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Absolute size-exclusion chromatography

Absolute size-exclusion chromatography (ASEC) is a technique that couples 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.

The definition of absolute used here is that it does not require calibration to obtain hydrodynamic size, often referred to as hydrodynamic diameter (DH in units of nm). The sizes of the macromolecules are measured as they elute into the flow cell of the DLS instrument from the size exclusion column set. It should be noted that the hydrodynamic size of the molecules or particles are measured and not their molecular weights. For proteins a Mark-Houwink type of calculation can be used to estimate the molecular weight from the hydrodynamic size.

A big advantage of DLS coupled with SEC is the ability to obtain enhanced DLS resolution. Batch DLS is quick and simple and provides a direct measure of the average size, but the baseline resolution of DLS is 3 to 1 in diameter. Using SEC, the proteins and protein oligomers are separated, allowing oligomeric resolution. Aggregation studies can also be done using ASEC. Though the aggregate concentration may not be calculated, the size of the aggregate can be measured, only limited by the maximum size eluting from the SEC columns.

Limitations of ASEC include flow-rate, concentration, and precision. Because a correlation function requires anywhere from 3–7 seconds to properly build, a limited number of data points can be collected across the peak.

 

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