I am trying to purify a small peptide using reverse phase HPLC. The peptide is soluble in isopropanol and insoluble in water. A solution of peptide in isopropanol produces precipitates on addition of even 0.1 mL of water to it. The peptide is insoluble in acetonitrile, methanol and ethanol. In this conditions, what would be the appropriate mobile phase combination for the purification of peptide.
Use buffers like acetate buffer ,or 20 to 30 percent acetic acid, or phosphoric acid or phosphate buffer and actotnitrle as mobile phase.
Hi Bishnu,
Perhaps this article could be help for you (Neue and Méndez. Selectivity in reversed-phase separations: general influence of solvent type and mobile phase pH. J Sep Sci 2007;30:949-63. PMID: 17566327). Common solvent choices are acetonitrile, methanol, and tetrahydrofuran, but if your peptide is insoluble in acetonitrile and methanol you could try with THF, but is important to use in a low concentration 2% in order to avoid any damage on your HPLC equipment as Dr Jandirk Sendker commented when answering a question here in Researchgate.
Hi Maria,
Thank you for the paper and your feedback. Of course, this paper seems helpful. I will try with THF and see how it works.
Use isocratic 50;50 THF:Acetonitrile and before using this mobile phase in hplc, try to see the solubility of your peptide in 50;50 THF:Acetonitrile.
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