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Reversed Phase Chromatography(外文书籍)
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下载地址http://www.namipan.com/d/Reverse ... 3e9f7057b6b0c7e0800 Contents 1. Introduction ..............................................................................................5 Theory of reversed phase chromatography ...............................................6 The matrix ................................................................................................9 The ligands ............................................................................................. 11 Resolution in reversed phase chromatography ........................................ 13 Resolution .............................................................................................. 13 Capacity factor ....................................................................................... 14 Efficiency ................................................................................................ 15 Selectivity ................................................................................................ 17 Binding capacity ..................................................................................... 18 Critical parameters in reversed phase chromatography........................... 19 Column length ...................................................................................19 Flow rate ............................................................................................19 Temperature ....................................................................................... 20 Mobile phase ..................................................................................... 20 Organic solvent .................................................................................. 20 Ion suppression .................................................................................. 21 Ion pairing agents .............................................................................. 22 Gradient elution ................................................................................. 23 Mode of use ............................................................................................24 Desalting ............................................................................................24 High resolution separations ............................................................... 25 Large scale preparative purification ................................................... 25 Stages in a purification scheme .......................................................... 26 Capture .............................................................................................. 26 Intermediate stages .............................................................................27 Polishing ............................................................................................27 2. Product Guide .........................................................................................29 SOURCE™ RPC ..................................................................................... 30 Product description ............................................................................ 30 High chemical stability ...................................................................... 32 Excellent flow/pressure characteristics ................................................ 34 High capacity ..................................................................................... 35 Availability ........................................................................................ 36 μRPC C2/C18 .........................................................................................37 Product description ............................................................................ 37 Chemical and physical stability .......................................................... 38 Flow/pressure characteristics ..............................................................38 Capacity ............................................................................................38 Availability ........................................................................................ 39 Sephasil™ Protein/Sephasil Peptide ......................................................... 39 Product description ............................................................................ 39 Chemical and physical stability .......................................................... 40 Flow/pressure characteristics ..............................................................40 Availability ........................................................................................ 40 3. Methods ................................................................................................. 41 Choice of separation medium .................................................................41 Unique requirements of the application .............................................. 41 Resolution .......................................................................................... 41 Scale of the purification ..................................................................... 42 Mobile phase conditions ....................................................................42 Throughput and scaleability ..............................................................42 Molecular weight of the sample components ......................................42 Hydrophobicity of the sample components ........................................ 43 Class of sample components ..............................................................43 Choice of mobile phase ........................................................................... 44 The organic solvent ............................................................................ 44 pH .....................................................................................................46 Ion pairing agents .............................................................................. 47 Sample preparation................................................................................. 49 Mobile phase preparation .......................................................................50 Storage of mobile phase ..................................................................... 50 Solvent disposal ................................................................................. 50 Detection ................................................................................................ 51 Ghosting ............................................................................................51 Mobile phase balancing ..................................................................... 51 Column conditioning .............................................................................. 52 Elution conditions ...................................................................................53 Column re-equilibration ......................................................................... 55 Column cleaning ..................................................................................... 55 Column storage ......................................................................................56 4. Applications............................................................................................57 Designing a biochemical purification ...................................................... 57 Naturally occurring peptides and proteins .............................................. 58 Purification of platelet-derived growth factor (PDGF) ........................ 59 Trace enrichment ............................................................................... 59 Purification of cholecystokinin-58 (CCK-58) from pig intestine ......... 60 Recombinant peptides and proteins ........................................................62 Process purification of inclusion bodies .............................................. 63 Purification of recombinant human epidermal growth factor ............. 63 Chemically synthesised peptides..............................................................65 Purification of a phosphorylated PDGF a-receptor derived peptide .... 65 Structural characterisation of a 165 kDa protein ............................... 66 Protein fragments from enzyme digests ................................................... 66 Protein characterisation at the micro-scale .........................................66 Protein identification by LC-MS ......................................................... 69 Chemically synthesised oligonucleotides ................................................. 70 5. Fault finding chart .................................................................................. 72 6. References ...............................................................................................81 7. Ordering information .............................................................................84 [ Last edited by zhaoyongqiang on 2008-3-17 at 12:07 ] |
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