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Theory
Understanding Light Scattering and Batch Mode

microCUVETTE

On occasion, it is necessary or desirable to make a light scattering measurement of an unfractionated sample. For example, the time dependent or concentration specific properties of a sample might be required. Such measurements are collectively termed batch measurements, and are made by either injecting a sample aliquot directly into the flow cell, or by introducing the sample in a container such as a scintillation vial or the microCUVETTE into the DAWN® HELEOS® II or miniDAWN™ TREOS® MALS detector.

The properties that may be determined in a batch light scattering measurement depend on the measurement technique. For Rayleigh scattering, the presence of species with different masses means that the measured quantities are averaged quantities. For example, the molar mass is a weight-averaged molar mass, and the size is averaged over a higher moment of the mass distribution. However, if batch measurements are made at multiple concentrations, it is possible to determine the second virial coefficient A2 for the solute-solvent interaction, very useful information in studies such as crystallography.

For QELS (Dynamic Light Scattering), batch measurements can determine semi-quantitative aspects of the hydrodynamic radius distribution. Using analysis techniques such as regularization (see the ASTRA® software), batch QELS data can reveal the distribution of sizes for broadly (i.e., over several orders of magnitude) polydisperse samples, and even differentiate between species with a size difference greater than a factor of five.

Although the chromatography mode is in general the best method for characterizing a sample, the batch mode is useful in those instances where the macromolecules cannot be easily separated by size exclusion chromatography, or the time or concentration-dependent properties of the sample are of interest. The latter might include studies of phenomena such as protein self-assembly, measurement of concentration-dependent aggregation, etc.