Empower Tip: Size Exclusion Chromatography with Multi-Angle Light Scattering Detection

In this tip, we will discuss Size Exclusion Chromatography with Multi-Angle Light Scattering Detection (SEC-MALS).

Let’s get started.

With the recent release of Empower 3.10.0 it is now possible to control the Wyatt Technology™ Multi-Angle Light Scattering Detectors, and in combination with a concentration detector, determine molecular weight and polydispersity of proteins, peptides and monoclonal antibodies via size exclusion chromatography.

Light scattering is proportional to molecular weight times concentration. We need to measure light scattering at a variety of angles to capture the complete scattering fingerprint and solve for molecular weight. Both the overall amount of light scattered, and the angular variations are important to capture for thorough analysis. This is analogous to how dust particles look in a projector beam depending on where you are standing relative to the projector. One of the advantages of this technique is that we do not need to run standards, nor calibrate the column, which makes this technique faster than conventional SEC.

A concentration detector is required, either UV or RI. We measure “LS” with the MALS, and separately we measure the “c” term with the concentration detector. Now we can solve for molecular weight. Since light scattering and concentration are measured in different places but are used together, Empower needs to know the volume delay between the concentration detector and MALS flow cells and how much peak broadening to expect between them as well. The inter-detector alignment and band broadening procedures will calculate these parameters, and the values are saved for correct processing of MALS results.

As we go through this series we will explore data acquisition, data processing and reporting of results.

It’s that easy!