I am trying to isolate an active compound from an HP20 water flow fraction by HPLC. This compound is not bound in HP20 resin or an SPE (solid phase extraction cartridge). When I inject this sample onto HPLC, the active compound eluted at near 5 minutes and other solvents also eluted and some other peaks. So at least 4-7 peaks eluted at the near the same retention time. This is a big problem for me because I can't isolate my compound in this situation. I used an Aqueous c18 column and my mobile phase is water (75%) and Methanol (25%). My sample is only water-soluble. Please give me any suggestions regarding this. This is my PhD research problem and very important to me.
Reduce your methanol content of the mobile phase to 5% or less to allow for more retention and longer retention time.
As your compounds are soluble in water you can try using a HILIC column instead of a C18. Don't forget to match your injection solvent with the initial mobile phase of the gradient.
Thank you Mr. Nelson and Mr. Paul for your nice suggestions.
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