19
allows direct comparison of retention times in this work (rather than a more formal use of retention factors, k, to be used).
Methods
The generic methods listed in Table 2 were used in this investigation. Columns were 100 x 2.1 mm i.d. (1.7 µm) and utilised a flow rate of 0.4 mL/min. Columns were thermostatted at 30o
C. 2 µL of test mixes (typically 0.1 mg/mL of each analyte) were injected, as well as individual stock solutions for peak tracking. While PDA detection was used, data was reprocessed and compared at 210 nm as a ‘worst-case’ scenario for observing system peaks and other baseline artefacts.
Potassium phosphate buffers have lower solubility in organic solvent-water mixtures than organic buffers. 10 mM dipotassium hydrogen phosphate (K2
HPO4
– pH 6.8) was
found to be compatible to a maximum methanol volume of 80% (v/v), while 10 mM potassium dihydrogen phosphate (KH2
PO4 –
pH 2.6) was soluble up to 85% (v/v) acetonitrile (both at room temperature). The same gradient was mimicked in both sets of experiments i.e. the same gradient slope was used in the phosphate experiments as the organic buffer experiments. However, when the maximum organic solvent level was reached in the phosphate gradients, the mobile phase was held isocratically for the remainder of the analysis.
Results and Discussion UV spectra
A comparison of the UV spectra for potassium dihydrogen phosphate and formic acid (pH 2.6), and dipotassium hydrogen phosphate and ammonium acetate (pH 6.8) are shown in Figure 1. Quite clearly the spectra for equivalent pH phosphate buffer exhibits much lower absorbance than the respective organic buffers. Significant absorbance is observed for formic acid from 240 nm and below, while ammonium acetate shows high levels of absorbance from 225 nm and below. The obvious result of this is significantly better signal to noise and baseline characteristics when using phosphate allowing lower levels of analyte quantitation. For example, up to 25- 30% increase in analyte response in the dipotassium phosphate buffer was observed compared to the ammonium acetate mobile phase. This exemplifies one of the reasons why phosphate is often the preferred buffer for chromatographic method development. Comparisons of separations using the different buffers are shown in Figures 2 and 3.
Retention The correlation coefficients for the test analytes retention and applying a linear relationship between the organic and
Figure 2. Overlay of one set of test analytes using method 1 with 0.1% formic acid (pH 2.6 – black line) and 10 mM potassium phosphate (pH 2.6 – blue line). The sloping nature of the formic acid baseline can make quantitation difficult and the increase in analyte response with the phosphate buffer is obvious. The signals were both collected at 210 nm and the same sample injected with both mobile phases.
Figure 1. A comparison of UV spectra for (a) 10 mM potassium dihydrogen phosphate and 0.1% formic acid (pH 2.6), and (b) 10 mM dipotassium hydrogen phosphate and 10 mM ammonium acetate (pH 6.8). In both cases, the phosphate spectra (dashed line) exhibits much lower absorbance from wavelengths above 225 – 250 nm.
Figure 3. Overlay of one set of test analytes using method 2 with 10 mM ammonium acetate (pH 6.8 – black line) and 10 mM potassium phosphate (pH 6.8 – blue line). This is an extreme case where retention (for one analyte) is significantly different between the two methods (corresponding to extreme top-right data point in Figure 5).
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