Hi there,

I finally have optimised my run for GFC using PBS as buffer. My protein, however, produced differently-shaped peak after 2 runs but still the same retention time. Somehow, the second chromatogram has a 'tail' and the peak base is wider. Furthermore, the peak height is shorter. What could possibly cause that? And is the addition of a metal ion causing anything to a protein with a his-tag attached?

Thank you

There are several possibilities. First, did you filter your sample, or centrifuge it, before injection? Particulates or precipitates can plug the inlet frit on the column, or the column itself, causing peak shape problems.

One other possible problem is that some protein material has deposited on your column. If it was less soluble in the mobile phase, then perhaps it precipitated inside the column, and is now affecting the surface characteristics.

Do you have a simple test compound that you could inject? Any small molecule that provides a detector response would be acceptable. If you get good peak shape from the test compound, then your column frit is probably OK. If not, then you have a serious column problem.

You could also try flushing the column with the same surfactant used to dissolve your sample.

You had a question about the presence of a metal in the system. What metal were you referring to? The sodium in the PBS will not cause any complexation problems. Is there something else?