The wide variety of equipment, columns, eluent and operational parameters involved makes high performance liquid chromatography (HPLC) method development seem complex. The process is influenced by the nature of the analytes and generally follows the following steps:
Mobile phase composition, for example, is the most powerful way of optimizing selectivity whereas temperature has a minor effect and would only achieve small selectivity changes. pH will only significantly affect the retention of weak acids and bases. A flow diagram of an HPLC system is illustrated in Figure 1.
Sample preparation. Does the sample require dissolution, filtration, extraction, preconcentration or clean up? Is chemical derivatization required to assist detection sensitivity or selectivity?
Types of chromatography. Reverse phase is the choice for the majority of samples, but if acidic or basic analytes are present then reverse phase ion suppression (for weak acids or bases) or reverse phase ion pairing (for strong acids or bases) should be used. The stationary phase should be C18 bonded. For low/medium polarity analytes, normal phase HPLC is a potential candidate, particularly if the separation of isomers is required. Cyano-bonded phases are easier to work with than plain silica for normal phase separations. For inorganic anion/cation analysis, ion exchange chromatography is best. Size exclusion chromatography would normally be considered for analysing high molecular weight compounds (.2000).
Gradient HPLC will also give greater sensitivity, particularly for analytes with longer retention times, because of the more constant peak width (for a given peak area, peak height is inversely proportional to peak width). Reverse phase gradient HPLC is commonly used in peptide and small protein analysis using an acetonitrile–water mobile phase containing 1% trifluoroethanoic acid. Gradient HPLC is an excellent method for initial sample analysis.
Column dimensions. For most samples (unless they are very complex), short columns (10–15 cm) are recommended to reduce method development time. Such columns afford shorter retention and equilibration times. A flow rate of 1-1.5 mL/min should be used initially. Packing particle size should be 3 or 5 μm.
Detectors. Consideration must be given to the following:
Fluorescence or electrochemical detectors should be used for trace analysis. For preparative HPLC, refractive index is preferred because it can handle high concentrations without overloading the detector.
UV wavelength. For the greatest sensitivity λmax should be used, which detects all sample components that contain chromophores. UV wavelengths below 200 nm should be avoided because detector noise increases in this region. Higher wavelengths give greater selectivity.
Fluorescence wavelength. The excitation wavelength locates the excitation maximum; that is, the wavelength that gives the maximum emission intensity. The excitation is set to the maximum value then the emission is scanned to locate the emission intensity. Selection of the initial system could, therefore, be based on assessment of the nature of sample and analytes together with literature data, experience, expert system software and empirical approaches.