Hi ,
I intend to separate a doublet of peak eluting from reverse phase HPLC column, along with the main peak, a shoulder peak appears which I want to get rid of the main peak. PDA detector at 245 nm.The column is semi prep C18. Mobile phase is acetonitrile: water = 90:10 (i am not adding formic or acetic acid or TFA 0.01% in water which people do generally) flow rate semi prep 5 ml/min, anal 0.5 ml/min
I have also tried with different flow rates 3,4,6 ml/min, at temp 26 and 35 C, with gradients from 50:50 to 90:10 (for 10 min hold and then 50:50 in next 20 min, 30 min, and 40 min.In all cases, elution is there but poor seperation, still able to collect ~90% pure peak despite some loss of sample. Plz. guide me how can I improve resolution. see pdf. File 1 purity of collected peak by anal c18 isocratic, file 2 is semi-prep isocratic run, file 3 is semi-prep gradient run
I would adjust the pH of your mobile phase with 0.1% Formic Acid. It looks like you have both a protonated form and deprotonated form of the same molecule. If this is successful I would regularly use pH 3 Phosphate Buffer since it is invisible in the UV.
It could be an especiation problem as pointed out by Bruce. It will helpful that you tells us the molecule you are purifying to discard this or not.
You did not provide any column dimensions or info about the particle sized used (Basic info about your method is needed to understand it). It appears that you may have very little retention (eluting close to tzero time). Though it is hard to tell from your chromatograms, it appears your sample elutes at very high organic concentrations (~ 90 %) so that would be the region you would want to work with when developing a method. Use MeOH or ACN vs acidified water starting at 80% running to 95% and see what you get. Try smaller particle size to improve resolution at any scale. Please ask your teacher for help too.
Dear
allway if you want to get a good separation and resolution of peaks you adjested a sutibil mobile phase, flow rate and column. I think you must added acid(acidified) to your mobile phase and decreas the flow rate to 1-2 ml/ min.
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