I have tried to purify a chemical sample. I performed multiple runs with the same method at specific wavelength. The first run gave me numerous significant peaks in the middle of the run. The second run of the exact same sample gave me a huge peak in the initial bit of the run and the peaks that were seen in the first run could not be seen the second run. I did a third run of the diluted sample and I got a spectrum similar to first run. Could somebody please explain what’s happening here.
Thank you!
Do you run isocratic or gradient elution? Are you sure that all the peaks under interest are eluted within the acquisition/run time?
Your story seems to refer to late eluting peak - the main one was not washed out of the column during first run and appeared in the second run.
However other explanations are possible as you gave not enough information about your test.
Vishal Bayya, I think you may have some confusion in terminology and an unfamiliarity with how HPLC works. If you use a valid HPLC method to collect data on the SAME sample multiple times, then you should obtain exactly the same results each time. If there is co-elution, the method is not selective, impurities are present or the the detector settings are not optimized, then the results may be of poor quality. Proper methods use / follow good chromatography fundamentals: IOW, the method is selective for the sample(s) analyzed, the sample has a good Kprime, is well resolved with good peak shape, a stable baseline is observed, detection settings used are valid, detector and the instrument used are fully serviced and checked for performance before use.
HPLC systems are complex. Without more detailed information about your HPLC method, instrument and sample, no constructive suggestions or comments can be made. If you can post more detailed information and a chromatogram or two with scales shown, then someone may be able to add more info. For the fastest resolution of the problem, please have the method used and data collected reviewed by an experienced chromatographer on-site. Questions of this type are often very easy to solve/explain when in front of the analytical instrument and user.
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