Absolute size exclusion chromatography

Absolute Size Exclusion Chromatography (ASEC) is a technique developed by Malvern Instruments which couples the Zetasizer Nano, a dynamic light scattering (DLS) instrument to a size exclusion chromatography system for absolute size measurements of proteins and macromolecules as they elute from the chromatography system.

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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 in the Zetasizer Nano.

Aside from the absence of costly and time consuming column calibration, a big advantage of DLS coupled with SEC is the ability to 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 study can be done using ASEC.

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 Stokes radius.

See also

• Dynamic Light Scattering
• Size Exclusion Chromatography

References

1. http://www.wyatt.com/solutions/hardware/wyattqels.cfm
2. http://www.wyatt.com/literature/appnotes/proteins.cfm
3. http://www.wyatt.com/literature/appnotes/biopolymers.cfm
4. http://www.wyatt.com/literature/appnotes/syntheticpolymer.cfm