All of the peptides (Hydrophobic, hydrophilic and neutral) give very strong peaks between 2-5 minutes in analytical HPLC, regardless of their properties. Even though I reversed the method, the peaks are still comes between 2-5 minutes. As a result of the analysis, it is expected that the peak will come after the 15th minute. How to overcome this problem? Solvents: A(Water+0.1%TFA) B(Acetonitrile + 0.085% TFA) Method: Fixed 1% B solution for 0-5 minutes B solution increasing from 1% to 100% between 5-35 minutes Column: Intersil ODS-3 5mm 46x150 mm (UP)
Hi,
are you sure you didn't swap the solvents?
And what you mean by saying "I reversed the method"?
It also occurred to me that the first solvents may have swap. I re-prepared the two solvents and carefully placed them. But I got the same result again.
I reserved the method means "During the same time as the first method, I tried solvent B with a new method to reduce it from 100% to 5%"
It belongs to the results obtained in two files. The black line on chromatograms provides information about the methods used.
Please contact someone at your school who has professional training in HPLC method development. The information you have provided is confusing and misdirected. Someone local could help you develop a method for your sample(s). Trying to work this out on your own, with no formal training as a student, would take many years just to reach a basic level of proficiency (a poor use of your time). The information you have provided is incomplete (please post an actual METHOD, using standard terms for these types of question, not phone pictures).
- The one chromatogram you attached has no information about the method (i.e. column dimensions, flow rate, composition, detection settings etc), but I do see an injection peak and also a peak at 15 minutes too. So something is being retained. Since no method or sample info was provided, there is no way to suggest anything else.
- "Reversed the method". No idea what that means.
Again, you probably do not realize that your question can not be solved by posting on the web. Please contact a local professional who has already been trained in HPLC method development. They will be able to assist you.
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