Chromatography Forum

OK here's a question. We have a "wacky" normal phase method which uses both a cyano column and a diol column in tandem. We injected some samples which contained trace amounts of water and the chromatography started to fall apart.

We rinsed with IPA but that didn't seem to help too much. Then we rinsed with our mobile phase B - which is 70/30 hexane/ethyl acetate. That seemed to work much better.

This surprised me. What is generally the best way to regenerate normal phase columns after they have come in contact with too much water.

Much Thanks in Advance

I need a bit more information to understand the issue here:
1. Besides mobile phase B, did you use other mobile phase? If yes, what is the composition?
2. What do you mean by "chromatography started to fall apart"? Bad peak shape, wandering retention, both, or something else.
3. After you rinsed the column with IPA, what the chromatography looked like compared to "good" one and "bad" one?

Assuming hexane is your initial mobile phase, to get rid of trace water trapped on the column, I imagine thorough IPA wash followed by thorough hexane equilibration should work. Acetone can also be used instead of IPA in this case, but should not be used on amino column.

We've found at least one company that has sodium sulfate cartridges that can be installed in line (like a guard column) to absorb water that may be introduced from the sample - before it gets to the column.

Has anyone had experience with this type of thing.

Xiaodong Liu,

I'm curious, why acetone could not be used to wash an amino column and could be used with other columns?

Acetone will chemically react with primary and secondary amines to form an imine.