Separation of peptides using off-line pH gradient-strong cation exchange chromatography

On the basis of theoretical pH calculation for buffer systems: an off-line linear pH gradient-strong cation exchange chromatographic method was developed for peptides separation. For the acetate buffer system, the peptides from tryptic digest of BSA were eluted by the linear pH gradient (pH 3. 7 -6. 0) with a low concentration of ammonium acetate salt gradient: whose salt is volatile and can be easily removed by lyophilization. As for the citrate buffer system, the peptides were eluted by a wider range of linear pH gradient (pH 3. 0 - 8. 5) with a even lower concentration of ammonium citrate salt gradient. Both methods are effective for the peptides separation and before mass spectrometric detection can simplify the desalting step; which is labor-intensive and may incur significant sample loss for the routine strong cation exchange chromatography.