Size exclusion chromatography would normally be considered for analysing high molecular weight compounds (.2000). 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.

validating an hplc method-87

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.

Analytical methods must be validated to provide reliable data for regulatory submissions.

These methods are essential for a number of purposes, including testing for QC release, testing of stability samples, testing of reference materials and to provide data to support specifications.

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: Depending on the overall requirements and nature of the sample and analytes, some of these steps will not be necessary during HPLC analysis.

The following must be considered when developing an HPLC method: 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.

p H will only significantly affect the retention of weak acids and bases.

HPLC technicians working in R&D laboratories, quality control laboratories and stability testing laboratories.

Managers with a responsibility for generating regulatory submissions.

For example, a satisfactory separation may be found during step 2, thus steps 3 and 4 may not be required.