Hello Colleagues,
I have several fractions (Hex:EtAc). In one small fraction (about 2mls), I got 6 peaks on HPLC. What do I need to do further or how do I need to separate these compounds, considering their small amounts? Further, I need to work on these individual compounds later.
Optionally, I was thinking of collecting the liquid as soon after each peak registers but HPLC has microliters of sample.
Please advise
Kindly,
Additional information to my question above, Hex:EtAc is at 80:20 and TLC showed well separated spots, no overlapping.
Column fractionation gave about 55 portions (about 2ml each) or less for those with distinct colours.
But each of these portions gave atleast 4 peaks on HPLC(Acetonitrile and separately with MeOH, 100% each).
Thank you and regards
A
After you get the initial fractions that have your demand compounds,
So you have two ways to purify these compounds:
1. Make a sub-fraction for the certain fraction by using other solvents. You can use the same solvent used by HPLC
2-Use preoperative- HPLC, in this devise you can injection more than 1-10 ml
Thank you Naser Jawad Kadhum I will try the sub fractions and see how far they take me.
Let me also explore prep-HPLC.
Thanks again
You may also track your compounds off the open column with TLC and visualize them with sulfuric acid char, water spray, iodine, or other spray techniques. Since it appears that you have access to a prep LC, that would be an easier way to run it since you have analytical LC informaiton.
Thank you Jack Silver , this helped alot. In addition, prep HPLC technique proved useful.
Andrew Mtewa , its not about the volume, i.e 2mls, rather, its about the amount of your mixture in mg. Since you have a working method which gave good separation on your analytical HPLC and you have access to Prep-HPLC also, then anything is possible. The only issue I can see is the amount, you said you need the compounds for further analysis but you did not give the amount of your compound in the 2mls, is it 5mg, 10mg or 30mg. The amount of the mixture will guide you on how to proceed.
Good luck.
Thank you Ayorinde Victor Ogundele ,
At first, it was too difficult/ frustrating to proceed with quantification of compounds in mg from a small fraction of 2ml that is showing 6 peaks. If 2 ml of fraction would give me 2 peaks for example, I would have the guts to proceed with quantification. Perhaps, I was too fast frustrated.
I know from the 6 peaks, it's easy to quantify even from peak areas, but my aim was beyond knowing the concentrations of the compounds. I was more interested in acquiring the compounds as sinlge isolates for further work with them individually.
But now, the pre-HPLC tech helped alot. This has been sorted out.
Thank you once again for your insightful input.
Have a great day
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