At 9:05 AM +0100 9/26/08, Meg wrote:
>i performed the HIC but the resolution was not good and my sample was
>diluted 10 fold and elution was near end of elution gradient.
HIC is generally not a high resolution technique. Ammonium sulfate it
the most efficient salt for HIC, but I've used high concentrations of
NaCl (e.g. ~1.5-2M) as well.
Use a less-hydrophobic media (low-sub instead of high sub; butyl vs
phenyl) to try to get your protein to elute earlier. Inclusion of an
organic modifier (e.g ethanol or methanol) could make your protein
elute earlier, but some proteins may not respond well to this
treatment. Also, if you are running at room temp, running at 4 deg C
may help it come out earlier.