I am currently working on the purification of methanol soluble compounds using HPLC. I have been using MeOH and water as a solvent system (gradient) and the eluted compounds are too close to each other to separate them further. I just want to know if there is any alternative solvent system i can use to separate the compounds from each other. I do not want to to TLC too many times, as i may end up losing on my compounds. Is ethyl acetate and hexane a good choice?
Obviously ethyl acetate and hexane are a good choice for normal-phase elution. But I think reversed-phase chromatography is the possible best solutipon in your regard. You can use a mobile phase of Acetic acid (H2O) and methanol for this purpose.
So you have problems in separation. You need to develop your method. Gradient elution is one factor to have good separation try my chapter 2 attached.
Good luck
Aly R Abdel-Moemin
Are you using a gradient? If so, could you please describe it? You may need a shallower gradient. Are the compounds tailing so that one peak tails (or fronts) into the other? If so, a modifier, such as acetic acid, formic acid, or trifluoroacetic acid may be helpful.
What solvent system separates your compounds in TLC? Consider using column chromatography with silica gel. Since the solvents and stationary phase are different, the selectivity will also be different, allowing improved separation.The Rf for your desired compound should be between 0.2 and 0.5 on TLC for good column chromatography, it will elite at 5 to 2 column volumes.
Reversed-phase chromatography using a mobile phase of acidified water (trifluoroacetic or acetic acid, like 3-5%) plus methanol works well I guess. You can begin with a higher percentage of acidified water and gradually increasing to methanol. A classical C18 column (5 micrometer) can be tried. Ethyl acetate or hexane are used for normal-phase elution.
Obviously ethyl acetate and hexane are a good choice for normal-phase elution. But I think reversed-phase chromatography is the possible best solutipon in your regard. You can use a mobile phase of Acetic acid (H2O) and methanol for this purpose.
Try Acetonitrile along with them and also keep check on pH , increase gap between your ramping
varying gradient solvent ratio of acetonitrile and phosphate saline buffer as mobile phase. (RP-HPLC). This can only give some clue...............
As everyone suggested try ACN instead of Methanol and once you find shoulders just increase the time in that particular interval. This should solve your problem. Start 30% ACN and move 80% in 30 mins. change intervals in between and bring it back to 50% at the end of the run.
Good luck cheers
You can use either Methanol or Acetonitrile along with a suitable buffer solution like phosphate buffer. pH adjustment of the mobile phase is also a critical factor in getting a proper separation.
Dear colleague,
I agree with Naciye Erkan. you should be used acidified water.
The Best
I am agree with Girijan
acetonitrile with acidify water or phosphate bugger as gradient solvent
but must be used spectroscopic grade solvent
Actually, it's based upon pH and pKa values of your molecules. I think you should try with different stationary phase with different mobile phase. For mobile phase, buffer pH is also important. Thank you and all the best.
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