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Phosphate buffer_part2_again problems!

Question asked by anto on Mar 25, 2019
Latest reply on Mar 29, 2019 by solyom

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......