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Introduction to Modern Liquid Chromatography, Third Edition part 11

Introduction to Modern Liquid Chromatography, Third Edition part 11. High-performance liquid chromatography (HPLC) is today the leading technique for chemical analysis and related applications, with an ability to separate, analyze, and/or purify virtually any sample. Snyder and Kirkland's Introduction to Modern Liquid Chromatography has long represented the premier reference to HPLC. This Third Edition, with John Dolan as added coauthor, addresses important improvements in columns and equipment, as well as major advances in our understanding of HPLC separation, our ability to solve problems that were troublesome in the past, and the application of HPLC for new kinds of samples. . | 56 BASIC CONCEPTS AND THE CONTROL OF SEPARATION 80 methanol 0.3 k 0.8 a I 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Time min 70 methanol 1 A 4 d JU I 1 1 0 2 Time mm 60 methanol c T61 h X a. 0 2 4 6 Time min 50 methanol d 4 fc 19 u w3 w4 0 5 10 15 Time min Figure 2.18 Separation 4 a function of mobile phase B. Herbicide sample 1 monolinuron 2 metobromuron 3 diuron 4 propazine 5 chloroxuron. Conditions 150 x 4.6-mm 5- im Cig column methanol-water mixtures as mobile phase 2.0 mL min ambient temperature. Recreated chromatograms from data of 7 . Rs reportedin thisbookarealways calculated from Equation 2.23 .Equation 2. 24 will be usedmainlyaoaan undatstandingot how reiolaiĩon depenot onvarious experimental coaiitione and as e mulde eyrsiüemaqic metVoAdeveloement. Aq alternativeexpreesionforRsin Emotion 2.f4 is al yo-er st ts n 2.aa The derivationsofEquaiions 2.24 and 2.25 arebasedondifferent approximations concerning the widths of the two peaks and each equation has a similar accuracy. Equation 2.24 has the advantage of greater simplicity for use in guiding method development. The development of an isocratic HPLC method proceeds by systematically adjustmg opsimizing expesimenial conditionsuntii udequate nusacatine is achieved bruforoblywidiaczitiealuesolutiupjhs isd. pquatioc 4.24tpcovidrsn 2.5 RESOLUTION AND METHOD DEVELOPMENT 57 useful guide for isocratic method development as will be explored in Sections 2.5.1 through 2.5.3. Each of terms a-c of Equation 2.24 can be controlled by varying certain separation conditions Table 2.2 . Usually the first step is to choose a column with a sufficient plate number that is likely to separate a sample of the required complexity. In many cases N a 10 000 is a good starting point and this can be achieved either withal50-mm longcolumn packed with5-pmparticles ora 100-mm 3-itm column. Tkeso-kent strength e 0Bi isnext variedto echieveantppron-ipterangr in values of t e.g. e 3 npd -cllowtd b optimiting seleetivity a .FmeUyi the colume pkatt umber N