The most common application for SEC is aggregate analysis, which requires “native”
conditions (i.e. physiological conditions). These conditions require a buffered solution
at around pH 7 with a moderate amount of salt. A good starting point for mobile
phase is a 100 mM Phosphate Buffer, pH 6.8. However, there are some instances
where it is appropriate to optimize the mobile phase to improve chromatography. If
you suspect that the protein is hydrophobic, a small amount of organic solvent (e.g.
methanol, acetonitrile, or isopropanol) can be added generally anywhere between
If you suspect that the protein may have strong ionic characteristics (e.g. basic protein
or protein with a high isoelectric point), the ionic strength of the eluent can be
increased to improve chromatography. A 2x phosphate buffered saline (i.e. 50 mM
phosphate, 300 mM sodium chloride, pH 6.8) is recommended to prevent secondary
interactions. Note that increasing the salt will also increase hydrophobic interactions.
Further, varying the salt may improve the separation of one critical pair, but result in a
loss of resolution of another.