USE OF IMPREGNATED LAYERS FOR TLC SEPARATION OF COMPOUNDS OF PHARMACEUTICAL AND ANALYTICAL IMPORTANCE

Abstract

Since 1956, when thin layer chromatography was first introduced by Stahl as a procedure for analytical adsorption chromatography large amount of work has been done on development of suitable solvent systems for the separation of various classes of compounds. Although a large amount of work on TLC separation of various classes of organic compounds and inorganic ions has been done and suitable separation schemes for plant materials like glycosides, alkaloids, sugars, anthractuinones, phenols and many materials of biological and biochemical importance e.g. amines, aminoacids, steroids, lipids have been worked out. However, an efficient separation of several such compounds and inorganic ions of pharmaceutical and analytical importance has been a challenging problem due to overlapping and tailing of spots. To resolve this problem the use of mixed adsorbents in chromatography was first tried by Morris in 1962, Carton and Bradbury in 1965 and then by Cheng-Hsia in 1968. Thus reagents, which will form salt, complex or chelate to a lesser or greater extent or have a tendency of hydrogen bond formation with the substance, to be separated, have been tried as additives to the main adsorbent material. The important work in this field has been done by Bark and coworkers in 1967 for the separation of phenols using polyamide and different amides 11 as impregnants. Later on Yasuda in 1971 used different metal salts as impregnant for improving the separation of aromatic amines. Brinkman et al in 1973 and Yeole et al in 1983 used high molecular weight amines as impregnants on silica gel layer and paper for the chroma tographic separation of inorganic ions. Inspite of a large amount of work done on the TLC separation of various groups of inorganic ions and different classes of organic compounds, much attention has not been paid for their separation on impregnanted layers. Since the separation and identification of several metal ions and compounds of pharmaceutical and analytical importance such as amino acids, dyes and antihistamines is very important and requires efficient separation methods, it was considered worthwhile to inves tigate the separation of such classes of compounds by TLC using different impregnants and to develop new solvent systems giving better resolutions. The present thesis is concerned with the TLC studies on inorganic ions including lanthanides, amino acids, dyes and antihistamines using different impregnants like organic ligands, liquid ion exchangers, metal salts and polyamide on silica gel or cellulose layers. The chromatographic behaviour of thirteen inorganic ions viz. Pb2+, Kn2+, Cd2+, Ni2+, Zn2+, Fe^, Cu2+, Mg2+, U02+, Th4+, V5+ and Pd2+ was studied on 8-hydroxy quinoline, dibenzoylmethane and 1-proline impregnated ill silica gel layers. It was found that silica gel G impreg nated with 0.5/i 8-OH-ouinoline and with 0.2^ debenzoyl methane acted as the best adsorbent systems using the solvent system BuOH-EtOAc-AcOH (40:10:10) and EtOAc-HCOOHH20- Pyridine (30:10:10:5) respectively, 1-Proline was not found as good impregnant. The hR„ values on 8-OH-quinoline and dibenzoyl methane impregnated layer were generally found higher than those on plain silica gel pla.te. The hR„(100) of iron on dibenzoyl methane impregnated layer also offered a method of the removal of traces of iron from silica gel G (Chapter II A). 2+ TLC separation of metal ions of interest viz. Cd , Cu2+, ^2+, Fe2+, m2+, U02t Co2+, Pb2+, Mg2+, V5+, Hg2+, As and Th44" was carried out on 0.50/ 2,2'dipyridyl and 0.75;* iminodiacetic acid impregnated silica gel layers using the solvent system Isoamyl alcohol-HjO-AcOH (20:10:10) and MeOH-Benzene-A cOH (20:10:5) respectively. The metal ions separated on the impregnated layer have been divided into two groups as 'metal ions in vivo' and 'toxic metal ions' (Chapter II B) . TLC studies of Th4+, Ti44", Zr4+, Y34" and seven lanthanides viz. Dy34-, Gd34", La3+, Nd34", Pr34", Sm34", Tb34" were carried out on dibutyl butyl phosphonate (DBBP) , bis(2-ethyl hexyl) orthophosphoric acid (HDEHP) and bis-(2-ethyl hexyl) orthophosphoric acid-trioctyl phosphine oxide (HDEHP-TO'PO) impregnated cellulose layers. The most suitable solvent system was MeOH-6N HNOo- Ace tyla ce tone IV (20:10:5) and microcrystalline cellulose powder impregnat ed with 1.0/ HDEHP-TOPO in EtOH-H20 (70:30) acted as the best adsorbent system. The spots were visualized by spraying with a 1.0/ alcoholic solution of alizarin and exposing to ammonia vapours. A tetranary separation of lanthanides was achieved (Chapter III). The different twenty amino acids studied for separation were : 1-leucine; d,l-iosleucine; d,l-tryptophane; d,l-methionine; d,l-valine; 1-lysine HCl; 1-histidine HCl; d,l-P phenyl alanine; d,l-threonine; d,l-alanine; d,l-serine; 1-tyrosine ; 1-glutamic acid; d,l-aspartic acid; 1-arginine HCL; glycine; 1-proline; 1-cysteine HCl; d,l-2 amino butyric acid and 1-ornithine HCl* TLC separa tion of these amino acid was carried out on 0.25/ impreg nated copper sulphate and polyamide mixed silica gel (5:1) layers. The best solvent system was found to be MeOH-BuOAc-AcOH-Pyridine (20:20:10:5) which travelled a distance of 10 cm in 35 minutes at 17+1°C. Therefore above solvent system offers a rapid method for the identi fication of amino acids on impregnated layers. The amino acids were visualized by spraying with 0.1/ ninhydron in acetone and then heating the chromaplate at 60 C for five minutes (Chapter IV). TLC separation of 30 synthetic dyes on copper sul phate and ammonium molybdate impregnated silica gel-G plates were tried. The different dyes were - chrysoidine, malachite green, rosaniline HCl, methyl red, crystal violet, fuchsine basic, auramine 0, bromophenol blue, eosine bluish, bromocresol purple, congo red, titan yellow, aluminon, alizarin red S, magneson, orange G, bromocresol green, phenol red, thymol blue, gentian violet, navilline brilliant pink, aniline blue, dichlorofluroscein, xylidine ponceau, benzo purpurine, methylene blue, nigrosin, fuchsine acid, light green and alizrin blue. Two solvent systems were developed for carrying out the separation of these dyes on ammonium molybdate and copper sulphate impregnated layers. The solvent system BuOH-AcOH-H^O (20:5:10) produced better separation on impregnated layers. All the dyes were self visualized (Chapter V). Seven antihistamines were separated on copper sul phate impregnated silica gel G layers using the solvent system Benzene-DMF-A cOH (20:10:5). The antihistamines taken were - diphenhydramine HCl, bromodiphenhydramine HCl, Carbinoxamine maleate, chlorpheniramine maleate, mepyramine maleate, promethazine HCl and triprolidine HCl- The anti histamines were visualized by spraying with Dragendorff reagent.