Chromatographic method for the determination of conditional equilibrium constants for the carbamate formation reaction from amino acids and peptides in aqueous solution

Article abstract of DOI:10.1021/ja00069a037

A novel and sensitive method has been developed and evaluated for the study of carbamate formation equilibria of amino acids and peptides in aqueous solution. The method is based on reversed-phase liquid chromatography with cetyltrimethylammonium bromide. The reliability of the method was established by comparing the results determined from the present study with the few data in the literature. The relaxation rate of the carbamate reaction was shown to be faster than the chromatographic distribution relaxation rate (seconds). As a result, the retention time of amine solutes is increased in the presence of CO₂. Carbamate formation constants and mole fractions of carbamates at physiological pH of eleven L-α-amino acids and peptides were determined. No correlation between the formation constant and the ammonium pK_a was found. There is significant dependence of the amount of a particular amino acid or peptide that exists as carbamate at pH 7.4 on the pK_a of the ammonium group, however. This is due to mass action rather than reflecting the influence of pK_a on the propensity of the amine to react with CO₂. It is suggested that amino acids and peptides with ammonium pK_a greater than 9.5 do not form significant amounts of carbamates in aqueous solution near neutral pH.