Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Gel Filtration of Proteins and Peptides
Protein separation by recycling gel filtration

Principle of the method. The material to be fractionated is passed through the same Column multiple times; this allows the Separation of even those mixture components that cannot be resolved on a standard-length column.

Applications. Isolation and Purification of Proteins.

METHODOLOGY

1. Equipment. The instrument set manufactured by LKB (Sweden) for continuous recycling Chromatography under the name "Recychrom" (Fig. 42) consists of the following main components: a chromatographic column, a peristaltic pump, and a flow-through absorptiometer.

2. Chromatography. The sample is applied to a Sephadex column, selected According to the Molecular Weight of the substances to be separated. The eluent flow, as shown in Fig. 42, is directed by turning a two-way distribution valve. A peristaltic pump is integrated into the system to feed the eluent onto the column in an upward direction. This system allows the fractionated sample to be repeatedly passed through the column. The optical density of the fractions is continuously recorded by a chart recorder. During protein separation, by appropriately turning the distribution valve, impurity (unwanted) fractions are first removed from the preparation, followed by the ISOLATION OF A completely homogeneous protein fraction (Fig. 43). The set includes columns measuring 3.2x105 and 3.2x65 cm. The peristaltic pump allows the flow rate of the solution to be adjusted from very low up to 80 ml/h.

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Fig. 42. Schematic diagram of the Recychrom system.

1 — chromatographic column; 2 — cooling jacket; 3 — thin-walled polyethylene capillary tubing; 4 — flow-through Cell of the absorptiometer; 5 — chart recorder; 6 — two-way distribution valve; 7 — peristaltic micropump; 8 — column filling funnel with coupling sleeve.

Fig. 43. Purification of ceruloplasmin component B using the Recychrom system.

Сб — discharge of unwanted impurities; Э — elution of the desired component.

Recommended Literature

Determann H., Gel chromatography, Springer Verlag, Berlin, Heidelberg, New York, 1968. (H. Determann, Gel Chromatography, Mir Publishers, Moscow, 1970.)

Leach S. I., Physical Principles and Techniques of Protein Chemistry, Academic Press, New York, London, 1969.

Williams C. A., Chase M. W., Methods in Immunology and Immunochemistry Vol. II, Academic Press, New York, London, 1968.