Capillary Electrophoresis Separation of Small Peptides: Effect Of pH, Buffer Additives, and Temperature

Abstract

The separation of dipeptides and small peptides by various modes of capillary electrophoresis was investigated in order to identify the best separation conditions and to compare the different charge-to-size parameters used in correlating peptide migration. For a series of equally charged polyalanines the best linear correlation was obtained when the electrophoretic mobility was plotted against q/(MW)^2/3. Deviations from linearity with other peptides are due to an imprecise charge calculation procedure. The best separations were achieved at low pH (∼2.5) when a large metal ion such as Zn⁺⁺ was added to the buffer. Under these conditions, peptides are positively charged and differences in charge are-maximized. The separation of peptides at pH 2.5 improved as temperature was decreased. A set of five 9-residue peptides with no significant difference in charge-to-size ratio were separated at pH 7.0 with a buffer composed of 50 mM Tris + 50 mM DTAB.