Prashant Hande, Lab Manager, Anchrom, gives an insight on how HP-TLC techniques offer numerous possibilities of detection
Traditional methods of detection, identification and quantification in HP-TLC are based on image documentation, scanning densitometry in UV absorbance and fluorescence, spectra recording and post chromatography derivatisation (PCD), as seen in earlier issue.
In HP-TLC, the chromatographed sample is retained by the layer, which is amenable for further analysis. By using multiple techniques, several ‘chromatograms’ are obtained from each sample.
After chromatographic separation, it is possible to generate as many as 15 ‘chromatograms’ by instrumental analysis (image documentation and scanning, both before and after PCD) and by bio-activity studies. HP-TLC plate, with multiple chromatograms of same sample, can be cut (!) and each ‘piece’ evaluated by a different detection mode to get different information about the sample. That’s a truck load of data, from just one ‘physical’ chromatogram, useful for QC as well as R&D! All this without repeating chromatography!!
Studies on molecules as potential new drug candidates are done on a micro or nano-scale. HP-TLC plates usually contain enough amount of unknown substances or a group of substances for in-situ bio-studies.
If a separated ‘fraction’ shows promise, that fraction may be further resolved by 2-D HP-TLC into more fractions for the same study. This narrows down the molecules with potential, within a day or two for further studies by MS, IR, NMR etc. Using 2mm thick layers, it is possible to isolate sufficient amounts of a pure substance.
In PCD, the plate is sprayed with the derivatisation reagent and heated to complete the reaction. However, for bio-assays, heating is usually not necessary. Here the suitably cut part of the plate is immersed in the reagent or a solution containing live organisms like bacteria or fungus. Then it is straight forward to observe the results, usually with naked eye.
Mass spectrometric detection is very widely used in chromatography, including HP-TLC. The ‘physical chromatogram’ is available on the plate and using an elution interface; the desired fraction(s) can be eluted and fed to a MS online. Such a cycle of band location and elution in to MS & obtaining m/z values takes 3-4 min. The interface is an ideal, very highly productive, off-line accessory for an existing LC-MS or a LC-MS-MS. Many R & D labs abroad have a standalone MS with TLC/ HP-TLC MS interface as common facility. As many as 70-80 mass spectra of selected fractions can be obtained in a day. The days of monitoring reactions by TLC and then wait for MS from LC-MS in next two to three days are history.
Some of the common bio-detection techniques in HP-TLC are as follows:
Toxicity study – Vibrio Fischeri is a bioluminescent bacterium. It is also very weak and easily killed in the presence of even mildly toxic substances. The plate is dipped in a solution containing V. fischeri & kept inside a completely dark chamber, mounted with a cooled, long exposure camera. The dark bands are observed where the bacteria die due to absence of luminescence. The test is practically instant, a very big advantage in drug discovery.
Anti-oxidant study – The chromatographed plate is dipped in a DPPH solution. Anti-oxidant fractions appear light on a dark background.
Effect directed analysis – It has been reported for estrogens, seed oils, insecticides
Anti-bacterial or Anti-fungal study – The chromatographed plate is similarly dipped in a suitable medium containing appropriate fungus or bacteria. Those fractions that have “anti” properties are evident due to zones of inhibition, against the normal growth all over the plate.
In conclusion, it can be said that preservation of ‘chromatogram’ is unique to HP-TLC. This allows multiple progressive detection of fractions by non-destructive and destructive detection modes.