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Quantitative two-dimensional thin-layer

chromatography using a diode-array detector

1955: Geboren in Remscheid
Prof. Dr. rer. nat. Bernd Spangenberg
Studium der Chemie und Philosophie an der Universitt Marburg
Fakultt Maschinenbau
1983: Diplom in Chemie, 1987 Promotion ber neue Reaktionen
und Verfahrenstechnik (M+V)
von Sulfenen am Pharmazeutischen Institut der Universitt Marburg
1988: Eintritt in die E. Scheurich Pharmwerk GmbH als Laborlei-
Badstrae 24
ter, Leiter der Abteilung analytische Forschung und Entwicklung,
77652 Offenburg
stellvertretender Kontrollleiter
Tel. 0781 205-101
1990: Wechsel zur EBULON AG,
Basel als Leiter der Entwicklungsanalytik
1991: Professor an der Hochschule Offenburg, Leiter des Labors
fr Analytik und Umweltanalytik
Seit 2007: Editor-in-Chief von Journal of Planar Chromatography
Lehrgebiete: Analytik, Umweltanalytik, Abfall- und Recyclingtechnik
Mitglied des Instituts fr Angewandte Forschung (IAF) der Hochschule Offenburg
Forschungsgebiete: Chemometrie, Trenntechniken, Dnnschichtchromatographie

3.3 Quantitative two-dimensional dimensional chromatographic method The separation of 12 sulphonamides with
thin-layer chromatography using as paper chromatography was reported similar structure is presented as an exa-
a diode-array detector in 1944 by Consden, Gordon, and Mar- mple. The method can be used to quan-
tin [1 3]. Since that time, this method tify sulphonamides in the effluent of
Barbara Milz B. Sc. has been mostly used for the separation wastewater treatment plants.
Prof. Dr. rer. nat. Bernd Spangenberg of a large number of compounds that
cannot be separated in a single dimensi- Results and discussion
Abstrakt on TLC experiment. In the first review Sulphonamides were the first synthetic
Wir prsentieren die weltweit erste Aus- on this topic G. Guiochon reported nu- drugs with antimicrobial properties and
wertung einer zweidimensional entwi- merous applications of this technique some of these compounds are still in use.
ckelten HPTLC-Platte (2D-HPTLC) mit [3]. The greatest dispersion of spots over Worldwide, large amounts of highly di-
Hilfe eines Diodenarray-Scanners. Das the TLC plate is obtained when the chro- luted sulphonamides are being released
System erreicht eine rumliche Platten- matogram is developed in two orthogo- into the environment. It is not easy to se-
auflsung von 250 m. Es knnen Ab- nal modes. That is the case when in the parate sulfonamides because they all
sorptions- und Fluoreszenzspektren im first direction a normal-phase (NP) sy- have very similar structures. That is the
Wellenlngenbereich von 190 bis 1000 stem is used, e.g. on silica gel with a reason why we decided to use 12 sulfo-
nm mit einer spektralen Auflsung von non-aqueous mobile phase, and in the namides as model compounds to show
besser als 1 nm gemessen werden. Eine second direction a reversed-phase (RP) the performance of the new 2D-scanner.
Trennzahl von 259 wurde erreicht. Da- system is applied [4]. This can be done
mit zeigt die Methode bessere Trennei- by using a single cyanopropyl coated si- For direct spectrophotometry of HPTL-
genschaften als die meisten HPLC-Sys- lica layer, on which TLC can be perfor- plates, a TIDAS TLC 2010 system (J&M
teme. Der Nachteil der 2D-Auswertung med either in the reversed-phase mode
ist der hohe Zeitbedarf von ber 3 Stun- (with an aqueous mobile phase) or in
den fr eine Plattenmessung. the normal-phase mode (using a non-
polar solvent) [4].
Generally all chromatographic methods The great advantage of 2D-TLC is its abi-
can be divided into two main separation lity to separate many compounds. The
groups: time and space. Thin layer chro- drawback is that quantitative evaluations
matography as a space separation me- are difficult and dont meet current stan-
thod is by far the simplest separation dards. Generally, TLC scanners which
technique considered in terms of equip- are available for densitometric measure-
ment and performance [1]. Multidimen- ments cannot be adapted easily to two-
sional chromatography is the most po- dimensional TLC.
werful tool for separations [2]. The
two-dimensional thin-layer chromato- The purpose of this publication is to pre-
graphy (2D-TLC) technique is one of the sent a method for sensitive quantification Fig. 3.3-1: Light fibre interface consisting of a
most versatile methods of TLC develop- of 2D-HPTLC separations, simultane- 200 m inner core, surrounded by nine light
ment. The first application of the two- ously measured at different wavelengths. fibres with a diameter of 100 m each

Aalen, Germany) was used with an re- Fig. 3.3-2: Example of a 2D-HPTLC se-
flection attachment of 9 identical optical paration of 12 sulfonamides on a cya-
fibers (Figure 3.3-1) with a diameter of nopropyl plate measured in absorption
100 m each (produced by TransMIT- at 276 nm. The sulphonamides are: Sul-
Centre for Fibre Optics and Industrial phacarbamide (1), sulphadiazine (2),
Laser Applications, Gieen, Germany). sulphadimidine (3), sulphaguanidine
(4), sulphamerazine (5), sulphametho-
These fibers were used for illumination xazole (6), sulphamethoxypyridazine
purposes and were circled around a sin- (7), sulphanilamide (8), sulphapyridine
gle fiber with a diameter of 200 m. This (9), sulphaquinoxaline (10), sulphaso-
fiber transports the reflected light from midine (11), and sulphathiazole (12)
the plate to the detector. A TIDAS-system
with a wavelength resolution of 0.8 nm
was used for detection. The interface is
fixed at a distance of 450 m above the
plate surface. During measurements, the
plate is moving underneath this inter-
face with constant velocity. The mea-
surement time for a single spectrum
(measured by a Tidas TLC 2010 device)
in the wavelengths range from 190 to
1000 nm is 100 msec.

For fluorescence evaluation an LED

(model: LEDMOD 365.1, produced by
Omicron Laserage, Rodgau, Germany)
was used instead of a deuterium lamp.
The plate was illuminated at 365 nm
using a measurement time of 0.1 se-
conds per spectrum.
Fig. 3.3-3: The same area of interest measured at different wavelengths in absorption (1-5)
The mixture of 12 sulphonamides was and fluorescence (6) shows that this method can improve the spot resolution
applied as a 3mm band on a cyanopro-
pyl-coated silica gel plate in the amounts wavelength. The date were evaluated This makes the method a quantitative
of 2 g per sulphonamide, each. In the using the following equation with a k- one, unique in 2D-TLC.
first direction the solvent mix of methyl factor of k = 1.
tert-butyl ether, methanol, dichlorome- References
thane, cyclohexane, NH3 (25%) [1] Spangenberg B., Poole C.F., Weins
(48+2+2+1+1, v/v) was used. This is a C.: Quantitative Thin Layer Chroma-
normal phase separation. The plate was k: backscattering factor (k0 and k1) tography. A Practical Survey, Sprin-
dried and in a second step the plate was a: absorption coefficient ger, Berlin, 2010
developed with a solvent mix of water, R: reflection = I/I0
acetonitrile, dioxane, ethanol (8+2+1+1, I: sample reflection [2] Mondello L., Lewis A. C., Bartle K.
v/v). This is a typical reversed phase se- I0: incident light D. (Editors): Multidimensional Chro-
paration mode. The dried plate was matography, Wiley, Chichester, UK
scanned as described with the position The program cursor can be moved over (2002)
of the application point at 342 rows the screen to show the on-line spectrum
consisting of 342 different data points. measured at a particular plate position. [3] Mona Zakaria, Marie-France Gon-
This resulted in a spatial resolution of The plot can be instantly changed by nord, Georges Guichon: Applica-
200 by 200 m. The measurement time choosing another wavelength. In this tions of two-dimensional thin-layer
was 342*342*0.1 sec = 3 h 25 min. A way you can resolve neighbouring spots chromatography, J. Chromatogr. 271
scanned TLC-plate needs 400 MB sto- which are not spatially separated. (1983) 127 192
rage space. These data were copied into In figure 3.3-3 the same area of interest
the computer memory to have rapid ac- is measured at different wavelengths, [4] Mirosaw A. Hawry, Anna Hawry,
cess to the data. The evaluation program showing that the method can improve Edward Soczewinski: Application of
was written in PureBasic (Ver. 4.50) and the spot resolution. The spectrum at a Normal- and Reversed-Phase 2D
needed a 64 bit computer (HP EliteBook chosen separation is available simply by TLC on a Cyanopropyl-Bonded Polar
2540p) to carry out the program. moving the cursor to its plate area posi- Stationary Phase for Separation of
tion. A spot at a given wavelength can Phenolic Compounds from the
Figure 3.3-2 shows a 3D plot (plate area be virtually encircled and the resulting Flowers of Sambucus nigra L., J. Pla-
and absorption intensities) at a given peak is integrated without any problems. nar Chromatogr. 15 (2002) 4 10