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

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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.