Chromatography Forum

I am quantitating homocysteine by HPLC and using a sperisorb reverse
phase column. I am at the moment having problems with the column. It
looks like it's contaminated by protein material from patient serum
from which I extract the homocysteine. The column is spherisorb, silica
3 um woth 90 Angstrom pore size. Phase is ODS-2 (S3-ODS2) and 11%
carbon. The length is 100mm with ID of 4.6 mm. Can anybody help me with
how to regenerate protein contaminated column. Thanks.


Jimmy

This has been discussed at length before (still available?).
In short, you need something which tends to dissolve proteins: The right % organic, pH, and/or chaotropes (certain salts or salt concentrations or better 4-6M urea, etc.), or even detergents with dithiothreitol as a last resort.

A better approach for the future might be to use a good sample preparation technique to clean up the sample. I can give you advice on proven SPE techniques.

Or precipitation of proteins (Na2SO4, ACN, ....) + solvating analytes, or ultrafiltration/dialysis (making sure that analytes stay solvated). Alas, in our experience these measures postpone the protein (and or lipids or whatever) problems, not eliminate them. You should still wash the column at the end of the day. Fortunatly, a good pre-cleaning seems to allow good washes with just H2O-org. solutions.
(Gee, we are repeating a lot, currently).