Simple steps to clarify a sample before applying it to a column will avoid the risk of blockage, reduce the need for stringent washing procedures and extend the life of the chromatography medium. Filter or centrifuge all solvents and samples before use.
- Refer to Table 6.4 to calculate the amount of dry medium required as the extent of swelling depends upon the solvent system. Swell Sephadex® LH-20 for at least 3 h at room temperature in the solvent to be used for the separation
- Prepare a slurry 75:25 settled medium:solvent and decant any ?ne particles of medium.
- Equilibrate all materials to room temperature.
- Resuspend and pour the slurry into the column in one continuous step (using a glass rod will help to eliminate air bubbles).
- Fill the column reservoir to the top with solvent. Seal, attach to a pump, and open the column outlet.
- Pack at 300 cm/h until the bed has reached a constant height. Stop the ?ow, empty, and remove the packing reservoir.
- Carefully ?ll the column with solvent and insert a wetted adapter into the column. Ensure no air bubbles are trapped under the net and adjust the adapter O-ring to give a sliding seal against the column wall.
- Connect all tubings, ensuring that there are no air bubbles in the ?ow path.
- Slowly slide down the adapter so that any air in the tubings is displaced by solvent and lock the adapter into position on the surface of the medium.
- Open the column outlet and continue packing until the packed bed is stable and a ?nal adjustment of the top adapter can be made.
In solvents such as chloroform, Sephadex® LH-20 is less dense than the solvent and the medium will ?oat. Pour the medium into the column and drain until the second adapter can be inserted. Lock the adapter in position at the surface of the medium and direct the ?ow of chloroform upwards. The bed will be packed against the top adapter and the lower adapter can be pushed slowly upwards towards the lower surface of the medium.
Close the column outlet when moving the adapter to avoid compressing the bed.
PERFORMING A SEPARATION
Start at a linear ?ow of 1 cm/h to check resolution. Low ?ow rate improves the resolution.
- Equilibrate the column with at least 2 column volumes of the solvent until a stable baseline is achieved.
- Apply a sample volume equivalent to 1% to 2% of the total column volume.
- Elute in 1 column volume. Re-equilibration is not needed between runs with the same solvent.
Cleaning
Wash the column with 2 to 3 column volumes of the solvent, followed by re-equilibration in a new solvent if changing the separation conditions.