Effects of mobile phase composition on the reversed-phase separation of dipeptides and tripeptides with cyclodextrin-bonded-phase columns.

The effects of mobile phase composition on the reversed-phase separation of several dipeptides and tripeptides with a gamma-cyclodextrin-bonded-phase column have been studied. The addition of organic modifier (i.e. methanol) into the aqueous buffer (pH 4.65) mobile phase causes a minimum capacity factor value to be observed for each peptide. This is interpreted to result from two retention mechanisms involved in the separation. The adsorption process causes the retention time to decrease as the water content in the mobile phase is increased. The inclusion process acts in the opposite fashion. The presence of Cu(II) salt in the mobile phase allows further modifications of separation selectivity. This is because the peptide conformation changes upon Cu(II) complexation which in turn alter the hydrophobicity and/or inclusion stability of the peptide. The effects of mobile phase pH (3.6-5.6) and ionic strength (0.001-0.06) were not significant in the present application. Studies with a beta-cyclodextrin column show similar results.