|Thin Layer Chromatography using Diode-Array Detection|
|Bernd Spangenberg, Karl-Friedrich Klein*|
Fachhochschule Offenburg, Badstrasse 24, D-77652 Offenburg
Phone (49) 781 205-231, Fax (49) 781 205-111; email: Spangenberg@fh-offenburg.de
*Fachhochschule Giessen-Friedberg, Wilhelm-Leuschner-Str. 13, D-61169 Friedberg
HPTLC (High Performance Thin Layer Chromatography) is a well known and versatile separation method which shows a lot of advantages in comparison to other separation techniques. The methode is fast and inexpensive and does not need time-consuming pretreatments. For registration of the sample distribution on a HPTLC-plate we developed a new and sturdy diode-array HPTLC-scanner. The scanner allows simultaneous registrations of spectra in a range from 198 nm to 612 nm with a spectral resolution of better than 0.8 nm.
The spatial resolution on plate is better than 160 µm. The measurement of 450 spectra of one separation track does not need more than two minutes. The new diode-array scanner (commercially available) offers a fast survey over a TLC-separation and makes various chemometric applications possible. The system delivers much more information than the commonly used TLC-scanner.
The „traditional“ Thin Layer Chromatography or the High Performance Thin Layer Chromatography (HPTLC) is a widely used and versatile separation method which shows a lot of advantages in comparison to other separation techniques. The method is easy to use, quick and inexpensive because no time-consuming pretreatments are needed. Therefore, it is suitable for screening tests in the environmental analeptics. Another wide field will be seen in pharmaceutical applications, especially in the analytical studies of plant ingredients. Especially, the HPTLC is suited as a method for monitoring the impurity of substances because the components of the separation will be visible „at first sight“: no substance deposited on the plate can be ignored . For registration of the sample distribution on a HPTLC-plate we developed a new and sturdy diode-array HPTLC-scanner. Typically, the HPTLC is using the normal phase separation. Therefore, it is an supplement in respect to the High Precision Liquid Chromatography (HPLC), using the reversed phase separation mainly. Comparing the two separation systems, the normal phase system shows a higher selectivity because nearly all mobile phases are usable; in the RP-HPLC the following substances for the mobile phase can be used: water, methanol, or in some cases THF [2/]. Because the measurements will be done in liquids, the substances with high intrinsic absorption in the wavelength region around 200 nm are not suitable for mobile phases.
Because of the lowest impurity level, methanol and other mobile phases are expensive. In contrast, the used mobile phase will be removed in HPTLC-technique before the test run; therefore, the disturbances are negligible. In addition, the amount is very small in comparison to the significant mass of mobile phase which is discarded after each separation. Anyhow, the HPLC-method will be used in much larger extent than the TLC-method. Although the separation quality is slightly worse and the detection response is a little bit more adverse, the biggest disadvantage in the past was the missing of fullautomated HPTLC-systems. In TLC, the three steps are discrete in time: deposition of the substance under test, separation and detection. This is the reason that the grade of automatization is low. On the other side, the flexibility of the HPTLC is much higher compared to HPLC. However, a spectral detection was never implemented in HPTLC. This is absolutely incomprehensible, because the step from single-wavelength to spectral detection using diode-arrays had been taken place in HPLC, 10 years ago. Now, the TLC method will follow lately in the same direction to improve the technique significantly.
2. Measurement equipment
Including a spectral detection system, we have developed a robust HPTLC-scanner, which is capable to measure directly on a thinfilm plate in the wavelength region from 198 nm to 612 nm ; an expansion up to 1050 nm is being planned. The new system has a great advantage: because of fiber-optics, lenses or mirrors are no longer needed. Therefore, the efforts for alignment can be significantly reduced. With this scanner, the spatial resolution on the plate is smaller than 160 m , parallel to the spectral resolution of approx. 0.8 nm. Measuring time for one HPTLC-track of approx. 45 mm length is typically between 1 to 3 minutes, depending on the required signal-to-noise-ratio. In one scan, 450 or 900 different spectra with 512 data points will be taken up.
The principal item of the developed scanner is an adjusted and optimized fiber-optic assembly, consisting of a set of 25 emitting and collecting optical fibers. The fibers in the fiber-array are improved UVM-fibers , without hydrogen-doping. The light of a deep-UV deuterium-lamp will illuminate the HPTLC-plate, approx. 5 mm in width due to the 25 fibers in line. The light can be either absorbed or scattered at the stationary phase of the HPTLC-plate. The light for detection will be collected by a parallel second fiber-array with the same amount of fibers and guided to the diode-array detector. Fig. 1 shows the endface of the fiber-optic sensing head, above the HPTLC-plate. For measuring, the HPTLC-plate will be moved computer-controlled with a fixed distance of 450 m below the sensing head, using a x-y translation stage.
With this HPTLC-separation system, a simultaneous measurement of absorption and fluorescence spectra is possible, in contrast to HPLC-method. The measurement data generated in one run can be analyzed separately.
Fig.1: Fiber-optic sensing head: each fiber array consists of 50 UV-improved fibers  with 100 µm core diameter
3. Chemicals and measurement conditions
All chemicals have been obtained by Merck (Darmstadt). Only de-mineralized water was used for dilution.
In table 1, the different parameters for the separation shown below are summarized.
|Plate material: ||Si60 without fluorescence indicator (Merck)|
|Application:||7 mm dash-like (CAMAG Linomat II), |
1mm distance between each line, 8 lines on one side
|Mobile phase||Isopropanol, Toluol, NH3(25%) [6,3,1 (V,V,V)]|
|Measurements:||450 single spectra|
Table 1 - Parameters of separation
|Applications mass:||50 ng caffeine||1 µg caffeine and|
2.5 µg purine
|Track:||45 mm||95 mm|
|Rf-value:||caffeine:50||uric acid: 1|
Cytosine: 27, Guanine: 38,
Caffeine: 51, Adenine: 59,
Uracile: 68, Thymine: 82
In Fig. 2, the separation of caffeine is shown. 50 ng of caffeine was developed over a separation distance of 45 mm and measured within 225 s. The spectral light absorption was determined in dependence of the separation distance. The symmetrical shape of the absorption peaks shows that no impurities are present.
Fig. 2: 3D-plot of the absorption spectra of a caffeine separation over 45 mm separation distance Using the same mobile phase, a separation with purine, cytosine, guanine, adenine, uracile and thymine is possible, whereas there are no entire separation between uracile and thymine. As shown in Fig. 3, uracile and thymine are very similar in their structure. However, adenine and guanine have chemical structures, which are similar to caffeine and uric acid.
Fig. 3: Chemical structures of urine acid, cytosine, guanine, caffeine, adenine, uracile and thymine
Using a multiple-component-analyses, uracile and thymine can be separated chemometrically, because the spectra of these compounds are slightly different at the wavelengths of 235 and 280 nm. Using cross-correlation technique /5/ the measured data can be well-defined assigned to one of both substances. For calculation, the spectrum of uracile at the separation distance of 51.1 mm and the spectrum of thymine at 56.3 mm were used. Two signal curves have been determined, as shown in Fig. 4 above the densitogram measured at 268 nm wavelength. The chemometrically separated signals of both compounds can be procured for quantification.
Fig. 4: Two-component analyze for chemometric separation of uracile and thymine at 268 nm
With the measurement parameters of the purine separation (table 1), a complete separation of the five purines parallel to caffeine and urine acid is possible, without additional calculations. In Fig. 5, a contour plot of this separation over 90 mm separation distance with 450 single spectra is shown. The densitogram at 269.4 nm wavelength is above the contour plot, while the spectra of caffeine at the separation distance of 50.5 mm can be seen on the left.
Fig. 5: Contour plot of a separation of urine acid (1), cytosine (27), guanine (38), caffeine (51), adenine (59), uracile (68) and thymine (82); the Rf-values are in shown in brackets;
on left side: caffeine spectrum at 50.3 mm; on the top: densitogram at 269.4 nm
The quality of the measured spectra is excellent; in combination with spectra libraries substance can be easily identified. It is remarkable that the solvents of the mobile phase have no disturbing effect, because they are totally removed before the measurement run. The contour plot gives a fast and comprehensive overview of the quality of the separation which can be used for an easy determination of the wavelengths for a more detailed quantification. In addition, these spectra are available for analyses of peak purity. The system is not distinguishable from commercially available diode-array detectors which are commonly used in HPLC. However, much more information can be expected for one HPTLC-track, in comparison to standard single-wavelength scanner used in TLC today.
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 V.R. Meyer: “Praxis der Hochleistungs-Flüssigchromatographie”. Verlag Salle+Sauerländer (1992),
 B. Spangenberg, K.-F. Klein: “ „Fibre optical scanning with high resolution in thin layer chromatography“. J.Chromatogr. A, 898 (2000), pp. 265-269
 M. Huebner, H. Meyer, K.-F. Klein, G. Hillrichs, M. Ruetting, M. Veidemanis, B. Spangenberg, J. Clarkin,
G. Nelson: „Fiber-optic systems in the UV-region“.
SPIE-Proc. BiOS‘00, Vol. 3911, pp. 303-312 (San Jose, Jan. 2000)
 B.Spangenberg, B.Ahrens, K.-F.Klein:“TLC-analysis in forensic sciences using a diode-array detector”. Chromatographia Supplement, Vol. 53, p.438-441 (2001)
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