UPLC sharp peaks but less response to HPLC ???

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I recently used NH4-formate (5mM)+ 0.1%Formic acid in aqueous solution plus ACN for organic (flow rate 0.3ml/min) for UPLC column 15cm length. The drug A peak width is 10 second , very sharp and positive mode intensity is 10E6 unit on Thermo Q-E machine. I re-run this drug on HPLC column (not UPLC) with (NH4)2CO3 10mM in aqeous solution plus ACN (flow rate 0.28ml/min). The drug A peak width is 60S, not sharp but good enough shape and intensity positive mode is 10E7 unit on the same machine Thermo-Q-E. The two runs are from the same machine and same vial of samples. How is possible that sharper peak has less peak intensity ( peak height 10E6 vs 10E7) ? The mass spec settings are similar for two columns. I am very very confused, can any one help?
The height of the peak is dependant on the injection volume or the mass placed on the column. Usually, for HPLC the injection volume is 1-10 uL while for a regular HPLC it is 50-250 uL.
HPLC chemist wrote:
The height of the peak is dependant on the injection volume or the mass placed on the column. Usually, for HPLC the injection volume is 1-10 uL while for a regular HPLC it is 50-250 uL.




Thanks for the reply. I injected same volume, 5uL. It is not the issue of injection. But I dont know why sharper peaks has less intensity (and peak area). It is weird.
Then my guess would be that your compound ionizes more efficiently in the aqueous mobile phase with ammonium carbonate compared to the formate buffer. This would be easy to check. I don't see a reason to use a different mobile phase though. The pH difference between the UPLC and the HPLC mobile phase is very large, which is possibly a bigger source of peak broadening than the column itself.
Rndirk wrote:
Then my guess would be that your compound ionizes more efficiently in the aqueous mobile phase with ammonium carbonate compared to the formate buffer. This would be easy to check. I don't see a reason to use a different mobile phase though. The pH difference between the UPLC and the HPLC mobile phase is very large, which is possibly a bigger source of peak broadening than the column itself.




Thanks for the answer. If you want to mention the pH, BEH amide condition is acidic and more protonated. However, HPLC method is basic and broader peaks. The sharper peaks under acidic conditions will generate more response for sure without doubt. But the reality is opposite, must something wrong?
Hi Tommylife1,

I agree with both Rndirk and HPLC Chemist--there are two phenomena occurring in this work:

1. The two sets of separation conditions are different. Would help to know the column dimensions for both columns, the UHPLC-type one under the acidic condition and the HPLC-type column under the basic condition. We know the injection volume was not varied (5 uL for both) and we know the flow rates...without knowing the column dimensions/particle sizes we cannot look into differences between linear velocities of the eluents in each case or know how much to roughly expect the band broadening of these columns may be.

2. The two sets of ionization conditions are different in each method of analysis.

You said,

The sharper peaks under acidic conditions will generate more response for sure without doubt. But the reality is opposite, must something wrong?


Why must the condition in the first statement be so? If the statement is so in this case, could there not be an opposite to the expected intensity per pH condition effect going on in positive ion mode? Exceptions to what one may intuitively expect regarding ionization efficiency are observed in LC-MS.

I'd say that the nature of what here is occurring would be interesting to look into...and no, it is not necessarily true that "something wrong" is going on. Using the same set of column dimensions/linear velocity may help a bit...as well as knowing the elution times/volumes of the peak of interest.
MattM
Peaks on HPLC method are looking good, not broadening, its a typical HPLC beautiful peaks. UPLC peaks are good too. I didnt see the bad peak shapes for both columns. I think it maybe a bit misunderstanding. However, not one metabolite have less response or intensity on UPLC, but many compounds are less response comparing to HPLC. It is not expected. Right?
tommylife1 wrote:
However, not one metabolite have less response or intensity on UPLC, but many compounds are less response comparing to HPLC. It is not expected. Right?


If you would like the switch of HPLC to UPLC to make more sense, then you have to keep everything (as much as possible) the same. Column phase, mobile phases (!), same injection from same vial, instrument,..

Basically you take the HPLC column but with a smaller ID, shorter and smaller particles, and use a method translator tool to change the flow. If you do it right you could expect similar retention times and hopefully more narrow peaks.

Since you're using a very different mobile phase in addition to HPLC => UPLC, you're changing too much at a time to make meaningful conclusions.
Hi Tommylife1,

Rndirk said what I am thinking--normalize the separation conditions (at least with regard to the column dimensions/particle sizes/eluent flow rates) and then you may find it easier to draw a conclusion to the nature of ionization strength between the acidic and basic eluents.
MattM
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