Optimized sample buffer for dispersed, high-resolution capillary zone electrophoretic separation of Escherichia coli B

Abstract

AbstractCapillary zone electrophoresis (CZE) is a powerful tool for high resolution chemical separations. Applying CZE to microbial samples may facilitate a deeper understanding of bacterial physiology and behavior. However, the study of complex microbial samples has been limited by the uncontrolled hetero-aggregation of bacterial cells under an applied electric field. We tested a wide range of sample buffers and buffer additives for the optimization of bacterial CZE separations using a 20 mM Tris–HCl background electrolyte. By modifying the sample buffer, but not the background electrolyte, we retain constant separation conditions, which aids in the comparison of the sample buffer additives. We report optimized methods for automated CZE separation and simultaneous fractionation of Escherichia coli B, which is one of the two most widely used wild-type strains. A modified sample buffer containing neutral salts and the addition of glycerol produced a 20-fold increase in loading capacity and a reduction in peak width/broadening of 86% in comparison to previously reported work. In addition, the glycerol-modified sample buffer appears to reduce the persistent aggregation and adhesion to the capillary walls during electrophoretic separations of complex environmental microbiota.