What are the advantages and disadvantages when adding additives to my mobile phase for analysis of proteins by SEC?
Additives are sometimes used in SEC mobile phases to promote the solubilization of proteins, to prevent secondary interaction of proteins with the stationary phase, or to denature proteins. Common additives used to analyze proteins by SEC include sodium dodecyl sulfate (SDS), urea and guanidine hydrochloride. All three of these additives enhance protein solubilization and each of them promotes denaturation by breaking non-covalent bonds that are part of the higher order structure of proteins. Adding a surfactant like SDS to the sample and mobile phase leads to a disruption of the native shape of a globular protein to form rod-like structures that are much smaller in size and thus chromatograph more efficiently. Guanidine hydrochloride and urea disrupt the protein structure to a randomly coiled (larger) structure. The use of additives in SEC of proteins should be considered in cases where protein solubility is limited, where non-specific interaction is suspected between the sample and c
