Comparison of an organic polymeric column and a silica-based reversed-phase for the analysis of basic peptides by high-performance liquid chromatography

The performance of a purely polymeric and a Type B silica-based C18 reversed-phase column was compared for the analysis of the basic peptide bradykinin and some analogues in order to assess the contribution of silanol interactions to peak shape. Good peak shapes were obtained for small masses of these peptides (0.1 μg or less) using acidic mobile phases on both columns; however, both showed a similar and serious deterioration in peak shape with increasing sample mass. Loss of efficiency on both columns as sample mass increased was considerably more serious when using formic acid rather than trifluoroacetic acid (TFA) as a mobile phase additive. For example, the peak capacity for a 2.5 μg load of one bradykinin on the polymeric column was reduced to only 0.38 times its value for 0.1 μg when using 0.02 M formic acid, compared with 0.77 times its value when using the same concentration of TFA. This result can be attributed to the ion pair effect of TFA and its higher ionic strength, which reduce mutual repulsion of charged peptides when held on the hydrophobic surface of the phase. Addition of salt (KCl) to the formic acid mobile phase caused dramatic increases in retention on the polymeric column, which can also be attributed to ion-pairing effects between halide ions and peptides. The increase in retention with salt addition also confirms that there are no ionic retention sites on the polymeric phase at low pH. The general similarity in behaviour between the polymeric and silica column suggests that silanol groups have little involvement in the retention and overload behaviour of these peptides when using highly inert Type B silica phases.