Separating basic and polar compounds

HPLC columns packed with silica-based sorbents are widely used in the pharmaceutical industry for drug discovery, quality control, and research and development. The separation of organic molecules by HPLC is based on the interaction of the functional groups of each analyte and the packing media. Due to the presence of residual acidic silanol groups in most commercial packings, secondary chromatographic effects are common and are usually troublesome. As a result, efficiency and peak shape for some analytes are usually unacceptable at neutral pH.

Basic and polar compounds, through ionic charges or hydrogen bonding effects, are affected more than neutral compounds. One possible way to reduce ionic interactions between basic molecules and acidic silanols is to use an acidic mobile phase. However, some acid-sensitive compounds may decompose and the method may not be robust.

This application demonstrates that good efficiency and peak symmetry for highly basic drugs are possible using a neutral pH mobile phase.

Instrumentation and methods

A separation of basic drugs was performed with a 150 x 4.6 mm Alltech HPLC column packed with 5 µm Prevail Select C18. The mobile phase consisted of 70% methanol and 30% of a 25 mM potassium phosphate buffer pH 7.0. The flow rate was set to 1.0 mL/min and UV detection was at 220 nm. A 10 µL injection of diphenhydramine and amitriptyline (0.15 mg/mL, each) was made and USP tailing factors were measured for each peak.

Column stability was tested with two 50 x 4.6 mm Alltech HPLC columns packed with 5 µm Prevail Select C18. For stability testing in acidic conditions, one column was flushed with a mobile phase containing 50% acetonitrile and 50% dilute H2SO4 at pH 1.0 for 24,000 column volumes at 60°C. For stability testing in alkaline conditions, a mobile phase of 50% acetonitrile and 50% ammonium hydroxide at pH 10.0 was flushed through a second column for 24,000 column volumes at 60°C.

Prior to and immediately following the washing, columns were tested with 5 µL injections of Alltech's RP-D4 test mix. The mix contains uracil, phenol, N,N-diethyl-m-toluamide, and toluene.

The mobile phase was 58% acetonitrile and 42% water at a flow rate of 1.0 mL/min.

Results and conclusion

Figure 1 shows excellent retention and resolution of two basic drugs, diphenhydramine and amitriptyline, at pH 7.0. These strongly basic drugs are capable of interacting with the surface-active acidic silanols through ionic interactions, and they are known to tail badly on many columns at neutral pH. Note the excellent separation and the USP tailing factor for both basic drugs is less than 1.35, which is difficult to achieve at neutral pH.

Column stability is an integral part of HPLC method development, which may require the use of a strongly acidic or strongly alkaline mobile phase. Alltech's Prevail Select C18 HPLC Column is based on innovative bonding technology, which permits exceptional long column life in both strongly acidic and basic conditions.

Figure 2 shows the chromatographic performance of the Alltech Prevail Select C18 columns after flushing with the acidic and basic mobile phases. In both cases, resolution, efficiency, and peak symmetry for all four compounds remain unaffected.