THIN-LAYER CHROMATOGRAPHIC STUDIES ON AMINO ACIDS AND THEIR PTH-, DNP-, AND DANSYL DERIVATIVES

Abstract

The choice of analytical method is clearly related to cost, the availability of instrumentation and to the sensitivity needed for the analysis. We considered TLC to have several advantages over other methods of chromatography because it is sensitive, inexpensive and simpler, allows easier detection of colorless compounds and densitometric scanning for quantitation, sample and standards can be run in parallel under identical conditions on the same plate, allows formation of a derivative without resorting to extensive purification and recrystallization and the components of the mixture can be eluted and used subsequently. The great advantages of TLC are the wide variety of development techniques allowing separation of complex mixtures and numerous detection procedures are possible because of the static nature of the detection process. Besides, various sorbents such as silica gel, cellulose, alumina, polyamide, ion exchangers and several other inorganic and organic sorbents are available for use in TLC which further enhance the versatility of the technique for analysing the types of substances. Various other chromatographic procedures are available, such as open liquid column chromatography, gas chromatography, and high-performance liquid chromatography (HPLC), these procedures are more complicated, require a greater investment in equipment and supplies, and more expertise in 11 instrumentation aspects than required for the performance of TLC. |Many a separation that requires several hours in the set up of instrumentation like GLC or HPLC can be accomplished in a few minutes on suitable layer by TLC. The importance of determination of primary structure of proteins namely the sequence of amino acids along the chain is well established. The protein or peptide is treated with a suitable reagent such as 2,4dinitroflurobenzene (Sanger's method) or 1-dimethylamino naphthalene-5-sulphonyl chloride (dansyl chloride) or phenylisothiocyanate (Edman method)which react with the N-terminal amino acid of the protein or peptide molecule and corresponding DNP-, Dansyl-, or phenylthiohydantoin derivative of N-terminal amino acid is separated and identified sequentially. These derivatives are generally identified by a suitable chromatographic method supplemented with another technique such as UV spectrophotometry. Literature reveals that GC requires derivatisation such as trimethylsilylation of the compounds to be resolved and is also not capable of resolving all the twenty derivatives of one kind or the other and therefore it could not acquire much prominence. HPLC is rapid and quantitative but involves a high equipment and running cost and many times encounters difficulties with the elution properties of PTH-Arg and His which chromatograph as broader peaks than other PTH-amino acids. Thus in view of the Ill importance of PTH-, DNP-, and dansyl derivatives in protein chemistry and greater advantages of TLC, present studies were undertaken. The work presented in the thesis is divided into following five chapters. I. General Introduction. II. TLC Separation of PTH-amino acids. III. TLC Separation of Dansyl amino acids. IV. TLC Separation of DNP-amino acids. V. TLC Separation of Racemic amino acids. Chapter I includes brief introduction about the importance, history, advantages of TLC and the importance of PTH-, 2,4 DNP-, and dansyl derivatives in protein chemistry along with the importance of resolution of racemic amino acids. Chapter II deals with the study of TLC separation of fifteen phenylthiohydantoin derivatives of amino acids. A brief introduction and an upto-date literature survey on these derivatives has been presented. Further, the literature survey indicated that attempts have not been made to resolve PTH-amino acids on metal ion impregnated TLC plates. Hence we used several transition metals, which are inexpensive and readily available, as impregnating reagents on TLC plates for the resolution of these PTH-amino acids. The various IV transition metal salts taken are MgSO., MnSO., FeSO., CoSO,, ZnSO., zinc acetate, zinc phosphate, zinc chloride and nickel sulphate in different concentrations. The various new solvent systems with use of these transition metal salts for the separation of this class of compounds are discussed in this chapter. Chapter III deals with the TLC separation of twenty 1-dimethylamino napthalene-5-sulphonyl (dansyl) derivatives on silica gel thin layers. A brief introduction and the literature review is presented. The dansyl derivatives have been divided into two groups depending upon their chromatographic behaviour on silica gel thin-layers in ten new solvent systems, five for each group* The optimization of chromatographic conditions and development of successful solvent systems have been described. Chapter IV deals with the TLC separation of ninteen dinitrophenyl (DNP) derivatives of amino acids. A brief introduction with the literature review is provided. The DNP-derivatives have been divided into two groups depending on their chromatographic behaviour on silica gel thin-layers in ten new solvent systems, five for each group. The optimization of chromatographic conditions and development of successful solvent systems have been described. Chapter V deals with the resolution of enantiomeric amino acids. These studies are important in view of the growing interest in D-amino acids in research on antibiotics, cellwall composition, testing of synthetic peptides having physiological properties, geochemical dating, and food and drug analysis. The enantiomeric purity becomes very significant when one of the enantiomers has a potential biological activity for example L-Dopa is very important drug for the treatment of Parkinson's disease. Chiral selectors such as metal complexes of L-proline were used as the impregnating agents with the inert support for the resolution of enantiomeric amino acids.