Chromatography Forum

hi What would be a proper mobile phase for the SEC analysis of protamine sulfate?

why SEC for such a small molecule?
it is a small peptide

anyway you can use a very small pore size column like Zenix 80 from sepax
conditions: 70%ACN, water 30% 0.1% TFA

70% ACN? Wouldn't that put you in the hydrophilic interaction chromatography mode? In that case the order of elution would be smallest to largest, not the largest to smallest that's typical of SEC, assuming that one was talking about analytes with varying numbers of identical subunits.

There is a method described in J. Pharm. Biomed. Anal. 1998, 18, 907, Snycerski A, Dudkiewicz-Wilczynska J, Tautt J: The assay was carried out on a diol-type column with a mobile phase containing 8% acetonitrile in 0.15% trifluoroacetic acid at pH 2.5. The flow rate was 1.0 ml/min.
You can use HMWP column, 7.8 mm X 300 mm (Waters) for this assay.

If you want to study purity of protamine sulfate there is a better method described here: J. Pharm. Biomed. Anal. 2005, 37, 551, Determination of protamine peptides in insulin drug products using reversed phase high performance liquid chromatography, Hvass A, Skelbaek-Pedersen B.

Yeah, 8% ACN is a completely different story than 70%. I would expect some kind of HILIC interactions from such column with 70% ACN.

Best regards