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Handbook of Methods and Instrumentation in Separation Science Volume 1

First Edition

Ian D. Wilson

AstraZeneca Pharmaceuticals, Macclesfield, UK

Colin F. Poole

Wayne State University, Detroit, USA

Boston  •  Heidelberg  •  London  •  New York  •  Oxford

Paris  •  San Diego  •  San Francisco  •  Singapore  •  Sydney  •  Tokyo

Academic Press is an imprint of Elsevier

Table of Contents

Cover image

Title page

Copyright page

Preface

Affinity Membranes

General Characteristics of Membrane Chromatography

Membrane Geometry

Membrane Material, Activation and Ligand Coupling

Affinity Ligands

Scale-up

Applications

Conclusions

Affinity Partitioning in Aqueous Two-Phase Systems

Aqueous Two-phase Systems in General

Affinity Partitioning

A Simple Theory for Affinity Partitioning

Experimental Results

Types of Affinity Ligands Used

Preparative Extractions

Countercurrent Distribution

Use of Dextran as a Ligand Carrier

Use of a Third Polymer as Ligand Carrier

Chiral Affinity Partitioning

Analytical Uses

Multiphase Systems

Semi-organic Systems

Affinity Partitioning of Nucleic Acids and Bioparticles

Future Prospects

Conclusions

Affinity Separations

Introduction

Biological Recognition

The Affinity Process

Matrices

Covalent Bonding

Intermolecular Binding Forces

Analytical Scale-Up

Affinity versus Traditional Media

Synthetic Ligands

Rational Design of Affinity Ligands

Combinatorial Libraries

Regulations and Drug Master Files

Alternative Affinity Approaches

Affinity Membranes

Conclusion

Analytical Ultracentrifugation

Theoretical Background

Instrumentation and Experimental Considerations

Data Analysis Methods

Sedimentation Velocity

Sedimentation Equilibrium

Conclusions

Biochemical Engineering Aspects of Affinity Separations

Introduction

Position in Process Flow Sheet – Direct Recovery

Choice of an Appropriate Affinity Ligand and Immobilization to a Support

Operating Protocols, Equipment, Monitoring and Control

Scale-up and Validation

Optimization

Theoretical Modelling

Economic Considerations

Conclusions and Future Prospects

Centrifugation

Introduction

Theory

Types of Separation

Centrifugal Equipment

Acknowledgments

Chromatography

Introduction

The family of chromatographic techniques

Mode of zone displacement

Chromatogram

Peak shape models

Flow through porous media

Zone broadening

Separation quality

General elution problem

Multidimensional and multimodal chromatography

Mode selection

Instrumentation

Conclusion

Countercurrent Chromatography: Extrusion–Elution

Introduction

Advantages of a Liquid Stationary Phase

Countercurrent Chromatographs

Band Broadening inside the Column

Resolution between Peaks inside the Column

The Elution–Extrusion Method

Practical Demonstration

Estimation of the Hydrophobicity of a Natural Extract

Conclusion

Countercurrent Chromatography: Instrumentation

Introduction

What is Countercurrent Chromatography?

Apparatus

Theory

Resolution

Solvent Systems

Other Chromatographic Approaches

pH-Zone Refining

Conclusion

CCC Literature

Countercurrent Chromatography: Large-Scale

Introduction

The Basic Principles of CCC and its Predictability

Humble Beginnings

The Engineering of Reliable Robust Centrifuges

The Development of the Advanced Bioprocessing Centre for the Rapid Scaleup from Drug Discovery to Phase 1 Trials

The Key Discoveries that Have Led to the Growth of CCC as a Viable Large-Scale Liquid–Liquid Chromatography System

A Developing Understanding of the Efficiency of the Process

Large-Scale Applications

The Next Phase of Development of the Technology

Countercurrent Chromatography: Overview

Introduction

The Two Basic CCC Systems

Hydrostatic CCC Systems

Hydrodynamic CCC Systems

Standard Procedure of CCC

Variations of Countercurrent Chromatography

Conclusions

Covalent Chromatography

Introduction

Development of the Technique

Applications of Covalent Chromatography

Concluding Comments

Decanters

Introduction

Description

Applications

Dye Ligands

Introduction

Development of Dye Ligands and Dye Affinity Adsorbents

Development of the Techniques

Instrumentation

Introduction to Applications

Gas Chromatography: Chiral Separations

Introduction

Methodology

Classification of Chiral Stationary Phases

Thermodynamics of Enantiomer Separation

Enantiomerization

Assignment of Absolute Configurations by Enantioselective GC

Method of Enantiomer Labelling

Precision and Sources of Error

Practical Considerations

Gas Chromatography: Column Technology

Introduction

Comparisons of Packed and Open-Tubular Columns

The Stationary Phase

Low-Bleed Columns

Conclusions

Glossary

Gas Chromatography: Derivatization

Introduction

Esterification

Acylation/Alkylation

Cyclization

Peralkylation

Conclusion

Gas Chromatography: Detectors: General

The Flame Ionization Detector

The Thermal Conductivity Detector

Gas Chromatography: Detectors: Infrared Spectrometry

Light-Pipe-Based GC-IR Instruments

Limits of Detection and Identification

Matrix-Isolation GC-IR

Direct-Deposition GC-IR

Prognostication

Gas Chromatography: Detectors: Ion Mobility Mass Spectrometry

Introduction

IMS Theory and Instrumentation

Application Areas

The Future

Gas Chromatography: Detectors: Mass Spectrometry

Introduction

Ion Formation

The Separation of Ions and Recording of Mass Spectra

Interfacing Mass Spectrometers with Gas Chromatographs

GC-MS Experiments

Other Techniques

Conclusions

Gas Chromatography: Detectors: Selective

Introduction

Element-selective detectors

Multi-element selective detectors

Other element-selective detectors

Compound-selective detectors

Conclusion

Gas Chromatography: Gas–Solid Chromatography

Introduction

Molecular Sieves

Alumina Adsorbents

Porous Polymers

Carbon Adsorbents

Silica

Metal PLOT Columns

Conclusion

Gas Chromatography: Headspace Gas Chromatography

Introduction

Fundamentals of Static HS-GC

Sensitivity of Static HS-GC

Instrumentation for Headspace Sampling

Quantitative Headspace Analysis

Classification of Sample Types

Practical Example: Determination of Ethylene Oxide in a PVC Tube

Applications

Gas Chromatography: High-Speed Gas Chromatography

Introduction

Volatile Organic Chemicals

Pesticides

PAHs

Solvent Purity

Miscellaneous

Future Developments

Gas Chromatography: High-Temperature Gas Chromatography

Introduction

Instrumentation for HTCGC

Applications of HTCGC

Conclusion

Gas Chromatography: Historical Development

Column Development

Instrumentation

Future Developments

Gas Chromatography: Large-Scale Gas Chromatography

Introduction

Principle

Interesting Characteristics

Limitations

Implementation

Variants

Prep GC: What Scale?

Application examples

Economy of Prep GC: Example

Conclusion

Gas Chromatography: Multidimensional Gas Chromatography

Multidimensional Concepts

Basic Instrumental Requirements and Considerations

Expanding System Capacity

MDGC for Trace Enrichment

Use of a Series of Parallel Heart-cut Reservoirs/Traps

Backflushing

Pressure Tuning

Comprehensive Multidimensional Chromatography

Comprehensive Gas Chromatography

Gas Chromatography: Pyrolysis Gas Chromatography

Introduction

Terminology

Modes of Pyrolysis

Sampling

Applications

Conclusions

Gas Chromatography: Sampling Systems

Introduction

Gas samples

Solid samples

Liquid samples

Packed Columns

Capillary columns

Split injection

Splitless injection with solvent trapping

Cold on-column injection

Programmed-temperature vaporizing injector

Cooled-needle vaporizing injector

Conclusion

Gas Chromatography: Theory of Gas Chromatography

Introduction

Stationary Phase

Mobile Phase

Methods of Chromatography

The General Elution Problem

Future Developments

Hydrodynamic Chromatography

Summary

Definition and General Features

Equipment

Experimental Parameters

Mechanism and Other Methods

Conclusion

Hydrophobic Interaction Chromatography

Introduction

Fundamentals of Hydrophobic Interaction Chromatography

Optimization of Hydrophobic Chromatographic Systems

Conclusions

Immobilized Boronates/Lectins

Applications

Problems or Pitfalls

Applications of Boronate Chromatography

Lectin Chromatography

Conclusion

Immobilized Metal Ion Chromatography

Introduction

Background

Components

Practical Considerations

Conclusion

Acknowledgments

Immunoaffinity Chromatography

Introduction

Antibodies

Immunoaffinity Supports

Retention and Elution in Immunoaffinity Chromatography

‘Analytical’ Applications of Immunoaffinity Chromatography

Protein Purification by Immunoaffinity Chromatography

Conclusions

Imprint Polymers

Introduction

The Imprinting Principle

The Preparation of Molecularly Imprinted Polymers

Physical Form of Imprinted Polymers

Applications in Separation Technology

Conclusions

Large-Scale Centrifugation

Introduction

Sedimentation centrifuges

Decanter centrifuges

Disc stack centrifuges

Solid bowl and tubular centrifuges

Hydrocyclones

Filter centrifuges

Summary

Liquid Chromatography–Gas Chromatography

Introduction

Purposes of Coupling LC to GC

Transfer of LC Fractions

Applications

Conclusion

Liquid Chromatography: Column Testing and Evaluation

Introduction

Properties Assessed in Testing

Column Tests

Liquid Chromatography: Derivatization

Introduction

General Approaches to Derivatization in Liquid Chromatography

Specific Recommendations for Successful Application of Derivatization in Liquid Chromatography

Problems and Pitfalls in Using Derivatization in Liquid Chromatography

Conclusions and Summary

Liquid Chromatography: Detectors: Evaporative Light Scattering

Physical properties of the nebulizer

Intensity of the scattered light

The performance of the Light-Scattering Detector

Conclusions

Liquid Chromatography: Detectors: Fluorescence Detection

The Fluorescence Detectors

The Multi-wavelength Fluorescence Detector

Conclusion

Liquid Chromatography: Detectors: Infrared Spectrometry

Introduction

LC/IR Transport Interfaces

The In-Line Flow Sensor

Conclusion

Liquid Chromatography: Detectors: Laser Light Scattering

Introduction

Alternative Light Scattering Detectors

Low Angle Laser Light Scattering Detector

Multiple Angle Laser Light Scattering (MALLS) Detector

Liquid Chromatography: Detectors: Mass Spectrometry

Introduction

Background

Ion Formation

Mass Analysis

Tandem Mass Spectrometry

Conclusion

Liquid Chromatography: Detectors: Nuclear Magnetic Resonance

Introduction

NMR Flow Cell Design

Experimental Set-up

LC-NMR Coupling

Continuous-flow Experiments

Stopped-flow Experiments

SPE-LC-NMR Coupling

GPC-NMR Coupling

SFC-NMR and SFE-NMR Coupling

Capillary Separations

Liquid Chromatography: Detectors: Refractive Index Detectors

Introduction

The Christiansen Effect Detector

The Interferometer Detector

The Thermal Lens Detector

Conclusion

Liquid Chromatography: Detectors: Ultraviolet and Visible Detection

Introduction

Measuring Concentration, Beer’s Law

Types of Absorbance Detectors

Design of Detectors

How to Get the Best Performance from Your Detector and How to Evaluate Manufacturer’s Specifications

Separation System Considerations

Liquid Chromatography: Electrochromatography

Introduction

Theory

Experimental Considerations

Applications

Conclusion

Liquid Chromatography: Flash Chromatography

Dry-column Chromatography

Vacuum Chromatography

Flash Chromatography

Stationary Phases

Sample Loading

Method Development

Detection

Future Developments

Liquid Chromatography: Historical Development

The Beginning

The First Followers

The Renaissance

From Empirism to Science

Towards HPLC

High Performance Liquid Chromatography

Liquid Chromatography: Instrumentation

The Instrumental Set-Up

Related HPLC Techniques

Liquid Chromatography: Large-Scale Liquid Chromatography

Introduction

Particular Effects Related to Column Overloading

Column Technology

Packing Materials

Optimization of Separation Conditions

Simulated Moving Bed

Conclusions

Liquid Chromatography: Mechanisms: Chiral

The Basis of Chiral Recognition

Chiral Recognition Mechanisms

Using Molecular Chiral Recognition to Select a HPLC-CSP

Conclusion

Liquid Chromatography: Mechanisms: Gradient Polymer Chromatography

Classical Precipitation Chromatography

High Performance Precipitation Liquid Chromatography (HPPLC)

Sudden-Transition Gradient Chromatography of Synthetic Polymers

Liquid Chromatography: Mechanisms: Ion Chromatography

Introduction

Stationary Phases for IC

Mobile Phases for IC

Detection in IC

Analytical Performance of IC

Liquid Chromatography: Mechanisms: Ion Exclusion Chromatography

Introduction

Background

Selected Applications

Neutral Compounds

Conclusion

Liquid Chromatography: Mechanisms: Ion-Pair Chromatography

Introduction to Ion Pair Chromatography

Retention Theories for Ion Pair Chromatography

Parameters Controlling the Retention factor in Ion Pair Chromatography

Choice of Experimental Parameters in IPC

Liquid Chromatography: Mechanisms: Micellar Liquid Chromatography

Introduction

Equations Describing Solute Retention in Micellar Liquid Chromatography

Efficiency

Elution Strength of Micellar Mobile Phases

Separation Selectivity

Applications

Conclusion

Liquid Chromatography: Mechanisms: Normal Phase

Introduction

The Nature of the Surface of Silica Gel

Mono-Layer Adsorption

Interactive Mechanisms on the Surface of Silica Gel in Normal-Phase Liquid Chromatography

Liquid Chromatography: Mechanisms: Reversed Phase

Overview

The Analyte

The Mobile Phase

The Stationary Phase

General Models

Liquid Chromatography: Mechanisms: Size-Exclusion Chromatography

Introduction

SEC – The Basic Technique

SEC with FTIR

SEC with Light Scattering Detection

SEC with Viscosity Measurement

Future Prospects

Liquid Chromatography: Medium Pressure Liquid Chromatography

Introduction

Instrumentation

Column Packing

Solvent Selection

Sample Introduction

Applications: MPLC in Natural Product Isolation

Conclusion

Liquid Chromatography: Monolithic Columns

Introduction

Silica-Based Monoliths

Polymeric Monoliths

Benefits and Limitations of Monolithic Columns

Liquid Chromatography: Multidimensional Chromatography

Basic Theory and Configurations

Scope of Multidimensional Liquid Chromatography

Future Developments

Liquid Chromatography: Theory of Liquid Chromatography

Stationary Phase

Mobile Phase

Modes of Liquid Chromatography

The General Elution Problem

Chromatographic Retention

Peak Shape and Broadening

Column Resolution

Future Developments

Liquid Chromatography: Ultra-Performance Liquid Chromatography

Introduction

History and Background

Speed and Performance of UPLC Columns

Instrument Design Considerations

An Application Example

Future Development

Macromolecular Interactions: Characterization by Analytical Ultracentrifugation

Sedimentation Velocity and Sedimentation Equilibrium

Ultracentrifuge Studies of Solute Self-association

Interactions Between Dissimilar Reactants

Studies of Ligand Binding by Sedimentation Equilibrium

Future Developments

Method Validation

Introduction

Terms and Definitions

System Suitability

Validation versus the Type of Method

Paper Chromatography

Introduction

The practice of paper chromatography

Modes of paper chromatography

Applications of paper chromatography

Paper electrophoresis

The practice of paper electrophoresis

Applications of paper electrophoresis

Detection and quantification of substances on paper chromatograms or electropherograms

Conclusions

Acknowledgment

Supercritical Fluid Chromatography: Chiral

Introduction

Formation of diastereomers by using a pre-column derivatization

Formation of labile diastereomers in the mobile phase

Use of chiral stationary phases

Applications

Conclusion

Supercritical Fluid Chromatography: Detection: Infrared Spectrophotometry

Flow Cell SFC/FTIR

Mobile-phase Elimination SFC/FTIR

Future Developments

Supercritical Fluid Chromatography: Historical Development

Discovery of Supercritical Phenomena

Early Developments

Capillary SFC

Developments in the Use of Fluids

Infrastructure Development

Future Developments

Supercritical Fluid Chromatography: Instrumentation

Introduction

Common Fluids

Hardware

Pumps

Other Desirable Pump Characteristics

Supercritical Fluid Chromatography: Large-Scale Separations

Introduction

Principle

Important Characteristics

Limitations

Implementation

Prep-SFC: What Scale?

Applications

Economics of Prep SFC

Lab-Prep-SFC

Conclusions

Supercritical Fluid Chromatography: Theory of Supercritical Fluid Chromatography

Theory of SFC

The Relationship between Speed and Diffusion in Chromatography

Effect of Physical Parameters on Retention

Method Development

Summary of Solutes and Elution Conditions

Open Tubular or Capillary SFC

Future

Theory and Development of Affinity Chromatography

Introduction

Early Developments in Affinity Chromatography

Spacer Arms

Some Quantitative Parameters

Types of Ligand

The Matrix

The Elution Step

Affinity Chromatography of Recombinant Proteins

Future Developments

Theory of Centrifugation

Introduction

Sedimenting Centrifuges

Filtering Centrifuges

Thin-Layer Chromatography: Chiral Separations

Introduction

Chiral Stationary Phases and Chiral Coated Phases

Chiral Mobile Phases

Retention and Resolution Data

Quantitative Analysis of TLC-Separated Enantiomers

Conclusions

Thin-Layer Chromatography: Detection: Densitometry and Image Analysis

Introduction

The Development of Modern Scanning Densitometry

Theory of Spectrodensitometry

Pre-scanning Considerations

Instrumentation

Applications

Video Densitometry

Future Trends

Thin-Layer Chromatography: Detection: Flame-Ionization Detection

General Considerations

Marine Lipids

Heavy Hydrocarbon Fractions

Conclusion

Thin-Layer Chromatography: Detection: Radioactivity Detection

Introduction

Detection and Measurement

Comparison of TLRC Detection Methods

Future Developments in TLRC

Thin-Layer Chromatography: Development: Conventional

Linear Development

Continuous Linear Development

Multiple Development

Radial Development

Conclusions

Thin-Layer Chromatography: Development: Forced Flow and Centrifugal

Introduction

Overpressured Layer Chromatography

Rotation Planar Chromatography

Comparison and Outlook of FFPC Methods

Thin-Layer Chromatography: Historical Development

History

Plate Material

Theoretical Foundation

Instrumentation

Thin-Layer Chromatography: Instrumentation

Introduction

Sample Application

Chromatogram Development

Densitometric Chromatogram Evaluation

Post-Chromatographic Derivatization

Combination of Planar Chromatography with Other Techniques

Future Trends

Thin-Layer Chromatography: Large-Scale Separations

Introduction

Parameters of PPC Separation

Stationary Phase

Mobile Phase

Vapour Phase

Development Mode

Separation Distance

Sample Application

Location and Removal of Separated Compounds

Selection of Appropriate PPC Method

Comparison and Outlook of PPC Methods

Thin-Layer Chromatography: Layer Properties

Silica Gel

Chemically Bonded Layers

Aluminium Oxide (Alumina)

Cellulose

Polyamide

Kieselguhr (Diatomaceous Earth)

Ion Exchangers

Impregnated Layers

Pre-scored, Channelled, Preconcentration Zone and Preparative Plates

Binders

Supports

Fluorescent Indicators

Conclusions

Thin-Layer Chromatography: Spray Reagents

Introduction

Non-destructive methods

Reversible reactions

Non-reversible reactions

Spraying versus dipping

Future Prospects

Thin-Layer Chromatography: Theory of Thin-Layer Chromatography

Introduction

Basic Equations

Flow Velocity of the Mobile Phase

Band Broadening and Plate Height Equation

Conclusion

Appendix: Symbols used

Index

Copyright

This book is printed on acid-free paper.

Copyright © 2009 Elsevier Ltd. All Rights Reserved.

Material in the work originally appeared in Encyclopedia of Separation Science, edited by Ian D. Wilson, Edward R. Adlard, Michael Cooke and Colin F. Poole (Academic Press 2000)

The following article is Canadian Crown Copyright

Large-Scale Centrifugation

Copyright © 2000 Minister of Public Works and Government Services, Canada

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press is an imprint of Elsevier

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Preface

Colin F. Poole, Editor

This handbook is directed to the needs of scientists requiring concise overviews and targeted summaries of the main methods employed for separations. It is a work derived from the successful Encyclopedia of Separation Science on-line edition with a new focus on the fundamentals of methods and instrumentation employed in separations. These days it is a simple task to obtain a massive amount of information on any separation method by a keyword search. For the busy professional the problem is fishing out just the information that one needs at that particular moment. This requires a more targeted resource and simplified search mechanism as provided by this handbook. The knowledge in any field has grown exponentially in modern times and the idea of a generalist in science has disappeared to be replaced by specialists with a detailed knowledge of a narrow spectrum of modern science. Outside our specialist area we all become novices and rely upon general sources of information for orientation before taking on assimilating the detailed information required for a research project. This is the interface that this handbook was conceived to fill. In a single source it affords access to general background material to enable the professional to quickly assimilate, sort, and interpret the dense information associated with a comprehensive literature search.

Separation science is a very diverse topic and to give the current work some structure and organization that is simple to follow the editor has adopted a general hierarchical structure arranged alphabetically for the primary separation methods. These methods are indicated as affinity, centrifugation, chromatography, crystallization, distillation, electrophoresis, extraction, floatation, ion exchange, membranes, and particle size. Each method has a general overview article associated with it for quick orientation of the main features of the method and its general uses. Again, arranged alphabetically, each method has associated with it a series of secondary articles that provide a spotlight on a specific topic. For example, from the general article on chromatography, the reader is guided to seek specific information on the main methods of chromatography identified as gas chromatography, liquid chromatography, supercritical fluid chromatography, thin-layer (planar) chromatography, and countercurrent chromatography. Through these associated articles the reader is guided to targeted articles on the historical development, theory, column chemistry, separation mechanisms, and instrumentation by different numbers of articles based on a comprehensive coverage of each topic. Thus, it takes little time to enter an unfamiliar field and quickly arrive at an article at the required detail for the readers purpose, be that basic or at the forefront of knowledge.

The diversity of separation methods also requires the input of many contributors who have just the expert knowledge required to authoritatively describe the core knowledge in their specialization. The handbook can be considered a compilation of the essence of separations written by individual specialists to enable fellow professionals to quickly launch projects in areas other than their own specialization and to share their knowledge in a condensed and readily accessible form. The success of this work is due entirely to the support and enthusiasm of the talented team of authors assembled for this work.

This handbook is a guide providing general information concerning its subject matter; it is not a procedures manual. The readers should consult current procedural manuals for state-of-the-art instructions and applicable government safety regulations. The publisher and authors do not accept responsibility for any misuse of this handbook, including its use as a procedural manual or as a source of specific instructions.

Affinity Membranes

K. Haupt    Lund University, Lund, Sweden

S.M.A. Bueno    Universidade Estadual de Campinas, Brazil

The rapid development in biotechnology and the large potential of biomolecules for applications in medicine, food industry and other areas, result in an increasing demand for efficient and reliable tools for the purification of proteins, peptides, nucleic acids and other biological substances. This situation is being additionally enforced by the increasing number of recombinant gene products that have arrived on the market or that are currently being investigated, such as insulin, erythropoietin and interferons. The recovery of fragile biomolecules from their host environments requires their particular characteristics to be taken into account for the development of any extraction or separation process. On the other hand, there is a demand for techniques that can easily be scaled up from laboratory to industrial production level.

In this context, the use of affinity methods has the advantage that coarse and fine purification steps are united through the introduction of a specific recognition phenomenon into the separation process. The most widely used method for preparative affinity separation of biomolecules is liquid chromatography on beaded resins (soft gels). Despite the commercial availability of many affinity ligands immobilized on to gel beads for use in column chromatography, there are some drawbacks in a large scale application of these supports. Flow rates and thus performance are limited by the compressibility of the resins and pore diffusion. Because of these intrinsic limitations, other chromatographic techniques, such as perfusion chromatography, or different separation techniques, such as affinity precipitation and affinity phase partitioning, have been suggested as possible alternatives. Another technique that is gaining increasing importance is membrane-based separation. Adsorptive membrane chromatography was introduced as a purification method in the mid 1980s. Microporous membranes have been successfully coupled with biological or biomimetic ligands, yielding affinity membrane chromatography supports. Several of them, with for example protein A and G, dye or metal chelate ligands, are commercially available. Affinity membrane chromatography is in fact a hybrid technique combining affinity gel chromatography and membrane filtration, with the advantages of the two technologies.

The purpose of the present review is to discuss relevant aspects and developments that are important for the design of an affinity membrane chromatography process, including the choice of the membrane material, coupling chemistry, affinity ligands, membrane configurations, operation modes and scale-up. In a wider sense, membrane-based affinity fractionation also comprises affinity filtration methods where the target molecule binds to an affinity ligand coupled to nanoparticles, which can then be separated by filtration through a membrane. However, this application will not be discussed here in detail.

General Characteristics of Membrane Chromatography

In contrast to chromatographic supports based on beaded resins with dead end pores, membrane chromatographic supports have through-pores and lack interstitial space. Mass transfer is mainly governed by forced convection and pore diffusion is negligible. The observed back-pressures are normally quite low, and high flow rates and thus high throughputs and fast separations become possible without the need for high pressure pumps or equipment. As the association time for an antibody–antigen complex is typically about 1 s or less, but the diffusion of a protein molecule to the centre of a 50 μm porous bead takes tens of seconds, in a membrane support, the low diffusional limitation leads to faster adsorption kinetics and higher throughput efficiency. Little deterioration of the separation efficiency occurs even at elevated flow rates. On the other hand, with affinity membranes the formation of the affinity complex can become the rate-limiting process at high flow rates.

A problem often encountered in membrane chromatography is extra-cartridge back-mixing, which can severely degrade membrane performance. This phenomenon is due to dead volumes outside the membrane, in tubing, fittings and valves, and leads to peak broadening and dilution. It is more pronounced in membrane chromatography systems compared to conventional columns packed with beaded supports, owing to the larger throughput/bed volume ratio.

Although the specific surface area of membranes is typically only 1% of that of conventional chromatographic resins, microporous membrane systems have high internal surface areas and reasonably high capacities. The open-pore structure of membranes increases the accessibility of affinity ligands and reduces steric hindrance compared to small-pore adsorbents.

Membrane Geometry

Just like filtration membranes in general, affinity membranes can be produced in different configurations, and membrane modules of various geometries are commercially available or have been manufactured in research laboratories (Figure 1).

Figure 1 Different geometries of affinity membranes. (A) Flat sheet; (B) stack of flat discs; (C) hollow fibre; (D) spiral-wound flat sheet; (E) continuous rod. The arrows indicate flow directions. (Adapted from Journal of Chromatography 702, Roper DK and Lightfoot EN, Separation of biomolecules using adsorptive membranes, pp. 3–26, Copyright 1995, with permission from Elsevier Science.)

Flat sheet or disc membranes can be mounted as individual membranes in specially designed cartridges or in commercial ultrafiltration units for use in dead-end filtration mode. This allows for the production of inexpensive single- or multiple-use devices for the rapid adsorption of a target molecule from dilute samples in batch or continuous recycling mode. Cartridges are also available that allow for operation in cross-flow filtration mode.

Stacks of flat membrane discs have been employed for affinity membrane chromatography in column-like devices, the main purpose being to increase the adsorption capacity. Another configuration is continuous rod-type membranes which can be directly cast in a chromatographic column. Both types of membrane columns are compatible with conventional high performance liquid chromatography or fast protein liquid chromatography systems and have advantages over columns packed with beaded resins, as described above. Being highly porous with a mean pore diameter of 0.1–10 μm, they allow for efficient separations even at high flow rates.

If the target molecule is to be recovered from complex feed solutions such as cell homogenates or blood plasma, or from solutions containing high molecular mass additives such as antifoam agents or even particulate material, the use of membranes in dead-end filtration mode is often impossible due to membrane fouling. A remedy to this problem is the operation in cross-flow filtration mode where the build-up of a polarization layer at the membrane surface is avoided or diminished. Hollow-fibre membranes are well adapted for such applications. They are usually mounted as bundles in tubular cartridges. Another configuration are flat-sheet membranes that are spiral-wound around a cylindrical core. Both systems have the advantage of high surface area/cartridge volume ratios and high operational capacities.

Membrane Material, Activation and Ligand Coupling

Membrane Material

Due to the specific properties of biomolecules, the membrane materials to be used for their separation should ideally possess the following characteristics:

• Macroporosity: This will allow biomolecules to cross the membrane and to access the affinity sites.

• Hydrophilicity: Using hydrophilic supports, nonspecific adsorption by hydrophobic interactions and denaturation of biomolecules can be avoided.

• Presence of functional groups: These are required for the coupling of an affinity ligand.

• Chemical and physical stability: The material has to withstand the sometimes harsh conditions during derivatization, operation and regeneration.

• Biocompatibility: This is particularly important if the membranes are used in extracorporeal devices, for example for blood treatment.

• Large surface area relative to membrane volume: This will allow for the construction of small, integrated devices with high operational capacities.

Cellulose and cellulose acetate were among the first materials that have been used for affinity membrane preparation. They are hydrophilic and biocompatible, and due to the presence of hydroxyl groups, ligand coupling can be easily achieved using for example CNBr or carbonyldiimidazole activation. In order to improve the mechanical and chemical stability of cellulose membranes, chemical cross-linking with epichlorohydrin is sometimes carried out. Cellulose membranes normally have a rather small pore size, resulting in a high pressure drop. Attempts to produce membranes with larger pores using coarse cellulose fibres have resulted in a less uniform membrane structure.

Polysulfone is another suitable membrane material which has good film-forming properties. It is of sufficient physical, chemical and biological stability, and ligands can be coupled after chloromethylation-amination or acrylation-amination.

Microporous polyamide (nylon) membranes have also been used for the preparation of affinity membranes. This material is mechanically stable and has a rather narrow pore size distribution. It contains only a small number of terminal amino groups for ligand coupling, which can, however, be increased by partial hydrolysis of the amide functions.

A suitable membrane material is polyvinyl alcohol, in particular because of its hydrophilicity and biocompatibility. Poly(ethylene-co-vinyl alcohol), which has a somewhat higher chemical stability, has also been used. Both materials contain hydroxyl groups and can be activated by the CNBr method, allowing immobilization of affinity ligands having an amino function. Ligands can also be coupled using epichlorohydrine or butanediol diglycidyl ether-activation.

Other materials that have been used for affinity membranes are poly(methyl methacrylate), poly (hydroxyethyl dimethacrylate), polycaprolactam, poly (vinylidene difluoride), poly(ether-urethane-urea) and silica glass. Table 1 shows a list of membrane materials and the appropriate ligand-coupling chemistries.

Table 1

Membrane materials and possible chemistries for ligand coupling

Composite Membranes

The main difficulty when choosing a membrane for affinity separation of biomolecules is sometimes to find a material that fulfils several or all of the abovementioned requirements. For example, a chemically stable material might be too hydrophobic and lead to nonspecific and irreversible adsorption of the protein to be separated, whereas a hydrophilic material that is compatible with the fragile protein molecules might not withstand the conditions required for ligand coupling and for regeneration and sterilization of the membrane. Therefore, the choice of a membrane material will sometimes be a compromise. The use of a composite membrane consisting of two or more different materials may often be the only solution to a particular separation problem. This approach consists of the grafting of hydrophilic polymers on to a chemically and mechanically stable microporous membrane. The result is an increased biocompatibility as well as the introduction of suitable functional groups for ligand coupling. One example is the radiation-induced graft polymerization of 2-hydroxyethyl methacrylate or glycidyl methacrylate on to a polyethylene hollow fibre membrane. This increases the hydrophilicity of the material and introduces active hydroxyl groups or reactive epoxy groups.

Activation and Ligand Coupling

From a practical point of view, apart from the chemical compatibility of the membrane material with the activation and coupling solutions, an important aspect is that these solutions need to access the pores of the membrane. In many cases it will therefore be necessary to do the activation in dynamic mode, that is, by forced convection. This is especially important if the membrane material is hydrophilic and the activation and coupling solutions are based on nonpolar solvents, since in that case the wettability of the membrane by the solutions will be low.

Spacer Arms

Occasionally, affinity membranes may show poor performance if the ligand, and in particular a small ligand, is coupled directly to the membrane. This is often due to a low steric availability of the ligand, a problem that can be overcome by the use of a suitable spacer arm. In that way, the ligand accessibility for the molecule to be separated is improved, resulting in an increase in membrane-binding capacity. For example, 1,6-diminohexane or 6-aminohexanoic acid are often used as spacers. In other cases, the coupling method itself provides a spacer, as is the case with butanediol diglycidyl ether. If composite membranes with crafted flexible copolymer chains are used, spacer arms are not normally required.

Affinity Ligands

Biologicial Ligands

Just like other affinity separation techniques, affinity membrane technology uses biomolecules as the affinity ligands, thus taking advantage of the specificity of biological recognition. One of the most common applications is the use of immobilized monoclonal antibodies against natural or recombinant proteins as the ligand for immunoaffinity separation. Another important example are membranes with covalently coupled protein A or protein G for immunoglobulin purification from plasma, serum or cell culture supernatants. Immobilized lectines have been used for the purification of glycoproteins. The use of inhibitors or coenzymes for the purification of enzymes is also possible. Although biomolecules are widely used as ligands for their selectivity, they do have drawbacks. Their poor stability and sometimes high price can make them problematic for use in large scale affinity separation. Drastic conditions are often necessary for elution of the ligate, for example with high affinity antibody–antigen interactions. This can lead to partial inactivation of the molecule to be purified. Ligand denaturation and inactivation, in particular with protein ligands, can occur during regeneration and sterilization of the membrane. Another important issue is the possible leaching of the affinity ligand, leading to a contamination of the final product, which is particularly problematic if the product is to be used in medical applications.

Pseudobiospecific Ligands

An alternative approach involves the use of biomimetic or pseudobiospecific affinity ligands. These are usually smaller and simpler molecules with higher chemical and physical stability than biomolecules. The working principle of pseudobiospecific ligands relies on the complementarity of structural features of ligand and ligate rather than on a biological function, whereas biomimetic ligands have a certain structural resemblance with a biological ligand. For example, textile dyes can be used for the separation of proteins, and in particular Cibacron Blue F3GA has been employed as ligand in affinity membranes for the purification of dehydrogenases, since it often binds specifically to the nucleotide-binding site. Other dyes may adsorb proteins less specifically, but by selection of the right dye (a large number of different dyes is currently available) and the appropriate adsorption and elution conditions, highly efficient separations can be obtained.

Proteins carrying accessible histidine residues on their surface have been shown to have affinity for transition metal–chelate ligands. Typical examples are the iminodiacetate–copper(II) complex (IDA-Cu(II)) and the nitrilotriacetate–nickel (NTA-Ni(II)) ligand widely used for purification of recombinant proteins with genetically attached poly-His tails.

A third group are amino acids such as phenylalanine, tryptophane and histidine. Being the least selective, they have nevertheless been successfully employed for protein purification. However, fine-tuned adsorption and elution conditions are necessary to achieve efficient separation. Mention should also be made of the thiophilic affinity system that has been used with affinity membranes. It is based on the salt-promoted adsorption of proteins via thiophilic regions (containing aromatic amino acids) on to sulfone or thioether-containing heteroaliphatic or aromatic ligands.

Molecularly Imprinted Membranes

A completely different approach for the preparation of affinity membranes is the use of molecularly imprinted polymeric materials. These are produced by polymerization of functional and cross-linking monomers in the presence of the target molecule (the molecule to be separated later), which acts as a molecular template. In this way, binding sites are introduced in the polymer that are complementary in shape and functionality to the target molecule, and that often have specificities comparable to those of antibodies. At the same time, the cross-linked polymeric material provides a porous, chemically and physically very stable support. Even though the technology is in principle applicable to larger biomolecules such as proteins, it has mainly been used for the separation of small molecules like amino acids and peptides. The molecular imprinting technique is reviewed in more detail elsewhere.

Scale-up

Process scale-up tends to be rather easy in adsorptive membrane chromatography, at least compared to the use of conventional beaded resins as the chromatographic support. It has been demonstrated that the diameter of a stack of disc membranes can be increased by up to one order of magnitude and more, with the dynamic capacity remaining constant. This allows for the processing of considerably larger sample volumes at higher flow rates. With radial flow membranes, when both the height and diameter of the cartridge were increased and the flow rate adjusted proportionally to the increased cartridige volume, the apparent specific capacity decreased only slightly.

Applications

Several different applications of affinity membranes have been described. Typical examples of their use for the separation and purification of biomolecules are shown in Table 2.

Table 2

Examples for the use of affinity membranes for isolation and purification of biomolecules

The most common application is the separation and purification of biomolecules and especially proteins for large scale production. A common example is the separation of immunoglobulins from blood-serum or plasma or from cell culture supernatants. Hollow-fibre cartridges with immobilized protein A or pseudobiospecific ligands have been used for this purpose. Figure 2 shows a chromatogram from a case study of immunoglobulin G separation from human plasma using a small, developmental-scale (28 cm² surface area) poly(ethylene-co-vinyl alcohol) hollow-fibre membrane cartridge. The pseudobiospecific affinity ligand histidine was immobilized on to the membrane after activation with butanediol diglycidyl ether, thus introducing a spacer arm. Serum was injected 10-fold diluted in cross-flow filtration mode. Weakly retained and entrapped proteins were then removed by washing the lumen and the outer shell of the fibres, as well as the pores in back-flushing mode. Adsorbed immunoglobulins were subsequently eluted with a buffered solution of 0.4 mol L−1 NaCl in back-flushing mode. The eluted fraction contained 93% immunoglobulins (82% IgG, 10.8% IgM). The dynamic binding capacity of the membrane for immunoglobulin G was determined to be 1.9 gm−2. The process could then be scaled up by using a cartridge with 1 m² membrane surface area.

Figure 2 Separation of immunoglobins from human serum using a poly(ethylene-co-vinyl alcohol) hollow-fibre cartridge with immobilized L-histidine. (a) Immunoglobulin adsorption in cross-flow filtration mode; (b) lumen wash; (c) shell wash; (d) back-flush wash; (e) back-flush elution. (Adapted from Journal of Membrane Science 117, Bueno SMA, Legallais C, Haupt K and Vijayalakshmi MA, Experimental kinetic aspects of hollow-fiber membrane-based pseudobioaffinity filtration: Process for IgG separation from human plasma, pp. 45–56, Copyright 1996, with permission from Elsevier Science.)

A related application is the final polishing of an already pure product. For example, the removal of bacterial endotoxins from contaminated solutions of monoclonal antibodies has been demonstrated using membrane-bound pseudobiospecific ligands.

Affinity membranes have also been suggested for use in extracorporeal circuits, for the removal of toxic substances such as certain metabolites or antibodies from blood. For example, exogenous human serum amyloid P component, a substance associated with Alzheimer’s disease, has been removed from whole rat blood in an extracorporeal circulation system. This model system used a polyclonal antibody coupled to cellulose flat-sheet membranes. The biocompatibility of the membrane was also demonstrated. A similar application is the removal of autoantibodies from human plasma, using membrane-bound affinity ligands in extracorporeal circuits.

Apart from preparative applications, small cartridges with membrane discs or continuous membrane rods should be useful for analytical-scale separations and affinity solid-phase extraction, for example for immunoextraction.

Conclusions

Affinity membrane separation techniques combine the specificity of affinity adsorption with the unique hydrodynamic characteristics of porous membranes. They provide low pressure separation systems which are easy to scale up and ideal for the processing of large volumes of potentially viscous feed solutions (e.g. microbial broth, bacterial cell extract, conditioned media) often involved in the production of recombinant proteins. The additional microfiltration effect of membranes allows for the processing even of unclarified, particle-containing feed solutions. The high performance of this separation technique is due to the presence of through-pores and the absence of diffusional limitations; mass transfer is mainly governed by forced convection. Affinity membranes are used in applications such as purification of biomolecules, final product polishing, removal of unwanted substances from patients’ blood in extracorporeal circuits, but also for smaller scale analytical separations. Biological affinity ligands and biomimetic or pseudobiospecific ligands are currently employed, as well as different membrane configurations such as flat sheets, hollow fibres or continuous rods. The technology is now in the process of being adapted more and more for large scale industrial separation and purification.

See also: Centrifugation; Countercurrent Chromatography: Large-Scale; Countercurrent Chromatography: Overview; Liquid Chromatography: Mechanisms: Gradient Polymer Chromatography.

Further Reading

Brandt S, Goffe RA, Kessler SB, O'Connor JL, Zale SE. Membrane-based affinity technology for commericial scale purifications. Bio/Technology. 1988;6:779.

Charcosset C. Purification of proteins by membrane chromatography. Journal of Chemical Technology and Biotechnology. 1998;71:95.

Klein E. Affinity Membranes: Their Chemistry and Performance in Adsorptive Separation Processes. New York: John Wiley; 1991.

Roper DK, Lightfoot EN. Separation of biomolecules using adsorptive membranes. Journal of Chromatography. 1995;702:3.

Suen S-J., Etzel MR. A mathematical model of affinity membrane bioseparations. Chemical Engineering Science. 1992;47:1355.

Thömmes J, Kula MR. Membrane chromatography – an integrative concept in the downstream processing of proteins. Biotechnology Progress. 1995;11:357.

Affinity Partitioning in Aqueous Two-Phase Systems

G. Johansson    Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden

Aqueous Two-phase Systems in General

The division of water into non-miscible liquid layers (phases) by addition of two polymers has led to the remarkable possibility of being able to partition proteins and other cell components between phases of nearly the same hydrophilicity. Proteins can be separated by partitioning if they have unequal distribution between the phases, i.e. when their partition coefficients, K (the concentration in top phase divided by the concentration in bottom phase), differ. Usually the difference in the K value of many proteins is not very large and then repeated extractions have to be carried out to get a reasonable purification. If, however, the protein of interest (the target protein) has a very high K value and is mainly in the upper phase and all the contaminating proteins have very low K values so that they are in the bottom phase, an effective and selective extraction can be obtained in a single or a few partitioning steps. This type of partitioning has been made possible by using affinity ligands restricted to the upper phase.

The composition of the phases when two polymers like dextran and polyethylene glycol (PEG) are dissolved together in water depends on the amount of the polymers and their molecular weights. The concentration of the polymers in two phases of a given system can be found in the phase diagram for the temperature being used. A typical phase diagram is shown in Figure 1.

Figure 1 ) give systems with more top phase (three to five times) than bottom phase.

The line that connects the points in the diagram representing the compositions of the top and bottom phases of a system is called the tie-line. Each system with a total composition (percentage of each polymer) belonging to the same tie-line will have the same phase compositions. The smaller the tie-line, the more similar are the two phases in their composition. The greatest difference in composition of the top and bottom phases is therefore obtained by using high polymer concentrations.

The partitioning of proteins and also of membranes and particles depends on the polymer concentration of the system. The K value of a protein will be the same for all systems belonging to the same tie-line. The partition coefficient will, in most cases, decrease with the length of the tie-line, i.e. by using higher concentrations of the two polymers the material will accumulate more in the lower phase. Another way to affect the partitioning of proteins is by addition of salts to the system. Their effect depends on the type of cation and anion introduced with the salt. Negatively charged proteins show increasing K values when the cation is changed in the series:

For the anion the partition coefficient increases in the following order:

The highest K value of negatively charged proteins will then be obtained with the salt tetrabutylammonium hydrogenphosphate and the lowest K value with potassium perchlorate. Proteins with zero net charge (at their isoelectric points) are not affected by salts while positively charged proteins behave in an opposite manner to the negatively charged ones. For a number of proteins the log K values are nearly a linear function of their net charge (Figure 2).

Figure 2 Log K of the protein ribonuclease-A as function of its net charge, Z , 50mM). System compositions: (A) 6.2% w/w dextran 500 and 4.4% w/w PEG 8000; (B) 9.8% w/w dextran 500 and 7.0% w/w PEG 8000. Protein concentration, 2gL −1 . Temperature, 20°C. (Reprinted from Johansson G (1984) Molecular Cell Biochemistry 4: 169–180, with permission from Elsevier Science.)

Affinity Partitioning

The principle of affinity partitioning is to localize an affinity ligand in one phase to make it attract ligand-binding proteins. Since the phase-forming polymers are in each phase, either one can be used as ligand carrier. The standard system for affinity partitioning has been the one composed of dextran, PEG and water. Dextran is then used for localizing the ligand in the bottom phase while PEG can be used to concentrate the ligand to the top phase. PEG has often been chosen as ligand carrier because bulk proteins can be effectively partitioned into the dextran-rich lower phase by using high concentrations of polymers and a suitable salt. Thus, the target protein is extracted towards the upper phase leaving contaminating proteins in the bottom phase. PEG has two reactive groups (the terminal hydroxyl groups) which can be used as points of ligand attachment. In many cases only one ligand molecule is attached per PEG molecule. If the ligand is a large molecule (e.g. an antibody protein) several PEG chains may be attached to the one ligand molecule. Normally, only a fraction (1–10%) of the PEG in the two-phase system has to carry the ligand to reach maximal extraction efficiency. The more extreme the partitioning of a ligand–polymer is toward a phase the more effective it will be in extracting a ligand-binding protein into this phase. The partitioning of the ligand–polymer should be in the same range as the non-derivatized polymer but it may, in some cases, be more extreme. The higher the polymer concentrations are in the system, i.e. the longer the tie-line of the system, the more extreme is the partitioning of PEG to the top phase and dextran to the bottom phase. This can be expressed by the partition coefficients of the two polymers:

where c is the respective polymer concentration in top or bottom phase. Table 1 shows the KPEG and Kdextran values for systems containing PEG 8000 and dextran 500. Dextran has a more extreme value of K than PEG, i.e. KPEG < 1/Kdextran. Dextran should therefore, in principle, be a better ligand carrier than PEG. The concentration ratio for dextran is roughly the square of the ratio for PEG in the same system.

Table 1

Partition coefficients of PEG (KPEG) and dextran (Kdextran) and their logarithmic values (log) at various tie-line lengths of the system in Figure 1

A Simple Theory for Affinity Partitioning

A basic theory for affinity partitioning was elaborated by Flanagan and Barondes in 1975. They analysed the combined binding and partition equilibria taking place in and between the two phases, respectively (Figure 3).

Figure 3 Scheme for affinity partitioning of a protein (P) with two binding sites for a ligand attached to PEG (L). The complexes between protein and ligand–PEG are PL and PL 2 , respectively.

In this scheme the ligand–PEG(L), the free protein (P) and the two complexes (PL and PL2) have each their own partition coefficient (KL, KP, KPL and KPL2). Furthermore, in both phases association between protein and ligand–PEG takes place which can be described by the association constants:

one set for each phase.

A total association constant for the equilibrium:

can also be used: Ktot = K1K2.

The association constants, Ktot, K1 and K2 may differ between the two phases. According to Flanagan and Barondes, the measured log K value of a protein, log Kprotein, will, theoretically, give rise to a saturation curve when plotted versus the concentration of polymer-bound ligand in the system (compare Figure 4).

Figure 4 Increase in the logarithmic partition coefficient of phosphofructokinase (PFK) from bakers’ yeast as function of the concentration of Cibacron blue F3G-A PEG (Cb-PEG). System composition: 7%w/w dextran 500, 5%w/w PEG 8000 including Cb–PEG, 50mM sodium phosphate buffer pH 7.0, 0.5 mM EDTA, 5mM 2-mercaptoethanol and 4 nkat g −1 enzyme. Temperature, 0°C. The inverse plot inserted is used to determine the Δ log K max .

The log Kprotein value reaches a plateau when the concentration of L–PEG is so high that practically all the protein is present as the fully saturated complex PL2. The protein molecule is then surrounded by two PEG chains and outwardly shows a PEG atmosphere.

, is related to KP, KL and the K values via the following equations:

or:

The maximum increase in the logarithmic partition coefficient, Δ log Kmax, is consequently given by:

If Ktot,T = Ktot,B then Δ log Kmax = 2 log KL.

From the values in Table 1 it may therefore be assumed that for proteins with two binding sites Δ log Kmax can be as high as 3.57 (an increase of 3700 times in K) when PEG is used as ligand carrier with KL = 61. If dextran is used as carrier, in the same system, the Δ log Kmax should theoretically be around – 8 corresponding to a one hundred million times increase in the affinity of the protein for the lower phase if KL is 0.0001. A higher number of binding sites (n) should then give strongly increasing Δ log Kmax values with Δ log Kmax = n log KL. However, the affinity extraction effect may be reduced by a reduction of individual binding strengths.

Experimental Results

The extraction curves of a protein, here exemplified with phosphofructokinase (PFK) from baker’s yeast, using Cibacron Blue F3G-A PEG, closely follows the predicted behaviour (Figure 4). The inverse plot makes it possible to estimate the value of Δ log Kmax.

The dependence of Δ log Kmax of PFK on the polymer concentration is shown in Figure 5. Increasing concentration of polymers corresponds to longer tie-line length (and greater KL value) and this makes the affinity partitioning, measured as Δ log Kmax, more efficient.

Figure 5 (A) Log K ), 3% of total PEG; or without Cb–PEG (Δ). (B) Δ log K ) and log K ) as function of the tie-line length. System composition: dextran 500 and PEG 8000 (including Cb–PEG) in weight ratio 1.5 : 1, 50mM sodium phosphate buffer pH 7.0, 0.5 mM EDTA, 5mM 2-mercaptoethanol, and 4 nkat g −1 enzyme. Temperature, 0°C.

In addition to the concentration of polymers and ligand–PEG the actual Kprotein obtained also depends on pH value, the salt added to the system and the temperature. Two salts which have little or no effect on the affinity partitioning are phosphates and acetates in concentrations up to 50mM. In the case of PEG the Δ log Kmax is reduced with increasing temperature.

The detachment of ligand from the enzyme can be achieved either by using a high concentration of salt or by the addition of an excess of free ligand. For PFK the addition of adenosine triphosphase (ATP) to the system containing ligand–PEG strongly reduces the partition coefficient of the enzyme (Figure 6).

Figure 6 The effect of adenosine triphosphase (ATP) and of ATP + Mg ²+ on the partitioning of phosphofructokinase from bakers’ yeast in a system containing Cibacron blue F3G-A PEG (Cb–PEG). Δ log K ). System composition: 7% w/w dextran 500 and 5% w/w PEG 8000 including 0.5% Cb–PEG (of total PEG). 50mM sodium phosphate buffer pH 7.0, 0.5mM EDTA, 5mM 2-mercaptoethanol, and 4 nkat g −1 enzyme. Temperature, 0°C.

Types of Affinity Ligands Used

A number of affinity ligands have been used and some are presented in Table 2. The attachment of ligand to polymers and the purification of the ligand–polymer differs from case to case. Some ligands such as reactive texile dyes can be bound directly to PEG and to dextran in water solution of high pH. Other ligands are introduced by reactions in organic solvent, such as the attachment of acyl groups to PEG by reaction with acyl chloride in toluene. PEG may also be transformed into a more reactive form such as bromo-PEG, tosyl-PEG or tresyl-PEG. Some reaction pathways are shown in Figure 7. A number of methods to synthesize polymer derivatives have been published by Harris.

Table 2

Examples of affinity partitioning

Figure 7 Some reactions used for the covalent linkage of ligands to polymers, preferentially to PEG. The encircled ‘L’ represents the ligand and the open circles the polymer chain.

Preparative Extractions

The following steps may be useful for a high degree of purification by affinity partitioning.

1. Pre-extraction in a system without ligand–PEG to remove proteins with relatively high partition coefficients. The target protein stays in the bottom phase by adjusting the choice of polymer concentration, salt and pH.

2. Affinity partitioning is carried out by changing the top phase for one containing ligand–PEG. The target protein will now be in the top phase.

3. Washing the top phase with bottom phase to remove co-extracted proteins.

4. ‘Stripping’ of protein from the affinity ligand by addition of highly concentrated phosphate solution (50% w/w) to the separated upper phase. This generates a PEG-salt two-phase system with PEG and ligand–PEG in the top phase and target protein in the salt-rich bottom phase. An alternative stripping procedure can be carried out by adding a new pure dextran phase to the recovered top phase and supplying the system with free ligand. In this case the target protein will be collected in the lower phase.

For each step the number of extractions and the most suitable volume ratios for yield and purity can be optimized. The procedure is summarized in Figure 8.

Figure 8 ) by using four partitioning steps and PEG–dextran two-phase systems with PEG-bound ligand. This approach has been used for the purification of lactate dehydrogenase (LDH) from meat juice by affinity partitioning with Procion yellow HE-3G PEG. The inserted SDS-PAGE patterns of the original meat extract and the final product (obtained in the phosphate-rich phase) show the removal of contaminating proteins. Recovery of enzyme = 79%. System composition: 10%w/w dextran 500 and 7.1% w/w PEG 8000 including 1% Procion yellow HE-3G PEG (of total PEG), 50mM sodium phosphate buffer pH 7.9, and 25% w/w muscle extract. Temperature, 0°C. (Reprinted from Johansson G and Joelsson M (1986) Applied Biochemistry Biotechnology 13: 15–27, with permission from Elsevier Science.)

The yield in the top phase, YT, can be calculated from the K value of target protein and the volumes of top and bottom phase, VT and VR, respectively, using the following equation:

and the yield in the bottom phase, YB

A considerable concentration of the target protein, in addition to purification, can be achieved by choosing an extreme volume ratio with a small collecting phase.

An example of preparative extraction of an enzyme by applying the method given in Figure 8 is the purification of lactate dehydrogenase (LDH) using a PEG-bound textile dye. Crude extract of pig muscle, cleared by centrifugation, is mixed with PEG, dextran and Procion yellow HE-3G PEG. After the first partitioning the top phase is washed twice with pure lower phases and then it is mixed with a 50% w/w salt solution (25% NaH2PO4 + 25% Na2HPO4⋅H2O). The protein content of the final product in the salt-rich phase compared with that of the initial extract is demonstrated by the polypeptide pattern in sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) shown in Figure 8. The L–PEG (and PEG) recovered in the final top phase is ≥ 95% of the initially introduced amount.

Purification of PFK in combination with a precipitation step with PEG before the affinity partitioning step greatly reduces the original volume of enzyme solution. The extraction included both preextraction and washing steps. The final polishing of the enzyme was made by ion exchanger and desalting with gel chromatography. The results can be seen in Table 3.

Table 3

Purification of phosphofructokinase from 1 kg (wet weight) bakers′ yeast

a In the presence of the protease inhibitor phenylmethylsulfonyl fluoride.

The effectiveness of affinity partitioning depends on the binding strength between ligand and protein. Good extraction is obtained with association constants of 10⁴ M−1 or more (Figure 9). The capacity, based on the amount of ligand in the system, is in the range of several hundred grams of protein per kilogram of system. Affinity extractions with 150 g of protein per kilogram of system have been carried out, and in these cases the two-phase systems strongly change the phase volume ratio while the bulk protein acts as a phase-forming component. In systems with high protein concentration the amount of dextran can be reduced or even excluded.

Figure 9 Affinity extraction into the top phase, by using increasing amount of PEG-bound ligand, calculated for an enzyme with the mole mass 100 000 g mol −1 , containing two binding sites for the ligand, and with K P  = 0.01. The value for the partition coefficient, K L , of the ligand is 100. The association constant, K , +) and 500gL −1 (Δ).

Countercurrent Distribution

A convenient way of multiextraction is countercurrent distribution (CCD). Here a number of top phases are sequentially moved over a set of bottom phases and equilibration takes place after each transfer. The process can be seen as a step-wise chromatography. The original two-phase system, number 0, contains the sample and after that a number (n) of transfers have been carried out n + 1 systems are obtained and the various proteins in the sample are distributed along the CCD train. The CCD process is visualized in Figure 10(A).

Figure 10 ). System composition: 7%w/w dextran 500 and 5%w/w PEG 8000 including ligand–PEG, 50mM sodium phosphate buffer pH 7.0, 0.2mM EDTA, and 5mM 2-mercaptoethanol. Temperature, 3°C. Systems in chamber 0–2 were initially loaded with yeast extract. (Reprinted from Johansson G, Andersson M and Akevland HE (1984) Journal of Chromatography 298: 485–495. With permission from Elsevier Science.)

The distribution of a pure substance can be calculated from the K value of the substance and the volumes of the phases, VT and VB. Assuming that all of the top phase volume is mobile and all bottom phase stationary, the fractional amount, Tn,i, in tube number i (i goes from 0 to n) after n transfers will be given by:

This makes it possible to calculate the theoretical curve for a substance and to make comparisons with the experimental distribution curve. Such an analysis may reveal the presence of several components even if they are not separated into discrete peaks. Figure 10(B) shows an example of a CCD of