I have MeOH plant crude extract, which has shown in-vitro anti-inflammatory activity (I am working on inflammation). I run on HPLC using 0.1 % Formic acid and Acetonitrile and got few peaks. I want to get maximum number of peaks in my chromatogram to move towrds identificaion of compounds by HPLC-MS. Which mobile phase system should I use?
If you are attempting to identify the anti-inflammatory factor then you are asking the wrong question. You have activity in the MeOH extract and then I assume you want to separate out the extract constituent(s) with a view to isolating the active component for subsequent identification. From the limited information supplied, it seems you have a bioassay of some description to detect the anti-inflammatory activity. If so, then the question to ask is; "Where is the anti-inflammatory activity in the chromatogram?" You need to collect a series of fractions of the HPLC eluent and test for the presence of the inflammatory activity. At this stage you know nothing about the nature of the active component so don't assume that your detector (UV/Vis?) will detect it (it may have no chromophore or it may be present below the limit of detection by your detector). Activity in must equal activity out + activity that remains on the column, unless there is degradation and loss of activity. How stable is the "activity"? Good Luck!
Hello Abdul,
the question is not so easy to answer. There are many factors involved. If you want to increase the chromatographic resolution, you can either use a longer column (R ~ 1/4*SQR(N) or R ~ 1/4*SQR(L/H) or R ~ 1/4*SQR(L/(h*dp) ) or use a smaller particle size. Also the selectivity of your stationary phase is important. Temperature, pH value and eluents also play an important role. When it comes to ionization, the polarity of your molecules plays an important role in the choice of ionization method (ESI, APCI, APPI, etc.). Which compound class are your compounds ?
Regards
Markus
If you are attempting to identify the anti-inflammatory factor then you are asking the wrong question. You have activity in the MeOH extract and then I assume you want to separate out the extract constituent(s) with a view to isolating the active component for subsequent identification. From the limited information supplied, it seems you have a bioassay of some description to detect the anti-inflammatory activity. If so, then the question to ask is; "Where is the anti-inflammatory activity in the chromatogram?" You need to collect a series of fractions of the HPLC eluent and test for the presence of the inflammatory activity. At this stage you know nothing about the nature of the active component so don't assume that your detector (UV/Vis?) will detect it (it may have no chromophore or it may be present below the limit of detection by your detector). Activity in must equal activity out + activity that remains on the column, unless there is degradation and loss of activity. How stable is the "activity"? Good Luck!
This is a very broad question. I think the answer lies what you are mostly interested in (polar or non-polar or both). Based on that the extraction, sample prep and mobile phase have to be selected. RP-HPLC with the solvent system you are using would be the first place I would start and definitely with a longer column, shallow gradient and long run time. Based on that I will adjust different parameters in order to optimize the method and conditions.
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