Capillary zone electrophoresis at subzero temperatures: III. Operating conditions and separation efficiency

Abstract

The advantages of carrying out capillary zone electrophoresis at subambient rather than ambient temperatures are examined. They include the possibility of using higher electric field strength and/or buffer concentration as well as wider capillary lumen without untoward effects of Joule heating on the separation. Furthermore, the separation efficiency is enhanced as molecular diffusivity decreases with temperature. A Beckman P/ACE unit with an auxiliary cooling system was used at temperatures down to −20°C and, based on the dependence of axial temperature gradient on the capillary length and the temperature of the coolant, an average capillary temperature was defined. The day-to-day migration times of proteins were reproducible with an R.S.D. better than 2.3%. The improvements in the separation efficiency upon lowering the temperature from 40 to 1°C are illustrated by the electropherograms of four closely related peptides. Generally, the plate efficiency is enhanced at low temperatures and the analysis time is prolonged due to the increased viscosity. However, the current at a fixed electric field also decreases with temperature and the capillary can be shortened to speed up the analysis. With benzenesulfonic acids, the rate of the generation of theoretical plates was two- to three-times higher using a 27 cm long capillary at −15°C than a 47 cm long capillary at 40°C. By using a 180 μm instead of a 50 μm I.D. capillary for the separation of the four peptides, the sample loading could be increased about 15 times without loss of resolution. In the capillary zone electrophoresis of proteins, it was found that protein interactions with the capillary wall can be significantly reduced by increasing buffer concentration at low temperatures where salt mediated hydrophobic interactions are attenuated. This is illustrated by the separation of β-lactoglobulins A and B in a raw fused-silica capillary using 400 m M sodium borate buffer, pH 8.4. The results suggest that at subambient or even subzero temperatures, the scope of CZE can be extended by several ways so that the benefits are great enough to justify the use of a cooling system and temperature control appropriate for psychroelectrophoresis with fused-silica capillaries.