metal contamination causing imp peaks at high pH eluent?

[kplc]

My problem started when I installed a new column….

Using an Xbridge 150 x 46 mm, 3.5 um column at pH 9.5 NH4Cl-acetonitrile mobile phase, I observed two (extra) very repeatable peaks in both standard and sample solutions. These peaks however, were not observed in any blank solution. Have you ever seen anything like this? Could the analyte be complexing with metals in the stationary phase? Also, the standards/samples themselves had been exposed to Pt/alumina catalysts, and in addition the main peak exhibited increased tailing.

I did not see the two peaks on the previous column and peak tailing was also less -but I had run with 0.1%H3PO4-acetonitrile mobile phase first, before I switched to the high pH method.

Keeping that thought I eluted (95:5) 0.1%H3PO4:acetonitrile through the new column overnight and the next day when I switched back to the high pH mobile phase, the two peaks were gone.

I would be very interested in any insight/feedback you might have.

Thanks!

[Uwe Neue]

While your description is not complete, it looks to me as if you are resolving two impurities at pH 9.5 that you did not see in 0.1% H3PO4. This is not unusual and actually quite common, since you have very large selectivity differences betwen the two pH values.

The other possibility is that your analyte is unstable at high pH, while completely stable under acidic conditions (assuming that you made up your samples in the high pH mobile phase). You can find this readily by letting the sample sit longer.

[kplc]

Clarification: hope this helps

1. The assay was NOT run at low pH conditions. 0.1% H3PO4 was used as a column wash only. The method was run at pH 9.5 ONLY.

2. The two peaks were NOT observed on other Xbridge columns which had been previously exposed to acid conditions. (When these columns were installed on the instrument the two peaks were NOT observed.)

3. The two peaks were observed ONLY on the 'new' Xbridge column which had been exposed to only basic conditions. (When the new column was installed on another instrument the two peaks WERE again observed).

4. The same standard and sample vials were used for these experiments.

5. The peaks were 1.3% and 1.4%, respectively.

[Uwe Neue]

OK, this clarifies things, at least some of them…

Peaks commonly do not come from the packing, especially if they are nice and sharp. It could be that something like this can be triggered by the inlet frit of the column, and that such a problem will disappear, once the frit is suitably deactivated, as it would happen by exposure to dilute phosphoric acid. Does your analyte form readily complexes with metal/iron?

I also do not (yet) understand your reference to the Pt/alumina catalyst. Is this something that is present in all your samples? Or only in the samples that you ran recently? Or is this something that could end up on the inlet frit?

[HW Mueller]

Did these peaks ever come back on the xbridge at pH = 9.5 after you washed the column with H3PO4? Seems to me that you had a transient, nonrepeatable problem. One can never find out what happened if one can´t repeat it. It is scientifically of little interest, thus a waste of time.

[kplc]

Repeatability of the experiment got me to thinking and I looked back over a previous XBridge column's history.

When that particular previous column was new the two peaks were present! However, since the same high pH method is used to monitor four different processing steps their presence was overlooked because they were not included in the peaks of interest at the time. Interestingly, the peak width of the standard of interest also appeared wider at the beginning of that column's life than it does when injected into that column now.

I will look into this in more detail.