Introduction to Modern Liquid Chromatography, Third Edition part 64. 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. . | 586 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Aqueous Component. The hydrophobicity and therefore retention of a polypeptide is heavily dependent on the ionization state of the amino-acid termini approximate pKa values of and and of the ionizable side chains of internal residues pKa values in Fig. . Thus the pH of the mobile phase can have a profound effect on polypeptide retention and it is necessary to control mobile-phase pH by the additionofa buffer . ln hu ti T separations ofproteins and peptides arsmost ofnsnperfosmedstlow t H where odilute aoidnanserve as buffer. Unto- these conditionssilsnoi ionirrtronis ruppsessodtsrducing ondsoirable interactions wiih protonatedsotutesand resiOOng osed organic acids onto sri uoonacas-caaid can rlooion-oair wisd protonatedtoSutcs resulting mmereaoed rrtrnoion Oir peniidesand improvfdneakshs estarprosdns. The ionization ortermiodl cndtiOe-chrincarCdnytgrouprts saoprssred atlownH which furiieeincretirsf nosention. The msfrstt r mod add-on theRPnsrnnrasion of prnridesandproscms is trifluorodsrtio addCTFA witnaconcentrariod nf . ao rr dhi tr 5-10 mM Thrstrangeracioitys-i TnAallswo nlawermoOiSe-pdate pH so j and lowetconarntrasinns arelTaoernre resid i r tr drmosred ttnloonoc dd acetir acid. A loworTFA odnurdfrationalso oeduces background absorbance when UV detection is useOos lcwwavrleegiOs. An nli c d Osnefit ofTFAioirs highdolasllity which fadlitdeecsoias t removaltrom c OettsO ssmdk Sracsionsin pneparrtidd application- however o rifiedpdordns -ypicollg retaia srgmacansamoudnnof bound TFAo wOic dy Oal OSOd diadhrasio .T r highorUV absorbance of ot aolds narsstitatesdetecsidncs OieOsrwacrlengdss with increased noosa aOofwAolfadnerrely affsrsstensitivr doteotios . For the traco snails ofpepsides pdosphorieadd whishlr fra rparentasn00nm and above canaeuted ooa c eof d osoamcndd bm s oi dns afteneWdbtrpoac peak shap-wiro phorphapicpdO. The
