30 Replies Latest reply on Feb 20, 2019 5:44 AM by anto

    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!




      Hi guys!

      i'm still here hope you can help me. i really don't know what I'm doing wrong.

      I have a new column (see previous part) and after more or less 40 runs it shows higher pressure: pressure rises from 300 bar (70%acn 0.5 ml/min) to 450 bar same conditions.

      Here what I did (I've used your suggestions)

      - prepare daily fresh phosphate buffer [for 0.5 ml of buffer, 0.6634g KH2PO4 (mw 136.08g/mol) + 1.328g K2HPO4 (mw 174.2 g/mol)], magnetic stirrer for 15 min, vacuum filtration 0.22um.

      - sample filtration with 0.22um

      - for cleaning the colum: I've created a method with these conditions (ACN & HPLC water)

           0 --> 60 min 15% ACN 0.1 ml/min

           0--> 7 hours slow gradient to 100% ACN with 30 min step (each step 5% ACN more)

           other 60 min at 100% acn 0.2 ml/min

      at the end the pressure was ok (less than 300 bar) but when I check the initial condition (70% acn 0.5 ml/min) the pressure became higher


      Where is the mistake??

      What should I do now? wash with isopropanol 100%? change flow? the column is too new......



      Messaggio modificato da ANTONELLA GALEONE