Problems with eluting peptide

[chichibabin]

Dear all,
I am currently trying to purify a 6kDa peptide which has poor aqueous solubility. I am using C12 Phenomenex Jupiter Proteo columns with water:Acn 0.1% TFA as mobile phase. The sample buffer is 20mM phosphate, 1mM EDTA, 5M urea, pH 6. The problem I am having is that after the peptide elutes the UV absorbance stabilises but does not return to baseline level (it's higher) suggesting the peptide is still slowly desorbing. If I perform a blank run afterwards the peptide elutes again with the same retention time and the UV absorbance is almost as strong as the initial run. Several blanks need to be performed to desorb the protein fully. Running a 95% Acn wash has no effect on desorbing the peptide. It seems that the peptide only desorbs at a an extrememly critical mobile phase composition and only does so partially at that. I have not had this problem before so was wondering if anyone had any suggestions. I was thinking of changing to a differnt organic modifier such as 50:50 Acn:isopropanol??
Cheers
Sat

[tom jupille]

In what is the peptide soluble? If you have better solublity in IPA than in ACN, then a switch in B solvent may help. Also, would the addition of detergent help solubility?

[HW Mueller]

What´s the reason for your sample solvent? Seems you should use that for the chromatography, maybe with some organic modifyer (chosen on the basis of what Tom said). I am not sure about small peptides, but most proteins are precipitated very well with high % ACN. You could "cement" the peptide to the column with that 95% ACN.

[ivanvins]

Such behaviour is not uncommon for proteins in reverse phase - they are eluted within extremely narrow composition window - just a few percent of organic may give the difference between total retention, elution and precipitation on column. With a long column and fast gradient, the protein may not have a chance to elute fully from the column before it is precipitated. Shorter column, longer gradient time or shellow gradient in the concentration range the peptide elutes may help.

[ivanvins]

Just another idea - in case you are starting your gradient at low organic concetration, the peptide may be precipitaded at the top of the column upon injection. Once precipitated, it may be difficult to dissolve it again. Try to mix the sample with the starting eluent and observe for precipitation - if it is the case, you may try to start gradient from higher organic concentration

[chichibabin]

Thanks for the response. This morning I was surprised to have the same problem with a different column which has not had the sample run through it. It therefore seems that the peptide, and maybe urea, is precipitating in the sample loop. I am using the mentioned sample buffer as this is what is required to elute the peptide initially using an FPLC based purification strategy. I could start the gradient at a higher organic concentration but then risk urea precipitation. I suppose the next thing to try is to perform buffer exchange in water-0.1% TFA and hope the peptide is soluble or a water/Acn-0.1% TFA mix.

[chichibabin]

Would it be safe to assume that my sample has precipitated in the injection valve and/or sample loop? If so does anyone have any suggestions as to how to clean the system? Is flushng with 95% Acn a good idea or is it likely to cement peptide to the column?
Cheers,
Sat

[Kostas Petritis]

If you are not interested in recovering the peptide, I do not see why connecting any column during cleaning. Also, are their any metal parts on your injection system? If you do not know the properties of your peptide/protein, maybe it could have high affinity for your metal parts of your system creating the problems you observe...

I would suggest several gradients from aqueous to organic and an injection of diluted phosphoric acid to try to desorb your peptide. Again, no need to use a chromatographic column during cleaning...

[HW Mueller]

If urea precipitated you would likely have flow stoppage. Again, it would be interesting to know what you could do with a mobile phase including buffered urea and organic modifyer. If you followed other chains you might have noted that many of us would use TFA as a last resort only.