Protein Applications for Advanced Multi-Detector Size-Exclusion Chromatography
In this application note, a mixture of four proteins was characterized in a single SEC measurement using multiple detectors. The generally consistent response to the RI to proteins makes it a useful total protein concentration detector, while the UV detector can be used to measure the concentration of individual proteins when the extinction coefficient is known. This is particularly useful for proteins that elute in the solvent peak making RI measurements unreliable. Finally, light scattering measurements allow each peak to be identified by its molecular weight.
sponsored by Malvern Panalytical
Size-exclusion chromatography (SEC) is a commonly used tool in the biological sciences. Typically, it is used to measure the molecular weight of an unknown sample by referencing the elution time of the unknown against that of standards of known molecular weight. This can be done with a single concentration detector such as UV or refractive index (RI). Advanced multi-detector SEC systems now include up to four detectors including UV, RI, light scattering and intrinsic viscosity. Light scattering detectors allow the measurement of molecular weight without the need for column calibration. Intrinsic viscosity is a measure of molecular density and enables structural changes to be assessed. A combination of light scattering and intrinsic viscosity allows size to be calculated.
Maintaining the detectors in series is essential for maintaining maximum sensitivity across all four detectors. SEC is generally used to measure pure samples.
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