ELSEVIER Journal of Chromatography A, 707 (1995) 77-85 JOURNAL OF CHROMATOGRAPHY A Free-solution electrophoresis of proteins in an improved density gradient column and by capillary electrophoresis K.D. Cole a'*, P. Todd b, K. Srinivasan a'l, B.K. Dutta a'2 aBiotechnology Division, Building 222, Room A353, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA bChemical Engineering Department, University of Colorado, Boulder, CO 80309, USA Abstract The electrophoretic mobilities of bovine serum albumin,/3-1actoglobulin A and B, a-lactalbumin and myoglobin were measured in free solution using an improved version of the Boltz-Todd vertical density-gradient electro- phoresis column. Dialysis membranes were used for the isolation of the side-arm electrodes from the column and large-volume electrode containers were connected to each other by a circulating buffer loop. The improvements increased reliability, facilitated removal of electrode gas, prevented proteins from contacting electrodes and allowed the use of low conductivity buffers without ion depletion. A low conductivity buffer (Tris-glycine) allows the use of high fields for rapid separations. The apparatus is modular and allows easy modification of column dimensions. We have also measured the electrophoretic mobility of these proteins in a coated capillary in the absence of significant electroosmotic flow. 1. Introduction The electrophoretic separation of proteins in free solution is important for several reasons. An obvious reason is to use differences in free- solution electrophoretic mobility as a preparative method for the isolation of proteins. A second reason is to measure electrophoretic mobilities of proteins for use in mathematical modeling of electrophoretic and electrokinetic separation (electrochromatography) processes to gain a * Corresponding author. Current address: Dionex 1228 Titan Way, Building 1, R&D, Sunnyvale, CA 94088-3603, USA. 2 Current address: University of Calcutta, Department of Chemical Engineering, 92 Acharya, PC Roy Road, Calcutta 7000009, India. better understanding of the separation methods. Predictive electrophoretic models are necessary for the efficient design and operation of indus- trial separation processes. The use of density gradients to stabilize elec- trophoretic separations has a long history [1-3]. Various configurations have been used to sepa- rate cells, organelles and proteins. Density gra- dient electrophoretic separations are simple in principle, however, in operation they can be cumbersome and unreliable. We have adapted a vertical column previously used for cell sepa- rations [4,5] for use in protein separations. This column is modular, flexible in dimensions, im- proved in reliability and ease of operation, simple to construct, and of low cost. Capillary zone electrophoreis (CZE) is known SSDI 0021-9673(94)01245-8