CAPILLARY-ZONE-ELECTROPHORESIS AT SUBZERO TEMPERATURES III - OPERATING-CONDITIONS AND SEPARATION EFFICIENCY

The advantages of carrying out capillary zone electrophoresis at subam bient rather than ambient temperatures are examined. They include the possibility of using higher electric field strength and/or buffer conc entration as well as wider capillary lumen without untoward effects of Joule heating on the separation. Furthermore, the separation efficien cy is enhanced as molecular diffusivity decreases with temperature. A Beckman P/ACE unit with an auxiliary cooling system was used at temper atures down to -20 degrees C and, based on the dependence of axial tem perature gradient on the capillary length and the temperature of the c oolant, an average capillary temperature was defined. The day-to-day m igration times of proteins were reproducible with an R.S.D. better tha n 2.3%. The improvements in the separation efficiency upon lowering th e temperature from 40 to 1 degrees C are illustrated by the electrophe rograms of four closely related peptides. Generally, the plate efficie ncy is enhanced at low temperatures and the analysis time is prolonged due to the increased viscosity. However, the current at a fixed elect ric held also decreases with temperature and the capillary can be shor tened to speed up the analysis. With benzenesulfonic acids, the rate o f the generation of theoretical plates was two- to three-times higher using a 27 cm long capillary at -15 degrees C than a 47 cm long capill ary at 40 degrees C. By using a 180 mu m instead of a 50 mu m I.D. cap illary for the separation of the four peptides, the sample loading cou ld be increased about 15 times without loss of resolution. In the capi llary zone electrophoresis of proteins, it was found that protein inte ractions with the capillary wall can be significantly reduced by incre asing buffer concentration at low temperatures where salt mediated hyd rophobic interactions are attenuated. This is illustrated by the separ ation of beta-lactoglobulins A and B in a raw fused-silica capillary u sing 400 mM sodium berate buffer, pH 8.4. The results suggest that at subambient or even subzero temperatures, the scope of CZE can be exten ded by several ways so that the benefits are great enough to justify t he use of a cooling system and temperature control appropriate for psy chroelectrophoresis with fused-silica capillaries. (C) 1998 Elsevier S cience B.V. All rights reserved.