IEX-HPLC for peptide mapping

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Diablo
Diablo's picture
IEX-HPLC for peptide mapping

Hi folks,

Im thinking about Ion-exchange (IEX) Chromatographie for a peptide map (tryptic digest). Is there any general rule with what kind of column (anion/kation exchanger) one should start?

greets
diablo

Dr. Analytical
Dr. Analytical's picture
You have several choices for

You have several choices for separating proteins and peptides. Ion exchange is one option. Your choice of pH will determine which type of ion exchange column that you should select. And this choice will depend on the pI value for your proteins - at lower pH, the protein has a positive charge, and cation exchange is the best choice. At high pH use an anion exchange column. The only problem with ion exchange is that the separations are slow and the efficiency of separation is low by modern HPLC standards.

There are many reversed phase columns that should also provide the separation that you need. Most column manufacturers offer several choices. Look for a C4 column with 300 A pore size as a good starting point. Use a mobile phase with 0.1 % TFA and varying amounts of acetonitrile. Your resolution should be much better on these columns compared to the ion exchange versions.

Write back if you have more questions.

Diablo
Diablo's picture
Thx, for your answer !

Thx, for your answer !

hm, yes i know that reverse phase chromatography seems to be the standard approach for HPLC peptide mapping, but i was thinking about IEX as an alternative...

Do you think that the resolution of an IEX peptide map can be as good as the resolution of an reverse phase peptide map or even better/worse ?