Enhanced performance of expanded bed chromatography on rigid superporous adsorbent matrix.
Rigid spherical macroporous adsorbent beads with surface hydroxyl groups were prepared by cross-linking of cellulose. These beads had diameter in the range 100-200 microm and a mean pore size of about 3 microm with about 60% pore volume. The matrix (bulk density approximately 1600 kg m(-3)) could be expanded into a stable bed and used for protein chromatography. Chromatographic runs were performed on a 10 mm diameter column under non-retaining and retaining conditions on the prepared matrix (called Celbeads) and performance of the runs was measured in terms of the height equivalent to a theoretical plate (HETP). The HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents, i.e. increasing and levelling with velocity. Unimpaired performance of the matrix at increasing flow-rates permitted expanded bed elution of adsorbed solutes without loss of efficiency in terms of purification factor and product concentration. As a model system, Celbeads was used to purify lactate dehydrogenase from porcine muscle homogenate by dye-affinity chromatography. The prepared matrix provided about 100 theoretical plates per meter for the enzyme system at a linear flow velocity of 1.27 cm x min(-1) in an expanded bed elution mode, and gave enzyme yields of 100% with a purification factor of 31 using an optimized procedure. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.
Pai
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Department of Chemical Technology, University of Mumbai, Matunga, India.
Gondkar
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Lali
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D000818:Animals; D002848:Chromatography, DEAE-Cellulose / Q000379:methods* / Q000592:standards; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification; D009132:Muscles / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D007770:L-Lactate Dehydrogenase