Fractionation and Separation by Continuous Liquid-liquid Chromatography

Abstract

Reviews are given of chromatographic theory with particular reference to gel permeation chromatography. The various process schemes that have been proposed for preparative and production scale chromatography have been discussed. A laboratory scale unit was made available in which a solute-rich liquid system is made to move counter-current to a liquid held in the pores of a solid porous support. Polymeric fractionation of dextran is achieved on the basis of molecular size. The counter-current movement is obtained by moving an annular bundle of 44 interconnected tubes (28.5 cm x 0.8 cm) about fixed liquid input and output ports. Cam-operated spring-loaded valves ensure the unidirectional flow of liquid counter-current to the direction of rotation of the bundle. The operation of the equipment is dependent on the reliability of a moving face seal. Mechanical difficulties were encountered with the moving face seal, which reduced the amount of experimental work that could be performed using the equipment. Experimental studies have been carried out in which the continuous chromatographic process has been applied to the fractionation of dextran. Dextran with a weight average molecular weight of 32,000 and a molecular weight distribution of about 2000 to 300,000 was used. The effect of increasing the feed concentration from 0.5% w/v to 20.8% w/v at a nominal feed rate of 0.5 cm3  min⁻¹ has been investigated. A concentration-dependent partition coefficient was shown to influence the process. Throughputs of up to 1.14 g hr ⁻1  have been achieved. The equipment has successfully been used to fractionate dextran to give a molecular weight distribution comparable with that produced by ethanol precipitation. The eluent and chromatographic packing used were distilled water and Spherosil X0B075 (200 µm-400 µm). Attempts have been made to elucidate the influences of process operating parameters using a synthetic binary mixture, dyed bovine serum albumin, and dyed pepsin. A decrease in the surface tension of the sealed fluid was shown to adversely affect the operation of the mechanical seal. A computer simulation of the process has been developed. This enabled the key parameters affecting the process to be theoretically determined.