How can i reduce the retention time to around 18 mins for the second major peak just by adjusting pH and mobile phase composition ratio only. With a C18 column and buffer, acetonitrile and methanol as mobile phase (225.5mL : 17mL : 7.5mL).I was able to have a seperation, with the adjustment of buffer using 10M NaOH to pH 3.5. Both peaks came out late but my challenge is how can i reduce the eluting time without changing the flow rate, column temperature or column. I need advice and suggestion.
1) Mobile phase
Try increasing the organic content of your mobile phase. You can also try using a different organic solvent. There are some cases when methanol will provide better selectivity, such as with biphenyl phases. Otherwise, acetonitrile may provide shorter retention times because it is more nonpolar. The solvent choice should be made on a case by case basis, though, as sometimes it may surprise you what works better.
2) Gradient mode
Try starting with higher organic and/or ramping up to more organic faster and see if that helps.
3) pH adjustments
Try to make the two components completely ionized to make them more polar to elute faster. use pH suitable for components according to their pKa.
you can know more from recently published papers about optimizing conditions for separation for example :
( K.M. Kelani, E.S. Elzanfaly, A.S. Saad, M.K. Halim, M.B. El-Zeiny, Different greenness assessment perspectives for stability-indicating RP-HPLC method used for the assay of isoxsuprine hydrochloride and four nephrotoxic and hepatotoxic photothermal degradation products, Microchem. J. 171 (2021) 106826. doi: 10.1016/j.microc.2021.106826.) .
Slightly change in ratio of acetonitrile portion of mobile phase. If possible remove the methanol I think one organic phase is enough.
Hi Tolu,
I think you would be very limited in the changes you can make to have any significant impact.
I think the suggestions given have been good and would recommend using a methanol mobile phase for your organic phase and changing your column. I doubt it will be the case but I think that if these are the 2 peaks of interest and they are eluting later, I would change columns to reduce the retention time and preserve the peaks.
Methanol will allow you to utilise other column chemistries such as PFP or phenyl columns which may help you reduce that retention time.
If you try and reduce it using gradient or organic percentage then you are going to merge the peaks before you significantly drop the retention time.
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