Ion Exchange
`Chromatography
`& Chromatofocusing
`Principles and Methods
`
`111-0004-21 I
`Edition AA
`
`• • •
`• ,--.~ Amersham
`• • • 81osc1enc s
`
`1 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Handbooks
`from Amersham Biosciences
`
`===---
`
`.... -... -CJ
`
`I =:·-
`
`--· -,,_ ---
`
`---
`
`Antibody Purification
`Handbook
`18-1037-46
`
`The Recombinant Protein Handbook
`Protein Amplification and
`Simple Purification
`18-1142-75
`
`Protein Purification
`Handbook
`18-1132-29
`
`Ion Exchange Chromatography
`& Chromatofocusing
`Principles and Methods
`11-00011-21
`
`Affinity Chromatography
`Principles and Methods
`18-1022-29
`
`Hydrophobic Interaction
`Chromatography
`Principles and Methods
`18-1020-90
`
`Gel Filtration
`Principles and Methods
`18-1022-18
`
`Expanded Bed Adsorption
`Principles and Methods
`18-112-1-26
`
`Microcarrier cell culture
`Principles and Methods
`18-1140-62
`
`Percell
`Methodology and Applications
`18-1115-69
`
`Ficoll-Paque Plus
`For in vitro isolation of lymphocytes
`18-1152-69
`
`GST Gene Fusion System
`Handbook
`18-1157-58
`
`2-D Electrophoresis
`using immobilized pH gradients
`Principles and Methods
`80-6429-60
`
`2 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Ion Exchange Chromatography
`& Chromatofocusing
`Pri nciples and Methods
`
`3 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Contents
`
`Introduction ............................................................................................................. 7
`Symbols ............ ... ... ... .. .. ... .. .. .... ......................... ........ .............................................................................. 8
`Common abbreviat ions .... ...... ..... ... .... ......... ... ................... ....... ....... ....... ....... ............................................. 9
`
`Chapter 1
`Principles of ion exchange ..................................................................................... 11
`Net surface charge and pH .. .. .... .... ............................................................................. 11
`Steps in an IEX separation ...... ......... ............. ............................................................. 12
`Resolution ...... .................... ...................................................................................... 14
`Efficiency ... .......... .. ... .... ............. ........ .... ..... ............. ...... ....... ....... ....... ....... ....... ....... ..... ....... ....... ........... 15
`Selectivity .... .... ... ..... . ... .... ... .... ... ... , .... ..... .............. ................ ....... ....... ....... ....... ................... ....... ........... 17
`Components of ion exchange med ia .... ........ ... ............................................................. 21
`Matrix, ....... ....... ..... .. .............. ............. .. .. .................. ...... ....... ....... ....... ....... ....... ................... ....... ........... 2 1
`Functional groups ............... .. ....... ....... .•... ........ ..... ... ... .... ...... ........ ........ ..... .. ..... ......... .............. .............. .. 23
`Binding capacity and recovery .... ..... ...... .... ....... .... .... .. ... ... .... .............. ........ ......... ... .... 25
`
`Chapter 2
`Ion exchange in practice ........................................................................................ 26
`Introduct ion .. .................. ...... ....... ...... ... .. ...... ..... .. ... ... ... ...... ......... .... ............. ..... ..... .. 26
`Media selection ............... ... ... ........... ....... ..... ...... ... ......... ......... ......... ....... ............. ... .. 26
`Capture .... .. ..... ...... .... ......... ..... ....................... ...... ... ....... ... .. ..... ................ .... ..... ....... ....... .. ....... .. ... ....... .. 26
`Intermediate puri licatio11 ... ... .......... ... ..... ............ ........ ......... ....... .......... ........ ......... ....... ...... ....... ...... ........ . 27
`Polishing .. , .................... ... ........... ..... ... .... ............. ......... ... ... .... .... ... .. ..... .. ..... ....... ...... ........ ........ ... .... ... ... 27
`Fast med ia select ion and method deve lopment .... ... .... ................ ... ......... .... .... ...... .. .... .. 30
`Automated media se lection . method development and optimization ...... ... ....... ... ............. ............. ... ........ ...... 30
`Man ual media selection, method development and optimization ... ... ....... ... .. ......... ....... ..... .. ..... .. ... .. .......... ... . 3 1
`Pract ica l considerations for IEX separation ..... .............. ................. ..... .. ........ ..... .... ... .... 34
`pH and ionic strength .................. ..... .. .. ... ......... .. ... ..... .. ................ ..... ....... .... .... ..... ..... .............. ....... ....... .. 34
`
`Anion or cation exchanger ···· ··· ····· ···•···· •·•····•· ···· ·•· ···· ·•···· ··•· •··•·• ·•···· •·• ·· ··• ······• ····· ·• ····• ·•···· .. . ·.·········· ······ ···· 35
`Strong or weak ion exchangers ... .. ....... ........... ... ... ... ....................... ....... ....... .... .... .... ...... ...... ..................... 36
`Buffer selection and preparation .. .......... ....... ...................... .... ........ ....... ....... ... .... ... .... ... .... ... ....... .... ....... .. 37
`Column and media preparation ..... ............. .......... ....... .... ..... ....... ... ... .. ..... ....... ....... ....... ... .. ....... .. ............ .. 39
`Sample preparation ... ....... .......... ....... ............. ..... ..... ... .......... ....... .... ... ... .... ... ...... ..... .... .............. .... ...... .... 39
`Sample application ...... .. ....... ...... .... ... ............ ..... .... .... ..... ....... ....... ....... ....... ....... ....... ....... ..... ....... ........... 40
`Sample load ...... ........... ....... ......... .... ... ............ .. ........ .. .... ....... ....... ....... ....... .......................... ....... ........... 40
`El ution ...... ............... ................... ..... ....... ..•.... ... .... ....... ....... ....... ....... ....... ....... ....... ....... ....... ....... ........... 42
`Linear gradient elution .... ....................... ... .... ................... ....... ....... ..... ....... ....... ....... ....... ....... ....... ........... 42
`Step elution .................... ........... ...... .. ... .... .. ..... ... ............ ....... ....... ....... ....... ....... ....... ..... ....... ....... ........... 44
`pH eluti on .. ............. ................ ......... ....... ... ....... .. ............ ....... ....... ....... ....... .......................... ....... ........... 46
`Flow rates ............. .. ... ..... ....... ............ ... ...... ........ ....... ... .............. ... ... .... .... ...... ....... ....... .... ..... .. ..... ...... ... 46
`Flow con trol ....... .. ..... ....... ... .......... ...... ...... .................... ... ....... ... .... .... .... .. ............ ............ .. ..... ....... .. ..... .. 47
`Wash and re-eq uilibration ........ ..... ...... ........ ....... ........•.... .... ... .... ... ................. .. .. ....... ..... ...... .............. ....... 48
`Detergents. denaturing agents and other additives
`.. ... .. ..... .... ... ... ........ ....... ................. .. ..... .. ....... .. ... ......... 48
`Ana lysis of resu lts and further steps ...... .. ........ .. ........ .... .. ................ .................... .... .. .. 50
`Scali ng-up ...... .... .. .... ........... .... ............ ... ... .. ............ ..... ........... ......... ............ ............ 5 1
`Equ ipment select ion ..... .... .. .... ....... ........ ........ ......................................... ..... ...... ........ 52
`Care of ion exchange media .... ................. .. ... ........................................................... .. 52
`Troubleshoot ing .. ......... .. ... .... ..... ........ ..... .... ..... ............................ ..... ..... .... ... ... .. ... .... . 53
`BioProcess Media - made for bioprocessing .. .................. ......... ..... ...... ... .. ... ..... .. .. ... .... . 58
`Custom Designed Med ia ....... .... .... ..... ... ... .... .. ........... .... .......... ...... ... ... .. ..... .... ..... .. .. .. .. 58
`
`4 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Chapter 3
`Ion exchange media ............................................................................................... 59
`Introduction ....... ........ ......... ................... ... .. ........ ..... .. .. .. ........................................... 59
`MiniBeads: purification or analysis of microgram-milligram quantities
`with highest reso lution ......................... ...................... ... ................................. ............ 60
`Purification options .............. ..................... ....... ....... .................. .... ...... ..................................................... 61
`Purification examples ......... .. .................... ............. .. ............................................................................... .. 62
`Performing a separation ... ........ ........................... ................ ...... .. ................... ............. ........ ....... ......... ... ... 63
`Cleaning ... ....... ....... .......................................................................................................... .............. ........ 65
`Media characteristics ...... ......................................................................................................................... 65
`Chemica l stability .... ..... .................... ........................ ............................................................................... 66
`Storage ... ..... ........ ... .................. ....... .. .................. .... ..... ........ ..................... ..................................... ..... ... 66
`MonoBeads: purification of mil ligram quantities with highest resolution ......... ................. 67
`Purification options ......................... ... ........ ................. .................. ....... ......... .... ......... ........ ....... .............. . 68
`Purification examples ........... .............. .. .................. ..... ..... .................. .... .......... .. ... .. ..... ...... .. ..... ....... ........ 69
`Performing a separation ............................................................... ........ ....... ................................. ............. 71
`Cleaning ................... ............................ ............................................................................ .. ..... ....... ....... . 72
`Media characteristics ............................................................................................................................... 73
`Chemical stability ... ............................................................................... ............. ............ .. .................... ... 73
`Storage ... ............. ......... ...... ................................... ............... .. ..... ....................... ...... ............... ..... ....... .. . 73
`SOURCE: purification at high throughput with high resolution and easy sca le-up .. .. ......... 74
`Purification options .. .. ....... .... .. ........ ..... ...... .... .... ....... ...... .. .......................................................... .... ......... 75
`Purification examples ........ ....... .. ....................... .................................................................... ... ... ..... .... ... . 76
`Performing a separation ... .... ..................................................................................................................... 80
`Cleaning ............................................. ... ........ .. .... .. ........................ .. ........... ......................................... ... 82
`Media characteristics ................................................................................................................. ...... .. ...... 82
`Chemical stability .......... .................. ........................................................................................................ 83
`Storage ........ ........ ..... ........ ...................................................................................... ..... ...... .... ................. 83
`Sepharose High Performance : purification with high resolution ................................... ... 84
`. ........ .......... ....................... .. ..... .. ..... .. ....... 84
`Purification options......................... ..... ...... ..... .........
`Purification examples ............ ....... ...... .... .......... ........ ................................................................... ............. 86
`Scal ing•up ...................................................................... .. ...... ...... ...................................... ..... ....... ........ 86
`Performing a separation ... ..... ............................ ........ ................................................................................ 89
`Cleaning .
`... .... .............
`.. ...................... ........ . 91
`Media characteristics ................................................... ......................................... ........................... ....... . 92
`Chemica l stability .. .................. .. .............................. ...................................... ... .... ... .. ............. ................. 92
`Storage ... ....... ...... ....... .. .... ..... ... .... .. ......... ..... .......................... .... ..................................... ..... ... .... ... .... .... 92
`Sepharose Fast Flow: purification with good resolution and easy sca le-up ........................ 93
`Purification options .......................................................................... ....... .. ......................... .. ..... .. ..... ....... . 94
`Punticatlon examples ........................................................................................................... ..... .............. . 9/
`Performing a separation .... .. ............................ .. ........................................... ..... .. ..... ..... .. ... .................... . 100
`Cleaning ............................. ............. ..................... ................................................................................ 102
`Media characteristics ............ .. ....................................................... ............... ... .... .. ... ........................... .. 103
`Chemica l stability ... ........... ... .. .......... ... ..... ............................................. ................ ...... ......................... . 104
`Storage ................................... ................................................................................................ .. .... ....... . 104
`Sepharose XL - for selected proteins that requ ire very high binding capacity
`to increase productivity, easy scale-up ..... ............ .................. .................................... 105
`Purification options .............. ...... ............ ..... .. ...... ..... ... .. .. .. ... ..... .. ..................... .. ..... .. ..... .. ... .. .. ... .. .. ........ 105
`Purification examples .................................................. .... .. ............................... .. ..... .. ..... .. ..... .. ............... 107
`Performing a separation .... ..... ................... ........... ........ ... ....... ....... ....... ................................................... 109
`Cleaning ................ ...................................... ................ .... ... .... ... .... ... .. ... .. .. ..... .. ..... .. ... .... ... .... ... .... ........ 111
`Media characteristics ................... ............................... .. .. .. ... ..... .. ..................... .. ..... .. ..... .. ... .. .. ... ..... .. ..... 111
`
`5 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Chemical stabi lity .. .. ..... .............. ............................................................ ................... .............. ............ .. 112
`Storage ..................................... ............................................................................................... .. .... .. ..... 112
`Sepharose Big Beads: purification from crude, viscous samples at large scale ...... ... ...... 113
`Purification options ............................. .............. ... ................ .. ... ...................................... .................... 114
`Performing a separation ................. ... ..... .. .............. ........... .. .. ..... .. .. ... .. .. ... .. .... ... .... ... .... .. ..... .... .. ..... .. ....... 115
`Cleaning ............................ .... ........................... ....... ... .. ....... ....... ....... ..... ....... ....... ....... ....... ....... ........... 116
`Media characteristics ................................................... ... .. ..... .. ..... .. ...... .... ... .. .. ... ....... ....... .... .. ..... .. ........ 116
`Chemica l stabi lity ....... ........ ........ ....... ....................... .... .. .. ..... .. ..... .. ..... .. ..... .. ..... .. ..... .. ........................... 117
`Storage ................ ......................................... ......... ..... .. ............ ........................................ ................... 117
`
`Chapter 4
`Ion exchange in a Purification Strategy (C1PP) ........................................................ 119
`App lying C1Pp ............................................................... ............................... ............ 120
`Selection and combination of purification techniques ................................................. 120
`Ion exchange as a capture step ...... ... .... .................................................................... 123
`Ion exchange for intermed iate purification .. .. ............................................................. 125
`Ion exchange as a polish ing step ............................................................................... 127
`Alternative tech niques for po lishing steps .................................................................. 128
`
`Chapter 5
`Chromatofocusing: principles and methods ........................................................... 129
`Introduction ... ............ ......... ...... .... .......................................................................... 129
`Media selection ..... ............... ................................................................................ .. ..... ........ .... ....... ... .... 132
`Buffer selection ... .... .. .............. ..................... ... ..... ........ ............... ....... ...... ......... ... .. ... ................. ... ........ 133
`Purification options ........................................... .... ... .. .................. .......................................... ................ 134
`Buffer options ....................... ............................................................................................................... . 134
`Purification examples ............................................... ... ..................................... ... .. ..... .. ............ ...... ........ 134
`Packing a column ........... ...... ........................ .. ........... .. .. ...... .. ..... ................................... ...... .... .. ............ 136
`Selection of pH gradient and buffers ............................................ ........................................................... 137
`Buffer preparation ......... .. ... ........ .......................... ... .... .... .. ........ ....... ................ .. ..... .. ..... .. ..... .. ..... .. ....... 140
`Sample preparation ............. ............................ ............. ...................... .. ..... .. ..... .. ..... .. ..... .. ...................... 141
`Performing a separation ........................................................ .... ... .... ... .. ... .. .... ... .... ... .... ....... ..... ... .... ........ 141
`Optimization ........... ..... ................ ..................................... ..... ....... ..... ....... ....... ..... ......... ... .... ....... ......... 143
`Troubleshooting ................ ......... .. .. .. ................... ... .... .. .... ....... ....... ....... ....... ..... ....... ....... ....... ....... ......... 144
`Removing Polybuffer .................................................... ...... ......................................... ..... ..................... . 148
`Clean ing .. .. ........................ .. ........................... .. .. .. ... ... .... .............. ....... ... ................ .. ..... ................... ... . 148
`Media charac teristics ........... .. ........... .. ............. ............... ... .. ........ .. .................. ... .... ..... ..... .................... . 149
`Chemica l stabil ity ............................................................................................................. ... .... ... .... ... .... 150
`Storage ............ .......................... ...... ............. ........... ... ................................................... .. .. ..... ....... ....... 150
`Chromatofocusing and CtPP ......... ........... ........ ........................................................... 150
`
`Appendix 1 .......................................................................................................... 151
`Sample preparation ............. .. .. ........ ........ ..... ........................................................... 151
`Sample ~tahility ........ ....................................................... ..... ..
`. ... .. ...... .. ..... .. ....... ... .............. . 151
`Sample clari fica tion ................................................................................ ....... .. .................. ...... ....... ....... 152
`Specific sample preparation steps ........ .. ............................................... .................... 153
`Resolubi lization of protein precipitates .... .. ........................................................ ......................... ....... .. .... 156
`Buffer exchange and desalting ... ........... .................................... ................................ 156
`Removal of lipoproteins ........... .......... .... .. ........... ..... ......... ................. ....................... 159
`Removal of phenol red .............................. ............... .... ............................................ 159
`Removal of low molecu lar weight contaminants .......................................................... 159
`
`6 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Appendix 2 .......................................................................................................... 160
`Non-volatile and volati le buffer systems ............... ........................................ .............. 160
`Non-volatile buffers for anion exchange chromatography ......... .. .... ........ .. .... ............... .. .................. .. ..... .. ... 160
`Non-volatile buffers for cation exchange chromatography ... .... ....... ....... ............... .................... ....... ....... ..... 161
`Volatile butter systems ................................... .......................................................... ....... .... .... .. ....... ... ... 161
`
`Appendix 3 .......................................................................................................... 162
`Column packing and preparation ........ ........... ................................ .... ............ ............ 162
`Column selection ............... ....... ....... .... .. ............................. .. ..... ..... .... ... ....................... .. .......... .... ......... 164
`Column packing and efficiency ........ .. ....... .. .......... ................... ........................ ....... .. ... .. ....... ..... .. ..... .. .... 164
`
`Appendix 4 .......................................................................................................... 167
`Selection of purif ication equipment ........................................................................... 167
`
`Appendix 5 .......................................................................................................... 168
`Converting from linear f low (cm/hour) to volumetric flow rates (m l/m in)
`and vice versa .......... .... ............. ......... ....... ..................... ......... ........ ...... .. ................ 168
`
`Appendix 6 .......................................................................................................... 169
`Conversion data: proteins, column pressu res .. ..................... ..... .................................. 169
`Colu mn pressures ........... .................... ......................................................... ..... ........................ ............. 169
`
`Appendix 7 .......................................................................................................... 170
`Table of am ino ac ids ............ ...... .... .. .. .......... ... .. .... ................. ................... ............... 170
`
`Appendix 8 .......................................................................................................... 172
`Analytical assays during purification .......................................................................... 172
`
`Appendix 9 .......................................................................................................... 174
`Storage of biologica l samp les .... .. .......... .......... ..... .. ............ ..... ........ ........ ................. 174
`
`Appendix 10 ........................................................................................................ 175
`Column cleaning .. ........... ..... .... ....... .. ... ....... .................... .. ............. ........ .......... ....... 175
`Removal of common contaminants ................................................................... ....... ............................ .... 175
`Removal of precipitated proteins, lipids, hydrophobica lly bound proteins or lipoproteins .............. ....... ... ...... . 175
`
`Additional reading ............................................................................................... 178
`References .......................................................................................................... 179
`Ordering information ............................................................................................ 180
`Ion exchange ......... ...... ............ .... ............. .... .. ........... ......... ..... .. .. ... ..... ....... .. .... ... ... 180
`Chromatofocusi ng ......................... .... ....... ........................................ ....... .... ... .. ....... . 183
`
`Product index ...................................................................................................... 184
`
`7 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`8 of 188
`
`Fresenius Kabi
`
`Exhibit 1036
`
`8 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Introduction
`Biomolecules are pm·ified using chromatography techniques that separate them according to
`differences in their specific properties, as shown in Figure 1. Ion exchange chromatography
`(IEX) separates biomolecules according to differences in their net surface charge.
`
`Property
`
`Charge
`
`Size
`Hydrophobfcfty
`
`Biorecognition (ligand specificity)
`
`Technique
`
`Ion exchange chromatography (IEX), chromatofocusing (CF)
`
`Gel filtration (GF). also called size exclusion
`Hydrophobic interaction chromatography (HIC)
`Reversed phase chromatography (RPC)
`Affinity chromatography (AC)
`
`-
`
`Gel filrrarion
`
`Hydrnphobic interaction
`
`Jon exchange
`
`Affinity
`
`Reversed phase
`
`Fig. 1. Separation principles in chromatographic purification.
`
`JEX for the separ ation of biomolecules was introduced in the 1960s and continues to play
`a major role in the separation and purification of biomolecules. Today, IEX is one of the
`most frequently used techniques for purification of proteins, peptides, nucleic acids and
`other charged biomolecules, offering high resolution and group separations with high loading
`capacity. The technique is capable of separating molecular species that have only minor
`differences in their charge properties, for example two proteins differing by one charged
`amino acid . These features make TEX well suited for capture, intermediate purification or
`polishing steps in a purification protocol and the technique is used from microscale purification
`and analysis through to purification of kilograms of product.
`
`This handbook describes both theoretical and pra-ctical aspects principles of the technique,
`the media available and how to select them, application examples and detailed instructions
`for the most common ly performed procedures. Practical information, w ith many tips and
`hints drawn from over forty years of experience in chromatography purification, guides
`beginners and experts towards obtaining the best possible results from the latest chxomato(cid:173)
`graphic media.
`
`7
`
`9 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`The final chapter includes information on chromatofocusing, another chromatography
`technique that separates biomolecules on the basis of charge, but, in this case, according
`ro differences in their isoelecrric points. This technique can provide very high resolution
`separations for specific applications. Proteins with a difference as small as 0.02 pH units
`in their isoelectr ic points can be separated in laboratory-scale applications.
`
`Amersham Biosciences offers a wide variety of prepacked columns and ready-to-use
`c hromatography media . A range of handbooks ensure that purification with any
`chromatographic technique becomes a simple and efficient procedure at any scale and
`in any laboratory.
`
`Symbols
`
`-chis symbol indicates general advice wh ich can improve procedures or provide
`recommendations for action under specific situations.
`
`this symbol denotes advice wh ich should be regarded as mandatory and gives a warning
`when special care should be taken.
`
`this symbol highlights troubleshooting advice to help analyze and resolve difficulties that
`may occur.
`
`t
`/
`
`chemicals, buffers and equipment.
`
`experimental protocol.
`
`10 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Common abbreviations
`
`In chromatography
`
`IEX: ion exchange chromatography (also seen as IEC in the literature )
`
`GF: gel filtration (sometimes referred to as SEC: size exclusion chromatography)
`
`AC: affinity chromatography
`
`RPC: reverse phase chromatography
`
`HIC: hydrophobic interaction chromatography
`
`CF: chromatofocusing
`
`OPP: Capture, Intermediate Purification and Polishing
`
`CV: colume volume
`
`p Ka: the pH at which an acid is 50% dissociated
`
`pf: isoelectric point, the pH at wbich a protein has zero net surface charge
`
`MPa: megaPascal
`
`psi: pounds per square inch
`
`SDS: sodium dodecyl sulfate
`
`A2sonm, A2140m: UV absorbance at specified wavelength
`
`M,: relative molecular weight
`
`N : column efficiency expressed as theoretical plates per meter
`
`R 5: resolution, the degree of separation between peaks
`
`Abbreviations found in product names
`HMW: high molecular weight
`
`LMW: low molecular weight
`
`Tricorn PE: column manufactured in PEEK (polyetheretherketone)
`
`Tricorn GL: column manufactured in glass
`
`HR: high resolution
`
`PC: precision coh1mn
`
`9
`
`11 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Ill
`10
`
`12 of 188
`
`Fresenius Kabi
`
`Exhibit 1036
`
`12 of 188
`
`Fresenius Kabi
`Exhibit 1036
`
`

`

`Chapter 1
`Principles of ion exchange
`This chapter provides a general introduction to the theoretical principles that underlie every
`ion exchange separation. An understanding of these principles will enable the separation
`power of ion exchange chromatography (JEX) to be fully appreciated. Practical aspects of
`performing a separation are covered in Chapter 2.
`Net surface charge and pH
`JEX separates molecules on the basis of differences in their net surface charge. Molecules
`vary considerably in their charge properties and will exhibit different degrees of interaction
`with charged chromarography media according to differences in their overall charge, charge
`density and surface charg