Switching over to a different mobile phase (method)

<p><span>Well I've decided to try using methanol instead of ACN on one of my methods to cut down on ACN use. </span></p><p></p><p><span>After changing the gradient I got decent chromatography though peak area is less. Anyway the question is what is the proper procedure when switching over from one mobile phase method to another? </span></p><p></p><p><span>We have a column manager that holds 4 columns. When I switched back to a method using ammonium acetate/ACN after running ammonium acetate/methanol the pressure climbed to 9000 psi before it came down to the usual 4000. It never use to do that. And that was using different columns. Should I do a prime before starting up different mobile phase methods? </span></p><p></p><p><span>TIA</span></p><p></p><p><span>Mike</span></p>


  • HI Mike,

    If you are using Empower, try setting up the sample set method with a wet prime for 3-5 minutes and a column equilibration for 1-2 minutes (pending the flow rate). The rise in pressure is due to the sharp differences in composition during the change and the residual MeOH in the column as well as in the system.

  • Thanks. We are using Masslynx. This time I primed the mobile phase before equilibrating the column and that helped.


  • Greetings MikeT (and everyone else),

    The one concept that we all have to remember during this temporary (possibly permanent) transition back to methanol is that the azeotropes of methanol and almost anything else will be more viscous than the azeotropes of acetonitrile. Viscosity translates into back pressure (all other parameters equal). I promise to post the viscosity curves of water/ACN vs. water/MeOH in the near future. What I can say, extemporaneously, is that the maximum viscosity for water/ACN is very close to 80/20 and the maximum viscosity for water/MeOH is 50/50 +/-5%. The methanol azeotropes are more viscous by a significant amount. So, as a system translates through the maximum viscosity of methanol and water (or a salt solution) pressure will climb to values never previously experienced in the acetonitrile world. The truth is, viscosity is disadvantageous with regards to the mass transfer equations that, mathematically, define chromatographic separations. This is why chromatographic practice migrated to acetonitrile over the past decade (or so). Peaks are sharper and secondary solvent effects such as UV background are reduced with the use of acetonitrile. One tip is that higher system (column) temperature will reduce viscosity effects. So, if your ACN applications ran at, oh say, 40 degrees C, consider running the methanol versions at 50 degrees C. You will get closer to your, original. pressure regime and you will be able to back off on the methanol composition. MikeT, I am a little concerned about you observation concerning the reduction in peak areas. Areas should be conserved (+/- 5%). If your areas are significantly less than 5% of their previous values, I would be concerned. When you switched over to methanol did you also characterize your system volumes? If not, you should. The only other explanation for reduced areas involves the detector and the selection of the, monitoring, wavelength.

    Best regards,


  • Thank you very much for the information.

    RE peak area no I did not characterize system volumes for methanol. Would I have to do this again after changing back to an ACN method? Is it possible each method could have its own characterized volumes? I am using Masslynx.

    The detector is a QQQ (TQ).

    I've been thinking of why else I have less peak area. One is the column. Rather than use the same column I've been using I pulled an old BEH C18 that was "retired" and sitting in a drawer for the last 8 months or so. I can't even remember why it was removed. I've since replaced it with a BEH C8 to see what kind of chromatography I get with that but haven't had a chance to try it yet.

    A second issue might have to do with the analyte itself - phosphatidylglycerol. It has been a very challenging thing to chromatograph as it has a polar end and a hydrophobic FA end. Maybe it doesn't like MEOH and I'm loosing it somewhere. I've tried getting help with this method (extraction and MS) but apparently I'm the first to try it and frankly no one from Waters knows much about lipids. Most of the LC/MS world is about proteins or pharmaceuticals and they want to get rid of phospholipids, not measure them.



  • Greetings Mike,

    Yes, we would always recommend that the system volumes be re-characterized when the solvent system (especially the weak wash solvent) is, significantly, changed. What is "significant"? Well, changing from one organic solvent to another (ACN to MeOH) would be considered significant. Also, large changes in composition of the weak wash solvent (say greater than +/- 10% organic) would also suggest that a re-characterization should be considered. Accurate system volume characterization is, somewhat, dependant upon weak wash solvent viscosity. Viscosity is dependant upon the type and concentration of organic solvent employed.

    It does not matter which Control Data System (CDS) is being utilized. The system volume characterization is accomplished from the ACQUITY Console which is available through either MassLynx or Empower. It is under the Maintain menu on the Sample Manager page.

    I am by no means a Mass Spec expert, but I seem to remember that methanol has a tendency to supress ionization when compared to acetonitrile. It was my understanding that this fact, along with the lower viscosity of acetonitrile, was a motivating force towards the adoption of acetonitrile as the, dominant, organic solvent in reverse phase chromatography. If this is true, it could explain your reduction in area. At a first approximation, it is unlikely that your column is the cause of the reduced area. All other things equal, peak areas should be conserved. That is, an older column might cause your peaks to be shorter and broader, but the areas (integration parameters not withstanding) should be about the same.

    I would re-characterize your volumes, with the methanol based solvent system, and investigate the potential ionization suppression of methanol to explain your loss of area.

    I wish I could be of more assistance with the Lipids questions, but there are others here who are far more knowledgeable on the subject than I. If they have not been able to advise you, I, certainly, will be of little help.

    Anyway, hope this helps,


  • Note to myself.

    Well I've tried a C8 column and while peak areas did increase they are still a tenth of what I was getting using ACN. I do not think it is a column issue. I think it must be the detector. I don't think I'm getting the same amount of ionization with MEOH as with ACN. Though I still haven't characterize system volumes for methanol I can't believe that would account for so much loss.

    I could try reducing the amount of reconstituent.

    I don't know what else to do. Ordered a 4 liter bottle of ACN a month ago (trying not to horde), still haven't received any.


  • Greetings to all,

    In my experience (pharmaceutical discovery), changes of ACN to MeOH give significant differences in the selectivity of the analites. Mike: have you seen differences also in UV signal? May be with one organic solvent there are coelution of analites (impurities,...) which don´t occur with the other solvent? This could explain the differences you observe in the peaks areas

    kind regards


  • MikeT

    I am using a TQD, not UV. I am assuming MeOH is not as volatile as ACN so I am not getting very efficient ionization. I am using neg ESI (ph10) so I can't add FA. I just thought of using a 50:50 solution of MeOH to ACN for my organic phase. Maybe that would give me better ionization.


  • mjvw


    I use 75:25 MeOH:ACN, I choose larger volume of MeOH after talking to an application chemist, I was told to use a larger volume of MeOH if I want to increase sensitivity and a larger volume of ACN for greater peek shape. I choose MeOH as the analytes in my last MRM dont ionise as well as others and sensitivity was more important than peek shape. Hope this is of help to you even though it contradicts what you found.

    What additives are you using?? I know that Acetic acid is good for negative mode but formic acid is not, it kills the signal. Play around with your source temp ect. we found increasing these resulted in increase in sensitivity.