Measuring molecular weight using the Zetasizer Nano

A number of methods are available to determine the molecular weight of a material such as a protein or polymer in solution. Some of them are called 'absolute', which in the accepted use of the term means that comparison of the sample with a material of known molecular weight is not required to calculate the result.

With this definition the following methods can be called 'absolute'.

• Mass spectrometry (MS)
• Osmometry
• Analytical ultra centrifugation (AUC)
• Total Intensity or Static Light Scattering

Some of these techniques may require significant amounts of sample, have measurements times from several hours to several days or require secondary detectors.

Size Exclusion Chromatography (SEC) separates materials according to their Hydrodynamic size, and the elution time can be used to determine their molecular weight if the column is calibrated with a range of molecules of known molecular weight. This calibration means that the technique is not absolute, and is also subject to error from a number of sources such as interaction of the sample with the column material for example.
However the benefits of this separation technique can be enhanced by connecting the technique with an absolute measurement technique such as dynamic light scattering (DLS) as performed by the Zetasizer Nano.

The Zetasizer measures the absolute size of materials such as proteins by Dynamic Light Scattering, and with the addition of a flow cell can be connected directly to the output of an SEC column. The size measured can then be converted into an estimate of the molecular weight using accepted models. The combination of Size Exclusion Chromatography and Dynamic Light Scattering has been termed 'ASEC' or absolute size exclusion chromatography, and gives an output of the absolute size and estimated molecular weight without the requirement for standards.

Benefits:

• Confirmation of the oligomeric state of the protein without using standards
• Highly sensitive detection of multimers and aggregates, much more sensitive than refractive index (RI) or UV detectors
• Confirmation of whether a peak in a chromatogram is an artifact or 'real' eluted material
• Measures the parameter that is actually used to perform the separation - hydrodynamic size
• Estimate of molecular weight without requirement for second concentration detector
• Can be converted from batch mode to flow mode in seconds

	Application Notes:
	On demand presentation on molecular weight in 60 seconds. The molecular weight of an unknown protein is quickly obtainable from a dynamic light scattering (DLS) measurement. An empirical size-mass relationship produces excellent agreement for a wide range of proteins. The example of insulin shows the use of this relation. When an equilibrium exists between monomer and higher oligomeric state then this technique can be used as a fast estimate of the ratio of the two contributions. Several additional commonly encountered proteins are also presented.
	Application note on Absolute Molecular Weight measurements with the Zetasizer Nano system Small ‘particles’ such as proteins scatter light isotropically, where the scattering pattern exhibits no angular dependence. Static light scattering (SLS) can then be used to determine the absolute molecular weight (MW) and the second virial coefficient of the scattering molecules, in particular protein solutions, at the single angle used by the Zetasizer Nano. An overview of the theory, experimental setup and discussion of the results for a range of proteins from 10 to 70 kDa (BSA, ribonuclease, lysozyme, ovalbumin, antibody fragment) is presented. There is a brief comparison with exclusion chromatography (SEC).
	Application note on Absolute Molecular Weight determinations of polymers and polysaccharides Static light scattering (SLS) is a non-invasive technique used for characterization of macromolecules in solution. SLS makes use of the time-averaged intensity of scattered light, from which the weight-average molecular weight and second virial coefficient can be determined. Single-angle Debye plots for two polymers (980 Da, 9.9 kDa and 96 kDa) and one polysaccharide (68 kDa) show good agreement between the measured and expected values. For the polysaccharide, the z-average radius obtained from dynamic light scattering may be used to estimate the molecular weight, and is found to be in close proximity to the expected value.
	Application note on Effect of Storage Conditions on IgG Antibodies are specialized proteins that function as an essential part of the immune system. They are members of a class of proteins known as immunoglobulins, typically with a molecular weight near 150 kDa. Here, the influence of different storage conditions on the size and quality of these proteins was investigated using both static light scattering and dynamic light scattering. With non-ideal storage, aggregates form in the sample and these can influence the result for the obtained molecular weight.