THE ABCs OF
FILTRATION AND
BIOPROCESSING
FOR THETHIRD MILLENNIUMEDITORSCONTRIBUTORSHarold W. Ballew

Harold W. Ballew

F. Jesus Martinez

Dennis Clark

Cassidy Markee

Geoff Crooks, Ph.D.

Roy T. Eddleman

Sa V. Ho, Ph.D.
Cassidy Markee
F. Jesus Martinez
David Milner, Ph.D.
Jason Partee
James Simmons

SPECTRUM America and Asia Pacific

SPECTRUM Europe

18617 Broadwick Street
Rancho Dominguez, CA 90220-6435
USA
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about this book...
...andSpectrum has published The ABCs of Filtration and Bioprocessing for the Third Millennium
to assist in the understanding and development of the most efficient methods for separating and bioprocessing liquids and gases using hollow fiber membrane technology.
Founded in 1970, Spectrum is a global leader in selected markets for hollow fiber and tubular membrane separation products that are used by research laboratories and biotechnology and pharmaceutical companies worldwide.
Hollow fiber separation is a dominant technology in various applications as demonstrated
by its success in hemodialysis, blood gas exchange and pharmaceutical filtration. That hollow fiber filtration and separation is the modality of choice is also subscribed to by such wellknown institutions as SRI International, National Institute of Standards and Technology(NIST), large pharmaceutical and biotechnology companies and millions of hemodialysis
patients around the globe. Spectrum is forecasting that many applications where membranes are used to separate, isolate and purify liquids and gases will upgrade to the performance and economy of scale of hollow fiber technology.
FILTRATION AND BIOPROCESSING
Spectrum has developed and patented a new generation of hollow fiber configurations that
greatly minimize membrane fouling and improves the filtration efficiencies of hollow fiber
modules by at least an order of magnitude. These products and technology will lower thecost of membrane bioprocessing of aqueous streams by fifty percent when compared toexisting products.
SELECTIVE MOLECULAR EXTRACTION
Spectrum has developed and acquired a novel class of membranes for the selective extraction, concentration and recovery of organic compounds from dilute aqueous solutions. This
novel class of polymeric liquid membrane technology will have significant application to
improving the taste and flavor of beer and wine, water remediation by the extraction of
VOCs from aqueous streams and the extraction and separation of industrial compounds
such as butanol, ethanol, acetic acid and others. Research indicates that this technology
reduces the requirement for processing energy by a significant degree when compared toexisting competitive technologies.
MODULAR, MULTI-PHASE HOLLOW FIBER TECHNOLOGY
Spectrum is a pioneer in the development of modular, multi-phase hollow fiber technology
where three independent membrane compartments, within the same modular structure,provide unique means of efficient triple streams processing of aqueous or non-aqueous
solutions. This unique multi-compartment module can be used for: mammalian cell expansion in bioreactors; to separate blood formed elements simultaneously in parallel streams
and in foods and beverages.
We welcome your inquiry.

Book Design and Illustrations: Jim Mingin© 2002, Spectrum Laboratories, Inc. All rights reserved. No part of this book may be reproduced or utilized inany form or by any means, electronic or mechanical, including photocopying, recording, or by any
information storage and retrieval system, without permission in writing from:Spectrum Laboratories, Inc, 18617 Broadwick Street, Rancho Dominguez, CA 90220
420-11345-000 Rev. 01 - 121907

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THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

TABLE OF CONTENTS

Section A: THE ABCs of FILTRATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Depth Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Screen Filters (Mesh) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Membrane Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
The Nature of Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Retention Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Plugging Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Fundamentals of Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Tangential (Cross Flow) Filtration vs. Dead End Filtration . . . . . . . . . . . . . . . . . . . . . . .9
Membrane Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Range of Filtration, Bioprocessing and Selection of Spectrum’s Membranes . . . . . . .12
Volume of Liquid Processed Based on Module Type . . . . . . . . . . . . . . . . . . . . . . . . .12
Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Electrodialysis (ED) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Reverse Osmosis (RO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Selective Gas Permeable (Permselective) Membranes . . . . . . . . . . . . . . . . . . . . . . . .15
Polymeric Membrane Extractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Integrity Testing of Membrane Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE ULTRAFILTRATION . . . . . . . . . . .19
Retentivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
MWCO Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Temperature and Chemical Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Filtration Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Concentration Polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Other Factors Affecting Membrane Flow Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE MICROFILTRATION . . . . . . . . . .29
FILTRATION MODES (CONFIGURATIONS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Membrane and Module Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Tangential (Cross Flow) Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Tangential Flow Membrane Technology using Hollow Fibers . . . . . . . . . . . . . . . . . . .35
Spectrum Hollow Fiber Membrane Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Scalability of Hollow Fiber Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
Sterilization of Spectrum Membrane Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Modes of Operation of Hollow Fiber Filtration Systems . . . . . . . . . . . . . . . . . . . . . . . .42
Extended Module Throughput by Forward, Reverse and Back Flushing . . . . . . . . . .44
MICROFILTRATION APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Cold Sterilization of Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Testing for Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Clinical and General Laboratory Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Cleaning of Particles and Clarification of Liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Cell, Bacteria and Virus Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47

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TABLE OF CONTENTS

Cell Recycle Perfusion of Animal Cell Bioreactors
with CellFlo® Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
ULTRAFILTRATION APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Desalting and Buffer Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
SYSTEM AND MEMBRANE SELECTION GUIDES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
Membrane Application Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
Industrial Applications for Hollow Fiber Membrane Systems . . . . . . . . . . . . . . . . . . . .57
Sizing Hollow Fiber Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
Representative Spectrum Hollow Fiber Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
Selecting the Optimum Membrane System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
MACROFILTRATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64

Section B: THE ABCs OF LABORATORY DIALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
DIALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
Dialysis Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
Dialysis Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
Factors that affect the Rate of Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
Dialysis Membrane Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
Chemical Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
MWCO Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
Tubular Membrane “flat width” Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
Closure Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
Representative Spectrum Dialysis Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74

Section C: THE ABCs OF POLYMERIC MEMBRANE EXTRACTION . . . . . . . . . . . . . .77
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
SeleXtrac™ Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Transport Rates of Organic Compounds through SeleXtrac™ Membranes . . . . . . . .79
SeleXtrac™ Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
Example - Extraction of Volatile Organic Compounds (VOCs) . . . . . . . . . . . . . . . . . .80
Example - Extraction of Water Soluble Polar Organic Compounds . . . . . . . . . . . . . . .80
DISCUSSION
SeleXtrac™ Membranes for Polymeric Extraction of Organics from Aqueous Streams . . . .82
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82
BASIC THEORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
SeleXtrac™ Supported Polymeric Liquid Membranes . . . . . . . . . . . . . . . . . . . . . . . . .83
Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
Characteristics of Polyglycol SeleXtrac™ Membranes . . . . . . . . . . . . . . . . . . . . . . . .84
Advantages of SeleXtrac™ Membrane Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85

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THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

TABLE OF CONTENTS

Mass Transfer Theory and Rate Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86
Mass Transfer Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86
Transport Rate Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .88
Key Variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .89
Effects of Polymeric Liquids and Permeating Molecules . . . . . . . . . . . . . . . . . . . . . . .89
Effects of Membrane Supports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90
Effects of Operating Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
PROCESS APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
Wastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
Phenolics (p-nitrophenol) Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
Carboxylic Acids Waste Stream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .93
Methyl Tertiary Butyl Ether (MTBE) Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96
Separation of Similar Organics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99
Flavor Recovery/Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100
General Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100
Low-Alcohol Beer Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .101
ANALYTICAL APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .104
General Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .104
Direct Analysis in Complex Mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .104
Detection of Phenol in Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .104
Field Detection of a Target Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .105
BIOMEDICAL APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106
In-Situ Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106
Detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106
Controlled Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106
ACKNOWLEDGEMENT AND LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . .106-107

Section D: THE ABCs OF CELL CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109
BIOREACTOR DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109
Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109
Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .110
Bioreactor Design Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112
Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113
Conversion of Existing Batch Systems to Continuous Perfusion Systems . . . . . . . . .113
Available Options for Conversion to Perfusion Operation . . . . . . . . . . . . . . . . . . . . .115
CellFlo® Technology for Cell Recycle Perfusion of Animal Cell Bioreactors . . . . . . .116
Results Obtained Using CellFlo® Hollow Fiber Cell Separators
for Converting Bioreactors to Continuous Perfusion . . . . . . . . . . . . . . . . . . . . .116
Converting a Batch Bioreactor to Perfusion
Using CellFlo® Cell Separators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117
Bioreactor with Steam Supply for Sterilization The Basic System Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117
CellGas™ Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119

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TABLE OF CONTENTSCELLMAX® CAPILLARY CELL CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
Conventional Cell Culture Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
Capillary Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
Advantages of Capillary Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122
Representative Applications of the CellMax® Cell Culture System . . . . . . . . . . . . . .123
The CellMax Capillary Cell Culture System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125
CellMax® Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .126
CELL IMPLANTS FOR DRUG SCREENING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .127

Section E: THE ABCs OF LIQUID CHROMATOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . .129
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .129
CHROMATOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130
The Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130
The Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .132
The UV Monitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .133
The Fraction Collector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137

Section F: APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143
GLOSSARY OF TERMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143
TRADEMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
CHEMICAL COMPATIBILITY CHART . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
FILTRATION AND BIOPROCESSING CHART - Figure A-5 . . . . . . . . . . . . . . . . . . . . . . . . . .154

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THE ABCs OF FILTRATIONTHE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

A-vii

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Section A

The ABCs of Filtration
Introduction
Filtration is the process of separating, fractionating or concentrating particles, molecules or ions within or from a fluid by forcing the material through a porous orsemi-porous barrier. The force can be pressure, vacuum, concentration gradient,valence or electrochemical affinity. The fluid can be either a liquid or a gas.

Figure A-1 The limitations of dead end filtration are overcome by tangential (cross flow) filtration.

Filtration is a cost-effective technology, generally requiring only the application of aforce. There is no need to heat the process material as in the case of evaporationor high temperature sterilization. The latter are energy expensive and can affect
the quality of the filtrate.
The main objective of filtration is to either achieve a clean liquid or gas, or to
extract, concentrate, blend or analyze matter separated from liquids or gases by
the filter. In most cases, the filtration efficiency depends primarily on the characteristics of the filter media, the properties of the fluid and the operating conditions.
Distinctive differences exist in the types of materials used to construct filter media.
Most modern filtration media are man-made from either natural or synthetic polymeric materials. These materials are fabricated into various filter materials with a
specifically designed structure, porosity, and performance characteristics. Filtration
media are often classified as depth filters, screen filters (mesh) or membranes.

Depth Filters
Depth media are mostly used for coarse filtration and fluid clarification. They arealso used to protect microfiltration, ultrafiltration, and reverse osmosis membranes.
Depth filters are most often manufactured from fibrous materials, woven or nonwoven polymeric material or inorganic materials.

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THE ABCs OF FILTRATION

Depth filters are not absolute and therefore do not have a precisely defined poresize or structure. Particles that are larger than the “aperture” or “pore size” of thefilter will be trapped more or less on the surface of the filter. In the case of smaller
particles, depth filters rely on the random entrapment and adsorption of matterwithin the structure, or interstices, of the media. In addition, the depth filter may
have an electric charge (zeta potential, described later), that will attract and trap
small particles. The result is efficient retention of a wide range of particle sizes bydepth filters.
Being of thicker construction and higher porosity, depth filters exhibit the advantage of higher flow rate and dirt loading capacity when compared to screens andmembrane filters. Depth filters are also less expensive than many screens and all
membrane filters.

Screen Filters (Mesh)
Particles are captured directly on
the surface of screen filters.
Therefore, screens retain with certainty only those particles the same
size or larger than the aperture sizeof the screen. Neglecting diffusion
and inertial impaction, most particles smaller than the aperture size
pass unimpeded through thescreen.
For that reason, screens are preferred if the user needs low nonspecific binding or low adsorption
or absorption of the filtrate.
Figure A-2 Retention characteristics of screens and depth filters

Should the user need maximum removalof all particles and/or a high binding
capacity, the depth filter should beselected. Since the depth filter has amuch larger available surface area than
the screen, it has a much larger particle
loading capacity and many more sites
where proteins, viruses and other small
particles can bind.
Figure A-2 shows the “remote cutoff” thatis characteristic of virtually all depth filters compared to the “sharp cutoff” characteristic of screen filters.
Figure A-3 Spectra/Mesh® screens areavailable from 5 µm through 1,000 µm

Membrane Filters

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INTRODUCTION A-3

Membrane filters are available in a variety of materials and configurations for a variety
of applications. They are used routinely in divergent applications such as purification,
diafiltration, desalination, particle fractionation and removal, molecular concentrationand separation, fluid sterilization, fluid clarification and many other applications.
Membrane filters are manufactured from polymeric as well as other materials byprocesses such as solvent casting, sintering, stretching or nuclear particle
track etching. The operating characteristics of membrane filters depend largely
upon the manufacturing process and thepolymeric materials used. Each of theseprocesses will produce a thin membranewith a relatively well defined flow rate,pore size, pore structure, pore density,
bubble point and tensile strength.
Due to these predefined characteristics,
the filtration efficiency of membrane filters
can be readily predetermined with a highdegree of accuracy for a wide range ofapplications and process conditions.
Microporous membrane filters function
primarily by surface capture or rejection
Figure A-4 Schematic of hollow fiber membrane showing tangentialof matter larger than the rated pore size of(cross flow) filtration
the membrane. However, random interstitial entrapment of particles smaller than
the rated pore size is also an important property of some membrane filters.
Microporous membrane filters exhibit lower flow rates and dirt loading capacity whencompared to depth filters. However, membrane filters offset this disadvantage with welldefined filtration characteristics and an ability to fractionate suspended matter of adefined size from a fluid. If cross flow filtration is used, the feed solution that sweeps
the filter surface continuously can provide throughput comparable to some depth filters.
Ultrafiltration and dialysis membranes that provide concentration, fractionation and othermolecular separations represent significant improvements over previously used methods
such as centrifugation, evaporation and heat treatment. The latter are energy expensive andmay have deleterious effects on both the concentrate/retentate and the permeate/filtrate.
Newer applications for microporous and ultrafiltration membrane filters include: protein
purification, cell harvesting, washing, clarification and concentration; cell perfusion; virus
clarification and concentration; cleaning latex particles and many others. Representativelaboratory and process applications are described in more detail in later chapters.
Membrane filters are primarily available in hollow fibers and flat sheets (pleated, stacked,
or spiral wound configurations). Hollow fiber modules are preferred in many applications
because of their superior flow dynamics, packing density (large membrane filtration surface area per unit of volume), continuous “sweep” cleaning of the membrane surface bycross flow, ease of handling, lower cost and disposability.

The Nature of Particles
Membrane filtration is used for a wide range of particle and molecular sizes as shown

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THE ABCs OF FILTRATION

in figure A-5 (inside back cover). Considering physical sieving only (excluding ions),
this range spans from molecules with a molecular weight of 100 (very roughly 10 Å insize) to particles upwardly of 20 µm in size, (200,000 Å), a 2,000 order of magnitude.
Particle shape is also a factor. Molecules can range in shape from essentially spherical or globular (such as gamma globulin) to linear (such as tropocollagen).
Particles can be long and slim (such as
glass and polymer fibers) or somewhat
spherical. They can be primarily rigid or
deformable; and if rigid, they can be crystalline or non-crystalline. Furthermore,they can have an electric charge.

Figure A-6A Schematic showing narrow particle size distribution

The electric charge possessed by some filter media is called its “zeta potential”. Zeta
potential can be either a positive or a negative charge. When it is positive, it will electrokinetically attract particles in the feedstream that have a negative charge and viseversa. Zeta potential allows some filters toremove particles that are much smaller thanthose ordinarily trapped by simple sieving.
Since the nature of the particles to be
removed or retained are often known, ahighly selective separation system can be
designed for the filtration objectives.
However, in most fluids, the particle load is
randomly dispersed and the filtration objective is to remove or retain 90% or more ofeverything above a given particle size.

Figure A-6B Schematic showing broad particle size distribution

As an example of the former case, the particle distribution and filtration objective may be
to remove or retain bacteria of a given typefrom a culture consisting entirely of the bacterial cells. In such a case, the particulate
distribution may be as shown in figure A-6A.
Far more common is the latter case. Figure
A-6B illustrates the typical distribution ofparticles in a fluid. N ote that the range ofparticle sizes is very broad with a higher percentrage in the submicron range.

Figure A-7A Rigid particles smaller than the pore size will go through the filter.

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Of equal importance to particle size is thenature of the particles themselves (refer tofigures A-7A and A-7B). Rigid particles are
generally much easier to filter than compressible particles. Since the latter candeform, they are much more likely to passthrough the filter. Thus, deformable particles
may behave similar to a smaller particle intheir separation characteristics. In addition,
compressible particles tend to compact on

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INTRODUCTION A-5

the surface of dead end filters, effectively
cutting off flow much sooner than would be
the case with rigid particles.
Some particles tend to agglomerate, i .e.
adhere to each other if they come in contact. The result is a much larger particle
that, if stable, is easier to filter than smaller
particles. In some cases, chemical agentscan be added to a solution to encourage(or discourage) agglomeration.

Retention Efficiency
Referring to figures A-8A, B and C the
upstream particle distribution previously
described is shown as a solid line. The
dashed lines show typical particle distributions downstream from a depth filter,a microporous membrane and a screen
filter, all rated at 1 µm.

Figure A-7B Compressible particles will often deform and go through the filter.

The most efficient filter for removing the
maximum number of particles, or
reducing the particle load is the depth
filter as shown in figure A-8A.
However, depth filters have the disadvantage that they are not absolute.
Microporous membranes are absoluteand are also quite efficient in reducing
the number of particles as shown in figure A-8B. Note that only those particles smaller than 1 µm pass throughthe filter.
Figure A-8A Downstream particle distribution for a depth filter

NUMBEROF
PARTICLES

downstream
m distribution of particles
partfrom typicala 1.0 µm absolute
microporouus membrane

PARTICLE SIZE (µm)
Figure A-8B Downstream particle distribution for a microporous membrane

Least efficient in terms of total numberof particles removed is the screen filter.
Figure A-8C illustrates the retention
characteristic of screens. All particles
greater than the screen pore or aperture size are trapped on the filter surface, while virtually all particles smaller
than the screen pore or aperture size
pass through the filter.
If the filter must be absolute and stillretain the maximum number of particles,
the filtration combination of choice
would be the microporous membranealone or in combination with a depth filter. The screen can also be used in
combination with a depth filter.

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THE ABCs OF FILTRATION

The data from these three idealized
curves have been replotted in figure A9 to illustrate the particle size cut off
rating of the three types of filters.
Characteristic of depth filters andmicroporous membranes is a gradualreduction in retention efficiency for particles below their rating. This is called“diffuse or remote cutoff”. Since
screens trap only those particles at orlarger than its pore size rating, they aresaid to have a “sharp cutoff”.

Figure A-8C Downstream particle distribution for a screen filter.

For removal of particles from a gas
stream, the retention characteristics forthe three types of media above areoften dramatically different than is thecase for liquid filtration.

Plugging Characteristics
Throughput as a measure of filter life has
been defined as that amount of fluid thatcan be filtered before an unacceptablepressure drop is reached across the filter.
It is not only affected by how the filtration process is managed, but also by theinteraction that takes place between thefilter media and the particles to be separated.

Figure A-9 Retention characteristics of depth filters, microporous membranes
and screen filters.

In the simple “sieving” mode of plugging,(Figure A-10A & B) particles smaller thanthe pores readily pass through the filter
and particles larger than the pores aretrapped on the surface of the filter.
Figure A-11 shows a phenomenon that
occurs in filtration if the particles in thefluid are considerably smaller than the
pores of the filter but are rigid. They may
collect on the surface of the filter andbegin to bridge over the pore openings.
Figure A-10A “Sieving” mode of plugging

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In time, these particles will completely

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

INTRODUCTION A-7

bridge the pores. These rigid particles do not
deform and the result is that a filter cake is formedon the surface of the filter that may be many times
more efficient as a filter than that of the filter
matrix itself. In dead end filters, these filter cakes
can build up to a considerable depth. As they
become thicker they offer more and more resistance to flow. Therefore, there is a practical limit
where the added filtration efficiency of the cake ismore than offset by the increase in flow resistance. An effective filter cake must be formed ofrigid particles since deformable particles will
nearly always squeeze into a compacted massthat will effectively reduce flow to an unacceptable level.
Figure A-10B “Sieving” mode of plugging

Throughput can often be dramatically increasedby analysis of the fluid to be filtered. In the caseof a very dirty liquid, for example, the liquid can beallowed to remain at rest for an extended period
during which time gravity will settle some of theparticles, particularly those of large size. In othercases, smaller particles can be made to agglomerate into larger particles that are much easier tofilter. However, even with these pre-treatments, itis nearly always necessary to additionally turn toother techniques to increase throughput. These
can include use of a suitable pre-filter in combination with the final filter, agitation, cross-flow, backwashing, serial filtration with two final filters, andcareful consideration of the proper installation of
the final filter media in its holder.

Fundamentals of Flow
Flow rate is defined as the volume of fluid thatflows past a given point per unit of time. It is
directly or inversely proportional to several parameters as shown below:
A ∆P
Q is proportional to ________
RÓ

Figure A-11 Plugging by pore bridging

Where:Q is the flow rate
R is the resistance to flow
A is the surface area of the filter∆P is the pressure differential
Ó is the viscosity of the fluid

The terms “flow rate” and “flux” are often used interchangeably. However, flux is
generally considered to be the flow rate through the filter per unit of filter surfacearea.
In dead end filtration, flow rate and flux, or flux rate, have essentially the same
meaning. The term flow rate is most commonly used however.

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THE ABCs OF FILTRATION

In common use, for tangential (cross) flow the rate of flow through the filter is often
called flux or flux rate, while the term flow rate designates the rate of flow acrossthe face of the filter.
Shown in figures A-12, A-13 and A-14 are the concepts of pressure drop and flux
rate as they relate to simple and compoundsystems. In a simple system (figure A-12), thepressure differential is simply the upstreampressure minus the downstream pressure.
When the filters are in series (figure A-13), thetotal pressure differential is the sum of the pressure drops across each of the three filters (orthe pressure dropFigure A-12 Simple flow across a module

Figure A-13 Series flow

between the first and last filter).
When the filters are in parallel (figure A-14), the pressure drop acrossthe three filters is equal, but the total
flux rate is the sum of the flux rates
through each of the three filters.
Other general considerations
include the following:
1. Increasing the pressure differential
increases the flux rate in direct proportion. However, if the increase inpressure drop causes compaction of
the filter or its cake, if the particles
are compressible, or if flow becomes
limited by the filter holder, flux rate
may decrease.
2. Increasing the cross sectional areaof the filter increases the flux rate indirect proportion.

Figure A-14 Parallel flow

3. Decreasing the viscosity of the fluidby increasing its temperature will
increase flux rate in inverse proportion.
In many cases, it is necessary to

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INTRODUCTION A-9

calculate the size of the filter module required for a specific filtration application.
This can be done as follows:A=

V___________(t) x (F)

Where:
A = required filter surface area in square meters (1 m2 = 10,000 cm2)
V = filtrate volume desired in liters (L)t = processing time in hours
F = steady state flux in liters per square meter hour (L/m2hr)
Example:900 liters of 0.2 µm clarified E. coli lysate is desired
The steady state flux is 50 L/m2hr
The desired processing time is 6 hours

.

A

=

900 liters________________________
6 hours x 50 L/m2hr= 3.00 m2

Tangential (Cross Flow) Filtration vs. Dead-End Filtration
Throughput can often be significantly improved by using a cross flow systemrather than a dead-end flow system. (The terms tangential flow and cross flow
generally have the same meaning; although tangential flow is sometimes used todesignate primarily laminar flow along the membrane surface and cross flow candesignate either laminar or turbulent flow along the membrane surface.)
As shown in figure A-15, in dead-end flow the flow rate gradually decreases as apolarized layer builds up on the surface of the filter. Eventually, an unacceptablepressure differential is reached.

Figure A-15 Tangential vs. dead-end flow

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A-10

THE ABCs OF FILTRATIONIn the case of the tangential or cross flow system configuration the particles ormolecules are continuously swept away from the surface of the membrane by theflow stream across the surface. (More details about dead-end and cross flow configurations are included in a later section.)

Membrane Filtration
Membrane filtration has been known almost 100 years, yet it has only been since
the mid-twentieth century that the membrane filter became a readily available
commercial product. Originally marketed for water bacteriology, it soon became
clear that membranes had a wide range of other applications.
Application of membrane filters to biochemistry was a major advance in the
1950’s. In the 1960’s membrane filters were used for the first time in nucleic acidhybridization. And in the 1970’s, development of recombinant DNA technology led
to the use of membranes in gene cloning.
In the pharmaceutical industry membrane filters are widely used for the cold sterilization of drugs, serum and large-volume parenterals. Membrane cartridges haveprovided particle and pyrogen free water for many industries, especially in microelectronics and pharmaceutical products. Membrane filtration is also used in air
filtration for contamination control, air pollution analysis, and gas sterilization.
Continuing development of membrane technology has led to membranes being
routinely used in a wide spectrum of research, laboratory and process applications. Descriptions of many of these applications follow in later chapters.

Figure A-16 Comparison of various membrane and filtration processes

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INTRODUCTION A-11

Disposable membrane modules are now used routinely for manufacturing biopharmaceutical products. This eliminates the need to requalify filtration systems thatutilize reusable modules and membranes.
Filtration membranes are often classified according to the size of the particle to beretained with or separated from the fluid. This is shown in figure A-16.
More complex separations might involve other membrane and feed fluid/particle
characteristics such as electric charge, valence and concentration gradient, forexample. In this case, in conjunction with the particle size, these additional characteristics might be the separation mechanism.
MACROFILTRATION is the separation of particles of one size from particles ofanother size where at least one of the particles is larger than 5 micrometers (µm).
MICROFILTRATION is the separation of particles of one size from particles ofanother size in the range of approximately 0.05-5.0 µm. The fluid may be either aliquid or a gas.
ULTRAFILTRATION is the separation of molecules of one size from particles
and/or molecules of another size. The size of the molecules may range from 10 to
500 angstroms (Å) (0.001 to 0.05 µm).
DIAFILTRATION is the removal or washing of molecules by the addition of solvent
directly to the solute being purified or retained. Constant volume and particle concentration is thus maintained. It is also used for desalting or buffer exchange.
DIALYSIS is the separation of solutes in a solution based on size exclusion acrossa semipermeable membrane where the driving force is the concentration gradient,with particles moving from an area of higher concentration to an area of lower concentration. Additional information about dialysis is contained in a later chapter ofthis book.
REVERSE OSMOSIS is a process that separates small molecules and ions, lessthan 10 Å in size, from a liquid such as water.
ELECTRODIALYSIS is the transport of ions through a semipermeable membraneas the result of an electrical driving force.
POLYMERIC MEMBRANE EXTRACTION is a new technology for the selective
extraction and concentration of organic compounds from dilute aqueous solutions
and complex processing mixtures based on molecular properties.

CALL 1-800-634-3300 FOR FREE FILTRATION AND BIOPROCESSING CATALOG

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

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A-12

THE ABCs OF FILTRATION

Guide to Filtration, Bioprocessing and Selection of Spectrum Membranes

Volume of Liquid Processed Based on Module Type10, 000 L
1, 000 L
100 L
10 L
1L
100 mL
10 mL

R & D Scale(Syringe Pump Driven)

R & D Scale(Syringe Driven)

R & D Scale(Peristaltic Pump Driven)

R & D Scale(Peristaltic Pump Driven)Laboratory
Scale
Laboratory
Scale

Figure A-17 Filtration characteristics of Spectrum products

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THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

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Pilot/Production
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INTRODUCTION A-13

DialysisPrimarily used in laboratories andprocess applications for concentrating,desalting and purifying proteins andenzymes, dialysis is a means of separating molecules of different sizes. As
shown in figure A-18, it involves placing
a solution to be dialyzed on one side ofa semipermeable membrane that has ahigher degree of selectivity for small
molecules and solvents than for larger
molecules (macromolecules).
While the retentate solution is on one
side or in the lumen of a tube or hollow
fiber module, a buffer solution is on theother side. Small molecules will passively diffuse through the membrane in
Figure A-18 Operation of hollow fiber dialysis membrane
both directions, driven by the concentration gradient until equilibrium isreached. The membrane retains macromolecules. Generally little or no pressure isused to assist in the transport of the smaller molecules into the dialysate buffer.
Dialysis membranes and hollow fiber modules are available for separating molecules with a range of molecular weights from 100 to 1,000,000 Daltons.
Dialysis separation is presented in much greater detail in a later chapter of thisbook.

Electrodialysis (ED)Electrodialysis is defined as the transport of ions through a semipermeable
membrane as the result of an electricaldriving force.
Although the most frequent use of electrodialysis is in removing salt fromwater, with the proper membrane it canbe used for separating electrolytes fromnonelectrolytes, cations from anions,
univalent ions from multivalent ions, etc.
Secondary only to the desalting ofwater is the use of ED in the productionof chlorine and caustic soda.
Figure A-19 illustrates the basic
process involved in the separation ofsalt from water, which has two cations,
H+ and Na+, and two anions, OH- andCl-.
Figure A-19 Principle of operation of electrodialysis

A voltage is established across a feed-

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A-14

THE ABCs OF FILTRATION

water solution separated by anion-permeable (but cation-impermeable) membranes, marked A, and cation-permeable (but anion-impermeable) membranes
marked C. The positive sodium and hydrogen cations are attracted toward thenegative cathode. They readily pass through the cation permeable membranes
but are rejected by the anion permeable membranes. If there is no membrane in
the path of the hydrogen cation, it is released at the cathode in the form of gas(H2). In reverse, the same thing happens to the oxygen and chloride anions. As aresult, in those sections of the electrolytic cell where there is a rejection of thehydrogen and sodium ions and transport of the oxygen and chloride ions, the ionspartially recombine to form saltier water. In those sections of the cell where thereis transport of both the hydrogen and chloride ions, only pure water will remain.

Reverse Osmosis (RO)Osmosis is a natural phenomenon that takes place when water passes from a less concentrated solution through a semipermeable barrier to a more concentrated solution.
Figure A-20 illustrates simple osmosis.
A semipermeable barrier separates adilute and a concentrated solution.
Flow occurs from the dilute to the moreconcentrated solution until the two solutions are equalized in concentration. At
equilibrium the head that develops iscalled the “osmotic pressure”. For
water this is equal to approximately 1
psi for each 100 parts per million (ppm)
total dissolved solids.

Figure A-20 Natural osmosis

Reverse osmosis occurs when a pressure is applied to the side containing
the more concentrated solution asshown in figure A-21. At a pressure
equal to the osmotic pressure, flow willcease. At a pressure greater than theosmotic pressure, flow is reversed.
In most reverse osmosis applications,tap water is forced through a membrane under pressure, generally in across flow configuration where the concentrate of tap water not transported
through the membrane goes to drain.
Reverse osmosis rejects not only a highpercentage (90-97%) of salts but also avery high percentage of organic matterand other particulates.

Figure A-21 Schematic of operation of reverse osmosis (RO)p 14

Figure A-22 shows the mechanism forsalt rejection in which a pure waterlayer of about 10 Å (0.00l µm) thickness
develops on the surface of the membrane due to the chemical nature of the

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INTRODUCTION A-15

membrane. Salts are repelled from thesurface of the membrane, with higher
valence ions being repelled to a greater
distance from the membrane than lower
valence ions.
Organic and other particulate matter isrejected entirely on the basis of the sizeand configuration of the particulate ororganic molecule as shown in figure A23. This is the familiar filtration mechanism except that the “pores” of the RO
membrane will filter down to very small
molecules. Since the typical RO membrane has 20 Å pores, nearly all molecules above a molecular weight (MW)of 200 are rejected and depending on
Figure A-22 Salts are repelled from the surface of RO membranes according
molecular shape, a substantial percentto molecular weightage of molecules of MW 100-200 are
rejected. Thus, sugar, with a MW 342 isrejected and formaldehyde with MW 30 is passed. This latter feature is importantsince it allows the RO equipment to be sterilized by a simple formaldehyde flush.
Upwards of 90-95% of all pyrogens, viruses and bacteria are rejected by the RO
membrane.
The most successful RO membranes for commercial use are spiral wound polymeric membranes with a very thin skinsupported by a porous substructure. In
these membranes, the substructureacts only as support for the active layer
and generally has no effects on the filtration characteristics of the membrane.
Since the actual discriminating barrier
is extremely thin, very high flow rates
can easily be achieved. Due to thehighly asymmetric structure, all rejectedmaterials are retained on the membranesurface and internal pore fouling is virtually eliminated.

Figure A-23 RO membranes reject most molecules with MW > 200

The largest application for reverse
osmosis has been for the production ofultrapure water for pharmaceutical,
medical, electronic and other industrial
applications. There is also a growing
market for household RO systems.

Selective Gas Permeable (Permselective) Membranes
Although the first reported observation that different gases permeate through media
at unequal rates occurred in 1831, significant work on permselective membranes

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A-16

THE ABCs OF FILTRATION

did not begin until the mid 1940’s with the availability of new polymeric materials.
Basically, permeation of a gas through a nonporous membrane involves adsorption of the gas on one side of the membrane; diffusion of the gas through themembrane; and desorption of the gas from the opposite side of the membrane.
Gas separation now ranks as one of the most important membrane applications.
Major applications include separation of helium from natural gas, oxygen enrichment from air, recovery of hydrogen from ammonia synthesis, enhanced oil recovery, separation of hydrogen and carbon dioxide from ammonia and methanol
purge gases and removal of sulfur dioxide from processing high sulfur coal.

Polymeric Membrane Extractions
A novel class of membranes has been recently introduced for the selective extraction and concentration of organic compounds from dilute aqueous solutions. The
pores of these advanced hollow fiber membranes are impregnated with a polymeric liquid having an affinity or attraction for the organic compound of interest. This
technology of separating compounds from a liquid or gas stream depends primarily on the chemical properties of the liquid polymer used and not on the conventional sieving or rejection of molecules or particles through the membrane pores.
These liquid membranes selectively transport and extract specific compounds byutilizing various molecular properties such as hydrophobicity, hydrogen bonding
capability, etc.
Figure A-24 illustrates a laboratory scale setup using syringes as pumps. The
SeleXtrac™ membrane module is designed with two sets of inlet and outlet ports
for circulating two different mixtures of a liquid or a gas or both. One set ofsyringes is used to circulate the “Feed” solution” (sample mixture). The other setof syringes is used to circulate the“Strip” solution” in a countercurrent flow
direction.
By chemical interaction, targetedorganic molecules are transported fromthe Feed solution through the liquid
polymer contained in the pores of thehollow fiber membranes and into theStrip solution that is being recirculated
through the fiber lumen. Either the
Feed solution or the Strip solution may
be a liquid or a gas mixture.
Larger scale extractions use either peristaltic or large volume pumps to circulate the two solutions.

Figure A-24 Schematic of syringe pumped SeleXtrac™ module

p 16

A complete description of polymeric
membrane extraction is included in alater chapter of this book.

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INTRODUCTION A-17

Integrity Testing Membrane Filters
It is often important to check for complete integrity of the filter mounted in its
holder. The most widely accepted test over the years has been the “bubble
point” test.
Referring to figure A-25, when capillaries are full of liquid, the pressure
required to force the liquid out of thecapillary must exceed the surface
tension of the liquid. The capillarypressure is higher in the case of asmall capillary than for a large capillary, i.e., a higher air pressure isrequired to force water from thesmaller diameter. The same is true ofthe pores in a membrane.

Figure A-25 Illustration of capillary pressure

Figure A-26 illustrates the operationof the bubble point test. When the
bubble point pressure is reachedbubbles will form downstream of thefilter. This is called the bubble point.
As pressure is further increased,
rapid bubbling will begin to occur. If
bubbling occurs before reaching the
bubble point pressure, the test has
failed.
In performing a bubble point test, it
must be considered that air has afinite diffusion rate through a liquid
and over a period of time small bubbles may form on the downstreamside of the membrane at pressures
well below the bubble point. For
example, a membrane rated at 0.22
micrometers might have a typical diffusion rate for air through water ofapproximately 0.04 ml/min/psi/sq. ft.

As a variation of the bubble point test,
Spectrum has developed the pressure decay test. In this test, the module is fully primed to ensure complete
wetting and a pressure of 5 psi of filtered air or nitrogen is connected tothe system feed inlet. An integral sysFigure A-26 Filter integrity test
tem and membrane will maintain 5 psiof pressure (less gaseous diffusion)
after shutting off the gas supply. To compensate for gaseous diffusion throughthe membrane, a pressure decay less than 0.1 psi/min indicates an integral
membrane and system.

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A-18

THE ABCs OF FILTRATION

If pressure decays more rapidly than 0.1 psi/min, the filtrate tubing is then submerged in water. If bubbles are seen, the membrane either has not been completely wetted or it has a defect and must be changed. If bubbles are notseen, then air is leaking from the system upstream of the membrane.
The equation for determining bubble point pressure is:Where:

4 Ó cos Ω
P = k ___________d
P = bubble point pressured = pore diameterÓ = surface tension of the liquidΩ = liquid to membrane contact angle
k = pore shape correction factor (the value of k is 1 for a perfect screen)
In the case of a hydrophobic membrane, a test similar to the bubble point test
is often used. Water is placed on one side of the hydrophobic filter and thewater pressure is increased until the filter
wets on the dry side of the filter. Thispressure is called the water intrusion pressure and is a good measurement of thehydrophobicity of a membrane. In general, the water intrusion pressure for ahydrophobic 0.2-micrometer membrane
will be on the order of 25 to 30 psi versus
the bubble point pressure of 70 to 90 psi
for a hydrophilic membrane.
Hydrophobic membranes are most often
used when filtering moist air since thepores of a hydrophilic membrane would
soon plug from condensation of the waterin its pores. A hydrophobic membrane,on the other hand, will condense water on
its surface but will not condense water
within its pores.
As shown in figure A-27, the contact angleof a liquid with a membrane is defined as
the angle between a line that is perpendicular to the exact point at which the liquidcontacts the filter and a line that is perpendicular to the surface of the filter.

Figure A-27 Contact angles for hydrophilic and hydrophobic membranes

p 18

Filters with water contact angles of 0 to
40° are considered hydrophilic. Contact
angles of 90° or more are defined ashydrophobic. The contact angles of membranes vary from nylon with a contact
angle near zero (very hydrophilic), throughmost hydrophilic membranes with typicalcontact angles of 25°, to PTFE with a contact angle of approximately 130° (veryhydrophobic).

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TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE MICROFILTRATION A-19

Tangential (Cross Flow) Hollow Fiber Module Ultrafiltration
Ultrafiltration (UF) separates dissolved particles and molecules according to sizeand configuration by flowing a solution that contains these molecules through amembrane. The membrane will retain most particles and molecules above its
retention rating and will allow most smaller molecules, along with the solvent, topass through the membrane.
Ultrafiltration is similar to reverse osmosis in that it filters on the basis of molecularsize, but it differs in that it does not separate on the basis of ionic rejection.
Another distinction is that UF operates at a moderate pressure, (on the order of 10
to 50 psi), while RO, depending on the concentration of the dissolved solids, may
require pressures upward of 1200 psi.
The main difference between ultrafiltration and microfiltration (MF) is the pore size.
MF separates micron and submicron sized particles. UF separates particles andmolecules from molecules of significantly lesser molecular weight. The principal
difference between UF and dialysis is that UF involves the application of pressure.
Dialysis does not. Dialysis depends entirely on the concentration gradient as its
driving force.
Although the UF membrane is not absolute, it does retain most macromolecules
above its molecular weight cutoff while allowing smaller molecules and the solvent
to pass through the membrane. As in the case of RO it does not involve phasechanges nor temperature extremes.
The selection of an ultrafiltration membrane requires the consideration of a numberof important performance parameters. These include retentivity, selectivity, molecular weight cutoff, filtration (flux) rate, adsorption, and temperature and chemical
resistance.

Retentivity
Ultrafiltration membranes are rated
according to the retention of a substantial percentage of molecules at or larger
than its pore size rating. This is defined
as its nominal molecular weight cutoff(MWCO). Since UF membranes are notabsolute, the shape of the molecule tobe retained (or passed) has a major
effect on retentivity. It can be seen in
figure A-28 that a linear molecule may
pass through a UF membrane while a
globular molecule of the same molecularweight may be retained.

Figure A-28 Retention of globular and linear molecules by UF membrane

In addition to the physical configurationof the molecule, the electrical charge ofa molecule will affect the separation
characteristics of the membrane.
Furthermore, retention varies because of
the pore size distribution that is characteristic of all membranes. Therefore, it is

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A-20

THE ABCs OF FILTRATION

difficult to precisely characterize themolecular weight rating for ultrafiltration
membranes.
In many cases, UF membranes are rated
at that MWCO where at least 90% of
spherical (globular) uncharged moleculesof that same molecular weight will be
retained. However, as previously noted, asubstantial number of linear moleculeswith a molecular weight equal to or
greater than this MWCO rating may passthrough the membrane. Figure A-29
shows solute retention efficiency versus
molecular weight for Spectrum
Laboratories’ ultrafiltration membranes.
Specific retention characteristics ofSpectrum hollow fiber UF membranes forvarious molecular weights is shown intable A-1. The bold retention percentage indicates the specified MWCO for thatparticular membrane.

Figure A-29 Solute retention of Spectrum UF membranes

Retention of Spectrum Membranes (percent)
Solute Size

SP-10 kD

SP-50 kD

SP-400 kD

SP-50nm

1 kd
3 kD
5 kD
10 kD
20 kD
30 kD
40 kD
50 kD
60 kD
100 kD150 kD
200 kD
300 kD
400 kD
500 kD630 kD
750 kD
800 kD
900 kD
1,000 kD
2,000 kD

20%
70%
90%
99%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%

0%
5%
15%
30%
57%
75%
85%
90%
94%
99%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%

0%
0%
0%
5%
16%
25%
33%
41%
48%
67%
80%
90%
97%
99%
100%
100%
100%
100%
100%
100%
100%

0%
0%
0%
0%
0%
2%
3%
5%
8%
18%
27%
39%
55%
66%
75%
83%
88%
90%
92%
94%
100%

Table A-1 Molecular retention by Spectrum UF membranes

MWCO Range
The retention efficiency, high selectivity and complete range of MWCO ratings
available with Spectrum Laboratories UF membranes give the user a precision of
separation previously unavailable.

p 20

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TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE ULTRAFILTRATION A-21

Selectivity
Also of importance in evaluating membranes is the degree of retention of molecules with molecular weights somewhat below the MWCO rating of the membrane.
If a membrane retains few of these molecules it has high selectivity (or a sharp
cutoff), a very desirable characteristic. If a membrane retains substantial numbersof molecules with a molecular weight somewhat below the membrane MWCO rating, it is said to have low selectivity (or a diffuse cutoff). Spectrum UF membranes
show a narrow pore size distribution which guarantees a relatively high selectivity
and sharp MWCO.

Adsorption
All UF membranes will adsorb nonspecifically some percentage of rejected substances. Such adsorption can cause erroneous and misleading results in criticalapplications, such as in clinical laboratories or in sensitive research areas.
Adsorption losses increase with membrane surface area, especially in the case ofproteins. In general, protein adsorption can be expected to be between 5 and 20µg/cm2.

Temperature and Chemical Resistance
Temperature and chemical resistance characteristics are important for non-aqueous solutions and for operating conditions higher than room temperatures.
Different membrane materials offer different resistance characteristics. UF membranes are typically made from polypropylene, polysulfone or cellulosic polymers.
Refer to Chemical Resistance Table on page F-150
In addition, sterilization can subject the membrane or system to high temperaturesor chemicals.

Filtration (flux) Rate
The membrane filtration rate will increase with increasing MWCO rating of themembrane and with increasing temperature. In some cases membrane flux will
also increase with increasing pressure, depending upon the molecular species
retained, the concentration of the solute and the hydrodynamic conditions at themembrane surface. In addition, the achievable flux rate depends on the concentration and heterogeneity of substances in solution. While membrane flux rates arenormally stated for ultraclean water, in practice the flux will be lower when processing biological solutions due to the phenomenon of concentration polarization.

Concentration Polarization
In addition to flow reduction caused by physical plugging of the membrane pores,
membranes can also experience filtration reduction, at least temporarily, by theformation at the membrane surface of a layer of concentrated macromolecules orparticles. Such a layer is called “concentration polarization” in the case of macromolecules. This layer will form to reduce flux even in the absence of physicalplugging.

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THE ABCs OF FILTRATION

Figure A-30 illustrates the principle ofconcentration polarization. Initially all
solute species are uniformly distributed
throughout the solution, as shown in figure A-30A. In figure A-30B, as pressure
is applied, the solution and small molecules pass through the membrane.
Macromolecules are retained at the surface of the membrane. Since the backdiffusion rate of these large molecules isslow due to their size, they accumulate
in a layer above the membrane. At thispoint, filtration is still limited only by thecharacteristics of the membrane and thepressure. This is known as “membrane
controlled filtration”.
As shown in figure A-30C, as pressure isincreased the number of these molecules in the boundary layer may
increase until a limiting concentration isreached. At this time further increasesof pressure may become ineffective, andthe concentration polarization itself may
control the filtration. This is often
referred to as “gel controlled filtration”.
The highest concentration occurs at themembrane surface and decreases exponentially toward the solution. In the caseof higher molecular weight substances,
the solubility limit is often reached at themembrane surface. The precipitatedlayer acts as a secondary membrane(the so-called “gel layer”), which meansthat the flux rate may decrease considerably. In addition, this “gel layer” mayhave a higher retention rating than themembrane itself.
Several factors may act to control formation of the gel layer. Most significant ismolecular diffusivity that is, among otherthings, a function of molecular weightand configuration. Linear molecules
tend to diffuse more slowly and thus forma gel sooner than is the case with globularmolecules as shown in figure A-31.

Figure A-30 Concentration polarization on surface of UF membrane

p 22

Agitation to increase the diffusion of themacromolecules back into the solution
partially or totally eliminates concentration polarization or gel limited filtration.
Figure A-32 illustrates the concept of

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TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE ULTRAFILTRATION A-23

using agitation to assist diffusivity and thus
filtration.
Sometimes flux rate increases quite dramatically with agitation as shown in figure
A-33.
Agitation includes not only physical movement of the solution such as stirring, shaking, etc., but also tangential or cross flowwhich is very effective in clearing the gellayer. However, the method of agitationmust take into consideration the effects on
the solutes such as denaturing, shearing,etc.
Figure A-31 Relative diffusivity of linear and globular membranes

Figure A-32 Agitation of the solution will often assist diffusivity of themolecules for higher flux

Figure A-33 Agitation of the solution will often dramatically increase theflow rate

Feed flow in Spectrum hollow fiber membranes is always through the center, or
lumen, of the membrane. This assureslaminar rather than turbulent flow along theinterior surface (or skin) of the membrane.
Although a gel layer may form on the membrane surface, a backflush will generally
break up and clear the gel layer, allowing
for extended throughput.
Tangential (cross flow) filtration shows particularly dramatic results when filtering a
fluid containing deformable particles. In
normal (dead-end) flow these particles
tend to compact on the filter surface andplug the filter very rapidly. In cross flow
fluid systems, these particles are continuously swept away by the feed stream,
greatly inhibiting plugging of the filter.
Figure A-34 illustrates a typical microfiltration application, the concentration of
deformable particles such as yeast, thatreflects the same phenomenon that occurs
in ultrafiltration. The cross flow fluid management technique clearly reduces theplugging of the filter when compared to thesame solution in dead-end flow.

CALL 1-800-634-3300 FOR
FREE FILTRATION AND
BIOPROCESSING CATALOG

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A-24

THE ABCs OF FILTRATION

Figure A-34 Decay of flow rates for concentration of yeast in cross flow and dead end filtration modes

Other Factors Affecting Membrane Flux Rates
TEMPERATURE - Water flux and process fluid flux will increase with increasing
temperature. Clean water flux varies linearly with changes of water viscosity. Overa range of 57° to 97° F changes in water viscosity varies with the ratio of the temperature change in degrees Fahrenheit.
( Ta )
Temperature Corrected Flux = (Flux At Standard Temperature) -------------( Ts )Where:
Ts = Standard temperature (e.g. 77°F)
Ta = Actual temperature (°F)

Process flux will also increase with temperature as illustrated in figure A-35.
However, the degree of process flux
improvement is less predictable thanwith clean water since both a “gel” layer
and a “fouling” layer on the membranesurface contribute to flux resistance.
As a general rule, operation should be at
the highest acceptable temperature, taking
into consideration temperature limitationsof the feed stream and the membrane.

Figure A-35 Flux increases with increasing temperature

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TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE ULTRAFILTRATION A-25

TRANSMEMBRANE PRESSURE (TMP)
Clean water flux will increase linearlywith increasing transmembrane pressure, as shown in figure A-36. Process
liquid flux will typically increase as afunction of transmembrane pressure.
However, depending on the circulation
rate, the improvement in flux may
become asymptotic since the “gel”
layer’s resistance to flux will increase
from compaction of the macromoleculesfrom the TMP.

Figure A-36 Flux will increase with increased transmembrane pressure until
gel limited flow is reached

Controlling the permeate back pressure(or permeate flux rate) may reduce thetendency of the membrane to foul in theinitial stages of a concentration, providing an overall higher average flux rate.

The transmembrane pressure may be calculated as shown below:
Pinlet + Poutlet
Transmembrane Pressure (TMP) = ---------------------------- - Ppermeate
2
CIRCULATION RATE - The circulation rate for clean water will have little or noeffect on flux since there is no gel layer nor fouling layer to restrict permeation.
In the case of process liquids, cross flow filtration will reduce gel layer formation.
Cross flow management will lower the resistance to filtration and will improve flux.
Thin feed flow channel devices (hollow fibers, spiral-wound cartridges, plate-andframe devices, etc.) all operate in laminar flow. Increasing the circulation rate will
increase the shear force and typically enhance the rate of filtration.
However, the pressure drop across thin channel devices will increase withincreased circulation rate. This limits thedegree to which feed velocity can beraised. Generally, to achieve high velocities with thin channel devices, the feedflow path should be as short as practical.

Figure A-37 Flux generally declines with increased solution concentration

CONCENTRATION - Process liquid filtration is highly dependent on feed components and the overall solute concentration. As would be expected, flux
declines with concentration. The rate of
decline generally decreases as shown infigure A-37.The volume concentration
factor (Cv) may be calculated as shownon the next page:

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A-26

THE ABCs OF FILTRATION

Where:

Cv = ------------------Vo
Vo – Vp
Vo = Original Feed Volume
Vp = Volume of Collected Permeate

The system conversion percentage (Sp) may now be calculated as follows:

Sp = (1 - ---------- ) 100
1
Cv
TIME - Filtration declines with time, even with “clean” water. The influence of timeon the rate of flux decline may, however, be insignificant compared to the effect ofconcentration. A rapid flux decline, while concentrating a solution indicates either
the circulation rate is too low or the stream contains bad foulants. Flux might also
decline over time due to gel layer compaction.
PARTICLE LOAD IN FEED STREAM – As previously shown, the feed stream flowrate has a major effect on permeate flux. However, consideration must also begiven to the feed stream particle load in determining the optimum flow rate.
Low fouling streams exhibit stable flux rates over time with low circulation rates.
The flux of low-fouling streams is basically concentration dependent. Please referto figure A-38.
A circulation rate that provides an intermediate shear force, between 4,000 to
8,000 sec-1, is a good starting point forprocessing low fouling streams.
However, for feed streams containing
fragile components that may be damaged by high circulation rates or hightemperatures, shear forces on the orderof 2,000 to 4,000 sec-1 are recommended.
If the stream is high fouling and there are
no fragile components in the stream,increasing the feed stream circulation rate
to at least 8000 sec-1 will generally reduce
gel layer thickness and increase flux.
Figure A-38 Variation of flux with tangential shear force for pure water andprocess liquids

Diafiltration

When using ultrafiltration for purification, desalting and/or buffer exchange, small
molecules and solvent pass through the membrane while macromolecules are
retained. The simplest method for this therefore is to replace the solvent that had
passed through the membrane with contaminant free solvent. This can be doneon an intermittent or continuous basis.
To illustrate the intermittent method, imagine that 90% of a solvent containing 1%
salt passes through a membrane. The feed solution is then brought back to its

p 26

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TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE ULTRAFILTRATION A-27

original volume by the addition of salt
free solvent. The feed solution would
now contain only 0.1% salt and the same
original number of macromolecules.
Repeating this process again, the resulting feed solution would then contain only
0.01% salt. Since the rate of removal of
the salt is proportional only to the pressure driven flux through the membrane,
and not to the impurity concentration,
low concentrations can be reached very
quickly.
Continuous diafiltration is a much more
efficient procedure in using ultrafiltration
for desalting and/or buffer exchange
than the simple one described above. In
this process, the solvent that is removed
is continuously replaced at the same rateby contaminant-free solvent. This allows
virtually complete purification. As shownin figure A-39, the wash fluid is pressurized by an external gas source or pulled
by vacuum, forcing this fluid into the cell
to replace the solvent volume that filters
through the membrane. The volume ofsolution and the number of macromolecules remain unchanged while themicromolecules are removed through themembrane.

Figure A-39 Schematic of continuous diafiltration system

In practice, a pressure balance or a liquid level controller may be used on thereservoir to insure dialysate addition at
the same rate as filtrate removal.

For constant volume diafiltration, each wash volume of filtrate removed reduces
the solute concentration by factor of e (2.718..). For example, a four fold constantvolume diafiltration will reduce the concentration of solute by a factor of e4, 50 foldor over 98%. Using this technique, the concentration of solute can be monitored
in the filtrate until the desired level of purification or product recovery is achieved.
The system set up is the same as for topped-off batch concentrations or clarifications except that the wash buffer enters the vessel instead of feed.
Figure A-40 shows a schematic of a Spectrum hollow fiber system operating in thediafiltration mode.
As permeate passes through the hollow fiber module from the processing reservoir, liquid flows from the feed or buffer reservoir to compensate for the removed
permeate.

Tangential (Cross Flow)

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THE ABCs OF FILTRATION

Figure A-40 Schematic of Spectrum diafiltration system using MiniKros® module with reservoir in a constant volume diafiltration mode.

Hollow Fiber Module Microfiltration

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TANGENTIAL (CROSS FLOW) HOLLOW FIBER MODULE MICROFILTRATION A-29

Microfiltration is the separation or removal of particles of more than 0.05 µm andless than 5 µm in size from a fluid or from particles of other sizes. The fluid may
be either a liquid or a gas. Particles include both rigid and deformable types.
Microfiltration media are available in a wide variety of materials and methods ofmanufacture, and are rated “absolute” or “nominal” depending upon the percentage of capture of particles of equal size or larger than the retention rating of themedia.
Nominal microfiltration media include fibrous mats and similar materials having aretention efficiency less than 100%. The performance rating for this media is often
specified as capture of 95% or 98% of all particles equal to or larger than the stated pore size of the filter.
Figure A-41 shows retention characteristics for a typical depth filter rated to capture 98% of all particles equal to or greater than 1 µm. The retention curve
approaches the 100% mark on an asymptote of performance eventually reaching
100% retention for very large particles. Although this curve shows no particle capture below 0.2 µm, in practice nearly all fibrous media have an electric charge thatis effective in the capture of very small particles. This charge is called zeta potential.
Only media that retains 100% of particles
at its pore size rating (within a finite timespan) are considered absolute. In general, only macrofiltration screens andmicroporous membrane filters fall within
this definition of absolute.
Microporous membrane filters have sometimes been classified by their pore structure, either capillary pore or tortuous pore.
Capillary pore membrane filters are true
screens, similar to macroporous screens.
They have round cylindrical pores, moreor less normal to the surface of the membrane, with even random pore dispersion
Figure A-41 Retention characteristic for a typical depth filter rated at 98%
over the surface of the membrane.
capture of 1 µm particles
Capillary pore screen membrane filters
are commercially available in thin films ofpolycarbonate or polyester, and are manufactured in a two step nuclear track andetch process.
Tortuous pore microporous membrane filters have a relatively rough surface where
there appears to be many openings considerably larger than the rated pore size.
These membranes are nevertheless absolute since they depend upon the random
tortuosity of the numerous flow paths to achieve their pore size rating. These
membranes are commercially available in various cellulosic compounds, nylon,
polysulfone, polyethersulfone, polyvinyl chloride, pure silver, polytetrafluoroethylene (PTFE), polyvinylidine difluoride (PVDF) and many other materials.

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

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FILTRATION MODES: CONFIGURATIONS A-31

Filtration Modes (Configurations)
Membrane and Module Configurations
Microfiltration membranes are primarily available in flat sheets, pleated cartridges,
and spiral wound, plate and frame or hollow fiber modules. Sheet membranes
and pleated cartridges are mostly used in dead end flow configurations.
Spiral wound modules and hollow fiber modules are generally in a tangential(cross flow) flow configuration.
Although dead end flow is much more widely used in filtration applications, plugging of the filter frequently becomes a major problem. Often throughput is significantly improved by using tangential flow instead of dead end flow.
Ultrafiltration membranes are primarily available in sheets, spiral wound modules
and hollow fiber modules. Because of the rapid plugging that occurs when using
UF membranes in dead end flow, tangential (cross) flow is used almost universallyin UF applications.
Figures A-42 and A-43 illustrate the flow paths for the two types of systems. The
dead-end flow system experiences molecular or particle concentration on the surface of the filter, reducing the permeation rate and eventually causing an unacceptable flow rate or pressure differential.

Figure A-42 Build up of polarized layer in dead end filtration mode

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A-32

THE ABCs OF FILTRATION

Figure A-43 Cross flow mode reduces concentration polarization and extends filter life

For dead end flow, the Transmembrane Pressure (TMP) may be calculated asfollows:

TMP = Pfeed - Pfiltrate

Tangential flow (or cross flow) separations are an efficient way to separate streams
that become quickly plugged using dead end techniques. When using tangential
flow techniques, most of the process fluid flows along the membrane surface
rather than passing through the membrane structure. Fluid is pumped at relatively
high velocity parallel to the membrane surface.
In tangential flow, except for water treatment applications, the permeate is only asmall percentage of the tangential flow along the membrane surface. In most celland particle separations, for example, only 1% to 5% of the feed flow becomes
permeate. The remaining 95% to 99% exits the membrane device as retentate.
Retentate is recirculated back to a feed reservoir or to the module inlet.
Various tangential flow membrane geometries include: stacked plate and spiraldevices that utilize flat sheet membranes; tubular devices; and shell and tube
devices that use hollow fiber membranes.
In the case of tangential flow separations, the driving force (transmembrane pressure, TMP) is the difference between the average of the membrane feed andretentate pressures, and the permeate pressure as shown below:
Pfeed + Pretentate
TMP = --------------------------------- - Ppermeate2

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FILTRATION MODES: CONFIGURATIONS A-33

Retentate flow results in a build up of components on the membrane inside surface. Generally these components are carried down the length of the membranes
and out the end of the module by the sweeping action of the recirculating fluid.
However, under certain conditions a cake layer accumulates on the surface of themembrane. This boundary layer is composed of solids and/or solute macromolecules which are retained by the membrane during the course of filtration. Thisphenomenon, concentration polarization, can affect module performance byreducing the apparent size of the membrane pore. In other words, the cake layer
becomes the membrane barrier, a “dynamic membrane”.
The extent of caking is influenced by such fluid variables as the degree of solvation, concentration and nature of the solids and solutes, fluid temperature andoperating variables such as solution velocity along the membrane and transmembrane pressure. Controlling this phenomenon is key to optimizing flux and solute
passage.
Caking can usually be controlled by ensuring adequate solution velocity at the liquid-membrane wall. Solution velocity, controlled by the pumping rate, varies
according to application. Generally it is necessary to test in laboratory scale modules before scaling up to large volumes.
When protein passage through the membrane structure is important, particularattention should be paid to feed (or recirculation rate). In general, high feed rates
allow more efficient protein passage. Depending on the characteristics of theretained components (cells, particles, cell debris), a caking layer can form on themembrane wall that is actually tighter than the membrane pores. In these
instances, high recirculation rates and low transmembrane pressures often help.
Other variables such as viscosity or shear sensitivity of the solution are also important considerations.

Tangential (Cross Flow) Configurations
Several physical configurations are used for cross flow fluid management, including flat media in an appropriate holder, plate and frame (also called “stacked or
stacked plate”), tubular modules, spiralwound modules and hollow fiber modules. Spiral wound modules are themost widely used configuration becauseof their widespread use in reverse osmosis, electronic and pharmaceutical water
applications.
However, the packing density, efficiency,
easy scalability, disposability and costsavings available with hollow fiber modules are gradually making them the preferred configuration.
Hollow fibers are manufactured by spinning or casting membrane material intofine fibers, having diameters of 80-1000µm with a wall thickness of approximately
30-200 µm (see figures A-44 and A-45.
Figure A-44 Micrograph of Spectrum mixed ester hollow fiber membrane

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THE ABCs OF FILTRATION

Most hollow fibers are asymmetric in
structure. The active surface (skin layer)of the membrane in contact with the feedstream is a high density skin cast onto ahighly porous substructure. The substructure acts only as support for the thin
active layer and generally has no effecton the filtration characteristics of themembrane. Since the actual discriminating barrier is extremely thin, very highflux rates can easily be achieved. Dueto the highly asymmetric structure, allrejected materials are retained on themembrane surface and internal pore
fouling is virtually eliminated.
Figure A-45 Schematic of hollow fiber membrane

Figure A-46 Schematic of a typical tangential (cross flow) hollow fiber module

As shown in figures A-45 and A-46,
pressurized fluid flows into the centers(or lumen) of the hollow fibers, then thepermeate flows through the walls of thehollow fibers and is collected in the
housing. The permeate then flows out of
the housing.
In the manufacture of hollow fiber modules, the two ends of a large bundle offibers are potted into headers. The
headers are sawed off to expose thecenters of the fibers and the bundle iscast into an outer housing forming a
module.
The hollow fiber module configuration
permits a very large amount of membrane surface area to be packed into asmall device.
In addition, hollow fiber modules havethe advantages of ease of handling,
direct scalability from lab to processapplications, and easy cleaning.
Spectrum hollow fiber modules are
unique in that they combine disposability
with high quality and exceptional performance. This eliminates the need forcleaning, retesting and revalidation of
the hollow fiber modules.
A schematic of a typical spiral woundmodule is shown in figure A-47.

Figure A-47 Schematic of spiral wound cross flow module

p 34

In this configuration alternate layers of filter media and separator material arerolled into a spiral configuration and the

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FILTRATION MODES: CONFIGURATIONS A-35

ends of the spiral “pack” are potted to form the proper flow channels.
Parameters

Hollow Fiber

Flat Sheets

Geometry
Cleaning
Module Configuration
Relative Membrane Surface area
Relative Module Size
Flow Path
Scalability
Relative Re-usability
Relative Material Cost

narrow tubes
disposablebundledlarge
small
uniform
directnot necessarylowbroad sheets
difficultstacked or spiral wound
small
large
non-uniform
difficultlowhighTable A-2 Comparison of tangential (cross flow) configurations, hollow fiber vs. flat sheets

Tangential Flow Membrane Technology using Hollow Fibers
Spectrum hollow fiber tangential flow membrane technology spans both microfiltration (MF) and ultrafiltration (UF) separations. Membrane technology for MF
separations include retention ratings of
0.5 µm, 0.2 µm, 0.1 µm and 0.05 µm.
UF technology includes membranes with
MWCO ratings of 1,000 kD, 400 kD, 50
kD and 10 kD.

PERMEATE

Figure A-48 Schematic of operation of a single hollow fiber

The hollow fibers are cast from either
polysulfone (PS), mixed cellulose ester(ME), polyethersulfone (PES) or polyvinyl
difluoride (PVDF) with fiber diameters of
0.5 mm and 1 mm. From these uniform
hollow fibers, as shown in figure A-48, filtration modules are constructed into several configurations with effective membrane surface areas ranging from 5 cm2
up to 3.3 m2.

Spectrum Hollow Fiber Membrane Modules
Spectrum membrane modules utilize advanced hollow fiber flow geometry inwhich every fluid path is identical in length and dimension. Uniform flow distribution makes it practical to process difficult streams such as those containing suspended solids, fibers, particles and cells.
Process liquid flows inside the fibers under pressure and is continuously recirculated to take full advantage of the benefits of tangential flow. The retained solutes(retentate) are progressively concentrated unless additional liquid is added as in
the case of diafiltration. Solvent and small solutes (permeate) flow through the hollow fiber pores and exit to the filtrate container.

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A-36

THE ABCs OF FILTRATIONAll Spectrum membrane modules are assembled in a certified clean room environment under Good Manufacturing Practices and ISO 9001 standards. They feature
low bioburden, meet all USP Class VI standards for biocompatibility and are nonpyrogenic by LAL test.
Spectrum tangential (cross flow) membrane modules differ from traditional tangential flow membrane devices in that they are supplied non-pyrogenic, ready for use
and are priced to be disposable. The initial investment is low compared to competitive devices that must be repeatedly cleaned, retested and reused to be costeffective.
This new approach closely fits industry standards. Single use disposable pleatedcartridges have been the industry standard for dead end membrane devices formore than thirty years.
Advantages of Single Use Disposable Membrane Modules:
1. Modules do not need to be cleaned subsequent to use.
2. Cleaning chemicals do not have to be rinsed from the modules after cleaning.
For parenterals, expensive pyrogen free water for injection must be supplied forrinsing non-disposable modules.
3. Module cleaning does not need to be documented.
4. Tests for effective removal (rinsing) of cleaning chemicals are not required.
5. Process development is simplified. Repeated and exhaustive cleaning and rinsing studies are not required to guarantee a successful scale up.
6. It is not necessary to preserve or store modules between processed batches.
7. Consistent initial performance and no loss of performance with repeated use.

Scalability of Hollow Fiber Filtration
Spectrum Laboratories hollow fiber filtration systems cover a complete range ofprocessed liquid volumes from hand pumped syringe disposable sampler products with liquid volumes as low as 2 mL to pilot and production scale systems tohandle liquid volumes greater than 10,000 liters (2,580 gallons). This range allows
the user complete scalability since the data that are taken with research anddevelopment systems testing is directly applicable to production volume levels.
Consideration of the expense and time span involved in bringing new biopharmaceutical products to market emphasizes the need to readily scale up fromresearch liquid volumes, through prototype liquid volumes to process liquid volumes. Further, it is important to have as much stability as is possible at all stagesof new product introduction. An important part of this stability is in the filtration
steps involved.
One of the greatest advantages of hollow fiber filtration over other filtration configurations is that hollow fiber filtration is directly scalable as long as the fiber path
length is held constant. A steady state flux rate (mL/min/cm2) is first determinedon a smaller, more manageable and less expensive scale. Then, by holding thesesame operating parameters constant and scaling up only the membrane surface
area and recirculation rate, larger scale volumes can be processed in the same

p 36

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FILTRATION MODES: CONFIGURATIONS A-37

amount of time as the small scale operation.
There are three ways to increase surface area for hollow fiber filtration.
1) Increase the length of the fibers
2) Increase the number of fibers
3) Increase both the length and the number of fibers
For a direct scale-up, the number of fibers are increased. If the user is scaling up
by increasing the fiber length, then the same recirculation rate must be usedwhich will result in different flux rates.
Figure A-49 illustrates the scalability of the range of Spectrum systems and theircorresponding range of liquid volumes.

Figure A-49 Scalability of Spectrum hollow fiber membrane filtration systems

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THE ABCs OF FILTRATION

MicroKros® Hollow Fiber Membrane Modules
MODULES: Mixed cellulose ester (0.1µm, 0.2 µm); polyethersulfone (0.1 um,
0.2 µm, 0.5 µm) and low protein binding
polysulfone (10 kD, 50 kD, 400 kD, 0.05µm) all in polysulfone housings
LIQUID VOLUMES: 2 mL - 50 mL(syringe); 20 mL – 200 mL (pumped)

Figure A-50 MicroKros® Modules with process volumes from 2 to 50 ml

MicroKros® Modules
Process Volumes: 2 - 50 ml
Diameter: 3.5 mm
Total Length: 102 mm to 232 mm

Figure A-51 Schematic of MicroKros® module

Designed for separations of extremely
small volumes, MicroKros® disposable
modules are the first practical tangential
flow devices suitable for processing volumes as small as 2 mL. (Figures A-50,
A-51 and A-52).
They are an ideal alternative to centrifugation for applications where pellet formation is undesirable. Flow can be supplied using either syringes or a peristaltic pump.
MicroKros® modules provide highly
advanced hollow fiber membranes in apolysulfone housing. They are autoclavable and, depending on model,
have a total membrane surface area
ranging from 5 to 20 cm2.
Due to their low cost and reproducibility
they are directly scalable to pilot plant or
production scale liquid volumes.
Applications of the MicroKros® membrane system include the following:
1. Diafiltering (washing) uniform latex
particles and other diagnostic particles
2. Clarifying Iysates to remove celldebris from soluble proteins
3. Washing and concentrating protein
precipitates
4. Recovering serum from whole blood
5. Media studies for hollow fiberbioreactors

Figure A-52 Schematic of MicroKros® syringe systemp 38

6. Protein concentration and washing

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FILTRATION MODES: CONFIGURATIONS A-39

MidiKros® Hollow Fiber Membrane Modules
MODULES: Mixed cellulose ester (0.1 µm,
0.2 µm); polyethersulfone (0.1 um, 0.2 µm,
0.5 µm) and low protein binding polysulfone(10 kD, 50 kD, 400 kD, 0.05 µm) all in polysulfone housings
LIQUID VOLUMES: 20 mL – 200 mL

Figure A-53 MidiKros® module
MidiKros® Modules
Process Volumes: 20 to 200 ml
Diameter: 7.8 mm Total Length: 132 mm to 258 mm

Figure A-53A Schematic of MidiKros® module

Designed for separations of small volumes,
MidiKros® disposable modules are practicaltangential flow devices suitable for processing volumes ranging from 20 mL to 200 mL.
(See figures A-53 and A-54).
These modules are an ideal alternative tocentrifugation for applications where pellet
formation is undesirable. Flow is supplied
using a peristaltic pump.
MidiKros® modules are hollow fiber membranes encased in a polysulfone housing.
They are autoclavable with a total membrane surface area of 20 to 145 cm2,
depending on model.
Due to their low cost and reproducibility
they are directly scalable to pilot plant or
production scale liquid volumes.
Applications for the MidiKros® module aresimilar to those listed for the MicroKros®
module above.

Figure A-54 Schematic of MidiKros® system set up

Volume Scalability from MicroKros® and MidiKros® Modules
to MiniKros® Sampler Module, MiniKros® Module and KrosFlo® Module
A direct scale up of MicroKros® and MidiKros® systems is achieved with the
MiniKros® and KrosFlo® modules and systems on pages 40 and 41.
Spectrum KrosFlo® Steam-In-Place (SIP) hollow fiber modules are a feasible alternative to expensive, reusable SIP filters. They can be sterilized by steam (maximum 142°C) or they can be autoclaved. Since their low cost allows modules to bediscarded after use, they have numerous advantages when compared to reusable
modules.
Reusable membrane modules require difficult and expensive chemical cleaning,storage and revalidation after each use.

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A-40

THE ABCs OF FILTRATION

MiniKros® Sampler Module
Process Volume: 50 ml to 4 liters
Diameter: 18.8 mm
Total Length: 185 mm

IDID
L
TL

Figure A-55 MiniKros® Sampler Module

MiniKros® Module
Process Volume: 300 ml to 100 liters
Diameter: 18.8 to 31.2 mm
Total Length: 276 mm to 510 mm

ID
L
TL

Figure A-56 MiniKros® Module

KrosFlo® Module
Process Volume: 3 to 1000 liters
Diameter: 70 mm
Total Length: 229 to 691 mm

ID

TL, L

Figure A-57A KrosFlo Module®

KrosFlo® Plus Module
Process Volume: 3 to 1000 liters
Diameter: 70 mm
Total Length: 298 to 760 mm

ID
L
TL

Figure A-57B KrosFlo® Plus Module

p 40

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FILTRATION MODES: CONFIGURATIONS A-41

INSUFLATOR BULB

SLIDE
CLAMP

VENT VALVERETENTATE
RETURNPROCESSING
RESERVOIRINSUFLATOR BULBFEED LINE
FILTRATE
OUTLET

VENT
VALVE
SLIDE
CLAMP

PRESSURE
GAUGE

BUFFER RESERVOIRCONSTANT VOLUME DIAFILTRATION MODE

Figure A-58 MiniKros® Lab System with reservoir used in aconstant volume diafiltration mode

Figure A-59 KrosFlo® large volume pilot system in a batch
concentration mode

Disposable SIP modules make validationeasier, require no cleaning or validationof cleaning procedures, do not require
special storage or storage and retestingof reprocessed modules, eliminate
potential product contamination associated with module reuse and are notaffected by the diminishing filtration performance that is characteristic ofreusable membrane modules.

Figure A-60 KrosFlo® SIP Module

KrosFlo® SIP hollow fiber modules fit into most standard new or installed stainlesssteel pressure housings. They are manufactured with hydrophilic, 0.2 and 0.5 µm
polyethersulfone (PES) hollow fibers potted in biocompatible epoxy resin. The
modules are available with 0.8 and 1.8 m2 total membrane surface areas. O-rings
assure leak-free inlet and outlet seals.

Sterilization of Spectrum Membrane Modules
For aseptic operation, Spectrum membrane modules may be sterilized as follows:
AUTOCLAVING - Autoclave at 121° C for 30 minutes. Do not expose to temperatures above 124°C.
IRRADIATION - Dry modules may be exposed to 2.5 megarads of gamma radiation or electron beam radiation.
ETHYLENE OXIDE - Dry modules may be sterilized by ethylene oxide.
CHEMICAL - Modules may be chemically treated using 1500 ppm of peracetic

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A-42

THE ABCs OF FILTRATION

acid (Minntech Corporation), 1500 ppm of sodium hypochloride (bleach) or gluteraldehyde solution. Follow supplier recommendation for each of these chemicalsterilants.

Modes of Operation of Hollow Fiber Filtration Systems
Most applications of crossflow microfiltration and ultrafiltration can be grouped into
five major operational modes:
BATCH CONCENTRATION of cells, cell debris, particles, precipitates or proteins.
BATCH CLARIFICATION of solutions containing cells, cell debris, particles, precipitates or proteins.
TOPPED OFF BATCH for reducing larger process volumes to smaller manageable
volumes for concentration or further process work.
CONSTANT VOLUME DIAFILTRATION (washing) of cells, cell debris, particles,
precipitates or proteins.
DEAD END FILTRATION to recover extra product following clarification.

Batch Concentration
Generally, the term “concentrate” is applied to applications where materials being
retained by the filter are (or contains) the desired product. As the process fluid
circulates through the filter, there is a loss of volume
in the process vessel due to the removal of filtrate.
As a result, product is concentrated in the process
vessel. This mode of operation is used, for example,
in fermentation recoveries where the desired productis the cell itself or as an initial process step where theproduct is intracellular.

Batch Clarification
The term clarification is generally used for applications where the desired product is in the filtrate suchas soluble proteins. This mode of operation is used,
for example, to harvest animal or bacterial cell cultures where the desired product is secreted by thecells or microbial fermentation where the desired
product has been released into solution by cell lysis.

Figure A-61 KrosFlo® Pilot System used in a batch
concentration modep 42

When operated in concentration or clarification
modes, the membrane quantitatively removes materials larger than the pores of the filter and allows thepassage of soluble materials that are smaller than themembrane pores.

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FILTRATION MODES: CONFIGURATIONS A-43

The system set-up is the same for both batch concentration and batch clarification
filtrations.
As filtrate is removed, the solution in the process vessel becomes more concentrated in the components that the membrane retains. In addition, the volume is
reduced. The degree of concentration, called the concentration factor (CF) or volume reduction factor (VRF), is given by the following equation where Vi is the initial
volume and Vf is the final volume:
Concentration Factor = Volume Reduction Factor =Vi
Vf

Topped off Batch Operation
PRESSURE AND FLOWMONITORPUMPINSUFLATOR
BULB
PUMP HEADVALVE

OUTLET PRESSURE
TRANSDUCER

VENT
VALVE

AIR VENT"CLOSE"INLET
PRESSURE
TRANSDUCER

SLIDE
CLAMP
FILTER

FLOW
TRANSDUCER

Referring to figure A-62, the small processing reservoir is kept full by continually adding feed material at
the same rate that filtrate is removed.

VALVE

FILTRATE
OUTLET

FEED RESERVOIRPROCESSING
RESERVOIR

Constant Volume Diafiltration

FILTRATE
RESERVOIR

Figure A-62 MiniKros® Sampler System in Topped Off Batch Mode

INSUFLATOR BULB

SLIDE
CLAMP

A disadvantage of batch operations is the relative
high flow rate needed (20 to 100 times the filtraterate) for efficient tangential flow. As a result, it is difficult to achieve high concentration factors without
foaming or vortexing in the processing vessel. A
way to avoid this problem is to operate the membrane in a “topped-off-batch” mode, where a smaller
processing reservoir is used.

VENT VALVERETENTATE
RETURNPROCESSING
RESERVOIRINSUFLATOR BULBFEED LINE
FILTRATE
OUTLET

VENT
VALVE
SLIDE
CLAMP

PRESSURE
GAUGE

BUFFER RESERVOIRCONSTANTVOLUMEDIAFILTRATION MODE
Figure A-63 Constantvolumediafiltration
mode using
MiniKros® Lab System with Reservoir

Materials that pass through the membrane can be
washed away from materials that are retained by themembrane (cells, particles, etc.). The technique isused to recover additional product in clarification
applications, and to achieve better product purity inconcentration applications. For best efficiency, thewash buffer should be free of the solute that is being
recovered or removed.
Diafiltration may be accomplished either by adding
buffer at the same rate as the filtration rate (constantvolume diafiltration), or by reducing the volume in thereservoir and re-adding buffer to regain the original
volume (intermittent diafiltration). The amount of
diafiltration performed can be expressed by theamount of wash buffer added divided by the batch
volume, i.e. the number of “wash volumes”.
During a constant volume diafiltration where solublecomponents pass freely through the membrane,each wash volume of filtrate removed reduces thesolute concentration by a factor of e (2.718…). For
example, a four-fold constant volume diafiltration will
reduce the concentration of solute by a factor of e4,

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A-44

THE ABCs OF FILTRATION

50 fold or over 98%. Using this technique, the concentration of solute can bemonitored in the filtrate until the desired level of purification or product recovery isachieved. The equipment set up is the same as for topped-off batch concentrations or clarifications except that the wash buffer enters the vessel instead of thefeed solution.

Dead End Operation
While the standard mode of operation involves
tangential flow, Spectrum cross flow membrane
modules can also be operated in the normal(dead end) mode. In this mode, shown in figure
A-64, the retentate line is capped or blocked by avalve so that all of the solution being filtered passes through the membrane wall. While the efficiencies of tangential flow filtration are lost, in certain
circumstances, such as at the end of a run where
maximum filtrate recovery is desired, running
dead end is advantageous.
Conventional tangential flow membranes cannot
economically be used in dead end flow. Theirhigh membrane costs require that they becleaned and re-used. Dead end techniques are
avoided because they interfere with membranecleaning.
Spectrum disposable membranes, however, lendthemselves to this mode of product recovery.
Since the membrane investment is substantiallylower than for reusable modules, cleaning a
Spectrum module subsequent to use can be
avoided and dead end techniques are economically feasible.

Figure A-64 Dead end operation

Extending Module Throughput by Forward, Reverse and BackFlushing
Forward Flushing
Shutting the permeate valve/clamp while recirculating sets up a backflushing condition in the downstream half of the module as shown in figure A-65.
The permeate pressure will rise and exceed the retentate pressure in the downstream half of the module. In the downstream region the permeate will flow fromp 44

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FILTRATION MODES: CONFIGURATIONS A-45

the outside of the membrane to inside. This
will loosen and carry away cake material.

Figure A-65 Forward Flushing of Module

The principle of forward flushing is further
illustrated in figure A-66. When the permeate is shut-off, the net permeation rate in themodule is zero. But permeation still occurs
internally. The inlet half of the membrane
module (the high pressure end) generates
permeate that backflushes the downstream
half of the membrane module (the low pressure end).
Normally forward flushing for 30 seconds is
sufficient to clean the downstream half of themembranes.

Reverse FlushingReversing the pump with the permeate
valve/clamp open will serve to backflush theother half of the module. In this case, thehollow fibers are flushed from outside in.

Back Flushing

Figure A-66 Forward flushing of module

Figure A-67 Back flushing of module

Back flushing the entire filter (both halves)
simultaneously can be achieved by reversing
the pump while the permeate valve/clamp is
open, as shown in figure A-67. Reversing
the pump will create a negative pressure in
the lumen side of the fibers drawing in filtrateor rinse buffer from the shell side. This will
act to clean the entire length of the fiberinternal surface. Note that the volume of theretentate solution will increase. An air vent
will be required to allow for this volume
increase.

CALL 1-800-634-3300 FOR
FREE FILTRATION AND
BIOPROCESSING CATALOG

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A-46

THE ABCs OF FILTRATION

Microfiltration Applications
Most applications of microporous membranes can be grouped into the following
broad categories: cold sterilization of fluids; testing for microorganisms; clinicaland general laboratory applications; harvesting of animal or bacterial cell cultures,
cleaning of particles; clarification of fluids and processing of cells, bacteria andviruses.

Cold Sterilization of Fluids
Membranes with pore sizes of 0.2 µm to 0.45 µm are used to remove microorganisms from pharmaceutical liquids and air, beverages, ultrapure water, air, etc. This
type of sterilization requires normal (dead end) flow in most applications since
100% of the organisms must be removed for complete sterility.

Testing for Microorganisms
Membranes with a pore size of 0.45 µm are used to test liquids and gases for thepresence and type of bacteria. This was the first application of microporous membranes used widely in Germany during World War II to test drinking water supplies. Membranes with a pore size of 0.65 and 1.2 µm are used to test for yeasts.

Clinical and General Laboratory Applications
Microporous membranes are used in numerous clinical and laboratory applications, such as cytology, cell culture, analysis, chemotaxis, blood studies, virus andparticle fractionations, and for general cold sterilization of fluids.

Cleaning of Particles and Clarification of Fluids
Cleaning Uniform Latex Particles to Remove Excess Free Protein
in vitro diagnostic tests using latex microparticles often require that the particles
be cleaned before and after an appropriate protein (e.g. albumin, antibodies, antigens or other ligands) is attached to the particle surface. After passive adsorption
or covalent binding, excess free protein must be quantitatively removed to ensure
reliable test results and optimum sensitivity.

Figure A-68 Effect of Solute Rejection on Constant Volume Diafiltration

p 46

Spectrum cross flow membranes are afast, effective and convenient alternative
to ultra-centrifugation for cleaning uniform latex particles. Using tangential
flow, latex particle preparations are
pumped along the membrane surface asshown in figure A-69. Fluid pressurecauses the particle solution to flow
through the membrane device tangential
to the membrane surface. The membrane retains particles while allowing solvents, unbound proteins and othersolutes to permeate the membrane.
Concentrated particles that are retainedby the membrane flow out of the device

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MICROFILTRATION APPLICATIONS

A-47

while particle free filtrate is removed separately.
Depending on the specific application, membranes with pore sizes of 0.1 µm, 0.2 µm and 0.5µm are used.
BATCH CONCENTRATIONS As particle free filtrate is removed, the concentration of latex particles is increased by a factor equal to the starting
volume divided by the ending volume.
DIAFILTRATION The processing volume can be
maintained by continuously adding buffer solution. Called constant volume diafiltration, thistechnique is used for buffer exchange of thelatex particles and for removal of surfactants orother unwanted soluble components. Using thistechnique, the concentration of solutes that passfreely through the membrane are reduced by afactor of e (2.718...) for each volume exchanged.
The influence of the membrane retention on thesolute washing is shown in figure A-68.
Constant volume diafiltration using Spectrum
MiniKros® and larger KrosFlo® disposable modules
is a fast and effective alternative to multiple centrifugation/resuspension steps. They offer several
Figure A-69 Typical Module Setup
advantages over filtration devices that must be
cleaned repeatedly. These advantages include
higher energy efficiency, improved quality and yield of product and lower cost.

Cell, Bacteria and Virus Processing
Recombinant Chinese Hamster Ovary Cell Separation
Many new biopharmaceuticals are being produced by mammalian cells. Hollow
fiber microporous filtration is a valuable technology for separation of a protein
secreted by mammalian cells.
In this example shown in figure A-70, the harvest
has an initial concentration of about 2 x 105
cells/ml in cell culture media. The extracellular
protein product is a 10 kD lymphokine similar toInterleukin-2 and is a potential cancer therapeutic. The result desired is removal of cells andparticulates without lysing the remaining cells,
while achieving high protein recoveries.
Processing time is critical because the product isthermal labile and must be kept below 10°C.

Figure A-70 Flux and Throughput vs. Time for Recombinant
Chinese Hamster Ovary Cell Separation

Two parallel Spectrum KrosFlo® modules with apore size of 0.2 µm and 3.3 m2 of membrane surface are used. As shown in figure A-71, thisallowed for separation of extracellular proteinsfrom 600 liters of recombinant mammalian cells
in 65 minutes with 94% product recovery and

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A-48

THE ABCs OF FILTRATION

insignificant flux decay.
KrosFlo® cellulosic membranes were usedbecause of their low protein binding capacity.
As single use modules, they allow consistentmembrane characteristics and eliminate therisk, cost and time associated with cleaning
procedures.

Figure A-71 System setup for Recombinant Chinese hamster ovary
Cell Separation

Escherichia coli Vaccine Cell Concentration
Fatal infant bovine diarrhea (calf scours) is prevented by an E. coli vaccine grownin batch fermentation. Once the culture reaches the desired density, cells are concentrated as the first step toward recoveringcell wall-associated antigens.
Selection of the proper filtration module andprocess conditions are essential for cost effective cell concentration. A fast processing timewith minimal membrane fouling is the key consideration.
This concentration process is often done bycontinuous centrifugation. Centrifuges havecertain disadvantages such as lengthy cleaning cycles, mechanical complexity and significant product losses in the supernatant. In
addition, unless special containment features
are incorporated, aerosol creation can be hazardous to operators (especially when processing pathogens).

Figure A-72 System Setup for E. coli Vaccine Cell Concentration

p 48

As shown in figures A-72 and A-73, a 0.2 µm
rated KrosFlo module with 2.1 m2 of membranesurface area was used. This setup concentrated 53 liters (with initially 1.8 x 109 cells/mL) to
just over 3 liters in only 24 minutes. This represented a 17-fold volume reduction at a steadystate flux of 58 L/m2/hr.

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

MICROFILTRATION APPLICATIONS

A-49

Single-use Spectrum KrosFlo® modules provide
consistent and economical run-to-run performance without risk, time loss and expensive
cleaning procedures.

Escherichia coli Lysate,
Cell Debris Removal

Figure A-73 Flux and Throughput vs. Time for E. coli Vaccine Cell
Concentration

A genetically engineered Escherichia coli wasfermented, mechanically lysed and the lysate
was processed to recover an intracellular protein, an interferon with cancer treatment potential. Choice of the proper filtration module andprocess conditions are essential for cost-effective separation of the component of interest
from cell debris.
As shown in figure A-74, a single 0.2 µm
Spectrum KrosFlo module containing 1.0 m2 ofmembrane surface was used. With an initialbatch of 245 liters of lysate, the system purified
237 liters of filtrate in 65 minutes, figure A-75.
The result was a 30-fold volume reduction andan equilibrium flux of 200 L/m2/hr. Passage ofthe desired protein was 99%.
Spectrum KrosFlo® filtration modules are aneffective and economical means of separating
soluble proteins from cell debris subsequent tolysis.

Figure A-74 System Setup for E. coli Lysate Cell Debris Removal

Single use modules offer several advantages
over multiple use filters. Starting each batchwith a disposable non-pyrogenic filter eliminates concerns about inadequate cleaning andrinsing of re-used filters. The filter is no longer
a possible source of batch-to-batch contamination. Finally, consistent high performance isassured during each run.

Figure A-75 Flux and Filtrate Flow vs. Time for E. coli Lysate Cell
Debris Removal

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A-50

THE ABCs OF FILTRATION

Cell Recycle Perfusion of Animal Cell Bioreactors with CellFlo® Technology
Figure A-76 illustrates the CellFlo® technology using hollow fiber membranes
arranged in a shell and tube configuration for increased bioreactor output. In thisapplication, designed to separate animal cells from spent media, cells are gently
pumped through the lumen of the hollow fiber membrane tubes. Secreted proteinspermeate the membrane structure and enter the shell side of the module. Using
this technique, spent media is removed (along with secreted proteins and unwanted metabolites) while fresh media is continuously added to maintain bioreactor
volume. CellFlo® technology allows control of the extracellular environment toachieve optimal conditions for product secretion and culture viability.
Using CellFlo® perfusion, ten-fold daily production increases have been achieved.
Other major benefits of perfusion culture include:
1. Increased total output
2. Less media usage per gram ofproduct recovered
3. Greater cell density
4. Simplified downstream purification
5. Longer useful culture life
6. Reduced in-reactor productdegradation
Spectrum CellFlo® modules are manufactured from non-cytoxic USP XXI class VI
materials. Large diameter hollow fiber
flow channels minimize plugging due tocell agglomeration.
They are non-pyrogenic by LAL testing.
CellFlo® modules can be sterilized by
autoclaving and are priced to be disposable. Single use disposability eliminates
the costs and risks associated withcleaning and rinsing, simplifies validationand ensures consistent optimum performance and protein passage.
Figure A-76 Typical Perfusion Setup. Arrows indicate the direction ofsolution flow

CellFlo® technology is included in greater
detail in a later chapter of this book.

CALL 1-800-634-3300 FOR FREE FILTRATION AND BIOPROCESSING CATALOG

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ULTRAFILTRATION APPLICATIONS

A-51

Ultrafiltration Applications
Most applications for UF may be grouped under three principal categories:
Concentration of a retained molecular species by the removal of a solvent
through the membrane.
Desalting or buffer exchange by passing microsolutes through the membrane.
Fractionation or clarification by retaining larger species and passing smaller
species through the membrane.

Concentration
Concentration will reduce large solvent volumes during isolation and purification
procedures, and/or will improve the ability to detect or analyze small quantities ofdissolved molecules. Ultrafiltration is often used for this purpose, replacing older
techniques such as precipitation, evaporation, pervaporation, dialysis and gel filtration.
Figure A-77 shows a schematic diagram of a typical laboratory configuration forconcentration.
UF offers these advantages:
Faster than dialysis.
Faster and less expensive than freezedrying.
Minimizes denaturation of molecules
compared to precipitation.
Produces higher yields than concentration by dialysis.
Faster than pervaporation and does not
require prior dialysis.

PERMEATE

Does not cause hyperconcentration ofsalts (maintains ionic strength).

Figure A-77 Schematic diagram of typical laboratory configuration for concentration

For larger scale process applications,
UF is rapid, simple, and relatively inexpensive as compared to the older techniques listed above.

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THE ABCs OF FILTRATION

Examples of the use of UF for concentration include:
Concentrate and/or desalt peptides and small molecules (MWCO 10kD)
Concentrate and/or desalt proteins, enzymes and viruses (MWCO 50kD)
Concentrate and diafilter albumin (MW~67,000) (MWCO 50kD)
Concentrate monoclonal antibodies and other macromolecules(MWCO 50kD or 400kD)

Concentrations in the Clinical Laboratory
CEREBROSPINAL FLUID. The concentration of CSF by UF makes possible
definitive electrophoresis patterns for diagnosis of proteins in the concentrate.
URINE. High protein levels in urine indicate an abnormality that can often be
detected by the analysis of the proteins. In most cases, protein concentrations inurine are too low for either electrophoresis or immunoelectrophoresis. Therefore,prior concentration of these proteins is essential.
SERUM. A membrane with 100,000 MWCO retains the antigen associated withserum hepatitis (HAA). Concentration of the HAA by the membrane will greatly
increase the test sensitivity, but will not interfere with the specificity of the test.

Virus Concentration and Purification
In medical research and in the manufacture of vaccines, UF has been used toremove viruses from process streams
and to concentrate viruses and viralfragments from crude cell lysates as well
as from partly purified solutions. In addition UF has been used to remove salts
and other low molecular weight contaminants from viral preparations which havepreviously been purified by other methods. UF has found this broad application in the concentration and purificationof viruses because of its ease of use, its
rapidity, and its gentle treatment of viralparticles.

Concentration, Purification andFractionation of Blood

Figure A-78 Comparison of typical virus sizes with Spectrum membranes
retention ratings

p 52

Bovine and fetal bovine serum are widelyused in research and in the manufactureof pharmaceuticals. The first step is
plasmaphoresis, or the removal of cells
from the blood by MF membrane separation. Then, it is desirable to concentrate
the serum albumin by “dewatering” theTHE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

ULTRAFILTRATION APPLICATIONS

A-53

serum (generally with a membrane with MWCO of 30,000-50,000) and then purifying by diafiltration or other UF desalting procedures in the same UF cell. UF is
ideal for accomplishing any of these procedures since UF does not denature theproteins, and it is also possible to purify each serum fraction by diafiltration.

Desalting and Buffer Exchange
There is often a need to purify solutions containing macromolecules either as anadditional step to concentration, or as an entirely independent procedure. This
purification generally involves the
INSUFLATOR BULB
removal of unwanted low molecularweight contaminants, such as salts or
VENT VALVE
SLIDE
solvents, from the solution without reducCLAMP
ing significantly the total number of
RETENTATE
RETURN
macromolecules in the solution. Or in
PROCESSING
some cases, it is desired that an entirely
RESERVOIR
different buffer replace the solvent or
buffer now present in the solution.
INSUFLATOR BULBFEED LINE
FILTRATE
OUTLET

VENT
VALVE
SLIDE
CLAMP

PRESSURE
GAUGE

The standard technique for most ofthese applications is dialysis. However,dialysis is slow and requires large volumes of dialyzing buffer to accomplish
purification. Additionally, since the rateof diffusion across a dialysis membrane
is proportional to the difference in concentration on either side of the membrane, dialysis is particularly slow at low
concentrations of microsolutes, the casein many solutions.

Ultrafiltration rapidly removes all thesecontaminants (under hydraulic pressure)
Figure A-79 Schematic of System Setup for Desalting and Buffer Exchange
while using less buffer than dialysis. In
CONSTANT VOLUME DIAFILTRATION MODE
addition, once these contaminants havebeen removed, concentration of the macromolecules can be accomplished in thesame filtration apparatus.
BUFFER RESERVOIRIn using UF for desalting and/or buffer exchange, small molecules quantitatively
pass through the membrane along with the solvent, while macromolecules are
retained by the membrane. The simplest method of desalting therefore would beto replace the solvent that had passed through the membrane with contaminant
free solvent. For example, suppose one began with a solvent containing 1% salt,passed 90% of the solvent through the membrane, and then brought the solution
to its original volume by the addition of salt free solvent. The solution would now
contain only 0.1% salt and the same original number of macromolecules. Since
the rate of removal of the salt is proportional only to the pressure driven flow rate
through the membrane, and not to the impurity concentration, low concentrations
can be reached much more quickly with UF than with dialysis. Additionally, much
lower concentrations can be reached with significantly less solvent. And since thesurface area of a UF membrane is less than that for a dialysis bag, less surface
denaturation of macromolecules occurs with UF than with dialysis.
Diafiltration is a much more efficient procedure in using UF for desalting and/or

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A-54

THE ABCs OF FILTRATION

buffer exchange than the simple one described above. In this process shown infigure A-79, the solvent that is removed is continuously replaced by contaminantfree solvent. In this method virtually complete purification can be achieved. The
dialysate fluid may be pressurized by means of an external gas source, forcing
this fluid into the cell to replace the solvent volume that flows through the membrane. The volume of solution and the number of macromolecules would remainunchanged while the micromolecules are removed through the membrane.
In diafiltration, 99% of the micromolecules are removed when the solvent volume
added is five times the original volume.

Fractionation
Fractionation is simply the separation of different size molecules. However, fractionation is normally performed by diafiltration of dilute solutions. (Gel polarizationinterferes with fractionation of more concentrated solutions). The dilute mixture ofcomponents with different molecular weights is filtered through a membrane rejecting the component with the higher molecular weight. Smaller molecules passthrough the membrane enriching the higher molecular weight component in theretentate.
If desired, the low molecular weight component can be recovered from the filtrateby ultrafiltration with a second membrane that rejects this component.
Applications for the use of UF in fractionation of molecular species include:

Deproteinization of Blood and Urine Samples
To analyze for creatinine, glucose, urea, nitrogen, or other diagnostically important
metabolites, proteins must be removed, otherwise turbidity, precipitates and otherreactions with test reagents will result.
The standard technique in removing proteins from blood and urine samples is toprecipitate these proteins with various reagents. The disadvantages of this technique include: ions may be added which become a potential source of interference and error; precipitation can change the concentration of solutes; some low
molecular weight serum constituents are co-precipitated; and different precipitation methods are required for analysis of creatinine, glucose, urea, nitrogen anduric acid.
UF adds no ions, does not change the concentration of solutes, does not precipitate low molecular weight molecules and can be used for all analyses.
Additionally, when working with urine or cerebrospinal fluid, the protein-enriched
concentrate from the same sample can be used for electrophoresis.

Binding Studies in Pharmacology
The therapeutic and toxic effects of drugs are directly related to the level of free(or unbound) drug in the blood. Blood plasma proteins bind significant fractionsof many drugs. With the bound fraction pharmacologically inactive, it is importantthat the level of unbound drug be determined.
The extent of binding varies from patient to patient. Recent studies have recommended routine evaluation of plasma binding of certain drugs, particularly anti-convulsants and heart-treatment drugs to determine the proper dosage for each patient.

p 54

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ULTRAFILTRATION APPLICATIONS

A-55

Low membrane adsorption is an absolute necessity in binding studies since“unbound species” must be freely permeable to the membrane. High adsorption
will give erroneous results with the membrane acting as a plasma protein andadsorbing “unbound species”.
UF is faster than equilibrium dialysis and gel filtration. The latter are slow, requiring 24 to 48 hours, during which time the drug binding components of plasma
may be altered. The bound complex may dissociate during this period as
unbound species diffuse away, thus a rapid separation method is desirable. UF
offers the advantage that weak binding interactions can also be assessed
because the fractionation occurs rapidly.

CALL 1-800-634-3300 FOR FREE FILTRATION AND BIOPROCESSING CATALOG

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A-56

THE ABCs OF FILTRATION

System And Membrane Selection Guides
Membrane Application Guide
Figure A-80 illustrates representative separation applications and the optimum
membrane module for each application.
The module should be matched to a system that will allow the maximum operational efficiency considering the nature of the feed stream, the objective of theseparation and other important operating parameters.

Application Guide based on Membrane TypeMF Applications

UF Applications

Application
Concentrate and/or desalt
peptides and small molecules

Membrane Selection
10 kD(PS) Polysulfone

10 kD, 50 kD(PS) Polysulfone

Concentrate and/or desalt
proteins, enzymes, viruses

50 kD(PS) Polysulfone

Concentrate and diafilter
albumin, (~MW 67,000)

50 kD(PS) Polysulfone

Concentration of monoclonal
antibodies and other macromolecules

50 kD, 400 kD(PS) Polysulfone

Bacculovirus and retrovirus
concentration and washing

400 kD, 0.05 µm(PS) Polysulfone

Clarification of cell lysates torecover proteins

0.1, 0.2, 0.5 µm(ME) Mixed Ester(PES) Polyethersulfone

Concentration and washing ofbacterial and/or mammalian cells

0.1, 0.2, 0.5 µm(ME) Mixed Ester(PES) Polyethersulfone

Continuous perfusion of cell culture

0.1, 0.2, 0.5 µm(ME) Mixed Ester(PES) Polyethersulfone

Clarification of viral cultures

0.1, 0.2, 0.5 µm(ME) Mixed Ester(PES) Polyethersulfone

Washing uniform latex
diagnostic particles

0.1, 0.2, 0.5 µm(ME) Mixed Ester(PES) Polyethersulfone

Washing and concentration ofprotein particulates

0.1, 0.2, 0.5 µm(ME) Mixed Ester(PES) Polyethersulfone

Depyrogenation

Figure A-80 Membrane Application Guide

p 56

Membrane Material

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SYSTEM AND MEMBRANE SELECTION GUIDES A-57

Industry

Application Examples

Pharmaceutical/
Biotech

Latex particle filtration
Protein purification, desalting and concentration
Enzyme isolation
Blood fractionation
Fermentation brothPyrogen removal
Monoclonal antibody production
Cell expansion for cell therapy

Chemical

MTBE removal from aqueous streams
Waste water processing
Dyestuff desalting
Latex concentration
Catalyst recycling

Environmental

Surface water purification
Filtration of waste water from car wash facilities
Treatment of waste water from oil mills
Printing ink removal
COD reduction (removal of organic matter)
Sludge concentration
Filtration and concentration of landfill leachates
Concentration and recovery of photo emulsions

MetalsSeparation of oil/water emulsions
Extension of life of degreasing baths

Paint

Filtration of electrocoat paintRecovery of water-based spray paint

TextilesRemoval and recovery of dissolved and suspended dyes
Recovery of sizing agent

Paper

Recycling of coating color
COD reduction

Food and Beverages

Concentration of milk and whey proteins
Cheese production
Wine and vinegar filtration
Fruit juice clarification
Starch and sugar concentration

Electronic

Prefiltration of ultrapure water

Table A-3 Industrial Applications for Hollow Fiber Membrane Systems

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A-58

THE ABCs OF FILTRATION

Sizing Hollow Fiber Systems
To properly size a system, use the following equation:V
A = ------------T•f
Where:A is the required membrane surface area in square meters
V is the filtrate volume desired in liters
T is the desired processing time in hours
f is the steady state flux in liters per square meter hour (L/m2hr)
Example:900 liters of 0.2 µm clarified E. coli lysate is desired
The steady state flux is 50 L/m2hr)
The desired processing time is 6 hours
900
A = ------------6 • 50
A = 3 m2

Representative Spectrum Hollow Fiber Systems
MicroKros® System For Low Liquid Volume Laboratory Separations
Concentration Operation Using Syringes
MicroKros® filters are designed for cross flow membrane separation of extremely
small volumes. These disposable filters
are the first practical tangential flow
devices for processing volumes as small
as 2 mL using a syringe as a pressuresource. MicroKros® modules are also an
ideal alternative to centrifugation for
applications where pellet formation isundesirable. Flow can also be suppliedwith a peristaltic pump.

Figure A-81 MicroKros® Module Setup with syringes for low liquid volume inlaboratory separations

p 58

In figure A-81, tangential flow filtration is
initiated by applying pressure on theinlet syringe containing the fluid to beprocessed, while allowing the other two
syringes to move freely. When the inlet
syringe is empty, pressure is applied tothe outlet syringe until it is empty. The
process is continued by applying pressure first to one syringe then to the otheruntil the desired concentration volume isobtained.

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

SYSTEM AND MEMBRANE SELECTION GUIDES A-59

The permeate is collected by the middle syringe.
Applications of the syringe pumped MicroKros® system include:
Concentration of proteins, antibodies and microbial cells
Diafiltering (washing) proteins or uniform latex particles and other diagnostic
particles
Clarifying Iysates to remove cell debris from soluble proteins
Recovering serum from whole blood
Media clarification for hollow fiber bioreactors
Virus separation

MiniKros® Sampler System (Process Volumes 50 mL-3 L)
The MiniKros® Sampler System is designed for rapid cell separation, concentration, diafiltration or diagnostic particle washing with aqueous based solutions atvolumes from 50 mL to 3 liters.
PRESSURE AND FLOWMONITORPUMPINSUFLATOR
BULB
PUMP HEADVALVE

OUTLET PRESSURE
TRANSDUCER

VENT
VALVE

AIR VENT"CLOSE"INLET
PRESSURE
TRANSDUCER

SLIDE
CLAMP
FILTER

FLOW
TRANSDUCER
VALVEFILTRATE
OUTLET

FEED RESERVOIRPROCESSING
RESERVOIRFILTRATE
RESERVOIR

Figure A-82 MiniKros® Sampler System in topped off batch mode

Referring to figure A-82, the pump circulates the fluid to be processed in aclosed loop with a pressure meter monitoring the inlet pressure. As filtrate is
generated, more feed or wash buffer ispulled into the loop automatically. The
system is particularly well-suited for processing shear sensitive solutions suchas mammalian cell cultures.
The MiniKros® Sampler System provides
an economical means of evaluating separation procedures that can be scaled
up to larger MiniKros® and KrosFlo® modules.
Membrane modules for the MiniKros®
Sampler System are available in retention ratings of 0.5 µm, 0.2 µm, 0.1 µm,
0.05 µm, 400 kD, 50 kD, and 10 kD andwith surface areas from 120 cm2 to 615
cm2.

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A-60

THE ABCs OF FILTRATION

Typical applications for the MiniKros® Sampler System include:
1. Latex washing
2. Single stage prefiltration of biologicals
3. Filtration of fluids with high suspended solids
4. Concentration of cellular suspensions
5. Cell washing and/or nutrient feeding
6. Sterilizing filtration
7. Mass culture of mammalian cells/cell perfusion
8. Protein isolation/purification
9. Virus concentration/removal
10. Desalting/buffer exchange

MiniKros® Lab System (Process Volumes 300 mL - 100 L)
MiniKros® Lab Systems are designed for laboratory scale tangential flow separation volumes of 300 mL to 100 liters. Applications include concentration of cells,
separation of insoluble components from suspensions, small batch filtration, protein concentration, diafiltration and scale-up studies for larger volume applications.
MiniKros® Lab Systems operate a single MiniKros® hollow fiber membrane modulewith lumen diameters of 0.6 mm and 1.0 mm, and surface areas from 170 cm2(0.16 ft2) to 6600 cm2 (7.1 ft2). The system is autoclavable with typical permeate
rates of 50 ml to 7.5 liters per hour.
The use of disposable MiniKros® modules
eliminates the uncertainties and costs associated with cleaning and rinsing and the needfor expensive and time-consuming validationof the membrane cleaning and rinse cycles.

INSUFLATOR BULB

SLIDE
CLAMP

VENT VALVERETENTATE
RETURNPROCESSING
RESERVOIRINSUFLATOR BULBFEED LINE
FILTRATE
OUTLET

VENT
VALVE
SLIDE
CLAMP

PRESSURE
GAUGE

BUFFER RESERVOIR

Figure A-84 Schematic of MiniKros® Lab Systemwith Reservoir
CONSTANT VOLUME DIAFILTRATION MODE

Figure A-83 The MiniKros® Lab Systemp 60

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SYSTEM AND MEMBRANE SELECTION GUIDES A-61

Experimental laboratory results with the MiniKros® Lab System indicate feasibilityof a ready scale-up to production level membrane modules and systems.

KrosFlo® Pilot System (Process Volumes 3 L-1000 L)
The CE certified KrosFlo® Pilot System is designed for production scale processingof pharmaceutical and other aqueous solutions. The system can process from 3
liter to 1,000 liters of solution, with permeate rates ranging from 20 to 1000 L/hour.
A wide range of membrane module configurations are available with the KrosFlo®
system. These include retention ratings of 0.5 µm, 0.2 µm, 0.1 µm, 0.05 µm, 400
kD, 50 kD and 10 kD and with membrane areas from 0.32 m2 to 5.1 m2.
The system is mobile with a carting design that allows the system to be wheeled inand out of storage or from one location to another.
The Pilot System is also Steam-In-Place (SIP) compatible.
Using disposable and autoclavable KrosFlo® modules, the KrosFlo® Pilot System iseasily converted to a SIP operating mode. The disposable KrosFlo® modules are
price competitive making them economically disposable after each use whencompared to reusable filters.

Figures A-85 Schematic of KrosFlo® Pilot System in batch
concentration mode.

Figure A-86 Photograph of KrosFlo® Pilot System

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A-62

THE ABCs OF FILTRATION

Applications of the KrosFlo® Pilot System include:
1. Diafiltering (washing) uniform latex particles and other diagnostic particles
2. Clarifying Iysates to remove cell debris from soluble proteins
3. Washing and concentrating protein precipitates
4. Recovering serum from whole blood
5. Media studies for hollow fiber bioreactors
6. Protein concentration and washing

The CE Certified KrosFIo® Pro Pressure and Flow Monitor
The KrosFlo® Pro Pressure and Flow Monitor shown in figure A-87, is a compact,
book-size monitor equipped with an
adjustable stand. The monitor can readand display inlet, outlet and permeatepressures simultaneously. Additionally, it
can calculate and display the transmembrane (TMP) pressure.
The monitor is equipped with an audible
and visual pressure alarm. Should thealarm mode be triggered, the monitor
sends a de-energized signal that can bedetected by the on-off LiquiSense® electronic cell. The pump then shuts off.
SYSTEM COMPATIBILITY. The KrosFlo®
Pro monitor is used with Spectrum tangential flow filtration systems such as the
MiniKros® Lab and KrosFlo® Pilot systems or as a stand alone unit.

Figure A-87 The CE Certified KrosFlo® Pro Pressure and Flow Monitor

The KrosFlo® Pro interfaces with
Windows 95/98. Through an RS-232
cable, the monitor can send pressureand flow readings to a computer that
displays and records the process data.

CALL 1-800-634-3300 FOR FREE FILTRATION AND BIOPROCESSING CATALOG

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SYSTEM AND MEMBRANE SELECTION GUIDES A-63

Selecting the Optimum Membrane System
Many factors must be carefully considered and technical and economic tradeoffs
made before one can select the optimum membrane system for their own application. Some of these factors and tradeoffs are shown in figure A-88, greatly simplified for illustration. Spectrum Laboratories maintains a staff of technically qualified
scientists and engineers to assist customers in examining their filtration and/or bioprocessing requirements and arriving at the optimum selection. This service
includes recommendations on other resources when Spectrum is unable to fill customers needs.

Figure A-88 Selecting the Optimum Spectrum Membrane and System

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A-64

THE ABCs OF FILTRATION

Macrofiltration

Figure A-89 Nylon screen filter (5 µm)

Macrofiltration has been defined as the separation of particles
of one size from particles of another size where at least one ofthe particles is larger than 10 micrometers. It is generallyachieved by the use of screens or depth media. Screens
have the major advantage that they have very low adsorption
and absorption of the filtrate. This feature is essential whenmaximum recovery of particles in the filtrate is required suchas in fractionation applications.
Macrofiltration screens are primarily available in electroetchedor sintered metals, woven stainless steel wire and woven polymeric materials. Woven polymeric screens are principally
available in fluorocarbon, nylon, polyester, polypropylene andpolyethylene.
Woven stainless steel screens are available in mesh openings
from 30 to 1000 µm. Woven polymeric screens are available inmesh openings from 5 to 1000 µm, depending on the polymer.
The selection of the optimum macrofiltration filter is based on
conditions of the intended application, including flow rate, particle size and the operating environment (pressure, temperature,abrasion resistance, resistance to corrosive chemicals, etc.)

Figure A-90 Fleaker® with macroporous screen filter

p 64

The characteristics of Spectra/Mesh® macrofiltration screensallow them to be used successfully in a wide variety of environmental, pharmaceutical, chemical and other industrial
applications as well as in research.

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

SYSTEM AND MEMBRANE SELECTION GUIDES A-65

These characteristics include:
1. Precise mesh openings for accurate particle
separations.
2. Most Spectra/Mesh® filters have negligible
adsorption/absorption for precision fractionationsof particles and similar applications. Nylon
Spectra/Mesh® filters feature non-specific highbinding capacity.
3. Essentially zero leaching eliminates contamination of the filtrate.
4. Excellent thermal and chemical resistance for
filtration of aggressive solutions.
5. Can be backwashed to significantly extend
filter life.
6. High flow rates permit lower initial capital
investment.
Figure A-91 Spectra/Mesh® macroporous filters range in poresize from 5 µm to 1,000 µm

Bureau of Standards
Sieve Mesh Size*

7. Excellent physical strength for high-pressure
differentials.

Mesh Opening
Microns (µm)

Bureau of Standards
Sieve Mesh Size*

Mesh Opening
Microns (µm)

4

4760

45

354

5

4000

50

297

6

3360

60

250

7

2830

70

210

8

2380

80

177

10

2000

100

149

12

1680

120

125
105

14

1410

140

16

1190

170

88

18

1000

200

74

20

841

230

63

25

707

270

53

30

545

325

44

35

500

400

37

40

420

* Specifications adopted by U.S. Bureau of Standards and American Society of Testing Materials.
Sieve Numbers are arbitrary designations and do not refer to the mesh count per inch.

Figure A-92 U.S. Standard Sieve Sizes – Specifications adopted by U.S. Bureau of Standards and ASTM. Sieve Numbers are arbitrary
designations and do not refer to the mesh count per inch.

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

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Section B

The ABCs of Laboratory Dialysis
Introduction
Dialysis is the process of diffusion of solutes through a semipermeable membranefrom a liquid with higher solute concentration on one side of the membrane to a liquid with a lower concentration on the other side. See figure B-1. The membranes
are semipermeable because they allow some molecules to pass while preventing
others from passing. The process has long been used for the molecular separationof small molecules from macromolecules.
This relatively simple process is desirable and is performance and cost effective forseveral reasons:
1) Wide Range of Sample Volumes2) Gentle Conditions
3) Inexpensive Membranes and Equipment

Figure B-1 Schematic of hollow fiber dialysis membrane

Common dialysis applications utilize tubular formsof membranes and involve placing a “sample”
inside the membrane and a “buffer” outside themembrane. The process is run until the desireddegree of separation is attained. Molecules
smaller than the pores will eventually be equallydistributed between the two solutions. Usually, avery large volume of buffer is chosen so that thepermeable species are greatly diluted and therefore reduced-to very small concentrations in theremaining sample solution. Commonly, dialysis
processes require several hours to complete.

Dialysis Membranes
The original type of dialysis membranes utilized for diffusion studies were cellulose
nitrate. These membranes were not ideal because they tended to have high protein
adsorption, thus limiting their use in the purification and separation of biologicalsolutions. Regenerated cellulose membranes were then used because they alleviated the protein adsorption problems. Regenerated cellulose also exhibited goodchemical and temperature resistance. The membranes, however, were limited in
Molecular Weight Cut Off (MWCO).
Further advances in dialysis membrane development were made as a result ofresearch to provide relief from renal disease by means of hemodialysis, a pressure
driven rather than concentration gradient driven process.
Greater membrane permeability was achieved through the use of cellulose ester.
These solutions could be formulated to yield a wider range of pore sizes. Cellulose
ester membranes are now widely used for clinical and laboratory dialysis.
Membranes used for dialysis have pore sizes ranging from 100 to 300,000 Daltons.
Sample volumes have also been greatly reduced to allow dialysis of small quantities of precious samples, particularly where maintaining enzyme activity is desired.

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B-68

THE ABCs OF LABORATORY DIALYSIS

Dialysis Applications
For the simple dialysis applications ofremoving salts or small molecules, theuse of membrane tubing is convenient,
efficient and inexpensive. This can beaccomplished with a length of membrane tubing in a container of buffer asshown in figure B-2. Stirring the buffer
is recommended to increase the dialysis rate. Dialysis closures are used toprovide an easy method for sealing theends of the dialysis tubing. In addition,a range of new products allow dialysis
of much smaller sample volumes.

Figure B-2 Dialysis using conventional tubular membranes

Typical Dialysis Applications
DesaltingConcentrating Plasma or Serum
Buffer and pH Change of Sample Solution
Preparation of Diluted Proteins prior to Electrophoresis
Concentration of Antibodies
Contamination Removal
Binding Studies
Flow Dialysis/Batch Analysis
Temperature Regulated Dialysis
Tissue Culture Extract Purification
Protein Removal from Gels after Electrophoresis
Removal of Oligosaccharides from Protein Solutions

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DIALYSIS APPLICATIONS B-69

Dialysis is Commonly Used in the Following Fields of Research
Biochemistry

Virology

Molecular Biology

Renal Biology

Biomedical Research

Neurology

Hematology

Physiology

Oncology

Genetics

Microbiology

Rheumatology

Pharmacology

Factors that affect the Rate of Dialysis
Molecular Weight Cut Off & Selectivity
Dialysis membranes are characterized by molecular weight cut off (MWCO).
MWCO is determined by testing the degree of permeability for several solutes ofdifferent molecular weights. The MWCO rating for the membrane is the molecularweight of the smallest solute that is 90% retained in a 17-hour dialysis test.
Molecular weight cut off ratings are used as a guide and not an absolute predictionof performance with every type of solute.
A membrane MWCO size rating should
be chosen as high as possible in order toachieve the maximum dialysis rate whilestill preventing the loss of the desired
solute.

Figure B-3 Retention of idealized dialysis membrane vs. solute molecularweight using a 10,000 MWCO membrane

Plotting the results of a MWCO test in theform of retention versus the solute molecular weight would ideally produce a sigmoid curve. The steepness of the curve
is a measure of the selectivity of themembrane. Figure B-3 illustrates anideal curve for a cellulose ester membrane with 10,000 MWCO. It is shown on
this curve that the central portion of thecurve is approximately a linear relationship that crosses the 0.9 retention axis at
10,000 molecular weight.

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B-70

THE ABCs OF LABORATORY DIALYSIS

Flux and Permeation Rate
The driving force for laboratory dialysis is the concentration difference across themembrane. The flux (or permeation rate) is directly proportional to the concentration difference, i.e. the greater the difference, the greater the rate. However, thedialysis rate is also influenced by other variables such as:
DIFFUSION COEFFICIENT. Different size molecules pass through a membrane atdifferent rates. Larger molecules have a smaller coefficient and a lower rate ofdiffusion across the membrane.
MOLECULAR SHAPE AND CHARGE. Linear molecules permeate faster thanglobular molecules. The pH and ionic strength also affect the rate of dialysis.
CONCENTRATION POLARIZATION. As molecules diffuse across a membrane,
they first move through the bulk of the sample solution to the surface of the membrane. The thin region next to the membrane has a higher concentration of solutes
than the bulk solution. This build up is termed “concentration polarization” and iscaused by depletion of small molecules at the surface of the membrane. This
polarized layer causes resistance to the movement of molecules across the membrane. Finally, after passing through the membrane, the molecule often meets athin layer of concentration higher than the bulk solution, further slowing the passage. These layers which form on either side of the membrane are called “fluid
boundary layers” or “gel layers”.
FLOW DIRECTION AND AGITATION OF THE SOLUTION. Sample and buffer thatflow perpendicular (or normal) might cause the membrane to plug. Sample andbuffer mixing during dialysis can reduce this phenomenon. Mixing can beachieved by either stirring or by passing the sample parallel (or tangential) to themembrane. Parallel flow promotes higher permeation rates. The higher the stirring
rate, the higher the dialysis rates (Concentration polarization is reduced byincreased stirring rates).
TEMPERATURE. Higher temperatures promote more rapid molecular movement
and therefore increase diffusion rate.
MEMBRANE THICKNESS. Membrane properties effect the dialysis rate. Thicker
membranes will require a longer time for molecules to pass through.
MEMBRANE SURFACE AREA. The larger the membrane area, the faster the
dialysis rate.
HYDRODYNAMIC PROPERTIES. Viscosity of the fluid and the membrane porosity
affect the permeation rate. Low viscosity and high porosity are ideal for higher rates.

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DIALYSIS MEMBRANE SELECTION GUIDE B-71

Dialysis Membrane Selection Guide
Spectra/Por® Dialysis Membranes
Spectrum offers three types of dialysis membranes:
SPECTRA/POR® REGENERATED CELLULOSE (RC) - good chemical compatibility for selective organic solvents, pH range of 2 to 12 and temperature stability of
up to 60°C. There are two types of RC membranes:
Spectra/Por® Biotech Regenerated Cellulose - ultra pure hydrophilic membranes
ready for use with precise MWCO selection of 3.5K to 60K.
Spectra/Por® Standard Regenerated Cellulose (Series 1 through 7) - hydrophilicstandard membranes widely used within the biotech community. However, there
are trace levels of heavy metals and sulfides contained in this series that arecommonly treated by “boiling” the membranes in chemical solutions.
Spectra/Por® 7, however, is chemically precleaned and ready to use.
SPECTRA/POR® BIOTECH CELLULOSE ESTER (CE) - ready to use membranes
with a wide selection of MWCO ratings ranging from 100 to 300,000 Daltons. Biotech
CE membranes are stable at pH 2 to 9 and temperature not exceeding 37°C. CE
membranes, however, are not compatible with organic and strong polar solvents.
SPECTRA/POR® POLYVINYLIDENE DIFLUORIDE (PVDF) - hydrophobic, ready touse, with excellent chemical resistance, three MWCOs (250,000; 500,000 and
1 million Daltons).

Table B-1 Specifications of Spectrum regenerated cellulose dialysis membranes

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THE ABCs OF LABORATORY DIALYSIS

B-72

Standard Regenerated Cellulose (RC) Dialysis Membranes
This series of membranes is composed of cellulose reconstituted from cotton linters. Spectra/Por® RC membranes carry no fixed charge and do not absorb mostsolutes. Standard RC dialysis membranes are used for general laboratory dialysis
functions, such as desalting, buffer exchange, or molecular separation.
Spectra/Por® RC Series 1 through 6 membranes contains small amounts of impurities that are easily removed by washing. Table B-1 shows the major characteristics of standard RC dialysis membranes.

Spectra/Por® Biotech Membranes
Spectrum’s Biotech membranes are manufactured in a process that eliminates theuse of metal salts in the manufacturing process. Thus, no boiling or soaking themembrane is required. Other advantages include precisely controlled Molecular
Weight Cut Offs (MWCO) for general dialysis applications and small tubing diameters for very small samples.
Biotech Regenerated Cellulose (RC) membranes are recommended when resistance to various organic solvents (such as DMSO, DMF or alcohols) is required.
Biotech Cellulose Ester (CE) membranes provide a wide selection of rigidly controlled MWCOs ratings ranging from 100 to 300,000 Daltons.
Biotech Polyvinylidene Fluoride (PVDF) membranes provide not only excellent
chemical resistance, but also the ability to heat-seal the membrane for encapsulation and implantation research.

Membrane Type:

Symmetric Regenerated Cellulose

Symmetric Cellulose Ester

Symmetric PVDF

MWCO:

3.5k, 8k, 10k, 15k,
25k and 60k Daltons

100, 500, 1k, 2k, 3.5k, 8k,
10k, 15k, 25k, 50k, 100k,
and 300k

250k, 500k and1 million Daltons

Physical Appearance:

Opaque, Flexible

Opaque, Rigid

Opaque, Rigid

Organic Solvent
Tolerance:

Good

Fair

Very Good

Packaging:

Dry with glycerin as a humectant(available irradiated wet indeionized water)

Wet with 1% hydrogen peroxide(available irradiated wet indeionized water)

Wet with 1%hydrogenperoxide

Flat width (mm):4, 8, 10 and 16

8, 10, 12, 16, 24, and 31

8, 12, 16, and 24

Disc/Flat Sheet:No

Yes, AsymmetricNo

Sample volume range:

0.5 ml to 30 ml

0.5 ml to 60 ml

0.5 ml to 30 ml

pH limits:

2 to 122 to 91 to 14

Suggested
Temperature Limit:

60 oC

37 oC

130 oC

Table B-2 Specifications for Spectrum Biotech dialysis membranes

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DIALYSIS MEMBRANE SELECTION GUIDE B-73

Applications for Biotech dialysis membranes include:
Removal of salts, surfactants, detergents and solvents
Buffer and pH adjustment of sample solutions
Concentration of proteins, peptides or antibodies
DNA electroelution
Preparation of proteins prior to electrophoresis, HPLC
Removal of contaminating micromolecules
Binding studies
Tissue culture extract purification

Chemical Compatibility
Refer to the chemical compatibility table as the first step in selecting the proper
membrane. Variables in temperature, concentrations, duration of exposure andother factors may also affect the performance of the membranes. PVDF membranes have the best chemical resistance. Generally, Regenerated Cellulose (RC)membranes have a better resistance to most chemical solutions than Cellulose
Ester (CE) membranes.

MWCO Selection
Selecting of the correct molecular weight cut off (MWCO) of the membrane isbased on the size of the molecular weight of the macromolecules to be retained
inside the membrane and the molecular weight of the molecules to be removed.
The ratio of the two molecular weights should be a minimum 25 to 1 to achieve themaximum 90% retention.

Tubular Membrane “flat width” Selection
Smaller tubing will dialyze more quickly than larger tubing. The latter will dialyze
more slowly due to the longer diffusion distances involved.

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B-74

THE ABCs OF LABORATORY DIALYSIS

Closure Selection
A closure 4 to 10 mm wider than the flat width of the membrane tubing should beselected. This will allow the closure to securely seal the tubing.
Spectrum recommends two types of closures: polypropylene and nylon
POLYPROPYLENE CLOSURES (SPECTRA/POR®): are autoclavable and are commonly used for RC membranes. There are 3 types of polypropylene closures:• Standard closures that seal at the top of the membrane tubing.
• Weighted closures that contain a stainless steel bar embedded in the standard
closures. The weighted closures are applied at the bottom of the membrane tubing
to keep it in a vertical floating position.
• Magnetic weighted closures that contain a magnetic stir bar. There is no need foran additional magnetic stir bar when the magnetic weighted closure is used at thebottom of the membrane tubing.
NYLON CLOSURES (UNIVERSAL) are specially designed for rigid membranes suchas CE and PVDF, although they can be used for all types of tubing membranes.

Representative Spectrum Dialysis Products
Spectra/Por® Float-A-Lyzer™
Dialysis for Sample Volumes from 300 uL to 10 mL
These floatable, presealed and ready-to-use tubes are available for dialysis of six
sample volume sizes: 300 uL, 500 uL, I mL, 3 mL, 5 mL and 10 mL. The Float-ALyzer™ contains Spectra/Por® Biotech Cellulose Ester or Regenerated Cellulose
dialysis membranes with 13 choices of Molecular Weight Cut Off ratings ranging
from 100 to 300,000 Daltons

Floatation
Disc

Dialysis
Membrane

Weighted
End Cap(Shows approximate
location only)

Figure B-4 Spectra/Por® Float-A-Lyzer™

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MWCO Color
Coded Dot

SPECTRUM DIALYSIS PRODUCTS B-75

The Float-A-Lyzer™ has an easy open cap for loading and recovering samples withthe provided pipette. Multiple samples can be processed in one reservoir
The high purity membranes yield excellent sample recovery for biological samples
such as proteins, peptides, antibodies, DNA, etc. In addition, removing small
molecular weight contaminants, desalting, buffer exchange and concentration arequick and easy to handle with the Float-A-Lyzer™.
There is no danger of puncturing the membrane when loading or recovering thesample from using hypodermic needles and syringes. The floatable cap makes it
easy to load and recover the sample with the provided pipette. It also allows forsamples analysis during dialysis.

Spectra/Por® Micro DispoDialyzer®
The Micro DispoDialyzer® is a dialysis device for processing ultra small sample volumes from 10 uL to 100 uL. It features easy handling using micropipettes for small
samples and set up, and Spectra/Por® Biotech Regenerated Cellulose dialysis
membranes
The Micro DispoDialyzer® is available in 3 colored sample volume sizes: 10 uL,
50 uL and 100 uL, and Molecular WeightCut Offs (MWCO): 3.5 kD, 8 kD, 15 kD,
25 kD, and 60 kD with a color-coded cap
for easy sample identification.
It incorporates a high membrane surface
area to volume ratio for a faster dialysis
rate and features maximum macro solute
recovery for both liquid and solid phasesamplesMultiple Micro DispoDialyzer® units canbe dialyzed in one dialysis reservoir
MWCO Color Coded Dot (top)Volume Color Coded Cap

No flotation device, syringe or needle
accessories are required.
Applications include:

Cap Post
Labeling Area (MWCO’s) Identification
Flotation Disk

Attached Seamless Regenerated
Cellulose Membrane

Figure B-5 Spectra/Por® Micro DispoDialyzer®

Removal of salts, surfactants, detergentsand solvents
Adjustment of sample buffer and pH
Purification of proteins, DNA, oligonucleotides, peptides, polymers, antibodies
and other biomolecules

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B-76

THE ABCs OF LABORATORY DIALYSIS

Spectra/Por® DispoDialyzers®
The Spectra/Por® DispoDialyzer® is a readyto-use dialyzer for small sample volumes
from 500 uL to 5 mL. It is available withboth regenerated cellulose and cellulose
ester membranes having a wide selection of
MWCOs ranging from 100 to 300,000. The
DispoDialyzer® is color coded by molecularweight cut off.
By allowing a small headspace during filling, the DispoDialyzer® will float verticallyand remains fully immersed during dialysis.
Multiple DispoDialyzers® can be simultaneously used in the same buffer solution.

Figure B-6 Spectra/Por® DispoDialyzer®

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Section C

The ABCs Of Polymeric Membrane Extraction
Selextrac™ Technology Summary
Spectrum Laboratories, Inc. has introduced SeleXtrac™, a novel class of membranes for the selective extraction and concentration of organic compounds from
dilute aqueous solutions.

Figure C-1 SeleXtrac™ membrane modules are designed with advanced hollow fiber membranes that have the pores impregnated with apolymeric liquid that has an affinity or attraction for the organic compound of interest.

SeleXtrac™ membrane modules are designed with advanced hollow fiber membranes that have the pores impregnated with a polymeric liquid that has an affinity
or attraction for the organic compound of interest. When membranes are prepared in this fashion, the ability of the membrane to separate compounds from aliquid or gas stream depends primarily on the chemical properties of the liquid
polymer used and not on the conventional sieving or rejection of molecules
through the membrane pores.
SeleXtrac™ liquid membranes selectively transport and extract specific compounds by utilizing various molecular properties, such as hydrophobicity, hydrogen bonding capability and the degree of dissociation as indicated by the pKa.
The housing of the SeleXtrac™ membrane module is designed with inlet and outlet
ports for recirculating two different mixtures of a liquid or a gas or both. Driven by
selective extraction, the SeleXtrac™ System operates with a dual head peristaltic
pump or syringes. One pump head recirculates the Feed Solution (sample mixture) through the inlet and outlet sideports located on the side of the module. The
other pump head recirculates the “Strip Solution” in a countercurrent flow direction
through an inlet and outlet port at each end of the module that provides access to
the lumens of the hollow fibers.
The Feed Solution, consisting of a liquid or a gas mixture, is recirculated on theoutside of the SeleXtrac™ hollow fiber membrane. By chemical interaction, targeted organic molecules are transported from the Feed Solution through the liquid
polymer contained in the pores of the hollow fibers and into the Strip Solution thatis being recirculated through the fiber lumen. The Strip Solution may be a liquid ora gas mixture. Its selection depends on the nature of the organic compounds to

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C-78

THE ABCs OF POLYMERIC MEMBRANE EXTRACTION

Figure C-2 Schematic of flow for SeleXtrac™ Hollow Fiber Membrane
Modulebe extracted. For example, water as theStrip Solution for soluble, neutral or nonvolatile organic compounds; acidic Strip
Solution (e.g. HCI) for basic organic compounds and basic Strip Solution (e.g. NaOH)
for acidic organic compounds; and gaseous
strip for volatile organics. The rejected ionized organic molecules remain in the Feed
Solution sample throughout the extraction
process. The Feed and Strip Solutions arerecirculated through the SeleXtrac™ System
until the desired concentration of the selected compound is achieved. The Strip
Solution containing the extracted compoundis collected as the “Extract”.

SeleXtrac™ Features
STRIP FLOW (OUT)
RECOVERY SOLUTION OF
EXTRACT ORGANICS

FEED FLOW (IN)
SAMPLE WITH POLAR ORGANICCOMPOUNDS/VOCS AND SALTS

FEED FLOW (OUT)
SALTS

Fast extraction procedure with no toxic
solvents or phase separation.

STRIP FLOW (IN)

Versatile extraction of liquid-Iiquid, liquid-gas
and gas-gas mixtures.
Available in two types of easy-to-use hollow
fiber modules:

LUER
TEE

LUER
TEE

Figure C-3 The SeleXtrac™ Module can be used with syringes for small
volume applications and for feasibility studies prior to scale-up

Type S for extraction of hydrophobic organic
compounds - more solvent resistant.
Type P is recommended for polar
compounds.
Hollow fibers provide a large surface area in
a compact module.
Three module sizes are available for processing volumes from 5 ml to several liters
depending on sample size and type.
Higher yields due to smaller sample
volumes.
Stable condition for biological samples.
Laboratory and prototype testing results cangenerally be scaled up to production levels.

Figure C-4 SeleXtrac™ Module in Pumped Configuration for prototype
and process scale quantities

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SUMMARY C-79

Transport Rates of Organic Compounds through SeleXtrac™ Membranes
Transport rates for selected organic compounds across SeleXtrac™ membranes
organic compounds are shown in figure C-5.

Figure C-5 Transport of selected organic compounds across Type S and Type P SeleXtrac™ Membranes

SeleXtrac™ Applications
Simultaneous extraction of hydrophobic organic compounds from biological samples, cell culture media, reaction solutions, fermentation broth, flavor and waste
water processing in Life Sciences and in bulk industrial applications.
Extraction of non-ionic acids, basic organic compounds such as phenol and phenolics, nitriles, esters, carboxylic acids, aromatic amines, chlorinated hydrocarbons and other related compounds.
Rejections of ionized organic acids and bases, Zwitterions, sugars, salts, proteins
and amino acids from aqueous solutions.
Extraction of volatile organic compounds such as alcohols, esters and chlorinatedsolvents using the SeleXtrac™ System in the pervaporation mode.

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C-80

THE ABCs OF POLYMERIC MEMBRANE EXTRACTION

Example - Extraction of Volatile Organic Compounds (VOCs)
Removal of MTBE from Contaminated Water

Figure C-6 The SeleXtrac Type S Membrane System is highly effective inthe removal of MTBE from contaminated water

Methyl Tertiary Butyl Ether (MTBE), a gasoline oxygenate additive, is also a groundwater contaminant present in many states,
especially in California. Due to its
hydrophilic properties, MTBE is highly soluble in water and has a low affinity for common absorbents. The SeleXtrac™ Type Shydrophobic hollow fiber membrane module
has proven to be an effective method to
extract MTBE from contaminated water in apervaporation mode. Once permeation
through the liquid polymer in the membranetakes place, MTBE can be effectively
removed by a “strip” gas such as air or
nitrogen or removed by applying a vacuum.
Water exhibits a very low permeation rate
due to the membrane’s hydrophobic nature.

In the experiments described in the discussion section below, the MTBE water mixtures used as the Feed Solution were recirculated through the lumen of the hollow fibers.
The water flow rates were varied to determine the significance of liquid film masstransfer resistance versus the liquid membrane resistance. Nitrogen was used as
the ventilating gas on the outside surface of the membrane. The high nitrogenflow rate was maintained constant to minimize gas film resistance and to achieve
the highest concentration gradient for MTBE.

Example - Extraction of Water Soluble Polar Organic Compounds
Removal of Para-Nitrophenol (PNP) From WastewaterIndustrial wastewater often contains phenolic compounds such as PNP and highconcentration of inorganic salts that can be removed and concentrated for recycling. Due to its extraction affinity for polar compounds, the SeleXtrac™ Type P
hollow fiber module can be used for removing PNP while rejecting the salts.
In the extraction process experiment described in the discussion below, 0.1 N
NaOH solution (pH 13) is used as an alkaline Strip Solution and recirculated
through the lumen of hollow fiber membranes. The PNP-containing wastewater at
pH 3.8 is used as the Feed Solution and is recirculated on the outside of the membrane surface. During the experiment, pH values in both solutions remained fairly
constant indicating no significant transport of H+ and OH- ions across the polymeric liquid membrane.
Discussion and Results: PNP removal was determined by measuring its concentrations in the Feed and Strip Solutions as a function of time. Figure C-7 showsthat PNP concentration on the Feed side steadily declined. The SeleXtrac™ mem-p 80

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SUMMARY C-81

brane module removed PNP in the Feed
Solution from an initial value of 1000 mg/L
to less than 1 mg/L. NaOH in the Strip side
reacted with the permeating PNP to form
sodium p-nitrophenate (Na+PNP-), which inthis ionic state will not permeate back into
the Feed side.
Thus simultaneous removal of PNP from theFeed and its concentration in the form of
sodium PNP on the Strip side can easily beachieved.

Figure C-7 The SeleXtrac™ Type P Membrane System is highly effective inthe removal of water soluble polar organic compounds fromcontaminated water

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C-82

THE ABCs OF POLYMERIC MEMBRANE EXTRACTION

Discussion
SeleXtrac™ Membranes for Polymeric Extraction of Organics
from Aqueous Streams
Introduction
The majority of industrial membrane applications involving liquid solutions are
based on size difference for the desired separation (1-4). Microfiltration membranes are widely used for removing particulates for solution clarification.
Ultrafiltration membranes with a range of molecular weight cutoffs (MWCO) are
typically used to retain dissolved compounds ranging from protein size (~100,000
MWCO) to smaller molecules (~1000 MWCO). Nanofiltration membranes get into
the range of small molecules (MWCO of a few hundred) and ions. Reverse osmosis membranes, while not strictly functioning based on size, complete the range byallowing primarily only water to pass through and are thus widely used for recovering fresh water from seawater and brackish solutions. Another type of membrane
applications is based on charge difference of the dissolved molecules or ions,such as electrodialysis.
There is a clear need for membrane separation based on the molecular propertiesof the dissolved compounds in addition to just size or charge, such as the wide
range of industrial separations accomplished with solvent extraction. This concept
has been cleverly employed in an approach called supported liquid membranes inwhich the extracting solvent resides (“supported”) inside the pores of a microfiltration or ultrafiltration membrane, allowing membrane contact with the feed solution
on one side and the strip solution on the other side. Consequently, all the separation capabilities of solvent extraction, including the use of selective carriers for
specific separations, are directly translated into a membrane configuration inwhich very little solvent is needed and two separate unit operation steps are combined into one.
Conventional supported liquid membranes typically employ common organic solvents like kerosene and alcohols as the liquid membranes along with some specific carriers for enhanced transport rate and selectivity. These membranes havebeen extensively studied for transport of ionic compounds such as salts and metal
ions (7,8) and for transport of organics (9-11). Despite their powerful and diverse
separation capabilities, these supported liquid membranes turn out to be not verypractical for industrial applications, primarily because of the instability of the membranes. This problem has been attributed to partial dissolution of the solvents,
leaching of the carriers, and displacement of the solvents from the pores of the
support matrix (12,13).
Spectrum has acquired from Monsanto Co. an interesting class of membrane
called SeleXtrac™, a supported polymeric liquid membranes in which the liquid isa functional liquid polymer rather than an organic solvent. SeleXtrac™ membranes have been found to be much more stable, especially under very highosmotic pressure differences (5,6,14), probably because of the typical physicalproperties associated with the polymeric liquids used, such as high viscosity,
extremely low volatility and insolubility in water. One unique application of thiscapability is for treating aqueous wastes discharged from chemical plants that typically contain low levels (a few percent or less) of small organic compounds(molecular weight <200) but also salts with concentrations reaching 30 wt% or

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DISCUSSION C-83

higher. The small size of the organics in these streams excludes the use of ultrafiltration and microfiltration, and the osmotic pressure limitation from the high saltcontent makes reverse osmosis impractical. The non-volatile nature of polymericliquids also allow SeleXtrac™ membranes to be operated in the pervaporationmode, thus having similar capabilities as hydrophobic (solid) membranes thathave been shown to work well for recovering relatively hydrophobic “volatile”
organics (VOCs) from dilute aqueous wastes (4).
SeleXtrac™ membranes using certain liquid polymers have also been found to
exhibit high permeability for flavors and aroma compounds, which could result in
important applications in the beverage and food processing areas. Other potential applications include analytical and biomedical. This chapter will cover theconcept and characteristics of SeleXtrac™ membranes, and the various key applications that have been identified.

Basic Theory
SeleXtrac™ Supported Polymeric Liquid Membranes
Concept

Figure C-8 Schematic diagram of SeleXtrac™ membranes

SeleXtrac™ membranes are prepared byfilling the pores of microfiltration or ultrafiltration membranes with polymeric(oligomeric) liquids having affinity for theorganic compounds of interest. These
polymeric liquids are polyamphiphilic, i.e.,they consist of repeating monomeric units
that contain both hydrophobic and polargroups. The chemical nature of thesegroups and the number of monomericunits in each polymer chain can be
manipulated to vary the chemical andphysical properties of the polymeric liquids, thus conveniently creating membranes of diverse separation capabilities.
Figure C-8 shows a schematic diagramfor SeleXtrac™ membranes, with theexample showing how ionizable organic
compounds can be extracted from ahigh-salt aqueous solution and concentrated in a caustic solution.

The use of functional polymers in SeleXtrac™ membranes provides the flexibility ofaltering the liquid physical properties (solubility, viscosity) by varying the polymer
size (average molecular weight) without compromising the chemical functionalityof the polymers. For example, polyglycols are available as polyethylene glycols(PEG), PPG, PBG and so on. The PEG series are not suitable as liquid membranes for treating aqueous solution since they are fairly water soluble up to MWof several million. In contrast, PPG-4000 in the polypropylene glycol series has
been found to remain stable as SeleXtrac™ membranes between 20% KCl and
0.1N NaOH aqueous solutions, thus withstanding the tremendous osmotic pressure difference (data presented below). Supported PPG-1200, however, lost its

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C-84

THE ABCs OF POLYMERIC MEMBRANE EXTRACTION

membrane integrity within a few hours between such solutions, i.e., allowing equalization of pH and salt concentrations between the two sides. PBGs being morehydrophobic and more viscous than PPG at the same MW were relatively stable as
SeleXtrac™ membranes at MW down to 1000.
The SeleXtrac™ membrane concept is equally applicable to other hydrophobic liquid polymers possessing different separating capabilities [6]. Other polymeric liquids that have functioned well as SeleXtrac™ membranes include various gradesof liquid silicone and its derivatives, liquid hydrocarbons, and liquid polybutadienes. Obviously, known liquid polymers can be chemically modified to alter thefunctionality, or new ones can be synthesized for a particular application.

Characteristics of Polyglycol SeleXtrac™ Membranes
The concept of SeleXtrac™ membrane was first developed using
polyglycols as the polymeric liquids.
The polyglycol that has been tested
extensively is polypropylene glycolwith an average molecular weight of4000 (PPG-4000), and to a lesser
extent, polybutylene glycol PBG-4800)
manufactured by Dow Chemical.
Therefore, it is worthwhile to review
the general properties of these
SeleXtrac™ membranes. Eachmonomeric unit in PPG has the formula -CH2-CH(CH3)-O- in which -CH2CH(CH3)- represents the hydrophobicgroup and the ether oxygen has therole of the polar group that can formhydrogen bonds (5). Due to its highlyhydrophobic nature, PPG appears toform an effective barrier to charged
species in solution. This has beenshown to include both small ions from
dissociation of inorganic salts, H+
and OH-, and larger charged molecules. Only neutral compounds canpass through. This characteristic
forms the basis for the use of PPG
SeleXtrac™ membranes to recoverorganics from an aqueous saline
waste illustrated in Figure C-8. For
effective separation across such amembrane, the strip solution should
provide an environment (a “sink”)
which limits the back diffusion of theorganic across the membrane into theTable C-1

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Partition and transport of organic
compounds with impregnated PPG
SeleXtrac™ membrane.

BASIC THEORY C-85

feed solution. This could be accomplished in several ways including pH change,
reaction, chemical complexation, biodegradation, pervaporation, etc.
Ions which have been shown experimentally to be effectively blocked by the PPG
membrane include H+, OH-, Na+, K+, Cl-, NH4+, NO3-, and SO4. Charged
organic molecules are also effectively rejected by the PPG SeleXtrac™ membrane.
Zwitterions, which by definition are ionized at any pH, such as amino acids, could
not pass through the PPG membrane at all.
For neutral compounds, the rate of transport through the PPG SeleXtrac™ membrane is sensitive to their chemical structures. The rate of transport for a compound appears related to its ability to form hydrogen bond with the ether oxygensof PPG, and to its hydrophobicity. Table C-1 shows the partition coefficients andthe transport rates for various compounds such as phenol, amines and carboxylic
acids. The partition coefficient is defined as the ratio of the concentration of theorganic compound in the polymeric liquid over that in water, thus an indication of
the interaction between the polymeric liquid and the compound. Rates are reported as the overall mass transfer coefficient (cm/s), which lumps the mass transfer
resistances of the two aqueous boundary layers on the sides of the membranewith the resistance due to permeation through the SeleXtrac™ membrane itself. In
these experiments, the pHs of the feed solutions were such that the organic compounds were un-ionized (pH about 2 units below pKa) and NaOH was used on therecovery side for weak acids, and HCl or HNO3 for weak bases. Note that allthese compounds have quite high partition coefficients, indicating a high degreeof affinity for PPG.
Table C-1 shows the partition coefficients and the corresponding mass transfer
coefficients for various organics with a polypropylene glycol supported
SeleXtrac™ membrane. As can be seen, ester compounds and higher alcohols
both partition very favorably into PPG from water and have very high mass transport rates compared to ethanol. It is this observation that let to the exploration of
the potential application of this membrane technology for enhancing the flavor of
consumable liquids such as beers, wines, fruit juices and soft drinks.

Advantages of SeleXtrac™ Membrane Systems
SeleXtrac™ membranes with polyglycols appear to exhibit relatively rapid transport rates for many organics including phenolics, aromatic acids and amines, carboxylic acids, alcohols and esters. Attractive features of this membrane processinclude the ability to recover the contaminants in concentrated form foreither recycle or more economical
disposal, low pressure (ambient)
operation, ease of regeneration, andsimple scale-up using commercial
hollow fiber modules.

Table C-2 Phenol permeability through various polymeric membranes

The magnitude and practical significance of the overall mass transfer
coefficients obtained with the PPG
SeleXtrac™ membrane shown in
Table C-2 can be appreciated bycomparison with other membrane
approaches for phenol transport.

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Klein et al [14] reported effective diffusion coefficient (D in cm/s = permeability x
membrane thickness) for phenol extraction using various polymeric films as membranes. With the PPG-polypropylene membrane (25µm thick) the permeability ofphenol is 6.0 x 10-4 cm/s (Table C-1), which gives an effective diffusion coefficientof 15 x 10-7 cm2/s. This is almost an order of magnitude higher than the best rateachieved by these workers (D = 2.06 x 10-7 cm/s) with the membrane made from apolymethylsilane-polycarbonate copolymer.
Another interesting comparison is with the hollow fiber contained liquid membrane
approach by Sengupta et al. [15]. In this approach, the shell side of a membrane
module that contained two sets of intermingled hollow fiber bundles was filled withan organic solvent that functioned as the liquid membrane. The feed solution
flowed through the lumen of one set of fibers, and the strip solution through theother. With methyl isobutyl ketone as the contained liquid membrane, they reported an overall mass transfer coefficient for phenol of about 3 x 10-4 cm/s. This isabout half of the rate obtained with the PPG-SeleXtrac™ membrane, which, incidentally, would utilize all of the hollow fiber membrane area available for transport
rather than only half of it as in the contained liquid membrane approach.
The PNP transport rates obtained with the polypropylene PPG SeleXtrac™ membranes also compare well with the transport of PNP through octanol-filled
SeleXtrac™ hollow fiber membrane [13]. The referenced work, which used themembrane-supported solvent extraction approach with octanol as the extraction
solvent, reported a mass transfer coefficient of 6.5 x 10-4 cm/s. This is within therange of 3.4 to 8.1 x 10-4 cm/s obtained with the PPG SeleXtrac™ membrane(Table C-1) The obvious advantage of the PPG SeleXtrac™ membrane is that the
separation can be accomplished in one step combining extraction and stripping,thus avoiding the additional step of stripping PNP from the solvent such as thecase with octanol.

Mass Transfer Theory and Rate Measurements
Mass Transfer Theory
In an extraction process using membranes, the three resistances to consider are
the membrane resistance and the two boundary layer resistances. The masstransfer theory for extraction processes using SeleXtrac™ membranes is briefly
summarized below.
The overall mass transfer coefficient, Kf, for steady-state operation is:
1 = 1 + 1 + 1
Kf
kf
kmKp
ksKo
Where:

p 86

(1)

Kf :
kf :

overall mass transfer coefficient in cm/s
feed-side mass transfer coefficient

km:
Kp:
ks:
Ko:

membrane mass transfer coefficientpartition coefficient of organic compounds between liquid polymer and feed
strip-side mass transfer coefficientpartition coefficient of organic compounds between strip and feed

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BASIC THEORY C-87

For reactions on the strip side such as acid-base reactions), the permeating compounds undergo rapid irreversible reactions as they enter the strip solution. Thus
Ko is very large and the resistance due to the strip boundary layer could be
ignored. For flat sheet, the overall membrane coefficient is:
km = DτmΖεWhere:

D m:
ε:
τ:
Ζ:

(2)

diffusivity of the organics in the polymeric liquid, cm2/s
membrane porosity
membrane tortuosity
membrane thickness, cm

For hollow fiber membranes, an area correction is added since the membrane
area and Kf are based on the outer diameter of the fibers
km = DmετΖ( dd )lmo(3)

The two parameters, Kp ad Kf, sufficiently describe each organic/liquid polymer
membrane system. Kp values indicate the relative solubility of the organic compound in the liquid polymer versus the aqueous solution. Higher Kp values would
favor the extraction of the organics from the aqueous solution and typically resultin higher transport rates through the membrane.
As indicated above, the definitions for Kp and Kf are:Kp =Concentration of the organic compound in the liquid polymer
Concentration of the organic compound in the aqueous solution

Kf is the overall mass transfer coefficient as defined in the following equation:
Membrane Flux = Kf (Cf - Cs)
Where:

Flux is in units of g compound per second per cm2 membrane area,
Kf : cm/s
Cf: concentration of the organic compound in the feed solution, in g/cm3
Cs: concentration of the organic compound in the strip solution, in g/cm3
Kf shows how fast the compound permeates through the SeleXtrac™ membrane,
which is a function of many variables such as temperature, membrane support
thickness, pore size and porosity, the partition coefficient Kf and possibly the liquidboundary layers on either membrane surface. The values for Kf were determinedusing a small stirred cell under conditions that minimize the resistance due to theboundary layers. However, for fast transport rates (Kf > 10-3 cm/s), the contributions of the liquid boundary layers may be significant. The significance of determining the Kf value for each system is that it can be used to estimate the total
membrane area required for applications at any scale.

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C-88

Transport Rate Measurements
STIRRED CELLS WITH FLAT SHEET MEMBRANES Transport measurementswith flat sheet membranes were conductedusing stirred cells. These cells are convenient for
rapid membrane screening as well as for determining the effects of various operational parameters on transport kinetics. Stirred cells weremade up of two glass compartments separated
by a sheet of membrane and held together by aclamp. Liquids in the compartments were magnetically stirred. The cell can contain approximately 30 ml of fluid in each cell compartment.
The surface area of the membrane in contact
with the fluids is 8 cm2 for the normal size cell.
Figure C-9 shows the picture of the small stirred
cell.

Figure C-9

For stirred cells the following equation can beused for analyzing the transport data, allowing
for back permeation of the solute from the pickup side to the feed side:

Small stirred cell for transport measurements with flat
sheet membranes[

Ln Cf (1 +Where:Vs) - CfoVf] = - K A (1V+ V /V ) t + Ln [C VV ]
f

m

s

f

s

fo

s

(4)

f

Am: membrane area, cm2
Kf: overall mass transfer coefficient in cm/s
Cf: solute concentration in the feed solution, mg/L, at time t
Cfo: initial solute concentration in feed solution, mg/L
Vf: liquid volume of the feed solution, mL
Vs: liquid volume of strip solution, mLt : operating time, s

[

]

Vs
The plot of Ln Cf (1 + V ) - Cfo vs. Time can be fitted to a straight line whosefslope is equal to and can be used to calculate the overall mass transfer coefficient
Kf.
HOLLOW FIBER MEMBRANE SYSTEMS With hollow fiber membranes, fluids are
circulated through the lumen side and the shell side. Organic concentrations of
the feed and strip solutions are measured as a function of run time, from which theoverall mass transfer coefficient can be calculated using the following analysis.
For hollow fiber membrane systems operating in a batch mode, the same equation
as the stirred cell (equation 1) can be used provided that:
AmKf / Qf < 0.1
Where:p 88

Qm: feed flow rate, cm3/s

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BASIC THEORY C-89

The term AmKf/Qf represents the ratio of the mass of material transported throughthe membrane to that passing through the module per single pass. For batch
operation in small modules, whose membrane areas are low relative to the circulation rate, the change in feed concentration per pass through the module is very
low and the whole system (feed reservoir and membrane) can be accurately treated as a perfectly mixed unit, therefore the applicability of equation 1. For large
modules and with relatively low circulation rate, the removal per pass through themembrane unit can be significant and more complex mathematical model is needed for the analysis:

Key Variables
Effects of Polymeric Liquids and Permeating Molecules
Transport characteristics of the polyglycols SeleXtrac™ membrane regarding
types of organics, effects of solid support, and type/molecular weight of the liquid
polymer have been described elsewhere (17,18). A brief summary is given below.
Due to its highly hydrophobic nature, PPG forms an almost impenetrable barrier tocharged species in solution. These include small ions (ions generated by salts,
H+ and OH-) and larger charged molecules. Only neutral compounds can passthrough. The driving force for transport includes concentration gradient, pH,chemical reactions, chemical complexation, biodegradation, etc.
Polar organics of intermediate solubility in water such as phenols, nitrophenols,
nitroanilines, and benzoic acid exhibit quite high partition coefficients (>100) andrelatively rapid permeation rates through the PPG SeleXtrac™ membranes (~ 4.0 x
10-4 cm/s or higher at room temperature). Partitioning and transport of several
monobasic carboxylic acids (C2 to C6) through the PPG liquid membrane showthat both the partition coefficients and the transport rates increase drastically with
the number of carbons. This behavior is probably a consequence of the different
solubility of these carboxylic acids in the PPG phase (hydrophobic interaction).
Effects of the number of hydroxyl groups on phenolic compounds were exploredwith resorcinol, hydroquinone, pyrocatechol, and phloroglucinol. More hydroxyl
groups attached to the ring make the compound more hydrophilic, which results inlower transport rates. Also very interesting is the behavior of amino acids and its
analogs. Being zwitterions, which are always ionized, amino acids, even the mosthydrophobic ones such as tryptophan, phenylalanine and tyrosine, are totally
rejected by the PPG membrane. Yet hydrocinnamic acid, which is phenylalanine
without the amino group, permeates very fast (comparable to p-nitrophenol)
through the PPG liquid membrane.
The above properties of SeleXtrac™ membranes make them very suitable fororganic/salt separation as well as for separating similar organic compounds that
may be difficult to achieve with other separation methods.

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C-90

Effects of Membrane Supports

Table C-3

Effects of the types of membrane support and their structure on the rates of transport through supported PPG SeleXtrac™ membranes are shown in Table C-3. For
the Celgard® flat sheet membranes,
increase in NaOH concentration in the
strip solution had no effects on the overall mass transfer coefficients (Celgard®
2400 and 2500). The mass transfer
resistance due to the boundary layer on
the strip side was thus not significant
due to the rapid chemical reaction
between PNP and NaOH. Doubling themembrane thickness resulted in half ofthe transport rate (Celgard® 2402).
Permeation through the PPG layer was,therefore, likely the rate limiting step,
partly due to slow diffusion through theviscous polymeric liquid and partly due
to vigorous mixing in both compartmentsof the stirred cells. It is not clear why the
Celgard® 2500 was twice as fast as theEffects of support characteristics on PNP transport through PPG
2400 with only small increases in pore
SeleXtrac™ membranes
size and porosity based on the data fromthe membrane supplier. With theassumption that the overall mass transfer coefficients obtained with flat sheet
membranes in stirred cells are practically the permeation rates of PNP through the
PPG SeleXtrac™ membrane, PNP diffusion coefficient in the PPG layer can beobtained by (see Appendix)
D = (Kf / Kp) • (l τ / ε)Where:(5)

l: membrane thickness
τ: tortuosity
ε: porosity
The different transport rates obtained for various flat sheet membranes could thus be
due to the differences in their physical characteristics. For Celgard® 2400, l is equal
to 25 mm or 25 x 10-4 cm and ε = 0.38. If we assume the tortuosity τ for Celgard®
2400 of about 2 [19], τ for Celgard® 2500 must then be 1.2 to account for the doubling in rate.. Similarly, the tortuosity for Gore-Tex® 0.02 µm would be 1.1, which is
not unreasonable considering the very open structure of the Gore-Tex® membranes[20]. However, the tortuosity for Gore-Tex® 0.2 µm would be only 0.6 to account for
the very high transport rate. For the tortuosity of this Gore-Tex® membrane to be 1,that of Celgard® 2400 must be at least 3.6, unless higher porosity and larger poresize also exerted some non-obvious secondary effects on the transport rates in PPG
SeleXtrac™ membranes. Note that these comparisons assume that the pores of different membranes were completely filled with the liquid polymer, which was likely thecase. It is clear from the results obtained in this section that characteristics of membrane supports such as material, thickness, pore size, pore structure, and porosity
have strong effects on SeleXtrac™ transport rates.

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BASIC THEORY C-91

Effects of Operating Conditions
Key operating parameters that impact the SeleXtrac™ performance are temperature, characteristics of the feed solution as well as of the stripping fluid (such as
gas vs. liquid; pH, ionic strength, % organic solvent), and to some extent the circulating flow rates through the membrane module. Temperature has a very strong
effect on the rate of permeation through the membrane, primarily due to the significant increase in the diffusivity as a consequence of lower viscosity of the polymeric liquid. The characteristics of the feed solution and the strip solution can affectthe transport rates as well as the obtained selectivity. Since only neutral compounds can permeate the membrane at a practical rate, pH of solution can be
adjusted to manipulate the charged characteristics of compounds in solution forthe desired selectivity, either extracted from the feed into the liquid membrane or
stripped from the membrane into the strip solution. Likewise, volatile compounds
can be selected over less volatile ones by using gas as the stripping fluid insteadof liquid. Subsequent reactions or physical removal (e.g., adsorption) of the permeating compounds provide another way to drive the transport process. Sufficient
circulating flow rates through the membrane module are necessary to minimize thetransport resistances due to the boundary layers at the membrane surfaces.
Some of these manipulations are evident in the applications discussed below.

Process Applications
The SeleXtrac™ technology has been used successfully to remove and concentrate organics from several actual aqueous waste streams. We will describe theprocess application for removing p-nitrophenol, carboxylic acids, and methyl tertiary butyl ether (MTBE) from contaminated water.

Wastewater Treatment
Phenolics (p-nitrophenol) Waste
This waste stream was generated from a chemical manufacturing plant and typically contains p-nitrophenol (PNP) as the major organic contaminant at <1 wt%.
The waste stream also contained high salt level (~20 wt%, mainly KCl). Existingtreatment options for this stream includes PNP removal followed by destruction or
biodegradation. The feasibility of using a SeleXtrac™ membrane process toremove PNP from the waste solution in a concentrated form for recycle was investigated.
The pKa value for PNP is 7.2. Thus at pH around 5 or lower, all dissolved PNP
molecules will be in the neutral (non-ionized) state and partition strongly into theliquid membrane phase (Kp from 100 to over 500 with PPG-4000 as the liquid
polymer). In order to concentrate the permeating PNP, the strip solution was held
at alkaline pH (pH>10) to convert PNP into its ionized form, which was blockedfrom going back to the feed side by the hydrophobic liquid.
Various radial flow commercial membrane modules had been previously tested in
the laboratory. Batch results obtained with 2.5 and 4 inch modules using a simulated PNP-containing feed solution were already reported (18). Here we
described the results obtained with a 4 inch module tested at the plant using an
actual waste stream.
A number of batch runs were first conducted to check out the physical integrity of

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the PPG SeleXtrac™ membrane and the module performance regarding PNP
removal. About 100 L of the PNP waste solution at pH 4 was processed per run.
The same strip solution (0.5 N NaOH) was used for the whole campaign. The feedsolution was circulated on the shell side at 11.4 to 45.4 L/min (equivalent to NRe of~1 to 4), and the strip solution on the lumen side at a fixed flow rate of 9.5 L/min.
Inlet pressure on the feed side was only 0.9 psi (6.2 kPa) at the highest flow rate,
and was higher on the lumen side, 7.5 psi (51.7 kPa), which is still relatively low.
The SeleXtrac™ module was found very effective for PNP removal. PNP concentration in the feed solution typically dropped from several thousand mg/L (ppm) to afew ppm in 1-2 hours. PNP concentration in the strip solution built up from zero
initially to over 20,000 ppm after seven runs. As discussed above, extraction of
the overall mass transfer coefficient, Kf, from batch data is quite complex andrequires a great deal of approximation. However, rough estimates from the fit to
the linear portion of the data show that Kf was not sensitive to flow rates in thetested range and was about 2.8 x 10-4 cm/s at 29°C and 4.5 x 10-4 cm/s at 41°C at
an average PNP concentration of ~2000 ppm. These values are in good agreement with earlier laboratory results for a similar 4 inch radial flow module using
clean, simulated PNP solutions. Note that the feed-side mass transfer coefficient
ranged from 3.8 to 6.7 x 10-3 cm/s at room temperature, which is more than anorder of magnitude higher than Kf.
Continuous runs were next conducted using the above module at a feed flow rateof 18.9 L/min (NRe ~1.7) and strip flow rate of 9.5 L/min. Steady state was typically achieved within 5 minutes of starting the feed flow. The degree of PNP removalfrom the feed solution through the membrane module remained constant throughout each run. The results obtained for two types of commercial fibers (X-10 and X20) are shown in the table below. It is noting that PNP removal ranged from ~30 toover 50% for a single pass. This is quite impressive considering the relatively highflow rate of 18.9 L/min and the compact size of the module (4 x 28 inches). The
feed (shell) side mass transfer coefficient kf at this flow rate was estimated to be
4.69 x 10-3 cm/s, from which the membrane coefficient km was found to be about
5.1 x 10-6 cm/s for the X-10 fibers, and 3.6 x 10-6 cm/s for the X-20 fibers. The
higher km values for the less porous X-10 fibers are probably an artifact of thehigh PNP concentrations in the feed solution for the X-10 runs, which overestimates the drop of Kp in this high concentration range. The highest PNP concentration in the data used to obtain the correlation for Kp was only 3000 ppm (14).

Table C-4 PNP removal from a waste stream

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PROCESS APPLICATIONS C-93

Note that the true membrane coefficient, kmKp, is over two orders of magnitude
higher than km, since the partition coefficient Kp varies from 200 to 600 for the
PNP/PPG system. At an average PNP concentration of 2000 mg/L, the overall
mass transfer coefficients from these continuous runs were found (using Eq. 1) tobe about 10 x 10-4 cm/s for the X-10 fibers and 7.6 x 10-4 cm/s for the X-20 fibers,
which are substantially higher than the batch values obtained above and the values obtained earlier with simulated waste solutions (18). It is very encouraging
that in the preferred continuous mode of operation and with an actual waste
stream, the SeleXtrac™ membrane process performed even better than expected.
Additional pilot testing is being conducted with various polyglycol polymers andfiber types that differ in pore size, porosity, and wall thickness to optimize theprocess with respect to both transport rates and liquid membrane/module stability.
Based on the rate data obtained with the PPG-4000 Celgard® X20-400 hollow fiber
membrane, we estimated that a membrane area about 15,000 ft2 is required toremove over 99.99% of PNP in a 15 gal/minute waste stream at 60°C (to get below
the 200 ppb needed to meet the regulation discharge). On the basis of 6 to 10
dollars per ft2 of membrane installed cost (21), the required membrane area translates into a maximum of $150,000 for the entire membrane module. This is contrasted with an alternate adsorption treatment using activated carbon, which costs
from $500,000 to $1 million a year for the carbon. Since the membrane module islikely to last at least a year or so, the membrane process looks extremely attractive, especially if the recovered PNP from the waste could be recycled for some
payback value. While PNP is a particularly attractive case, it should be emphasized that the ability to control rate through control of the membrane structure canpermit the application of this approach to many cases where the rates based on
existing membrane supports are much lower than for PNP.

Carboxylic Acids Waste Stream
This waste stream was generated from a chemical manufacturing plant. The
organic content of the waste comprises mainly C2-C6 monobasic carboxylic acids(about 3800 mg/L organics or 2000 mg/L TOC). The stream also contains 1%
nitric acid (pH 1.2) and trace metals and is at 60-70°C. Our study involved using
the membrane process to simultaneously remove and concentrate the carboxylic
acids from the waste solution to facilitate further treatment. At pH 1.2, all the carboxylic acids in solution are in their neutral (uncharged) state and would permeate
through the membrane. Nitric acid andmetal ions also present in the solution
exist as charged species, hence can not
go through the membrane. The development of the SeleXtrac™ membraneprocess to remove the organic acids
leading to significant reduction in the
TOC of the solution is described below.

Table C-5

Transport of Carboxylic Acids Through Celgard® 2400/PPG-4000
SeleXtrac™ Membrane

FLAT SHEET MEMBRANES Partitioning
and transport rates of C1-C6 carboxylic
acids were measured individually using
the stirred cell with flat sheet membranes. The results (Table C-5) wereobtained with the PPG impregnatedmembrane sandwiched between a feed

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solution containing each organic acid and a 0.1 N NaOH strip solution to neutralize the acid. Note that the feed solutions also contained 20% KCl, which imposeda very high osmotic pressure difference across the membrane and would rapidly
destabilize conventional SeleXtrac™ membranes employing low molecular weightorganic solvents (12,13). The transport rates were found to correlate with thenumber of carbons in the carboxylic acids. Shown in Table C-2, caproic acid (C6)
was the fastest followed by smaller acids in the order of size. Kf for caproic acidwas actually over 70 times higher than for formic acid. The strong correlation
between the overall mass transfer coefficients and the corresponding partition
coefficients suggests that the membrane resistance was probably a controlling
factor.
The relative rates of transport observed for the individual carboxylic acids seem tohold for the mixture in the waste solution with caproic acid permeating through themembrane first, followed by valeric acid and butyric acid. Because of this characteristic of the process, the rate of organic removal steadily decreased as its concentration in the waste solution dropped. It was found, however, that up to 90%
organic reduction the transport process could be approximated by two average
rates. Linearized plot of concentration vs. time gives two rate constants: onetermed Kf initial is valid up to 50-60% organic removal, the other is Kf final and isgood up to about 90% removal. Kf final is roughly half the value of Kf initial. This
mathematical approximation allows the membrane area required for any degree of
TOC removal to be estimated in a simple but accurate fashion.

Table C-6

Higher temperatures significantly enhanced the rate of TOC removal. This is anadvantageous feature of the membrane process since the waste stream was at
65°C at the plant. Shown in Table C-6 for SeleXtrac™ ( Celgard® 2500/PPG-4000)
membrane, the rate went up 2.6 times as temperature increased from 25 to 65°C.
The effects of temperature on the transport rates with SeleXtrac™ membrane were
investigated in more detail by Ho et al. (16) with p-nitrophenol as the permeant. In
that study, two opposing effects were found to occur with increasing temperature:
the partition coefficient decreases but isaccompanied by an increase in the diffusion rate through the membrane.
Assuming the diffusivity is proportional totemperature and viscosity-1, as in the
Wilke-Chang equation (22), the increasein diffusivity was almost seven-fold from25 to 65°C, which can more than accountfor the 2.6-fold increase in the overall
transport rate. This strong effect comes
Effect of temperature on rate of TOC removal using a SeleXtrac™
mainly from the drastic drop in viscosity of(PPG-4000/2500) membrane
the polyglycol at higher temperatures.
HOLLOW FIBER MEMBRANES Several small modules (256 cm2 membrane area)
were prepared in our laboratory for testing with the CA waste solution. The abilityof the hollow fiber membranes to remove the organic acids from the solution wasshown with 10% sodium carbonate (soda) solution, pH 9.8, as the strip solution toneutralize the organic acids permeating through the membrane. At the end of therun, TOC in the waste had dropped from the initial value of 2000 ppm to about 50
ppm, equivalent to over 97% TOC reduction. Organic content in the soda solution
built up to about 6%, which is equivalent to over 15-fold concentration.

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PROCESS APPLICATIONS C-95

A large module was purchased from a supplier for the scaleup study. It contained
9000 fibers (polypropylene X20-240), with a membrane area of 3.72 m2, thus ascaleup factor of over 100-fold compared to the small modules. A total of eightruns were made with the large module, five at room temperature and three at
60°C. Two liters of fresh CA solution was used for each run. The same 2L of 10
wt% soda solution was used as the strip solution throughout the campaign. The
strip flow rate was maintained constant at 2.65 L/min; its pH was 9.85 initially,
dropping gradually to 9.22 after eight runs due to the accumulation of the permeating carboxylic acids. The pH of the feed solution hardly changed after each run(from 1.28 initially to 1.30-1.33) during which 92 to 94% organic removal from thefeed side occurred. These pH results are consistent with neither nitric acid nor
salts being transported through the membrane at any significant extent.
The effects of feed flow rates on transport rates at 25°C and 60°C were investigated. It is clear that for this large module the liquid film mass transfer on the feedside (lumen) was very efficient at room temperature with the maximum transport
rate achieved at the Reynolds number of about 6. The initial mass transfer coefficient was, however, only about 0.3 x 10-4 cm/s, which is less than one half of thatobtained with the small modules. This lower value probably reflects the poor liquid
distribution on the shell side given the large number of fibers packed in this shelltube configuration. More recent work with a different waste stream using radialflow hollow fiber modules gave much better scale-up results (16). The new radialflow design was introduced recently by a supplier for better fluid distribution on
the shell side.
The transport rates increased substantially with temperature. At 60°C, the rates
were 3 to 4 times higher than at room temperature (Kfi = 1 x 10-4 cm/s). This relationship seems to hold for the initial as well as the final mass transfer coefficient.
With the large module, some transport of water through the membrane could beobserved from the feed side to the strip side due to the osmotic pressure difference. At room temperature, a volume increase of about 50 ml was observed on
the strip side over a three-hour period, which translates to a water flux of about 0.4
ml/hr-cm2. The water flux at 60°C was about 0.9 ml/hr-cm2. These relatively small
amounts of water transport did not seem to affect the efficiency of the process.

PROCESS ECONOMICS CONSIDERATION Since this study was at an exploratory stage, no detailed cost analysis was conducted. However, we did look into theapproximate capital cost. For membranes, the cost is based on the total membrane area required, which is inversely proportional to the overall transport rate.
As presented earlier, the organic acid removal rate was quite rapid up to about
60%, followed by a slower rate up to about 90%. While removal over 97% wasachievable, the final rate was much lower. Thus, the targeted organic removal
has a major impact on the membrane cost.
Consider a waste stream with a flow rate of 385 gallons/min (1457 L/min) and arequired 90% TOC reduction. Based on the highest mass transfer coefficientsobtained (Celgard® 2500 at 65°C, Table C-3), a total membrane area of 150,000 ft2(about 14,000 m2) would be needed. With approximately $10/ft2 membrane area
for the largest membrane module (about 1500 ft2) from Hoechst-Celanese (18), thecapital cost for the membrane portion would be around $1.5 million. For handlingthe soda strip solution (assumed ~ 6 wt% organics), a process option evaluatedby the plant involved thermal destruction of the organics and recycle of the salts.

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THE ABCs OF POLYMERIC MEMBRANE EXTRACTION
This would add another $1.2 million. Thus, the total capital cost would be around$3 million, considering additional auxiliaries needed. This cost was judged promising since it was about one half of the estimated cost for an alternate biotreatment
process considered by the plant. Also, on the basis of $/gpd (gal per day ofwaste solution), the capital cost estimated for the SeleXtrac™ based process(~$5/gpd) seems to compare well with pervaporation, which ranges from $5-8/gpd
for the easy cases (hydrophobic, volatile organics) to over $100/gpd for the moredifficult ones (hydrophilic, water soluble organic mixtures) (4).
With the membrane cost constituting a major portion of the total capital cost, themembrane module’s lifetime is obviously a major factor in the overall economics.
Other key areas for step-change cost improvements include a) increasing theorganic concentration in the strip solution, which reduces the liquid volume going
into the thermal treatment step, b) recycling the recovered organics to regainsome value rather than destroying them, which incurs additional cost, and c)
selecting a polymeric liquid containing functional groups that exhibit much higher
selectivity for carboxylic acids than PPG does, such as sulfoxide or phosphineoxide (6), thus substantially increasing the transport rates, especially for the smaller carboxylic acids.

Methyl Tertiary Butyl Ether (MTBE) Removal
The use of MTBE as a gasoline additive has generated a serious, widespreadgroundwater contamination problem in the US, especially in California. The primary source of contamination in groundwater is leaking underground fuel tanks(LUFTs). There are thousands of such sites in California, some resulting in nearby
groundwater concentrations of MTBE as high as 6,000,000 ppb. Groundwater
MTBE migrated from LUFTs to drinking water supplies. The current maximum contaminant level of MTBE in drinking water ranges from 5 ppb (recommended) to
13 ppb (acceptable).
In addition to the currently investigated technologies for removal and/or destruction of MTBE in groundwater, such as oxidation, air stripping and activated carbon
adsorption, membranes could provide an effective means for this treatment. The
key issue is clearly that of economics: the process has to be cost effective, as wellas practical on a large scale. These organics have relatively high solubilities, low
Henry’s constant (23), low sorption and low biodegradability, such that common
treatment technologies such as air stripping, granular activated carbon or bioremediation are not very cost-effective (24,25).
The diversity of the contamination situations, which range from levels at low ppb tothousands of ppm, will require combination of complementary technologies toachieve the best economical approach for each situation. Membranes could playa key role in this combined approach. For instance, activated carbon adsorption
tends to be very efficient at treating organic contaminants at very low levels, sincecarbon binds tightly to many organics, and the lower the contaminant level themore water volume can be treated for the same amount of carbon. Membranes,
however, tend to be more cost effective at percent or high ppm levels, because
they work by removing a certain fraction of the initial contaminant level; the sameamount of membrane area (thus the same cost) will be required to remove, say,
90% of the initial contaminant amount, be it 1% or 0.01%.
LIQUID POLYMER SELECTION Based on our previous work with existing commercially available liquid polymers, silicone 350 cst (Si-350) and polybutylene glycol

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MW 4800 (PBG-4800) represent the two best ones for the removal of methyl tertiary
butyl ether (MTBE) from contaminated water. These are non-toxic, have extremely
low solubility in water, and have been shown to be stable as SeleXtrac™ membranes
in contact with pure water. Quantitatively, two main parameters are typically used tocharacterize a new liquid polymer with respect to its utility in the SeleXtrac™ membrane configuration. One is the partition coefficient; the other is the transport rate.
The partition coefficient Kp, defined as (MTBE in polymer/MTBE in water) at equilibrium, is a direct measure of the polymer’s affinity for extracting MTBE from water. For
both polymers at room temperature, the partition coefficient was found to be about 9,
which is quite good considering the high solubility of MTBE in water.
For measuring transport rates, we used our standard stirred glass cell with a flat
sheet membrane. This system allows
tight control of the membrane preparation and the operating conditions, as wellas accurate determination of the membrane area. The results are shown in
Figure C-10. It is clear that MTBE accumulated more rapidly in the strip solution
for the silicone membrane than for the
PBG one. Figure C-10 also shows the fitof a mathematical model for extracting
the overall mass transfer coefficient, Kf,from the concentration-time data. Kf is aquantitative measure of transport rate.
As can be seen, MTBE transport ratewith Si-350 is more than two times fasterthan that with PBG-4800 (Kf = 9.65 x 10-4
Figure C-10 Transport of MTBE across supported liquid membranes
cm/s and 4.24 x 10-4 cm/s, respectively).
HOLLOW FIBER MODULE TESTING With silicone selected as the liquid polymer
of choice for this application, we next studied the use of SeleXtrac™ membrane ina process for MTBE removal from contaminated water. A SeleXtrac™ membrane
hollow fiber membrane module (MiniKros® Sampler) whose small size and compact design makes it very convenient for laboratory testing was used. The hollow
fiber SeleXtrac™ membranes are of ultrafiltration type, made of polysulfone andhaving a 400,000 molecular weight cutoff. The characteristic dimensions of thissize module are shown in Table C-7.

Table C-7 Characteristics of Spectrum Modules Used

In order to assess the utility of SeleXtrac™
membranes for removal of MTBE fromwater, two membrane modules wereused. One was a standard, uncoated, i.e.
membrane pores empty; in the other, thepores were filled with Si-350 using a centrifugal method to apply the polymer tothe membrane. About 2.75 g of silicone
was loaded on the coated module.
Studies using hollow fiber membranes(HFM) under pervaporation indicate that
this is a viable option for removing MTBE
from an aqueous solution (26).

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The SeleXtrac™ membrane system was operated in a pervaporation mode, with a gas (nitrogen or air) sweeping on one side of the membrane and MTBE-containing water circulatingon the other side. The water circulating flow
rates were varied to determine the significanceof liquid film mass transfer resistance versus
the liquid membrane resistance. The gas
sweeping flow rate was deliberately set very
high to minimize the gas film resistance. Mostof the runs were made with the water circulating through the lumens of the fibers, which represents a more well-defined fluid flow situation.
A few runs were made with the water circulating through the shell side for comparison.
In a typical run, MTBE concentration in thewater was monitored as a function of time at a
certain circulating flow rate. A set of data for
one run is shown in Table C-8.

Table C-8 MTBE Removal with Silicone-Coated SeleXtrac™ Module

Figure C-11 MTBE transport with silicon coated SeleXtrac™ module

Table C-9 MiniKros® Sampler (PS 400K, 680 cm2)

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Figure C-11 shows the excellent data fit using asimple transport model. The transport rate
expressed as the magnitude of the overall
mass transfer coefficient, Kf, for a particular run
can be easily calculated from the slope of thefitted line.
Table C-9 summarizes all the results obtained
for the two modules, one uncoated (control
sample) and one coated (SeleXtrac™ test
model), with water through the lumen. It canbe seen that the transport rates clearly
increased at higher water circulating rates, fromabout 4 x 10-4 cm/s at 200 ml/min to 6 x 10-4
cm/s at 1000 ml/min. This typically means thatthe liquid-side mass transfer resistance (assupposed to membrane resistance) is important in this flow regime and needs to be considered during scaleup. The transport rates started to level off above 500 ml/min, corresponding
to a linear velocity of about 15 cm/s and a
Reynolds number of about 80.
It is interesting that the uncoated module actually exhibited lower transport rates (2.2-2.6 x
10-4 cm/s) than the SeleXtrac™ module. Thiswas unexpected since the presence of the liquid polymer instead of just open pores should
retard the transport of MTBE through the membrane, which can occur by volatilization. We
suspected that water vapor may have condensed inside the open pores of the un-coated

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fibers, effectively creating a water film that MTBE had to diffuse through. Actually,
based on an estimated MTBE diffusivity in water of 8.67 x 10-6 cm2/s and the
known membrane physical characteristics, the resulting mass transfer coefficientthrough a water-filled membrane would be 2.7 x 10-4 cm/s, which is quite close to
the measured values. Incidentally, we also observed a significant amount of moisture collected in the tubing on the gas side during the runs with the un-coatedmodule, but none with the coated ones. Thus the liquid polymer coating results ina double benefit; it not only forms a physical barrier to ensure separation between
the contaminated water and the other side, but also unexpectedly enhances theoverall transport rate by preventing possible condensation of water vapor in themembrane pores.
We have also tested earlier with two other modules in which water was circulatingon the shell side and gas sweeping through the lumen. At the highest flow of1000 ml/min, the obtained mass transfer rate ranges from 5.7 to 7.5 x 10-4 cm/s,
which is comparable to the 6.1 x 10-4 cm/s shown in Table C-9. For well-engineered designs, such as radial flow or cross flow, the mass transfer on the shellside can be better than in the lumen. However, with the simple shell and tube
configuration, the mass transfer on the shell side tends to drop significantly uponscaleup due to poor contact between the circulating liquid and the large numberof fibers in big modules. This aspect will need to be carefully considered in themodule design for the eventual large-scale operation.
A follow-up study by an independent investigator (Keller, University of California at
Santa Barbara) evaluated the SeleXtrac™ membrane process with a Spectrum
pilot module. The membranes used in this study were about 31cm (12.3 in.) longand the module has an overall external diameter of about 6.5 cm (2.5 in.). Basedon the information provided by Spectrum Laboratories, Inc., each module contained 5,414 polysulfone (PS) fibers, with a molecular weight cutoff of 400 kD. The
outside surface area is 3.5 m2, while the inside surface area is 2.7 m2. The fibers
were arranged along the length of the HFM module and were coated with Si-350.
The experimental setup was similar to that described above, with the MTBE solution flowing upward through the lumen side of the module and the sweep gas flowing counter-currently downward on the shell side.
An aqueous solution containing ~50-100 ppm MTBE was used as the feed solution
and a pressurized or vacuum sweep gas passed through the shell side. Liquidflow rates were in the range from 0.5 to 8 L/min. The results indicate that MTBE
can be readily removed from the solution using these membranes, and that Kf is afunction of liquid and gas flow rates, as well as the overall operating pressure.
The highest Kf (3.86 x 10-4 cm/s) was obtained under high vacuum conditions(0.23 atm), compared to 2.3-3.3. x 10-4 cm/s for the pressurized sweep gas or low
vacuum conditions (0.73 atm).

Separation of Similar Organics
The SeleXtrac™ membranes can be used to not only recover but also separateorganic compounds based on differences in their physical properties such ashydrophobicity, hydrogen-bonding capability, and degree of dissociation as indicated by their pKa’s. The example here demonstrates the separation achieved
between p-nitrophenol (pKa=7.1) and benzoic acid (pKa=4.25) based on the difference in the pKa’s by varying the solution pH. The results are shown in Table C10. As pure component at pH several units below their pKa’s, these compounds

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pass through the membrane at fairly high rates. As a mixture at a solution pH of
5.7, PNP exists in solution mainly in the non-ionized form whereas benzoic acid ispractically all ionized. PNP was found in this case to readily pass through themembrane, as expected, resulting in about 99% PNP removal from the feed solution overnight. Benzoic acid, however, was virtually blocked by the membrane:
overnight only trace amount of benzoic acid (<1% of feed concentration) was
detected in the strip solution. It should
be noted that this remarkable separation
was accomplished in the presence ofvery high ionic strength (20wt% KCl),
which would render ion-exchange methods practically inoperable for this separation. To further demonstrate the effectof pH and ionization on the transport
rates, the same experiment was repeated but this time at solution pH of 2.6,
which is below both pKa’s. Overnight,
both PNP and benzoic acid were virtually gone from the feed, having beentransported completely to the strip side,and the measured transport rates for thetwo as a mixture were fairly comparable
Table C-10 Separation of p-Nitrophenol and Benzoic Acidto those obtained with pure components.

Flavor Recovery/Modification
General Approaches
Based on the transport characteristics of Spectrum’s SeleXtrac™ technology, we
identified three general ways that the technology can be utilized in thebeverage/flavor area:
A. Selective transport of complex flavor profiles directly from a natural source(such as peels slurry or extract) into the liquid of choice (such as a finished beverage) across the membrane, thus bypassing many intermediate steps while preserving the natural complex flavor. We have demonstrated this in the laboratory
with peels from lemon and grapefruit, as well as with wines and grape juice.
B. Selective removal of objectionable components from a finished product or flavor
source. This applies to the removal of the vegetative flavor in O’Doul’s discussed
on page 101.
C. A tool for product development by food/beverage/flavor companies taking
advantage of the membrane’s capability to impart complex flavors into a target
solution without carrying along other components like sugars, proteins, ions, etc.
The next section describes the work done on improving the flavor/taste of lowalcohol beers using approaches (a) and (b).

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Low Alcohol Beer Applications
As mentioned above, polyglycol SeleXtrac™ membranes can transport esters very
rapidly, much faster than ethanol. Other membranes that have similar properties
are SeleXtrac™ silicone (polydimethylsiloxane) and dense silicone membranes,
and asymmetric hydrophobic pervaporation membranes. With an appropriate
pickup fluid (strip) on one side of the membrane in contact with a regular or concentrated beer on the other side, sufficient flavor components could pass throughthe membrane while little ethanol does, resulting in a strip solution that has fullbeer flavor but with little alcohol, essentially the desired compositions for a low
alcohol beer (<0.4% ethanol).
BENCH-SCALE TESTS Stirred cells with flat sheet membranes were used for
rapid feasibility test of the approach and evaluation of key operating parameters
affecting the process. These include membrane type, temperature and feed beer
type. For these feasibility tests the concentrations of key flavor compounds (e.g.,aldehydes, esters and higher alcohols) in the treated solutions were used to judge
the effects of various operating parameters. To be qualified as low-alcohol (ornon-alcohol) beers, the final ethanol concentration has to be below 0.5%.
EFFECTS OF LIQUID MEMBRANE TYPE The SeleXtrac™ membranes used inthis test were polypropyleneglycol (PPG, MW 4000), polybutyleneglycol (PBG, MW4800) and silicone (polydimethylsiloxane, MW 12500). Michelob Light beer wasused as the sample feed solution and HPLC grade water was used on the strip(permeate) side. Representative compounds of three types of organic moleculesin the solutions were analyzed to determine their relative transport rates throughthe membranes: alcohols (C2 through C5), esters (ethyl acetate and amyl
acetate), and an aldehyde (acetaldehyde). Table C-11 shows the results as thepermeate to feed ratios for these compounds after 24 hr operation. A value of 1
indicates equal concentration between permeate and feed, i.e., equilibrium was reached. The
lower the ratio for a compound, the slower its
transport across the membrane.

Table C-11 Beer extraction with various SeleXtrac™ membranes.

It’s clear that the two esters (ethyl acetate andisoamyl acetate) permeate very rapidly throughthese membranes, reaching equilibrium in lessthan 24 hr. Of the alcohols, the relative permeation rates are: C5 > C4 > C3 > C2 (i.e. amyl
alcohol > butyl alcohol > propanol > EtOH).
Only the amyl alcohol appears to have reachedequilibrium after 24 hours. Acetaldehyde permeation rate lies between those of ethanol andpropanol. These results show that the polyglycols and silicone membranes tested could be
used for separating esters from ethanol.

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EFFECT OF TEMPERATURE The relative transport rates of the organic compounds through the SeleXtrac™
(PBG-4800/Celgard® 2500) membrane
as a function temperature were studied. Table C-12 shows thepermeate/feed ratios for these compounds at 21°C and 4°C after 24 hr
exposure. The lower temperatureappears to enhance the selectivity ofother components over ethanol. The
drawback of low temperature operations would be slow transport rates.
PILOT-SCALE OPERATIONS To
enable the preparation of sufficientbeer solution for taste testing, a largepilot unit was installed and operated
at the A-B pilot brewery. The initial
Table C-12 Permeate to feed ratios for key compounds in membrane-treated beers
approach was to impart flavor compoat two temperatures.
nents from regular beers into O’Doul’s
to improve its taste and aroma. Thiswas done by contacting the O’Doul’s with a regular beer separated by the
SeleXtrac™ membrane. The operation was done in a batch mode, i.e., the twosolutions were circulated across the membrane for various times. The second
approach was to remove the objected “beany” or vegetative flavor from the
O’Doul’s. This was done by using just water on the other side of the membrane topick up the bad flavor. In this case, the O’Doul’s was fed continuously through themembrane module in a single pass while the water was circulated.
FLAVOR ADDITION APPROACH Several pilot runs were made in a second campaign in which the air introduction was minimized and several types of feed beer
were used. Figure C-12 shows typicalconcentrations for various key components in the treated O’Doul’s as afunction of run time. As shown, levelsof ester compounds and higher alcohols increased significantly while thatof ethanol remained low. Based on asensory testing with an expert sevenperson panel, the treatment was
found to add top notes to the O’Doul’s.
However, while one member gave themembrane-treated sample the highest
score in preference tasting, the rest
gave it rather poor scores in comparison to the control O’Doul’s. Thus
there was not a consensus whether
the flavor-enhanced beer would be abetter product, probably due to thecomplex interactions between the new
Figure C-12 Transport of key beer components from an English Ale feed beerflavors and those already in theacross a treated hollow fiber membrane into O’Doul’s® pickup solution
O’Doul’s.

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FLAVOR REMOVAL APPROACH The above approach had focused on improving
O’Doul’s flavor by selectively adding more “beery” flavor from a regular beer.
However, it was also noticed that the objectionable “beany” flavor of O’Doul’s wasgone from the membrane-treated products. Apparently, while flavors were movingfrom the feed source across the membrane into the O’Doul’s, there was simultaneous movement of the unappealing vegetative flavor in the opposite direction.
To test this hypothesis, the membrane process was operated with O’Doul’s on one
side and only carbonated water on the other side. With a feed solution pumped
through the shell side of the module at about 500 ml/min and carbonated water as
the pick-up fluid circulated through the lumen side of the module at a rate of 3
liters/min, a tasting panel found no beany note present in the treated O’Doul’s.
When the feed flow rate was raised to 1000 ml/min, however, the panel did detectsome residual beany note in the treated O’Doul’s. This simple process resulted ina significantly preferred O’Doul’s with strong consensus from the panel.
Additional pilot-scale testing of various liquid polymers led to polybutylene glycol(PBG-4800) as the best for this application in terms of membrane stability in thecold room and effectiveness of the beany flavor removal. Unfortunately, this polymer has not been approved by the Food and Drug Administration for use in foodprocessing. A PBG’s close cousin (polypropylene glycol) and one silicone liquid(200 Fluid from Dow Corning) are approved for use in food processing, but PPG is
not stable in contact with cold water (high solubility) and silicone is not effective
for the vegetative flavor removal. Consequently, obtaining FDA approval of PBG4800 for use in low-alcohol beer processing appears to be the only path for commercializing the membrane technology for this application.
PROCESS DESIGN AND COST CONSIDERATIONS Cost estimates for the
SeleXtrac™ membrane process have not been done. One piece of relevant information is that the membrane cost for 50 modules is about $500,000 ($10,000 per
module). So if the modules lasted only one year instead of the typical two to five
years for this type of operation, the membrane cost alone would amount to an
additional $1 per barrel of the beer produced. According to our A-B collaborators,
an added cost of 1 to 2 dollars per barrel would be preferable, while $5/barrel
would be too high. While we did not pressure test this criterion, it certainly indicates that the membrane process for this application can be economically viable.
One major upside is that the membrane area required could be much lower thanestimated based on the beer flow rate of 300 ml/min. Our modeling calculations
and data obtained with other systems have shown that, at this low flow rate
through the shell side, the liquid film mass transfer is severely limiting the overallrate. The correlation obtained for liquid transport on the shell side of HoechstCelanese radial flow modules (16) shows that the mass transfer coefficient varies
to the 0.4 power of liquid flow rate: NSh = 1.28 NRe0.4Sc1/3. Thus, the transport
rate can increase several fold by raising the flow rate from 300 ml/min to, say, 3
L/min or higher and stacking the modules in series to obtain the needed residence
time.

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Analytical Applications
General Methodology
To date, the analytical applications of SeleXtrac™ membrane technology are muchless developed than process applications. However, based on the capability of
SeleXtrac™ technology to selectively extract organic compounds from a complex
mixture, it is not difficult to come up with many interesting and enabling analyticalapplications. The availability of many natural as well as synthetic polymeric liquidsof diverse extractive properties and the ease of membrane preparation makeSeleXtrac™ technology particularly attractive here. The general approach is to
expose the complex mixture to the membrane, which extracts the compound ofinterest that can then be analyzed without the interference of the complex matrix.
The analysis can be direct via a detector on the other side of the membrane, or
through a sweeping fluid (mobile phase) that carries the extracted compound to
the appropriate detector. Two specific examples will be discussed below, one foreach type of detection.
Another potential usage of SeleXtrac™ membrane technology for analytical purposes is sample preparation. One could utilize the SeleXtrac™ technology to
extract a particular organic compound from a complex mixture and concentrate itin the liquid polymer. The support matrix in this case can be membranes but
could also be porous materials, porous beads or tubing. This mode of application
is similar to the popular solid phase extraction (SPE) method utilized by analyticalchemists. While not as robust as (SPE), one advantage of SeleXtrac™ membrane
technology for certain applications is that the liquid polymer along with the dissolved organic compound could be completely removed from the support matrix
with an appropriate solvent, thus accomplishing 100% recovery rapidly.

Direct Analysis in Complex Mixture
Detection of Phenol in Urine
This example covers the application in which small molecules are extracted rapidly from a complex mixture, possibly in concentrated form, so very low detection
limits can be attained without the intermediate time-consuming sample preparation.
The sample can be water, urine, blood plasma, or any liquid compatible with the liquid
membrane. Contact time and pH of thesample can be adjusted to maximize theextraction of the organic compound of interest into the liquid membrane. Since ioniccompounds will not be extracted by the liquid membrane, this method is effective forextracting metabolites from urine or bloodplasma leaving proteins and salts behind.
The injection liquid on the other side of themembrane can also be pH adjusted to maximize the extraction of the organic compound from the liquid membrane. This
Figure C-13 Schematic diagram for direct analysis of a trace chemical
added separation increases the overallcomponent in a complex mixture
selectivity of the method.

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In the determination of phenol in urine as an example was demonstrated in principle using the stirred cell described in the experimental section. Urine solution at pH
neutral or lower was in contact with a PPG-polyproylene liquid membrane and an
alkaline solution (either NaOH or an amine buffered solution) was on the other sideof the membrane. The experiment showed that phenol was selectively extracted
into the alkaline solution without the accompanying proteins and salts.
Phenol analysis was then done either with simple spectrometriclight absorption or using HPLC. For an actual analytical setup,10 ml of urine could be passing through a liquid membrane loop. The phenol getting into the liquid membrane
can then be extracted into an HPLC 0.1 ml injection
loop by the alkaline buffered solution, thus accomplishing both sample cleanup and concentration.

Figure C-14 Schematic diagram of HPLC setup for analysis with aliquid membrane sample loop

Over the past few years HPLC-mass spectrometry
interface using electrospray techniques has
become a powerful tool for combining the identification capability of mass spectrometry with the separating capability of HPLC. This has enabled analytical chemists to directly identify unknown compounds present in complex mixtures. An important
limitation of this approach is the compatibility of the
HPLC mobile phase with the electrospray process.
The SeleXtrac™ membrane technology method
could potentially solve some of these problems
since the injection solution can have any buffer thatis compatible with the electrospray process.

Field Detection of a Target Enzyme
In this example the presence of a particular enzyme in a complex mixture is detected by the action of the enzyme on an added substrate that releases a coloredproduct. Normally the color generated will be either masked by the matrix or difficult to measure accurately. SeleXtrac™ membrane technology can be used to
extract the colored compound from the matrix enabling more accurate detection.

Figure C-15 Schematic diagram for detection of potatin directlyin crude plant samples

Figure C-15 shows the schematic diagram for thedetection of potatin, a tyrosine kinase in plants. A
fiber optic probe is inserted in the lumen of a hollow fiber liquid membrane, with sample inlet andoutlet capillaries at each end of the hollow fiber for
introducing the strip solution. A chromogenic
enzyme substrate such as peptide-paranitroaniline(P-PNA) is typically added to a field sample todetect the presence of potatin that would release
the colored PNA from the peptide. The hollow fiber
membrane, polypropylene impregnated with PPG,
will extract PNA from the sample solution surrounding the membrane. The fiber optic probe encasedin the hollow fiber can be used to detect and quantify the amount of PNA present in the strip solution,
which correlates with the original amount of PNA
released in the sample.

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Biomedical Applications
There has not been any experimental work done in this area, even though thepotential for various biomedical applications of SeleXtrac™ membrane technology
has been recognized and discussed with several technical groups. The objectiveof this section is to briefly summarize the main thoughts for future reference.

In-situ Monitoring
Similar to the use of SeleXtrac™ for analytical applications, the possibility of
extracting specific compound from a complex mixture to the other side of themembrane for detection leads to the idea of in-situ monitoring of the target compounds in animals or humans. One can envision a hollow fiber membrane device
not unlike kidney dialysis unit but whose fibers are impregnated with the appropriate liquid polymer. Blood or urine can circulate through the lumens of the fibers
and an appropriate strip solution on the shell side of the fibers will bring thepicked-up target compound(s) to a detector for the analysis. Thus the detection is
not intrusive and can be both quantitative and continuous. The possibility ofdetecting phenol present in urine has already discussed in the analytical section.
Similarly, phenol or other metabolites of drugs or drugs themselves in the bloodstream could, in principle, be monitored continuously after injection or taken orally.

Detoxification
Whereas kidney dialysis simply allows the rather nonselective removal of ions andsmall molecules from the blood stream, SeleXtrac™ devices could be used to
selectively extract larger and more complex organic compounds that are considered toxins in the blood stream, thus accomplishing the detoxification objective
using the same procedure. In tandem with kidney dialysis, SeleXtrac™ membranes can lead to treatment of diseases that is not currently possible.

Controlled Delivery
The controlled delivery idea comes from the recognition that the liquid polymer
supported in a solid matrix can store certain drug compounds that release slowly
into the surrounding blood or tissues. Or the liquid membrane can act as a selective barrier that meters the drug at a controlled rate to the body.

Acknowledgement
The development of the SeleXtrac™ membrane technology received great intellectual as well as financial support from many organizations and the author’s colleagues.
In particular, the author would like to acknowledge his colleagues: Drs PhilipBrodsky and Philip Needleman for their enthusiastic management support, P. Wayne
Sheridan for his first-rate technical support and collaboration throughout the development effort, and Drs. Robert Orth and Leanna Levine for their contributions to theanalytical applications development. Great appreciation is extended to the late Dr.
Alan Michaels for his invaluable intellectual contribution to the discovery, understanding, and development of this technology. Lastly, the interest and technical collaboration from Spectrum Laboratories have made a difference in solving numerous
practical problems associated with the commercialization of this technology.

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LITERATURE CITED C-107

Literature Cited
1. Hestekin, J. A., et al., “Membranes for Treatment of Hazardous Wastewater,” in“Encyclopedia of Environmental Analysis and Remediation,” R. A. Meyrs, ed., John Wiley &Sons, Inc., New York, pp. 2684-2708 (1998).
2. Paul, D. H., “A review of membrane water treatment technologies,” Ultrapure Water,
14(3), pp. 39-40 (1997)
3. Noble, R. D. and S. A. Sterns, eds. “Membrane Separations Technology: Principles andApplications,” Elsevier, New York (1995).
4. Wijmans, J. G., et al., “Treatment of Organic-Contaminated Wastewater Streams by
Pervaporation,” Environ. Progress, 9, pp. 262-268 (1990).
5. Ho, S. V., et al., “Supported Polymeric Liquid Membranes for Removing Organics from
Aqueous Solutions - I. Transport Characteristics of Polyglycol Liquid Membranes,” J Mem.
Sci, 112/1, pp. 13-27, (1996).
6. Ho , S. V., “SeleXtrac™ Membrane and Separation Process Employing Same,” US Patent
5,507,949 (1996).
7. Misra, B. M. and J.S. Gill, “Supported Liquid membranes in metal separations,” in ACS
Symp. Ser. “Chemical Separations with Liquid Membranes,” 642, pp. 361-375 (1996).
8. Chiarizia, R., “Application of SeleXtrac™ membranes for removal of nitrate,technetium(VII) and chromium(VI) from groundwater,” J. Mem. Sci., 55(1-2), pp. 39-64(1991).
9. Schaefer, A. and M.M. Hossain, “Extraction of organic acids from kiwi fruit juice using a
SeleXtrac™ membrane process,” Bioprocess Eng., 16(1), pp. 25-33 (1996)
10. Molinari, R., et al., “Coupled transport of amino acids through a SeleXtrac™ membrane,” J. Mem. Sci., 73(2-3), pp. 203-215 (1992).
11. Urtiaga, A.M., et al., “Supported liquid membrane for the separation-concentration ofphenol,” Ind. Eng. Chem. Res., 31(7), pp. 1745-1753 & 31(3), pp. 877-886 (1992).
12. Danesi, P.R., et al., “Lifetime of Supported Liquid Membranes: The Influence ofInterfacial Properties, Chemical Composition and Water Transport on the Long-Term Stabilityof the Membranes,” J. Mem. Sci., 31(2-3), pp. 117-45 (1987).
13. Fabiani, C., et al., “Degradation of supported liquid membranes under an osmotic pressure gradient,” J. Mem. Sci., 30, pp. 97-104 (1987).
14. Klein, E., J. K. Smith, R. E. C. Weaver, R. P. Wendt, and Shyamkant V. Desai, “Solute
separations from water by dialysis. II. Separation of phenol by downstream conjugation,”
Separation Science, 8(5), (1973) 585 .
15. Sengupta, A., R. Basu, and K. K. Sirkar, Separation of solutes from aqueous solutions
by contained liquid membranes, AIChE J., 34 (1988) 1698 .
16. Harriott, P., and S. V. Ho, “Mass Transfer Analysis for a Supported Polymeric Liquid
Membrane,” J. Mem. Sci., 135, pp. 55-63 (1997).
17. Ho, S.V., “A New Membrane Process for Recovering Organics from Aqueous
Wastes,”Industrial Environmental Chemistry, eds. D.T. Sawyer; A.E. Martell, 229-245 (1992).

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C-108 THE ABCs OF POLYMERIC MEMBRANE EXTRACTION

18. Ho, S. V., P. Wayne Sheridan, and E. Krupetsky “Supported Polymeric Liquid
Membranes for Removing Organics from Aqueous Solutions. I. Transport Characteristics of
Polyglycol Liquid Membranes” Journal of Membrane Science, 112/1, 13-27 (1996).
19. Tompkins, C. J., A.S. Michaels and S.W. Peretti, “Removal of p-nitrophenol from aqueous solution by membrane-supported solvent extraction,” J. Membrane Sci., 75 (1992) 277292.
20. W. L. Gore & Associates, Inc,. Publication Literature (1990).
21. Hoechst Celanese Product Literature, 1991.
22. Reid, R.C., et al., “The Properties of Gases and Liquids”, McGraw-Hill Book Company,
New York, pp. 573-576 & 587-588 (1977).
23. Bierwagen, B and Keller, AA (2001). Measurement of Henry’s Law Constant for Methyltert-butyl-ether (MTBE) using Solid-Phase Microextraction. J. Soc. Environ. Toxicol. &Chem., 20(8).
24. Keller, AA, Sandall, OC, Rinker, RG, Mitani, MM, Bierwagen, B and Snodgrass, MJ(1998). Cost and Performance Evaluation of Treatment Technologies for MTBEContaminated Water. UC Toxic Substances Research & Teaching Program, UC Davis, CA.
Report to the Governor of California.
25. Keller, AA, Sandall, OC, Rinker, RG, Mitani, MM, Bierwagen, B and Snodgrass, MJ(2000). An Evaluation of Physicochemical Treatment Technologies for Water Contaminatedwith MTBE. Ground Water Monitoring and Remediation, 20(3):114-134.
26. Keller, AA and Bierwagen, B (2001). Hydrophobic Hollow Fiber Membranes for treating
MTBE-contaminated water. Environmental Science and Technology, in press.
27. Miller, B., CELGARD® LLC, price quote May 27, 1998.

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Section D

The ABCs Of Cell Culture
Bioreactor Design
Introduction
Most pharmaceutical fermentation products are derived from bacteria and fungigrown in stirred tank bioreactors ranging in size up to tens of thousands of liters.
Most of these older processes employ microbial cells.
However, many products can be produced by mammalian cells. Mammalian cells
are significantly different and much more difficult to grow than microbial cells.
Some of the disadvantages of using mammalian cells include: slow growth, sensitivity to shear and interfacial forces, complex nutritional requirements and anchorage-dependency.
With the advent of recombinant DNA techniques and the emergence of thebiotechnology industry, new technology has had to be developed to meet therequirements of these new processes.
The most successful developments have been the manufacture of monoclonal
antibodies by in vitro mammalian cell culture using hollow fiber membrane modules.

Monoclonal Antibodies
All mammals produce antibodies as part of their immune response system.
Antibodies are also called immunoglobulins (Igs). They are large glycoprotein
molecules composed of polypeptide chains linked by disulfide bonds. Five maintypes of circulating antibodies are known: IgG, IgA, IgM, IgD, and IgE.
IgGs are the most abundant immunoglobins in human serum. They provide
defense against bacteria, viruses, and bacterial toxins. The molecular weights of
IgGs are about 150,000 Daltons.
Monoclonal antibodies are produced commercially in large volume bioreactors.
The major uses of monoclonal antibodies are for:in vivo diagnostics to detect human and animal health problems; contamination offoods, drugs, or water; and the presence of plant pathogens
Therapeutic drugs and drug delivery agents
Purification of high-value molecules by affinity chromatography
Miscellaneous diagnostics or catalysts

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Bioreactors
A large number of technologies and types of bioreactors have been developed toaddress the problems associated with growing mammalian cells. These include
modified stirred tanks (including airlift); porous and nonporous microcarrier materials; entrapment by microencapsulation techniques; fluidized beds; porous ceramic
matrices and membrane bioreactors. All have been used in applied research andprocess development and some have been scaled-up for production.
As discussed above, with the advent of recombinant DNA techniques and theemergence of the biotechnology industry, it was assumed that production of the
new class of pharmaceuticals would use these same bioreactors. When the firstrecombinant products were produced in bacteria, it was discovered that many of

Figure D-1 Biopharmaceutical Development Steps

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BIOREACTOR DESIGN D-111

them were not biologically active. This has led to the development of new technology for mammalian cells.
Bioreactors can be generally classified into two categories: batch systems andexchange systems.

Batch Bioreactor Systems
The simplest form of bioreactor is the single batch-fed type in which cells are inoculated into a growth chamber in a fixed volume of culture medium. The cells areallowed to expand over time to a desired Ievel and then the cells or cell product(s)are harvested. With single batch fed systems, there is no culture medium
exchange from the start-up to the termination of the culture.
The single batch fed bioreactor is usually simple to operate, and because of lessmanipulation steps poses relatively low risk from contamination. However, themethod often gives less than optimal cell expansion due to nutrient deprivation,and the accumulation of inhibitory catabolic waste products, such as ammonium
ion, lactic acid and other growth inhibitors. Often there is inadequate oxygenationand pH control, which adversely affect the quality and quantity of harvested cells.

Exchange Bioreactor Systems
Another type of bioreactor operation is one in which nutrients consumed by cells
as they expand are replenished by the exchange of nutrient-depleted culture
medium with fresh culture medium. Medium exchange may be accomplished byeither sequential batch exchange or continuously.
For both the batch and continuous exchange modes, medium exchange is best performed when the growing cells are confined in a small compartment of the system,with the bulk medium perfused either unidirectional or by recirculation from a large
medium reservoir. The restriction of cells to a small volume of medium, but allowing
them access to the total nutrient available in a large volume of perfusing culture
medium in the bioreactor is best achieved by the use of semipermeable membranes. A greatly simplified schematic of such a system is shown in Figure D-2.
Since operation in the continuous mode allows the continuous diffusion or bulk
transfer of medium and products between the perfusing medium compartment
and the cell compartment, the efficiencyof exchange is highest when there is
maximum membrane surface area perunit volume across which medium andproducts can flow. Referring to figure D2, cells are gently pumped through theinside diameter of the hollow fiber membrane tubes. Cells are retained in thelumen of the membranes to be recirculated back to the reservoir. Spent media,
secreted proteins and unwanted
metabolites permeate the membrane
structure and enter the shell side of themodule to be harvested. As media is
Figure D-2 Schematic of exchange type bioreactor system using Spectrum’s
depleted, fresh media is added to mainCellFlo Module
tain bioreactor volume.

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Bioreactor Design Considerations
The main components of a continuous exchange system using hollow fiber membrane modules include the reservoir, pumps, valves, the gas exchanger and themodule.

The Hollow Fiber Module
Several factors must be considered in the design of the membrane module.
These include:
The pore size of the membrane
Other physical characteristics of the membrane, such as lumen diameter, length, etc.
The ability to support attachment of anchorage-dependent cells
Ability to sterilize the module
Biocompatibility of the materials used

The latter four characteristics above areestablished in the basic design of themodule. However, a variety of membrane pore sizes are available to fit theneeds of the user.

Figure D-3 Schematic of a hollow fiber membrane showing cross flow filtration

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As shown in figure D-3, the medium flows
through the lumen of the hollow fibers that
separate the cells from the medium.
N utrients and oxygen are supplied fromthe perfusing medium via diffusion and/or
bulk transfer through the wall of the hollow
fibers. Products produced by the cells
traverse in the opposite direction. The
membrane wall material and its porosity
will therefore greatly influence how well
cells expand in the module.

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The Gas Exchanger
An adequate supply of oxygen is critical for proper growth and productivity of
mammalian cells. Most commonly, commercial production of products from mammalian cells is carried out in stirred tanks that were originally designed to support
bacteria and yeast. Oxygen delivery to these stirred tanks is usually accomplished by sparging directly into the liquid media. The combination of vigorous
stirring and gas sparging will deliver adequate levels of oxygen to bacteria andyeast.
Mammalian cells, however, do not have a cell wall and usually cannot withstandthe same levels of stirring and sparging. Also, serious problems with foaming canoccur in sparged cultures containing serum or high levels of proteins.
In addition oxygen demand increases significantly in perfusion mammalian cell
culture over that for bacteria and yeast.
The Spectrum CellGas™ module is designed as an internal gas exchange device
to be used for increased oxygen demand and to eliminate foaming and shear
related problems characteristic of sparging.
CellGas™ is a modified hollow fiber module without the “shell” of the standard hollow fiber module. When mounted properly within the bioreactor, it allows for maximum exposure of the gas exchange surface of the hollow fibers to the liquid contents of the bioreactor.
A more detailed description of CellGas™ is included in a later section.

The Pumps
The pumping device, other than its output capacity, must generate very low heat,yield no leachables and be autoclavable in the parts exposed to medium or product.

The Medium Reservoir and Tubing
The most important considerations regarding the reservoir and tubing is that they
be constructed of material that does not yield leachables into the culture medium
and are autoclavable.

Sterilization
Sterilization by autoclaving is generally preferred over other methods. Howevergas sterilization (ethylene oxide) or irradiation may be used.

Conversion of Existing Batch Systems to Continuous Perfusion Systems
The long history of pharmaceutical production in fermenters and a lack of bioreactors that are custom designed for mammalian cells, has led to interest in converting existing stirred-tank bacterial fermenters into mammalian cell bioreactors.
Most microbial fermenters are designed for batch operation. Batch operation of a
microbial fermenter is economical due to high cell densities (>10 9 cells/ml), rapid
doubling times (apx. 20 min for many bacteria) and to the hardy nature of thecells, which allows the use of aggressive agitation and gassing. Typical microbial
batch operations last 5 to 10 days. In this short period of time, vast quantities of
product can be produced.

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Mammalian cells, on the other hand, grow to densities 10-1000 times lower thanbacterial cells, even under optimal conditions. They have doubling times in therange of 18-36 hours, much more complex nutritional requirements than microbial
cells and they are far more sensitive to minor perturbations in their environment. A
mammalian cell culture operated in a 5 to 10 day batch mode will reach reasonable densities only near the end of the run, so product yields are generally low.
Variations of batch operation (such as fed-batch or semi-continuous batch) can
improve the run length, cell density and productivity, but are still far from optimal.
Therefore, optimization of bioreactor designs and culture processes for mammalian cells has become a high priority within the biopharmaceutical industry.
Optimization efforts share common goals: maximizing cell density; cell viability;
productivity per cell and culture length. To achieve these goals, all aspects of theculture process must be examined and carefully controlled. Factors to be considered include: cell line construction; culture media formulations; feeding strategies;
bioreactor system design and process control strategies.
There are three compelling reasons to convert bacterial fermenters into mammalian cell bioreactors:
There is a long history of use of stirred tanks and their design and operation are
well understood.
There are a large number of fermenters being used for bacterial production thatcan be converted to mammalian cell production, resulting in enormous capital
equipment savings.
Many custom designed mammalian cell culture bioreactors have been reported as
being non-scalable for use as production systems.

Modifications to existing fermenter designs to convert them to mammalian cellbioreactors include tank design, impeller design, impeller drive design, gas transfer system design and pH and DO controller designs. In addition to hardware
designs, changes sometimes are made to the culture process itself.
Optimization of mammalian cell production requires a high degree of control overthe extracellular environment and the ability to run batches for several weeks.
Control of the extracellular environment is possible with continuous perfusion culture. In perfusion culture, fresh media is continuously delivered to the cells andwaste products are removed. Under these conditions mammalian cells grow todensities as much as ten-fold higher than in batch or fed-batch culture, leading toproportional increases in production.
The higher cell densities also result in reduced serum dependence, thus lowering
operating and purification costs.

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Available Options for Conversion
to Perfusion Operation
A chemostat-like environment can be achieved by simply pumping the contents ofthe bioreactor to a harvest bottle while also feeding, however this techniqueremoves cells and dilutes the culture, severely limiting the cell density and thus,
total production. The optimal continuous perfusion process is one where the cells
are retained within the bioreactor, while fresh media is added and waste media isremoved, allowing higher cell densities and production.
Retention of cells within the bioreactor requires a separation process of some type.
Methods for converting fermenters to continuous perfusion bioreactors include theuse of disc filters, spin filters, flat sheet filters and hollow fiber filters. Due to thecell density and the presence of cell debris, filters used for continuous perfusion
clog or foul after a period of time. As a result, in order to maintain continuous perfusion, the filter must be replaceable while the system is operating.
A perfusion filtration system can be mounted inside or outside the bioreactor.
Spin filters and hollow fiber filters have the proper dimensional characteristics to fit
within most bioreactors. Flat sheet and hollow fiber filters can be mounted outside
the bioreactor and connected to the bioreactor via an external recirculation loop.
There are advantages and disadvantages to both types of setups. Placement of
the filter inside the bioreactor eliminates the need for pumps to move the bioreactor contents through the filter and return them to the bioreactor. The disadvantageof this setup is that there is no access to the filter once the culture is started. If
there are any problems with the filtration setup (such as clogging), the bioreactor
must be shut down, ending the production run. In addition, spin filters are specialized devices that require modifications to the impeller and headplate of the bioreactor that can require custom engineering.
External filtration setups on the other hand, connect to the bioreactor throughexisting ports and do not require specialized hardware. They do require that thecells be pumped through an external loop. The major advantage of an external
perfusion filter is it can be changed if it becomes clogged (or other problems
occur), allowing long-term continuous operation.
Optimal performance of a perfusion filtration system requires efficient retention ofcells within the bioreactor, a convenient filtration system that can be readilychanged when it becomes clogged and a membrane porosity sufficiently large toallow removal of the desired secreted product as part of the perfusion process.
Low product residence time minimizes exposure to proteases, reduces the potential for feedback inhibition and improves overall product stability. Of the various filtration options available, microporous hollow fiber filters best meet the requirements for optimal performance of a perfusion filtration system. They have a highfilter surface area to volume ratio, are compact, easily incorporated into a fluid
loop configuration and can be changed during a run if necessary. A filter with
0.2 um to 0.45 um pore size will allow high flux rates, large volume throughput andthe continuous harvest of spent media containing cell-free product. Since the harvest is cell free, the initial cell separation step is eliminated, thus, simplifying downstream purification. In addition, a continuous cell-free harvest is compatible withexisting continuous affinity purification techniques.

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CellFlo™ Technology for Cell Recycle Perfusion of Animal Cell Bioreactors
Spectrum has developed the CellFlo™ line of hollow fiber cell separators specifically for the conversion of stirred or airlift bioreactors to continuous
perfusion. CellFlo™ modules utilize large internal
diameter membranes, allowing recirculation ofcells without damage and reducing plugging withcell debris.

Process volumes > 10,000 liters.
Diameters: 18.5 to 70 mm
Total Lengths: 276 to 689 mm
Figure D-4 Schematic of CellFlo™ hollow fiber cross flow module
used for production scale process volumes andcontinuous external loop, cell-recycle perfusion ofdeep tank reactors.

Manufactured from non-cytotoxic USP XXI class VI
materials, Spectrum CellFlo™ modules are supplied non-pyrogenic by LAL testing. They can besterilized by autoclaving and are priced to be disposable.
Single use disposability eliminates the costs andrisks associated with cleaning and rinsing, simplifies validation and ensures consistent optimum
performance and protein passage.
CellFlo™ modules are available in a variety of surface areas from 800 cm2 to 3.3 m2.

Results Obtained Using CellFIo™ Hollow Fiber
Cell Separators for Converting Bioreactors to Continuous Perfusion
Recent comparisons of fed-batch versus continuous perfusion operation of a 40Lairlift bioreactor for the production of monoclonal antibodies showed several differences between the two modes of operation. Both the product concentration anddaily production increased 5-7 fold in perfusion culture (figures D-5 and D-6).
Cumulative production also increased proportionally (D-7). In perfusion culture,
the production per liter of media consumed was 25 fold higher than in batch culture, thus reducing
the overall media cost per gram of product produced (Figure D-8). In addition, the ability to produce 5-10 fold more product from the existingbioreactor reduced the length of the production
run required to produce a given quantity of product. Perfusion can also reduce capital equipment
costs by extending the capacity of existing bioreactors and reducing the size of new bioreactors.

Figure D-5 Comparison of antibody concentration in continuous
perfusion reactor vs. fed batch reactor

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Although there are a wide variety of different bioreactors being used for mammalian cell culture, thesetup and operation of a perfusion loop does not differ significantly from one bioreactor type to another.

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BIOREACTOR DESIGN D-117

Converting a Batch Bioreactor to Perfusion
Using CellFlo™ Cell Separators

Figure D-6 Comparison of antibody production in continuous
perfusion reactor vs. fed batch reactor

Figure D-7 Comparison of cumulative production incontinuous perfusion reactor vs. fed batch reactor

Virtually any stirred or airlift bioreactor can be converted to perfusion operation using CellFlo™ cell
separators. The conversion process and the materials used differ slightly from one reactor type toanother and with increasing bioreactor size. The
essential components required are three fluid metering pumps, a CellFlo™ cell separator, tubing, valvesand connectors. The hardware setup and connections differ somewhat between systems and with orwithout a steam source.
Although there are several different options for operation, a basic system is presented that illustrate thegeneral operation of the CellFlo™ conversion system.

Figure D-8 Efficiency of continuous perfusion reactor vs. fed batch
reactor in terms of production per liter of media consumed

Bioreactor with Steam Supply for Sterilization –The Basic System Setup
A basic system setup is illustrated in figure D-9. The essential components are
the bioreactor, tubing for recirculation, the CellFlo™ module, sanitary valves foraseptic connection of the loop to the bioreactor, a media feed line, a harvest line
and three pumps. The system illustrated is one of numerous possible setup configurations, depending upon the needs and desires of the end-user.
This setup was designed to meet GMP requirements and to simplify the setup andon-line changing of CellFlo™ modules.
The setup shown in figure D-9 provides maximum flexibility in the connection of
the perfusion loop and for changing of CellFlo™ modules during a continuous culture. As detailed in figure D-9, the dual valve sets at the outlet and inlet to thebioreactor permit sterile changing of the entire perfusion loop, including all tubing.
The dual valve sets at the inlet and outlet of the CellFlo™ module permit sterile
replacement of just the CellFlo™ module.

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D-118 THE ABCs OF CELL CULTURE

Figure D-9 Schematic of continuous perfusion reactor using the CellFlo™ module

Component Parts of the Basic Bioreactor System Design are listed below:
1. BIOREACTOR. Any stirred or airlift will do. Bioreactor size will affect the sizeof the tubing and the CellFlo™ module used but not other components.
2. DIAPHRAGM VALVE. Sanitary valve with access port for steam inlet.
3. SMALL DIAPHRAGM VALVE. Valve for steam inlet or outlet at the connection
point.
4. DIAPHRAGM VALVE. Second valve to serve as a sterile disconnection point
for the entire perfusion loop.
5. SMALL DIAPHRAGM VALVE. For steam inlet or outlet.
6. FLEXIBLE PUMP TUBING (suitable for extended operation).
7. FLOWMETER with digital readout (optional-to monitor the recirculation rate.)
8. DIAPHRAGM VALVE. For on-line sterile connection of replacement CellFlo™
Modules.
9. SMALL DIAPHRAGM VALVE. Steam inlet outlet for sterile connection.
10. DIAPHRAGM VALVE. Second valve for sterile connection of replacement
CellFlo™ Modules.
11. SMALL DIAPHRAGM VALVE. Second steam inlet outlet valve.

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BIOREACTOR DESIGN D-119

12. CELLFLO™ MODULE. The size of the CellFlo™ module will depend upon
the size of the bioreactor, the serum content and desired module life.
13. PRODUCT HARVEST LINE.
14. PRESSURE TRANSDUCER (optional). For monitoring transmembrane pressure. This transducer is useful for determining when the filter is fouling and needs
to be changed.
15. DIAPHRAGM VALVE. For sterile connection of the top end of the CellFlo™
module.
16. SMALL DIAPHRAGM VALVE. Steam inlet/outlet for sterile connection.
17. DIAPHRAGM VAIVE. Second valve for sterile connection of the top end of
the CelIFlo™ module.
18. SMALL DIAPHRAGM VALVE. Second steam inlet/outlet for sterile connection.
19. FLEXIBLE PUMP TUBING.
20. DIAPHRAGM VALVE. For sterile connection of the upper end (bioreactor
inlet) of the perfusion loop.
21. SMALL DIAPHRAGM VALVE. Steam inlet/outlet for sterile connection.
22. DIAPHRAGM VALVE. Second valve for sterile connection of perfusion loop.
23. SMALL DIAPHRAGM VALVE. Second steam inlet/outlet for sterile
connection.
24. MEDIA INLET TUBING LINE.

CellGas™ Module
The CellGas™ module is designed as an internal gas exchange device to be used
when traditional means of oxygenation are insufficient and/or cause foaming or
shear related problems. As pictured in figure D-10, CellGas™ is a modified hollow
fiber module without the “shell” of the standard hollow fiber module. When mounted within the bioreactor, it allows for maximum exposure of the gas exchange surface of the hollow fibers to the liquid contents of the bioreactor.
The efficiency of the CellGas™ module is determined by the gas concentrations at
and immediate to-the gas exchange surface on the outside of the hollow fibers.
The higher the concentration differential of any gas across this surface, the higher
the gas transfer rates.
Thus, the gas exchange rates are affected by controllable variables on both theinside and outside of the fiber bundle. The primary variables on the inside of the

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fibers are the feed gas(es) concentrationand the gas flow rate. On the outside, theprimary variables are the dissolved gas(es)concentration and the liquid flow rate overthe surface of the fibers. Gas flow rate,
impeller speed and CellGas™ module orientation then become the controllable variables.

Figure D-10 Schematic of CellGas™ oxygenator

The module has two endcaps that containthe potted fibers and form the gas inlet andoutlet for the module. The fiber bundle iscomposed of unsupported, individual,hydrophobic fibers of varying lengths andnumbers in accordance with the surface
area rating of the module.
The CellGas™ module can be steam sterilized in place inside the bioreactor.
In general there are two recommended orientations of the CellGas™ Module. Figure
D-11 shows the “Ball” orientation and the“U” orientations.

Figure D-11 CellGas™ oxygenator configurations

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CELLMAX® CAPILLARY CELL CULTURE D-121

Cellmax® Capillary Cell Culture
Conventional Cell Culture Technologies
Cell culture in the laboratory, as contrasted with applications that are oriented
more toward the process level previously described is a tool used primarily for the
production of laboratory scale cells and cell products. For many applications,conventional cell culture technology has proven inefficient, cumbersome, costly
and labor-intensive, all too often hindering rapid progress in the laboratory.
Cells have a natural tendency to grow in three dimensions. Two-dimensional substrates, such as T -flasks and roller bottles, are not designed for layered cultures.
As cells pile up, cells in lower layers can quickly perish from slow diffusion of oxygen, nutrients and metabolic waste.
Nutrient medium must be frequently replaced, and cell populations must be minimized to prevent overcrowding and layering. Medium replacement causes sudden, frequent changes in the microenvironment immediately adjacent to the cells.
These changes often prevent the accumulation of beneficial cell secreted growth
factors.
In addition, autocrine and conditioning factors are lost when cells are fed and thepH of the growth medium can become very acidic which is non-physiological.

Suspension Cultures
In suspension cultures, cells are continuously agitated in nutrient medium. Somecells are too fragile to tolerate this environment. Culture density is also severely
limited. A high-density culture does not permit adequate agitation, feeding andoxygenation of cells due to the higher viscosity of the medium. In order to continually divide and function, most cells also require costly medium comprised of 520% serum or serum proteins. Moreover, this technology requires that the desired
cell secreted products be concentrated from very large volumes of medium.

Capillary Cell Culture
To overcome the disadvantages above, Spectrum developed CellMax® to duplicate
the in vivo process as closely as possible. This will produce the optimal pericellularenvironment for growth as well as maximal secretion
and accumulation of cell product.
In the CellMax®, cells are grown on and around a network of hollow fiber artificial capillaries encased in a
cartridge shell. Cells suspended in tissue culture
medium are inoculated into the extra-capillary spaceof the cartridge. These cells settle onto the outer surface of the capillaries as shown in figure D-12.

Figure D-12 Schematic of CellMax® configuration with cells

Cells remain in the extra-capillary space during culture, protected from the shear of the rapidly perfusing,continually circulating medium. Since the flow rates
for each cartridge can be independently controlled,
both new and ongoing cultures can be maintained
simultaneously.

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Nutrients and oxygen in the circulating medium readily diffuse through the capillary walls to nourish the cells. Metabolic waste products similarly diffuse awayfrom the cells and are diluted in the circulating medium.
To remain viable, cells require a significant amount of oxygen. The entire CellMax®
system-operates in a standard CO2 incubator where oxygen from the incubator
atmosphere diffuses through the walls of the system’s silicone tubing.

Advantages of Capillary Cell Culture
Metabolic waste and inhibitory factors are dialyzed away from the cells
Autocrine and conditioning factors concentrate around the cells reducing serum
requirements. In addition, the long term culture, stable microenvironment and cell
specific autocrine factors allow cells to resemble tissue and organs
pH of growth medium is stable and physiological
Large surface area for cell attachment and provision of oxygen and nutrients
Secreted products (protein & antibodies) concentrated in the small volume (10 ml
to 60-ml) extracapillary space (ECS)
Large cell numbers (up to 5 x 1010 can be conveniently handled
Endotoxin load reduced
Cells can be cultured for over 6 months. Cells reach confluence and stayattached and alive
Closed system allows safer handling of pathogenic cultures
High cell densities (108/mL) permit developmental interactions to become observable in cell co-cultivation cultures
Stable microenvironment allows in vivo like cell growth
Advantages of capillary cell culture over older methods are shown in the table on
the next page.

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Advantage

Description

Size

The Cellmax® has a surface area equivalent to eight large
tissue culture flasks. The CellMax® cartridge also has asurface area equivalent to two roller bottles.

Space

The CellMax® system occupies the same incubator space
as a single large flask. In addition, the CellMax® unit fits
inside a standard incubator – roller bottles do not. A
CellMax® Quad has the equivalent surface area of eightroller bottles and fits inside a standard cabinet incubator –the eight roller bottles do not.

Cost

One CellMax® cartridge can last up to six months of continuous culturing. Regular tissue culture flasks need to bechanged monthly. Over time, the CellMax® system uses
20% less culture media than regular tissue culture flasks orroller bottles.

Convenience

CellMax® artificial capillary cartridges are ready to use
right out of the package. All that is needed are cells andmedia. Roller bottles require a temperature-controlled roomthat is inconvenient and expensive. The CellMax® System
can be placed in easy-to-use cabinet incubators.

Time

Changing the media for a CellMax® artificial capillary systemtakes only five minutes. This minimizes manipulation of thecell culture and system contamination. The CellMax® system
reduces cell seeding culture time from hours to minutes!

Table D-1 Advantages of the CellMax® System over Flask or Roller Bottle Culturing

Representative Applications of the CellMax® Cell Culture System
Lymphocyte Expansion
Repeated culture and harvest of primary lymphocytes can be achieved with the
CellMax® Capillary Cell Culture System.
Modules are available for the expansion of lymphocytes up to 1010 per module. This method
can be used to produce and collect significant
amounts of lymphocytic growth factors.
CellMax® modules have also been used toincrease the efficiency of viral transductions.

Endothelial Cell Culture

Figure D-13 Culture and harvest of primary lymphocytes with
CellMax® Capillary Cell Culture System

Spectrum has developed several Capillary Cell
Culture Cartridges specifically designed for theculture and study of endothelial cells under flow.
The system provides a means to evaluate
endothelial cell function in a more physiologicalenvironment when compared to other methods.

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As a result, typical cell morphology is maintained, allowing the investigator to generate results that more closely reflect the in vivo situation.
CellMax® provides a reproducible level ofshear force that emulates venous or arterial
shear conditions. In addition, the microporous nature of the capillaries creates anideal format for co-cultures with perivascular cells, such as smooth muscle or brainneuroglia.

Figure D-14 Culture and study of endothelial cells with CellMax®
cell culture system

Figure D-14 is a scanning electron micrograph of endothelial cells cultured in a
CellMax® Endothelial Module under conditions of arterial shear stress of 13-15
dynes/cm2. Individual fibers were sectioned longitudinally revealing an adherent
monolayer of cells attached to the lumenalsurface of a polypropylene capillary. As an
artifact of preparation, one edge of themonolayer has partially detached from theinner capillary surface revealing the contiguous nature of the endothelial layer.

Monoclonal Antibody Production
The CellMax® Artificial Capillary system is the method of choice for the in-vitro production of 50 mg to several grams of monoclonal antibody. A cellulosic fiber witha MWCO of 30kd is used. This permits the inhibitory factor TGF beta to freely diffuse away while trapping the higher molecular weight immunoglobulins along withthe hybridoma cells in the extra capillary
space.

Figure D-15 High Titer Monoclonal Antibody Production using the Spectrum
CellMax® System

Cell density can become higher than 108/ml
and antibody concentration is in the rangeof 0.5 mg to 5 mg per ml. Harvest volume
is only 10 to 20 ml and each cartridge willproduce antibody for up to 4-6 months.
Serum requirements can be reduced by asmuch as 5 fold and the adaptation toserum free medium is facilitated. Chimericand non-murine antibodies can be easily
produced and biological contaminants
such as lipids, endotoxins, proteins orviruses are reduced or eliminated.
Purification is simplified and overall yields
are improved.

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Secreted Protein Production
Current methods for the production and harvest of secreted biologicals such as
growth factors, recombinant proteins and antibodies involves the use of inefficientin-vitro culture systems or animals. Spectrum artificial capillary cell culture systems permit the culture of large numbers of cells (up to 5 x 1010 cells) within asmall volume extra capillary space (10 ml to 100 ml). Selection of the appropriate
fiber molecular weight cut-off can trap the secreted product in this small volume
while allowing lower molecular weight nutrients and waste products to diffuse into
the circulating medium. Both total concentration of product and product secreted
per cell can be increased by a factor of 10 or more using the CellMax® system.
The reduced harvest volumes and the use of reduced serum or serum free mediums simplify post-harvest purifications.

Figure D-16 BeWo Choriocarcinoma hCG Production – T-Flask vs. Spectrum CellMax® Artificial Capillary Cell Culture

The CellMax® Capillary Cell Culture System
Referring to figure D-17, culture medium is drawn from the medium reservoir bottle(A) through silicone rubber tubing (B).
The adjustable pump compresses the
thick walled tubing (C) causing the medium to flow through the oxygenator (D)where oxygen and carbon dioxide areexchanged. The medium flows into theartificial capillary cartridge (F) at the inlet
end port (E). As shown in the enlarged
inset, medium flows through the lumen ofeach fiber within the artificial capillarybundle where it perfuses the cells growing on the outside of the capillaries withfresh nutrients and oxygen, and removes
metabolic waste products. There is no
direct medium flow within the extracapillary space between the capillaries(enlarged inset). The medium is thenFigure D-17 Schematic of operation of the CellMax® Capillary Cell Culture System
returned to the reservoir bottle.

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CellMax® Systems
Single Station Cell Culture System
The CellMax® single station cell culture system
is designed to support cell culture in one capillary cartridge. The system has a positive pressure displacement pump system that can generate high culture medium flow rates without
the use of a peristaltic pump. Peristaltic pumps
can cause particulates to shed from the wall of
the pump tubing, clogging the pores of thefibers and shortening cartridge life.
With this system monoclonal antibodies havebeen produced for up to six months in thesame cartridge. The CellMax® system also
supported a glioma cell line for over two yearsof continuous culture.

Figure D-18 CellMax® single station cell culture system

The single station system features three flow
rates and takes up about the same area in an
incubator as a T225 flask. The system comescomplete with all components required to operate one independent culture module, and permits the culture of up to 5 x 10 9 cells per module and produce 5 to 50 mg of monoclonal antibody per harvest.

Four Station Cell Culture System
The Spectrum CellMax® QUAD Cell Culture System is designed for the culture of avariety of mammalian cell types.
It features a proprietary positive pressure displacement pumping mechanism that permits
long term culture in artificial capillary modules
but has 12 precisely controlled flow rates. The
range of available flow rates is especially usefulfor endothelial cell studies under conditions ofdefined shear stress.

Figure D-19 CellMax® Quad four station cell culture system

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The system comes complete with all components to simultaneously operate up to four separate, independent culture modules. The system-allows the simultaneous culture of several
cell lines, or for repetitive studies of a single
cell type in one system.

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Cell Implant for Drug Screening
New technology has been developed by the National Cancer Institute for thescreening of anti-cancer compounds. This in vivo method of identifying compounds with potential chemotherapeautic activity against cancer and HIV is now
routinely used by many researchers.
The technology allows human cells to be implanted into host animals and subsequently harvested en masse. While in the host, cells can be exposed to potential
chemotherapeutic agents and the impact of that exposure can be measured after
retrieval.
As shown in figure D-20, target cells are macroencapsulated in biocompatible hollow fibers that are then implanted into the subcutaneous or intraperitoneal compartments of laboratory rodents. After selecting a subset of compounds with thehollow fiber encapsulation assay, the activity of the compounds is confirmed using
the classical testing models currently used for evaluating these types ofchemotherapeutic agents.
This methodology results in significant savings of time, labor, amount of compound
required and the number of animals used in the evaluation of the potential therapeutic activity of these candidate compounds.
The hollow fiber assay is a unique in vivo model permitting the simultaneous evaluation of compound efficacy against six cell lines at subcutaneous (s.c.) and peritoneal (i.p.) sites (Hollingshead, et al. Life Sci. 57; 131, 1995).

Figure D-20 Procedure for CellMax® implant assay

As an example, this model was used toinvestigate the relationship between celldensity and compound activity.
Additionally, the model was adapted formechanism of action studies. A racemic
mixture of flavorpiriodol (YZ149, a literature standard and a cyclin-dependent
kinase inhibitor) was used. HCT116 cells(human colon carcinoma) were loaded
into hollow fibers at cell densities of 1.0,
5.0 or 7.5 x 106 cells/mL, and implanteds.c. or i.p. into nude mice. YZ149 was
dosed daily by i.p. bolus at 6, 12, 18 and
25 mg/kg on days 3 through 6 after
implantation. Cell growth was determined by the MTT dye conversion assay.
In controls, increasing cell density
decreased net cell growth with maximal
cell growth at 1 x 106 cells/mL, indicating
the compound is more active against thehigher cell density. After dosing, cells
were removed from fibers and changes
in expression of cell cycle related proteins such as cdk’s, cyclins and Rb weremonitored by Western analysis. The protein changes were correlated with in vivo
growth inhibitory activity.

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These data demonstrate that the efficacy of a compound is a function of cell density, suggesting that the degree of stringency for drug activity can be targeted withthe manipulation of cell density using this assay.
Additionally, the model was adapted for mechanism of action studies. This technology is now used routinely by the National Cancer Institute of NIH for testing
compounds for anti-cancer activity. It is also being employed to look for antiviralcompounds for HIV.
CellMax® implants can also be used with established cancer cell lines, virus
Infected cells, hematopoletic cells, bacteria, fungi and specific organ cells (liver,kidney etc.). The membrane capsules are non-porous to immune system cells,
viruses and mycoplasma.
CellMax® Implants have a biocompatible inner and outer surface.
CellMax® Implants have the following advantages over the current xenographic
model: 10 day assay versus 60 day assay; smaller assay variability; fewer animals
needed; uses small amounts of test compounds; multiple cells lines can be tested
in the same animal model and they are compatible with different cell lines.
When the Spectrum CellMax® Implant in vivo assay is used to screen compoundswith poor pharmacokinetics they are rejected and those compounds with pro-drug
activity are accepted.

Figure D-21 Cells growing within the lumen of a CellMax® hollow fiber membrane

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Section E

ABCs Of Liquid Chromatography
Introduction
Liquid Chromatography is a tool used by Biologists, Microbiologists, Biochemists,
Chemists, and others interested in separating or purifying proteins or other biologicalmolecules. In principle the method is quite straightforward. One simply injects atest sample into a flowing stream of solvent (the mobile phase), another stream
passes through a bed of media in a column (the stationary phase). As the sample
passes through the column the components of the sample spend time attached tothe stationary phase and time free in the mobile phase. Since these times are different for the different molecules, the components of the sample exit the column at different times. The separated components of the sample are then collected as they
exit the column.
In practice, however, development of the technique can require some patience. The
stationary phase is made of small, spherical beads, usually between 10 and 100 µm
in diameter. These beads need to be packed into the chromatography column so
the material is stable and without voids. The art of packing the column can be difficult to learn. It may be the most difficult part of the separation. Fortunately, once acolumn is properly packed it can provide several years of use. There may also besome trial-and-error effort involved in the selection of the mobile phase.
The three most commonly used methods in Low Pressure Liquid Chromatography
are Gel Permeation Chromatography (GPC), also called Gel Filtration Chromato-graphy, Ion Exchange Chromatography (IEC), and Affinity Chromatography (AC).
These three methods are similar, though the stationary phase and the mobile phaseused may be different, but they all can use the same pump, column, monitor, andfraction collector.
Gel Permeation Chromatography (GPC) is used to separate protein molecules bysize or molecular weight. The stationary phase is usually beads made of agarose or
polyacrylamide. The beads have small pores through which the smaller moleculesof the sample can move. It is difficult for the larger molecules to enter the pores. As
the molecules pass through the column, the smaller molecules take a longer, more
tortuous path into and out of the pores on the beads while the larger molecules takea more direct route around straight through the column. In a GPC separation thelarger molecules elute before the smaller molecules.
If a molecule is larger than the size of the pores in the beads, it will not be able toenter any of the beads. Any of these large molecules that are excluded from thebeads elute first, followed by the smaller molecules that can get into the beads. This
simple binary separation, of molecules that are too big from molecules that are not
too big, is performed commonly enough to get it’s own name. It is referred to as
Size Exclusion Chromatography. It is simply a specialized application of GPC and iscommonly used to desalt protein solutions.
Since GPC separates molecules based primarily on their size and not on their chemical characteristics, the chromatograms are generally run with no change in solvent
during the chromatogram (an isocratic separation). Many other types of chromatography make use of a change in solvent to control the separation. These solvent gradients can be gradual changes in solvent (usually produced with a dedicated solvent mixer) or they can be step gradients that can be produced by simply switching
the pump inlet from a flask of one solvent to a flask of another.

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Ion Exchange Chromatography (IEC) is used to separate biomolecules basedon their net charge. The stationary phase used is either a resin with a positively
charged functional group attached (an anion exchange resin) or a resin with anegatively charged functional group attached (a cation exchange resin).
Which type of resin is used depends on whether the molecules to be separated
are negatively charged (anionic) or positively charged (cationic). As the sample components pass over the stationary phase, those appropriately charged
are attracted to and bound by the charged resin. If the binding is weak, thechromatogram may be run isocratically with the less highly charged molecules
eluting first followed by the more highly charged. If the binding is strong, as is
usually the case, the bound sample components must be eluted from the column by changing the composition of the mobile phase; usually a sharp, step
gradient is used for this. The change in solvent releases the bound biomolecules and allows them to pass through the column.
Affinity Chromatography (AC) makes use of active ligands attached to agarose
beads to bind specific proteins and antibodies. The ligands are generally tailored to the specific protein(s) or antibody(s) to be purified and may be proteinaceous themselves. As in IEC, the sample components to be purified bindto the stationary phase as the sample passes through the column. A properly
designed affinity ligand results in very tight binding of only the protein to be
purified, requiring a change in mobile phase to elute it from the column.
A typical low pressure chromatographic system is shown in figure E-1.
Performance and selection criteria for each of the components are discussed
in the remainder of this chapter.

The Column
Important considerations in the selection of the column are the method of separation, the sample size, the required resolution, and the solvents to be used.
Unlike Affinity Chromatography and IEC, GPC is commonly used both for the
purification of biomaterials as well as for their characterization. In analytical
GPC, the molecular weight of an unknown can be determined. To make thisdetermination, a calibration chart of the volume to wash a molecule through thecolumn vs. the logarithm of its molecular weight is made for several known
compounds. This should produce a linear calibration plot. An unknown canthen be applied to the column and its elution volume used to determine its
molecular weight.
Analytical GPC can provide very accurate results. Best results are obtained
when the pore size of the packing media is similar to the size of the unknown.
The pore size distribution is usually provided by the manufacturer of the media
as a “linear fractionation range” indicating the range of molecular weights overwhich a linear relationship between the elution volume and the log of themolecular weight can be expected. The length of the column affects the precision of the molecular weight determination; the resolution of the elution volume
will increase with the column length.
Figure E-1 A typicalchromatographysystemp 130

When doing preparative GPC, one needs to carefully trade off the resolution ofa longer column against the increased dilution of the separated material that it
causes. The total volume of a peak increases with the square root of the column length. If the peak volume increases for the same amount of material

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E-131

being purified, dilution occurs. Increasing the column length also linearly increases
the time required for a separation.
GPC is usually done in long, small diameter columns. For analytical work, a column
diameter of 6 to 25 mm is usually suitable. Column lengths can range from 10 to
200 cm. A sample size of about 1 to 2% of the total column volume generally produces good analytical results. As mentioned above, there are other factors that willaffect the results, including the length of the column, the media selected and thesize of the proteins. Samples sizes of up to 20% of the column volume can be used
for preparative separations with columns as large at 15 cm in diameter and as longas 200 cm.
IEC sample sizes can be larger than those used for GPC, especially in the case ofstrong binding. As the sample is loaded onto the column, the active sites on theresin nearest the column inlet will fill with the charged components of the sample.
As more and more sample is loaded the boundary between the filled resin sites andthe available resin sites will move toward the column outlet. When all of the active
sites on the resin are filled, some of the material that should be bound will begin topass out of the column. If an appropriate detector is in the column outlet, this breakthrough volume can be determined and recorded for future runs.
After all of the sample has been loaded into the column, an appropriate change insolvent can be made to elute the bound material from the column. It is not necessary to fill all of the active sites, but ensure that there are enough sites available tobind all of the sample. Generally the solvent change involves a solution that contains solutes that bind more tightly to the resin. This allows the new solutes to displace the material that is being purified.
Because the resolution in this type of IEC is mostly independent of the column flow
characteristics, short, large diameter columns are usually used. Short, large diameter columns can be used at higher flow rates than long, small diameter columns of
the same volume and ion exchange capacity. This reduces the time required for the
separation.
The sample loading and operation considerations for Affinity Chromatography are
similar to those for IEC. One generally has better initial knowledge of the column’s
capacity. Because of the high cost of the media, column sizes are usually small, but
any short, large diameter column is suitable.
When selecting a column, be sure that the column has a non-clogging bed support
that does not broaden or mix the bands as they elute. If a pump is being used andthe bed support becomes clogged, the pressure rating of the column may be
exceeded causing it to leak or crack. When using gravity flow, the clogging of the
bed support may stop flow completely in the column. In either case clogging
requires packing the column again. Some non-clogging bed supports broaden the
bands as they elute from the column. This results in a degradation of the resolutionthat was desired.
Many of the chemical interactions used in chromatography are temperature dependent. Choose a column that can be fitted with a water jacket. This allows the use of atemperature controlled water bath to maintain the temperature of both the column
packing and the liquid flowing through it.
Another, often overlooked consideration is the compatibility of the column material
with the solvents to be used. In biological laboratories, most chromatography

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processes are done with rather benign aqueous buffers. Generally almost any material (except for many metals) can be used without risk. Spectrum manufactures aline of columns with less expensive polypropylene endplates for these applications.
Other scientists use less benign solvents in their separations, including organic solvents and concentrated acids and bases. More inert materials are required in theseapplications. Spectrum manufactures a line of columns with more inert PTFE endplates for these users. The only wetted materials in these columns being the PTFE
endplates and the borosilicate glass bodies.
A glass column is frequently superior to a column made of metal or other opaquematerial, since it allows the condition of the media to be observed. Voids or separations which may develop can be easily discovered and corrected. The transparencyof a glass column also makes it easier to layer the sample on the top of the media
bed without dismantling the column.
Spectra/Chrom® columns all feature transparent glass bodies and non-clogging bed
supports. They are available with either polypropylene endplates for lower cost or
PTFE endplates for additional chemical compatibility. They are available in diameters ranging from 0.6 cm (about 0.25 inch) to 15 cm (about 6 inches) and in lengths
to 200 cm.

The Pump
Low Pressure Liquid Chromatography can be performed using only gravity to supply
the energy to drive the mobile phase through the column. As long as the solvent
container is located above the top of the column hydrostatic pressure can move fluid
through the column. Step gradients can be generated using two or more solvent
jugs and switching the column inlet from one to another. This method is inexpensive, simple and pulse free. Drawbacks to using this method are:As the fluid level falls, the hydrostatic pressure decreases resulting in a decreasing
flow rate during the separation.
Gradients can only be step-gradients.
The separation takes longer because of the low pressures involved (a fluid level 3
feet above the top of the column is less than 2 psi).

A peristaltic pump is an instrument that forces fluid along tubing by using rollers tosequentially squeeze the tubing against a platen, trapping the fluid in small packets
between the rollers. The rollers continuously move the packets of fluid across theplaten releasing one downstream as each roller lifts off the platen. As each packet
of fluid is released a small pressure pulse is generated. This pulse is a small sudden drop in pressure caused by the increase in tubing volume that occurs when thepump roller lifts off of the tubing. Under some conditions, these pressure pulses candisturb the top of the column packing. These pulses may be dampened by using alength of soft tubing between the pump and the column minimizing their effect on
the column packing.
The big advantage of a peristaltic pump over a piston pump is that the fluid is confined inside a piece of tubing. No other parts of the pump are ever in contact withthe mobile phase. By changing the size and composition of the pump tubing, thep 132

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available flow rates and chemical compatibility of the pump can easily be customized to a particular application.
Some peristaltic pumps have additional features
such as flow rate calibration and digital display ofthe selected flow rate. They are promoted as precise and accurate after user calibration.
Calibration involves measuring the volume delivered at a particular flow rate for a fixed period oftime. The flow rate of a pump is dependent not
only on the speed of the pump motor, but also on
the operating pressure, elasticity, and exact inside
diameter of the tubing. To maintain the quoted
accuracy of these pumps, they need to be calibrated every time the tubing is changed, and periodically as the tubing in the pump ages. The digital display is no more useful than a simple knob,
since it is just displaying the flow rate that has
been set.
Most low pressure separations are very forgiving
in terms of pump flow rates. Ion exchange andaffinity separations, where a step gradient is used
to effect elution, does not require exact flow rates,
since nothing will elute from the column until thestep is made. The flow rate is of more significance for gel filtration separations, since the elution process is continuous. Even when doing gel
filtration chromatography, knowing the flow rate tobetter than ±3% is of little value.

Figure E-2 Spectrum MP-2 Peristaltic Pump

Figure E-3 Generated a linear gradient with 3 Pump Heads

The Spectra/Chrom MP-1 & MP-2 Peristaltic
Pumps (figure E-2) are general purpose peristaltic
pumps. The pump motors are fitted with digitaltachometers to maintain motor speeds within 1%of the user set point. The only flow rate changes
that occur are caused by the tubing and the column. Up to three pump heads can be mounted
on the pump drive. This allows the user to easily
generate both linear and non-linear continuous
gradients (figure E-3).

The UV Monitor
The UV Monitor is the heart of the chromatography system. It is an instrument that isable to detect when a component of the sample is eluting from the column because
bio-molecules absorb UV radiation. It monitors the transmittance of a narrow beamof ultraviolet radiation passing through the effluent stream. A flowcell with two tubing
fittings is connected in the effluent stream. The flowcell has two quartz windows forthe ultraviolet beam to intersect the effluent stream and exit to strike a photocell or
photodiode. The signal is then converted to an absorbance and displayed in
Absorbance Units (AU).

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When radiation is passed through a substance in solution the radiation is absorbed
depending upon the absorbancy, and the concentration of the substance in thesolution. If L0 is the light entering the solution and L is the light leaving the solution,then the transmittance (T) is defined as:
T= (L/L0)
Absorbance is defined as:
A = log10 (1/T)
A = log10 (L0/L)
Because this is a log function AU is dimensionless. One AU is equal to a reductionof the incident radiation by a factor of 10, while 2 AU is a reduction of the radiation
by a factor of 100. A more useful equation is the Beer-Lambert law, which states theabsorbance, is equal to:
A=a
Where:k l

a is the absorbtivity in liter/mol•cm,
k is the concentration in mol/liter, andl is the path length in cm through the solution.
This is a linear equation rather than logarithmic, and it is easier to use to determine
concentrations. Modern UV Monitors display results in AU.
The variable wavelength, the scanned array, and the fixed wavelength are the threebasic types of UV Monitors. The first two are very expensive and are used in mosthigh performance liquid chromatography (HPLC). Most biological molecules aredetected at two wavelengths (280 nm and 254 nm) so fixed wavelength monitors
such as the Model 280 UV Monitor from Spectrum are most often used for low pressure liquid chromatography.
The variable wavelength monitor uses a Deuterium light source to generate a continuous spectrum from about 190 nm to 400 nm. A diffraction grating is then used to
select the wavelength to be used for detection. Drawbacks to this system are themechanical linkage to the grating and the resolution of the grating/optical system.
There may be “play” in the linkage between the knob that is used to set the wavelength so that the center wavelength may not always be repeated. The user must
approach the selected wavelength from the same direction each time to minimize
this affect. The grating/optical system has a bandwidth as high as 20nm. Thismeans that if the user selects 254 nm as the center wavelength the monitor willdetect absorbance as low as 245 nm and as high as 265 nm. If the user is attempting to quantify two different components of the sample and the absorbance profile atthe selected wavelength is changing, the ratio of the areas will give an erroneous
indication of the relative quantities of the two components. A discrete wavelength
monitor used at 254 nm would give the correct quantities since it has a bandwidth of
0.1 nm at 254 nm.
Scanned array monitors also use Deuterium lamps and diffraction gratings, but in

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E-135

these monitors the position of the diffraction grating is fixed. The diffraction grating isused to disperse the radiation from the Deuterium lamp onto an array of photodiodes
instead of just one photodiode. This eliminates the need to rotate the grating, removing one of the drawbacks of the variable wavelength monitor. A scanned array monitor usually has 256 diodes in the array. The bandwidth is about 1 nm, which makes this
instrument useful for quantization.
A scanned array monitor is very rarely required in low pressure liquid chromatography,
but is a useful tool in analytical HPLC. It is able to produce a 3 axis display ofabsorbance plotted against wavelength and time facilitating the identification of
unknown compounds in a sample. These very computer controlled monitors have features that allow both the chromatogram and the individual spectra to be observed invarious formats. They also include algorithms that can be used to identify samples
components from their empirical spectra. The scanned array monitor is ideal for work
involving the identification of unknowns, but they generally add unneeded complexity
to preparative low pressure liquid chromatography.
Discrete wavelength detectors are the ideal detector for low pressure liquid chromatography. They are compact, inexpensive, and easy to use. They provide an output in Absorbance Units based on the quantity of a compound in the effluent stream.
This type of detector generally uses a low pressure Mercury arc lamp as its light source
providing a radiation line at 254 nm. Other longer wavelengths can be generated using
this line to excite rare earth phosphors.
When selecting a discrete UV Monitor one important feature to require is a display ofthe output. This enables a quick determination of the status of the chromatogram.
Another time saving and frustration reducing feature is an autozero. With this feature
the user can press a button to zero the baseline on the absorbance range to be used.
Monitors without this feature must be manually zeroed at the highest available range
and then switched to lower ranges, re-zeroing at each step, until the desired
absorbance range is reached.
Changing the wavelength can be a time consuming chore on some discrete monitors.
Some instruments require replacing lamps and/or filters which usually requires removing and replacing the cover, then waiting for an extended time for the baseline to settle down. Select an instrument in which changing wavelengths is a simple step requiring no tools and very little time.
Extremely sensitive absorbance ranges are not generally needed for preparative chromatography. Because proteins and most other interesting biomolecules have broadand deep UV absorbance bands, most low pressure liquid chromatography is done atsensitivities ranging from 0.1 to 1.0 AU.
Instruments with usable ranges more sensitive than 0.01 AU full-scale are usually quite
expensive. (Some less expensive instruments include more sensitive ranges, but havenoise figures that would preclude operation at these more sensitive ranges.) Two specifications that are of more significance than the lowest absorbance range available are
the detection limit and the noise level of the monitor. It is important that the wavelengthand the measurement conditions are specified with these parameters. Typically thenoise at 254 nm with a dry flowcell is much less than the noise that will be experienced
at 280 nm with solvent flowing through the flowcell.
If the UV Monitor is the heart of the chromatography system, then the flowcell is theheart of the UV Monitor. The design of the flowcell is critical to the performance of the
UV Monitor. When selecting a UV Monitor for low pressure liquid chromatography

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make sure that flowcells are available to span all of your liquid chromatography needs.
Four parameters to consider in selecting a flowcell for a particular separation are:
What is the range of specific absorbtivity and concentrationof the components of the sample to be separated?
What is the flow rate that will be used?
What is the size of the sample?
Will gradient separations be used?
The absorbtivity and concentration of the sample determine the path length of theflowcell that should be used. Most UV Monitors use 5 mm as the path length for theirstandard flowcell, and this is satisfactory for most applications. If the concentration
and absorbtivity will be very low, then a 10 mm flowcell may be required. As the BeerLambert law shows, this will double the sensitivity of the monitor. If the absorbtivity andconcentration is very high, then a short path length flowcell, such as 2 mm path length
flowcell may be needed. This increases the absorbance range by a factor of 2.5.
Preparative liquid chromatography may be done with flow rates that are quite high, ashigh as several liters per minute. You should be sure to select a flowcell that will handle the highest flow rates you plan on using. For a given pressure, a short path length
flowcell will generally have a lower flow rate than a long path length flowcell. Larger
columns may have pressure limits as low as 15 psi, limiting the pressure available atthe flowcell to something less than 15 psi. A graph of the pressure drop vs. flow rate
can tell you if a particular flowcell can accommodate your anticipated flow.
If the volume of the sample is small, then a flowcell with a large internal volume may
affect the resolution of the separated components. If the flowcell is to be used in HPLC
then this is an especially important factor. Flowcells with internal volumes of less than
2 microliters per millimeter path length are sometimes necessary to preserve resolution.
Flowcells that are designed for low pressure liquid chromatography and preparative liquid chromatography have large internal volumes, which are small, compared to the
preparative sample size, but still allow for the higher flow rates that are necessary.
One of the most important considerations in selecting a UV Monitor and flowcell iswhether there is the possibility of gradient separations being used. With a two component gradient the different absorbtivityof the solvents can cause the baseline tochange considerably. This makes it difficult to accurately determine the area ofpeaks when quantitating the results. In
some cases the chromatogram will go off
scale. Select a UV Monitor that contains
dual flow path flowcells. Not only does thisgive a stable reference when doing isocratic separations, but flowing the gradientthrough the reference path of the flowcell
prior to the column will subtract baselinechanges caused by the solvent.
Figure E-4 Model 280 UV Monitor

p 136

The Spectrum Model 280 UV Monitor (seefigure E-4) has been designed with all of

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THE UV MONITOR

E-137

the features that a discrete wavelength UV Monitor should have. It has a digital display of the output, an autozero, and three dual flow path prep flowcells of 2 mm, 5 mm,
and 10 mm available (the 5 mm path length flowcell is included with the monitor). In
addition it has an event mark pushbutton allowing the user to place positive going
spikes on the chromatogram to signal events such as injection. It is compact and has
an optional bracket that allows mounting on a mast close to the column exit. It comescomplete with both 254 nm and 280 nm capability. To change wavelengths it is merely necessary to flip the filter holder. No tool is required and the time expended is lessthan 10 seconds. The Model 280 has a fast warm-up with drift becoming negligibleon the 0.05 absorbance range within 10 minutes after turning on the power. The Model
280 UV Monitor is the best value on the market for a low cost state of the art monitor
for low pressure Liquid Chromatography.

The Fraction Collector
Fraction collection is the art of dividing the liquid eluting from the column to maintainthe separation of the sample that occurred in the column. The separated components
are collected in individual containers (usually test tubes). The scientist collects thesefractions for further study as in analytical chromatography or uses them as a step
toward an end product as in preparative chromatography.
A typical modern fraction collector is shown in figure E-6. It can not only collect fractions by volume, but also collects by time or drop. Using the absorbance signal froma UV Monitor, the modern fraction collector can be programmed to discriminate against
the peaks detected by the monitor so that only the desired parts of the sample are collected. The effluent that is of no interest is diverted into some other container or towaste.
There are four basic types of fraction collectors. The simplest is just a set of valvesattached to a manifold that are sequenced to divert the effluent into large containers.
A rectilinear fraction collector, moves collection vessels under the effluent stream. The
X-Y fraction collector moves the effluent stream over the collection vessels. The roundfraction collector moves both the tubes and the effluent stream. All four of these basic
fraction collectors have their advantages and disadvantages and have options thatenhance their utility for specific applications.

Which Type Fraction Collector Should Be Used?
A number of questions need to be answered before selecting which of the four typesof fraction collector to use.
What is the size of the fractions to be collected?
What is the flow rate of the effluent stream?
How many fractions need to be collected?
Is band spreading important?
Is cross contamination of samples important?
Are the peaks to be collected in separate tubes?

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E-138 THE ABCs OF LIQUID CHROMATOGRAPHY

Usually the only time one uses valves to collect fractions is in large scale preparatory
chromatography where the sample is very large and the time that the peaks of interest will elute from the column is precisely known. The fraction size may be many liters
in size, and most of the time only one peak is collected. The valve(s) can be controlled
by a sequential timer that starts either when the mobile phase is started (if the sample
has been layered on top of the column), or when the sample is injected into the flowing mobile phase stream. The only other fraction collector that can collect extremely
large fractions is the X-Y. This is accomplished with an option that replaces the normal test tube rack with an array of funnels connected to tubing that leads to large collection vessels. This type of scheme is used when collecting more than three or fourlarge peaks.
For moderate sized fractions the other three fraction collectors can be used although
the researcher needs to consider certain features of the round and the X-Y fraction collector that might be detrimental to the collection desired.
Because the Round fraction collector moves the drop head along the radius of theround test tube rack and the X-Y over the whole test tube rack, the tubing coming fromeither a valve or the detector must be long enough to accommodate the traverse. As
the peaks travel along this extra length of tubing they can spread out reducing the resolution that was obtained coming out of the column. For very large fractions this may
not be a problem, but for small fractions one must keep the length of tubing between
the column outlet and the collection tube to a minimum.
Another problem with Round and X-Y fraction collectors is their tendency to “fling”
hanging drops when they are moving from one tube to another. This causes drops
from one peak to be deposited in tubes meant for other peaks. The manufacturers ofthese types of fraction collectors have tried all types of design schemes to prevent thisproblem but nothing works. The only solution is a stationary drophead, which only the
Rectilinear fraction collector has.
The X-Y fraction collector is the only system that can collect very small fractions. Thisis because it can be programmed to deposit the peaks in a microplate. Of course theproblem of the drops falling where they are not supposed to can be a serious problem.
The Rectilinear fraction collector has another useful feature. The user can remove thetest tube racks as the peaks are collected without interrupting the rest of the collection.

Time, Drop or Volume?
If the flow rate is low enough (less that 6 ml/min), the best method of collection for mostseparations is counting drops. This approach eliminates variations in collection volume
caused by changes in pumping speed, or changes to the flow rate due to levelchanges in the solvent vessel when using gravity flow. There will still be changes in thevolume collected caused by different size drops due to the different physical propertiesof components of the sample. In most cases these will be minimal. The Spectrum CF1 Rectilinear fraction collector has a drop counter as standard equipment.
For higher flow rates the time collection mode is usually used. As the name implies,
effluent is collected into each collection vessel for the same time period. The user
should select a collection time so that with constant pumping speed the fraction collector will change collection vessels when they are filled to 75% so there will be no
danger of them overflowing.

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THE FRACTION COLLECTOR

E-139

Some fraction collectors (the Spectrum CF-1) allow collection by volume. In thismode the fraction collector is controlled by the pump, changing tubes when a preset number of pulses from the pump indicate that the desired collection volume has
been reached. This is very useful when collecting equal aliquots of a liquid in aseries of test tubes to be used, for instance, as an incubation media. When using
this collection method with a column separation there will still be changes to the volume collected in the test tubes due to the changing characteristics of the column
during the separation.
Type of Fraction Collector

Requirement

VALVES

Very Large Fractions > Literl

Large Fractions > 75 mll

RECTILINEAR

Standard Fractions < 75 ml

X-Y

ROUND

l
l

Small Fractions < 50 µl

l

l

l

High Flow Rate > 100 ml/minlFlow Rates < 100 ml/min

l
l

l

l

l

l

More Than 3 Fractionsl

No Cross Contamination of PeakslMinimum Band Spreadingl

Table E-1 Requirements and the Fraction Collectors that will adequately satisfy them

Custom Fraction Collector
When a simple fraction collector is used to collect the separated parts of a sample,
the size of the fraction will remain relatively constant throughout the collection. The
test tubes containing the peaks will have the same amount of liquid in them as thetest tubes containing the liquid from between the peaks. If the fractions fill the tubes
to 75%, some of the peaks will probably be diluted with non-peak effluent. If thefractions are kept small to keep the peaks as pure as possible one may run out oftubes.
A “smart” fraction collector such as the Spectrum CF-1, using the signal from a UV
Monitor, presents the scientist with four options: (1) Collect peaks and non-peaks at
the same volume, (2) Collect peaks at one volume and non-peak effluent at anothervolume, (3) Collect only the peaks, shunting the non-peak effluent either to waste ora large collection vessel, (4) Collect only the peaks of interest. With the CF-1
Fraction Collector one can customize the collection and retain in memory up to four
different collection protocols.

CALL 1-800-634-3300 FOR FREE FILTRATION AND BIOPROCESSING CATALOG

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E-140 THE ABCs OF LIQUID CHROMATOGRAPHY

Time Windows
Most smart fraction collectors utilize time windows to allow the user to customize a collection. The CF-1 provides up to 10 time windows, which allow the user to select intervals
from the start of a separation in which the fraction collector will:
Collect all the effluent in the test tubes.
Collect only when the UV detector signal
indicates that there is a peak eluting fromthe column.
Shunt all of the effluent to waste or a separate
container.

The time windows and peak detector, usedwith the signal from the UV Monitor, will allow
the user to collect only the peaks of interest.
Figure E-5 shows five time windows used tocollect two peaks while shunting the unwanted
effluent and the other peaks to waste. The
peak detector is used in the slope mode inthis example.

Figure E-5 Time Windows

NOTE. It is always a good idea to collect the “waste” rather than letting it flow into adrain on the off chance that a valuable sample may have inadvertently been included in the waste either through a UV Monitor or Fraction Collector malfunction or aprogramming error. If all the peaks are collected and the separation is isocratic, the“waste” is pure solvent and may be recycled.

Peak Separation
There are three sensing modes in which smart fraction collectors may use the signal
from a UV Monitor to identify peaks. They are level sensing, slope of the leadingedge of the peak, and both level and slope sensing.
Most smart fraction collectors have at least level sensing. With this sensing modethe fraction collector will determine that a peak is present anytime the UV Monitor
signal is above a level preset by the user. The big disadvantage to this method isthat baseline drift can cause the fraction collector to think there is a peak present.
When using two or more solvents with different absorbtivity during a gradient separation, the base line can change significantly. For this reason level sensing is almost
useless unless the separation is isocratic. Most gradient separations require that thefraction collector recognize a peak by the slope of the UV Monitor signal.
The Spectrum CF-1 Fraction Collector has a sophisticated slope detector as well asa level detector. Not only does the user have the option of selecting whether the
peak signal has a fast or slow rise time, but also whether the peaks will be large(over 50% of full scale), narrow or broad (can enter the expected range of widths ofp 140

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THE FRACTION COLLECTOR

E-141

the peaks). Specifying these criteria insures that all of the peak will be collected.
Since during lengthy separations peaks that take a long time too elute the column isbroader than fast eluting peaks, the user can specify that the fraction collector adjust
the criteria to expect some broadening.
If a user wants to collect peaks in the highest concentration possible it is necessary
to be able to use both the slope and level peak sensing together. In this mode theuser sets the slope appropriate for the desired peaks, and then the level is set 10%
to 15% above the baseline. This will eliminate the portion of the peak in the tail thatis not very concentrated while retaining the main body of the peak.

The Intelligent Fraction Collector
Using a fraction collector with the features of the Spectrum CF-1 (see figure E-6),
gives the scientist the opportunity to collect fractions in a way best suited to eachseparation. The fraction collector can be programmed to collect one peak or one
hundred peaks; all the non-peak effluent or none of the non-peak effluent. Peaks
can be collected at one volume while non-peak effluent can be collected at another.
Using a time window the void volume from the column can be shunted to waste so
as not to use collection tubes unnecessarily. Test tubes from 10 mm diameter toscintillation vials of 28 mm may be used.
The Spectrum CF-1 is easy to programand can retain four different programs for
future use. It has an easy to read LCD
display that leads the user through theprogramming sequence. The membrane
key-pad allows the selection of one of four
options displayed each step of the programming sequence. At each step help isavailable by pressing the “help” key.
A mast package which allows the mounting of a column and a UV Monitor is available as an option. This allows the effluent
flow path to be as short as possible, keeping band spreading to an absolute minimum.

Figure E-6 CF-1 Fraction Collector

The Spectrum CF-1 is a low priced unitthat will satisfy all possible collection
requirements.

CALL 1-800-634-3300 FOR FREE FILTRATION AND BIOPROCESSING CATALOG

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p 141

Section F

Appendix
Glossary of Terms
ABSOLUTE -A degree of filtration, usually referring to 100% removal of rigid
particles greater than a stated size.
ABSORB -To take up or drink in, as a sponge imbibes water.
ACTIVATED CARBON -Carbon activated by high temperature steam or carbon
dioxide to form a material of high adsorptive capacity.
ADSORB -Attracting and holding a gas, vapor, or liquid on the surface of a solid.
ADSORBENT -A solid material that adsorbs, such as clay, carbon, activatedalumina, etc.
AMORPHOUS -Non-crystalline, having no determinable form.
ANGSTROM (Å) - Unit of measure (10-10 m)
ANISOTROPIC - Materials that vary from one side to the other, asymmetric or
non symmetric.
AREA -The surface exposed to the flow of a fluid.
ASBESTOS - A natural group of magnesium silicate materials found in fibrous
form.
ASYMMETRIC – See anisotropic.
ATTRITION - Loss of material due to wear caused by rubbing or friction.
BACKWASH - To reverse flow of air, liquid, etc., through the filtration media toeffect solids removal.
BAFFLE - A plate protecting filter elements from the velocity of flow entering vessel.
BIND - To adhere to another object.
BLIND SPOTS - A place in the filter media where no filtration takes place.
BLINDING - Reducing or shutting off of flow due to closing of pores in thefilter media
BLOWDOWN - The use of pressure to remove liquids and/or solids from a vessel.
BRIDGING - Particles being removed arch over individual openings in the filter
media or between the individual filter septa.
BUBBLE POINT - The point at which gas pressure applied to a membrane overcomes the surface tension of a fluid wetting the membrane. The bubble point correlates to pores size, as the pore size increases the bubble point decreases.

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F-144

APPENDIX

CAKE - Solids deposited on the filter media.
CARTRIDGE - Self contained filter media. Usually applied to dead-ended filters.
CASTING - Process for making a membrane. Typically a polymer mix is allowed toform a membrane as solvents evaporate out of the mix. May be a wet or a dry process.
CE - Cellulose ester. Common material used in manufacture of membranes.
CONCENTRATE - The unfiltered solution on the upstream side of a filter that
becomes more concentrated as filtration progresses.
CELLULOSE - fibrous material of vegetable origin.
CLARITY - Clearness of a liquid measured by the amount of contaminants remaining.
CLAY - A natural occurring material usually being activated and used as an absorbent.
COMPRESSIBILITY - Degree of physical change in a filter cake when subjected
to pressure.
CONCENTRATION GRADIENT – the force generated by atomic valence.
CONCENTRATION POLARIZATION - The formation of a thin layer of material notable to pass through the filter pores on the upstream side of a filter. Acts to inhibit
permeate flow.
CONTACT ANGLE – the angle between the perpendicular at which watercontacts a filter surface and the perpendicular to the filter surface.
CROSS FLOW FILTRATION - Filtration method in which the flow path of the fluid
being filtered is parallel to the membrane surface so that fouling agents and particles are swept away by the flow. Recirculation flow keeps material from being
driven into the membrane while trans-membrane pressure drives materials thatare able to pass through the membrane.
CYCLE - Filtration interval; length of time filter operates before cleaning.
DALTON OR “D” - Unit of measure for the molecular size of molecules. One
Dalton refers to the size of one hydrogen atom.
DEAD ENDED FILTRATION - method perpendicular to the filter surface in which
there is no recirculation or bypass. As fluid enters the filter it is either passed orretained by the filter media.
DELTA P (∆P) - The difference in pressure between two points in a system.
DENSITY – mass per unit volume (in filtration terms a measure of compactness
or thickness.
DEPTH FILTRATION – Filter media with many paths to entrap particles.

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GLOSSARY OF TERMS

F-145

DIAFILTRATION - A filtration method where molecules are being removed or
washed by the addition of solvent directly to the solute being purified to maintain aconstant volume.
DIALYSIS - The use of concentration gradient to separate solutes of differing sizes
across a semipermeable membrane.
DIALYSATE - The solution (buffer) used for receiving the solutes which diffuse(dialyze) through a dialysis membrane.
DIFFUSION – the passage of a fluid through the intermolecular structure.
DIFFUSIONAL FLOW - The rate of gas diffusion flow from the high-pressure side
to the low-pressure side of a filter.
DIATOMACEOUS EARTH - Absorbent earth.
DIFFERENTIAL PRESSURE - The difference in pressure between two given
points.
DOCTOR KNIFE (BLADE) - A scraper for filter cake removal. Normally found on
drum type filters. Also, a knife for forming thickness of cast membrane filters.
EDGE TYPE FILTER - A filter that entraps particles on the edges of the medium.
EFFLUENT - The discharged liquid from a filter.
ELEMENT - Usually another term for septum.
FILTER - A device used to remove contaminants.
FILTER AID - Diatomaceous earth, etc.
FILTER MEDIA - Material mounted on a support for separation of particulates ormolecules from liquids or gases.
FILTRATE - The portion of material that has passed through the filter element andappears on the downstream side of the filter. The liquid that has passed throughthe filter. See also permeate.
FILTRATION - The process of removing particles from a fluid.
FINES - Particles smaller than a specified size.
FLAT WIDTH - The measurement of the tubing width in a flat position (1/2 x thecircumference). Diameter = (2 Flat Width) x 3.14
FLOW RATE - The amount of material passing a given point per unit of time.
Typically measured in gallons per minute or hour, or liters per minute or hour.
FLUX - The amount of filtrate flow rate per unit area. Usually stated as, for example, liters per hour per square meter.

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F-146APPENDIXFULLER’S EARTH – Clay, a hydrous aluminum silicate.
GEL LAYER - A layer of fouling agents, particles or both that builds up on the
upstream side of the filter to inhibit flow. See also concentration polarization.
GPH - Gallons per hour.
GPM - Gallons per minute.
GSFD - Gallons per square foot per day.
HYDROPHILIC - Water wetting. To have an affinity for water.
HYDROPHOBIC - Water rejecting. To repel water.
INTEGRITY TEST - A test designed to ensure that filter pore size has not been
compromised by any form of physical damage.
ISOTROPIC - Material having a uniform physical structure through the material. In
the case of a membrane, the pore size is uniform from one surface to the othersurface.
LEAF - A support for the filter medium.
LIQUID - Material to be filtered.
LUMEN - The cylindrical open space in the middle of a hollow fiber.
M - A unit of measure for molar concentration, given to be moles of material per
liter.
MEMBRANE - A thin, porous material with a specific pore size that allows the passage of solutes with sizes smaller than the pores and the retention of solutes withsizes larger than the pores.
MESH - Number of openings in a lineal inch of wire or woven cloth.
MICROMETER (MICRON) - Unit of length: 10 -6 meters.
MODULE - A term for a membrane and its housing. Often used to describe a hollow fiber cross flow unit.
MOLE - The fundamental Standard International (SI) unit used to measure themass of a given substance containing a specific number (Avagadro’s number) ofentities. Used to determine the molar mass of a given substance.
MOLECULAR WEIGHT - The sum of the atomic weights of all the atoms in a molecule.
MOLECULE - The smallest unit of matter, consisting of two or more atoms bonded
together, that retains all the physical and chemical properties of that substance.
ml or mL - A unit of measure for volume, milliliter.

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GLOSSARY OF TERMS

F-147

mm - A unit of measure for distance, millimeter.
MWCO - “Molecular Weight Cut Off” is an indirect determination of the pore sizeof a membrane. The membrane will retain 90% of spherical, uncharged target
macromolecules that have a molecular weight equal to the membrane MWCO.
NEGATIVE PRESSURE - Vacuum or suction.
NORMAL – Perpendicular to a surface.
PARTICLE SIZE DISTRIBUTION - The distribution obtained from a particle countgrouped by specific micron sizes.
PARTICLE - A non-soluble piece of material suspended in solution or air.
PERMEATE - The portion of a solution that passes through the membrane andappears on the downstream side of a filter. See also filtrate.
PORE - An open passageway through any filter element.
PORE DENSITY - The number of pores per unit area of a membrane surface.
PORE SIZE - Approximate diameter of individual pores.
PORE SIZE DISTRIBUTION - Range of pore sizes within a membrane.
POROSITY - The fraction of a filter membrane that is open for flow. Usually
expressed as a percentage.
PPB or ppb - part per billion, a measure of particle or substance concentration.
PPM or ppm - part per million, a measure of particle or substance concentration.
PSI - A unit of measure for pressure, pounds per square inch.
PVDF - polyvinylidene difluoride.
PTFE polytetrafluoroethylene
PREFILT - Material to be filtered.
RECYCLE - The return of filtered liquid for another filtering.
RC - regenerated cellulose
RECIRCULATION RATE - The amount of flow which passes through the lumen of
the hollow fibers in a cross-flow filter.
RETENTATE - The solution or solid containing large molecules that is retained by
the membrane.
RPM - A unit of measure for centrifugal force, revolutions per minute.

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p 147

F-148APPENDIXSEPTUM - The material mounted on a support; support for filter aids.
SOLVENT - A liquid into which other materials are contained or dissolved.
SOLUTE - Substances dissolved or suspended in a solvent.
SKINNED - Used to describe a membrane that has a relatively dense surface witha much less dense underlying structure.
SYMMETRICAL - Having the same physical characteristics from one side to theother. The pores have the same diameter from one surface of the membrane tothe other.
SURFACE AREA - The area of a filter available for filtration, usually measured in
cm2 or ft2.
SUPERNATANT - Liquid above settled solids.
TANGENTIAL FLOW - Flow parallel to the upstream membrane surface. Also
known as cross flow.
TANGENTIAL FLOW FILTER - A filter with the fluid flowing parallel to the surfaceof the membrane.
THRUPUT - The amount of solution that passes through a filter or membrane
before it plugs.
TRANS-MEMBRANE PRESSURE - In a cross flow filter, the average of the inlet
and outlet pressures less any backpressure on the filtrate. TMP -[(Pin + Pout/2] PfiltrateµL - A unit of measure for volume, microliter.
WETTABILITY - The ability of water to wet out a membrane.
VOIDS - The openings in a medium or filter cake.

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TRADEMARKS

F-149

Trademarks
CellFlo is a trademark of Spectrum Laboratories, Inc.
CellGas is a trademark of Spectrum Laboratories, Inc.
CellMax is a registered trademark of Spectrum Laboratories, Inc.
Clear Bath is a registered trademark of Spectrum Laboratories, Inc.
CulturedGard is a registered trademark of Spectrum Laboratories, Inc.
Dispodialyzer is a registered trademark of Spectrum Laboratories, Inc.
Drawer/Ganizers is a registered trademark of Spectrum Laboratories, Inc.
DynaGard is a registered trademark of Spectrum Laboratories, Inc.
Eppendorf is a registered trademark of Eppendorf NethelerHinz, GmbH
FilterPic is a registered trademark of Spectrum Laboratories, Inc.
Fleaker is a registered trademark of Spectrum Laboratories, Inc.
with special licensing to Corning Glass Works
Float-A-Lyzer is a registered trademark of Spectrum Laboratories, Inc.
KrosFlo is a registered trademark of Spectrum Laboratories, Inc.
KrosTrac is a registered trademark of Spectrum Laboratories, Inc.
LiquidSpy is a registered trademark of Spectrum Laboratories, Inc.
MediaKap is a registered trademark of Spectrum Laboratories, Inc.
Medidroppers is a registered trademark of Spectrum Laboratories, Inc.
Microgon is a registered trademark of Spectrum Laboratories, Inc.
Micro Dispodialyzer is a registered trademark of Spectrum Laboratories, Inc.
Micro-Pro/DiCon is a registered trademark of Spectrum Laboratories, Inc.
MicroKros is a registered trademark of Spectrum Laboratories, Inc.
Micro-ProDiCon is a registered trademark of Spectrum Laboratories, Inc.
MidiKros is a registered trademark of Spectrum Laboratories, Inc.
MiniKros is a registered trademark of Spectrum Laboratories, Inc.
Molecular/Por is a registered trademark of Spectrum Laboratories, Inc.
Oxford is a registered trademark of Sherwood Medical Industries, Inc.
Refrig/Arrangers is a registered trademark of Spectrum Laboratories, Inc.
SeleXtrac is a registered trademark of Spectrum Laboratories, Inc.
SilentSwirl is a registered trademark of Spectrum Laboratories, Inc.
Spectra/Gel is a registered trademark of Spectrum Laboratories, Inc.
Spectra/Mesh is a registered trademark of Spectrum Laboratories, Inc.
Spectra/Por is a registered trademark of Spectrum Laboratories, Inc.
Spectrum is a registered trademark of Spectrum Laboratories, Inc.
Transfertube is a registered trademark of Spectrum Laboratories, Inc.
Vacu/trol is a registered trademark of Spectrum Laboratories, Inc.

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p 149

F-150APPENDIX

This chemical resistance chart is intended foruse as a guide, not as a guarantee of chemicalcompatibility. Variables in temperature, concentrations, durations of exposure and other factors
may affect the use of the product. It is recommended to test under your own conditions.

Chem
Resist ical
ance

The following codes are used to rate chemical resistance:
R . . . . . . . . .Recommended
L . . . . . . . . . .Limited Exposure
NR . . . . . . . .Not Recommended
U . . . . . . . . .Unknown

Membrane Compatibility Table

p 150

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CHEMICAL COMPATIBILITY CHART

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F-151

p 151

F-152APPENDIX

Index
A
Absolute Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Application Guides . . . . . . . . . . . . . . . . . . . . . .56,57,68,91,123

B
Backwashing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
Batch Clarification Operation . . . . . . . . . . . . . . . . . . . . . . . . .42
Batch Concentration Operation . . . . . . . . . . . . . . . . . . . . . . .42
Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . .109,110,112,117
Bioreactor Conversions . . . . . . . . . . . . . . . . . . . . . . . . . . . .114
Bioreactor Design Considerations . . . . . . . . . . . . . . . .109,112
Bubble Point Integrity Testing . . . . . . . . . . . . . . . . . . . . . . . . . .
Buffer Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53

Filters, Mesh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2,64,65
Filters, Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2,64,65
Filter Operational Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Filtration, Cross Flow . . . . . . . . . . . . . . . . . . . . .See Cross Flow
Filtration, Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Filtration, Tangential Flow . . . . . . . . . . . . . .See Tangential Flow
Filtration Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Flavor Recovery Modification . . . . . . . . . . . . . . . . . . . . . . . .100
Flow Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54

G
Gas Exchange in Bioreactors . . . . . . . . . . . . . . . . . . . . . . .113
Gas Permeable Membranes . . . . . . . . . . . . . . . . . . . . . . . . .15
Gel Controlled Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
Gel Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

H

C
Capillary Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
Capillary Pore Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Carboxylic Acid Removal from
Wastewater Streams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .93
Cell, Bacteria and Virus Processing . . . . . . . . . . . . . . . . . . . .47
Cell Implants for Drug Screening . . . . . . . . . . . . . . . . . . . . .127
Cell Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
Cell Processing . . . . . . . . . . . . . . . . . . . . . . . . .47,116,117,118
CellFlo Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .116
CellFlo Reactors . . . . . . . . . . . . . . . . . . . . . . . . . .116,117,118
CellGas Modules . . . . . . . . . . . . . . . . . . . . . . .113,116,119,120
CellMax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .127
CellMax Capillary Cell Culture . . . . . . . . . . . . . . . . . . .125,127
CellMax Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .126
Chemical Resistance . . . . . . . . . . . . . . . . . . . . . . . . .21,73,150
Chromatography Columns . . . . . . . . . . . . . . . . . . . . . . . . . .130
Chromatography Fraction Collectors . . . . . . . . . . . . . . . . . .137
Chromatography Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . .132
Chromatography UV Monitors . . . . . . . . . . . . . . . . . . . . . . .133
Clarification of Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Concentration Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43
Concentration Polarization . . . . . . . . . . . . . . . . .21,22,23,24,70
Constant Volume Diafiltration . . . . . . . . . . . . . . . . .11,26,42,43
Contact Angle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Conversion of Batch Reactors
to Continuous Perfusion . . . . . . . . . . . . . . . . . . . .114,115,117
Cross Flow . . . . . . . . . . . . . . . . . . . . . . . .1,9,19,29,32,33,34,35

D
Dead End Operation . . . . . . . . . . . . . . . . . . . . . . . . . .1,9,42,44
Depth Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1,2,5
Desalting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11,26,42,43
Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11,13,67

E
Electric Charge on Filters (zeta potential) . . . . . . . . . . . . . .4,28
Electrodialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11,13
Extending Membrane Life . . . . . . . . . . . . . . . . . . . . . . . . . . .44

Hollow Fiber Membranes, Modules
and Systems . . . . . . . . . . . . . . .34,35,38,39,40,41,58,59,60,61,

I
Integrity Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

K
KrosFlo Membranes and Modules . . . . . . . . . . . . . . . .40,41,61
KrosFlo Pilot System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
KrosFlo Pressure and Flow Monitor . . . . . . . . . . . . . . . . . . . .62

L
Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . .129
Liquid Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

M
Macrofiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11,64,65
Membrane and Module Configurations,
General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Membrane Controlled Flow . . . . . . . . . . . . . . . . . . . . . . . . . .22
Membrane Filters, Definitions . . . . . . . . . . . . . . . . . . . . . . .3,10
Membranes, Dialysis . . . . . . . . . . . . . . . . . . . . . . . . .7,71,72,73
Membranes, Gas Permeable . . . . . . . . . . . . . . . . . . . . . . . . .15
Membranes, Hollow Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . .See Hollow Fiber Modules and Systems
Membranes, Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .3,10
Membranes, Liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Membranes, Microfiltration, Definitions . . . . . . . . . . . . . . . .3,10
Membranes, Reverse Osmosis . . . . . . . . . . . . . . . . . . . . .11,14
Membranes, Ultrafiltration, Definitions . . . . . . . . . . . . . . .11,19
Mesh Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2,64,65
Microfiltration, Definitions . . . . . . . . . . . . . . . . . . . . . . . . .11,29
Microfiltration Applications . . . . . . . . . . . . . . . . . . . . . . . . . . .46
MicroKros Modules and Systems . . . . . . . . . . . . . . . . . . .38,58
MidiKros Modules and Systems . . . . . . . . . . . . . . . . . . . . . . .39
MiniKros Modules and Systems . . . . . . . . . . . . . . .40,41,59,60
Modules and Module Configurations, General . . . . . . . . . . .31
MTBE Removal from Wastewater Streams . . . . . . . . . . . . . . .96
MWCO Cutoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20,21,69,73

N

F

Nominal Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Factors Affecting Membrane Flow Rates . . . . . . . . . . . . . . . .24
Filters, Depth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1,2,5
Filters, Membrane, General . . . . . . . . . . . . . . . . . . . . . . . . . .3,5

P

p 152

Particle Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

INDEX

F-153

Permselective Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Phenolics Removal from Wastewater Streams . . . . . . . . . . . .91
Plugging Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Polymeric Membrane Extraction . . . . . . . . . . . . . .11,16,77-106

R
Retention Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . .5,19,20
Reverse and Forward Flushing . . . . . . . . . . . . . . . . . . . . .44,45
Reverse Osmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11,14

S
Scalability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36,37,39
Screen Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2,64,65
Selection of the Optimum System . . . . . . . . . . .37,56,57,63,71
Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
SeleXtrac Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
SeleXtrac Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . .78,83
SeleXtrac Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Separation of Similar Organics . . . . . . . . . . . . . . . . . . . . . .100
Spiral Wound Modules . . . . . . . . . . . . . . . . . . . . . . . . . . .33,34
Steam-In-Place (SIP) Modules . . . . . . . . . . . . . . . . . . . . .41,61
Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41,46,113

T
Tangential Flow . . . . . . . . . . . . . . . . . . . .1,9,19,29,32,33,34,35
Temperature Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Topped Off Batch Operation . . . . . . . . . . . . . . . . . . . . . . .42,43
Tortuous Pore Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Transmembrane Pressure . . . . . . . . . . . . . . . . . . . . . . . . .32,35

U
Ultrafiltration, Definitions . . . . . . . . . . . . . . . . . . . . . . . . . .11,19
Ultrafiltration Applications . . . . . . . . . . . . . . . . . . . . . . . . . . .51

W
Wastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
Woven Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2,64,65

Z
Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4,28

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

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F-154

APPENDIX

Figure A-5 Filtration and Bioprocessing Chart

Hollow Fiber Tangential Flow Filtration

Spectra/Porr® Dialysis Membranes

Relative Sizes
ofCommonMaterials

SeleXtrac® Molecular Extraction

CellMaxx® Cell Expansion

Separation Process

p 154

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

FILTRATION AND BIOPROCESSING CHART

Myoglobin

F-155

Pigments 100kD - 1µm

Cytochrome C
Vitamin B12

Collagen

Hemoglobin

Salts

0.2 - 30µm Bacteria

Viruses 0.005 - 0.1µm
Deoxyribonucleases

Antibiotics

Mycoplasm

Pyrogens 0.003 - 0.2 µm

SugarsInsecticide Dusts

Y
Yeast
Cells 0.3 - 8 µm

Pollens 10 - 100 µm

Carbon Black

Ribonucleases

Apoferritin

Trypsin

Glycoprotein (Human Plasma)
BSA

Red
Blood
Cells

Sand

Polio Virus

Aprotinin

Pseudomonas diminuta
AlbuminThyrogloblin

Insulin

IgG

IgM

Human Hair
T
TobaccoMosaic Virus

Colloidal Particles
Carbohydrates (Starch - Cellulose)
Dialysis / Ultrafiltration

E.coliMicrofiltration

Macrofiltration

THE ABCs OF FILTRATION AND BIOPROCESSING FOR THE THIRD MILLENNIUM

p 155