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

  • I'm working for 2 years with this column, according to this Application Note, water soluble vitamins with much more troublesome matrix: animal feed premixes.

    I've modified it to 50 mM (even more concentrated, than Apllication Note, it is not very good for column in general) Sodium Phosphate pH=7, I have to because of my extraction process.

    It turned out impossible to prepare a buffer with sodium phosphate salts and to dissolve them fully. Maybe I mixed not long enough, but magnetic stirrer for 2 hours seems quiet enough, maybe my salts were not quality enough. Even after filtration it was clogging the column.

    I prepare it with NaOH (50%) solution and H3PO4 (85%) with no problem since then. I recommend for that ChemBuddy BufferMaker software if you have troubles with general chemistry calculations or just as lazy as I am.


    I would suggest you 30 ml each solution on a reversed column (0.1-0.4 ml per minute, you can't go 0.4 ml/min with viscous IPA and DMSO below 400 bar): start with acetonitrile-water (50:50), 1% H3PO4 washing, 3% H2O2 washing, DMSO washing, 0,5 g/l EDTA in 3% NH4OH (I have lots of inorganic salts in animal feed premixes), IPA-Hexane-IPA (you absolutely have to go with IPA, hexane is immiscible in the system with no IPA), 3% HCl washing, finish with acetonitrile-water (50:50) as I did it myself with 3 Agilent Poroshell columns EC-C18 2.7um 150mm 3mm with a guard 5mm 3mm 2.7um and it helped me with my matrix and didn't ruined all this 3 columns. But you should not do it regularly, if you have to, you should run a better sample preparation. You should try a solution or a set which is best dissolving your clogging material, there is no silver bullet, you should know your sample best.

    But the columns are pretty reliable as I see now.

  • Hi, if you can pump liquid through the column, the column is not dead yet anyway. Crystallization of buffer may crush sorbent particles in it, but may not. If the column is fully blocked, you should place it in some rinsing solution for some time, hoping the blockage is somewhere near the frit and it will be dissolved by diffusion of rinsing solution.

    The best option is to always have a columns stock for your supply time.

  • Hi! It seems the column with 2.7 um particles has same good old 2 um frits, just as most 5 um columns:

    see page 5:

    and page 29:

    So it is most likely not a solution, as I did filtered with both 0.45 um and 0.2 um when I had issues with this method and it was no difference in clogging.

  • This may be the problem - 0.45µ filters are not quite adequate for sub 5µ column packings (which you are using).

    Try 0.22µ filters for the buffer and samples. This should at least slow down the clogging of your next column.


    Also, when you clean out your columns with 5% ACN in water, do you turn them around so flow is backwards? This helps speed up the cleaning process sometimes.

  • Hello,


    Filter your buffer with 0.45 um and wash your column with water for 2 hours to get remove the salt from column, gradually wash column  with  water and acetonitrile,( likewise, 90.0 % water , 10.0 % Acetonitrile increase the Acetonitrile percentage and decrease  the water percentage )and last wash the column through 100% Acetonitrile for 30 min.

  • How are you filtering your samples?  A slow build up in pressure might be due to particles in the sample.

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