Prashant Hande, Lab Manager, Anchrom, in the second part of the series, gives an insight about how HP-TLC offers numerous possibilities of detection which can provide insight about the sample
Part I of the article dealt with the traditional methods of detection, identification and quantification in HP-TLC i.e. image documentation, scanning densitometry in UV and fluorescence, spectra recording and post chromatography derivatisation (PCD).
In HP-TLC, because there are two chromatograms to begin with viz. the physical chromatogram on the plate and the instrumental chromatogram(s), one can have the cake and eat it too!
In fact, after chromatographic separation, it is possible to generate as many as 15 “chromatograms” by instrumental means (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 anything!!
Studies on effectiveness of a molecule as a potential new drug candidate 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.
One physical “Chromatogram”
Multiple detection methods can be used
If a separated ‘fraction’ shows promise, that fraction may be further resovled 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.
By far, mass spectrometric detection is most popular in chromatography. HP-TLC too can be very simply hyphenated with it. The “physical chromatogram” is available on the plate and using an interface, any desired fraction(s) can be eluted and fed to a MS. Such a cycle of band location and elution 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 2-3 days are history.
Some of the common bio-detection techniques in HP-TLC are as follows:
- Anti-oxidant study – Dip the plate in __% DPPH solution. Anti-oxidant fractions appear light on a dark background in __ hrs.
- 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-bacterial or Anti-fungal study – The plate is similarly dipped in a suitable medium containing bacteria or fungus. Those fractions that have “anti” properties are evident due to zones of inhibition, against the normal growth all over the plate.
- Effect directed analysis – It has been reported for estrogens, seed oils, insecticides
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.
It may be concluded that HP-TLC offers numerous possibilities of detection that can give tremendous possibilities of detection that can give detailed insight about the sample. This is feasible due to separated fractions on the plate, are available for further analysis.