Chromatography Forum

Hi guys,

There is some dilemma going on here.
A chemist made an API and a method needs to be developed.

MPA is 10mM ammoniumbicarbonate pH adjusted to 10 with amm. hydroxide.
MPB is Acetonitrile.

The column which is used is a Waters Xbridge C18 column, 2.1x150mm, 3.5µm
We are seeing some impurities on this used column.

Now comes the funny part:
With another used Waters Xbridge C18 column, 2.1x150mm, 3.5µm with exact same partnumber and S/N number gives the same impurities but these impurities are much lower in area.

This was analyzed with the same buffer/mobile phase just right after the first column.

Is it possible that there is some on column degradation of the compound? because there is no carry-over observed in the blanks after analyzing the API.
And why is it that some impurities are higher than on the other column?

If two different columns give the same peak, it can be possible the impurities are there from a unknown source.

Ammoniumbicarbonate degradation ? It decompose at 36 Celsius.
Buffer/mobile phase contamination ? Did you tried to change the buffer and the mobile phase from a new batch ?

When passing the blank, did you use the same MP ?

It possible the 2 equals columns have different sensibility. You told us that both are used, that can explain.

It would be easy to find where the impurities come from if you know what is this impurity.

If two different columns give the same peak, it can be possible the impurities are there from a unknown source.

Ammoniumbicarbonate degradation ? It decompose at 36 Celsius.
Buffer/mobile phase contamination ? Did you tried to change the buffer and the mobile phase from a new batch ?

When passing the blank, did you use the same MP ?

It possible the 2 equals columns have different sensibility. You told us that both are used, that can explain.

It would be easy to find where the impurities come from if you know what is this impurity.

Hi John,

the two different (same) columns give the same peak but the area is a bit lower with the column which gives more impurities. Also the blank was run with after the sample with the same MP.

I changed the buffer but it gave the same impurity profile.

I ordered 2 new same columns and will test them both.

I also tested 25mM of buffer and compared the chromatogram with the 10mM buffer.
Less impurities are being seen now.
It seems that a higher buffer strength gives less degradation.

pH 10 is somewhat uncommon. Can you try a lower pH?

pH 10 is somewhat uncommon. Can you try a lower pH?

Xbridge is stable in the pH range 1-12 so I think that's no problem. Do you set pH of your sample before injection? Is it also pH 10? I suppose that you use LC MS or DAD for your analysis. You can check if these impurities have something common with your analyte - similar spectra, characteristic neutral loss etc. (you will see if it is the matter of degradation )