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Figure 2: Effect of mobile phase pH on analyte retention.


Column: ACE Excel 3 SuperC18, 50 x 2.1 mm; Mobile phase: A: 20 mM ammonium formate pH 3.0, 4.5, 6.0, 7.5, 9.0 and 10.5 (aq), B: 20 mM ammonium formate pH 3.0, 4.5, 6.0, 7.5, 9.0 and 10.5 in MeCN/H2


O 9:1 v/v; Gradient: 3 to 100% B in 5 minutes; Flow Rate: 0.6 mL/min; Injection Volume: 1 µL; Temperature: 40°C; Detection: UV, 214 nm.


tolerate high pH are commercially available (such as those shown in Figures 2, 3 and 4) and are typically manufactured from a hybrid organo-silica material, polymer based, or utilise modified bonding technology.


Using pH to control analyte selectivity


From Figures 2 and 3, it is clear that when ionisable analytes are present in a sample, the selectivity between analytes can vary significantly with mobile phase pH. For such samples, it is highly recommended that mobile phase pH is explored during initial method development, to determine the most suitable option for the sample. When beginning any method development, it is useful to consider analyte structures and properties, if known, to anticipate any acidic/basic behaviour. If unknown, screening the sample on a generic gradient with low and high pH mobile phases can be a productive starting point. Figure 4 shows an example gradient separation of a set of acidic, basic and neutral analytes at low and high pH; analysed on a novel stationary phase with extended pH compatibility (pH 1.5-11.0). Neutral analytes, or analytes whose ionisation state remains unchanged within the pH range examined, show little change in retention with pH (e.g. peak #11). In contrast, all the acidic analytes show a sharp decrease in retention as mobile phase pH increases, whereas the opposite is observed for basic compounds. Clear differences in selectivity are observed: for example, peaks 5 and 12 show complete reversal in elution order. Mobile phase pH can also provide useful selectivity for samples containing just acidic or basic components: for example, the resolution between the bases carvedilol (9) and trimipramine (12) increases dramatically from low to high pH.


. Often this may not be possible, e.g. for complex samples or for multifunctional analytes containing moieties with overlapping pKa


For method robustness, it is generally recommended to work at a mobile phase pH at least 2 pH units away from the analyte pKa


values. In these situations, it is important to accurately control and document mobile phase preparation to ensure consistent pH between batches of mobile phase. Examining the effect of small changes in pH on the separation is also highly recommended to assess the method robustness.


Additional benefits of changing mobile phase pH


Careful selection of mobile phase pH can also provide several other benefits for the chromatographer. For LC/MS applications,


pH can provide enhanced sensitivity in some cases. For example, using a high pH mobile phase to ionise acidic components may enhance detection in negative ion mode. For preparative applications and analyses involving one or more overloaded peaks (e.g. impurity testing), working at a pH where the analyte is neutral can be beneficial. Figure 5 summarises a loading study for amitriptyline at 2 different pH’s. At low pH, amitriptyline is positively charged and shows peak tailing, whereas at high pH (non-ionised form), peak shape is vastly improved. As the sample load on column is increased, the peak width at low pH increases significantly. This could lead to loss of resolution or difficulty in observing/quantifying smaller impurity peaks. At high pH, however, the peak width is more constant as the mass of analyte on column is increased.


Figure 3: Chromatograms of carvedilol on an ACE Excel 3 SuperC18, 50 x 4.6 mm column with (A) low pH and (B) high pH mobile phases.


Figure 4: Separation of a range of acidic, basic and neutral analytes on a high pH compatible solid-core column with novel encapsulated bonding. Column: ACE UltraCore 2.5 SuperPhenylHexyl, 100 x 3.0 mm; Mobile phase (low pH): A: 15 mM ammonium formate pH 3.0 (aq), B: 15 mM ammonium formate pH 3.0 in MeCN/H2 B: 0.1% NH3


O 9:1 v/v; Mobile phase (high pH): A: 0.1% NH3 in MeCN/H2 O 9:1 v/v; Gradient: 5 to 100% B in 10 minutes; Flow Rate: 1.2 mL/min; Injection Volume: 1 µL; Temperature: 40°C; Detection: UV, 260 nm (aq)


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