- Absolute size exclusion chromatography
Absolute Size Exclusion Chromatography (ASEC) is a technique which couples a
dynamic light scattering(DLS) instrument to a size exclusion chromatographysystem for absolute size measurements of proteins and macromolecules as they elute from the chromatography system.
A measurement is said to be absolute if it doesn’t require calibration. In ASEC, there is no instrument or column to calibrate. The sizes of the macromolecules are measured as they elute in the flow cell of the DLS instrument.
Aside from the absence of costly and time consuming column calibration, a big advantage of DLS coupled with SEC is the ability to obtain enhanced DLS resolution. Batch DLS is simple and provides a direct measure of the protein size but the resolution of DLS is 3 to 1. Using SEC, the proteins and proteins oligomers are separated allowing oligomeric resolution. Aggregation studies can be done using ASEC.
In addition, size vs. mass relationships for
globular proteinsand polymeric systems are well known, and can be used to calculate the molecular mass from the DLS measured Stokes radius.
Dynamic Light Scattering
Size Exclusion Chromatography
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