tailieunhanh - Practical HPLC method development

This revision brings the reader completely up to date on the evolving methods associated with increasingly more complex sample types analyzed using high-performance liquid chromatography, or HPLC. The book also incorporates updated discussions of many of the fundamental components of HPLC systems and practical issues associated with the use of this analytical method. This edition includes new or expanded treatments of sample preparation, computer assisted method development, as well as biochemical samples, and chiral separations | PRACTICAL HPLC METHOD DEVELOPMENT SECOND EDITION Lloyd Snyder Joseph J. Kirkland Joseph Qlajch 1 GETTING STARTED Introduction What is Known Before Starting Nature of the Sample Separation Goals Sample Pretreatment and Detection Developing the Separation Selecting an HPLC Method and Initial Conditions Getting Started on Method Development Improving the Separation Repeatable Separation Completing the HPLC Method Quantitation and Method Validation Checking for Problems Method Ruggedness INTRODUCTION Every day many chromatographers face the need to develop a high-performance liquid chromatography HPLC separation. Whereas individual approaches may exhibit considerable diversity method development often follows the series of steps summarized in Fig. . In this chapter we review the importance of each of these steps in preparation for a more detailed examination in following chapters. Our philosophy of method development is based on several considerations. There exists today a good practical understanding of chromatographic separation and how it varies with the sample and with experimental conditions. Any systematic approach to HPLC method development should be based on this knowledge of the chromatographic process. In most cases a desired separation can be achieved easily with only a few experiments. In other cases a considerable amount of experimentation may be needed. A good method-development 1 GETTING STARTED FIGURE Steps in HPLC method development. strategy should require only as many experimental runs as are necessary to achieve the desired final result. Ideally every experiment will contribute to the end result so that there are no wasted runs. Usually this requires that the results of each chromatographic run be assessed before proceeding with the next experiment. Sometimes the chemical structures of the sample components are known other times this is not the case. The .