Abstract
For the purpose of separation and analysis of peptides by high speed liquid column chromatography using volatile eluents, the effects of pH and polarity of eluents on retention of amino acids on octyl-silica were investigated. The use of lower pH eluents resulted in the increase of retention time. The ratio of capacity factor of an amino acid at pH 2.3 to pH 3.3 and at pH 10.3 to pH 3.3 were 7.3 and 0.1 respectively. The use of lower polar eluents such as 50% MeOH-0.01 M formic acid resulted in the reduction of retention time of hydrophobic amino acids, on the contrary in the increase of retention time of hydrophilic amino acids. Logarithmic plot of partition coefficient (of amino acids in octanol-water system) against the capacity factor gives a straight line with several eluents. In order to increase the affinity of rather polar substances, introduction of some substitutional groups gave some improvements. The ratios of capacity factor of an amino acid against its derivatives indicates a constant value φ which is specific for the substitutional group. Value φ is 2, 3, or 21 for N-glycyl, O-methyl, or N-acetyl, respectively. The retention time of peptides could be predicted from summation of the logarithm of capacity factor of each constitutional amino acid, using Pardee equation. On the other hand, from retention time of peptide and from the amino acid composition, molecular weight of a peptide was calculated. This method was successfully applied to an acidic peptide purified from bovine brain, and the peptide was assigned to be γ-glutamyl-cystinyl-glycine.
