Connecting columns in a series

[DJ]

I'm trying to elute a peptide from a plolyhydroxyethyl HILIC column directly on to a polycat wcx. After initially binding the HILIC, I step down to a % MeCN that is just above the approx % for eluting the peptide, then connect a polycat column down-stream of the hilic, and begin pumping starting mobile phase for the polycat (50% MeCN, 10 mM NH4OAc.) which is low enough % MeCN to elute the ligand from the HILIC, however, subsequent salt gradient on the polycat produced awful "peak" shape (compared to direct injection on to the same polycat column, same mobile phase program).

I'm wondering if this is because the peak volume from the HILIC = to large a sample volume for the the polycat column, or because prior to the peptide eluting from the HILIC to wcx, a column volumes-worth of mobile phase left in the hilic disturbs the equilibrated polycat as it passes through.

Mobile phase for both steps in this sequence contained 10 nM NH4OAc + MeCN. The difference is the % of MeCN used to equilibrate the polycat (50%), is less than the % MeCN left in the HILIC prior to eluting the peptide.

If I were instead to load the sample on to a hilic trap column, step elute weakly bound material, then invert, place up-stream of the polycat, am I more likely to get descent peak shape from the second column?

[tom jupille]

If I were instead to load the sample on to a hilic trap column, step elute weakly bound material, then invert, place up-stream of the polycat, am I more likely to get descent peak shape from the second column?

Yes.

[DJ]

Thanks.

Could you elaborate some on what exactly causes poor separation in the second column-- Is it the peak volume/sample volume from the first column or the difference in mobile phase composition (70-40% MeCN)

I've loaded massive peptide/protein sample volumes on to RP columns, and it seems very insensitive to total sample volume, composition (so long as the organic is below % required for elution). Is this because broadening does not occur even with multiple large-volume injections, or, else the band is compressed during the gradient?

[tom jupille]

If I had to guess, I'd say that even though what's coming off the HILIC column is weaker than your catx mobile phase, the strengths are close enough that you're not getting much reconcentration when you go on to the second column.