Hey! You can try change the mobile phase and if this not solve the problem you can change the injection volume, flow rateand and other parameters likewise. Also the problem can be your matrix...
I really can not change the mobile phase due to the chemistry of my sample :( for injection volume and flow rate i followed manual for my instrument (320 Lc-MS-Ms Agilant ) Some body suggest me to use degasser for solvent. now I am wondering if there is another cheaper solution or not!!?
Ok, I understand! I never worked with this instrument. Also, I never used degasser for solvent. Can you dilute your sample?
I did dilution but with decreasing concentration the sharpness of peak reduces and also we have much so shorter peaks.
If I see a doublet, I tend to think it's more of a column issue. I'd try a different column packing.
If your peak shape is better after dilution there could be two (?) answers for this phenomena: first, with higher concentration you have overload LC column or saturated detector in MS system. I have observed even 3or 4 peaks with our Qtrap system, when concentration of analyte was too high.
Are you sure that it is only an LC problem? Could it be that your resolution in the mass spectrometer is not what it should be due to some reason? It would be nice if you could make sure that the MS is ok, perhaps finding a way of running standard ESI -without chromatography- of a well known sample (lets say substance P). Check then that your resolution is ok and if it is you can move to the LC section in search for improvements.
Yes...I agree with Vladimir. Also, you have to make sure that your peaks are from your compound...because one of them can be related with some other thing. So...its better to try what described above. Good luck!
These undefined peaks that I am talking about are related to the blank because I see them in the blank as well so it is not my sample!!!
Aysana,
if your peaks show up in the blank, they are probably a contamination in the system. This can come from former samples or the solvent that you are using. Are you using MS-grade solvents for your mass spectrometers?
Without further information (e.g. what masses do you have, when do the peaks elute, at what percentage organic/aqueous, what solvent system are you using etc), it will be difficult to troubleshoot your problem.
I do think that this presentation might help you narrow down your problem.
http://www.chem.agilent.com/Library/slidepresentation/Public/Tips_and_Tricks_HPLC_Troubleshooting.pdf
Assuming your detector (MS) is working fine, there are multiple reasons why peaks can split and you probably have to test them one by one to identify your specific problem.
Are you running RPLC or HILIC?
Make sure your sample solution matches the initial mobile phase condition.
Sample solution mismatch usually give you split peak on chromatogram.
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