Preliminary results of 2-D separation of test dye mixture using high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) are demonstrated. The advantage of 2-D HPTLC/PPEC separation is based on different separation selectivities obtained in both HPTLC and PPEC systems. HPTLC RP18 W plates of 5x20 cm from Merck were used in the investigations. In the first dimension, a HPTLC process was performed using 5 cm length of the plate and in the second dimension PPEC separation was obtained applying plate of 20 cm length. PPEC process followed prewetting the chromatographic plate with sample zones on it, which were partly separated after first dimensional (HPTLC) separation. In the experiments, the modified version of PPEC device for 20 cm long chromatographic plate and the reservoir for prewetting the adsorbent layer were applied.
Because chemical and pharmacological studies of Eleutherococcus species growing in Asia showed their biological activity depends on glycosides known as eleutherosides, we used TLC to search for eleutherosides in ethanol extracts of the roots of E. senticosus, E. setchuensis, E. sesiliflorus, E. henryi, E. gracilistylus, and E. divaricatus cultivated in Poland. Eleutherosides B, E, and E 1 were identified in all the roots investigated and isofraxidin was identified in the roots of E. senticosus. Satisfactory separation of the eleutherosides from other components of the extracts was achieved by use of two mobile phases in two-step elution. The identity of the resolved compounds was confirmed by comparing their R F values and UV spectra with those of standards.
A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP + /NBT/PMS (b-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H 37 Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H 37 Ra PGI was 226 mg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H 37 Ra PGI inhibition by PEP.
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