When is Single Angle Light Scattering (LALS/RALS) the right detection technique to use, and when do you need Multi Angle Light Scattering (MALS)?
Size Exclusion Chromatography (SEC) is a well-established tool for the measurement of the molecular weight and purity of proteins and polymers. It is based on the separation of molecules based on their hydrodynamic volume and size – not their molecular weight.
In its simplest form, sometimes called Conventional Calibration, SEC uses an RI or UV detector, and the elution volume is calibrated against known standards. Sounds pretty easy, however, the fundamental assumption is that your unknown molecule is very similar to the standards which were used.
So let’s take a real example: If you use a common size calibration standard like for example typical protein molecular weight marker kits, these may contain bovine serum albumin, alcohol dehydrogynase and β-amylase – and while these are relatively common they also are fairly globular. What happens if your protein is not globular? It will elute at a different position, most likely it will elute at an earlier volume corresponding to a larger molecular weight than its actual molecular weight. This is solely due to its hydrodynamic volume being larger than that of a globular protein of the same molecular weight. This is even more important for polymers where there is the additional issue of conformation changes due to branching.
What if we don’t know the shape of our polymer or protein…?
Fortunately there is a method of measuring the molecular weight directly: light scattering. The technique involves detection of the intensity of the scattered light from the sample in conjunction with a concentration detector, and the different types can be classified under the general term SEC-LS, however the ‘LS’ part more specifically comes in various forms with the acronyms:
- LALS = low angle light scattering
- RALS = right angle light scattering
- MALS = multiple angle light scattering
Which technique is appropriate for my application?
The most commonly known version is SEC-MALS (sometimes also referred to as SEC-MALLS for multiple angle laser light scattering). MALS measures the light scattered by the samples at a range of angles and then extrapolates back to zero degrees. It allows you to measure molecular weight and also, thanks to the change in light scattering intensity with angle, Rg (for molecules greater than approximately 10 nm, which exhibit these differences). LALS measures close to zero degrees and therefore measures molecular weight without the need for extrapolation but also loses the ability to measure Rg. RALS measures at 90 degrees which offers sensitivity for small molecules but is unable to accurately measure the molecular weight of larger molecules (> approx. 10-15 nm). In our protein example, RALS would actually cover almost all applications but some prefer the confidence afforded by having more angles in the measurement.
The accuracy of the measured molecular weight by the different techniques is actually comparable over a wide range of molecular weights. While we combine RALS and LALS detectors to achieve both sensitivity and accuracy for large molecules, MALS covers the full range of sizes and has earned strong acceptance in some industries, in particular, for protein measurements. It is however dependent on choosing the right model and fit order to accurately fit the angular data. The most appropriate technology is often therefore down to preference as much as application dependence. By offering a choice of technologies, we hope to work with our customers to find the best solution for you.
Photos of Malvern’s SEC-LS systems: Single angle light scattering systems like the Viscotek 270 (left), Zetasizer μV (center), and the Viscotek TDA (right) are an efficient choice for advanced protein purity characterization.
Photo of Malvern’s most recent SEC-MALS system: Multi angle light scattering systems like the Viscotek SEC-MALS 20 are an ideal choice when information on the radius of gyration for advanced polymer characterization is of interest.
- 5 steps to improve SEC-LS data
- Demonstration of Chromatography software (3minutes)
- Notes from a recent GPC training