Chromatography Forum

Hi all,

I have a chromatographer/mass spec experience question/issue.

I'll try to describe following phenomena:

Running a compound on RP-UPLC with pure water (no additives) and an organic modifier seems to result in an extra created on column peak/compound.

Running under exactly the same chromatographic conditions, but only changing the pure water to an 10mM Ammonium formate (Aq) seems to inhibit the formation of this extra (on column) created compound.

With MS results, I can assing a mass to this extra formed peak under the conditions with pure water as mobile phase A. With 10mM ammonium formate (aq) as mobile phase A, I don't find this extra peak at all.

I tested already a lot of envolving things, such as blancs, selectivity changing issues etc., resulting in a remaining hypothesis that in this case, there is difference between the use of pure water and the use of Amm.Formate(Aq) which triggers this on-column-formation of this extra compound.

Anyone familiar with this phenomena?

Thnks for your reaction.

The phenomenon you are seeing is most likely due to the system peak formation. Whenever a sample is injected into a column whose composition is different from the mobile phase, it disturbs the local equilibrium of the mobile phase and the stationary phase. Most likely there is no chemical reaction with water and your sample. If it were indeed a chemical reaction with the sample, one would see the same effect with aqueous ammonium formate. Could you describe the MS experiment in little bit more detail " I can assign a mass to this extra formed peak under the conditions with pure water as mobile phase A"?

(1) What is the mass of the corresponding "new" peak?

Regards.

I'll try to outline a little bit

The peak of the analyte elutes at 15 min.
With pure water as mobile phase component A, the peak of the analyte elutes at 15 min and an extra formed peak elutes at 17 min. With MS, I can assign a m/z of e.g. 600
With amm. formate (aq) as mobile phase component A, no extra peak is observed. The peak of the analyte still elutes at 15 min. With MS, over all the chromatogram, no trace of something with a m/z of 600 can be found back.

Blancs are checked: not the cause
For information: there are other analytes (but not interfering) present as well. They are labeled with a m/z through MS. No selectivity issues. Pure water vs amm. formate (aq) does not change retention and selectivity ( => perfect overlay).

So it seems, that the use of water vs ammonium formate triggers the formation of this extra peak with m/z 600.

Which mass does the "right" peak have?

I have frequently the problem of modifying analysts on my HPLCs, but every time it looks like this:


(My Paintskills are awesome, right? )

Depending on whether it is a fast or slow reaction the front peak gets higher or lower. But I always face the "connection" between the both peaks because the reaction happens the whole time in the system/on the column.

I'll try to outline a little bit

The peak of the analyte elutes at 15 min.
With pure water as mobile phase component A, the peak of the analyte elutes at 15 min and an extra formed peak elutes at 17 min. With MS, I can assign a m/z of e.g. 600
With amm. formate (aq) as mobile phase component A, no extra peak is observed. The peak of the analyte still elutes at 15 min. With MS, over all the chromatogram, no trace of something with a m/z of 600 can be found back.

Blancs are checked: not the cause
For information: there are other analytes (but not interfering) present as well. They are labeled with a m/z through MS. No selectivity issues. Pure water vs amm. formate (aq) does not change retention and selectivity ( => perfect overlay).

So it seems, that the use of water vs ammonium formate triggers the formation of this extra peak with m/z 600.

Is the mass of the water induced peak really 600 or was it just an example? As I said earlier, it cannot be a chemical reaction of your analyte with water because the same reaction does not occur with aqueous ammonium formate.

(0) Does your sample have formate ions as anion?
(i) Check if the sample pH is drastically different from the blank and the mobile phase i.e. water?
(ii) I would be curious to see what happens when you inject ammonium formate as a sample.

I can think of a ionzable molecule that will be retained with pure water, and won't be retained under the buffered conditions. Does the "extra" peak retention changes at different pH?