Absolute Size Exclusion Chromatography

1st April 2013

Size Exclusion Chromatography (SEC) is a common technique in biochemistry laboratories and routinely used for purification, identification, and quantification of protein mixtures. Dynamic light scattering (DLS) is traditionally used for pre-column size & polydispersity measurements, along with aggregate quantification.

Aside from the enhanced sensitivity, a big advantage of DLS coupled with SEC is the ability to perform Absolute SEC (ASEC), or size exclusion chromatography in the absence of costly and time consuming column calibration. DLS provides a direct measure of the protein size, as opposed to the SEC derived size extracted from a calibration curve. In addition, size vs. mass relationships for globular proteins and polymeric systems are well known, and can be used to calculate the molecular mass from the DLS measured hydrodynamic radius.

* Samples are measured directly after elution from a chromatography column
* The scattering intensity, hydrodynamic size, and molecular weight of the protein are displayed in real time, under flow
* Absolute method requires no column calibration or secondary detectors for molecular weight determination
* Size distribution resolution is enhanced through combination with chromatography





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