HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF PEPTIDES USING SALT SOLUTIONS AS MOBILE PHASES

The purpose of this research was to develop new analytical techniques for the analysis of low molecular weight peptides in biological samples, using reversed phase high performance liquid chromatography. First, several neutral and ionic compounds were investigated as solutes to determine the effect of pH on the use of carboxylic acids as ion pairing reagents in RP-HPLC. It was found that the ion-pairing properties of carboxylic acids in mobile phases could be controlled by adjusting the pH of the solution. These results, coupled with the use of soft acid cation salts of carboxylic acids, provided a new system of static retention for peptides in HPLC which could be controlled by adjusting the pH of the eluent. It was found that increased retention of peptides was influenced by the soft acid character of the cation of the carboxylic acid salt used, and by the chain length of the acid. Longer chain length and softer acid character provided increased retention. Proton decoupled (\u2713)C-NMR spectroscopy was used to investigate the interaction of these salts with a series of model peptides. Silver(I) acetate was found to mainly interact with the amide carbonyls and terminal amino group on these peptides. An ammine type complexation was hypothesized for this interaction. The use of silver(I) valerate solutions for the analysis of peptide mixtures was attempted. By employing a split column technique and a pH and methanol gradient system, good separation of a series of model peptides, including Phe-(Gly)(,n), where n = 1-4, was obtained. Increased resolution of these peptides using this technique was obtained compared to isocratic elution. This is due to concentration of the sample at the head of the analytical column prior to elution, and the use of the gradient system. This concentration-elution system presents itself as an improved method of injection in HPLC. Preliminary results for the analysis of peptides in complex biological samples indicated that this technique suffers from poor resolution and low sensitivity for these samples. The use of other solvent or gradient systems, coupled with other methods of detection may increase the sensitivity and resolution of the present technique