Initial HPLC Method Development

Degradation samples are analyzed at the initial phases of high-performance liquid chromatography (HPLC) method development using... 

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

As with all HPLC method development, it is important to start work with a new colunm and fresh eluent at an optimum flow rate. Frequently, the HPLC separation is initially optimised using a UV or other detector that relies on a physical property of the analyte and is therefore independent of the performance of the ED. Transferring the analysis to an electrochemical detector and then to real samples often brings problems. Firstly, the operational sensitivity may be completely different and it may be necessary to compare separations of standards performed at maximum sensitivity using a UV detector with those done with an ED at almost minimal sensitivity. Although the selectivity of the ED using real sample extracts will probably be better than with a UV detector, there is likely to a substantial frontal peak caused by electrode disturbance and/or the presence of other electroactive compounds in the extract, such that the HPLC conditions have to be adjusted. 

The selection of starting conditions is the first step in HPLC method development. Instead of starting with arbitrary conditions or conditions derived from the chromatographer s experience or intuition, the software can predict suitable initial conditions for reversed-phase methods from analyte structures and properties of the sorbent/solvent system [1-3]. If structures are known, the theoretical approach has the potential to save time and effort, since in this way the experimental method development process will start under the theoretically predicted optimum conditions. 

As we have seen so far, libraries of hydrogenation catalysts are never composed of more than a few dozen members, up to 100 to 200 at the most. Consequently, modern analytical equipment such as gas chromatography (GC) or high-performance liquid chromatography (HPLC) equipped with an auto-sampler or even flow-through NMR systems are sufficient to handle the analysis of the entire library. Nevertheless, a few groups have initiated research towards the development of fast, sometimes parallel, analytical procedures. A few reviews have appeared on this subject [59]. Here, we will concentrate on the methods developed to analyze hydrogenation reactions, or methods that could likely be applied. 

HPLC techniques were initially developed as liquid-liquid chromatographic methods and difficulties in maintaining the stationary phase were resolved by chemically bonding it to the particulate support. Subsequently a whole range of column materials have been developed that enable the basic HPLC instrumentation to be used for the major chromatographic techniques. 

It is normally not too difficult to find a suitable column which will give desirable retention and peak shape for the analyte. One of the reasons why method development is often difficult is that satisfactory retention and peak shape alone are not enough for a method to be suitable. As discussed in Chapter 1, specificity is very important. In pursuing a separation using one particular mode of HPLC, it may be that, despite all the changes in experimental variables that take place in the method optimisation process, it is not possible to separate the analyte from all interferences present in the sample. In considering the initial stages of method development, it is therefore necessary to bear in mind as many approaches to the problem as possible, in case the first option proves not to be successful. 

A method that uses high performance liquid chromatography/ mass spectrometry (HPLC/MS) for the analysis of chlorinated phenoxyacid herbicides is described. During method development different techniques were used to increase both the sensitivity and the specificity of thermospray HPLC/MS for chlorinated acid herbicides. These included the operation of the instrument in the negative chemical ionization (NCI) mode initiated by discharge and the use of a wire-repeller in the ion source for efficient extraction of positive ions. Single quadrupole repeller-induced and multiple quadrupole collision activated dissociation (CAD) experiments were also performed to increase the structural information of the mass spectra. 

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