I am using HPLC for compounds isolation and purification, with methanol and water as solvent system. The problem, i am facing is that for the same sample, in the same solvent system (keeping the same ratio), i get different peaks.
I wish to ask, whether this is common with HPLC, as mostly we concentrate on retention time. If this is not the case, so how could i resolve this problem.
Did you mean you injected same sample (same concentrations) using same column, same mobile phase, same flow rate, same solvent on > 1 replications, you have different peaks? I mean, do you have peak (s) with different Rts of same sample with identical HPLC conditions? Did you use UV, DAD, ELSD, MS or others as detector? If you used DAD or MS, have you measured the UV or MS of the peak(s)? Let me know, maybe I can recommend you something to solve the problems
Dear Gunawan Indrayanto,
yes the same.. I am going to identify two compounds whether they are same or different.. but time and again for the same one compound, the peaks are different, in case of their retention time and peaks appearance. I did not used other techniques, only the rate, solvent system, column specification, rate flow are same. for these two compounds i also used simple and reverse phase TLCs. At one time their Rf values were same, but another time different. one of the compound is flavonoid, while the other is also hopefully the same. previously, for other compounds a also got this problem with TLC, and later on by HNMR those appeared same. so this time i am more cautious about these, and i want to analyze by HPLC, before NMR. but again i got this problem....
Hi, is your HPLC equipped with DAD or MS detector?, or do you have TLC Scanner? If yes, than you can measured the UV spectra or MS spectra of the peak(s), to know whether your compound(s) were stable or not. Did you apply same fresh solution for each measurements? It could be your compound(s) were not stable; Did you ever tested their stability on chromatography systems (mobile phase, stationary phase/TLC plate)?
dear Gunawan Indrayanto,
our HPLC is connected to UV detector. I used 208 wavelenght for their detection. I also faced another problem that the flavonoid was confirmed by HNMR pure, but for that compound on HPLC, i have 4 peaks. indeed, i think i should go for MS, but i will be thankful to you if you give me any idea to compare their similarity on HPLC.
Thanx for your time.
Hi, How could you know that your sample is pure? It could be the other peaks from solvent that you used for dissolving the sample, and its impurities. Have you analyzed the solvent, that you used, by HPLC (at 208 nm)?, please compare the chromatogram of solvent only, and chromatogram of sample in solvent? Have you confirmed that your sample is stable in the solvent, and mobile phase? It will be nice if you analyze your sample using LC (qTof)MS, you will know the identities of the peak(s).
Dear Gunawan Indrayanto,
I am very thanful for your valuable suggestions. hopefully, i will find it helpful.
Thanx for your help, and your time.
Hameed Shah.
You may check pH, I believe you mentioned it is flavanoid. may be different forms; protonated /non-protonated. Ratio might depend on how long you leave sample sitting.
Also I have had equilibria of two tautomeric forms that were visible in HPLC, but not in NMR (same the other way around)
Halla Hameed.
I believe you may have more than one species here that can not be detected with HNMR. I am working with vanadium and if concentration and pH allow you can detected 2 species.
can you please share photos of the chromatograme of your samples , then we may guess where is the problem
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