surements. The recoveries were corrected for the values
ment of the sample. This is demonstrated by comparing obtained in the procedural blanks.
the values obtained for solvent, 0.14 ng mL1 for the sum
of 18 PCB, compared with 24.6 ng/sample for a proce-
dural blank (purification steps included), in which a max- PAH
imum of 30 mL solvent were used.
To verify these results a procedural blank was performed Sixteen PAH were found in Fraction 2; their concentra-
in another building on the University campus (School of tions and recoveries are given in Table 3. Some of the
aIndicative
Total 113.5315.21 13.4 116.098.36 102.4 7.2
concentration only
compounds are given as sums of two or three individual quantification by use of other surrogate standards does not
compounds because of co-elution from the gas chromato- significantly change the result. The standard deviations
graphic column (chrysene + triphenylene; benzo[b]fluor- calculated on three measures are comparable with those of
anthene + benzo[j]fluoranthene + benzo[k]fluoranthene; the certified concentrations, except for PCB 44. The re-
dibenz[a,h]anthracene + dibenz[a,c]anthracene). Recov- sults are an improvement on previously published results
eries range from 58% to 114%, with an average of 85% [21] for some congeners, because of the extra HPLC pu-
for all the compounds (Table 3). Relative standard devia- rification step. This removes the polar pesticides which
tions are quite high for the lighter compounds (phenan- can interfere with the PCB, e.g. 4,4-DDT which co-elutes
threne, anthracene, fluoranthene, and pyrene) ranging from with PCB 138.
17 to 34%, for phenanthrene. The heavier compounds, from
chrysene to benzo[g,h,i]perylene have relative standard
deviations equivalent to or lower than those given with Pesticides
the certified values. It is possible that the acid purification
step leads to slight degradation of the smaller tri-aromatic Six chlorinated pesticides are certified in the marine sedi-
PAH, leading to the low recovery for anthracene. The ment SRM 1941.a, and an indicative concentration is given
lowest recoveries were found for the PAH anthracene and for 4,4-DDT. The recoveries and relative standard devia-
dibenzanthracenes. These compounds are also the least tions are shown in Table 5. The recoveries of hexachloro-
abundant in the matrix with concentrations of only 117 benzene, 4,4-DDE, 4,4-DDD and 4,4-DDT were good,
and 184 ng g1. The recoveries of the PAH are, however, and ranged from 102% to 124%.
comparable with those in other studies [17, 18], and the The relative standard deviations are quite high for this
results are reasonable for environmental analysis. class of compound, except for hexachlorobenzene. This is
probably because of the low concentrations of some of
these pesticides in this matrix. The average recovery for
PCB the sum of the pesticides is 118.5%. The recovery of 2,4-
DDE is too high at 278%. This compound is not totally
All the PCB are found in Fraction 1 except for PCB 128, separated from interference, and its low concentration in
which is in Fraction 2. For most of the PCB, and using the this matrix, 0.73 ng g1, leads to a chromatographic peak
surrogate standards described, the recoveries are high (usu- which is small relative to those of other closely eluting
ally >80%); the results are presented in Table 4. The av- compounds such as PCB 101, the concentration of which
erage recovery of all the PCB is 102.4%. Only for PCB 87 is 11.0 ng g1. To improve the recovery of this compound
is the recovery low 53%; the relative standard deviation a larger amount of certified material should be extracted.
of 10% is, however, not very high compared with those of Recovery of cis-chlordane and trans-nonachlor from
the other PCB congeners. The recovery of PCB 52 is high this matrix are very high, 420% and 250%, respectively.
124%; this can be explained by the close elution of a When analysed without addition of PAH surrogates re-
large interfering peak which is not removed by the HPLC coveries of these compounds are much improved 130%
purification step. High recoveries, approximately 120% to and 70%, respectively. cis-Chlordane and trans-nonachlor
150%, were obtained for PCB 170 to PCB 209. The peaks are not entirely separated in the chromatogram and are co-
of these congeners are well-resolved in the chromatogram eluted with deuterated pyrene and pyrene. The certified
and co-eluting impurities do not seem to be present. Their values of these compounds are determined by mass spec-
204
trometry, which can clearly distinguish between the com- Acknowledgements Prolabo is acknowledged for the loan of the
pounds as a result of their different molecular mass, m/z= microwave apparatus. NIST is thanked for the SRM samples.
202, pyrene; 212, pyrene-d12; 373, cis-chlordane; 409, trans-
nonachlor. In this matrix pyrene is present at a concentra-
References
tion of 81124 ng g1, compared with 2.330.56 ng g1 for
cis-chlordane and 1.260.13 ng g1 for trans-nonachlor. 1. Lafaurie M, Narbonne JF, Galgani F (1992) Analusis Magazine
To avoid interference problems GCMS is the preferred 20:6, 27
method for detection of these compounds, although the 2. Garrigues Ph, Burth H, Wallcer CH, Narbonne JF (2001) Bio-
markers in Marine Organisms. Elsevier, Amsterdam, 500 p
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7. Mullin MD, Pochini CM, McCrindle S, Romkes M, Safe SH,
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27:938
9. Fuoco R, Colombini MP, Samcova E (1993) Chromatographia
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Conclusion 10. Jarnberg U, Asplund L, De Wit C, Grafsom A, Haglund P,
Jansson B, Lexen K, Strandell M, Olsson M, Jonsson B (1993)
The analytical procedure combining microwave-assisted Environ Sci Technol 27:1364
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man A (1993) J Chromatogr 634:79
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14. Alcock RE, Halsall CJ, Harris CA, Johnston AE, Lead WA,
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GCECD chromatogram can lead to over-estimation of J High Resol Chromatogr 15:763
18. Grob K, Wagner C (1993) J High Resol Chromatogr 16:464
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of the PAH are low compared with those of chlorinated Spectroscopy 13:71
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Anal Bioanal Chem (2002) 372 : 205215
DOI 10.1007/s00216-001-1125-6
O R I G I N A L PA P E R
Received: 19 June 2001 / Revised: 17 August 2001 / Accepted: 20 September 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract A multi-channel detection system utilizing fiber liquid chromatography (HPLC) method as the unknowns.
optics has been developed for the laser-induced fluores- The percentage errors of the retention times (RTs) deter-
cence (LIF) analysis of chromatographic eluents. It has mined using RAFA compared to the known RTs measured
been applied to the detection of polycyclic aromatic hy- with a standard absorbance detector were between 0 and
drocarbons (PAH) in a chromatographically overlapped 11%. For the standard PAH mixture, all 17 components
standard mixture and to a complex soil sample extract ob- were identified correctly and for the soil extracted sample,
tained during fieldwork. The instrument utilizes dual-fiber all 8 analytes present were correctly identified with only
optic arrays, one to deliver multiple excitation wave- one false positive. Overall, the system achieved excellent
lengths (258342 nm) generated by a Raman shifter, and qualitative performance with semi-quantitative results in
the other to collect fluorescence generated by the sample the concentration predictions of both the standard mixture
at each excitation wavelength; the collected fluorescence and the real-world sample. Electronic supplementary ma-
is dispersed and detected with a spectrograph/CCD com- terial to this paper can be obtained by using the Springer
bination. The resulting data were arranged into excitation LINK server located at http://dx.doi.org/10.1007/s00216-
emission matrices (EEM) for visualization and data analy- 001-1125-6.
sis. Rapid characterization of PAH mixtures was achieved
under isocratic chromatographic conditions (1.5 mL min1 Keywords Hydrocarbons, polycyclic aromatic
and 80% acetonitrile in water), with mid g L1 detection Fluorescence, laser-induced Fiber optic array
limits, in less than 4 minutes. The ability of the instrument Chromatography HPLC
to identify co-eluting compounds was demonstrated by
identifying and quantifying analytes in the rapid analysis
of a 17 component laboratory-prepared PAH mixture and Introduction
a soil extracted sample. Identification and quantification
were accomplished using rank annihilation factor analysis The analysis of complex mixtures by HPLC is a powerful
(RAFA) using pure component standards and the EEMs technique for compound identification and quantification.
of mixtures measured during the rapid high-performance Traditionally, compounds are chromatographically sepa-
rated and then identified by one of several means, includ-
ing refractive index, absorbance (single/dual wavelength
Electronic supplementary material to this paper can be obtained responses, or full absorption spectra with a photodiode ar-
by using the Springer LINK server located at ray (PDA) detector), and fluorescence (single excitation
http://dx.doi.org/10.1007/s00216-001-1125-6. and emission wavelength) measurements. Of these detec-
S.J. Hart () G.J. Hall J.E. Kenny tion techniques, fluorescence is the most sensitive, while a
Tufts University, Chemistry Department, PDA detector provides the most information (a full ab-
Medford, MA, 02155, USA sorption spectrum at each time point) for compound iden-
e-mail: shart@ccf.nrl.navy.mil tification. Numerous other techniques have been devel-
Current address: oped, including fluorescence lifetime [1, 2], fluorescence
S.J. Hart excitation-emission matrix (EEM) [3, 4, 5, 6, 7], low-an-
Naval Research Laboratory, Chemistry Division,
4555 Overlook Ave, Washington, DC 203755342, USA gle light scattering (LALLS) [8], chemiluminescence [9,
10], mass spectrometry [11], and electrochemical tech-
Current address:
G.J. Hall
niques [12]. Advanced detection systems for HPLC can
Department of Science, US Coast Guard Academy, offer several benefits, including accurate and rapid com-
15 Mohegan Ave, New London, CT 06320, USA pound identification and/or quantification, faster analysis
206
Table 1 Standard compounds, abbreviations, regression coefficients and LODs (3 of the baseline)
Compound Abbre- Slope y-intercept r2 ex max em max LOD
viation (nm) (nm) (g/L)
Naphthalene N 183.3 6.1 0.99998 266 322 131.6
p-Xylene X 66.5 3.3 0.99997 266 286 286.3
1-Methyl-naphthalene 1-MN 102.1 121.9 0.99786 266 354 186.6
2-Methyl-naphthalene 2-MN 197.1 10.9 0.99997 266 354 109.5
Acenaphthene ACE 857.4 111.9 0.99979 299 322 31.1
Fluorene FLE 1285.1 51.4 1.00000 266 304 22.7
Phenanthrene Ph 53.7 8.6 0.99836 289 347 236.5
Anthracene A 111.2 13.2 0.99987 342 380 205.6
Fluoranthene FLA 45.9 4.8 0.99992 289 445 415.2
Pyrene P 525.2 28.4 0.99998 315 372 38.7
Benz[a]anthracene BA 307.5 64.4 0.99978 289 385 61.9
Chrysene C 219.6 19.9 0.99995 266 362 92.5
Benzo[b]fluoranthene BBF 88.8 2.4 0.99995 299 446 286.1
Benzo[k]fluoranthene BKF 487.5 231.1 0.99856 299 409 41.7
Benzo[a]pyrene BAP 521.5 28.4 0.99998 299 403 43.8
Dibenz[a,h]anthracene DA 188.8 13.4 0.99850 289 394 114.4
Benzo[ghi]perylene BP 173.1 72.1 0.99830 299 404 154.1
times, lower detection limits, elucidation of peak overlap, was applied to bimodal chromatographic data from a stan-
and the ability to measure more complex samples. dard PDA [21, 22]. This method involved the analysis of
Several researchers have developed various systems heavily overlapped components on two different columns
intended to exploit the information contained in EEM data under different conditions (HPLC mobile phase composi-
to enhance HPLC measurements. A lamp-based video flu- tion). The separation of components occurs differently in
orometer was developed by Christian et al. [13] and uti- each case (e.g., a sample having complete overlap on one
lized by Hershberger and Christian with a laminar flow column will have only partial overlap on the other). Using
cell to perform EEM analysis of HPLC separations [3]. the two separations and single component standards mea-
During the chromatographic separation, EEMs were mea- sured on the same columns, the overlap can be deconvo-
sured (10 s total time required per EEM) and component luted using GRAM. Results for concentrations of three
identification and quantification were achieved by least PAHs (BBF, BKF and perylene; for acronyms employed,
squares fitting and rank annihilation analysis. An updated see Table 1) in a laboratory-prepared sample were in
version [4] of this system, using a dye-laser source capa- agreement with known values, with errors between 3%
ble of rapidly scanning excitation wavelengths for opti- and 15%.
mum sample excitation with an intensified array detector, Our group has been involved with the in situ LIF de-
has been developed. This was used to measure fluores- tection of PAHs using fiber optics. LIF-EEM measure-
cence emission spectra during chromatographic elution. ments are made through sapphire windows in a probe that
Data analysis was accomplished using least squares fitting is hydraulically pushed by a cone penetrometer testing
of emission intensity-time matrices for 4 PAHs with quan- (CPT) vehicle into the subsurface [23, 24]. Our motiva-
titative results. Jalkian and Denton developed a variable tion for developing a dual-fiber optic detector for HPLC
excitation wavelength detection system for HPLC [5]. was to enable us to use on-site multidimensional LIF-
The system was capable of collecting an EEM in 30 s, in HPLC measurements for the calibration and verification
a static mode, but could only vary the excitation wave- of in situ results. Dual-fiber optic measurements made in
length for optimum emission during an HPLC separation, situ are subject to soil matrix effects that can alter the in-
and did not attempt quantification of overlapped analytes. tensities and spectral properties of the analytes of interest.
The measurement of EEMs during a chromatographic These effects include dependence of penetration depth on
separation using a multiple-wavelength laser system and particle size, medium-induced shifts in both absorption
fiber optics has been accomplished in our laboratory using and emission spectra, energy transfer effects, and contam-
an aluminium flow cell to spread out the eluent as it ination by fluorescent species not of interest (e.g., humic
passes the excitation and emission fibers [14]. materials). The correlation of in situ measured EEMs with
The analysis of EEM data has received much attention those measured by LIF-EEM HPLC after soil extraction
in the literature beginning with Warner et al., with least could aid in the identification and correction of matrix ef-
squares [15] and rank annihilation [16] methods of analy- fects. In addition, a comparable measurement on a sample
sis. This first attempt at rank annihilation was later im- extracted from a known quantity of soil could allow cali-
proved by Lorber with RAFA [17,18]. The use of the gen- bration of the in situ fluorescence measurements to pro-
eralized rank annihilation method (GRAM) [19, 20] has vide concentrations (mg of analyte per kg of soil). During
been demonstrated by Kowalski and coworkers. GRAM the development of the LIF-EEM HPLC system it became
207
Fig. 3 EEMs of standard compounds used for RAFA. a) X, b) N, and emission. There can be cross-talk between channels at either
c) 1-MN, d) 2-MN, e) ACE, f) FLE, g) Ph, h) A, i) FLA, j) P, k) the CCD detector or the fiber optic array. In our system, excitation
BA, l) C, m) BBF, n) BKF, o) BAP, p) DA, q) BP light from each channel is detected in neighboring channels be-
cause of charge blooming on the CCD chip, which is the spilling
over of photon-generated electrons from pixels whose well capac-
ity has been exceeded by excess excitation light [35]. While filters
LC-235C). The column used was a 3.3 cm diameter, 5 m particle are often used to reject scattered light in traditional single wave-
size, C18 column (Perkin-Elmer, Brownlee Columns, Norwalk, length fluorescence experiments, in a multi-channel LIF system a
CT). A C18 guard column (Alltech, Waukegan, MI) was used be- different long-pass filter would be required to efficiently remove
fore the analytical column. The separation conditions were as fol- the scattered light from each channel while not absorbing any of
lows: flow rate of 1.5 mL min1, isocratic elution (80% acetoni- the fluorescence. The placement of multiple filters is not readily
trile/20% water) for 5 min. All injection volumes were 25 L and possible in such a system while maintaining optimum sensitivity
the column oven temperature was set at 30 C. This system and [6]. However, we have found that subtraction of a blank EEM re-
method were used to measure the standard EEMs (single compo- moves the majority of the scattered light resulting in a 94% de-
nents), the 17 component mixture, and the soil extract. crease in the height of a contaminating scattered light peak. Data
Another HPLC method was used for the purposes of determin- post-processing routines can remove any residual scattered light
ing the LIF-EEM systems sensitivity towards the various PAHs artifacts. The cross-talk of fluorescence from channel to channel
and the conventional analysis of the unknown soil extract sample. would represent a greater challenge, because of the contamination
This required complete separation of the analyte peaks and was ac- of the analytical information sought. Measurements were per-
complished by using more rigorous separation conditions coupled formed to determine whether fluorescence cross-talk between
with a more selective column (Green PAH, Hypersil, UK). The channels was significant. This was done by exciting the fluores-
separation conditions were isocratic elution (50% acetonitrile/50% cence of quinine sulfate (0.1 g L1 in ethanol), static in the capil-
water) for 5 minutes, and then a linear gradient elution (to 100% lary, using one excitation channel at a time while collecting data
acetonitrile) for 25 min. A column equilibration time of 5 min was from all the emission fibers. The fluorescence cross-talk, defined
used between runs. All other parameters were the same as in the here as the peak fluorescence level in the emission channel being
other method. The detection limits obtained using the isocratic elu- tested divided by the peak fluorescence level in the emission chan-
tion method are expected to be somewhat higher due to the greater nel corresponding to the excitation, was <0.9% for all cases.
band broadening associated with isocratic separations. However,
the retention times with the rapid method are short (less than
4 min) and this tends to reduce the band broadening effect.
Data processing
Detector characterization The LIF-EEM data were acquired in the autostore mode of the
CCD software. In this mode, the software collects data continu-
An important consideration regarding detector performance in a ously and saves each frame, consisting of seven emission spectra,
multi-channel instrument is channel separation, both for excitation one at each of seven excitation wavelengths, i.e., an EEM. A pre-
210
Fig. 4 Relative intensity plots for rapid HPLC of 17 component 365 nm) in detector counts on a per channel basis. In order to iden-
mixture. a) PDA UV chromatogram (255 nm) (dashed line), total tify temporal regions of interest, the data are reduced by summa-
summed fluorescence (solid line), summed fluorescence at differ- tion in the emission wavelength dimension. A C++ program has
ent excitation wavelengths b) 258 nm, c) 266 nm, d) 278 nm, e) been written to read the processed files (background, scattered
289 nm, f) 299 nm, g) 315 nm, h) 342 nm light removed, and normalized) and sum the intensities at all emis-
sion wavelengths, outputting channel (excitation wavelength)
sums versus time or frame number. The total fluorescence chro-
viously measured file containing the CCD dark current and any matogram is simply the plot of the sum of the normalized excita-
other possible light reaching the detector, excluding the laser exci- tion channel sums as a function of time. The RAFA calculations
tation lines, was subtracted in real time by the software on a per [17, 18] were programmed in MatLab [37] (MathWorks Inc., Sher-
frame basis. In this application the integration time was 3 s and the born MA), and were run on a Pentium 166 MHz PC. RAFA was
file processing/writing was accomplished during the subsequent selected as the analysis method of choice as the added complexity
integration period. During the typical 5 minute HPLC separation, of multi-analyte GRAM analysis was deemed unnecessary for this
100 files were collected at 37 kB each, totaling 3.7 MB of data. application. The RAFA calculations on the PAH mixtures were
The raw data were processed using programs written in C++. performed sequentially using EEMs of the pure components as
Certain functions (standard deviation and smoothing) were called standards.
upon from a commercial C++ function library package (Science,
Engineering, and Graphics Tools, Quinn-Curtis, Inc., Needham,
MA). The programs were designed to allow rapid background sub- Reagents
traction and scattered light removal for the large number of files
generated during each measurement [25, 36]. This was accom-
plished by first subtracting a blank EEM, i.e., that of pure mobile The following PAHs were of 98% purity or higher, with the ex-
phase measured when no analytes were present in the detection re- ception of 1-MN (95%), and were used without further purifica-
gion. Small residual scattered light peaks are left behind after tion: N, ACE, FLE, Ph, A, P, BBF, 1-MN, 2-MN (Aldrich Chem-
blank subtraction, due to variations in the mobile phase composi- ical Co., Milwaukee, WI), FLA, BKF, BP, BA, C, DA, and BAP
tion. These are removed using an automated C++ program, which (Acros Chemical, NJ) (See Table 1 for compound abbreviations).
scans the derivative spectrum to find transition points that define p-Xylene (98%) (J.T. Baker, Phillipsburg, NJ) was also used with-
scattered light peaks (<10 nm), which can then be removed and in- out further purification. The mobile phase used was HPLC grade
terpolated data substituted [25, 36]. acetonitrile (Fisher Scientific, Fairlawn, NJ) with distilled water
Each EEM was power normalized using the peak calibrant prepared in house. All PAHs were prepared in the mobile phase
(0.10 g L1 quinine sulfate in ethanol) fluorescence intensity (at used for analysis (80% acetonitrile/20% water).
211
Fig. 5 Selected EEMs from the rapid separation of the 17 compo- tional to the excitation energy. In order to account for
nent mixture. The position of the EEM in the chromatogram is variations in the source intensity during the approximately
given by the lettering of the normalized summed fluorescence
point that corresponds to that EEM (excitation wavelength is Ex , 4 minute HPLC analyses, three measurements of quinine
emission wavelength is Em ) sulfate (0.10 g L1 in ethanol) fluorescence were made be-
fore and after each analysis. The average of all six mea-
surements served to normalize the excitation energy de-
An unknown mixture of significant complexity was chosen to
pendence of the fluorescence sums and EEMs for a given
highlight the capability of the instrument to identify analytes with analysis. The % standard deviation for all six measure-
significant spectral overlap and fluorescence contamination. The ments was roughly 7% for all cases. The variation in any
sample chosen was an extract of a soil sample from Otis Air Na- given channels excitation energy over several days was
tional Guard Base (OANGB) in Cape Cod Massachusetts. The 30%. While the variations between measurements are
sample was extracted according to a procedure using ultra-sonica-
tion and reversed-phase column clean-up described elsewhere accounted for by the normalization procedure, short-term
[36]. Conventional HPLC with PDA detection was performed on variations occurring in between the calibration measure-
the sample to determine the identities and concentrations of the ments remain unaccounted for and may contribute to er-
PAHs present. rors in the subsequent analysis. Due to the spatial and
temporal variations of the Raman shifted beams [36] and
our fiber launch conditions (overfilled to prevent dam-
Results and discussion age), the variations may be greater than measured. These
unaccounted variations may be responsible for the degra-
Continuous monitoring of the source intensities over half- dation of quantitative information produced by the sys-
hour periods was performed to ascertain excitation inten- tem. Further work is being performed to improve the sta-
sity stability. This was accomplished using three second bility of this novel excitation source.
exposures of the CCD (each integrating the results of 60
laser shots) with a standard solution of quinine sulfate
present in the capillary, the fluorescence being propor-
212
Fig. 6 Selected EEMs from the rapid separation of the ONAGB ues to within 11% in all cases. It should be noted that the
soil sample extract. The position of the EEM in the chromatogram RTs of the individual analytes were unknown (refer to Fig.
is given by the lettering of the normalized summed fluorescence
point that corresponds to that EEM (excitation wavelength is Ex , 4 a) and had to be determined through 17 separate HPLC
emission wavelength is Em ) analyses (described above) of pure standards injected
into the same system under identical conditions (requiring
85 min of analysis time).
EEM is dominated by BKF with a smaller amount of BBF Overall, the system achieved semi-quantitative perfor-
present, obvious by observing the same doublet with mance, with RAFA-calculated concentrations generally
peaks at 420 nm and 435 nm. (22 out of 25) being within a factor of 2 from the known
There are two performance comparisons to be made: values and many (18 out of 25) being within a factor of
between the measured analyte RTs and concentrations and 1.5. All but three concentrations determined by RAFA
the known RTs and concentrations. Table 2 lists the were within 50% of the known concentrations, with the
known RTs and analyte concentrations and compares RAFA results being generally higher. This is not unex-
them with the RAFA determined values. The RAFA RTs pected, since there is no constraint in RAFA against at-
are the times at which the peak concentrations of analytes tributing the observed fluorescence sequentially to more
were determined. All 17 analytes were correctly identified than one analyte. Three of the values (P, DA, and BP)
and the RAFA-predicted RTs agreed with the known val- were determined to within 10% error (ratio, 0.91.0).
214
Table 3 Comparison of
known retention times and Analyte PDA RTa RAFA RTb Ratioc PDA (Known) RAFA Calculated Ratiod
concentration values with (min) (min) Conc (mg L1) Conc (mg L1)
RAFA determined values for X nd nd nd nd
the ONAGB soil sample ex-
tract N nd nd nd nd
1-MN nd nd nd nd
2-MN nd nd nd nd
ACE nd nd nd nd
FLE nd 0.75 nd 0.13
Ph nd nd nd nd
A 0.95 1.00 1.05 1.45 1.46 1.01
Symbols: nd = non-detect, FLA 1.07 1.00 0.93 1.05 1.95 1.86
RT = retention time; aphoto- P 1.23 1.20 0.98 1.99 2.33 1.17
diode array detector deter- BA 1.56 1.55 0.99 0.27 0.94 3.48
mined RTs by single analyte
injection, bpeak concentration C 1.57 1.55 0.99 0.79 2.11 2.67
time from rank annihilation BBF 2.09 2.10 1.00 1.47 0.94 0.64
factor analysis, cratio of RAFA BKF 2.27 2.20 0.97 0.62 1.07 1.73
determined RT to PDA deter- BAP 2.54 2.55 1.00 0.31 0.40 1.29
mined RT, dratio of RAFA DA nd nd nd nd
determined concentration to
known value BP nd nd nd nd
A linear regression of measured and RAFA calculated Table 3. The PDA and RAFA determined RTs for the
concentrations versus known analyte concentration shows OANGB soil extract agreed to within 9%. All eight ana-
a general linear trend, as indicated by a r2 value of 0.97. lytes present (determined by HPLC) were correctly iden-
The rapid separation technique with RAFA analysis of tified with one additional compound (FLE) found that was
EEMs may therefore be said to provide semi-quantitative not detected using the standard HPLC analysis. All RAFA
results for specific chemical substances. determined concentrations were within a factor of 2 of the
A soil extract sample collected at OANGB was ana- known values with the exception of BA and C, ratios of
lyzed to determine the systems ability to characterize a 3.5 and 2.7, respectively (the poorer result may be a result
complex real world sample. The normalized summed flu- of spectral overlap). The other analytes had ratios ranging
orescence chromatogram and selected EEMs are shown in from 0.6 to 1.7. Four (A, P, BKF, and BAP) out of eight
Fig. 6. The summed fluorescence chromatogram reveals a analytes had ratios that were within 0.51.5. It is reason-
region of elevated baseline from 0.5 min to 2.5 min with able to expect that the performance of the system should
several peaks overlaid. The first EEM at 0.65 min (Fig. 6 a) be poorer for BA and C due to spectroscopic overlap [36].
contains broad fluorescence emission (350490 nm) that
does not match any EEM in the library (Fig. 3). The sec-
ond EEM at 0.95 min (Fig. 6 b), contains the characteristic Conclusion
anthracene signal similar to the standard EEM shown in
Fig. 3 h. There is additional, unaccounted, fluorescence There are relatively few chromatographic detection tech-
emission from 313 nm to 427 nm (324 nm peak) at exci- niques that can determine analyte identity and concentra-
tation wavelengths 266288 nm. The third EEM at tion in an unseparated or partially separated mixture; most
1.15 min (Fig. 6 c), contains the fluorescence profile of require complete chromatographic resolution of neighbor-
fluoranthene with long wavelength emission from 356 nm ing peaks. Our dual-fiber optic LIF-EEM detector has
to 506 nm. There is additional fluorescence near the same proven effective in the rapid characterization of complex
region as in the previous EEM: 310340 nm (peak at mixtures. Optical fibers as channel separators and light
323 nm). This may indicate a common type of interfer- collection devices are beginning to be used in commercial
ence that was co-extracted from the sample with the PAHs analytical devices. In a commercially available UV-VIS
that is contributing to the elevated baseline. The peak of PDA detector (Thermo Separation Products, UV6000LP,
the summed fluorescence plot is defined by EEM d, which San Jose, CA), fibers are used to transform the light
shows the fluorescence profile of pyrene, similar to the emerging from a reflective light pipe flow cell (circular
standard in Fig. 3 j. The complete chromatographic over- beam profile) into the rectangular shape required by the
lap of BA and C can be seen in Fig. 6 e, where there is sig- diode array detector, thus minimizing losses associated
nificant spectral overlap (refer to standard EEMs, Fig. 3 k with a circular beam incident on a slit [38].
and 3 l). The spectral signature of BBF can be seen in The dual-fiber optic capacity makes our detection sys-
EEM f in Fig. 6 with additional emission signal (possibly tem flexible and potentially applicable in several areas.
due to a co-eluting unknown) in the 290330 nm region at These include FIA (flow injection analysis) [25], CE
excitation wavelengths 258278 nm. (capillary electrophoresis), and manufacturing process
The RAFA results and comparison with conventional control [39] (e.g., fuel production, natively fluorescent or
HPLC for the OANGB soil extract sample are given in tagged bio-polymeric products). The use of dual-fiber op-
215
O R I G I N A L PA P E R
Received: 8 June 2001 / Revised: 28 July 2001 / Accepted: 25 August 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract For measurement of biomarkers from poly- food prepared by grilling, smoking, or storage in charred
cyclic aromatic hydrocarbon (PAH) exposure, an analyti- wooden casks. PAH exposure is also caused by cigarette
cal method is described quantifying hydroxylated PAH and cigar smoking [2]. Biomarkers of human exposure to
(OH-PAH) in urine samples. This method determined PAH are necessary for the accurate determination of ex-
monohydroxy metabolites of naphthalene, fluorene, phen- posure and body burden from the above-mentioned
anthrene, fluoranthene, pyrene, chrysene, benzo[c]phen- sources. By measuring metabolites of PAH (PAHm) in
anthrene, and benz[a]anthracene. The sample preparation urine, short term (1 to 3 days) exposure may be evaluated.
consisted of enzymatic hydrolysis, solid-phase extraction A widely adopted method was reported by Jongenee-
and derivatization with a silylating reagent. Five carbon-13 len and coworkers for the quantification of 1-hydroxy-
labeled standards were used for isotope dilution. Ana- pyrene from urine [3]. Their protocol includes enzymatic
lytes were separated by gas chromatography (GC) and hydrolysis of the conjugated metabolites, solid-phase ex-
quantified with high-resolution mass spectrometry (HRMS). traction (SPE) with C-18 media, separation with high-per-
This method produced good recoveries (4170%), linear- formance liquid chromatography (HPLC), and detection
ity, and specificity. Data were corrected for blank levels by fluorescence emission. Recent exposure assessments
from the naphthalene, fluorene, and phenanthrene metab- using this method include workers at a gasworks plant,
olites. Method detection limits ranged from 2 ng L1 for firefighters at a training facility, and children from an in-
1-hydroxypyrene to 43.5 ng L1 for 1-hydroxynaphtha- dustrial region [4, 5, 6]. Another HPLC method that de-
lene. Using quality control charts from two urine pools, termines hydroxypyrene, hydroxyphenanthrene, hydroxy-
the method can be readily applied to biomonitoring PAH benz[a]anthracene, and hydroxybenzo[a]pyrene has been
exposure. developed by Angerer and coworkers [7, 8]. They applied
their method to monitor occupational exposure of workers
Keywords Polyaromatic hydrocarbons Isotope dilution in a fireproof stone-producing plant. Exposures to PAH
High resolution mass spectrometry Human urine were found with the conclusion that the workers expo-
Solid-phase extraction sure was too high when compared with other industrial
plants and the exposure limits for benzo[a]pyrene.
Several groups have worked on the analysis of PAHm
Introduction using GCMS techniques and incorporate varying sample
preparation methods. With solid-phase microextraction,
Many classes of chemicals are thought to be carcinogenic Gmeiner and coworkers developed a screening method
or genotoxic. Among these, PAH are one of the most sig- for PAHm with GCMS to detect 10 monohydroxy PAHm
nificant priority pollutants based upon the amounts pro- in urine samples [9]. Korfmacher and coworkers have
duced by combustion of biogenic and anthropogenic ma- used GCMS with chemical ionization to analyze metab-
terials [1]. Human exposure to PAH is generally caused olites of nitro-PAH from microsomes [10]. Incorporating
by airborne particulates and by dietary consumption of liquidliquid extraction and fractionation with a silica gel
column, Wilson and coworkers have used GCMS to
monitor PAH exposures in children of low-income fami-
lies [11]. A productive method has been applied by Grim-
C.J. Smith () W. Huang C.J. Walcott W. Turner mer and coworkers who have used liquid extraction and
J. Grainger D.G. Patterson Jr
Centers for Disease Control and Prevention,
GCMS to determine metabolites of phenanthrene, fluo-
4770 Buford Highway, MS F-17, Atlanta, GA 30341, USA ranthene, pyrene, chrysene, and benzo[a]pyrene in urine
e-mail: cps6@cdc.gov [12, 13, 14, 15]. They have demonstrated that the relative
217
GCMS conditions
Recovery
O R I G I N A L PA P E R
Received: 23 July 2001 / Revised: 24 September 2001 / Accepted: 13 October 2001 / Published online: 12 December 2001
Springer-Verlag 2001
Abstract Glass fragments dating from the seventh and Co(II) ions. In some instances glass colour can arise from
eighth century AD were excavated in the Crypta Balbi in light scattering, because of the presence of very minute
Rome. They were studied to detect agents involved in particles dispersed in the glass matrix; as an example, trans-
colour development and opacification. Reflectance spec- parent ruby glass is coloured by gold or copper particles
tra recorded on powdered samples revealed the contribu- [1]. Depending on their size, particles which segregate
tion of Fe(II), Fe(III), Mn(III), Cu(II), and Co(II) ions in from a transparent matrix can cause opacity of the glass
determining colour hues. The effect of the Mn/Fe atomic elemental copper or copper(I) oxide particles are found in
ratio on glass colour is discussed. It is apparent that me- red opaque glass [2, 3]. Apart from these, opacifying
dieval glassmakers in Italy could obtain a wide range of agents exploited since ancient times include calcium anti-
colours by exploiting the presence of iron and manganese monate and tin dioxide (which generate a white opaque
as contaminants of sand and flux and controlling the glass), and lead antimonate and lead stannate (which gen-
amount of oxygen let into the furnace. X-ray diffraction erate a yellow opaque glass) [1, 4].
and scanning electron microscopy coupled with energy- Fe(II) and Fe(III) ions are the most frequent colouring
dispersive X-ray analysis were used to study opaque frag- agents in ancient glass, because iron is normally present
ments. The presence of calcium antimonate was detected in a glass batch as a contaminant of sand; otherwise, addi-
in white, blue, and blue-green fragments, and elemental tional iron can be added intentionally to enhance colour
copper was detected in a red glass. saturation. Depending on the relative amount of Fe(II)
and Fe(III) ions, different colour hues can be obtained [1,
Keywords Ancient glass Colourants Opacifiers 5, 6]. Glassmakers could, therefore, produce glass of dif-
Reflectance spectroscopy x-raydiffraction SEM-EDS ferent colours by modifying the redox conditions which
Archaeometry determine electron transfer between the two iron oxida-
tion states. This could be achieved by controlling the
amount of oxygen present in the furnace, or by exploiting
Introduction the presence of other redox systems in the batch. In this
respect the most important equilibria are those involving
Coloured and opacified glass has been produced since an- manganese ions, which could be established as a conse-
cient times. Glass colour can be determined by the pres- quence of the introduction of this element either occasion-
ence of metal ions dissolved within the glass matrix, ally, as a contaminant of raw ingredients, or intentionally.
which absorb visible light as a result of electronic transi- Oxidation of Fe(II) to Fe(III) by oxygen, Mn(III) ions, or
tions [1]. The most common colouring agents present in other suitable agents (for instance Sb(V)), causes a colour
ancient glass are Fe(II), Fe(III), Mn(III), Cu(II), and shift from bluegreen towards green, yellowgreen, and
yellow. Fe(II) renders a glass blue whereas Fe(III) colours
it pale yellow and the combined presence of the two ions
in different ratios determines the actual tint of the glass. In
P. Mirti () P. Davit M. Gulmini addition, a colourless or nearly colourless glass can be ob-
Dipartimento di Chimica Analitica, tained when iron is prevalently present in its oxidised
via P. Giuria 5, 10125 Torino, Italy
e-mail: mirti@ch.unito.it
form and its total concentration is low, because of the very
low absorption coefficient of Fe(III) ions; in such circum-
Present address:
P. Davit
stances the presence of Mn(III) in moderate excess can
Dipartimento di Chimica IFM, Universit di Torino, add to the colourless effect, because its purple hue coun-
via P. Giuria 7, 10125 Torino, Italy terbalances the pale yellow hue of Fe(III). Finally, a pur-
222
ple glass is generated when Mn(III) is present in greater flectance spectroscopy is the considerable diminution of
excess. colour saturation, and its dependence on sample grain
Apart from iron and manganese, copper and cobalt size. Hue, however, is not affected by sample grinding.
were important colouring agents in the ancient glass in- Opacifying agents can be identified by X-ray diffrac-
dustry. Both Cu(II) and Co(II) ions generate a blue glass, tion, because their crystal structure generates peculiar dif-
but the former determines in a rather greenish hue and the fraction patterns. X-ray powder diffractograms can be
latter a rather purplish hue. Co(II) ions, moreover, have a recorded using of the same powder used for reflectance
definitively higher absorption coefficient than Cu(II) ions, spectroscopy, because both techniques are analytically
so cobalt still plays a major role in colour development non-destructive. Whenever powders are difficult to ob-
even when its concentration is significantly lower than tain, diffraction patterns can be recorded on small samples
that of copper, as is frequently found in blue ancient glass. by use of a microdiffractometer. Apart from minute items,
Occasionally the ubiquitous presence of iron can some- which do not provide enough sample for X-ray powder
how affect the actual hue of a blue glass, in particular diffraction (XRPD), microdiffractometry (MXRD) is par-
when copper is the main blue colourant. ticular useful for studying non-destructively glass frag-
Calcium antimonate, either Ca2Sb2O7 or CaSb2O6, was ments bearing particular decorative features, for example
the first opacifying agent used in glass production [1, 4]. vessels decorated with filaments of opaque glass.
It is formed by reaction of an antimony-containing ingre- Scanning electron microscopy (SEM) is another tech-
dient with calcium present in a soda-lime matrix and nique which is successfully used to study ancient glass.
causes the development of a white opaque glass. Since By taking images in the secondary electron (SE) or back-
Roman times antimony has gradually been replaced by scattered electron (BSE) mode one can observe particles
tin, which forms tin dioxide as a white opacifier, but its dispersed within the glass matrix. Coupling with an en-
use is still documented at the end of the first millennium ergy-dispersive detector (SEMEDS) also enables the
AD [4, 7]. Both antimony and tin could be used to pro- analysis of selected areas of the glass sample. Because of
duce a yellow opaque glass, when added to a lead glass, their small size (down to 1 m or even less), opacifying
because of the precipitation of yellow lead antimonate particles might not always be reliably analysed quantita-
(Pb2Sb2O7) and lead stannates (Pb2Sn2O4 or PbSnO3) re- tively, because the electron beam can also excite X-ray
spectively [4]. Other colours could be obtained in opaque emission in the surrounding matrix. Usually, however, the
glass as a result of the joint effects of opacifying and
colouring agents; for instance, blue and green opaque Table 1 Date, form, and colour of the studied fragments
glass can be obtained in the presence of Cu(II) with a
white or yellow opacifier, respectively. Finally, a red Sample Century Typology Colour
opaque glass is obtained by segregation of elemental cop- CB1 7th Foot of chalice Blue
per or Cu(I) oxide particles. CB4 7th Foot of chalice Blue
UVvisibleNIR spectroscopy, X-ray diffraction, and CB7 7th Foot of chalice Blue-green
electron microscopy are suitable techniques for studying CB10 7th Foot of chalice Green
colourants and opacifiers in glass. UVvisibleNIR spec- CB13 7th Foot of chalice Yellow
troscopy enables assignment of absorption bands to spe- CB15 7th Rim of lamp Blue-green
cific colouring agents [6, 8, 9, 10, 11]. Fe(II) ions gener- CB18 7th Base of lamp Yellow
ate a wide absorption band centred between 1050 and CB19 7th Base of lamp Yellow-green
1100 nm, with a tail in the visible region, and Fe(III) ions CB21 7th Base of lamp Green
are characterised by absorption at approximately 380 nm. CB24 7th Window pane Colourless
In addition, Fe(III) also gives bands at approximately 420 CB26 7th Window pane Yellow-green
and 435 nm, where bands from Mn(II) may also occur. In CB29 7th Window pane Green
contrast, Mn(III) is recognisable from characteristic ab- CB31 7th Decorative inlay Blue, translucent
sorption at approximately 490500 nm. Cu(II) ions are CB33 7th Decorative inlay Blue-green, opaque
recognised by a band centred at approximately 800 nm, CB34 7th Decorative inlay White, opaque
and Co(II) gives a characteristic triplet between 530 and CB35 7th Decorative inlay Pale blue, opaque
650 nm; the same ion also gives absorption bands in the CB36 7th Decorative inlay Red, opaque
NIR region, at approximately 1250, 1490 and 1750 nm. CB41 8th Foot of chalice Red-purple
Studies on ancient glass fragments generally deal with CB42 8th Foot of chalice Blue
samples with curved surfaces, of irregular thickness, CB43 8th Foot of chalice Blue-green
and/or with weathering products, which prevent the cor- CB45 8th Foot of chalice Yellow
rect collection of transmitted light for recording electronic CB46 8th Foot of chalice Blue-green
spectra. This problem is easily overcome by collecting re- CB47 8th Foot of chalice Green
flected instead of transmitted light, which also enables the CB49 8th Rim of chalice Green
recording of spectra for samples of opaque glass. Re- CB51fil 8th Chalice decoration White, opaque
flectance spectra may be recorded on abraded surfaces, or, CB52 8th Rim of lamp Green
when the form of the glass is complex, on a powdered CB53 8th Rim of lamp Blue
sample. The major drawback of grinding samples for re- CB56 8th Window pane Yellow-green
223
detection of key elements can suffice to disclose the na- filament current 2.43.0 A, working distance 25 mm. SEM images
ture of the opacifier, which can be confirmed by X-ray were obtained by use of either secondary or back-scattered elec-
trons. EDS analyses were performed on opaque inclusions, by fo-
diffraction. In this respect, a positive aspect of the joint cusing the electron beam on 5-m diameter areas; as already men-
use of SEMEDS and MXRD is the possibility of using tioned, this usually enabled the identification of the opacifier by
the same sample for both kinds of investigation. detection of key elements. Analytical data were obtained by use of
This paper reports the use of reflectance spectroscopy, the Oxford QX 2000 software and a ZAF-4 correction program,
using pure elements or oxides as standards. Cobalt was used for in-
X-ray diffraction, and SEMEDS to study colourants and strument calibration.
opacifiers in glass fragments dating from the seventh and
eighth century AD. The fragments were excavated in the
exedra of the Crypta Balbi in Rome [12, 13]. Transparent X-ray powder diffraction and X-ray microdiffraction
and translucent glass included fragments of chalices,
These techniques were used to detect crystalline phases in the
lamps, window panes, and inlays; they were blue, blue opaque fragments. XRPD was performed with a Siemens D5000
green, green, yellowgreen, yellow, redpurple or colour- diffractometer, with graphite-monochromatised copper radiation
less. Opaque glass was represented by fragments of deco- and collecting patterns in the 550 2 range. MXRD was per-
rative inlays, coloured white, pale blue, bluegreen or red. formed on the same samples prepared for the SEMEDS study, af-
ter removal of the graphite layer with silicon carbide paper. Dif-
Some of the eighth century chalices and lamps were, fractograms were obtained by use of a Rigaku PSPC/MDG dif-
moreover, decorated with filaments of white opaque glass; fractometer equipped with a rotating copper anode; X-rays were
a tiny fragment was sampled from one of these for study- collimated on to 100 m diameter areas, and patterns were ob-
ing the opaque decoration. tained in the 2080 2 interval.
Table 1 gives date, form, and colour of the studied frag-
ments.
Results and discussion
ions (present as 0.25% CuO), but further reveals the pat- As for copper, relatively high levels in the bluegreen
tern of Co(II) ions. This pattern is also appreciable in the and green glasses examined are frequently matched by
spectra of CB52, where the band of copper (present as relatively high levels of both antimony and lead. It seems
0.30% CuO) is masked by the Fe(II) band. plausible that copper was not added intentionally to affect
A typical spectrum of a glass coloured green by iron is the final hue, but that it might be present as a consequence
shown by CB29, with bands of similar intensity for Fe(II) of the addition of mosaic tesserae as recycled cullet to the
and Fe(III) ions. Still different, and more similar to the batch [13, 14, 15]. Indiscriminate use of white, blue, yel-
spectrum of the blue-green fragment CB46, are those of low, and green tesserae might have caused the contempo-
CB10 and CB47. Here again there is intense absorption rary presence of copper, antimony and lead in the final
due to Fe(II), with the pattern between 380 and 440 nm of glass, and Cu(II) ions to affect the resulting colour. In this
significantly lower intensity. The greater contribution of context, one might further consider that Sb(V), present as
the Fe(III) pattern compared with CB46 would account a white or yellow opacifier in the recycled tesserae, might
for the development of the green hue. have added its contribution to those of manganese and/or
The spectra of the yellow-green fragments CB19, CB26, oxygen in oxidising Fe(II) to Fe(III).
and CB56 (Fig. 5 and Ref. [12]) are all dominated by the Apart from sample CB35 discussed above, spectra of
bands of the Fe(II) and Fe(III) ions, and resemble that of blue glasses (Fig. 6 and Ref. [12]) are generally charac-
the green fragment CB29. The increased contribution by terised by the prevalent contribution of Co(II) relative to
Fe(III) ions relative to Fe(II), however, accounts for the Cu(II) ions, despite the lower concentration of the former;
development of a yellowgreen rather than a green hue. only the spectrum of CB53 reveals a comparable contri-
The spectra of the yellow glasses (Fig. 5) are finally bution of the two chromophores. The cobalt content of
characterised by still more prevalent Fe(III) absorption blue fragments ranges from approximately 0.01% (CB1
relative to Fe(II). The colour of fragment CB18 is more and CB4) to 0.11% (CB31) CoO (apart from CB35, in
shifted towards orange hues compared with CB13 and which it was not detected), whereas copper content
CB45, and this might be ascribed to the absence of appre- mainly varies between 0.10% (CB1) and 0.92% (CB53)
ciable absorption by Fe(II) ions. Similar to those of the CuO (but reaches 2.85% in CB35). Its higher absorption
yellow fragments are the spectra (not reported) of CB24, coefficient makes the Co(II) ion a more effective chro-
colourless, and CB34, opaque white; here a low total iron mophore than the Cu (II) ion; the increase of the copper to
content, and the prevalence of the oxidized form, account cobalt ratio, however, usually causes a shift from more
for the colourless effect an opaque white, which turns into purplish towards more greenish hues. Fe(III) can further
in CB34 because of the presence of calcium antimonate contribute to the more greenish hue of some samples.
(see below). Fig. 7 finally shows the spectra of the red and redpur-
As observed above, cobalt and/or copper ions can ple samples. The latter is dominated by the intense ab-
modify the hue of a glass mainly coloured by Fe(II) and sorption band of Mn(III) ions. The former is characterised
Fe(III). In this context, it is worth noting that the typical by the removal of the wavelengths below 600 nm as a re-
pattern of Co(II) is discernible in a large number of the sult of light scattering by copper particles (see below);
recorded spectra; this is because of the high absorption spectral features between 800 and 1200 nm also suggest
coefficient of this chromophore, which enables recogni- the presence of Fe(II) and Cu(II) ions. Fe(II) ions would
tion of its pattern even when the element could not be de- have formed from metal iron added to create reducing
tected by ICPOES analysis. conditions in the system (see below), but the presence of
227
Fig. 7 Reflectance spectra of red and
redpurple samples
Opacifiers
Calcium antimonate has been found as the main opaci- the Italian Ministry for University and Scientific Research
fier in white, blue, and bluegreen glass. Its presence at a (MURST) is acknowledged.
rather late date during the first millennium AD suggests
continuity with Roman glass production; in fact, at this
date tin dioxide was already used as an alternative opaci- References
fying agent, even though use of calcium antimonate has 1. Newton R, Davison S (1996) Conservation of glass. Butter-
been documented down to at least the end of the millen- worth-Heinemann, Oxford
nium. Air bubbles and devitrification products add to the 2. Freestone IC (1987) Composition and microstructure of early
overall opacifying effect, and this might account for the opaque red glass. In: Bimson M, Freestone IC (eds) Early vit-
reous materials. British Museum, London, pp 173191
concentration of antimony, which is not particularly high 3. Brill RH, Cahill ND (1988) J Glass Stud 30:1627
compared with levels in other ancient opaque glasses. In- 4. Henderson J (1985) Oxford J Archaeol 4:267291
deed, antimony could derive from more ancient opaque 5. Newton RG (1978) Glass Technol 19:5960
cullet; in contrast with transparent glasses, however, lead 6. Sellner C, Oel HJ, Camara B (1979) Glastechn Ber 52:255264
was found at a significant level in only one of the exam- 7. Freestone IC (1993) Compositions and origins of glasses from
Romanesque champlev enamels. In: Stratford N. (ed) Cata-
ined opaque fragments, and copper only in the blue and logue of medieval enamels in the British Museum. British Mu-
bluegreen inlays, to which it was added as a colouring seum, London, vol II, pp 3745
agent. The occurrence of tin in the latter two fragments, 8. Sanderson DCW, Hutchings JB (1987) Glass Technol 28:99
and the tin to copper ratios observed, might suggest that 105
9. Mirti P, Ferrari RP, Laurenti E, Casoli A (1993) Spectrochim
glassmakers used bronze scraps to introduce copper into the Acta 49A:13611371
melting batch. 10. Orlando A, Olmi F, Vaggelli G, Bacci M (1996) Analyst
A red opaque glass has been found to owe both colour 121:553558
and opacity to the presence of small particles of elemental 11. Bartecki A, Burgess J (2000) The colour of metal compounds.
Gordon and Breach Science Publisher, Amsterdam, chap 8,
copper, less than 1 m across. This indicates that glass- pp 195209
makers well knew how to create strongly reducing condi- 12. Mirti P, Lepora A, Sagu L (2000) Archaeometry 42:359374
tions; in this instance these were almost certainly obtained 13. Mirti P, Davit P, Gulmini M, Sagu L (2001) Archaeometry
by adding iron to the final batch. 43:491502
14. Freestone IC (1992) Theophilus and the composition of me-
Acknowledgements Thanks are given to Dr Lucia Sagu (Dipar- dieval glass. In: Vandiver PB, Druzik JR, Wheeler GS, Free-
timento di Scienze Storiche, Archeologiche e Antropologiche stone IC (eds) Materials issues in art and archaeology III. Ma-
dellAntichit, Universit di Roma La Sapienza) for providing terials Research Society, Pittsburgh, pp 739745
the glass fragments and discussing their archaeological features. 15. Henderson J (1995) Le verre de Dorestad. Continuit tech-
The authors are also grateful to Mr Dario Vaudan (Soprintendenza nologique ou innovation? In: Foy D (ed) Le verre de lantiquit
ai Beni Culturali, Aosta) for the MXRD analyses and to Professor tardive et du haut Moyen Age. Typologie-chronologie-diffu-
Giacomo Chiari (Dipartimento di Scienze Mineralogiche e Petro- sion. Muse Archologique Dpartemental du Val dOise,
logiche, Universit di Torino) for XRPD analysis; to Dr Emanuele Guiry-en-Vexin, pp 5155
Costa, Dr Raffaella Ruffini, and Mr Carmelo Sibio (Dipartimento 16. Cable M, Smedley JW (1987) The replication of an opaque red
di Scienze Mineralogiche e Petrologiche, Universit di Torino) for glass from Nimrud. In: Bimson M, Freestone IC (eds) Early
help in the use of the SEMEDS equipment. Financial support vitreous materials. British Museum, London, pp 151156
from the Italian National Council of Researches (CNR) and from
Anal Bioanal Chem (2002) 372 : 230231
DOI 10.1007/s00216-001-1171-0
I N D U S T RY N E W S
Ralph G. Weyandt
Abreviations BCYE buffered charcoal yeast extract Until now these standardized and, because of the rela-
BSE bovine spongiform encephalopathy ELISA enzyme- tively low technical requirements on the equipment, wide-
linked immunosorbent assay GMO genetically modified spread methods are among the most important routine pro-
organism GVPC BCYE-agar with glycine and three cedures in private and government laboratories and author-
antibiotics (vancomycin, polymyxin, cycloheximide) ities for investigation of foodstuff microbiology around the
LIA lumino-immuno-assay PCR polymerase chain world.
reaction Times have, however, obviously changed abruptly
because of increased knowledge about molecular biology
and biochemistry in the last ten years, application-friendly
solutions for sensitive and mostly rapid determination of
In the field of drinking water and the production and stor- germs have developed from research activities. These so-
age of foodstuffs a variety of bacteria and fungi play a lutions have revolutionized the laboratory landscape for
considerable role with regard to reduction of quality and, providers of analytical services and will continue to do so.
even more, with regard to health risks. Various species of PCR-based rapid determination of hygienic strains is al-
fungi produce so-called mycotoxins in cereals that have most part of the standard repertoire, just as are the varied
been harvested and stored. These mycotoxins are suspected immunochemical determination methods via ELISA or
of causing tumours in man and animals. Fresh egg prod- LIA. Because of new types of biomarker, biochip, and mi-
ucts, fresh mayonnaise, and desserts containing egg can cro-array, fully automated robotic systems, and multiplex
suffer from a mass increase in Salmonella, which cause methods, these techniques are just the beginning.
fatal infections. Types of cheese contaminated with Liste- A variety of conditions have led to this development:
ria have repeatedly led to deaths. Hygienic strains, viruses,
widespread availability of highly pure biochemicals and
and pathogenic protozoa, which are responsible at least lo-
enzymes,
cally for severe epidemics, are frequently spread via drink-
the decoding of genetic target sequences,
ing water, the most important foodstuff. The list of exam-
tailor-made production of gene probes, primers, markers,
ples of serious microbial contamination of foodstuffs and
and antibodies,
drinking water is very long and, as media reports show, is
industrial production of robust laboratory equipment
increasing almost daily.
which guarantees automated and miniaturised process-
As a method for identification of such often-occurring
ing of many samples simultaneously,
pathogens and putrefiers, culture techniques with subse-
development of a new branch of industry operating
quent biochemical and/or serologic diagnostics partially
world-wide, which, via its R&D activities, its coopera-
established over many decades have proven themselves.
tion with universities, and, finally, via a large number of
Progress in this classical field of application of microbiol-
spin-off-companies, can produce new ideas and imple-
ogy was mainly achieved by optimization of the demands
ment them rapidly, and
on nutrients and cultures by means of selective enrichment
changes in the foodstuffs field have led to changes in
of the respective organisms and by (partial) automation of
foodstuff analysis, e.g. the introduction of GMOs and
the steps necessary for identification.
the effects of the on-going BSE-crisis.
The emerging revolution can be seen by way of the ex-
R.G. Weyandt ()
Institut Fresenius GmbH,
ample of the newest methods for Legionella analysis.
Im Maisel 14, 65232 Taunusstein, Germany Whereas up until 3 years ago the only method of determi-
e-mail: weyandt@rud.fresenius.com nation available to the routine laboratory was the classical
231
Legionella analysis according to ISO 11731 [1] (including on the part of the manufacturer, but has also contributed to
the steps: further propagation of the technology itself, to which, on
the other hand, new methods of determination were adapted.
1. membrane filtration,
Finally, and we are now in this exciting phase of develop-
2. treatment with an acid buffer, and
ment not only hard competition among the new prod-
3. cultivation on, e.g., GVPC-agar and on cysteine-free
ucts but also integration of individual determinations in
nutrient agar (e.g. BCYE-Cys); incubation period takes
automated system solutions led, after an initial phase, to
7 to 10 days.
considerable price cuts, so that the new products now
Today the provider of analytical services can choose from hardly need to fear competition from classical methods.
a wide range of detection equipment available on the mar- An obstacle that unfortunately still exists, and that has
ket: for a long time prevented more rapid introduction of the
new technologies, is the usually conservative legitimiza-
optimised classical determination,
tion structures at national and international levels deter-
determination by means of PCR after enrichment via
mination procedures that have once been proven and stan-
filtration [2],
dardized become the gold standard. As long as the new
determination microscopically (epifluorescence) after
procedures are not expressly accepted into the official col-
treatment of the filter residue with gene probes [3], and
lection of methods, acceptance by foodstuffs manufactur-
immunochemical determination in accordance with the
ing, by foodstuffs marketing, and by short-sighted providers
ELISA principle; even suitable as a fast on-the-spot de-
of analytical services remains comparatively low. It is,
termination [4].
therefore, desirable that, with such rapid development of
The same applies currently to Salmonella, Listeria, Campy- new analytical techniques (particularly in the field of hy-
lobacter, Escherichia coli O157, Clostridium spec. or Cryp- giene microbiology) and in view of the urgent need for
tosporidium. The list of manufacturers is also very long, quick, reliable methods, administrative and legislative ne-
and it is clear that the availability of additional methods cessities do not become a bottleneck hampering develop-
depends only on the needs of, and acceptance by, the mar- ment.
ket.
It is encouraging that these ready-made methods of de-
termination usually only guarantee the manufacturer a References
monopoly for a short time, if at all. As long as the analy-
sis is commercially relevant alternatives bringing further 1. ISO 11731 Water quality-detection and enumeration of legionella,
1998
refinement or time-saving are developed quickly. 2. http://www.iqproducts.nl
In any case this development has led not only to differ- 3. http://www.vermicon.com
entiated and problem-related application from the provider 4. http://www.siemens.de/gebaeudemanagement
of analysis services, or to corresponding niche occupation