For the best peak shape, retention and loading of basic analytes in reversed-phase HPLC, the pH of the mobile phase should be at least 2 units higher than the pK
a of the molecule. For many bases, this means the pH of the mobile phase should be 10 or higher. Volatile buffers and additives such as ammonium bicarbonate and ammonium hydroxide are commonly used in high pH mobile phases, but their upper buffering capacity is pH 11. Phosphate buffer, a favourite of chromatographers because of its three pK
a's of 2.15, 7.2 and 12.3 and transparency at low UV wavelengths, is rather aggressive at pH 12. Standard silica-based column packings are unusable at pH 12 with phosphate buffer, and many columns designed to work at high pH suffer from reduced lifetimes. With the introduction of the new XBridge™ family of HPLC columns, phosphate buffer at all three buffering ranges can now be routinely used.
Table 1: Gradient profile for selectivity study.
Chromatographic Conditions Stability Testing
Column: XBridge™ C18 5µm 4.6 x 150 mm
Mobile Phase A: 200 mM K3PO4 (pH 12):MeCN (80:20)
Mobile Phase B: Water
Mobile Phase C: Acetonitrile
Isocratic Mobile Phase Composition: 10% A; 37% B; 53% C
Flow-rate: 1.0 mL/min
Injection Volume: 10µL of 440µg/mL (total concentration)
Detection: UV @ 254 nm
Column: XBridge™ C18 3.5µm 4.6 x 100 mm
Mobile Phase A1: 30 mM Potassium phosphate, pH 2
Mobile Phase A2: 30 mM Potassium phosphate, pH 7
Mobile Phase A2: 30 mM Potassium phosphate, pH 12
Mobile Phase B: Acetonitrile
Flow-rate: 1.4 mL/min
Gradient: See table 1.
Column Temperature: 30°C
Injection Volume: 20µL of 5µg/mL (each) standard
Detection: UV @ 210 nm (pH 2, 7); 220 nm (pH 12)
Instrument (both studies): Waters Alliance® 2695 with 2996 PDA