Chromatography Forum

This is a weird problem....
We use Aminex HPX-42A column to separate sugar oligomers from biomass hydrolysates. Mobile phase is water. RI detector @ 410 nm. General conditions are 0.2ml/min, 85oC.

Recently we are facing problem with separation- even with stds. This problem persisted even after changing the columns twice!!!

Nature of the problem: 1) Negative peaks
2) Change in retention time- all compounds elute at the same time (60 min) and have same peak area, irrespective of concentration and difference in chemical structure (for different xylose DP stds)

Troubleshooting:
1) Injecting without guard column --> no improvement
2) Injecting straight to the RI detector --> positive peaks for all xylose DP stds (1 to 6). i.e. The detector seems fine.
3) We checked the stds in HPAEC-PAD ----> stds are not degraded
4) Bio Rad shipped us a new column in April (apparently from a different batch) ----> still no change.

The same HPLC system (Waters 2695) works fine for monomer separation (we use Shodex SP0810 column). So where is the problem?

Recently we are facing problem with separation

I interpret this to mean that the separation formerly worked for you. In that case, the question is "what changed?".

Was the change abrupt or gradual? If it was abrupt, did it coincide with some other event (instrument service, new column, . . . )?

The symptoms you are seeing (long retention, no resolution, RI going the wrong way) are consistent with a temperature problem, so another thing to check is the temperature controller.

Most of the mobile phase issues I can think of (low pH, presence of Cl-, SO4=, or HCO3-) would affect the SP0810 column (which is a Pb++ form resin) the same way as the HPX42A (Ag+ form).

In addition to Tom can you tell us what water you use, DI water, fresh distilled water, HPLC grade water?

As I understand the description of the problem , the only place to look at is the column.
If a positive peak is observed without a column, the water can’t be the problem. The same goes for the temperature.
New column = negative peak  Something’s eluting continuously, thus changing the refractive index value of the mobile phase (the water).
Suggestion: Equilibrate the column for longer time than (until the preservative or whatever has eluted completely and then try again. Monitor the baseline during the equilibration/flushing.

Best Regards

Recently we are facing problem with separation

I interpret this to mean that the separation formerly worked for you. In that case, the question is "what changed?".

Was the change abrupt or gradual? If it was abrupt, did it coincide with some other event (instrument service, new column, . . . )?

The symptoms you are seeing (long retention, no resolution, RI going the wrong way) are consistent with a temperature problem, so another thing to check is the temperature controller.

Most of the mobile phase issues I can think of (low pH, presence of Cl-, SO4=, or HCO3-) would affect the SP0810 column (which is a Pb++ form resin) the same way as the HPX42A (Ag+ form).

Yes, the change was sudden and it happened after an instrument service (it was a short circuit problem in the computer. nothing to do with the columns). We lost the old software and had to set-up a new software (& instrument mtd from memory. We use NREL mtd for sugar oligomer separation).

But the Shodex column works fine with the same tubing, RI detector and old instrument mtd.

In addition to Tom can you tell us what water you use, DI water, fresh distilled water, HPLC grade water?

Thanks! We use Millipore (DI) water. Made fresh every time.

Again the same solvent set-up works fine for the other column (Shodex SP0810).

Dear Guest,
RI detector at 410 nm!!!!!!!!!!!!
please clarify about your instrument configuration,

also try to analyse as per COA of the column!!

also why need Ag form for this analysis, i believe Na+ will serve this purpose,
http://www.shodex.com/english/dc030324.html



upamaniah

We found out the problem.... what "changed" was the instrument method. The injector depth was not configured properly. Previously it was set at 19 mm!!!! Now it is at 0 mm.


Thank you all for the discussion.

Thanks for the update! Amazing how those can be "hair-pulling" problems!