Ion Exchange Chromatography Principles

✍ Scribed by Amersham Pharmacia

• Publisher: Amersham Pharmacia Biotech
• Year: 1999
• Tongue: English
• Leaves: 163
• Category: Library

✦ Synopsis

Adsorption chromatography depends upon interactions of different types between solute molecules and ligands immobilized on a chromatography matrix. The first type of interaction to be successfully employed for the separation of macromolecules was that between charged solute molecules and oppositely charged moieties covalently linked to a chromatography matrix. The technique of ion exchange chromatography is based on this interaction. Ion exchange is probably the most frequently used chromatographic technique for the separation and purification of proteins, polypeptides, nucleic acids, polynucleotides, and other charged biomolecules (1). The reasons for the success of ion exchange are its widespread applicability, its high resolving power, its high capacity, and the simplicity and controllability of the method.

This handbook is designed as an introduction to the principles of ion exchange chromatography and as a practical guide to the use of the media available from Pharmacia Biotech. The handbook is illustrated with examples of different types of biological molecules which have been separated using ion exchange chromatography and different ways the technique can be used. For information on specific separations, the reader is recommended to consult the original literature.

✦ Table of Contents

Front Page......Page 1
Contents......Page 4
1. Introduction......Page 11
The theory of ion exchange......Page 12
The matrix......Page 13
Resolution in ion exchange chromatography......Page 15
Capacity factor......Page 17
Efficiency......Page 18
Selectivity......Page 19
Capacity......Page 20
SOURCE......Page 22
STREAMLINE ion exchangers......Page 23
Equipment......Page 24
MonoBeads......Page 25
Chemical stability......Page 26
Physical stability......Page 27
Capacity......Page 29
Recovery......Page 30
Reproducibility......Page 31
MiniBeads......Page 32
Chemical stability......Page 33
Physical stability......Page 34
Availability......Page 35
5. SOURCE......Page 36
Chemical stability......Page 39
Flow rate......Page 40
Recovery......Page 42
Availability......Page 43
Chemical stability......Page 44
Sepharose High Performance ion exchangers......Page 45
Capacity......Page 46
Sepharose Fast Flow ion exchangers......Page 48
Capacity......Page 49
Flow rate......Page 51
Sepharose Big Beads ion exchangers......Page 52
Availability......Page 53
STREAMLINE SP and
STREAMLINE DEAE......Page 54
Capacity......Page 55
Physical stability......Page 56
Capacity......Page 57
Flow rate......Page 58
Availability......Page 59
Chemical stability......Page 60
Capacity......Page 61
Availability......Page 62
Chemical stability......Page 63
Capacity......Page 64
Availability......Page 66
The required resolution......Page 67
Scaleability......Page 68
The molecular size of the sample components......Page 69
Choice of exchanger group......Page 70
Test-tube method for selecting starting pH......Page 71
Electrophoretic titration curves (ETC)......Page 72
Chromatographic titration curves (retention maps)......Page 76
Choice of buffer pH and ionic strength......Page 78
Choice of buffer substance......Page 79
Test-tube method for selecting starting ionic strengths......Page 81
Column design......Page 82
Pre-packed ion exchange media......Page 83
Column Packing Video Film......Page 84
Checking the packing......Page 85
Equilibrating the bed......Page 86
Sample viscosity......Page 87
Sample preparation......Page 88
Sample application with an adaptor......Page 89
Sample application under the eluent......Page 91
Change of pH......Page 92
Choice of gradient type......Page 93
Resolution using a continuous gradient......Page 95
Choice of gradient shape......Page 96
Sample displacement......Page 97
Gradient Mixer......Page 98
Batch separation......Page 99
Expanded bed adsorption......Page 100
Basic principle of operation......Page 101
Auxiliary equipment......Page 102
Sterilization......Page 103
MonoBeads and MiniBeads columns......Page 104
Prevention of microbial growth......Page 105
Determination of the available and
dynamic capacities......Page 106
Calculation......Page 108
11. Process considerations......Page 109
Defining the purpose......Page 110
Capture......Page 111
Polishing......Page 113
Selection of chromatography media......Page 114
Regulatory support......Page 115
Binding conditions......Page 116
Elution......Page 117
Sample load......Page 118
Flow rate......Page 119
Selecting a column......Page 120
Sanitary design......Page 121
Column configuration......Page 122
Scale-up......Page 123
The design of a biochemical separation......Page 126
Enzymes......Page 129
Isoenzymes......Page 130
Nucleic acid separation......Page 131
Polypeptides and polynucleotides......Page 132
Antisense phosphorothioate oligonucleotides......Page 134
Areas of application......Page 135
Purification of a recombinant Pseudomonas aeruginosa exotoxin A, PE553D......Page 138
Strategy......Page 143
13. Fault finding chart......Page 145
14. Ordering information......Page 153
15. References......Page 157