Continuous fractionation of protein mixtures by p H parametric pumping: Experiment

Abstract

Continuous pH parametric pumping separations of a haemoglobinalbumin-CM Sepharose system were experimentally investigated. The parapump has a feed containing the protein mixture to be separated, introduced alternately to the top and bottom of the chromatographic column. The top and bottom products are withdrawn from the apparatus, respectively, during the bottom and top feed. It is shown that under certain conditions the pH driven parametric pump has the capacity for removal of protein components from one product fraction and large enrichment in the other fraction. Moreover, the continuous process can be operated with a large feed throughput. Parametric pumping represents a new development in separation science. It has attracted considerable attention both because of its novelty and the possibility of continuous operation in small equipment with very high separation factors. Much experimental and theoretical work has been done on thermal and pressure parametric pumping. By contrast, very little work has been done on pH parametric pumping. The pH parapumping involves reciprocating flow of the protein mixture to be separated through a bed of ion exchanger and, simultaneously, synchronous cyclic variation of the pH. The change of pH displaces the interphase equilibrium and, in combination with the reciprocating flow, causes preferential movement of the sorbable components of the mixture towards one end of the bed, leading to a buildup of the separation from cycle to cycle.Recent experimental results obtained by Chen et al. (1977) have shown that parametric pump has the capacity for separating a mixture of haemoglobin and albumin. The pump considered had a center feed between an enriching column and a stripping column and was operated batchwise during upflow and continuously during downflow.In the present paper, the feed is alternately introduced at the top and bottom of the column, while the top and bottom products are withdrawn from the apparatus during the bottom and top feed, respectively. Emphasis is placed on the operating conditions necessary to achieve high separations and high product rates. The system studied is haemoglobin-albumin on CM Sepharose. Contrary to the system with the center feed for which the haemoglobin initially present at the top of the stripping section above the point of high pH liquid penetration is immobilized there permanently, no haemoglobin immobilization problem occurs for the present arrangement. CONCLUSIONS AND SIGNIFICANCEThe pH parametric pumping is extended to protein separation. The new continuous process developed is possible to cause certain proteins in a mixture to migrate toward one end of a chromatographic column, thereby effecting separation. Experimental data are obtained for the system of haemoglobin-albumin on CM Sepharose.Correspondence concerning this paper should be addressed to H. T. Chen. The experiments show that after an initial transient, the product concentrations reach a limiting condition and remain constant as the number of cycles continues to increase. Thu...

Effect of Feed Concentration in Equilibrium Parametric Pumps

“…(2,2), was later modeled by several researchers (3-6). Different versions of the process were described (7)(8)(9)(10)(11)(12)(13)(14).…”

Separation of Proteins via pH Parametric Pumping

“…By contrast, very little work has been done on pH parametric pumping. Included are the studies of Sabadell and Sweed (6), Shaffer and Hamrin (3, and Chen et al (1,2).…”