Size exclusion chromatography (SEC) is a non-invasive technique that provides
information on the molecular size of proteins and polymers. SEC requires a column
with a uniform porous material with a defined pore size and a mobile phase
appropriate to the solute. Mobile phase components, such as salts, can affect SEC
separations. The purpose of adding salt to the eluent is to minimize secondary
interactions of the sample components with the resin. The presence or absence of
sodium chloride (NaCl) in aqueous eluents influences the elution volume of proteins
by altering their hydrodynamic radius as a function of salt strength.
It is important to note that relatively minor changes in protein structure may affect
protein solubility and encourage secondary hydrophobic interactions causing
similarly sized proteins to elute at different times. In such cases, it is important to
modify the mobile phase composition to regain a separation based on molecular
size alone.
The retention behavior of three globular proteins (γ-globulins, bovine serum albumin
and ovalbumin) was investigated using a Agilent ProSEC 300S column in low ionic
strength mobile phases of varying NaCl concentration. The dependence of ‘nonideal’
protein-protein and protein-matrix interactions on NaCl was determined by
comparison of UV responses at each salt concentration.