UPLC compatible ion pair reagents

Hi,I'm a new user to UPLC and am seprating a mixture of water-soluble bases and organic soluble compounds. I've seen some lit. papers using HPLC and hexafluorpropanol or heptafluorobutyric acid, but I'm not sure of they're appropriately compatable with my UPLC (analysis @280nm). Does a list exist of UPLC compatible ion pair reagents and would anyone have experience with relative retention of basic compounds (pKa~9.9) using them, (i.e. more or less retentive than TFA?).Thanks

Answers

  • Hello:

    If you are doing a search for HPLC methods, so you have the opportunity not to make the mistakes of the past. The BEH small particle columns are very robust and pH rugged. May we suggest that you start trying to develop the method at high pH without the ion-pairing agents first, because that mode of separation always adds complications.

    You can run ammonium bicarbonate buffer @ pH 11 (should be far enough above the pKa) and see what the retention and peak shape look like. Once you get up to pH 11, I wouldn't have him run the temp any higher than 40 degree C or the column lifetime may start to be affected.

    However, to answer your real question the system is compatible with those ion-pair agents.

    Hope you do not mind the advice, best regards and luck with the method.

    Liz and the ACQUITY Team

  • Thanks for the suggestion, Liz. One question: don't bicarb buffers suffer from pH decrease over time due to formation/build up of carbonic acid?

    I will try your suggestion and report back.

  • Hello

    Still no answer on your question about bicarb buffers please bear with me.

    Liz

  • Over time, bicarbonate buffers can adsorb carbon dioxide from the air, causing the pH to slowly shift towards neutral. This reaction, however, is somewhat slow and depending on the concentration of the buffer, may change by 0.5-1 pH units over the course of 2 weeks. The magnitude of the impact this may have on your assay will be dependent on the sensitivity of the analytes to this change. It is therefore recommended that a fresh buffer is prepared on a weekly basis to avoid any shifts in retention time and maintain the integrity of the assay.

  • Hi,

    I would like to recommend that you avoid the use of ion-pair reagents on your Aquity system. We used methods very similar to those that you have mentioned......the methods worked really well, but unfortunately left us with severe contamination problems that took quite a while to resolve.

    Kind Regards,

    Jonathan

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