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Introduction to Modern Liquid Chromatography, Third Edition part 34

Introduction to Modern Liquid Chromatography, Third Edition part 34. 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. . | 286 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES b Choice of Separation Conditions 1. Initial separation 2. Separation problems Sections 2.4.2 9.3.1.1 1a. Vary B for1 Æ 10 Section 6.2.1 or 1b. Start with gradient run Section 9.3.1 3. Reconsiderinitialconditions RPC Chapter 6 or Section 7.3 IPC Section 7.4 IEC Section 7.5 NPC Chapters 4. Confirm peaksas. acid base or aeuCral Section 7.2 6. Adjust column conditions A Section 2.5.3 5. Optimize selectivity Figure 6.21 Continued can be varied step 6 for the purpoae oa either increasing resolution orreducing run time. Samples rhara rda.i.dy easy to separate may requite uq mote then the selection of a final value of B which involves only a few experiments in which B is varied. If gradient elution is used during initial method development experiments only asingleexperiment is neededin orderto select avalueof B for 1 k 10 Section 9.3.1 . Many samples will require a further improvement of separation selecticety prercSicg Sfetcon6.30for samesampleschis mayinvotvethe simiiltaiu-oiiodiaagcol twnoemoraseparatiynronditient. Varinusproceduees Sot such multi Btraricl h opoim tian willba descoirednext. 6.4.1 Mu Iti p It-VariaOleOptimization Multiple-v.trable oasimiration tn h e tite on an aa . ng. y ydan for the required extsenmenle at 1ltysftefedmF1pureO.nO hircertaiccombinations of conditions that affect selectivity for neutral samples. In each case it is assumed that B has been eat edmitrally.sq as to atange iu B that provides adequate retention o fthe sdmpkg toraxamp c 40-5O B so that 0.5 k 20 for every peak wheoxafying nfor rchannetn fdectiwenia wiOorS-cangetiiantheusual 1 k lOisregommondcd . By way of iiiuitcation fiosfeonsiOar nigure n.2ta. Experimentsi aa are ooeeled outfirrt i.e.x chanyein f only . These two 6.4 METHOD DEVELOPMENT AND STRATEGIES FOR OPTIMIZING SELECTIVITY 287 a b Figure 6.22 Experimental designs for the simultaneous optimization of various separation conditions for optimum selectivity a Solvent .