Please help me figuring out what is going on with my RP-HPLC protocol.

I am purifying a small peptide out of E.coli Culture, heat stable enterotoxin, its amino acid sequence is:


I am collecting culture supernatant and using amberlite XAD2 to concentrate and desalting the peptide in 80% and 99% methanol fractions. These fractions were subjected to flash evaporation. Then I am using on MCI gel CHP20P (glass column) for further concentration and removing unwanted proteins (polar and less hydrophobic ones) using stepwise elution with 20%, 40%, 60%, 80% and 100% methanol fractions. Each fraction is subjected to flash evaporation. A sample (1 ml) from each fraction (60, 80 and 100%) was then loaded to RP-HPLC using xbridge C8 preparative column (19x25 mm,10 micron, 130 angstrom) on a gradient system (solvent A: water/ solvent B: 80%methanol) initial-5 minutes 100 A, 5-10 minute 30% B, 10-65 minute 100% B, 65-80 minute 100% B at flow rate 2 ml/minute. The chromatogram showed a single large peak (3 AUF) at 25 minutes, B solvent around 45-50%.

I am wondering that the retention time is lower than that was reported before, 35 minute at 55-60% B (I followed almost the same protocol 5 years ago except using MCIgel methacrylate resin and Vaydc C8 column instead of mci gel CHP20P and xbridge C8 column). It is also very frustrating that I loaded 0.5 ml of the collected peak on Waters analytic C8 coulumn, 5 micron particle size and it elutes very early at 5-6 minutes (almost in the mobile phase A: water). My frustration comes from that I am using highly hydrophobic resin particle (MCI gel CHP20P) to capture hydrophobic molecules and loaded these fractions (60%, 80% and 100% methanol MCI gel) on C8 column and eluted so early.

I am sorry for this long message but I rely on your understanding. Any answer would be highly appreciated. Thank you in advance

  • 1
    $\begingroup$ "I am sorry for this long message" -- it would be much easier to read if it were in paragraphs $\endgroup$
    – jonsca
    Commented Jul 6, 2015 at 1:32
  • 1
    $\begingroup$ What evidence is there that the peaks you are seeing have anything to do with your target peptide? Do you have an activity assay for that peptide? That peptide has a lot of cysteines. Is its chromatographic behavior affected by inter- or intramolecular disulfide formation? How are you assessing the function of all the columns you are using? Do you have a standard mix for column capacity and separation factor estimation? $\endgroup$
    – Curt F.
    Commented Jul 6, 2015 at 3:23
  • $\begingroup$ Thank you for your response. I am using in vivo suckling mouse model to test its biological activity. I am just wondering how it comes that 60-100% methanol fractions from MCI gel CHP20P eluted very early on C8 column?! $\endgroup$
    – NAAN
    Commented Jul 6, 2015 at 13:51
  • $\begingroup$ Is there any reason why you're growing such a small peptide in culture instead of producing it through solid phase peptide synthesis? You'd have a lot less crap to remove and could probably use a simpler reverse phase purification strategy. And do you have access to LC-MS? You could directly test the mass of each peak and confirm it matches the peptide rather than rely on a suckling mouse model. $\endgroup$
    – user137
    Commented Jul 7, 2015 at 17:58
  • $\begingroup$ As far as I know that cystiene-rich peptide is a problematic on SPPS. Do you know trusted company for synthesis this peptide efficiently. Thank you $\endgroup$
    – NAAN
    Commented Jul 7, 2015 at 22:15

1 Answer 1


I am just wondering how it comes that 60-100% methanol fractions from MCI gel CHP20P eluted very early on C8 column?!

The gel you are using seems to be a porous divinyl benzene / styrene resin. The aromatic groups of the resin may interact more strongly with aromatic side chains on your molecule (e.g. Y residues) than does the aliphatic C8 groups of the reversed-phase HPLC column you are using.

"Hydrophobic" vs. "hydrophilic" is a very useful axis for understanding solute interactions with stationary phases, but unfortunately not everything can be collapsed into this single-variable scale.

Another possibility is that residual solvents or very large amounts of co-solutes are interfering with the RP-HPLC method you are using, causing premature elution. As an extreme example of what I mean, imagine if you injected a few μL of chloroform along with your sample in the RP-HPLC method. The chloroform would prevent column binding and cause premature elution. Now, it's unlikely your samples have chloroform, but they could have large amounts of methanol or other "solvent-like" impurities.

These are just general points; unfortunately I can't be sure that either is the precise source of your particular problem, without more (and more clearly presented) information on your former method from five years ago, your method today, and the differences in observed vs. expected results.

  • $\begingroup$ Actually, I have methanol in my loaded samples with varying concentration. I have 3 fractions from MCI gel CHP20P namely: 60%, 80% and 100% methanol. The last 2 fractions still have some methanol after flash evaporation (they are not turn solid upon freezing) but the first fraction did not. I have only one large peak (AUF=2+) on preparative C8 from the three fractions at the same retention time. $\endgroup$
    – NAAN
    Commented Jul 6, 2015 at 19:57
  • $\begingroup$ My previous protocol had the same steps, however I used MCI gel methacrylate based resin instead of divinyl benzene / styrene resin (MCI gel CHP20P). Thus actually I used AmberliteXAD2 followed by MCI gel methacrylate resin, then Vydac preparative C8 (300 Angstrom) column. The chromatogram showed some peaks at first then my target peak at RT 36 minutes B % 55. Thank you for your help $\endgroup$
    – NAAN
    Commented Jul 6, 2015 at 20:13
  • $\begingroup$ Another piece ofinformation from my previous protocol is that my target peptide was present mostly in the 60% MCI gel methacylate based resin fraction. $\endgroup$
    – NAAN
    Commented Jul 6, 2015 at 20:18

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