tailieunhanh - Introduction to Modern Liquid Chromatography, Third Edition part 65

Introduction to Modern Liquid Chromatography, Third Edition part 65. 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. . | 596 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Table Initial Conditions for RPC Method Development Condition Values for Different Samples Peptides Proteins Sample 1 M 5kDa 5 M 20kDa M 20 kDa Sample treatment prior to injection None Add 8 M urea store for 30 min Add 8M urea store for limn Column 150 x type-B Cig 8-12 nm Pore-diameter 3- im particles 150 x type-B Cig 12-30 nm P e-diame terM 3- xm particles 50 x type-B C4 30 nm -di arnet. 3- im particles Solvent A TFA water TFA watei TFA water 50 8 TFA ACN 0-10 TFA ACN 0-Od TFA-A GN Gradient range 0-60 B d-100 B Ou 100 B Temperature 30-35 C 30-35 Cfe c o .b Flow rate mL min Gradient time min 25 50 50 k 0 1 1 B min 2A Value of S assume- 25 40 ehh Columns shouldbe stable at low pH and temperatures 6o C other column lengths diameters and particle sizes can be used in which case gradient time and flow rate should be adjusted to maintain similar values of k with acceptable pressure drop. The choice of ligand length C8 C18 is less critical. Higher temperaturee . 6o-8o C can be desirable for some protein samples especially Shose with M 20 V a columnstamn or ohese conditions should be verified before the use 5o C and pH . Figure theinitial four rum eo-dcan beusedto predict thebestcombination of temperatureaod gradieot iunctoro ttunalrcsolueioii 10e .Once accootablo peak spacin0 h cnln cvud tho gradient tomj c coin Ire trimmed to shorten overall separation time. Fcrexamph tha gradcntcanta imtiodfd at a B-aalue justprioc to elution of thefirst peak and terminated at the B-value just after elution of the last peak Fig. c If no dembinatconofi odient dmeand bumpecataaejeeldsaaiepeabhe resc-lution thenext niep uoiOdbeachange io thccolumn ortfiecomddscoon ofdre A- or B-soCneni b c e eeamdlei anienacatoin UOA eoneeettatiou a rhangeanpH or the subtticution of iroprooadciforaaecombriie a B-soCvena. After ane urmoae of the lattercdcugesin .