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
- step 1 - selection of the HPLC method and initial system
- step 2 - selection of initial conditions
- step 3 - selectivity optimization
- step 4 - system optimization
- step 5 - method validation.
Depending on the overall requirements and nature of the sample and analytes, some of these steps will not be necessary during
HPLC analysis. For example, a satisfactory separation may be found during step 2, thus steps 3 and 4 may not be required.
The extent to which method validation (step 5) is investigated will depend on the use of the end analysis; for example, a
method required for quality control will require more validation than one developed for a one-off analysis. The following
must be considered when developing an HPLC method:
Figure 1: A flow diagram of an HPLC system.
- keep it simple
- try the most common columns and stationary phases first
- thoroughly investigate binary mobile phases before going on to ternary
- think of the factors that are likely to be significant in achieving the desired resolution.
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.
HPLC method development
Step 1 - selection of the HPLC method and initial system. When developing an HPLC method, the first step is always to consult
the literature to ascertain whether the separation has been previously performed and if so, under what conditions - this will
save time doing unnecessary experimental work. When selecting an HPLC system, it must have a high probability of actually
being able to analyse the sample; for example, if the sample includes polar analytes then reverse phase HPLC would offer both
adequate retention and resolution, whereas normal phase HPLC would be much less feasible. Consideration must be given to the
Table I: HPLC detector comparison.
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. This is only a requirement for complex samples with a large number of components (.20–30) because the maximum number of peaks
that can be resolved with a given resolution is much higher than in isocratic HPLC. This is a result of the constant peak
width that is observed in gradient HPLC (in isocratic HPLC peak width increases in proportion to retention time). The method
can also be used for samples containing analytes with a wide range of retentivities that would, under isocratic conditions,
provide chromatograms with capacity factors outside of the normally acceptable range of 0.5–15.
Table II: The basic types of analytes used in HPLC.
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:
- Do the analytes have chromophores to enable UV detection?
- Is more selective/sensitive detection required (Table I)?
- What detection limits are necessary?
- Will the sample require chemical derivatization to enhance detectability and/or improve the chromatography?
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.