Method development optimization

Achievement of good resolution between analytes in complex chromatogram is the main goal in HPLC method development. Optimal resolution could be achieved by optimization of system efficiency, or selectivity (or... 

If the method does not meet run time criteria or robustness requirements, it may require further optimization. Method development optimization is typically continued until the objectives outlined in the development plan are met. Method development experts often have the experience necessary to adequately optimize the methodology others may benefit from the documented and organized development approach of available software packages. Several commercial packages are available that combine classical chromatographic theory with statistical design to predict optimum separation conditions with a minimum number of experiments. 

Weinberger, R. Lombardi, R. Method Development, Optimization and Troubleshooting for High Performance Capillary Electrophoresis Simon and Schuster Custom Publishing Needham Heights, MA, 1997. 

The scope of this five-step program for effective method development/optimization is based on a worst case scenario, and depending on the individual situation, the five steps could be modified and shortcuts taken. Usually, some information about a sample is available, perhaps also about its area of use. Such information must be used in order to shape the testing conditions, e.g. 

From today s point of view, the following trends in HPLC in the context of method development/optimization can be discerned ... 

Method Development and Optimization of Enantiomeric Separations Using Macrocyclic Glycopeptide Chiral Stationary Phases... 

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

Method development remains the most challenging aspect of chiral chromatographic analysis, and the need for rapid method development is particularly acute in the pharmaceutical industry. To complicate matters, even structurally similar compounds may not be resolved under the same chromatographic conditions, or even on the same CSP. Rapid column equilibration in SFC speeds the column screening process, and automated systems accommodating multiple CSPs and modifiers now permit unattended method optimization in SFC [36]. Because more compounds are likely to be resolved with a single set of parameters in SFC than in LC, the analyst stands a greater chance of success on the first try in SFC [37]. The increased resolution obtained in SFC may also reduce the number of columns that must be evaluated to achieve the desired separation. 

Identification of sources of analytical bias in method development and method validation is another very important application of reference materials in geochemical laboratories. USGS applied simplex optimization in establishing the best measurement conditions when the ICP-AES method was introduced as a substitute for AAS in the rapid rock procedure for major oxide determinations (Leary et al. 1982). The optimized measurement parameters were then validated by analyzing a number of USGS rock reference samples for which reference values had been established first by classical analyses. Similar optimization of an ICP-AES procedure for a number of trace elements was validated by the analysis of U S G S manganese nodule P-i (Montaser et al. 1984). 

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