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May/June 2011
while providing reduced baseline noise (and hopefully additional buffering capacity to increase separation robustness). The reason for requiring a cleaner baseline is simply that many of these impurities can co-elute with real compound related impurities (which are often present at similar levels) making quantitation difficult.
High pH additive – UV performance A UV spectral overlay of two of the comparator mobile phase additives (0.1% (v/v) 1-methyl piperidine and 0.1% (v/v) TEA) with 0.1% (v/v) ammonium hydroxide is shown in Figure 7. The figure clearly shows that ammonium hydroxide has a much reduced absorbance at the lower end of the UV spectrum compared to the other two solutions. This was also observed for the other high pH mobile phase additives and illustrates that from a signal-to-noise and sensitivity perspective, ammonium hydroxide is a good choice as a mobile phase additive. Additionally, with these solutions and all the other mobile phase additives investigated, none gave superior sensitivity and at best only a similar baseline artefact count to ammonium hydroxide.
High pH additive – retention correlation Correlation coefficients for the test analyte retention in the ammonium hydroxide and the other high pH mobile phases were calculated and are listed in Table 5. Of the alternative mobile phases, only triethylamine (as acetate salt) gave similar retention to the baseline ammonium hydroxide method. Two values for this particular correlation are listed in Table 5. The first value (0.8290) is for all 33 test analytes. The second value is the correlation from 32 analytes as one data point exhibited much reduced retention (over 3 minutes less retention) in the triethylamine acetate mobile phase which significantly skewed the correlation. No reason can be attributed for this observation at this time. Significant deviations from linearity were observed for all other mobile phases. An example comparison of one of the test analyte sets with 10 mM triethylamine as acetate salt and 0.1% ammonium hydroxide is shown in Figure 8.
After triethylamine acetate, CAPS provided the closest retention correlation to ammonium hydroxide, but exhibited poorer analyte sensitivity and peak shape. While the baseline artefact count for CAPS was equivalent to ammonium hydroxide, CAPS is not MS compatible and is therefore a poorer choice for SIM development. This was a similar finding for all other high pH mobile phases tested.
The findings of this study suggest that from a
Figure 7. Comparison of some UV spectra for high pH buffers investigated in this study. Ammonium hydroxide is the additive used as the reference mobile phase to which all other high pH systems were compared.
Figure 8. Comparison of analyte response in 0.1% ammonium hydroxide (black line) with 0.1% triethylamine acetate (pH 10.6 – blue line). While triethylamine acetate provided the most similar orthogonality to ammonium hydroxide and a slight improvement in baseline artefacts, this mobile phase gave a much lower analyte response and generally poorer peak shape for the test analytes.
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