Operation of the chromatography-

1. Column packing- The column material is allowed to swell in the mobile phase. It is poured into the glass tube and allow the beads to settle without trapping air bubble within the column. Once the matrix is settled to give a column, it can be tested for presence of air channel and well packing by flowing a analyte with Kd=1, it is expected that the elution volume (Ve) in this case should be V_o + V_i .

2. Sample Preparation- The sample is prepared in the mobile phase and it should be free of suspended particle to avoid clogging of the column. The most recommended method to apply the sample is to inject the sample with a syringe.

3. Elution- In gel filtration column, no gradient of salt is used to elute the sample from the column. The flow of mobile phase is used to elute the molecules from the column.

4. Column Regeneration- After the analysis of analyte, gel filtration column is washed with the salt containing mobile phase to remove all non-specifically adsorb protein to the matrix. The column is then equiliberated with mobile phase to regenerate the column. The column can be store at 4°C in the presence of 20% alchol containing 0.05% sodium azide.

Figure 30.4: Protein kinase assay using ion-exchange chromatography.