Performance test of gel chromatographic column

1. Swelling

Commercial gel are dry particles, usually 40~63um. The gel needs to be fully dissolved in the eluent for one to several days before use. If the wet gel is gradually heated to near boiling in a boiling water bath, the swelling time can be shortened to 1 to 2 hours. The swelling of gel must be complete, otherwise it will lead to uneven chromatographic column. The thermal expansion method can also kill the bacteria generated in the gel and remove the bubbles in the gel.

2. Column installation

Since the separation of gel depends on sieving, the filling requirements of gel are very high. The whole packing column must be very uniform, or it must be refilled. Before the gel is loaded into the column, the monomer, powder and impurities in the gel can be removed by water flotation, and the bubbles in the gel can be discharged by vacuum pump* The glass or plexiglass gel hollow column is easy to buy, and there are flat screens or sieve plates at both ends of the column. Fix the column vertically, add a small amount of mobile phase to eliminate bubbles at the bottom of the column, and then add some mobile phase to a height of about 1/4 of the column. A funnel is connected to the top of the column, and the diameter of the neck is about half of the column neck. Then the degassed gel suspension is slowly, evenly and continuously added under stirring. At the same time, the capillary outlet of the chromatographic column is opened, and the appropriate flow rate is maintained. The gel particles will rise horizontally layer by layer, and deposit evenly in the column until the required height* Then remove the funnel, cover the surface of the gel bed with a small piece of filter paper, and wash the gel bed with a large amount of eluent for a period of time.

3. Column uniformity inspection

The separation effect of gel chromatography mainly depends on whether the chromatographic column is loaded evenly. Before the sample separation, the chromatographic column must be checked for uniformity. Since the gel is translucent in the chromatographic column, a fluorescent lamp parallel to the column can be placed next to the column to check whether there are "lines" or bubbles in the column with naked eyes. Colored macromolecules can also be added to the chromatographic column. The molecular weight of the added substances should be within the separation range of the gel column. If narrow, uniform and flat spectral bands are observed in the column, the chromatographic column performance is good; If the color band is irregular, disordered and very wide, the gel column must be refilled.

4. Sample loading

After the gel column is installed, the sample can be loaded only after the mobile phase of the column is well balanced. The loading of gel column is also a very important factor. The general principle is to make the sample plunger as narrow and flat as possible. To prevent some sediment in the sample from contaminating the chromatographic column, the sample is generally filtered or centrifuged before loading. The concentration of the sample solution should be as high as possible, but if the solubility of the sample is temperature dependent, the sample must be appropriately diluted and the sample temperature should be consistent with the temperature of the chromatographic column. When everything is ready, open the piston of the chromatographic column, make the mobile phase just parallel to the gel bed, and close the outlet. Use a burette to absorb the sample solution and gently add it to the chromatographic column along the column wall. Open the outlet to allow the sample solution to penetrate into the gel bed.