How to ensure good light scattering SEC chromatography data?

noisy light scattering chromatogram with an overlay of "5 tips for better SEC" signWhen using a light scattering (LS) detector with your size exclusion chromatography (SEC) system, data quality can be a concern. Occasionally, noise in the light scattering signal makes interpretation of the result challenging. Is it possible to get information from the ‘noise’? How can you improve chromatography LS data? Here are a few points to consider in your measurement method.

And these tips may, of course, also be of interest for good chromatography in general, not “just” for light scattering.

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 you should store buffer 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. This should be the first step to improve chromatography LS data.

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 columns for light scattering, like the P4000 protein column. 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 to improve chromatography LS data

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. So consider this as a potential item to improve your chromatography LS data.

Step 4: Load enough to get clean signals and separation, for better chromatography

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. For example frits, o-ring, and membrane for aqueous mobile phase are available. 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 it will catch any column debris and avoid spikes in the scattering intensity, that could lead to a noisy chromatogram. So this is also a potential opportunity to improve chromatography, and especially light scattering LS data.