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

  • Hi Anto,


    Are you certain that the high pressure is coming from the column?  There could just be a blockage in the needle/needle seat.

  • Hi Abdur,

    nice to ear you!


    I’m quite sure that the problem is in the column. Here what I did before say thist statement:

    -when I saw that the pressure was getting higher and higher, after had washed the column, I’ve change the frit and the golden seal cap

    -after that I had a problem with the purge valve: it leaked but the pressure was really high (500bar)

    -I’ve removed the column and put the union part: with a 1 ml/min flow 100% MeOH the pressure was 5-6 bar

    - I’ve switched several times mainpass-bypass: no differences

    - then I tried to wash the column, first of all with milliQ water 50°C, then I change the flow of the column and wash all the system for all weekend long with 95% h2O-5% ACN 0.1 ml/min, the pressure fallen down from 300 bar to 120bar

    - I did all the washes with no collection from column to flow cell

    Now the instrument is working with another column (Agilent C18 zorbax) with ACN and 0.1% formic acid: with 0.5 ml/min flow is 30 bar, chromatogram is ok


    What do you suggest to me?

    1. I’ve ordered another column, but I’m so sad.. and think my boss isn’t so happy for this disaster L




  • Hi Anto,


    After preparing your phosphate buffer, do you then filter the buffer before using it?

    As long as the buffer is continuously moving in the system you should be ok.  Then if you need to turn the system off, you should make sure to wash with a mixture of water.

    You can try to reverse the flow throw the column, by flipping the column in the other direction to see if what is in there can come out the other end.

    You might also try looking into a guard column. 

  • Yes, I filter the buffer with 0.45um sartorius filter and i prepar it fresh every day.

    I reverse the flow by changing the direction of the column and let wash for 36 h in 95% water and then by gradient to 100% acn but the pressure still remain high


    How is the best way to wash if I have to turn off the instrument?


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