Why aren my peptides retained on the PolySULFOETHYL A column?
Your sample solvent or starting mobile phase contains TFA or HFBA. Use 5-10 mM K2HPO4, 0.1% formic acid, or 0.1% acetic acid instead. b) The salt concentration is > 30 mM. Dilute or dialyze your sample. c) You performed a tryptic digestion at pH 8 and haven’t adjusted the pH to 2.7-3.0. It’s easier to do this if you use NH4HCO3 as the trypsinization buffer instead of Tris. NOTE: If using a SpeedVac®*, it is necessary to take a sample to dryness 3x in succession in order to get rid of excess NH4HCO3. d) You performed an iTRAQ® derivatization and didn’t adjust the pH to 2.7-3.0 and desalt adequately. e) You overloaded the column by more than 6x the recommended maximum loading limit.
