Capillary electrophoresis of peptides in isoelectric buffers

Peptide maps in capillary zone electrophoresis are typically obtained in 80 mM phosphate buffer, pH 2.0, since at this pH value essentially all surface silanols of silica are protonated and thus one obviates to the need of chemically treating the inner capillary surface. When developing a peptide map of a tryptic digest of bovine beta-casein under these conditions, good separations were obtained, but at the expense of a rather long running time (80 min) due to the low maximum voltages applicable (only 110 V/cm) under experimental conditions utilizing relatively large bore capillaries (100 mu m I.D.) for increased sensitivity. When resorting to isoelectric aspartic acid as the sole buffering ion [pH=isoelectric point (pI)=2.77 at 25 degrees C] much higher voltages could be applied (up to 800 V/cm) with a concomitant reduction of total running time (down to only 6-7 min). However, at this operative pH, strong adsorption of larger peptides to the capillary wall was noticed. This inconvenience was fully eliminated when finally adopting a buffer of the following composition: 50 mM Asp, pH 2.77, added with 0.5% hydroxyethylcellulose number-average molecular mass (M-n) of 27 000 and with 5% 2,2,2-trifluoroethanol. Ln this buffer, very high resolution could be achieved, due to the very low band spread at such greatly reduced running times and to the absence of wall adsorption. The most important parameter, when selecting amphoteric buffers, is their buffering power, which is a function of their pI-pK values. The buffering power, for isoelectric amino acids, is very strong for Lys and Asp (27 and 26 mequiv. I-1 pH(-1), respectively, for 50 mM solutions), intermediate in the case of Glu (20), and rather minute for His and Arg (6 and 5.5, respectively). It is suggested that the minimum buffering power for a background electrolyte in CZE should be kept in the order of greater than or equal to 10 mequiv. 1(-1) pH(-1).