Liquid chromatographic separation of basic analytes is often hampered by problems, such as insufficient retention, wide and asymmetric peaks, etc, especially if carried out at acidic pH. Many modern HPLC coulmns can stand pH values up to 10 without problems and separation at a higher pH is often a good approach for liquid chromatographic separation of basic analytes, such as different amines. Unfortunately, the choice of buffer systems in the range of pH 7-10 suitable for LC/ESI/MS work is very limited. The main requirement for the electrospray ionization mass spectrometry is that besides suitable pH all the components of the buffer system should be volatile. This immediately excludes phosphate, carbonate, borate, etc buffers. On the other hand, the buffer should not compete with the analytes in the electrospray ionization process. This excludes most organic bases. The ammonia/ammonium acetate or ammonia/ammonium formiate buffers are the most used ones. Nevertheless, also use of these buffers and mobile phase pf pH aroun 9 often results in less than ideal separation and peak shape. Thus new LC/MS-compatible volatile buffer systems would be highly welcome.

Karin Kipper from UT Institute of Chemistry has made a significant contribution in this field by introducing two new buffer compounds, two fluoroalcohols – 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFTB) (Journal of Chromatography A, 2011, 1218, 8175–8180). She has found that these compounds offer interesting possibilities to adjust retention behavior of different analytes by using high pH values (ammonia is used for pH adjustment) and expand the currently rather limited range of ESI-compatible buffer systems for basic mobile phases. Good separation of different compounds was observed with excellent peak shape. The fluoroalcohols did not suppress the ionization of the analytes in the electrospray source and for several analytes ionization enhancement was observed. Thus, these compounds are suitable for electrospray mass spectrometry.

All trends in retention of the acidic and basic analytes can be interpreted by the following model: the neutral fluoroalcohols are quite strongly retained by the stationary phase whereas their anions are less retained, thus their amount on the stationary phase is dependent on mobile phase pH; the anions of the fluoroalcohols form ion pairs in the mobile phase with the basic analytes; the fluoroalcohols on the stationary phase surface compete with acidic analytes thereby hindering their retention; the fluoroalcohols on the stationary phase bind basic analytes thereby favoring their retention.

The suitability of these fluoroalcohols as buffer components for LC/ESI/MS analysis has been verified on the example of simple organic compounds (bases, such as amines, pyridines, anilines) as well as several antibiotics – three fluoroquinolones: ciprofloxacin, norfloxacin, ofloxacin; and two sulfonamides: sulfadimethoxine, sulfamethoxazole (Analyst, 2011, 136, 4587–4594).

We hope that this novel approach will eventually gain popularity and we welcome any feedback on its practical use.


One Response

  1. I am very much impressed with work carried out on basic buffers
    As it would be a breakthrough if this type of buffers are marketed in
    Industries on trail basis with protocol to use buffers.
    I hope limitations with ammonia alone will be eliminated,
    Only concern is that phospolipid profiling should also be checked

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