Size exclusion chromatography (SEC) is widely used for characterizing the size and composition of aggregates or fragments in a biomolecular sample. Conventional SEC relies on reference standards to calibrate column elution time as a function of molar mass. However, these standards often do not accurately represent the conformation of the sample, or the sample may exhibit column interactions that change its elution properties relative to the standard. Coupling a light scattering detector, such as the miniDAWN® TREOS®, to SEC enables absolute quantification of a molecule’s molar mass, irrespective of elution time.
Ultra-high performance liquid chromatography (UHPLC) enables fast, efficient separation of biomolecular samples. Compared to standard SEC, UHP-SEC provides improved resolution, higher throughput, and lower solvent and sample consumption for analysis of precious biological samples. Detectors designed for standard HPLC, however, have proven incompatible with this technology. The Wyatt μDAWN® multi-angle light scattering (MALS) detector and Optilab® UT-rEXTM refractive index (RI) detector, designed specifically for UHPLC applications, combine to measure the absolute molecular weight (molar mass) and size of eluting species in UHPLC.
