An introduction to HPLC文献.pdf

1 Introduction 1.1 HPLC: A POWERFUL SEPARATION METHOD A powerful separation method must be able to resolve mixtures with a large number of similar analytes. Figure 1.1 shows an example. Eight benzodiaze- pines can be separated within 70 s. Such a chromatogram provides directly both qualitative and quantitative information: each compound in the mixture has its own elution time (the point at which the signal appears on the recorder or screen) under a given set of conditions; and both the area and height of each signal are proportional to the amount of the corresponding substance. This example shows that high-performance liquid chromatography (HPLC) is very efficient, i.e. it yields excellent separations in a short time. The ‘inventors’ of modern chromatography, Martin and Synge,1 were aware as far back as 1941 that, in theory, the stationary phase requires very small particles and hence a high pressure is essential for forcing the mobile phase through the column. As a result, HPLC is sometimes referred to as high-pressure liquid chromatography. 1.2 A FIRST HPLC EXPERIMENT Although this beginner’s experiment described here is simple, it is recommended that you ask an experienced chromatographer for assistance. It is most convenient if a HPLC system with two solvent reservoirs can be used. Use water and acetonitrile; both solvents need to be filtered (filter with 1 mm pores) and degassed. Flush the system with pure acetonitrile, then connect a so-called reversed-phase column (octadecyl ODS or C 18, but an octyl or C 8 column can be used as well) with the correct direction of flow (if —————————— 1 A. J. P. Martin and R. L. M. Synge, Biochem. J., 35, 1358 (1941). Practical High-Performance Liquid Chromatography, Fourth edition Veronika R. Meyer # 2004 John Wiley Sons, Ltd ISBN: 0-470-09377-3 (Hardback) 0-470-09378-1 (