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

Protein chromatography
Protein chromatography on a DEAE-cellulose column
Chromatography on a DEAE-cellulose column in the OH- form

DEAE-Cellulose, owing to its functional group—the diethylaminoethyl residue—possesses The properties of a weak anion exchanger. It is primarily used for the Separation of natural high-molecular-weight polymers, especially those sensitive to abrupt changes in pH and Temperature, i.e., chiefly for protein fractionation. Chromatography on DEAE-cellulose features high resolving power. This ion exchanger is suitable for both analytical and preparative fractionation of Proteins on columns of appropriate size.

a) Preparation of DEAE-cellulose. Before packing the Column, DEAE-cellulose is treated as follows:

1) The required amount of DEAE-cellulose is stirred in a 5-fold volume of 0.5 N NaOH for 15 min at room temperature and allowed to settle. Unsettled fine particles of the ion exchanger are decanted along with the supernatant.

2) DEAE-cellulose is washed several times with distilled Water to pH 8–9. Washing is conveniently performed by centrifugation, which allows the cellulose particles to settle much faster, reducing the overall washing time.

3) The washed DEAE-cellulose is mixed with a 3-fold volume of 96% ethanol, allowed to settle, and then the supernatant is decanted.

4) The Treatment with 0.5 N NaOH is repeated once more.

5) The cellulose is washed with distilled water to a neutral pH using centrifugation.

DEAE-cellulose treated in this manner can be stored as an aqueous suspension at 4°C. Storing ion exchangers at room temperature should be avoided due to the risk of microbial contamination.

b) Column size Selection. Proteins with an average molecular weight (about 50,000) are usually separated on long, narrow columns. For the fractionation of 100 mg of protein on DEAE-cellulose with a capacity of 0.7–0.8 meq/g, a 1.5x35 cm column is required. Depending on the Properties of the protein being fractionated, its impurity content, the elution method, etc., the column dimensions may vary in both diameter and length.

By conducting appropriate preliminary experiments, one can calculate the optimal column dimensions for the preparative separation of large amounts of protein. To ensure an optimal flow rate through the column and thus sufficiently rapid chromatography, the column volume should be increased primarily by increasing its diameter. For the fractionation of 1.0 g of a medium-molecular-weight protein, a 3.5 x 45 cm column is sufficient.

c) Column flow rate. The target flow rate through the column is determined by the column dimensions. For micropreparative chromatography on a 1.5x35 cm column, the optimal rate is 15–20 ml/h. Preparative chromatography on a larger diameter column is carried out at a flow rate of 25–30 ml/h; at higher rates, separation deteriorates and the fractions become more diluted.

d) Selection of chromatographic conditions. Column chromatography on DEAE-cellulose can be performed using stepwise or gradient elution. Stepwise elution is advisable only when the components being fractionated elute at widely differing pH and molarity values, i.e., when there is no risk of individual fractions mixing.

Regardless of the elution method, the optimal chromatographic conditions for a given material are selected based on three well-known alternatives: 1) increasing the molarity of the eluent, 2) increasing the pH of the eluent at constant molarity, and 3) simultaneously increasing both the pH and molarity of the eluent.

Chromatography on DEAE-cellulose is typically initiated in a low-molarity buffer at pH 8. The material to be fractionated is dissolved in a 0.05 M buffer at pH 7.8–8.2, and after loading it onto the column, the eluent molarity is increased stepwise or via a gradient, first to 0.1, then to 0.5, and finally to 1.0 M.

The choice of buffer is determined solely by The Nature of the material being fractionated. If chlorides do not react with the latter, it is recommended to perform chromatography on DEAE-cellulose in the Cl^- form.