Can Phosphate buffer (25mM, pH 2.6) and AcN mix on line?

We try to convert the same HPLC method to UPLC method. It appeared mixing online gave the better resolution for 6 impurities in 4 mins. If we premixed the mobile phase (phosphate buffer pH 2.6 and AcN 75%:25%, isocratic), we have to extend our run to 4.5 min at least. Is that possible we mix phosphate buffer and AcN on line? I haven't had problem during the method development when I take caucious cleaning the buffer line.

Answers

  • Can you do this? Of course.

    Is there a distinct possibility that you will get bacterial growth in the 100% phosphate buffer line at some time? Yes.

    I do not understand why a pre-mixed mobile phase yields longer retention times than online mixing.

    --Doug

  • Thanks! Maybe mixing on line is more accurate than premixed mobile phase by graduate cyclinder. When we did another UPLC method validation (no buffer), the retention time of premixed mobile phase is 0.2 minute more than mixing on line. We also experience the premixed buffer mobile phase has a negative peak at 1.5 min but I didn't see it in mixing on line. Should we always put 100% buffer or pre-mixed mobile phase on A1 or A2?

  • It should not matter which mobile phase you put into which line, so long as you keep track of where they are and make your gradient program carefully.

    You are wise to watch your phosphate buffer carefully for contamination. An occasional (weekly or monthly) flush with acetonitrile or methanol will kill most bugs that try to take up residence. Just a quick prime into organic and back should be sufficient. Swirling the bottle containing your buffer daily and looking for floaters is a very good practice. Once you see them, toss the buffer. Do not rely on the filter stones. If you have growth, you will have metabolites and ghost peaks.

    As to why you see a retention time difference between premix and online mixing may have to do with how you are premixing your mobile phase. If you were to take separately measured buffer and ACN at 75mL and 25mL and pour them together into a third graduated cyllinder, you would see that you had less than 100mL of combined liquid.

    This is because of a colligative property of ACN and water that is a little like pouring sand and golf balls together. The sand fills in between the golf balls.

    So if you measure first your Acetonitrile and add buffer to the mark of the same cyllinder, you will have more buffer in your premix than you may have in your online mix, and a slightly weaker mobile phase. Measure separately for best results.

    Matt B the FSE

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