Journal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Chromatopraph~, 415 (1989) 177-l 85 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands CHROM. 21 512 HIGH-PERFORMANCE CHROMATOFOCUSING OF PROTEINS ON AGA- ROSE COLUMNS II. DEFORMED NON-POROUS 12-15 pm BEADS STELLAN HJERTEN*. JIN-PING Ll” and JIA-LI LIAO Institute qf Biochemistrl;, University of Uppsala, Biomedical Center, P.O. Box 576, S-7SI 23 Uppsala (Swe- den) (Received March 14th, 1989) SUMMARY By shrinkage and cross-linking in organic solvents, macroporous agarose beads were rendered impermeable to proteins. Beads derivatized with polyethyleneimine in an aqueous solution gave a relatively linear pH gradient upon elution with Polybuf- ferTM. The titration capacity was 667 and 334 mequiv. per 100 ml agarose gel at basic and acidic pH, respectively. The columns were packed at pressures high enough to deform the beads, which increases the resolution owing to the decrease in the distance between the beads. The resolution has been determined as a function of the flow-rate, concentration of eluting buffer (Polybuffer) and sample load. Interestingly, the reso- lution increased with an increase in flow-rate. The importance of choosing an opti- mum concentration of Polybuffer was also demonstrated in a series of experiments with human growth hormone, which was fractionated into four components when the Polybuffer was diluted 1:80, but not at a dilution of 1:40. The mass recovery was 96% for human serum albumin and 93% for ovalbumin, whereas the recovery of the activity of fl-galactosidase was 90%. In comparison with macroporous beads, the deformed (compressed) non-porous beads have the advantage of permitting a more rapid separation and regeneration and to give a resolution which is independent of or even increases with the flow-rate. The protein capacity of the latter beads is not much lower than that of the macroporous beads. INTRODUCTION Recently we have described the preparation of chromatographic columns of deformed non-porous agarose beads and their application to the separation of pro- teins by high-performance hydrophobic interaction chromatography1,2, ion-ex- change chromatography’,3 and adsorption chromatography based on interaction ’ Permanent address: Institute of Radiation Medicine, Beijing. China 0021-9673/89/$03.50 c 1989 Elsevier Science Publishers B.V.