LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY
Liquid
Chromatography/Infrared
Spectroscopy
Govert W. Somsen
University of Groningen, Groningen,
The Netherlands
Tom Visser
National Institute of Public Health and
Environmental Protection, Bilthoven,
The Netherlands
1 Introduction
2 Coupling Modes and Operating Principles
2.1 Flow-cell Approach
2.2 Solvent-elimination Approach
3 Flow-cell Techniques
3.1 Cell Types and Infrared Detection
Modes
3.2 Use in Liquid Chromatography
3.3 Use in Flow-injection Analysis
4 Solvent-elimination Techniques
4.1 Deposition Substrates and Infrared
Detection Modes
4.2 Early Interfaces
4.3 Spray-type Interfaces
5 State of the Art
6 Perspective and Future Developments
Abbreviations and Acronyms
Related Articles
References
Analytical techniques that combine liquid chromatography
(LC) and infrared (IR) spectroscopy have been developed
primarily to permit specific detection and/or identification
of sample constituents. LC is an important and extensively
used method for the separation of mixtures into their
individual components. IR spectroscopy is very useful for
the characterization of functional groups and has strong
compound-identification capabilities which are especially
suited for the differentiation of structural isomers. Over the
past years the coupling of LC and IR spectroscopy (LC/IR)
has been accomplished by two different approaches. The
first and simplest approach is to use a flow cell through
which the effluent from the LC column is passed while
Encyclopedia of Analytical Chemistry
Edited by Robert A. Meyers.  John Wiley & Sons Ltd, Chichester. ISBN 0471 97670 9
1
the IR spectra are continuously measured. The merits of
flow-cell IR detection include ease of operation, real-time
detection and low maintenance, but its main disadvantage
lies in the significant IR-absorption of the solvents
commonly used in LC. These absorptions seriously limit
both the detection sensitivity and the obtainable spectral
information. The second approach involves elimination
of the LC solvent prior to IR detection. In this approach
an interface is used to evaporate the eluent and deposit
the separated compounds onto a substrate suitable for IR
detection. The primary advantages of solvent-elimination
LC/IR are the possibility to obtain full spectra of
the analytes and the considerably enhanced sensitivity
when compared to flow-cell detection. Unfortunately,
common LC solvents, and particularly aqueous eluents,
are not easily removed and therefore the evaporation
interfaces are often rather complex. This article reviews the
1 developments, practical aspects, applications and current
2 status of LC/IR, covering both coupling approaches. It
2 follows that despite the unfavorable detection limits, flow-
3 cell LC/IR can be useful for the specific and quantitative
4 detection of major components of mixtures. However,
solvent-elimination-based IR-detection should be used
4 when small amounts of sample constituents have to be
6 characterized with a high level of confidence. In general,
7 the practical use of IR detection in LC is still limited,
but the advent of various (commercial) flow-cell and
8 interface designs shows that LC/IR is more and more
being recognized as a feasible and rewarding technique.
8
9
10
15 1 INTRODUCTION
17
IR spectroscopy has a high potential for the elucidation
18
of molecular structures. The IR spectrum of a poly-
19 atomic molecule is based on molecular vibrations, each
19 specifically dependent on atomic masses, bond strengths
and intra- and intermolecular interactions. As a conse-
quence, the entire IR spectrum of an organic compound
provides a unique fingerprint, which can be readily
distinguished from the IR-absorption patterns of other
compounds including isomers. In other words, when ref-
erence spectra are available, most compounds can be
unambiguously identified on the basis of their IR spectra.
Moreover, characteristic absorption bands can be used
for compound-specific detection.
LC is a powerful and versatile separation technique,
which can handle a wide range of sample types and
compound classes. Because of the widespread use of LC
and the (growing) need for analytical procedures that
provide confirmation and/or identification of sample con-
stituents, much effort has been – and still is – devoted to
the coupling of LC with spectrometric techniques such
2 INFRARED SPECTROSCOPY
as mass spectrometry (MS), IR and nuclear magnetic
resonance (NMR) spectroscopy. Today, with modern
Fourier transform infrared (FTIR) instrumentation rou-
tinely available, spectra can be recorded from nanogram,
or even picogram, amounts of pure substance so that IR
detection, in principle, is suited for molecular recognition
at analyte levels frequently met in LC. Unfortunately,
because of the (spectral) characteristics of the mobile
phase, the coupling of LC and IR spectroscopy (LC/IR)
is not straightforward and often requires the construction
of special flow cells or the development of rather complex
interfaces. Therefore, compared with other LC detection
modes such as ultraviolet/visible (UV/VIS) absorption
spectroscopy or MS, the use of IR detection in LC is still
rather limited. Nevertheless, progress in interfacing tech-
niques during the last decade has brought LC/IR to a stage
of analytical utility which suggests that LC/IR may well
become a commonly available and applied technique.
2 COUPLING MODES AND OPERATING
PRINCIPLES
In the first LC/IR systems.1,2/ flow cells were used in a
fashion analogous to LC with on-line UV/VIS absorption
detection. In order to circumvent interfacing difficulties
related to the IR absorptions of the mobile phase, in
1979 Kuehl and Griffiths.3/ developed the first solvent-
elimination based LC/IR set-up in which the eluent
is evaporated prior to IR detection. Since then two
approaches can be discerned in LC/IR, namely, the flow-
cell approach and the solvent-elimination approach. In
the contemporary practice of LC/IR both approaches
are applied, although the detection limits and spectral
information obtained with either approach may differ
considerably. The principles, applications, merits and
limitations of flow-cell and solvent-elimination LC/IR
have been reviewed in a number of books and papers..4 – 8/
2.1 Flow-cell Approach
The simplest way to couple LC and IR is to let the column
effluent pass directly through a flow cell suited for IR
measurements. The IR absorption of the LC effluent is
continuously monitored and spectral data are collected
on-the-fly and stored throughout the chromatographic
run. During or after the run, the spectra and/or IR
chromatograms are computed and absorption due to the
mobile phase is subtracted. In a flow-cell design, band
broadening caused by detection is easily minimized.
Unfortunately, the absolute sensitivity of IR spec-
troscopy is relatively poor compared to spectrometric
techniques like MS, UV/VIS and fluorescence spec-
troscopy. Moreover, solvents suited for LC generally
have many absorption bands in the IR region, which
leads to serious limitations of flow cell LC/IR interfacing.
Firstly, absorption bands of analytes may be obscured by
eluent absorptions. In other words, in flow-cell LC/IR, the
spectral information that can be obtained is limited and
depends on the window provided by the mobile phase
used. Moreover, ill-considered subtraction of solvent
absorption bands may lead to the erroneous conclu-
sion that there is no absorption of the analyte in the
corresponding spectral regions. Secondly, gradient elu-
tion can hardly be applied as the changing composition
of the mobile phase frustrates proper subtraction of the
background. To some extent, the second derivative of a
spectrum can be used to compensate for this problem,
but accurate spectral correction is virtually impossible.
Thirdly, the pathlength of the flow cell has to be limited
to ensure a certain spectral window and sufficient energy
reaching the IR detector. For organic solvents commonly
used in LC/IR, the effective pathlength rarely exceeds
1 mm which, bearing in mind Beer’s law, seriously reduces
the analyte detectability. For aqueous eluents, the largest
tolerable pathlength is even shorter. As can be seen from
the spectrum of water in Figure 1, most of the mid-IR
region between 4000 and 1000 cm 1 is opaque, even at a
very short pathlength of 10 µm. It implies that a practical
combination of reversed-phase liquid chromatography
(RPLC) and IR via a transmission flow cell is restricted to
specific applications. Finally, the use of signal averaging
to improve the signal-to-noise ratio (S/N) of the spectra is
limited owing to the short time that is available for analy-
sis under dynamic conditions. Occasionally, the principle
of stop-flow can be used to enhance the S/N without too
much loss in chromatographic performance.
The choice for a certain mobile phase and flow-cell
properties is primarily determined by chromatographic
considerations and the desired spectral information. In
principle, the spectral window of the solvent can be
very small for quantitative analysis as the measurement
Transmittance (%)
20
10
0
4000 3000 2000 1000
Wavenumbers (cm−1)
Figure 1 IR transmission spectrum of water recorded using a
flow cell with silver bromide windows and an optical pathlength
of 10 µm.
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY 3

of a single wavenumber, e.g. the band maximum, is analysis (FIA). An important feature of the flow-cell
sufficient. Contrary, qualitative information desires IR LC/IR technique is the ability to monitor the elution
transparency over a much wider spectral region in order of compounds with specific structural characteristics, i.e.
to determine the presence or absence of functional functional groups. Besides, apart from regions of solvent
groups, or to identify a compound by its IR fingerprint opacity, spectra can be obtained instantaneously from
region (1300 – 600 cm 1 ). Obviously, the spectral window any point in a chromatographic peak.
of the mobile phase is inversely proportional to the
cell thickness and thus, an excess of sensitivity can be
traded for more spectral information and vice versa. 2.2 Solvent-elimination Approach
The experimental parameters in LC/IR are, therefore, a The compatibility and time-domain difficulties connected
compromise between chromatographic and spectroscopic to flow-cell IR detection can be circumvented by coupling
considerations giving maximum cell thickness and solvent LC and IR spectrometry via a substrate suitable for
transparency..5 – 8/ IR detection. In this indirect approach, the eluent
In order to minimize the problems associated with is eliminated and the chromatographically separated
eluent absorption, the choice of the solvent in flow-cell compounds are immobilized on the substrate prior to
LC/IR is generally restricted to chlorinated alkanes and the collection of IR spectral data. The immobilization of
deuterated solvents. These solvents leave relatively wide the chromatogram is accomplished by using an interface
windows in the spectrum, although even these eluents which evaporates the eluent and continuously deposits the
inevitably obscure parts of the spectral functional group column effluent onto the moving substrate. In this way,
region (4000 – 1300 cm 1 ) and the fingerprint region. The interference-free IR spectra of the deposited compounds
use of a small percentage of a more polar modifier in
can be recorded independently from the LC conditions
the eluent, as is quite common in normal-phase liquid
and the sensitivity of the FTIR spectrometer can be fully
chromatography (NPLC), may already prohibit effective
exploited. A schematic of a solvent-elimination LC/IR
detection. Owing to the short optical pathlength, the
set-up is depicted in Figure 2.
absolute limit of detection in on-line LC/IR often is in
Besides the possibility to acquire full spectra, there are
the order of a few micrograms, which implies that analyte
some additional advantages of the solvent-elimination
concentrations of 1 – 10 g L 1 have to be injected to obtain
approach with respect to flow-cell IR detection. By careful
identifiable spectra.
control of the interface performance and the speed of the
Tran et al..9/ proposed a completely different flow-
substrate, concentrated deposits may be obtained which
cell LC/IR technique with, potentially, higher sensitivity.
will enhance analyte detectability. Spectral analysis can
Different from the conventional way of measuring the
be performed without any time constraints since the
IR absorption over a certain wavenumber region, this
chromatogram is stored on the substrate. This implies
technique, called IR thermal lens spectrometry, is based
that signal averaging can be used and analyte spots can be
on the measurement of the temperature rise that is
analyzed repeatedly. For instance, after rapid screening
produced in an illuminated sample by non-radiative
of the deposited trace applying low spectral resolution
relaxation of the energy absorbed by molecules upon IR
and only one scan per spectrum, high-resolution spectra
laser excitation. Detection is carried out either directly,
with a high S/N can be recorded for a few interesting parts
i.e. at a specific wavelength where only the analyte of
of the chromatogram. Moreover, spectrometric analysis
interest absorbs, or indirectly, i.e. at a wavelength where
of the stored chromatogram in principle can be carried
only the mobile phase absorbs. Obviously, no spectral
out at any convenient time or place, which may be helpful
information is obtained with this quantitative technique,
when suitable IR facilities are limited and spectrometers
and the wavenumber region that can be covered with
have to be shared.
IR-tunable lasers is limited.
In conclusion, flow-cell IR detection is not a widely
applied technique in LC as result of the interference of
absorption bands of the mobile phase and the limited LC
sensitivity of IR spectroscopy. Applications largely focus
on dynamic systems where low detection limits are
IF
not crucial and solvents appropriate for IR detection
,,,,

T
S
can be selected more or less freely, that is, without Sub
having unacceptable detrimental effects on the analytical
performance. Examples of such techniques are gel Figure 2 Schematic representation of a set-up for sol-
permeation chromatography (GPC), also referred to as vent-elimination LC/IR; LC, liquid chromatograph; IF, inter-
size-exclusion chromatography (SEC), and flow-injection face; Sub, deposition substrate; T, transfer; S, spectrometer.
4 INFRARED SPECTROSCOPY
Evidently, the solvent-elimination approach to LC/IR is
(technically) more complicated than on-line IR detection.
It requires an interface which should adequately effect
the evaporation of the eluent and, at the same time,
maintain the chromatographic resolution during the
deposition process. In this respect, the LC flow rate,
the composition of the eluent, the nature of the analytes
and the substrate material are important factors. For
example, small volumetric flows of a volatile solvent may
be readily evaporated, while rapid elimination of aqueous
eluents will require an interface with enhanced solvent-
elimination power. Elimination of the eluent may also
be hampered by the presence of non-volatile additives
such as buffer salts. Next to eluent evaporation, ideally
the interface also should provide compound deposition
into narrow spots in order to minimize band broadening
and achieve optimum IR sensitivity. However, complete
eluent evaporation and compact analyte deposition may
well be irreconcilable goals. Therefore, in solvent-
elimination LC/IR reduced flow rates, non-buffered
eluents and/or eluents with a low (or even zero) water
percentage are frequently used.
The compounds analyzed by solvent-elimination
LC/IR, of course, should be considerably less volatile
than the eluent to accomplish their deposition. Since LC
is used for non-volatiles in particular, this condition is
generally met. The quality of the used substrate should
not be affected by either the eluent or the deposited com-
pounds. The substrate also must be compatible with the
selected IR mode without introducing additional inter-
ferences. Furthermore, the physico-chemical properties
of the substrate may influence the efficiency of ana-
lyte immobilization. For instance, residual eluent easily
spreads over a substrate with a hard and smooth surface,
while it may be effectively sorbed by a powder.
During the last decades, the combination of LC
and IR via solvent elimination has been pursued by
several research groups, which designed quite a number
of different interface concepts with varying success.
The common goal of all these LC/IR systems is to
sensitively acquire IR spectra of mixture constituents
that are free from spectral interferences and can be
used for identification purposes. Through the years, the
Table 1 Optical materials for use in IR flow cells
Material Spectral window (cm 1 )
Silver chloride 4000 – 350
Calcium fluoride 4000 – 1100
Quartz 4000 – 2400
Potassium bromide 4000 – 400
Zinc selenide 4000 – 450
KRS-5 4000 – 250
Polyethylene 630 – 30
IR detection limits obtained with solvent-elimination
LC/IR have improved gradually from the microgram to
the low- or sub-nanogram range. Despite the progress,
today there is no single ‘‘perfect’’ solvent-elimination
interface available: every described system has its specific
limitations with respect to, for example, flow rate,
composition of the eluent and/or achievable sensitivity.
The research activities in the field, which until now
have been dominated by the problem of simultaneous
eluent-evaporation and compound-deposition, are still
on-going. Nevertheless, during the last years, several new
commercial interfaces based on the solvent-elimination
approach have been introduced..10,11/
3 FLOW-CELL TECHNIQUES
3.1 Cell Types and Infrared Detection Modes
A variety of flow cells, differing in optical material,
pathlength and cell volume, is available for LC/IR
detection purposes. The cells, including corresponding
beam condensing optics, generally fit in the standard
optical bench of the IR spectrometer. The distance
between the flow cell, i.e. the IR spectrometer, and
the LC system is kept as short as possible in order
to minimize deterioration of the chromatography. As
noted in section 2.1, the selection of a certain cell type
and, particularly, the optical pathlength, depends on the
required information.
The choice for a specific window material is mainly
determined by the properties of the LC eluent and the
spectral region that has to be monitored. A fully IR-
transparent material such as potassium bromide (KBr),
for instance, cannot be used with RPLC. Instead, more
expensive water-insoluble materials such as calcium
fluoride and zinc selenide (ZnSe) have to be chosen.
The optical and physical properties of some commonly
used IR-window materials are presented in Table 1. In
all cases, in order to obtain an identifiable IR spectrum
a minimum amount of analyte has to be present in the
detection cell during the time of measurement. According
to Beer’s law, the minimum identifiable concentration
Solubility in water Sensitive to
slightly soluble complexing agents
insoluble ammonium salts; acids
insoluble hydrofluoric acid; hot sulfuric acid
very soluble water; methanol
insoluble acids; strong alkalis
slightly soluble complexing agents
insoluble organic solvents
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY
decreases when the pathlength of the cell is increased.
However, extending the pathlength also results in an
increase of the eluent absorbance, thus limiting the
spectral window. Commonly, the volume of the flow
cell has to be minimized to such an extent that only a
small part (1% or less) of an LC-peak volume will fill the
cell. Obviously, this places a significant limitation on the
obtainable sensitivity. Through the use of microbore LC
columns, the volume of the flow cells and LC peaks can
be made more compatible and, for that reason, micro-
LC/IR is often preferred. Besides a reduced solvent
consumption, microbore LC may also offer higher peak
concentrations in the cell. At the same time, however, it
should be noted that the sample capacity (both in mass
and volume) of micro-LC columns is rather limited..12/
Two types of commercially available flow cells can
be distinguished for LC/IR: transmission cells and
attenuated total reflection (ATR) cells. The principle
of a transmission flow cell is depicted in Figure 3. The
basic part of the cell consists of an IR-transparent cavity
or two IR-transparent windows separated by a metal
spacer. The optical set is mounted into a metal body
between flexible rings to prevent breakage. The LC
effluent enters and exits the cell via capillary tubes
connected to an assembly of universal fittings and the
IR beam passes perpendicularly through the LC flow.
Additional equipment can be purchased for operation
at elevated temperature and pressure. Zero-dead-volume
(ZDV) flow cells for IR detection have been developed
to minimize the volumes needed to connect the cell to
the column, which is very important when microbore LC
is used. In this type of cell the LC effluent is directly led
LC-tubing
Back plate
,,,, Fitting assembly
, transmission detection.
,,,,,,,
,,,,,,,
Cavity cell
,,,, Front plate
, Gasket
,,,,
Figure 3 Cavity flow cell for IR transmission detection.
5
into the cavity of the optical element (Figure 4). Flow
cells, micro-flow cells and ZDV-cells can be purchased
in many variations, rigid and demountable, differing
in window material, pathlength (0.001 – 0.5 mm) and
internal volume (0.1 – 10 µL). Micro-cells are generally
used in combination with a beam condenser to obtain a
sufficiently high energy throughput and thus enhance the
S/N of the recorded signal and/or spectra.
The second type of flow cell is based on the phe-
nomenon of ATR and is called cylindrical internal
reflectance cell or CIRCLE cell..13/ The principle of
the CIRCLE cell is depicted schematically in Figure 5.
The cell consists of a cylindrically shaped IR-transparent
rod crystal with cone shaped ends that is incorporated in
a boat-type cell body made of stainless steel (SS) or glass.
The effluent of the LC column flows around the optical
crystal while the interrogating IR beam enters the crystal
at one end, reflects off the internal surfaces of the crystal
and then exits at the other end. The cell body fits into a
small optical bench with special input and output optics.
Several crystal materials can be used but ZnSe is com-
monly preferred because of its high IR transparency, high
refractive properties and insolubility in water. CIRCLE
cells can be equipped with heating or cooling jackets
IR-transparent O-ring
window
LC-fitting
LC-tubing
Cell body
Figure 4 Schematic representation of a ZDV flow cell for IR
LC-flow (in)
IR-beam Detector
Rod crystal
LC-flow (out)
Figure 5 Schematic representation of an IR flow cell for
cylindrical internal reflectance detection (CIRCLE cell).
6 INFRARED SPECTROSCOPY
too. The cell design tends to involve a relatively large
sample volume and efforts have been made to reduce the
internal volume of CIRCLE cells to 1 – 25 µL in order
to allow their efficient application in LC. The effective
pathlength of a CIRCLE cell is defined by the number
of reflections in the optical element and, therefore, sensi-
tivity can be enhanced by using longer optical elements.
These, however, also imply an increased cell volume and,
thus, extra broadening of the LC peaks. CIRCLE cells
cannot be used for quantification in a straightforward
manner since the penetration depth of the IR radiation in
the LC eluent is limited (typical 1 – 5 µm) and wavelength
dependent. Special algorithms are used to compensate
for this.
3.2 Use in Liquid Chromatography
As outlined in the previous sections, flow-cell IR detection
is not the principal choice in LC. Yet, it is a valuable
alternative and additional method to obtain specific
quantitative and structural information on analytes. In
view of the relatively poor sensitivity, IR detection is
restricted to applications in which low detection limits are
not required. In GPC, for example, column capacities and
sample concentrations are usually high. Gel permeation
chromatography/infrared (GPC/IR) is well suited for
the characterization and quantification of compositional
changes throughout a (bio)polymer mass distribution.
Besides, the structural differences between the polymer
components (usually homologues) often are small which
implies that one or two spectral windows will suffice for
specific detection.
Applications of on-line LC/IR, including automatic
subtraction of the solvent background, had already been
described in the mid-1970s..1,2/ Injected amounts at sub-
microgram level were found to be feasible for detection
in both NPLC and RPLC separations. In subsequent
studies, Taylor et al..13,14/ demonstrated that microbore
LC (column i.d., less than 1 mm) offers improved
sensitivity compared to conventional LC (column i.d.,
4.6 mm) because of the higher sample concentration
in the detector cell. Furthermore, use of halogenated
hydrocarbons as eluent was shown to offer better spectral
specificity owing to the higher IR-transparency of these
solvents. A reasonable number of applications of flow
cell LC/IR in a variety of disciplines has been developed
since. Saunders and Taylor,.15/ for example, applied on-
line GPC/IR with tetrahydrofuran (THF) as mobile phase
for the determination of the nitrogen content of cellulose
nitrates. A cylindrical micro flow cell with a pathlength
of 1 mm and an internal volume of 4 µL was employed
in combination with a beam condenser. The extinction
of the O N O asymmetric stretching band was used
for the quantification of primary and secondary carbon
nitration. An improved resolution and quantification
was achieved by spectral derivatization. On-line GPC/IR
is also a viable analysis method in the polymer field.
One method employs high-temperature GPC with flow-
cell IR detection to characterize the molecular weight
distribution of high-density polyethylenes..16/ Tri- and
dichlorobenzene were used as mobile phase as these
solvents do not exhibit interfering absorption in the CH2 -
stretching band region (3000 – 2700 cm 1 ). Furthermore,
a quartz flow cell with a relative long pathlength of
1 cm could be used in order to enhance the sensitivity.
The method compares favorably with gradient elution
fractionation combined with 13 C-NMR spectroscopic
characterization.
The strong IR absorption of water, methanol and
acetonitrile, limits the application of on-line reversed-
phase liquid chromatography/infrared (RPLC/IR) to
the analysis of samples with relatively high analyte
concentrations, such as wines, beverages and cellulose
solutions. Various examples of this type of analysis have
been described in the literature. Recent applications are
the identification and quantification of sucrose, glucose
and fructose in soft drinks,.17/ and the specific analysis of
monosaccharides, alcohols and organic acids in wine..18/
In both studies, separations were achieved with ion-
exchange columns using an aqueous eluent. IR detection
was performed with a 25-µm pathlength flow cell and
minimal identifiable concentrations of typically 1 g L 1
were obtained.
When a high sensitivity or selectivity is required, con-
ventional RPLC solvents cannot be used with IR flow
cells. Therefore, alternative methods have been devel-
oped to circumvent the IR-opacity problems imposed
by the aqueous mobile phase, while maintaining the
advantages of on-line detection. One approach is the
extraction of the analytes into a more IR-transparent sol-
vent. Another approach is the use of deuterated solvents
which virtually have the same chromatographic proper-
ties but do not absorb in the spectral region of interest. An
example of the first method is the dynamic extraction of
the analytes from the aqueous effluent into chloroform or
carbon-tetrachloride..19/ The on-line liquid – liquid extrac-
tion (LLE) is carried out in an extraction coil followed
by continuous separation of the aqueous and organic
phases by a hydrophobic membrane. Subsequently, the
organic phase, carrying the extracted analytes, is mon-
itored in a common IR flow cell. A basically different
post-column extraction method has been developed by
Messerschmidt..20/ In this method, the RPLC effluent
is diluted with water, and the analytes of interest are
trapped on several small solid-phase extraction (SPE)
columns. These columns are dried with a flush of nitro-
gen, and sequentially eluted with a small volume of
tetrachloromethane into an IR flow cell allowing the
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY
individual spectra of the analytes to be recorded. An
additional advantage of this method is the improved
minimal identifiable concentration as a result of ana-
lyte concentration on the SPE column. This technique
has been successfully applied by DiNunzio.21/ for the
separation and identification of active compounds and
degradation products in pharmaceutical samples.
In on-line LC/IR, deuterated solvents can be attractive
substitutes for conventional (hydrogenated) solvents. The
absolute absorbance of deuterated solvents is usually
smaller and, more importantly, their IR absorption bands
are shifted to different spectral regions. Additionally,
deuterated solvents and their hydrogenated counterparts
show very similar elution properties. With respect to
on-line extraction procedures, the use of deuterated
eluents has advantages in terms of simplicity, speed
and maintenance of the chromatographic resolution. A
major drawback is their high price. A detailed study on
the utility of deuterated eluents in micro-column RPLC,
NPLC and GPC with flow-cell IR detection was carried
out by Fujimoto et al..22/ It follows that the problem
of interfering solvent absorption bands can be (partially)
solved by a deliberate choice of a deuterated eluent. Chen
and Kou,.23/ for instance, used deuterated methanol and
water instead of the non-deuterated solvents to overcome
the strong interfering absorptions that hinder the effective
detection and quantification of lipid fractions by on-line
LC/IR. Remsen and Freeman analyzed proteins using on-
line gel permeation chromatography/Fourier transform
infrared (GPC/FTIR) with a deuterium oxide eluent
to remove water and other detection-interfering low-
molecular-weight compounds and to achieve a rapid
hydrogen exchange..24/ Amounts of 50 µg per protein
could be detected in the amide-I region between 1600
and 1300 cm 1 , and important information on the protein
conformation could be obtained.
3.3 Use in Flow-injection Analysis
FIA is a well-established analytical technique, based
on the automated injection of a series of samples in
a continuous carrier stream. In FIA various detectors
are used, mainly for quantitative purposes. Amongst
these detection modes, flow-cell IR is not a frequent
choice because of the interfering solvent absorptions
and the relative poor sensitivity compared to UV/VIS
and fluorescence detection..25/ In several cases, however,
on-line flow-injection analysis/infrared (FIA/IR) is an
appropriate alternative for the rapid determination and
quantification of a specific analyte in a simple mixture. In
FIA, there is no need for an eluting solvent and, therefore,
in contrast with LC, a carrier solvent suitable for IR
detection can be selected quite easily. In this respect,
FIA/IR has a wider application range than LC/IR. Still,
7
the flow cells used in FIA/IR are not different from the
ones used in LC/IR and the resemblance in detection
approach is evident. Commonly, FIA/IR is applied for
single-component analysis but since IR spectra comprise
a range of absorption frequencies, multi-component
analysis can in principle be carried out as well. Obviously,
the IR absorption bands of the carrier should not
spectrally interfere with the marker band of each analyte.
Guzman et al..26/ compared the characteristics of
transmission and CIRCLE cell detection in FIA/IR
for multicomponent analysis of ternary solvent mixtures.
With tetrachloromethane as carrier, the transmission cell
provided better sensitivity in the continuous-flow mode.
The shorter effective pathlength of the CIRCLE cell was
compensated for by applying the stopped-flow technique
which allowed data averaging and, thus, enhancement of
the S/N of the ATR spectra. Analyte concentrations of
1 – 2% (v/v) could still be measured.
The principles and applications of flow-cell FIA/IR
have been studied extensively by the group of de la
Guardia..27 – 33/ The effects of flow rate, injection volume,
cell volume and optical pathlength on the performance
of the FIA/IR analysis of o-xylene in solutions of hexane,
were investigated..29/ Further optimization was achieved
in the development of a method for the automated
determination of benzene in gasoline..30/ In a further
study, incorporation of an analyte-enrichment step was
proposed to improve the detection limits of on-line
FIA/IR..31,32/ Preconcentration was carried out by trap-
ping the analytes on an SPE cartridge. After drying of the
cartridge with nitrogen gas, the analytes were eluted with
dichloromethane and detected by IR transmission spec-
troscopy using a micro flow cell. For aqueous solutions
of the pesticide carbaryl and its major metabolite 1-
naphthol, detection limits of 0.36 mg L 1 and 1.6 mg L 1 ,
respectively, were obtained after preconcentration of
100-mL samples. Quantification was achieved by spe-
cific detection of the bands at 1744 cm 1 (carbaryl CDO)
and 1276 cm 1 (1-naphthol C O) (Figure 6). FIA/IR is
also a useful alternative for the quantitative analysis of
mineral oil and grease..33/ Analogous to the Interna-
tional Organization for Standardization (ISO) procedure
for the quantitative analysis of mineral oil in water and
soil, this method is based on measurement of the aro-
matic, olefinic and aliphatic C H stretching bands in
the 3200 – 2700 cm 1 region. The samples were extracted
with tetrachloromethane by LLE or microwave-assisted
extraction. Next, the extracts were analyzed by FIA/IR
at a sampling frequency of 60 h 1 using a quartz flow
cell with a 10-mm pathlength. The detection limit was
1 mg L 1 when 300 µL of the extract was injected.
Miller et al..34/ used FIA/IR for the simultaneous detec-
tion of succinylcholine chloride and bethanechol chloride,
8 INFRARED SPECTROSCOPY
0.80
A B
0.60
Absorbance
0.40
0.20
0.00
1850 1650 1450 1250
Wavenumber (cm−1)
Figure 6 Absorbance spectra of carbaryl ( ) and 1-naphthol
(- - - ) dissolved in CHCl3 showing peaks at (A) 1741 cm 1
and (B) 1276 cm 1 . (Reproduced by permission of Elsevier
Science from Y. Dagbouche, S. Garrigues, M. de la Guardia,
Anal. Chim. Acta, 314, 203 – 212,  1995.)
two pharmaceutically important compounds. The com-
pounds were specifically monitored at 1075 cm 1 and
953 cm 1 , respectively, using a CIRCLE cell. The detec-
tion limit was about 0.02% (w/v) at a throughput of 60
samples per hour. FIA/IR was also applied to the quantifi-
cation of acetylsalicylic acid, caffeine and paracetamol in
pharmaceutical formulations..27,28/ Using a chlorinated
solvent the extinction of a characteristic absorption
band could be measured for each of the analytes in
the 1800 – 1500 cm 1 region. Commercially available flow
cells with an optical pathlength of 0.117 mm (5 µL) or
0.17 mm (7.2 µL) were used. Two FIA/IR methods for
automated analysis in clinical and process chemistry have
been developed by Kellner et al..35,36/ The first method
aimed for the determination of glucose and urea after on-
line enzymatic digestion to gluconic acid and ammonium
carbonate, respectively. A 25-µm pathlength transmis-
sion cell was used to monitor the aqueous flow from the
enzyme detector. The performance of the system was
satisfactory for relative clean samples such as standard
solutions, fruit juices and soft drinks. However, for rou-
tine application, for example in blood analysis, further
improvement of the reproducibility is still required. The
second FIA/IR method was used to determine the amy-
loglucosidase activity during starch hydrolysis processes.
The IR flow cell (pathlength, 49 µm) was constructed of
two different window materials in order to compromise
between high transparency (ZnSe) and low reflectiv-
ity (calcium fluoride). Because of the high viscosity of
the starch solutions, a stopped-flow method was used
to obtain maximum reproducibility and sensitivity. The
changes in the IR-spectra of the process mixture appeared
to be directly correlated with the enzyme activity.
4 SOLVENT-ELIMINATION TECHNIQUES
4.1 Deposition Substrates and Infrared
Detection Modes
The solvent-elimination approach in LC/IR involves the
use of an eluent-evaporation interface that deposits the
LC-separated compounds onto an IR-compatible sub-
strate. With most described set-ups, after immobilization
of one or more chromatograms, the substrate is trans-
ferred to the IR spectrometer where spectra from the
deposited spots are recorded. The deposited traces on
the substrate may be moved (stepwise) through the inter-
rogating IR beam so that, when scanning is complete,
continuous IR chromatograms can be reconstructed by
computer software. In some designs, IR detection is exe-
cuted during the immobilization process, within 5 – 20 s
after deposition, allowing spectra to be obtained in real
time. Obviously, such a design requires a dedicated detec-
tor set-up. Dependent on the type of substrate used (see
below) and/or size of the deposited spots, often special
optics, such as a (diffuse) reflectance unit, a beam conden-
sor or an IR microscope, are used to scan the deposited
compounds.
Basically three types of substrates and corresponding
IR modes are used in solvent-elimination LC/IR: pow-
der substrates for diffuse reflectance Fourier transform
infrared (DRIFT) detection; metallic mirrors for reflec-
tion/absorption (R/A) spectrometry; and IR-transparent
windows for transmission measurements. In early solvent-
elimination LC/IR designs DRIFT detection of analytes
on potassium chloride (KCl) powder was used, but today
other, more convenient, detection modes are preferred.
DRIFT as such is one of the most sensitive IR modes
and sub-microgram identification limits can be achieved
when the residual solvent is evaporated quickly from
the powder. If the eluent is not highly volatile, it can
draw analyte away from the KCl powder surface into
the substrate. This will limit the sensitivity, because the
effective penetration depth of a DRIFT measurement is
not more than 100 µm. To overcome this problem, diffuse
transmittance spectrometry has been applied instead of
DRIFT, using a layer of KCl powder on an IR-transparent
substrate..37/ The main limitations of DRIFT detection in
LC/IR, show up during application. Reproducibility is
hard to control since factors such as sample homogene-
ity, sample load and compactness of the powder layer
significantly influence the DRIFT analysis. Reorientation
of the DRIFT matrix as a result of sample deposition
may lead to a poor background compensation. Careful
filling of cups or trays with the powder substrate is very
time-consuming and has to be repeated for every analysis.
Finally, common substrates such as KCl powder cannot
be used in combination with aqueous eluents. In view of
the overriding importance of RPLC, this is a very serious
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY
restriction. Some authors have used diamond powder as a
water-resistant DRIFT substrate, but it is expensive (and
thus not disposable) and not easy to clean.
Front-surface aluminum mirrors, which are suitable
for R/A detection, are compatible with aqueous elu-
ents and are relatively easy to handle. Compound
deposition on this type of substrate requires efficient
solvent-elimination interfaces because residual solvent
will easily spread over the hard and smooth reflective
surface. The band intensities in the R/A spectroscopy are
largely governed by a double-pass transmittance mecha-
nism, so that spectral data analogous to transmission data
are obtained. Some useful results of solvent-elimination
LC/IR using mirrors have been reported, although several
authors.38 – 40/ have reported evidence of band asymmetry
and spectral distortions. Aspects such as specular and
diffuse reflection from the analyte, thickness and micro-
crystallinity of the spot, and optical characteristics of the
substrate affect the shape and intensity of R/A spectra
obtained from analytes on aluminum mirrors. The use
of an IR-transparent germanium disc with a reflective
backing has been proposed in order to reduce spectral
distortions. This type of disc is used in the commer-
cially available LC-Transform LC/IR interfaces..10/ The
cleaning of aluminum mirrors in between analyzes is quite
delicate: the thin metal layer is fragile and can be damaged
easily by rubbing.
Most favorable results in solvent-elimination LC/IR
are obtained with IR-transparent deposition substrates
that allow straightforward transmission measurements.
So far mainly KBr and ZnSe windows have been
applied in experimental LC/IR set-ups. These substrates
have a hard and smooth surface and, therefore, eluent
elimination has to be fast to achieve proper depositions.
ZnSe is a water-resistant, inert material, while KBr
usually cannot be used in combination with RPLC.
Deposited compounds on ZnSe can be removed simply,
with water or alcohol, so that one window can be
used repeatedly. With ZnSe, good-quality IR spectra
with symmetrical band profiles can be recorded from
deposited spots. When the size of the sample spots is
small and microscopic optics are used for measurement,
the sensitivity of ZnSe transmission measurements is
higher than the sensitivity of DRIFT measurements..40/
ZnSe windows also cause fewer spectral artifacts than
mirror substrates for R/A detection..39/ Many LC/IR
studies demonstrated that spectra obtained using ZnSe,
closely resemble conventional KBr-disk transmission
spectra. Consequently, existing spectral libraries and
search programs can be used for identification purposes,
which is very important for the acceptance of IR as a
valuable detection technique.
The quality and appearance of spectra obtained with
solvent-elimination LC/IR will be influenced by the
9
morphology of the deposited analytes. The morphology
will depend primarily on parameters such as eluent
composition, evaporation rate, temperature and nature of
the substrate and the analytes. During solvent elimination
some compounds will form nice crystals while others
will deposit as an amorphous layer. Also, some analytes
will deposit as a smooth film, whereas others may form
irregular clusters. When the spot thickness exceeds a
certain level, the effect of light scattering may become
apparent. A compound may also exhibit polymorphism so
that mutually (slightly) different spectra can be obtained
for the same compound. In general, IR detection of
deposited compounds on IR-transparent substrates does
not pose serious problems. However, analyte morphology
should always be taken into consideration during spectral
interpretation.
In solvent-elimination LC/IR the identification limits
usually improve when the width of the analyte spots
is decreased. A prerequisite for this gain is the use of
the appropriate detector and sampling optics. Optimum
solvent-elimination interfaces can produce analyte spots
with a width as small as 100 – 300 µm. For these deposits,
the focus of a conventional beam condensor is too large
and the use of an IR microscope is indicated. Frequently,
the sensitivity enhancement is rationalized by considering
the relatively increased spot thickness only (Beer’s law!),
but this approach is too simple. From more complete
S/N considerations it follows that the good sensitivity of
IR microscopic detection essentially results from the low
noise level of the IR detectors in IR microscopes..4/ In
other words, to achieve the most sensitive IR detection in
LC, the width of the analyte deposits should have the same
dimensions as the microscope detector area (typically,
100 – 200 µm). Of course, as with any IR experiment,
the S/N ratio also can be improved by increasing the
measurement time (signal averaging). As outlined in
section 2.2, this advantage can be exploited to its full
extent in solvent-elimination LC/IR, although at the cost
of an increased time of analysis.
4.2 Early Interfaces
The solvent-elimination systems that were developed in
an early stage, generally used KCl-powder substrates for
DRIFT detection or flat KBr windows for transmission
detection. The first working interface for the coupling of
NPLC and IR was designed by Kuehl and Griffiths..3,41/
The organic eluent was led through a heated concen-
trator tube and dropped into a series of cups filled
with KCl powder suited for DRIFT analysis. A carousel
rotated the cups into the IR spectrometer where iden-
tifiable spectra could be recorded for sub-microgram
amounts of analyte. In order to allow for RPLC sepa-
rations, the aqueous effluent was first on-line extracted
10
with dichloromethane which, after continuous phase sep-
aration, was directed through the concentrator to the KCl
cups..42/ For extracted compounds good-quality spectra
were obtained. The carousel – DRIFT method was also
adopted for narrow-bore NPLC (use of 1-mm i.d. column)
by reducing the size of the KCl cups and by omitting the
concentrator tube..43/ Using a similar set-up, Kalasinsky
et al..44,45/ coupled both narrow-bore NPLC and RPLC
with DRIFT. The KCl-powder substrate was held either
in a ‘train’ of compartments or in a continuous trough.
Aqueous eluents could be used by on-line conversion of
the water into methanol and acetone via a post-column
reaction with 2,20 -dimethoxypropane (DMP). The identi-
fication limits of these systems were 1 – 3 µg, typically.
The early DRIFT-based systems for the first time
demonstrated that solvent-elimination LC/IR can provide
(much) better sensitivity and spectral quality than
flow-cell based LC/IR. However, the systems were
mechanically complex and tedious to work with, and
DRIFT detection appeared to be strongly affected by
small disturbances of the KCl-powder surface and by the
presence of (residual) water.
Jinno et al..46 – 47/ proposed the use of micro-LC
columns (i.d., 0.3 mm) in solvent-elimination LC/IR in
order to alleviate the problem of the evaporation of large
eluent volumes. The effluent (5 µL min 1 ) from either
a GPC or an NPLC was led directly to a moving KBr
plate which was covered by a stream of heated nitrogen.
Subsequently, the plate with the deposited track was
scanned by IR transmission spectroscopy using a 3 ð
beam condensor. The potential of the approach (termed
‘‘buffer-memory’’ technique) was illustrated by the
analysis of a mixture of dithiocarbamate metal complexes
by three spectroscopic techniques..47/ In a modified set-
up the linearly moving KBr plate was replaced by a
rotating KBr disk which, after the chromatographic run
was finished, was transferred to a special rotation module
in the IR spectrometer..48/ In order to permit the use of
RPLC, a SS wire net (WN) was used instead of an KBr
window..49/ IR transmission measurements were possible
because after deposition and drying the analytes were
partly suspended in the metal meshes.
The ‘‘buffer-memory’’ technique demonstrated the
usefulness of the storage of a continuous chromatogram
on a flat substrate. Besides, it was considerably simpler
than the DRIFT methods. However, at least several
micrograms of analyte were needed for a positive IR
identification. These amounts often exceeded the sample
capacity of the micro-columns and required unrealistically
high concentrations to be injected.
4.3 Spray-type Interfaces
When using the ‘‘buffer-memory’’ technique for com-
pound deposition on flat substrates, it is not possible
INFRARED SPECTROSCOPY
to eliminate organic or aqueous eluents at flow rates
higher than about 5 µL min 1 without spreading the com-
pounds over a large area of the substrate surface. To
achieve a more viable coupling of LC and IR, the use of
interfaces with enhanced evaporation power is essential.
The solvent-elimination interfaces developed in the last
decade all use some kind of spraying to induce rapid
eluent evaporation. Heat, gas, electric potential and/or
ultrasonic vibrations are used to break up the LC elu-
ent stream into small droplets. Some designs incorporate
existing (commercial) equipment, while others have been
built from scratch.
In the thermospray (TSP) interface, originally devel-
oped for liquid chromatography/mass spectrometry
(LC/MS), a directly heated tube evaporates part of
the column effluent to an expanding vapor causing
nebulization of the remaining effluent. As a result, a
mist of desolvating droplets emerges from the tube.
In the TSP-based LC/IR systems, nebulization is per-
formed at atmospheric pressure and the spray is directed
towards a deposition substrate. Eluent flow rates of up
to 1 mL min 1 can be handled. Griffiths and Conroy.50/
reported preliminary results on the use of TSP for LC/IR,
but the first really working interface was introduced by
Jansen..51/ With a home-made TSP, the LC effluent was
sprayed on a SS IR-reflective tape which moved through
an optical accessory in the IR spectrometer (Figure 7).
Most of the eluent was eliminated directly by the TSP
and residual solvent was evaporated off the tape by
heating. The immobilized chromatogram was monitored
continuously and full IR spectra were recorded. Sev-
eral simple polymer samples (20 – 80 µg injected) were
analyzed by GPC/IR using an organic eluent at a flow
rate of 0.5 – 1 mL min 1 , but some low-molecular-weight
monomers were too volatile to be deposited. The charac-
terization of two Irganox-type polymer additives (100 µg
each), which were separated by RPLC using an eluent
2
3 4
,,,,,
,,,,
,,
,,
,,
,,
,
1
Figure 7 Schematic of TSP – moving belt interface for LC/IR;
1, moving SS tape; 2, TSP; 3, heater; 4, IR reflectance cell
mounted in the IR spectrometer.
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY
(0.5 mL min 1 ) with 30 vol% water, was also shown.
Robertson et al..52/ further optimized the TSP– moving
belt interface by studying the effect of the TSP tempera-
ture and distance to the substrate. The system was used
for the analysis of amino acids, saccharides, carboxylic
acids, antioxidants and polymers,.53,54/ and analyte iden-
tification could be achieved down to concentrations of
50 µg mL 1 or about 2.5 µg injected.
The main advantage of the TSP-based systems is
that relatively high flow rates (0.5 – 1 mL min 1 ) of
both organic and aqueous eluents can be handled and
conventional-size LC can thus be used. Furthermore,
spectral data are acquired during the run, which gives
the IR detector an essentially on-line character. On the
other hand, the high temperature of the TSP may induce
analyte losses by evaporation or thermal degradation and
the analyte spots on the moving tape are still quite large
which results in a moderate IR sensitivity.
The particle beam (PB) interface was modified for
LC/IR by de Haseth et al..55,56/ and Wood..57/ In this
interface the LC eluent is nebulized by helium and
directed into a desolvation chamber where most of
the liquid is vaporized. The mixture of gas, vapor
and condensed analyte molecules (i.e. particles) is
accelerated into the momentum separator where the
analytes travel straight through the skimmer cone,
while the gas and vapour are pumped away. For
IR detection an IR-transparent substrate is placed
in the particle-beam path to collect the analytes of
Rotary
pump
Helium supply
Desolvation chamber
Liquid
,,,,
,,,,
,
,
flow
Ball Ball
valve valve
Rotary
pump
(a) Momentum separator
KBr plate located in
0.5-in. glass O-ring fitting
slot cut into glass
Interface
High vacuum
10−5 torr
0.75-in. o.d. glass tube
0.5-in. o.d. glass tube
(b)
Figure 8 Schematic of PB interface for LC/IR.
11
interest (Figure 8) and after deposition, the substrate is
transferred to the IR spectrometer for analysis. Until
now stationary substrates have been used in liquid
chromatography/particle beam/infrared (LC/PB/IR), that
is, no complete chromatograms, only fractions, were
analyzed. The particle beam/infrared (PB/IR) interface
can effect the elimination of aqueous eluents at flow
rates of up to 0.3 mL min 1 as was demonstrated by the
analysis of erythrosin B and p-nitroaniline (50 µg each)
by RPLC/IR..56/ Since the PB interface has strong eluent-
elimination capacities, it was believed that interference
caused by buffers might be small..58/ The interface
indeed appeared to be able to process a 0.3 mL min 1
flow of buffered eluent, but the buffer salts were never
completely eliminated. Best results were obtained with
eluents buffered with ammonium acetate, although buffer
bands were clearly present in the IR spectra recorded
from microgram amounts of analyte. When phthalate
or phosphate buffers were used, the analyte spectra
were completely dominated by absorption bands of the
buffer salts. Spectral subtraction procedures could be
used to recover spectra from 130-µg depositions but were
unsuccessful at the 13-µg level. PB/IR has been used as a
tool for the determination of protein structures..59 – 61/
For b-lactoglobulin and lysozyme it was shown that
their structural integrity is maintained during the PB
desolvation process and the subsequent deposition on the
substrate. In addition, lysozyme appeared to retain its
biological activity. The sample loads in these experiments
generally were quite high (5 – 500 µg).
The PB interface can effectively remove both organic
and aqueous solvents. However, relevant applications in
LC/IR would still require the construction of a device
that allows the continuous deposition of a complete chro-
matogram on a moving substrate. The PB/IR analysis of
compounds at the nanogram level has been indicated,.55/
but the reported sample quantities mainly are in the (high)
microgram range. The modest analyte detectability no
doubt is related to the fact that the efficiency of analyte
transfer in the PB interface probably is 5 – 10% only.
The potential of electrospray (ESP) nebulization for
micro-LC/IR was studied by Raynor and co-workers..62/
A high electrical potential is used to form a spray
of charged droplets at the end of a capillary filled
with flowing liquid. As a result of charge density, the
initial droplets break up into smaller droplets which
facilitate solvent evaporation. Use of low flow rates
(typically 1 – 20 µL min 1 ) is indicated in order to obtain
a stable ESP. The ESP is formed under atmospheric
conditions and a sheath flow of nitrogen gas is applied to
enhance eluent evaporation (Figure 9). The ESP interface
was used to deposit the effluent from a micro-RPLC
column onto a ZnSe plate..62/ After LC separation,
20-ng amounts of caffeine and barbital could be analyzed
12 INFRARED SPECTROSCOPY

25-µm capillary HPLC effluent

High-voltage
cable (+3 KV)
Gas
Tee Nitrogen gas

Reflective surface

(Evacuated
Tapered chamber)
stainless steel
electrospray tip
ZnSe plate (Vacuum Drive shaft
Stage pump) (a) Gearbox

(a) Earthed cable

N2 Solvent
Sample track

25-µm fused silica Effluent

High-voltage capillary tubing deposit
(b)
cable (+3 KV) (0.25-mm o.d.)
Figure 10 Schematic of a narrow-bore NPLC and IR system
with pneumatic nebulization; (a) side view during deposition;
(b) top view of collection mirror. (Reproduced by permission
from J.J. Gagel, K. Biemann, Anal. Chem., 58, 2184 – 2189, 
Tapered stainless steel 1986, American Chemical Society.)
tubing (0.3-mm i.d.)
Focused stream of
is heated. Pneumatic nebulizers have been used in sev-
,,,
,,,

electrostatically charged
droplets Solvent stream forms
Taylor cone at tip
(b) Earthed disc
Figure 9 Schematic of ESP interface for (a) micro-LC/IR
(b) with the ESP tip in detail. (Reproduced by permission of
Hüthig Publishing from M.W. Raynor, K.D. Bartle, B.W. Cook,
J. High Resolut. Chromatogr., 15, 361 – 366,  1992.)
successfully using an IR microscope. Identification of 2 ng
caffeine appeared to be possible, although subtraction
of the interfering bands from a siliceous impurity was
required. Stable ESP conditions were achieved with
hexane, dichloromethane, acetonitrile, methanol and
several aqueous solvents, but problems were reported for
pure water. Until now there have been no further studies
on electrospray/infrared (ESP/IR) detection in LC.
During pneumatic nebulization a high-speed gas flow
is used to disrupt the liquid surface and to form small,
fast-moving droplets. Organic solvents can be rapidly
evaporated by pneumatic nebulization, while removal
of aqueous solvents is possible when the nebulizer gas
eral solvent-elimination LC/IR designs, among which are
the most successful so far. One of the first nebulizer-
based LC/IR methods involved the continuous spraying
of the effluent from a narrow-bore NPLC column on
a rotating IR-reflective disk (Figure 10)..63/ The efflu-
ent was mixed with nitrogen gas to form a fine spray
and the immobilized chromatogram was analyzed by
rotating the disk through a 3 ð -condensed IR beam
while recording R/A spectra. The system was tested with
polycyclic aromatic compounds (200 – 800 ng each) which
were separated using hexane – dichloromethane as elu-
ent (30 µL min 1 ). Good-intensity spectra were obtained,
although some spectral deviations were observed. This
pneumatic nebulizer design was improved to accomplish
elimination of aqueous solvents..38/ A heated nitrogen
flow served as an evaporation-enhancing and spray-
focusing sheath gas. Eluents containing up to 55% water
could be handled at 30 µL min 1 and a number of isomeric
naphthalenediols (500 ng each) were separated and iden-
tified. Again the recorded R/A spectra showed anomalies.
These spectral problems could be partially solved by using
an IR-transparent germanium disk with a rear surface
of aluminum as substrate..64/ This pneumatic nebulizer
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY 13

LC/IR design is commercialized by Lab Connections..10/ B

The instrument consists of a sample-collection module A
and an optics module for R/A analysis. So far the com-

,, ,
mercial interface has been applied mainly in the field of

,,
,,
,,
GPC/IR..65,66/ It was also used for the identification of
triclosan, an antibacterial agent, in toothpaste..67/
C
A simple but effective concentric flow nebulizer (CFN)

,,,,
for the coupling of narrow-bore LC and IR spectrometry

,
was constructed by Lange et al..68/ The interface consists E
D
of two concentric fused-silica capillaries. The LC column
effluent is led through the inner capillary and heated
helium gas through the outer capillary (Figure 11a). The
hot gas facilitates the evaporation of the solvent and the
focusing of the spray emerging from the inner tube. To
enhance the elimination of aqueous eluents, the CFN and
the ZnSe substrate were placed in a vacuum chamber Solvent
(Figure 11b). IR microscopy was used for optimum trap
detection. The CFN can handle eluents with up to 100%
water at a flow-rate of 50 µL min 1 and identifiable spectra
of analytes can be recorded down to the low-nanogram F
range. The CFN was also installed in an evacuated Vacuum
compartment which included the IR-microscopic optics system
G

and a motor to translate the ZnSe window..69/ With
this system, an RPLC effluent (50 µL min 1 ) could
be continuously deposited on the moving substrate.
After immobilization of the chromatogram, spectral
data could be collected without the need to transport
the substrate from the Chromatograph to the IR
spectrometer. To further improve the on-line character of
the system, a modified CFN was installed on the optical
bench of a Tracer (Biorad, Düsseldorf, Germany)
gas chromatography – IR interface which allows spectral
acquisition in real time..70/ So far, only some preliminary
results with this on-line LC/IR system have been
reported. Unfortunately, proper analyte spectra cannot
be obtained with the CFN/IR system when using non-
volatile buffers, because of strong co-deposition of
buffer salts..71/ However, if sufficient vacuum pump
capacity is applied, a 1 mM ammonium acetate buffer can
be completely eliminated, although higher ammonium
acetate concentrations cause interference.
Somsen et al..39/ proposed a spray-jet interface for the
coupling of narrow-bore RPLC and IR spectrometry.
In this interface a heated nitrogen flow provides pneu-
matic nebulization of the column effluent (20 µL min 1 )
which is led through a SS needle that protrudes through
a spray nozzle (Figure 12). With ZnSe as substrate and
IR microscopy for detection, identification limits in the
10 – 20-ng range were achieved for quinones and poly-
cyclic hydrocarbons. The narrow-bore RPLC/IR system
was used for the impurity profiling of a steroids,.72/
for the isomer-specific characterization of chlorinated
pyrenes,.73/ and for the identification of additives in
polymer samples..74/ Furthermore, the suitability of the
,,
,,
,,
H
Figure 11 CFN for (a) narrow-bore LC/IR and (b) interface
chamber; A, electric connections; B, LC effluent inlet; C, helium
gas inlet; D, heating wire; E, concentric fused-silica tubes; F,
stage; G, magnet for stage rotation; H, vertical positioner.
(Reproduced by permission from A.J. Lange, P.R. Griffiths,
D.J.J. Fraser, Anal. Chem., 63, 782 – 787,  1991, American
Chemical Society.)
interface for GPC/IR was demonstrated by analyzing
polystyrene oligomers..74/ When RPLC is applied, the
spray-jet LC/IR system is limited with regard to the LC
flow rate, the water percentage of the eluent and the han-
dling of buffered eluents. In order to take away these lim-
itations, a post-column LLE module (phase segmentor,
extraction coil and phase separator), was inserted on-line
and the organic phase, carrying the extracted analytes,
was sent to the evaporation interface..75/ The resulting
liquid chromatography/liquid – liquid extraction/infrared
(LC/LLE/IR) system can handle eluents with high
water percentages (20 – 100 vol%) at flow rates up to
0.2 mL min 1 and provides identification of compounds
at the sub-milligram per liter level. Since the salts are
not extracted, non-volatile buffers can be used without
14 INFRARED SPECTROSCOPY

Fused N2
Injection silica
valve capillary

LC Absorbance
Pump detector Heater
Column

Fused LC Flow
silica
50-µm i.d. Spray jet
Union assembly Movement
250-µm i.d. Heated
,,, ,,,,,,,,,,,,
,,, ,,,,,,, N2 Translation table

,,, , , ,
Stainless steel
needle Needle protrusion distance
100-µm i.d. Needle to substrate distance
,,,,
,,,
,,,
,,,

,,
,,,, ZnSe Substrate

Figure 12 Schematic of the spray-jet interface for narrow-bore LC/IR.

causing interference. The detectability of the analytes
was further improved by incorporation of on-line SPE
for analyte enrichment (Figure 13). With such a system,
triazine herbicides, including several isomers, could be
identified at the low-microgram per liter level in river
water (Figure 14)..76/
In an alternative approach to improve the compatibility
of the spray-jet interface with RPLC, the eluent flow rate
was reduced to 2 µL min 1 ..77/ To obtain a useful spray,
a make-up liquid (20 µL min 1 of methanol) was added
to the micro-LC effluent. As a consequence, the perfor-
mance of the interface became independent of the water
content of the eluent, so that gradient elution was possible.
A micro-precolumn for on-line trace enrichment was
applied to improve detection limits. With a 40-µL sample
volume, good-quality IR chromatograms and analyte
spectra were recorded at the low-milligram per liter level.
In an ultrasonic nebulizer a spray is formed by deposit-
ing the LC effluent on a transducer that is vibrating at
ultrasonic frequencies. For LC/IR purposes, the spray
is directed towards a substrate by a carrier gas. Castles
et al..78/ used ultrasonic nebulization for the deposition
of compounds separated by narrow-bore RPLC on a
diamond-powder substrate suitable for DRIFT detection.
Spectra of satisfactory quality were obtained for injec-
tions of 3 µg of analyte. In some instances, the complete
and direct evaporation of the eluent by the ultrasonic
nebulizer was not achieved because the vibrating sur-
face was not uniformly effective and occasionally large
droplets were formed which wetted the diamond powder.
Dekmezian and Morioka developed an interface for
high-temperature GPC/IR which involved an ultrasonic
nebulizer..79/ The nebulizer was placed in a vacuum
chamber and sprayed the column effluent on a set

EC Waste
P4 T R N2
PS
P3
V3 UV H
Anal

Recycle
P2
Waste I
Waste V1 V2 ZnSe
P1 TrT
Pr

S "Inject" FTIR
"Load"

Figure 13 Set-up for LC/IR with on-line SPE and post-column LLE; P1, pump for sample loading and SPE-column rinsing; P2
and P3, eluent pumps; P4, organic phase pump; S, solvent selection valve; V1-V3, six-port injection valves; Pr, SPE column; Anal,
analytical column; T, T-piece; EC, extraction coil; PS, phase separator; R, restrictor; UV, UV absorbance detector; I, spray-jet
interface; H, gas heater; ZnSe, ZnSe deposition substrate; TrT, translation table; FTIR, FTIR microscope.
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY 15

Absorbance units
0.025
1

0.10
GS-units
0.05

0.000
0 10000 20000 30000 40000 3500 2500 1500 1000
L(µm) Wavenumber (cm−1)
10 20 30

(a) t (min)
Total absorbance

Absorbance units
0.050
3
5.0

0.025
2.5
0.0

0.000

0 10000 20000 30000 40000 3500 2500 1500 1000

L(µm) Wavenumber (cm−1)
10 20 30
(b) t (min)
0.000 0.025 0.050 0.075
Total absorbance

Absorbance units

5
3 4 5
3
2

1 2
1
0
−1

0 10000 20000 30000 40000 3500 2500 1500 1000

L(µm) Wavenumber (cm−1)
10 20 30
(c) t (min)
Figure 14 SPE and LC/LLE/IR chromatograms of river Meuse water samples spiked with five triazines: (a) 20 mL (30 µg L 1 ),
(b) 50 mL (6 µg L 1 ) and (c) 100 mL (2 µg L 1 ). IR spectra of peaks 1, 3 and 5. Chromatogram representation, (a) Gram – Schmidt
or (b and c) spectral window (1650 – 1500 cm 1 ). Peaks: 1, simazine; 2, atrazine; 3, sebutylazine; 4, propazine and 5, terbutylazine.

of heated KBr discs, which were subsequently ana- quantitative analysis of copolymers by GPC/IR.80/ and
lyzed by IR transmission spectrometry. The system was for steroid analysis by RPLC/IR..81/
applied to the determination of compositional changes
of ethylene – propylene rubbers. An interface comprising
an ultrasonic nebulizer in a vacuum chamber is used by
Lab Connections in their LC-Transform 300 Series..10/ 5 STATE OF THE ART
This commercial device sprays the chromatographic efflu-
ent on a rotating germanium collection disk suited for In the contemporary practice of LC/IR both flow-
R/A analysis (see above). The system was used for the cell and solvent-elimination approaches are applied.
16 INFRARED SPECTROSCOPY

Since the flow-cell procedure cannot get around the
detection limitations imposed by the LC eluent, it has
developed into a special-purpose method with restricted
applicability. The IR absorptions of any solvent invariably
take up parts of the mid-IR spectral region and, therefore,
the main power of IR spectroscopy, i.e. the reliable
identification of compounds, cannot be fully exploited
in flow-cell IR detection. Nevertheless, making use of the
spectral windows of the eluent, flow-cell IR spectroscopy
can serve as a moderately sensitive, compound-specific
detection technique. Occasionally, it is used as a universal,
fast and low-cost method to obtain quantitative and
structural information on major constituents of samples.
Various types of flow cells are commercially available, and
the experimental set-up and practice of flow-cell LC/IR is
relatively simple and well-suited for routine applications.
When the objective of IR detection in LC is the
unambiguous identification of (low-level) constituents of
mixtures, coupling via solvent elimination should be the
approach of choice. Solvent elimination procedures offer
the possibility
ž to record spectra over the entire mid-IR region
without interference from the eluent;
ž to perform signal averaging in order to improve the
S/N of the spectra; and
ž to contain a relatively large part of the chromato-
graphic peak within the IR beam.
As a result, the solvent-elimination approach provides a
set-up which features increased sensitivity and enhanced
spectral quality, two important conditions for effective
analyte identification. The most recent commercial
LC/IR systems which are presently available,.10,11/ are
solvent-elimination devices. Interestingly, also in gas
chromatography and supercritical-fluid chromatography
analyte-deposition-based IR detection has proven to be
more sensitive and versatile than flow-cell-based tech-
niques. On the other hand, one should realize that the
vibrational information obtained after solvent elimina-
tion is different from the vibrational information obtained
with flow-cell detection (condensed-phase spectra against
solution-phase spectra). Recently, this difference was
used to determine subtle molecular features of drug
metabolites which were analyzed by both flow-cell and
solvent-elimination LC/IR..82/
Today, the vast majority of LC separations is carried
out by means of RPLC and, not surprisingly, research in
the field of LC/IR has concentrated on the development
of interfaces that are suitable for the elimination of aque-
ous eluents. Table 2 summarizes the characteristics of the
various solvent-elimination reversed-phase liquid chro-
matography/Fourier transform infrared (RPLC/FTIR)
systems which have been developed during the last years.
The systems based on TSP, PB and ultrasonic nebuliza-
tion can handle relatively high flows of aqueous eluents
and allow the use of conventional-size LC, which evi-
dently is an advantage. However, these systems often
exhibit moderate, or even unfavorable identification lim-
its and, therefore, their analytical applicability is limited.
Until now, the most favorable results have been obtained
with pneumatic nebulizers which essentially represent the
state of the art in solvent-elimination LC/IR.
The pneumatic interfaces combine rapid solvent
elimination with a relatively narrow spray. This implies

Table 2 Characteristics of solvent-elimination RPLC/IR systemsa

Type of interfacing IR mode Substrate Eluent flow rateb Limit of identificationb Reference
(µL min 1 ) 1
mass (ng) conc (mg L )
Concentrator after LLE DRIFT KCl powder 800 100 10 42
Deposition after DMP DRIFT KCl powder 50 1000 1000 44
Buffer memory trans SSWN 4 1000 10 000 49
TSP R/A SS tape 500 10 000 – 51
R/A SS tape 1000 1000 25 52
PB trans KBr window 300 1000 200 56, 58
ESP trans-micr ZnSe 4 1 10 62
Pneumatic nebulizer R/A Al mirror 30 30 30 38
trans-micr ZnSe 50 1 17 69
trans-micr ZnSe 20 5 3 39, 73
. . .after LLE trans-micr ZnSe 200 30 0.2 75
. . .after SPE C LLE trans-micr ZnSe 200 50 0.001 76
. . .after SPE C make-up trans-micr ZnSe 2 20 0.02 77
Ultrasonic nebulizer DRIFT Diamond powder 40 1000 1000 78
R/A Ge disk 500 100 20 81
a DMP, on-line reaction with DMP; make-up, addition of excess methanol; trans, transmission; trans-micr, transmission with IR microscope;
– , concentration and injection volume not stated.
b Typical values.
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY
that analytes can be deposited, for example on ZnSe,
in a narrow track of spots and IR microscopy can be
applied effectively to achieve mass sensitivities in the
low-nanogram range. Bearing in mind that often only
part of the injected amount of analyte is actually ana-
lyzed, this means that the mass detectability of these
LC/IR systems approaches a level close to the minimum
that can be identified by IR spectroscopy. The systems
based on pneumatic nebulization are limited with regard
to flow rate (2 – 50 µL min 1 ) and water percentage of the
eluent. The tolerable water content of the eluent depends
on the flow rate. Flow rates of 2 – 5 µL min 1 of even pure
water can be eliminated, but for 20 – 50 µL min 1 flows of
aqueous eluents, enhancement of the solvent evaporation
efficiency is required, for example by mixing the effluent
with nitrogen gas.38/ or by placing both the nebulizer and
the deposition substrate inside a vacuum chamber..68/ The
tedious evaporation of water can also be circumvented
by applying on-line LLE of the aqueous effluent with an
organic solvent. Such a system allows much higher flow
rates (0.2 mL min 1 ) and percentages of water (up to 100
vol%)..75,76/ Of course, the required LLE module adds to
the complexity of the system and the analytes must have a
sufficiently high extraction efficiency. Solvent-elimination
RPLC/IR with gradient elution poses the problem of the
efficient evaporation of an eluent with a changing water
content. One solution involves the increase of the temper-
ature of the nebulization gas during the gradient run,.38/
another the addition of excess methanol to a micro-
LC effluent in order to mask the changes in its water
content..77/
The injection volumes that can be handled with micro-
and narrow-bore LC columns, are at most 1 – 2 µL.
The identification limits in terms of concentration units
of the pneumatic nebulizer-based systems therefore
are in the low-milligram per liter range, which is
sufficient for a number of analytical applications..72 – 74/
By using the LLE-pneumatic nebulizer combination, the
detectability can be improved to sub-milligram per liter
levels because larger LC columns – and, thus, increased
injection volumes – can be used. With an adequate trace-
enrichment procedure such as on-line SPE, the LC/IR
detection limits can be further improved down to the
low-microgram per liter level..76/ The use of buffered
eluents is generally avoided in solvent-elimination LC/IR,
since buffer salts may seriously affect the deposition and
detection of the analytes. With pneumatic nebulizers even
volatile buffer salts are rarely completely eliminated. In
fact, until now the LLE-pneumatic nebulizer combination
is the only described LC/IR system which allows the use of
non-volatile buffer salts without introducing interfacing
disturbances and/or spectral interference..75,76/
Effective solvent elimination by the pneumatic nebu-
lizers allows the use of deposition substrates with a hard
17
and smooth surface such as ZnSe windows. With these
substrates interference- and distortion-free transmission
spectra are obtained which can be readily compared
with conventional KBr-disk IR spectra. This implies that
libraries of condensed-phase reference spectra can be
used for spectral recognition and identification. Com-
pounds of various nature such as quinones, steroids, drugs,
polymer additives and herbicides have been analyzed suc-
cessfully by pneumatic-nebulization-based LC/IR. The
interfaces can handle most types of analytes, although too
volatile compounds will be evaporated by the nebulizer
gas and therefore will not be deposited on the sub-
strate. Thermal degradation of analytes is commonly not
observed during pneumatic nebulization, despite the fact
that the nebulizer gas is heated to rather high tempera-
tures (70 – 180 ° C). Probably, due to the rapid evaporation
of the solvent, the spray droplets are cooled considerably.
6 PERSPECTIVE AND FUTURE
DEVELOPMENTS
In the last fifteen years, LC/IR has emerged as a
potentially powerful tool for the specific detection
of major components (flow-cell approach) or for the
identification of (minor) constituents of complex mixtures
(solvent-elimination approach). With respect to common
LC detection techniques such as UV/VIS absorption
detection, the sensitivity of flow-cell IR detection is rather
poor and its merits therefore mainly lie in the ability
to quantitatively monitor absorptions that are specific
for the analyte or for a certain functional group. Since
the limitations of flow-cell LC/IR are inherent in the
technique, no vast improvements can be anticipated in
the future. Some gain in performance may be achieved by
optimization of the flow-cell design and use of advanced
FTIR spectrometers, but these improvements will be
modest and not essentially change the applicability of
flow-cell IR detection. Employment of chemometrical
techniques, however, may be useful particularly when the
signal or spectrum is the result of the absorbance of two
or more substances.
The usefulness of solvent-elimination LC/IR to provide
structural information and/or identification of unknowns
has been demonstrated convincingly since 1990. Unfor-
tunately, the development of coupling techniques pro-
ceeded, and still proceeds, quite slowly and until now most
interfaces have been used only by their designers. Nev-
ertheless, the difficulty of solvent-elimination LC/IR, i.e.
simultaneous eluent evaporation and analyte deposition,
seems to be a technical rather than a fundamental prob-
lem. In other words, the development of an overall
effective and routinely applicable interface probably is
18 INFRARED SPECTROSCOPY

a matter of time, effort and technological innovations. design the LC column effluent is deposited on a moving
Of course, solvent-elimination LC/IR has to compete ZnSe window which instantaneously passes through the
with other identification techniques of which today on- focused beam of the IR spectrometer allowing spectra and
line LC/MS undoubtedly is one of the most important. IR chromatograms to be recorded in real time. The place-
Quite a number of LC/MS interfaces have been devel- ment of the chromatograms on the substrate is controlled
oped and commercialized, but still each interface has its by computer which also keeps a record of the posi-
specific limitations. Furthermore, MS techniques cannot tion of deposited compounds. The IR Chromatograph
discriminate between isomers. Hence, even with adequate seems promising but, as it has been introduced only
LC/MS techniques available, there is a need for alter- recently, it is still too early to assess its merits. The han-
native and complementary detection techniques which dling of the obtained spectral data also is a matter of
independently confirm MS-based identifications and dif- concern in solvent-elimination LC/IR. The identification
ferentiate between structurally highly similar compounds. of analytes on the basis of their IR spectra often is a
A recent study demonstrated that LC/IR can make a difficult operation. Therefore, the automated retrieval
viable contribution to identification analysis in a research of spectra in reference collections, and the computeri-
setting that includes MS and NMR..82/ Prerequisite for zation of spectral interpretation are important. Several
the successful implementation of IR detection was a good such procedures have already been introduced in the
understanding of the relative strengths and weaknesses vibrational-spectroscopic field and high priority should
of each technique, and the integration of analysis in the be given to their implementation in the separation field.
total research program. Finally, it should be noted that the solvent-elimination
In order to enhance the acceptance of solvent- approach in LC is not restricted to IR detection but
elimination LC/IR, several items of interest should can, in principle, be applied to any spectrometric
be considered. The practicality of the technique for technique which requires the compounds of interest
real-life samples should be demonstrated more exten- to be present as deposits..83/ An example of such an
sively. The applications described so far.72 – 74,76/ indicate analyte-deposition-based detection technique is matrix-
that LC/IR can indeed be used for the characteri- assisted laser desorption/ionization (MALDI) MS. From
zation and unambiguous identification of target and a technical viewpoint the collector systems developed
unknown compounds. LC/IR is particularly useful for the for the coupling of LC or capillary electrophoresis
distinction of isomeric compounds.77 – 79/ which cannot with MALDI/MS show a strong similarity with solvent-
be distinguished by LC/MS. Another item of atten- elimination LC/IR systems.
tion is the development and use of appropriate on-
line sample-treatment procedures to improve analyte
detectability. Despite the low-nanogram identification ABBREVIATIONS AND ACRONYMS
limits, the detectability in concentration units of even
the best LC/IR systems will not be sufficient to meet ATR Attenuated Total Reflection
current demands in bio- and environmental analysis. On- CFN Concentric Flow Nebulizer
line SPE can improve the concentration detectability DRIFT Diffuse Reflectance Fourier
by one or two orders of magnitude. Obviously, such Transform Infrared
an improvement is unlikely to be obtained by opti- DMP Dimethoxypropane
mization of interfacing and/or IR detection only. The ESP Electrospray
first studies indicating this advantage in both flow-cell ESP/IR Electrospray/Infrared
and solvent-elimination IR-detection have already been FIA Flow-injection Analysis
reported..31,32,76,77/ FIA/IR Flow-injection Analysis/Infrared
Concerning the viability of solvent-elimination LC/IR, FTIR Fourier Transform Infrared
the availability and use of commercial interfaces also GPC Gel Permeation Chromatography
is essential. The LC-Transform interface (Lab Con- GPC/FTIR Gel Permeation Chromatography/
nections) has been available now for several years, Fourier Transform Infrared
but unfortunately few applications have been reported. GPC/IR Gel Permeation Chromatography/
Because this solvent-elimination system uses a mirror Infrared
substrate and standard IR equipment, both the IR sen- IR Infrared
sitivity and spectral quality are limited. In a more viable ISO International Organization for
approach an IR-transparent substrate should be used Standardization
together with microscopic IR detection. Such a configu- LC Liquid Chromatography
ration is used by the Infrared Chromatograph interface LC/IR Liquid Chromatography/Infrared
(Bourne Scientific). In this commercial and automated Spectroscopy
LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY 19

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