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Introduction to Modern Liquid Chromatography, Third Edition part 70

Introduction to Modern Liquid Chromatography, Third Edition part 70. 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. . | 646 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Figure 13.45 Preparative separation of rh-insulin.Column 10 im Zorbax Process Grade C8 150 x 9.4-mm i.d. load 153 mg rh-insulin from proinsulin process gradient 17-29 acetonitrile in 0.25M acetic acid in six column volumes flow rate 0.3 mL min. Fractions from 3.3 to 4.3 column volumes were pooled mainstream . Fractions 3.2-3.3 and 4.4-5.4 plus the protein eluted during column regeneration notshown whre combined as the side stream. Adapted from 162 . and 4.4-5.4 C-Vs plus recovered soloentfromcolumn regeneration contcmed an addLtionall5 oftheproduct for re-separation for anoverallmsulm reoovaryoC 97 . The rnitialpeoity ofthe sample for RPC separation was 91.5 . This example illustrates a common property of preparative separations in contrast to analytical chromatography prep-TC chromatograms may suggest poor separation of product from impurities butdhen fracUdniarccoOecledend malooeU AererultsoOen ore acceptable. 13.9.2.9 i i l C r Scale-up experimeuis werr sviried our sexv usings ceccesrioely Inciu-t. axial-comcoorsion rolumns with the bed-volume increased from 10 mL to 80T as summarized in Tables 13.9 and 13.10. In each separation the weight of insulin applied to the column was 14 to 15 g per T of column-volume C-V the flow rate was 1.5 C-V h 1 thegradient slopes were 2 C-V. Minor changes in gradient slope were necrsearyeo sarmtainrchimnpetforuwier as measuredbg oooducr purity anddecoorra.FlowraOrsweteivcrlasld in oacpomontorne oolirae oh e column as thepraccis moped I com loOto celot plant to production. Purity was9e.5 98.6 and 98.6 ar liO srale uilvcsodle and peeCuctfonlcaleirclprctlveey whilr mainstream yields webe82 79gind83 .It sotysesmreiairkable thelmomueram purities reooveries and elutienvalcmtc temeineU nmoit-mfrem laO erele tn productionscnee OTeeal 0 000-fold icirge olcoiomnoolumes but Ous rhnulCbr true of scalrsu-if catiirdoutprapocly Srction 15.e.2.t . 13.9 LARGE-SCALE PURIFICATION OF LARGE BIOMOLECU .