If you are using a light scattering detector with your size exclusion chromatography system, consider these points in your measurement method:


Step 1: Start with clean buffers and an equilibrated system

It should be obvious that the buffer should be clean, ideally prepared not more than a few days ago, and stored away from sunlight. A good general recommendation is to filter all buffers at 0.2 microns before use in your chromatography setup. The best data are obtained from a well equilibrated system, make sure the system is run for a few hours before sample injection to make sure there are no pressure fluctuations.

Step 2: Use an appropriate column

The most general advice for this is to talk to the applications team from the manufacturer of your SEC-LS system. This lets you draw on their expertise and potentially avoids a few pitfalls on the way. Some columns give more stable baselines when using a light scattering detector. At Malvern we have a set of generic aqueous columns as well as a special protein column set. For a very general use protein column, one of the most popular is probably the GE Superdex S200 column.

Step 3: Consider a guard column

If there is any doubt about the quality of the sample, use a guard column to help protect the main column. This is likely only a concern for labs which routinely measure a range of unknown samples, or have a large number of different operators using the system.

Step 4: Load enough to get clean signals and separation

For lower molecular weights, the injected amount needs to be increased in order to detect enough light scattering. The concentration detector used also needs to have an output significantly above the background. As a very rough guide, an injection of 20μL of 5mg/mL Bovine Serum Albumin (BSA) should give clear peaks for the monomer and dimer. For larger molecular weights, you can reduce the concentration, for smaller molecular weights, you may have to increase it.

Step 5: Consider a post column light scattering filter

The main purpose of a post column filter is to catch column debris and column bleed. There are different filter membranes available, and while there may be some concern about protein binding, convince yourself experimentally by checking the protein recovery in the data analysis. The maximum size of particle that can pass through a typical SEC column frit is between 100nm and 200nm. Therefore, a filter of 200nm pore size will not interfere with any molecular species eluted, however will catch any column debris and avoid spikes in the scattering intensity, that could lead to a noisy chromatogram,