Hello.  As I understand, your chromatic peaks are overlapping on your MS signals (and not on your UV detector), and you wish to have baseline peptide separation. Two points:

1. The sensitivity of your mass spec is most likely several magnitudes greater than the sensitivity of your UV system. It, therefore, might be that you actually have overlap on your UV detector as well.

2. The "spreading out" of chromatographic peaks is, amongst many things, caused by long transfer-lines (as Fabrizio Donnarumma also is pointing out above). As your mass spec is located at the down-flow from the UV detector (which also might have a large-volume detector chamber), I would try and bypass the UV detector and make the shortest transfer line possible from the column to the mass spec.

If 2) isn't resolving the issue, I would try and optimize the LC gradient, as the problem you are experiencing in this case likely is caused by my point in 1). For starters, you can try and flatten the gradient which should increase your chromatographic resolution.

Hope this helps, and good luck with your research!

Best Regards

Hello. Could you please tell us some more about the molecules you are analysing and which MS instrument and detector you are using.

I think this problem may be either due to poor ionization of your compounds which may require the addition of some acid (TFA, Formic) to enhance the ionization, or you can try APCI (atmospheric pressure chemical ionization) if your molecules are less than 1500 Da.

you can also check the flow rate and the split ratio of the splitter.

you can also adjust the injection volume and concentration.

Make sure you have no dead volume within the transfer lines from UV to mass spectrometer - this will be the main cause for overlapping peaks.  If you are using API, the position of your probe will also be important - make sure this is optimised by directly infusing a standard peptide.

Just to avoid confusion, the resolution you are talking about is NOT mass resolution, right? Are you running your sample through the UV detector and then into the MS?

If your UV chromatogram displays well separated peaks  and your TIC (or BPC) is not, then I would suggest you follow Pavel indication. It really sounds like a connection problem.

Remember that normally we do not couple UV and Mass Spec. Actually, we try to reduce as much as possible the distance between the column outlet and the ESI source of the mass analyzer.

Hello.  As I understand, your chromatic peaks are overlapping on your MS signals (and not on your UV detector), and you wish to have baseline peptide separation. Two points:

1. The sensitivity of your mass spec is most likely several magnitudes greater than the sensitivity of your UV system. It, therefore, might be that you actually have overlap on your UV detector as well.

2. The "spreading out" of chromatographic peaks is, amongst many things, caused by long transfer-lines (as Fabrizio Donnarumma also is pointing out above). As your mass spec is located at the down-flow from the UV detector (which also might have a large-volume detector chamber), I would try and bypass the UV detector and make the shortest transfer line possible from the column to the mass spec.

If 2) isn't resolving the issue, I would try and optimize the LC gradient, as the problem you are experiencing in this case likely is caused by my point in 1). For starters, you can try and flatten the gradient which should increase your chromatographic resolution.

Hope this helps, and good luck with your research!

Best Regards

Of course, one has to minimize post-UV spreading caused by multiple connections between the detectors.

Sometimes hydrophobic peptides do not elute in sharp chromatographic peaks, but elute over longer time. Only looking at the extracted ion chromatograms for the corresponding peptide ions one will realize this behavior. As Tue Bjerg Bennike mentioned the MS is more sensitive than UV and provides additional capability to follow the elution of multiple components.

If the problem appears only with the late eluting peptides in the reverse phase HPLC, adding isopropanol to the mobile phase (solvent B) can sharpen the chromatographic peaks as well as provide higher mass spec response.

Hello. I also consider Tue suggestions are in the correct way. One possible reason is much higher sensitivity of the MS detector (so you are able to "see with MS more than you see with UV", and a second one is peak widening due to long path between UV and MS detectors. Conversely, I am not sure adding isopropanol is a nice choice. Good luck with research 

Adding to those that have been suggested, you also want to make sure the scan speed of MS is compartible with UV peak width.  Too slow of a MS scan speed will artificially broaden the peak, resulting in an apparent poor peak resolution.

Hello,

Is your lcms application an existing method or do you want to switch from UV  to MS detection. Very important here can also be the source interface.