CHROMATOGRAPHIC STUDIES OF AMINO ACIDS, THEIR DERIVATIVES AND COMPOUNDS OF BIOLOGICAL IMPORTANCE

Abstract

Chromatography in its various forms such as High performanc liquid chromatography (HPLC), Thin layer chromatography (TLC), Gas chromatography (GC), Paper chromatography (PC) and column chromatography (CC) is regarded as one of the most important tech nique for separation, purification, identification and isolation of organic and inorganic compounds. Of the various forms of chro matography, TLC has assumed an ever increasing importance since 1956, when this technique was first introduced by Stahl as a procedure for analytical adsorption chromatography. Thin layer chromatography has attained the status of routine analytical tech nique because it is an inexpensive and provides excellent sharp ness of separation, high sensitivity and great speed. The sample and standard can be run in parallel under identical conditions on the same plate, allow the formation of the derivatives without resorting to extensive purification and recrystallisation. Tne inert character of thin layer material makes it ideally suitable for the study of corrosive compounds, for performing several kinds of reactions on the plate and for impregnation of the adsorbent layers with a variety of compounds. Amino acids, phenylthiohydantoin amino acids, vitamins and alkaloids are very important classes of compounds. They have received a very significant attention in chemistry, biochemistry, medicinal science and forensic science. Therefore, the separation and identification of these compounds is very important and requ ires an efficient method. since a search of literature reveals that impregnation of thin layers has been resulted into the e iii improved resolution of a variety of compounds (Chapter - IV and VI) and not much work has been done on these compounds using imp regnated systems. It was considered worthwhile to investigate the separation of such compounds by the above cited simple, rapid, more sensitive and efficient technique i.e. impregnated Thin layer chromatogrphy. In view of this, TLC studies of such classes of compounds were carried out. The present work includes the TLC studies of amino acids, their phenylthiohydantoin derivatives, vitamins and alkaloids, besides, adsorption and partition coefficients of amino acids were determined on untreated and impregnated systems and these coefficients were correlated with' the hRf values of amino acids. TLC resolution of enantiomers of amino acids and PTH-amino acids has also been carried out. During these studies use of alkaline earth metal ions, halides, zinc acetate, cadmium sulphate,tartaric acids and nickle chloride was made as the impregnating agents for the better resolution of these compounds, and new, rapid and effective solvent systems were developed. The present thesis comprises six chapters. The first chapter is an introdutory one, dealing with the historical development, significance and use of the technique of thin layer chromatography. Special references have been cited towards the present position regarding the use of •impregnated layers. The second chapter concerns with the TLC studies of amino acids. Amino acids exist as anion; (P ) and cations below i1 is above their isoelectric point •t. These ions of amino acids may combine with a cation (metal ion) or anion (halides) respectively and IV thus may show different chromatographic behaviour. Attempts have been made to study some aspects of the theory of amino acids sepa ration based on such ion behaviour. The thin silica plates were impregnated with alkaline earth metal ions under different condi tions and halides and two new solvent systems were developed for these studies. Third chapter describes the separation of enantiomeric mix ture of amino acids and their 'phenylthiohydantoin derivatives. All the DL-amino acids and DL-phenylthiohydantoin amino acids were tried but a few could resolve. DL-phe, DL-try, DL-val and DL-ile were resolved successfully on cellulose layer using the solvent system n-butanol-8-hydroxy quinoline (1.0%) in 50% ethanol (8:10, v/v). Cellulose provides chiral environment and is believ ed to form molecular inclusion complexes with the two enantiomers under varying conditions of hydrophobicity leading to their reso lution. Phenylthiohydantoin derivatives of DL-met, phe, val, lie, tyr, thr, ala and ser were resolved on (+)-tartaric acid (0.3%) impregnated silica gel layer using the solvent system: chloroform-ethyl acetate-water (28:1:1, v/v/v).(+)-Tartaric acid and (-f-)-PTH-amino acids formed (+-) and (++) diastereoisomeric salts having different solubilities, as expected and were separat ed in the mobile phase. The spots of amino acids were located by ninhydrin and of PTH-amino acids by exposing the chromatoplate to iodine vapours. The fourth chapter incorporates TLC studies of vitamins B-complex group (water soluble vitamins) . The separation of this group is a challenging problems due to tailing and overlapping of spots. Attempts were made to resolve this problem. The use of zinc acetate impregnated silica gel layers and the solvent systems BuOH-HOAc-H2 0 (9:4:5, v/v) and BuOH-C6 Hg -HOAc-H 0 (8:7:5:3, v/v) resulted in the better resolution of vitamin B , B2 ' B6' B12 and nicotinic acid. The fifth chapter deals with separation of alkaloids viz. atropine, brucine, berberine, ephedrine and quinine. The TLC of these alkaloids was carried out over untreated and cadmium sulphate impregnated silica gel and a new solvent system BuOHHCOOH (10:10, v/v) was developed when all of these above alkaloids were separated. The spots of these alkaloids were located by exposing the chromatoplate to iodine vapours. Chapter Vlth deals with the determination of adsorption and partition coefficients of amino acids on silica gel, water and solvent systems used in the thin layer chromatographic experi ments of this work. Attempts have been made to correlate the hRf values of amino acids with their adsorption and partition coefficients.