Coupling HPLC-HPTLC

F. 6 Schematic representation of HPLC-HPTLC coupling by means of the OSP-2 system (Merck) for post-column enrichment of the column eluate fractions. 

The term multimodal has been used in two ways in TLC, to designate layers such as bonded cyano sorbents that can operate with two or more mechanisms (see Section IV.C) or, in the context of this section, to specify multidimensional separations that are performed by coupling TLC, HPTLC, or OPLC (223) with another technique, such as gas chromatography, supercritical fluid chromatography (224), countercurrent chromatography (225), and, most commonly, HPLC (145, 226-228), in order to improve the separation capacity available from either of the individual methods. For example, the combination of adsorption AMD-HPTLC and partition HPLC for water analysis produced as many as 700 individual densitometric peaks (49). Multimodal TLC separations have been reviewed (229-231). 

HPTLC is a very fast and convenient assay to separate samples components and is often used in Organic Chemistry and in Synthetic approach. Unknown substances, after different display assay, were generally scraped off from the TLC/HPTLC plate, diluted into a tube and transferred into the MS system for structural elucidation and characterization. Now, a TLC-MS interface was developed by CAMAG, which can semi-automatically extract zones of interest and on-line direct them into any brand of a HPLC-MS system. The TLC-MS interface is connected by two fittings to any HPLC instrument coupled with mass spectrometer, without other system configuration adjustments or mass spectrometer modifications. By this way, the unknown substances can be directly extracted from a TLC/HPTLC plate, eluted and resolved by HPLC system and sensitive and selective mass spectrometric signals are obtained within a minute per substance zone [33],... 

Densitometric methods. In situ densitometry is an often-used technique for lipid quantitation and has been extensively reviewed by Prosek and Pukl (1996). Lipids are generally sprayed with reagent and their absorption or fluorescence can be measured under UV or visible light by means of a densitometer. The method needs to be standardized and suitable calibration curves need to be constructed to avoid errors. There are several models of densitometer available and some of them are highly automated and coupled to computer systems. Apart from these the use of CCD (charge-coupled device) cameras and colour printers have further improved the densitometric capabilities for accurate quantitations (Prosek and Pukl, 1996). A recent review by Ebel (1996) compares quantitative analysis in TLC with that in HPTLC, including factors that can effect quantitation, the need for careful calibration and errors in quantitative HPTLC analyses. Ebel is of the opinion that as both HPTLC and HPLC are based on the same absorption and fluorescence phenomena they should obtain similar results with respect to quantitation. 

By nature, thermal desorption techniques are limited to lower mass species amenable to vaporization and thermally stable compounds. Several approaches are not commercially available so far. New ion sources working under ambient conditions are not yet status quo in the most MS laboratories and must be bought specifically for TLC/HPTLC-MS, which might be hindered by an expensive price. These circumstances lessen the impact of the new development. On the other hand, having a forced-flow technique in the laboratory or the TLC-MS interface, coupling is readily feasible with any HPLC-MS system. 

New developments in the TLC of vitamin E have been concerned mainly with refinement of detection and quantitation (e.g. densitometry), rather than with new chromatographic systems, although a few HPTLC procedures as well as a separation of D and L isomers (50) have been reported. This trend toward increasing sophistication hardly makes these new TLC approaches attractive to poorly equipped laboratories as routine techniques for vitamin E determination. As shown above, the opposite may be true for assays of vitamin D metabolites, in which TLC can conveniently replace HPLC as a sample purification method. The explanation for this difference in the position of TLC with respect to both vitamins lies in the availability of suitable techniques for quantitation. In case of vitamin D metabolites, TLC can be easily coupled with a simple and yet highly specific radioligand assay. In contrast, for vitamin E no such off-line determination of equally powerful performance exists, the older colorimetric or gas chromatographic procedures being obsolete. 

The direct coupling of HPLC and HPTLC seems to be a very powerful method in the multiresidue analysis of pesticides. An instrument was developed for the direct connection of these chromatographic methods. The effluent obtained from a HPLC column was transferred directly to a TLC plate widi this device. According to the Camag AMD method, the plates were developed by a 20-step universal elution gradient from methanol-dichloromethane to n-hexane. The compounds investigated in this system were benomyl, 2,4-D, etrimfos, atrazine, phenylmercury acetate, and linuron. Densi-tometric evaluation was carried out with a computer-controlled Camag TLC scanner n, with HP 9816 S and TLC evaluation software 86. This method opens a new way in the automated multiresidue analysis of pesticides (134). 

---