I want to try basic conditions for LCMS in negative mode to try to satisfy the different pKa values of my 5 analytes which gear towards acidic and basic. pH5 is fine but the basic analytes can resolve better if I try to ionise them to see if I can sacrifice the performance of my resolution or acidic analytes. What column material have you tried in HILIC mode chromatography. I wanted to try charged phases but will only do cation or anion exchange. neutral phases not applicable. I am worried if that pH will degrade the material. Such as pH 6.5-8. Thank you for your support.
I read that HILIC columns are not so sensitive to pH change but the production HILIC materials in columns is more difficult to attach the hydrophilic ligands.
J Chromatogr Sci. 2013 Aug;51(7):684-93. doi: 10.1093/chromsci/bmt015. Epub 2013 Mar 13.
Main interactions and influences of the chromatographic parameters in HILIC separations.
Greco G1, Letzel T.
I am also wondering which pH range is most suitable for acidic analyte +ve mode ionisation and which pH range for negative mode ionisation for electrospray ionisation and atmospheric pressure APCI ionisation.
The pH of 6.5 to 8 is a grey zone in silica based HILIC. Don't worry too much about pKas in HILIC for screening purposes (for the time being). The reason is that HILIC mode is multi-modal, i.e. there are so many interactions which are responsible for retaining the analytes. Do a screening test with a typical ACN / ammonium acetate pair (85% ACN) and see what the separation looks like for your individual standards. Buffer conc. change also helps in increasing retention. You did mention what is your current problem? Are the analytes not retaining enough or it is too long of a retention. What is the nature of your compounds? Bare silica is also a decent HILIC material, it will retain acidic, basic, and neutral compounds (mostly small molecules). If the ligands are bonded, pH 8 would destroy the column.
I am analysing small molecules. Using that pH may show improvement in the resolution and the ligands are bonded so beyond pH 6.5 might be problematic. There was not much difference in resolution after running 5-15mM buffer concentrations; after best pH established with no difference from pH 4 to pH 6.5.
It could just be a column selectivity issue, i.e. the given column cannot resolve that pair easily. Bare silica can offer different selectivity with respect to other HILIC columns esp. with small acids. What is you current column chemistry? We have done comparative charts (grouping) of 33 HILIC/ RPLC columns. The whole text is available on RG.
http://pubs.rsc.org/en/content/articlelanding/2016/ay/c6ay01246a/unauth#!divAbstract
Another thing is that a you can try buffer conc. upto 25 mM (total). The more the buffer, the more retention you will see in general.
I echo Farooq's answer, with a short comment. I have not had much success affecting resolution with pH in the ranges that you are talking about. However, being slightly above the pKb of your compound will radically alter your negative ion responses so from that standpoint it is definitely worth a shot. Silica gets really iffy above a pH of around 8 so be aware that you may trash your column.
From an ESI+ standpoint I prefer working in the formate buffer range (pH around 3); plus, the ammonium ion will sometimes give you a nice ammonia adduct to play with. APCI is not as sensitive, in my experience, to the pH since the ionization mechanism is not dependent upon solution charge state. For ESI- I typically try to work around a pH of 7.5-8.5, but I use RPLC that is stable in that range (like an aqueous-type phase). I do almost exclusively small molecule work, and I have found over time that I get better results using high aqueous phases than I do with HILIC.
High pH (>pH 9) mobile phases have proven useful for efficient retention and resolution of ionizable compounds especially in combination with negative MS. I suggest you look up studies by
David Watson et al. at Strathclyde, UK.
Journal of Chromatography A, 1362 (2014) 168–179
You may also look at the study by Lucy et al.
Journal of Chromatography A, 1458 (2016) 82–89
and Teleki and co-workers
Analytical Biochemistry 475 (2015) 4–13
You will need to work with a basematerial that is pH stable at high pH and here is the polymeric phases recommended (keep in mind though that a polymeric particle is more sensitive to over-pressure and you should make sure not to work at pressures over 200 bar). You should also keep in mind that charged phases are pH dependent and they will alter their behavior with pH, thus not only your ionizable analytes. As a recommendation for buffer salt, a carbonate system will provide better buffer capacity than using only ammonia/ammonium hydroxide but both have been used in the literature.
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