Peptide Emulsion - UPLC?

<p><span>Hello all,</span></p><p style="height: 8pt"/><p><span>I am working on a sample which is comprised of a very hydrophobic peptide in an emulsion (oil:water:detergent mixture). There's about 1% oil (olive or castor oil) and 1% detergent (polysorbate 80/tween 80) with some other additives as well. I am trying to develop a fairly rapid UPLC method for assay and impurity (related peptides, degraded peptides) analysis. If I simply dilute the sample in organic and inject onto a BEH C18 column, the separation is initially pretty good, but the resolution quickly diminishes with time. I guess all the lipid and detergent fouls the column. If I have to add extensive column washing and/or sample preparation steps, I might as well go back to the older HPLC method that we have. Anyone have any suggestions or experiences? Thanks for your help.</span></p><p style="height: 8pt"/><p><span>Steve</span></p>

Answers

  • Hi

    There are several possibilities to consider, but I would like to have some more detailed information before proposing any testing experiments, possible causes, or solutions. In your older HPLC method, what kind of column do you use? Is it a C18? Do you know anything about the underlying surface chemistry? What are the sizes of the HPLC and the UPLC columns that you tried? When you dilute the sample before analysis on the BEH C18, do you dilute in the same way with the same solvent as with your traditional HPLC method? How does the injection volume on the HPLC compare to that used on the UPLC? How does the actual equivalent sample amount injected on the two columns compare. In your ACQUITY UPLC injector method, there is provision for a strong and a weak needle wash. What solvents did you use for those two washes and how much volume did you specify for each? What ACQUITY injection mode did you choose? What kind of injector d you use on your HPLC? Could I please know the details of both your HPLC and your UPLC methods? We find that we have to be particularly careful when transferring methods to use the same solvent profile. While discrepancies arise in the separation part of the method, we also pay close attention to the number of column volumes used for column regeneration and for column equilibration. I am also particularly curious about the kind of column heater and the temperature used for the HPLC method. Did you also use the column stabilizer in the UPLC heater? Finally, I don't want to assume that you use low wavelength UV detection. What detection do you use?

    I will try to check back to see if you have had a chance to answer And to see what suggestions and thoughts I may have.

    Thanks.

    Tom

  • Hi Tom.

    Thanks for your interest, you certainly have a lot of questions! Do you have any general comments? I don't wish to get into great detail on this forum, is there any way for me to answer your questions privately? It will take me a while to go back in my notes and answer all those questions anyway. On a general note, I'm not worried about transferring an HPLC method directly to UPLC exactly. I just want to see if I can develop a UPLC method, whether or not it is similar to the HPLC method.

    Thanks for you help.

    Steve

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