is my peptide retaining in the column?

[vijayak]

Hi all,
I am using phenomenax Rezex ROA H+ ion column for analysing acids in my fermentation samples. The stationary phase of the column is polysterene divenyl benzene. I have peptide compund(98% pure) wherein we are using H3PO4 while purification.So, using this column and RI detector i have made a method for quantifying H3PO4. The standard peak comes clean and i have H3PO4 peak in sample also but now my worry is, i prepared 5mg/ml of the peptide sample in 0.089N H2So4 and injected in to my method. I am getting only H3PO4 peak in chromatogram and there are no other peaks. Is the peptide retaining in the column? or it doesnt bind in to my column?
I think RI will give response to whatever we inject even if the peptide dosent get bound to the column.
I am confused and worried how i can check whether peptide is retaining or not? Kindly help me if you have any suggestion.
regards
vijayak

[tom jupille]

I suspect that your peptide is irreversibly retained.

A bit of explanation about that column and it's mechanism is necessary here. What you are doing with phosphate is classed as "ion exclusion chromatography", where the analyte (phosphate) does not enter into the packing because it has the same charge (as a gross oversimplification, "like charges repel"). At pH 1 (your mobile phase), phosphate (with a pKa of 2.1) should elute shortly after the total exclusion volume. Unless you have a *very* unusual peptide, it will be positively charged at that pH and probably bind to the cation exchange resin.

[vijayak]

Hi tom,
Thank you, as you said the OPA elutes excatly after the column volume. Assuming my peptide got bound to column, if i increase the concentration of H2So4 in my mobile phase from 0.089N to 0.25N(some thing like that) will it help in pulling out the peptide from my colum?.

[tom jupille]

H+ is a relatively weak driving ion, and your peptide is probably bound both by cation exchange and by hydrophobicity. You can always try; it won't hurt the column, but I wouldn't count on it working. Possibly the higher acid concentration plus 10% or so acetonitrile might work better, but again, no guarantees.