THIN LAYER CHROMATOGRAPHIC STUDIES ON AMINO ACIDS AND SOME IMPORTANT PHARMACEUTICALS

Abstract

The wide spread use of thin layer chromatography (TLC) reflects a rare combination of characteristics in an analytical procedure : tremendous versatility, exceptional performance and marked simplicity of technique, apparatus and interpretation. The usefulness of TLC has been further multiplied many folds by various significant advances such as, an increased variety of precoated layers including chiral phases, bonded phases, improved instrumentation for spotting, developing and scanning layers and for applying detection reagents and development of optimization strategies and special coupled techniques viz. TLC/MS. In modern chromatographic methods such as high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), supercritical fluid chromatography (SFC), gas chromatography (GC) and so often in fields of analytical chemistry, the available commercial instruments are frequently far more complicated and consequently far more expensive than is necessary for routine chemical application. TLC has rapidly developed into an indispensable tool in chemistry, biochemistry, medicine, biology and pharmacy because of rapid and inexpensive nature. Despite the trend towards increasing use of modern TLC with expensive instrumentation, it is still undoubtedly true that most work with TLC is being done throughout the world with relative inexpensive basic equipment and manual techniques. TLC involves concurrent processing of multiple samples and standards on the same open layer developed by a mobile phase in a variety of modes, including simple one dimensional, multiple, circular and multidimensional. It allows (i) formation of the derivatives without resorting to extensive purification and recrystallization. To enhance separation and identification characteristics further, thin layer is impregnated with some suitable organic or inorganic compound and thus several kinds of reactions can be performed on a plate. Reagents used for impregnation often affect the behaviour of adsorbent by •interacting with sample through coordination, chelation or inclusion complexation. The range of application of TLC is considerably expanded through the use of different impregnation reagents added to layer or to the mobile phase in various concentrations. The detection is done statically with an assortment of diverse possibilities. Amino acids, their derivatives such as dansyl amino acids, antihistamines and antibiotics chosen for thin layer chromatographic studies have wide applications in the fields of protein chemistry, medicinal science, forensic science, toxicology etc. Amino acids are building block of proteins. In addition to protein bound amino acids, the cellular tissue and fluid of living organisms contain a permanent reservoir of free amino acids. Therefore free amino acids take part in metabolic reactions. Their importance in determining structure of peptides and proteins and establishing nutritional value of protein is ever increasing. Enantiomers of amino acids and other bioactive molecules often act as two different molecules with different biological activity and hence gaining significance in chiral synthesis, protein structure function relationship studies, dating ol fossil materials and above all in the development of safer, more potent chiral drugs. Not only biological activity but also potency, toxicity, transport mechanism and routes of metabolism of enantiomers are different. This has led their industrial manufacture for applications in (ii) pharmaceutical industry, food industry, animal husbandry and poultry farms. Antihistamine agents are primarily used in the management of certain allergic disorders. Antihistamines separation and identification in body fluids or in tablets, is of great significance in medicine and toxicology. Aminoglycoside antibiotics discovered during early age of antibiotics research, have evolved into a major therapeutic armamentarium for clinicans for the maintenance of human, animal and plant health. This group comprises over 100 compounds produced by diverse genera of microorganisms having typical chemical and antimicrobial features. Dansyl amino acids owing to their fluorescent nature are preferred for study of protein sequencing. They are easily detectable'] In view of above it was considered worthwhile to study TLC separation of amino acids, dansyl amino acids, antihistamines, antibiotics and work out more simple and effective separation schemes than earlier reported. Various impregnation reagents such as suitable chiral selector for enantiomeric separation and metal ions have been used for improving such systems wherever required. The present thesis comprises of four chapters. The first chapter is an introductory one dealing with historical developments, theory and mechanism, nature of adsorbent, advantages and applications of TLC. The literature, related to TLC studies of the classes of compounds selected, has been cited in subsequent chapters. Second chapter has been divided into three sections. Section A is related with studies of amino acids using plain and impregnated silica gel thin layers. Chloride, nitrate, acetate, sulphate and oxalate salts of ammonium ion have been used for impregnation after working out two new

solvent systems viz., n-butanol-methanol-acetic acid (8:l:3;v/v) and n-butanol-carbontetrachloride-acetic acid (8:3:l;v/v). Effect of variation of concentration of impregnation reagents on chromatographic behaviour of amino acids was studied. Efforts have also been made to propose mechanism of separation by studying both, pH of sample solution and the solvent system. Section B deals with TLC separation of enantiomeric amino acids on silica gel layers. Use of chiral selector during slurry preparation resulted in resolution of certain amino acids. Application of Cu(Il)-L-proline in adsorbent for the enantiomeric separation of DL-Try and DL-Ser was studied. Solvent systems developed were different ratios of acetonitrile-methanol-water. Spots of amino acids were visualized with the help of ninhydrin.*Section Cdescribes separation of mixture of dansyl amino acids on plain plates. Three solvent systems, dichloromethanebenzeue- acetic acid ( 15:4: I;v/v), chloroform-ben/ene-acetic acid (2:15:2.5;v/v) and heptane-n-propanol-propionic acid (14:1.5:2;v/v), have been developed using silica gel thin layers for ten dansyl ammo acids viz., Ala, Phe, Leu, He, Val, nor-Val, Pro, Tyr, Try, Met. Also difficult combinations of certain dansyl amino acids were resolved on untreated plates in the newly worked out solvent systems. UV detection was employed. Third chapter gives detail account of seven antihistamines namely triprolidine. mebhydrolin, promethazine, trimeprazine, cyproheptadine, pheniramine and diphenhydramine, separation. Three new solvent systems n-bulanol-water-acetic acid (4:1:1 &4:0.5:1 ;v/v) and benzene- n-butanolwater- acetic acid (7:8:2:5;v/v) were systematically worked out for separation on untreated and impregnated silica gel layers. Metal ions used for impregnation were Mn++, Fe++, Co++, Ni++, Cu++, Zn++ and Cd++. (iv) Solvent system n-butanol-water-acetic acid (4:0.5: l;v/v) gave best results on plain plates only. Detection was performed by exposing chromatoplate to iodine vapours in an airtight chamber. Fourth chapter incorporates results of TLC separation studies of six aminoglycoside antibiotics viz. kanamycin, neomycin, sisomycin, streptomycin, tobramycin, and amikacin. Fifteen solvent systems comprising dioxan-ammonia, methanol-ammonia and n-propanol-ammonia (each having five ratios) were systematically developed giving successful separation of aminoglycosides. Effect of chain lengthening of alkyl group, present in the alcohol content of solvent system, on hR,. values, was studied. Detection was performed by iodine vapours. Thus various important classes of compunds stated above were resolved and detected in the conditions mentioned.