Overcoming the negative effect of protein structure on molecular weight measurement by Size Exclusion Chromatography
In this application note we will look at two examples where the retention volume of a sample could not be used to obtain an accurate measure of the sample MW. We will also describe the additional information that can be obtained using these advanced detectors. This work has been performed in conjunction with the Institut Pasteur, Paris, France
Overcoming the negative effect of protein structure on molecular weight measurement by Size Exclusion Chromatography
In this application note we will look at two examples where the retention volume of a sample could not be used to obtain an accurate measure of the sample MW. We will also describe the additional information that can be obtained using these advanced detectors. This work has been performed in conjunction with the Institut Pasteur, Paris, France
Introduction
Size exclusion chromatography (SEC) is a well-established technique for measuring the molecular weight (MW or molar mass) of proteins. Traditionally this method involves comparing the column retention volume of a sample to that of a series of known MW standards. Importantly the separation process in SEC is controlled by the hydrodynamic volume of a molecule, not the molecular weight, therefore the results produced by this method are only relative to the standards used to calibrate the system. How close the "relative" MW is to the real MW of a sample is controlled by how similar the standards and analyte structures are. This means any deviation in the structure of the sample protein from that of the protein standards will negatively influence the accuracy of the result. In addition to this, some proteins can interact with the column matrix or change their hydrodynamic volume during elution. In those situations, it is impossible to rely on the retention volume to calculate the MW of the measured molecule.
The addition of advanced detectors to chromatography systems allows the absolute MW of a sample to be measured independent of any standards or sample elution volume.
In this application note we will look at two examples where the retention volume of a sample could not be used to obtain an accurate measure of the sample MW. We will also describe the additional information that can be obtained using these advanced detectors.
