sample pH in anion exchange

[MK]

This is the first time I'm doing anion exchange and intresting thing came out on running pre-test for acid/base forced degradation. Could anyone please explain whats going on?

-analyte pI is around 6
-mobile phase and sample pH is about 7.5
-gradient is from 300mM to 1500mM NaCl

For base degradation I did sample pH's 8.5, 9.5 and 10.5. RT increased and peak area decreased from 8.5 to 9.5 and to 10.5.

For acid degradation sample pH's 7, 6 and 5 were tested. This resulted to constant RT but peak area decreased from 7 to 6 and increase again with 5.

I can understand the base: The analyte is more negatively charged above the pI and thus has more retention. Peak area decreases logically as the analyte gets unstable on alkaline conditions.

But why doesn't the RT decrease when sample pH goes to acidic below the pI? Why the peak area drops around the pI and the comes back up on pH 5?

Many thanks
MK

[Mark Tracy]

MK,
You didn't mention whether or not the mobile phase is buffered (it ought to be). If it is not (or only weakly) the sample pH is probably perturbing the column. What happens if you adjust the pH of the samples (after the degradation experiment) to match the mobile phase?

[MK]

Mark Tracy,

Yes, MP is buffered by 50 mM Hepes (pKa = 7.5 useful pH range = 6.8-8.2). I did dilute the base degradation (pH 10.5) to MP and then got the same RT.

I agree I'm probably perturbing the MP.

Does this make any sense:

My base samples pH 8.5, 9.5, 10.5, were all outside the buffering range
and thats why I saw the increase in RT.

First acid sample pH 7 was in the buffering range. pHs 6 and 5 were also
quite close. These were better mixed and buffered back to MP and thus no RT shift?

But whats happening with the peak area on acid? It drops a lot from pH 7
to 6 and increases againg on pH 5? Can the analyte be stable again on acidic conditions outside the buffer range?

thanks
MK

[Mark Tracy]

I'll admit it is a bit confusing to me too. Is your analyte a simple molecule with well behaved pH equilibria, or something that isomerizes with slow equiliibrium? Many compounds have shifts in their UV spectra with pH. Usually, by the time the analyte leaves the column, it is at the pH of the mobile phase, regardless of what it was in the sample vial, so that is why I'm confused too.