26 Replies Latest reply on Feb 18, 2019 12:14 AM by dmarchini

    Phosphate buffer in HPLC



      since few months I am using phosphate buffer for my analysis (method from Agilent Application Solution "Analysis of water-soluble vitamins from multivitamin tablets"), I've never used it before. I have an Agilent 1260 HPLC binary pump and the column is Agilent poroshell EC-C18 2.7um, mobile phases are phosphate buffer pH 7 (channel A) and acetonitrile (channel B). I am looking for a washing protocol to prevent salt precipitation.

      This is what I did untill now, but I don't know where I did a mistake: the column pressure is still too high (more than 400bar with 99% H2o and 1% ACN instead of 270 bar, I've already try to restore the column with different washes,..), I have to change it (but is quite new, I've bought it last august..)

      -never stop analysis letting buffer in the system (es. overnight or during the weekend)

      -after analysis wash the entire system with colum with Water (HPLC grade) 90% + ACN 10% for 20 min, then return to 100% ACN

      Maybe 20 min is not enough?


      Thank you!