Protein band spreading in capillary zone electrophoresis Effects of sample zone length and presence of polymer

Abstract

It is commonly accepted that intra-column zone dispersion in CZE rests on multiple mechanisms, viz. diffusion, interaction of analyte with the capillary walls, Joule heat and conductivity differences between sample zone and the surrounding buffer. The most important extra-column contributor to bandwidth is thought to be the starting zone width. The present study shows that the length of the starting zone above 10 mm is linearly related to the bandwidth of R-phycoerythrin ( M r290·10 3). Below that length, bandwidth demonstrates a plateau preceded by a slight rise. Within the ‘plateau range’ the ratio of bandwidth to effective capillary length is close to constant while it is independent of electric field strenght in the range of 37 to 370 V cm −1 and of protein concentration in the range of 0.1 to 1000 μg ml −1. The experimental observations support the notion that the analyte-wall interaction is the determining source of intra-column zone dispersion. A slight rise observed at initial zone lengths of less than 2 mm was accounted for by a diffusion model taking into account a non-local initial concentration of analyte. The presence of polyethyleneglycol in the buffer within a concentration range up to 6% does not affect bandwidth. Above that concentration, the level of constant bandwidth is raised.