I am facing difficulties in purifying Histidine based peptide by HPLC, I tried gradient method with C-18 Column and mobile phase condition 10 % ACN water to 90 % ACN water system any suggestions would be appreciated.
Thank you
The most selective way to purify His containing peptides is
Immobilized-metal affinity chromatography, when iminodiacetic acid bonded phases loaded with copper can specifically retain His containing peptides from very complex mixtures.
I'm sorry, but I don't see enough information in your question for us to be able to diagnose the problem and give a simple answer.
- Do you see the peptide at all? That is a wide gradient range (10 to 90% acetonitrile). It should come off in that range, though I would consider going to 100% acetonitrile- it won't harm a C18 column.
- Related to above, how are you detecting the peptide? If UV, what wavelength(s) are you using? Make sure that is set correctly.
- If the peptide comes off the column, is the problem because the peak is broad? Is it eluting with other compounds?
- I'm assuming C18 chromatography since that is most commonly used in HPLC and and also from the direction of your gradient. It is possible to purify peptides with HILIC with those solvents, but going from 10% to 90% acetonitrile suggests reverse phase.
The one thing I do notice is that there is no modifier listed in the mobile phase in the question. The pKa of the imidazole group conjugate acid is pH 6. A small difference in mobile phase pH can cause differences in chromatography. I find most water seems to be at that pH (even fresh deionized water). I suggest that, if the peptide is stable in acid, adding trifluoroacetic acid ( TFA, 0.1% usually works).
Do you have any additives in your mobile phase? Addition of 0.1% formic acid in both your water and acetonitrile solvents can dramatically improve peak shape and resolution. If addition of formic acid doesn't provide the requisite improvement, try using trifluoroacetic acid (0.1%) in both solvents.
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