What should I do if I don’t get the resolution needed with my size exclusion chromatography column?

There are several things one can do if they have insufficient resolution with their size exclusion chromatography column. 

• One thing that can be changed is the viscosity of the sample. The sample may be too viscous, and thus it should be diluted in a buffer. It is ideal to maintain protein concentration at 50 mg/mL. 
• The sample may contain particles. The column should be re-equilibrated, and the sample should be filtered with a low protein binding filter. 
• The sample volume may be too large, and one should decrease it properly as recommended by the protocol. 
• There also may be contamination of the column, and it should always be properly cleaned before being used. 
• It is important to check the selectivity curve in the recommended guides for the experiment to make sure the correct resin type is being used. 
• The flow rate may also be too high and contribute to insufficient resolution. One should check the recommendations for the experiment and lower the flow rate. 
• Lastly, one should minimize volumes in the system, as the sample may be diluted between the injection valve and column inlet, and/or between column outlet and UV flow cell. The volume can be minimized by decreasing tubing diameter and minimizing length. 
• One can also mount the column directly to the UV cell (without column valve) and remove all unnecessary particles in the flow path.