Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters.

Abstract

R-phycoerythrin (PHYCO, Mr 240 000), glucose-6-phosphate dehydrogenase (GPD, Mr 104 000) and two charge isomers of recombinant green fluorescent protein (GFP-1 and GFP-2, Mr 27 000) were subjected to capillary zone electrophoresis (CZE) in capillaries of 50, 100 and 150 microm inner diameter at various sample concentrations, electric field strengths, and lengths of the initial zone with the purpose of testing the hypothesis that protein - capillary wall interactions rather than thermal effects are predominantly responsible for the peak spreading of proteins in CZE. The efficiency of CZE was expressed in terms of the number of theoretical plates, N, or the plate height corrected by subtracting the contribution from initial zone length, H'. The latter has the advantage of solely reflecting contributions to the separation efficiency arising from intracolumn peak spreading in capillaries of different diameters. The separation efficiency measured varied widely, by two orders of magnitude, for these proteins under identical conditions, with GPD exhibiting the highest and PHYCO the lowest values of N. H' was found to be independent of sample concentrations within the concentration ranges studied, 1-100 microg/mL for PHYCO and 100-1000 microg/mL for GPD, while exhibiting a decrease with sample concentration for GFP, especially in 150 microm diameter capillaries, within the concentration range 1-100 microg/mL. H'was also found to be independent of electric field strength up to 300-400 V/cm for PHYCO and GFP. In all experiments, the CZE of proteins in 100 microm diameter capillaries provided a higher or, at least, equal efficiency, compared to that in 50 or 150 microm diameter capillaries. It may be concluded that the protein - capillary wall interactions and protein microheterogeneity are the dominant sources of peak spreading and their specific combinations are thought to be responsible for the wide variation in separation efficiency between proteins in CZE observed under identical conditions.