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May/June 2011
phosphate buffers is shown in Table 3. Correlation coefficients for the low and mid- pH data demonstrate excellent correlation (see Figures 4 and 5 for the low and mid-pH plots respectively). However, the absolute retention times between the two methods can vary. In the largest variation observed (1.18 and 1.20 min under method 1 and 2 conditions respectively), this equates to approximately an 11% deviation in retention over the length of the whole analysis.
The lower pH formic acid buffer demonstrates a larger maximum retention range variation than the pH 6.8 systems. This is most often observed with early eluting polar or charged analytes. This is due to significant ion-pairing of basic analytes with the phosphate counter- ion leading to longer retention. For acidic and neutral analytes (18 out of 33 compounds) at pH 2.6 where no ion-pairing with phosphate would be anticipated to occur, near perfect correlation of retention was observed (r2 0.9997) supporting this conclusion.
=
In the low pH mobile phase comparison, only two analytes (from all 33 analytes analysed) were found to change elution order when switching from formic acid to potassium dihydrogen phosphate. At pH 6.8, no changes in elution order were observed and the observed retention correlation is superior to that at low pH. As may be expected, ion pairing effects for the basic analytes were much less pronounced at this higher pH, possibly due to less ionisation of the bases under these conditions. Again, this reinforces the premise that retention order and peak selectivity is largely maintained when switching mobile phases.
Peak shape
A qualitative assessment of peak shape was undertaken as part of this study. Generally peak shapes were consistent when switching between organic and phosphate buffers. On occasion where different peak shapes were noted, the organic additives were generally found to give worse peak shapes than their phosphate equivalent. At low pH, this might be slightly surprising as the potassium diphosphate buffer has lower ionic strength than the formic acid mobile phase (Table 4) and higher ionic strength mobile phases is one factor that generally provides better peak shapes [17,18]
Methods Organic additive Phosphate buffer
1 2 3 0.1% Formic
acid (pH 2.6) KH2 10 mM
10 mM Ammonium
hydroxide (pH 10.6)
Table 3. Correlation coefficients for test analyte retention of MS compatible and phosphate methods. 10 mM PO4
Ammonium K2HPO4 acetate (pH 6.8)
(pH 2.6) 10 mM (pH 6.8) N/A (see text)
0.8290 (0.9837 N/A (see text) – see text)
0.9826 -1.20 to +0.08 Correlation coefficient (r ) 2 0.9492 Maximum
retention time variation/min
-1.09 to +1.18
Figure 4. Comparison of analyte retention times for method 1 with 0.1% formic acid (pH 2.6) and 10 mM potassium phosphate (pH 2.6). Note the higher retention with phosphate buffer for early eluting analytes which is believed to be due to ion-pairing effects. Full method details are provided in the Experimental section. The correlation coefficient was 0.9492.
. As noted above, a likely factor for
the improved peak shape with the phosphate buffer is phosphate ion-pairing with the basic analytes. This prevents secondary interactions with silanol groups on the stationary phase and leads to improvements in peak shape.
At pH 6.8, peak shape for the dipotassium
Figure 5. Comparison of analyte retention times for method 2 with 10 mM ammonium acetate (pH 6.8) and 10 mM potassium phosphate (pH 6.8). Full method details are provided in the Experimental section. The correlation coefficient was 0.9826.
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