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Scale Up and Optimization in Preparative
Chromatography Principles and Biopharmaceutical
Applications Chromatographic Science 1st Edition Ajoy
Velayudhan Digital Instant Download
Author(s): Ajoy Velayudhan
ISBN(s): 9780824708269, 0824708261
Edition: 1
File Details: PDF, 3.10 MB
Year: 2002
Language: english
 Scale-Up and Optimization in
Preparative Chromatography
Principles and Biopharmaceutical Applications

edited by
Anurag S. Rathore
Pharmacia Corporation
Chesterfield, Missouri, U.S.A.

Ajoy Velayudhan
Oregon State University
Corvallis, Oregon, U.S.A.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

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 CHROMATOGRAPHIC SCIENCE SERIES

A Series of Textbooks and Reference Books

Editor: JACK CAZES

1. Dynamics of Chromatography: Principles and Theory, J. Calvin Giddings

2. Gas Chromatographic Analysis of Drugs and Pesticides, Benjamin J. Gudzinowicz
3. Principles of Adsorption Chromatography: The Separation of Nonionic Organic
Compounds, Lloyd R. Snyder
4. Multicomponent Chromatography: Theory of Interference, Friedrich Helfferich and
Gerhard Klein
5. Quantitative Analysis by Gas Chromatography, Josef Novák
6. High-Speed Liquid Chromatography, Peter M. Rajcsanyi and Elisabeth Rajcsanyi
7. Fundamentals of Integrated GC-MS (in three parts), Benjamin J. Gudzinowicz, Mi-
chael J. Gudzinowicz, and Horace F. Martin
8. Liquid Chromatography of Polymers and Related Materials, Jack Cazes
9. GLC and HPLC Determination of Therapeutic Agents (in three parts), Part 1 edited
by Kiyoshi Tsuji and Walter Morozowich, Parts 2 and 3 edited by Kiyoshi Tsuji
10. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L.
Hawk
11. Chromatography in Petroleum Analysis, edited by Klaus H. Altgelt and T. H. Gouw
12. Biological/Biomedical Applications of Liquid Chromatography II, edited by Gerald L.
Hawk
13. Liquid Chromatography of Polymers and Related Materials II, edited by Jack Cazes
and Xavier Delamare
14. Introduction to Analytical Gas Chromatography: History, Principles, and Practice,
John A. Perry
15. Applications of Glass Capillary Gas Chromatography, edited by Walter G. Jennings
16. Steroid Analysis by HPLC: Recent Applications, edited by Marie P. Kautsky
17. Thin-Layer Chromatography: Techniques and Applications, Bernard Fried and Jo-
seph Sherma
18. Biological/Biomedical Applications of Liquid Chromatography III, edited by Gerald L.
Hawk
19. Liquid Chromatography of Polymers and Related Materials III, edited by Jack
Cazes
20. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L.
Hawk
21. Chromatographic Separation and Extraction with Foamed Plastics and Rubbers, G.
J. Moody and J. D. R. Thomas
22. Analytical Pyrolysis: A Comprehensive Guide, William J. Irwin
23. Liquid Chromatography Detectors, edited by Thomas M. Vickrey
24. High-Performance Liquid Chromatography in Forensic Chemistry, edited by Ira S.
Lurie and John D. Wittwer, Jr.
25. Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca
26. HPLC Analysis of Biological Compounds: A Laboratory Guide, William S. Hancock
and James T. Sparrow
27. Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins,
Herbert Schott
28. HPLC in Nucleic Acid Research: Methods and Applications, edited by Phyllis R.
Brown
29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman and E. J. Levy
30. Modern Chromatographic Analysis of the Vitamins, edited by André P. De
Leenheer, Willy E. Lambert, and Marcel G. M. De Ruyter
31. Ion-Pair Chromatography, edited by Milton T. W. Hearn
32. Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography, edited by
Steven H. Y. Wong
33. Affinity Chromatography: Practical and Theoretical Aspects, Peter Mohr and Klaus
Pommerening
34. Reaction Detection in Liquid Chromatography, edited by Ira S. Krull
35. Thin-Layer Chromatography: Techniques and Applications. Second Edition, Re-
vised and Expanded, Bernard Fried and Joseph Sherma
36. Quantitative Thin-Layer Chromatography and Its Industrial Applications, edited by
Laszlo R. Treiber
37. Ion Chromatography, edited by James G. Tarter
38. Chromatographic Theory and Basic Principles, edited by Jan Åke Jönsson
39. Field-Flow Fractionation: Analysis of Macromolecules and Particles, Josef Janca
40. Chromatographic Chiral Separations, edited by Morris Zief and Laura J. Crane
41. Quantitative Analysis by Gas Chromatography, Second Edition, Revised and
Expanded, Josef Novák
42. Flow Perturbation Gas Chromatography, N. A. Katsanos
43. Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto, Kazuhiro Naka-
nishi, and Ryuichi Matsuno
44. Countercurrent Chromatography: Theory and Practice, edited by N. Bhushan Man-
dava and Yoichiro Ito
45. Microbore Column Chromatography: A Unified Approach to Chromatography, edi
ted by Frank J. Yang
46. Preparative-Scale Chromatography, edited by Eli Grushka
47. Packings and Stationary Phases in Chromatographic Techniques, edited by Klaus
K. Unger
48. Detection-Oriented Derivatization Techniques in Liquid Chromatography, edited by
Henk Lingeman and Willy J. M. Underberg
49. Chromatographic Analysis of Pharmaceuticals, edited by John A. Adamovics
50. Multidimensional Chromatography: Techniques and Applications, edited by Hernan
Cortes
51. HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M.
Gooding and Fred E. Regnier
52. Modern Thin-Layer Chromatography, edited by Nelu Grinberg
53. Chromatographic Analysis of Alkaloids, Milan Popl, Jan Fähnrich, and Vlastimil
Tatar
54. HPLC in Clinical Chemistry, I. N. Papadoyannis
55. Handbook of Thin-Layer Chromatography, edited by Joseph Sherma and Bernard
Fried
56. Gas–Liquid–Solid Chromatography, V. G. Berezkin
57. Complexation Chromatography, edited by D. Cagniant
58. Liquid Chromatography–Mass Spectrometry, W. M. A. Niessen and Jan van der
Greef
59. Trace Analysis with Microcolumn Liquid Chromatography, Milos KrejcI
60. Modern Chromatographic Analysis of Vitamins: Second Edition, edited by André P.
De Leenheer, Willy E. Lambert, and Hans J. Nelis
61. Preparative and Production Scale Chromatography, edited by G. Ganetsos and P.
E. Barker
62. Diode Array Detection in HPLC, edited by Ludwig Huber and Stephan A. George
63. Handbook of Affinity Chromatography, edited by Toni Kline
64. Capillary Electrophoresis Technology, edited by Norberto A. Guzman
65. Lipid Chromatographic Analysis, edited by Takayuki Shibamoto
66. Thin-Layer Chromatography: Techniques and Applications: Third Edition, Revised
and Expanded, Bernard Fried and Joseph Sherma
67. Liquid Chromatography for the Analyst, Raymond P. W. Scott
68. Centrifugal Partition Chromatography, edited by Alain P. Foucault
69. Handbook of Size Exclusion Chromatography, edited by Chi-San Wu
70. Techniques and Practice of Chromatography, Raymond P. W. Scott
71. Handbook of Thin-Layer Chromatography: Second Edition, Revised and Expanded,
edited by Joseph Sherma and Bernard Fried
72. Liquid Chromatography of Oligomers, Constantin V. Uglea
73. Chromatographic Detectors: Design, Function, and Operation, Raymond P. W.
Scott
74. Chromatographic Analysis of Pharmaceuticals: Second Edition, Revised and
Expanded, edited by John A. Adamovics
75. Supercritical Fluid Chromatography with Packed Columns: Techniques and Appli-
cations, edited by Klaus Anton and Claire Berger
76. Introduction to Analytical Gas Chromatography: Second Edition, Revised and Ex-
panded, Raymond P. W. Scott
77. Chromatographic Analysis of Environmental and Food Toxicants, edited by Taka-
yuki Shibamoto
78. Handbook of HPLC, edited by Elena Katz, Roy Eksteen, Peter Schoenmakers, and
Neil Miller
79. Liquid Chromatography–Mass Spectrometry: Second Edition, Revised and
Expanded, Wilfried Niessen
80. Capillary Electrophoresis of Proteins, Tim Wehr, Roberto Rodríguez-Díaz, and
Mingde Zhu
81. Thin-Layer Chromatography: Fourth Edition, Revised and Expanded, Bernard
Fried and Joseph Sherma
82. Countercurrent Chromatography, edited by Jean-Michel Menet and Didier
Thiébaut
83. Micellar Liquid Chromatography, Alain Berthod and Celia García-Alvarez-Coque
84. Modern Chromatographic Analysis of Vitamins: Third Edition, Revised and
Expanded, edited by André P. De Leenheer, Willy E. Lambert, and Jan F. Van
Bocxlaer
85. Quantitative Chromatographic Analysis, Thomas E. Beesley, Benjamin Buglio,
and Raymond P. W. Scott
86. Current Practice of Gas Chromatography–Mass Spectrometry, edited by W. M. A.
Niessen
87. HPLC of Biological Macromolecules: Second Edition, Revised and Expanded,
edited by Karen M. Gooding and Fred E. Regnier
88. Scale-Up and Optimization in Preparative Chromatography: Principles and Bio-
pharmaceutical Applications, edited by Anurag S. Rathore and Ajoy Velayudhan
89. Handbook of Thin-Layer Chromatography: Third Edition, Revised and Expanded,
edited by Joseph Sherma and Bernard Fried

ADDITIONAL VOLUMES IN PREPARATION

Chiral Separations by Liquid Chromatography and Related Technologies, Hassan

Y. Aboul-Enein and Imran Ali
 To Csaba Horváth
Mentor and Friend

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Preface

Preparative chromatography is arguably the most widely used purification

technique in the pharmaceutical and biotechnological industries. Scaling up a
preparative separation, however, continues to be seen as a difficult and time-
consuming task. While this perception may have had some validity a genera-
tion ago, it is not accurate today. In fact, many industrial separations can be
scaled up using one of the simple methods that have been described in the
literature. The goals of this book are to bring wider recognition to these simple
methods and to show that they are effective in many practical problems.
Another perception that persists is the view that results obtained in aca-
demia are either inapplicable or not applied to the day-to-day problems faced
by industrial professionals. While there is no doubt that a greater degree of
collaboration between academia and industry is desirable, it is nevertheless
true that useful results obtained in academia have been, and are being, used
in industry. Equally, the results and experience gained by industrial experts
have informed and refined the academic approach to these problems. The first
six chapters of this book describe the state of the art in methods and approaches
to scale-up and optimization of preparative chromatography. These chapters
are followed by a set of industrial case studies, which show how scale-up is
carried out for a variety of important separations.
The editors contribute an overview that includes a simple quantitative
approach as well as a discussion of the various practical aspects of scale-up.
Signposts are provided to later chapters in which more detail is provided on
specific topics that are discussed in earlier chapters.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Lightfoot et al. contribute an incisive overview to mass-transfer effects
in the current chromatographic context. In addition to laying out problems
that are currently being addressed, the chapter outlines fruitful lines of future
study.
There has been a great deal of work on numerical optimization of nonlin-
ear chromatographic separations, aimed at making it possible to obtain the
production rates and yields for every operational mode for a given separation,
thereby allowing a rational choice of the best operational mode for that separa-
tion. Felinger summarizes such work and indicates how these results can be
used to advantage in guiding the scale-up process.
Simulated moving beds (SMBs) are becoming increasingly popular in
preparative work, and may even have become the method of choice for some
enantiomeric separations. Antia’s chapter describes clearly and succinctly the
issues involved in designing and controlling SMB units.
Watler et al. give a detailed analysis of the theoretical issues that must
be addressed in successfully scaling up ion-exchange separations. The various
theoretical issues—selectivity, bandspreading, optimization—are presented in
a way that allows direct application to other separations.
Levison describes a variety of ways in which ion-exchange adsorbents
are used at large scale, going beyond batch and column modes to suspended
and fluidized beds. This detailed assessment provides a valuable complement
to Watler et al.’s analysis of theory applicable to ion-exchange chromatog-
raphy.
The second section of the book contains a set of industrial case studies
that provide the practical approach taken in industry to scale-up of realistic
separations. Fahrner et al. describe in detail the development of the crucial
cation-exchange step in the kilogram-scale purification of a recombinant anti-
body fragment. Useful discussions of appropriate analytical methods and
scouting for stationary phases are followed by the development of an opti-
mized stepwise elution protocol. The purification of supramolecular assembl-
ies is a topic of great current interest. Sagar et al. report their experiences
with large-pore adsorbents in the purification of plasmid DNA. The entire
purification sequence for producing monoclonal antibodies to tumor necrosis
factor (TNF) is detailed by Ng. A variety of ion-exchange steps as well as a
size exclusion step were used in this process. Miller and Murphy report on a
normal-phase separation of a synthetic intermediate that was scaled up to the
kilogram level. Guhan and Guinn describe the removal of a trace impurity
from the parenteral drug alatrofloxacin. Rathore provides a detailed develop-
ment of how a chromatographic process was implemented and optimized,
based on the simple approach to scale-up described in Chapter 1.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 The book as a whole is intended to provide a useful summary of the
methods and approaches current in preparative chromatography, enabling the
reader to transfer the insights gained here to her or his specific separation
problem. In addition to serving as a reference for industrial practitioners of
preparative chromatography, the material should be readily accessible to grad-
uate students in many disciplines including chemistry, pharmacy, bioengineer-
ing, and chemical engineering.
As the editors of this book, we wish to express our indebtedness to
Professor Csaba Horváth of Yale University, in whose research group we both
received our doctorates, and to whom the book is dedicated. There is little
doubt that science is one of the areas in which apprenticeship to an expert
continues to be the premier way to gain, both explicitly and osmotically, in-
sight into the field. Discussing scientific problems with Csaba—how to
choose, approach, and solve them—was a vital part of our training in becom-
ing independent researchers.

Anurag S. Rathore
Ajoy Velayundhan

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Contents

Preface
Contributors

Part I Methods and Approaches

1 An Overview of Scale-Up in Preparative Chromatography

Anurag S. Rathore and Ajoy Velayudhan

2 Interaction of Mass Transfer and Fluid Mechanics

Edwin N. Lightfoot, John S. Moscariello, Mark A. Teeters,
and Thatcher W. Root

3 Optimization of Preparative Separations

Attila Felinger

4 Engineering Aspects of Ion-Exchange Chromatography

Peter Watler, Shuichi Yamamoto, Oliver Kaltenbrunner,
and Daphne N. Feng

5 A Simple Approach to Design and Control of Simulated

Moving Bed Chromatographs
Firoz D. Antia

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 6 Large-Scale Ion-Exchange Chromatography: A Comparison
of Different Column Formats
Peter R. Levison

Part II Case Studies

7 Development and Operation of a Cation-Exchange

Chromatography Process for Large-Scale Purification
of a Recombinant Antibody Fragment
Robert L. Fahrner, Stacey Y. Ma, Michael G. Mulkerrin,
Nancy S. Bjork, and Gregory S. Blank

8 Case Study: Capacity Challenges in Chromatography-Based

Purification of Plasmid DNA
Sangeetha L. Sagar, Ying G. Chau, Matthew P. Watson,
and Ann L. Lee

9 Case Study: Purification of an IgG1 Monoclonal Antibody

Paul K. Ng

10 Case Study: Normal Phase Purification of Kilogram

Quantities of a Synthetic Pharmaceutical Intermediate
Larry Miller and James Murphy

11 Case Study: Development of Chromatographic Separation

to Remove Hydrophobic Impurities in Altrafloxacin
Sam Guhan and Mark Guinn

12 Case Study: Process Development of Chromatography Steps

for Purification of a Recombinant E. coli Expressed Protein
Anurag S. Rathore

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Contributors

Firoz D. Antia, Ph.D. Department of Chemical Engineering Research and

Development, Merck & Co., Inc., Rahway, New Jersey, U.S.A.

Nancy S. Bjork Department of Analytical Chemistry, Genentech, Inc.,

South San Francisco, California, U.S.A.

Gregory S. Blank, Ph.D. Department of Recovery Sciences, Genentech,

Inc., South San Francisco, California, U.S.A.

Ying G. Chau Department of Chemical Engineering, Massachusetts Insti-

tute of Technology, Boston, Massachusetts, U.S.A.

Robert L. Fahrner Department of Recovery Sciences, Genentech, Inc.,

South San Francisco, California, U.S.A.

Attila Felinger, Ph.D. Department of Analytical Chemistry, University of

Veszprém, Veszprém, Hungary

Daphne W. Feng, B.S. Department of Process Development, Amgen, Inc.,

Thousand Oaks, California, U.S.A.

Sam Guhan, Ph.D. Department of Bioprocess Research and Development,

Pfizer Global Research and Development, Groton, Connecticut, U.S.A.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Mark Guinn, Ph.D. Department of Bioprocess Research and Development,
Pfizer Global Research and Development, Groton, Connecticut, U.S.A.

Oliver Kaltenbrunner, Dipl. Ing. Dr. Department of Process Develop-

ment, Amgen, Inc., Thousand Oaks, California, U.S.A.

Ann L. Lee, Ph.D. Merck Research Laboratories, Merck & Co., Inc., West
Point, Pennsylvania, U.S.A.

Peter R. Levison, B.Sc., M.B.A., Ph.D. Department of Science and Tech-

nology, Whatman International Ltd., Maidstone, Kent, England

Edwin N. Lightfoot, Ph.D. Department of Chemical Engineering, Univer-

sity of Wisconsin–Madison, Madison, Wisconsin, U.S.A.

Stacey Y. Ma, Ph.D. Department of Analytical Chemistry, Genentech, Inc.,

South San Francisco, California, U.S.A.

Larry Miller Department of Global Supply Early Process Research and De-
velopment, Pharmacia Corporation, Skokie, Illinois, U.S.A.

John S. Moscariello Department of Chemical Engineering, University of

Wisconsin–Madison, Madison, Wisconsin, U.S.A.

Michael G. Mulkerrin, Ph.D. Department of Analytical Chemistry, Genen-

tech, Inc., South San Francisco, California, U.S.A.

James Murphy Pharmacia Corporation, Skokie, Illinois, U.S.A.

Paul K. Ng Purification Development, Department of Biotechnology, Phar-

maceutical Division, Bayer Corporation, Berkeley, California, U.S.A.

Anurag S. Rathore, Ph.D. Department of Bioprocess Sciences, Pharmacia

Corporation, Chesterfield, Missouri, U.S.A.

Thatcher W. Root, Ph.D. Department of Chemical Engineering, University

of Wisconsin–Madison, Madison, Wisconsin, U.S.A.

Sangeetha L. Sagar, Ph.D. Merck Research Laboratories, Merck & Co.,

Inc., West Point, Pennsylvania, U.S.A.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Mark A. Teeters, B.S. Department of Chemical Engineering, University of
Wisconsin–Madison, Madison, Wisconsin, U.S.A.

Ajoy Velayudhan, Ph.D. Department of Bioresource Engineering, Oregon

State University, Corvallis, Oregon, U.S.A.

Peter Watler, B.S., M.S., Ph.D. Department of Process Development, Am-

gen, Inc., Thousand Oaks, California, U.S.A.

Matthew P. Watson, B.S. Merck Research Laboratories, Merck & Co., Inc.,
West Point, Pennsylvania, U.S.A.

Shuichi Yamamoto, Ph.D. Department of Chemical Engineering, Yama-

guchi University, Ube, Japan

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 1
An Overview of Scale-Up in
Preparative Chromatography

Anurag S. Rathore
Pharmacia Corporation, Chesterfield, Missouri, U.S.A.

Ajoy Velayudhan
Oregon State University, Corvallis, Oregon, U.S.A.

I. INTRODUCTION

Preparative chromatography continues to be the dominant purification tech-

nique in the production of biological compounds, especially in the pharmaceu-
tical and biotechnological industries. However, the conceptual complexity of
a purely theoretical approach to preparative chromatography is formidable,
because we are dealing with systems of highly coupled, nonlinear partial dif-
ferential equations [1,2]. Although theoretical work is progressing, it can cur-
rently capture predictively only a few aspects of realistic biotechnological sep-
arations, especially given the extremely complex biochemical feedstocks often
used in these applications. It is not entirely coincidental that the current ap-
proach to scale-up and optimization in industry is highly empirical. Although
this is natural, especially given the constraints of process validation, the first
few chapters of this book attempt to show that current theoretical understand-
ing does give insight into the practical issues involved in scale-up and optim-
ization. These chapters show that a careful combination of basic theory with
experiments can reduce the time needed to achieve an effective scale-up of a
realistic chromatographic separation.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 It will be convenient to introduce some terminology [3] to clarify the
ensuing discussion. The various kinds of physiochemical interactions that are
used in chromatography to produce selectivity are called modes of interaction.
Examples include electrostatic interactions in ion-exchange or ion chromatog-
raphy, hydrophobic interactions in reversed-phase and hydrophobic interaction
chromatography, and specific interactions in affinity chromatography. Once a
mode of interaction has been chosen, the various ways in which a separation
can be achieved (isocratic or gradient elution, stepwise elution, displacement,
frontal analysis) are called modes of operation. For many separations, the best
mode of interaction is easily specified, and scale-up or optimization focuses
on the choice of mode of operation.
Finally, when the concentrations of all adsorbable components are low
enough to lie within the linear or Henry’s law region of their respective adsorp-
tion isotherms, the separation is called linear. Even if one component’s con-
centration reaches the nonlinear region of its (multicomponent) adsorption
isotherm for some fraction of the separation, the process is called nonlinear.
The basic ideas for scale-up and optimization given in the beginning
chapters are applied to real separations in the subsequent chapters in which
industrial case studies are presented. An issue of practical importance in a
separation sequence is that of how to achieve the global optimum in the param-
eter of interest (typically maximum productivity or maximum recovery or min-
imum cost; mixed or combined optimization criteria are also possible). This
issue is not discussed in detail in this chapter, because Chapter 3 deals with
it comprehensively. Further, the case studies in subsequent chapters often al-
lude to constraints from one separation step limiting or otherwise affecting
the choice of conditions in other steps.
The structure of this chapter is as follows. An introductory section on
method development places in perspective the various steps involved in arriv-
ing at an effective separation protocol at the bench scale. This is, of course,
a necessary preliminary to scale-up, which by definition seeks to maintain
upon scale-up the quality of a separation that has already been developed.
Section III begins with heuristic rules for scale-up and then develops a simple
quantitative model that clarifies when such heuristic rules can be used with
reasonable accuracy. The issue of bed heterogeneity and its implications for
scale-up are also discussed. In Section IV practical considerations characteris-
tic of the various modes of interaction and operation are described briefly.
Although considerations of space preclude the full discussion of all these is-
sues, key points are brought out and important references in the literature are
highlighted.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 II. METHOD DEVELOPMENT

Method development is a multistep process that precedes scale-up and opera-

tion at large scale. The general practice is to perform optimization at small
scale due to relatively smaller requirements of material and resources as well
as the ease of performing several runs in a parallel fashion.

A. Decoupling of Thermodynamics from Kinetics

A variety of parameters—choice of stationary and mobile phases, the particle
size of the stationary phase, the column dimensions, the flow rate, the feed
loading—affect the production rate and recovery obtained in a preparative
separation. Trying to understand the interplay of all of these parameters simul-
taneously is a daunting task. In addition, testing all the various possibilities
experimentally is likely to be extremely tedious and is impractical under typi-
cal industrial constraints. However, the following simplification is available
to us at little cost. It is likely that equilibrium parameters (the choice of station-
ary and mobile phases, leading to selectivity) can be selected independent of
“kinetic” parameters such as flow rate, feed loading, and particle size. Such
a decoupling of thermodynamic and kinetic parameters is probably rigorously
justifiable only in linear chromatography, but even in nonlinear chromatogra-
phy it is likely that choosing the mobile and stationary phases first does not
significantly decrease the attainable production rates and recoveries.
The first step is therefore to choose the most effective mode of interac-
tion. This is often clear from the fundamental properties of the feedstock or
the product. Other important factors include the objective and nature of the
separation problem, literature precedents, and prior experience with the prod-
uct. Inputs from the vendors of chromatographic media and instrumentation
may also be useful at this stage. The strategy is depicted schematically in Fig.
1 [4].

B. Optimization of Thermodynamics at Bench Scale

We present here a simple and rapid approach to the thermodynamic component
of method development. We take the view that for many separations the choice
of stationary phase is far more important than the choice of mobile phase (this
is particularly true of ion-exchange runs, where standard salts are used as
mobile phase modulators). Of course, there are many cases where specific
binding of various kinds can require the use of special additives for the mobile

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Figure 1. Strategy for optimization of a chromatographic separation.

phase, but we ignore these situations in order to make the general approach
clear. We therefore intend to use a standard mobile phase, and we wish to
screen a variety of stationary phases rapidly and equitably, i.e., we have re-
duced the problem to one of resin screening.
Once a list of resin candidates has been prepared, screening is performed
to select the best resin to perform a particular separation. Selection of the
resin for a chromatography step is perhaps the most important step in method
optimization [4–9]. A resin screening protocol is illustrated in Fig. 2. In most
cases the primary criterion for resin screening is selectivity. However, other
screening criteria may also be identified and used depending on the particular
separation problem.
The general approach is as follows (the specifics in what follows are
for ion-exchange chromatography in the gradient mode of operation, but the
arguments can easily be generalized to other contexts). The process takes place
in two stages.
Stage 1. All stationary phases are packed into columns of identical
size. If possible, all columns should be run at the same flow rate. This is not
always practical (e.g., if the particle sizes available for different stationary
phases are markedly different, then pressure drop constraints may limit the
range of flow rates). Run a test gradient that spans a wide range of modulator
levels, so that feed retention is facilitated. Make the gradient as shallow as

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Figure 2. Resin screening protocol. (Reprinted courtesy LCGC North America, Ad-
vanstar Communications, Inc.)

practicable, in order to simultaneously get as much resolution as possible un-

der these conditions. Stationary phases that exhibit little or no retention of the
product are excluded at this stage. In addition, if almost no resolution is found
between the product and the primary impurities, these stationary phases may
be excluded. This latter decision should be made carefully, because the test
gradient may not be a fair indicator of a sorbent’s resolution. In other words,
a sorbent may provide poor resolution of the product under the test gradient
but high resolution under another gradient. Thus, the latter decision is to be
made only if there is good reason to believe that this sorvent is unlikely to
be effective.
Stage 2. For each of the stationary phases remaining, determine a
tailored comparison gradient that is intended to show each sorbent under its
most effective conditions for the given feed mixture. Parameters such as the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 feed loading and equilibration buffer should be kept the same for all sta-
tionary phases. If the flow rate was the same for all runs in stage 1, then it
should be maintained in this stage. If different sorbents were run with differ-
ent flow rates in stage 1, then use the same flow rate for each sorbent in this
stage.
The comparison gradient is centered around the modulator concentration
at which the product eluted in the test gradient in stage 1. Then, making the
assumption that the band spreading of the peaks is inversely proportional to
the gradient slope, all other parameters being constant, we have
α w ⫽ βm (1)
where α and w are respectively the gradient slope and product peak width in
the test gradient, and β and m are the corresponding parameters in the compari-
son gradient. If we require that all comparison gradients have the same time
(for standardization), then the starting and ending modulator concentrations
(cx and cy, respectively) can be determined from the equations
αntwF
cx ⫽ celution ⫺ (2)
2mV
and
αntwF
cy ⫽ celution ⫹ (3)
2mV
where celution and tW are respectively the concentration and time at which the
center of the product peak eluted in the test gradient, n is the number of column
volumes over which the comparison gradient is run, F is the flow rate, and V
is the column volume. Note that if the beginning concentration cx is found to
be negative from Eq. (2), it is set to zero.
The comparison gradient provides an equitable way of comparing differ-
ent stationary phases for the given feed mixture, because each stationary phase
is provided with a gradient that is optimized for its particular retention behav-
ior. Now the usual quantitative parameters of production rates and recovery
and purity can be used to determine which stationary phase is best.
The simple approach described above provides a rapid way to choose
the best resin. However, if the chromatography step is intended for operation
at preparative scale, particulary for commercial manufacture, several other
issues must be addressed before final resin selection. These include the cost
of the resin, the physical and chemical stability of the resin at the bed height
and the number of cycles to be used at the manufacturing plant, media avail-
ability with respect to the demand at commercial scale, resin lifetime, leaching

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 of ligands, regulatory support files offered by the vendor, batch-to-batch vari-
ations in resin quality, etc.
It should be noted that the assumption that peak width is inversely propor-
tional to gradient slope is an approximation and is not always expected to be
valid (e.g., significant competition among the product and impurities for bind-
ing sites on the adsorbent could cause the assumption to fail). However, it is
likely to be a reasonable approximation for many realistic separations. More
detailed methods of this kind can be established (see, e.g., Quarry et al. [10])
but would usually require more data for each sorbent. Similarly, Jandera et al.
[11] and Jandera [12] determined optimal gradients in normal and reversed-
phase systems through numerical optimization of the governing equations; this
is a significant advance in the field but is not yet at the level of accessibility
where industrial practitioners would use it routinely. The method outlined here
was chosen for its simplicity and ease of use in an industrial context.
This approach to resin screening is now demonstrated in detail for a
practical separation problem. Rathore [4] showed that the stationary phase of
choice for an anion-exchange separation was found rapidly using this ap-
proach.

1. Resin Screening for an Anion-Exchange Chromatography Column

This case study presents data obtained during the optimization of an anion-
exchange chromatography column used in the process of purifying a protein
molecule derived from microbial fermentation.
Nine anion-exchange resins—BioRad High Q, BioRad DEAE, Phar-
macia DEAE FF, Pharmacia Q FF, Pharmacia Q HP, Whatman Q, Whatman
QA52, Whatman DE53, and TosoHaas Q650M—were chosen for screening.
All chromatography experiments were performed using an Äkta Explorer
(Amersham Pharmacia Biotech). The buffer and other operating conditions
were chosen on the basis of prior experience with the molecule. (Pre-equilibra-
tion buffer: 1 M Tris, pH 8.5. Equilibration buffer: 50 mM Tris, pH 8.5. Protein
loading: 10 mg/mL resin.) Because the objective of this chapter is to lay out
an efficient resin screening protocol and not to recommend a particular resin,
the resins that were used will be referred to as resins 1–9 (not in the order in
which they are named above). The optimum resin is expected to vary with
the separation problem.
Columns were packed with 1 mL of resin and equilibrated for 30 min
with the equilibration buffer. Equilibration was followed by loading of protein
solution containing 1–2 times the intended protein loading for the respective
column (mg protein/mL resin). After 30 min, a wash was performed with 5
mL equilibration buffer and the “flow-through” stream was collected. Protein

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Table 1 Final Comparison of Resins Considered

Recoveryb,c Pool Final

a b b
Resin Binding Selectivity (mAU/mL) purityc (%) decisionb

Resin 1 X
Resin 2 ⻫ X
Resin 3 ⻫ X
Resin 4 ⻫ X
Resin 5 ⻫ X
Resin 6 ⻫ ⻫ X
Resin 7 ⻫ ⻫ 75.4 95.6 X
Resin 8 ⻫ ⻫ 25.3 100 X
Resin 9 ⻫ ⻫ 81.5 97.7 ⻫
a
Resins included BioRad DEAE, BioRad High Q, TosoHaas Q650M, Whatman Q, Whatman
QA52, Whatman DE53, Pharmacia DEAE FF, Pharmacia Q FF, and Pharmacia Q HP, not num-
bered in this order.
b
⻫ ⫽ Satisfactory column performance; X ⫽ unacceptable column performance.
c
Based on measurements by anion exchange HPLC (AE-HPLC)
Source: Reprinted courtesy of LCGC North America, Advanstar Communications Inc.

was eluted with 10 mL of elution buffer (1 M NaCl in the equilibration buffer),

and the eluant was collected separately. The flow-throughs and the eluants
were analyzed for protein by UV absorbance at 280 nm and for its purity
by anion-exchange high performance liquid chromatography (AE-HPLC). As
shown in Table 1, it was found that most resins showed satisfactory binding
characteristics with the product. Only resin 1 showed anomalous behavior in
that the product was not retained under these conditions, so resin 1 was not
considered further.
Next, columns were packed with 10 mL of the remaining eight resins,
and separations were performed using an identical test gradient of 0–500 mM
NaCl in 20 column volumes (CV) of equilibration buffer. Peak fractions were
analyzed by AE-HPLC. Figure 3 illustrates the performance of resins 3, 4,
and 5 under a test gradient of 0–500 mM NaCl in 20 CV. The Y axis in Figs.
3 and 4 denotes the peak area obtained upon analysis by AE-HPLC (mAU)
per unit injection volume (µL). The flow velocity and the fraction sizes are
given in the figure legends. It is evident that running identical gradients with
different resins leads to very different elution profiles in terms of the peak

Figure 3. Column performance under test gradients. (Reprinted courtesy LCGC

North America, Advanstar Communications, Inc.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
 Table 2 Calculation of the Comparison Gradients

Test Comparison
Migration gradient, Gradient Column Flow gradient,
time Elution start–end volume volume velocity start–end
Resina (min) (mM) (mM) (CV) (mL) (mL/min) mM

Resin 7 5.21 110 100–200 20 10 1.7 90–130

Resin 8 6.61 109 100–200 20 10 0.9 80–130
Resin 9 7.35 146 100–300 20 10 1.7 100–200
a
Same as in Table 1.
Source: Reprinted courtesy of LCGC North America, Advanstar Communications Inc.

width and peak position in the overall gradient. The poor selectivity obtained
with resins 3–5 led to their elimination from further consideration.
As listed in Table 1, it was found that only resins 6–9 showed satisfac-
tory selectivity between the product and the impurity. Moreover, because resin
9 exhibited better resolution than resin 6 and they had identical matrix and
ligand chemistry, the former was chosen over the latter for further consider-
ation.
Comparison gradients were calculated according to the procedure de-
scribed above for resins 7–9. Product recovery was defined as the sum of
product peak areas (in mAU) in the pooled fractions (having ⬎90% purity by
AE-HPLC) per milliliter of injected sample. Pool purity was defined as the
purity of the total pool formed by mixing the fractions that meet the pooling
criteria. Table 2 shows the calculation of the comparison gradient for these
three resins, and Fig. 4 illustrated the protein and impurity profiles that were
obtained after fraction analysis by AE-HPLC.
Figure 4 reinforces the understanding that performing separations with
the designed “comparison gradients” yields very similar elution profiles with
different resins and leds to a fair comparison of resin performance. It also
follows from Fig. 4 that resin 8 showed good purity but poor recovery. Resins
7 and 9 showed comparable recovery and pool purity. However, because of
its better selectivity, resin 9 was chosen as the resin for this purification process
and selected for further optimization of buffer pH, protein loading, feed flow
rate, elution flow rate, gradient slope, and column length.

Figure 4. Column performance under comparison gradients. (Reprinted courtesy

LCGC North America, Advanstar Communications, Inc.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
 C. Optimization of Kinetics (Operating Conditions)
at Bench Scale
Once the stationary and mobile phases have been chosen, we turn to the deter-
mination of optimal operating conditions, i.e., determination of the kinetic, as
opposed to thermodynamic, contributions. Thus, the particle size and column
dimensions are determined in these studies, along with the optimal gradient
slope and feed loadings. The following general approach is suggested.
First, experiments are performed to evaluate the effect of various op-
erating parameters that affect resin performance in terms of the selectivity and
protein loading. These parameters may include the mobile phase conditions
(pH, organic content, buffer composition, etc.) and the gradient slope and de-
sign. Optimum mobile phase conditions and the gradient design are chosen
from the experimental data obtained.
Next, the effect of flow velocity and protein loading on the quality of
separation is evaluated and, on the basis of resin performance, the bed height,
protein loading, and flow velocity are chosen to obtain satisfactory resolution
and cycle time. It is desirable that laboratory experiments be done at the bed
height that will be used at pilot scale in order to obtain comparable column
performance at large scale.
A detailed analysis of the interaction among these kinetic parameters is
complicated and is not described here. Many of the underlying issues are
brought out clearly by Felinger in his chapter on optimization (Chap. 3). In
industrial practice, a heuristic approach similar to the one just described is
often used. It is likely to produce effective, if not necessarily optimal, op-
erating conditions in the hands of an experienced practitioner. More details
of these practical approaches are given in several of the industrial case studies
in this book.
This separation of very large molecules and particles such as viruses is
an important industrial topic and is beginning to be addressed in the literature
[13,14]. However, the field is still in its infancy and is likely to change rapidly.
We therefore do not feel that it would be appropriate to attempt a summary
here, and we refer the reader to the growing literature on this subject.

III. THEORETICAL CONSIDERATIONS IN SCALE-UP

A. Physical Overview
The performance of a chromatography column depends on a variety of design
and operating factors. In order to have a successful scale-up it is desirable to

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Figure 5. Three different scales of columns used frequently during process develop-
ment.

maintain kinetic (particle size, pore size, ligand chemistry, temperature, mo-
bile phase) and dynamic (bed height, flow velocity, packing density) equiva-
lence between the chromatography columns used in the laboratory and the
pilot plant. This objective can be accomplished by using identical stationary
and mobile phases in the two columns and operating them at identical bed
height, linear flow velocity, protein loading (mg protein per mL of resin), feed
conditions, gradient length, and gradient slope [6]. To handle the increased
volume of load at pilot scale, the most common procedure used to increase
column volume is to increase the column diameter so that the column volume
increases proportionately [8,15]. This keeps the residence time of the product
constant and avoids causing any product stability issues.
Figure 5 is a schematic illustration of the three sizes of columns that
are often used at laboratory, pilot plant, and commerical scales. Scouting ex-
periments in the laboratory are mostly done in small columns to conserve
the materials and also because several experiments can be done in parallel
simultaneously at lab scale. However, as discussed above, it is extremely im-
portant to maintain bed height constant while scaling up, so the best approach
is to perform the final optimization steps at the bed height that will later be
used at the pilot plant and commercial scale. This approach is illustrated in
Fig. 6 and 7.
These general considerations are frequently used in industry as the basis
for scale-up. In the next section, a quantitative analysis is given that shows
when such simple “volumetric” scale-up can be used and describes alternatives
that are appropriate when the column length must be changed on scale-up.
The van Deemter equation is widely used to characterize band broaden-
ing in a chromatography column and is expressed as

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Figure 6. Scaling from laboratory (or bench) to pilot-plant scale.

Figure 7. Scaling up from pilot-plant scale.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 H ⫽ A ⫹ B/u ⫹ Cu (4)
where u is the linear flow velocity, H is the plate height of the column, and
A, B, and C are constants. The plate height H is equal to the length of the
column divided by the total number of plates N, so H is smaller for a more
efficient column. A reflects the quality of the packing of the column and is
independent of the linear flow velocity. A is small when the column is packed
well and is homogeneous throughout its length. B is a measure of the band
broadening due to longitudinal diffusion of the sample components along the
edge of their respective bands as they travel across the column. It decreases
with increasing linear flow velocity because the sample components spend
less time undergoing diffusion inside the column. C includes contributions
from the binding kinetics (adsorption/desorption) as well as the mass transfer
of the sample components to and from the packing particles.
Preparative chromatography is usually carried out at high flow velocity
in order to increase throughput. Then the C term usually dominates Eq. (4),
leading to the simplified form
H ⫽ Cu, or, equivalently, N ⫽ L (1/Cu) (5)
In an ideal case, when the column packing and operating conditions are
kept the same while scaling up (C is a constant), the scale-up involves just a
volumetric increase in column dimensions. For such a case, Eq. (5) can be
rewritten as
L/u ⫽ CN (6)
To preserve the efficacy of separation, the total number of plates is to be kept
constant, so it follows from Eq. (6) that if the bed height needs to be increased
or decreased for some reason (e.g., pressure drops too high), the linear flow
velocity might also be altered appropriately so as to keep the ratio of L/u
constant. This ensures a constant number of plates in the column (N), and
the column performance is maintained. This simple analysis is expanded and
generalized in the following section. In particular, if the particle size needs
to be changed upon scale-up (for economic or other reasons), the more general
treatment must be used.
This very simple physical introduction to scale-up sets the scene for a
straightforward quantitative analysis of the problem in the next section.

B. Simple Scale-Up Calculation

The basic idea behind scale-up is to preserve the quality of the separation
achieved at small scale [16,17]. Implicit in this approach is the admission that

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 we are not yet able to determine optimal operating conditions a priori for
different scales of operation. Thus, we settle for determining effective, near-
optimal operating conditions at bench sale. Effective scale-up rules should
then produce comparable results at larger scale. (The alternative approach of
finding optimal operating conditions is currently practicable for some impor-
tant classes of separation problems; this approach is discussed in Chapter 3).
A typical scale-up from laboratory to pilot plant is on the order of 50–
100-fold. This is frequently followed by a 10–50-fold scale-up from pilot plant
to final commercial manufacturing scale.
The usual approach is to hold the plate count constant upon scale-up
and increase the feed volume and column volume proportionately. This ap-
proach was originally based on the assumption of linear adsorption. Later in
this section we discuss how this assumption can be relaxed.
If the subscripts b and l are used to describe parameters at bench and
large scale, respectively, we have

Nl ⫽ Nb (7)

Vfeed,l V
⫽ feed,b (8)
Vcolumn,l Vcolumn,b

If band spreading is dominated by pore diffusion, as is often the case

in realistic separations [18–20], then the plate count can be described by

L
N⬃ (9)
ud 2p

where L is the column length, u the mobile phase linear velocity, and dp the
particle diameter. The proportionality constant includes geometrical factors
such as the phase ratio and thermodynamic factors such as the retention factor.
This result can be derived from the van Deemter equation [16]. Combining
Eqs. (7) and (9) we get

Ll Lb
⫽ (10)
2
ul d p,l ub d 2p,b

This represents one constraint on the three variables Ll, ul, dp,l. Recall that this
approach is based on mimicking bench-scale results at large scale; the vari-
ables Lb, ub, dp,b are therefore assumed to be known.
Quantifying the pressure drop across the columns give another result;
we use Darcy’s law in the form

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 d 2p ∆p
u⫽k (11)
µ L
where k is the permeability without the dependence on particle diameter, which
has been factored out, and µ is the mobile phase viscosity. In general, best
results are obtained at the maximum allowable pressure drop [2]. If the maxi-
mum permissible pressure drop is different at bench and large scales, there
follows
ul Ll uL
⫽ P b2 b (12)
d 2p,l d p,b
where P is the ratio of maximum pressure drops at large and bench sale. In
the common case where P ⫽ 1, the result becomes
ul Ll ub Lb
⫽ 2 (13)
d 2p,l d p,b
Dividing Eq. (10) by Eq. (13) gives the simple expression:
ul ⫽ ub (14)
Thus equality of plate counts and maximum pressure drops leads to equality
of mobile phase velocity across scales. Substituting Eq. (14) into either Eq.
(10) or Eq. (13) gives the familiar result
d 2p,l d 2p,b
⫽ (15)
Ll Lb
Typically, the choice of particle size at the large scale is limited by cost or
availability. Once a particle size is chosen, equation (15) specifies the column
length. An approximate theoretical calculation for the optimal d 2p /L is given
in Guiochon et al. [2]; this can also be used to give another estimate of the
column length, given the particle size. The chapter by Felinger (Chap. 3) dis-
cusses such optimal calculations in detail.
Finally, in order to determine the column diameter at large scale, Eq.
(8) can be rewritten as
Vfeed,l
Vfeed,b
V
⫽ σ ⫽ column,l ⫽ l
冢 冣
L D2c,l
Vcolumn,b Lb D2c,b
(16)

Here, σ is the scale-up factor (which must be specific before scale-up can
begin) and Dc is the column diameter. Because the column length at large

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 scale has been determined from Eq. (15), the column diameter is obtained
from the equation

冢 冣
1/2
Dc,l L
⫽ σ l (17)
Dc,b Lb

When the particle diameter is kept constant on scale-up, we obtain the results
for scale-up by “volume overloading”: From Eq. (15), the column length re-
mains constant (in addition to the particle diameter and the mobile phase ve-
locity); thus, scale-up consists simply of increasing the column diameter by
a factor of √σ.
These results have been obtained for pore diffusion as the process that
dominates band spreading. Analogous results for the case where external mass
transfer (film diffusion) is controlling are given by Pieri et al. [21], Grushka
et al. [16], and Ladisch and Velayudhan [22]. The case where pore diffusion
and film diffusion are comparable has been considered briefly by Ladisch and
Velayudhan [22]. A more general treatment including the effects of axial dis-
persion (which is typically negligible for liquid chromatography) is found in
Lee et al. [23].
Although the approach taken above was based on linear adsorption
[which allowed the specialization of the van Deemter equation to obtain
Eq. (9)], extensions to nonlinear adsorption are possible. Knox and Pyper [24]
showed that Eq. (15) applied for scale-up of noninteracting compounds whose
bands in isocratic elution can be approximated as right triangles (i.e., band
spreading is dominated by isotherm nonlinearity). Wankat and Koo [25]
showed that Eq. (6) holds for single compounds that have nonlinear single-
component isotherms. Golshan-Shirazi and Guiochon [26,27] demonstrated
that Eq. (12) also holds for two compounds with binary Langmuirian
isotherms. Wantak [28] presents a general scale-up argument based on the
constancy of plate count at both scales, which also results in Eq. (12) for
pore-diffusion-controlled runs and applies even for arbitrary multicomponent
isotherms. In fact, it is quite possible that similar results hold for multicompo-
nent adsorption as long as the isotherms are locally concave downward (so
that the leading edge of a band is self-sharpening and the trailing edge shows
a “proportionate pattern”). The approach described above is therefore a good
starting point for scale-up, although variations due to more complex adsorption
behavior should be kept in mind.
There has been some discussion in the past about whether small particles
are needed in scale-up, particularly when the column is highly overloaded.
This is based on the view that under conditions of strong overloading the band

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 spreading caused by isotherm nonlinearity dominates that caused by “kinetic”
factors such as film and pore diffusion that depend on particle size. There is
an important distinction to be drawn here. Under such conditions that the
product band can be separated from its nearest impurities “thermodynami-
cally,” i.e., when a sequential stepwise elution schedule can be found such
that no impurity elutes in the step in which the product elutes, then it is clear
that plate count and its causal kinetic factors are unimportant. Such thermody-
namic separations are sometimes possible when on–off binding of the feed
components occurs, i.e., the feed components are either very strongly bound
or almost completely unbound. This kind of “all-or-nothing” adsorption often
occurs for macromolecules [29,30]. The same mechanism is often exploited
in solid-phase extraction protocols. Under these conditions, it is clear that large
particles can be used with impunity, at both bench scale and process scale.
The only problem in scaling up such a separation is the possibility of overload-
ing the column too heavily. Because the product is selectively displaced from
binding sites by more retentive impurities, it is possible that the product may
move faster than expected and thus emerge in more than one step of the step-
wise elution schedule. This problem can be avoided by reducing the loading
or increasing the column length.
However, there are many separations of practical importance for which
such stepwise elution protocols cannot be found. Then the co-migration of
the product peak with one or more impurities must be considered, and now
kinetic factors play a vital role in determining the extent of mixing between
adjacent peaks in the chromatogram [2,31] and thus recovery and production
rates. In such cases, isocratic elution, gradient elution, and displacement chro-
matography are used and are reasonably well described by the general scale-
up equations developed above. More detailed models, especially for gradient
elution, are presented in Chapter 4 of this volume by Watler et al.

C. Constancy of Phase Ratio with Scale

An important assumption in the calculations above was that the phase ratio
(and therefore the interstitial and intraparticulate porosities) remained constant
upon scale-up. This is not always a good assumption when the overall scale-
up factor is very large (above 100). A recent example of such variations in
phase ratio is given in Heuer et al. [32]. In practice, it is worthwhile to estimate
the phase ratio experimentally at each scale and use the results above with
caution if the phase ratio changes appreciably with scale.
Experimental techniques to avoid such changes in packing structure as
the column diameter increases include axial compression, radial compression,

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 and annular expansion (mixed radial and axial compression). Useful reviews
of these packing methods are found in Jones [33,34] and Colin [35]. In many
cases, these approaches have results in improved column performance.
Fundamental and applied work on packing continues, and results from
soil physics and other fields are being applied to the chromatographic problem
(e.g., Bayer et al. [36] and Cherrak and Guiochon [37]). It is too early to say
to what extent packing heterogeneities can be removed by further improve-
ments in technology, but such observations raise the question of whether a
lumped parameter like the phase ratio is sufficient to capture packing geome-
try. Close collaboration between manufacturers, industrial users, and academ-
ics is needed to arrive at highly effective designs for process columns. The
next section includes some practical observations on packing properties as a
function of scale under the heading of bed stability.
An assessment of these and related issues from a fundamental viewpoint
is presented in Chapter 2 of this book by Lightfoot et al.

IV. PRACTICAL CONSIDERATIONS IN SCALE-UP

A. Practical Guidelines
In practice, several issues must be kept in mind when attempting to scale up
a separation. In this subsection, these issues are dealt with briefly. They will
recur constantly in the case studies discussed in subsequent chapters.

1. Bed Stability (Physical)

In a laboratory scale column the column wall offers support to the column
bed and contributes to the stability of the column. However, when the col-
umn is scaled up and its diameter increases, the wall support contribution
to bed stability starts to decrease. For column diameters greater than 25–30
cm, the lack of wall support may become an issue and could cause redistri-
bution of packing particles and settling of the bed. The total drag force on
the packing particles is a function of the liquid velocity, the liquid viscos-
ity, and the bed height. The supporting force that keeps the particles in
place decreases with increasing column diameter as a smaller fraction of
the particles are supported by the column wall. Therefore, for large-scale
columns with compressible packings, the maximum velocities are restricted
and decrease with increasing column diameter under identical bed height
and pressure drop, and the situation worsens with duration of column use
[38–41]. This phenomenon is more prominent for nonrigid gel materials

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 and is often reversible within limits but almost always with a marked hys-
teresis [39,42]. As illustrated in Fig. 6, the issue of physical stability of the
column bed becomes particularly significant at large scale because the bed
height is larger than in the lab and the column is put to more frequent reuse
during commercial manufacturing. The bottom of the column is most vul-
nerable because it feels the pressure drop across the column as well as that
due to the weight of the column itself, which can be appreciable for large
columns. These effects must be taken into account for robust column de-
sign. For cases where bed compression is a problem and the maximum per-
missible bed height is smaller than the minimum required to obtain satisfac-
tory separation, a suggested solution is to use stacked columns [42]. This
reduces the pressure difference across any single section of the column and
permits the use of smaller particles and nonrigid gels to obtain enhanced
resolution.

2. Bed Stability (Chemical)

Chemical stability of the packing material includes any factors that may result
in deterioration of the column performance over a period of use. It may be the
leaching of ligands into the mobile phase as often in affinity chromatography
destruction of the matrix in the mobile phases used for column operation,
regeneration, or storage (e.g., silica packings at high pH) or irreversible bind-
ing at the packing surface [41]. The issue of chemical stability of the column
bed becomes particularly significant when the column is reused many times
during commercial manufacturing.

3. Product Loading
The product loading (milligrams of product loading per milliliter of resin) is
generally held constant during scale-up. In most cases the resolution is found
to decrease with increasing product loading after the loading has reached a
certain level. Further, this behavior is more prominent when the paricle size
is small. To ensure a successful scale-up and successful operation at large
scale, studies must be conducted at lab scale to determine the maximum prod-
uct loading with which satisfactory resolution can still be achieved. It is com-
mon to operate the column at 80–90% of this maximum loading.

4. Gradient Separations
Gradient elution is widely used owing to its ability to provide higher effi-
ciency, reduced process times and solvent consumption, and a concentrated

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 product stream. However, as process scale increases, buffer volumes increase
also, and it becomes increasingly difficult to form accurate and reproducible
gradients. Thus, either the possibility of performing step gradients should be
explored or the ability of the chromatography skid to perform adequate buffer
mixing and form controlled gradients must be evaluated [15].

5. Flow Distribution
For large diameter columns, uniform flow distribution at the column head may
become difficult to achieve. This may result in deviations from the desired
plug flow and lead to peak tailing. Use of a flow distributor at the column
inset is generally found to be the most effective way of ensuring uniform flow
distribution in the column [41]. The rational design of inlet and outlet headers
to ensure uniform distribution is discussed at length in Chapter 2.

6. Packing Quality
Packing large columns such that the resulting packed column is homogeneous
is very critical for obtaining uniform flow distribution. Channeling inside the
column often leads to peak tailing and/or peak splitting. A variety of ap-
proaches have been developed by the different chromatographic equipment
vendors to alleviate this problem. The most popular technique at preparative
scale is axial compression and the use of self-packing columns [35]. Some
degree of compression has been known to enhance resolution [41]. The con-
nection between packing and the phase ratio used in quantitative scale-up was
touched upon in the previous section.

7. System Design
The system dead volume arising from the piping and other support equipment
for chromatography columns such as the valves, flow meters, air sensors, and
tubings is much larger at pilot and particularly manufacturing scale than at
lab scale. This leads to dilution effects and higher pressure drops as well as
additional band broadening, so the impact these factors may have on the over-
all column performance must be evaluated. The general guidelines are to keep
the system dead volume to the minimum, to have bypasses through devices
such as air traps and filters for use during sample load, and to choose tubing
diameter to achieve turbulent flow so as the reduce undesirable axial mixing
[43]. Further, the chromatographic system should be designed such that all
inlet sources are at or above the level of the column, whereas all outlet sinks

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 are at or below the level of the column. This ensures that the column is not
being operated against a hydrostatic pressure head.

8. Fraction Collection
The peak width and shape as seen in the chromatogram depends on several
factors such as the column dimensions, extracolumn effects, operating condi-
tions, and sample volume [42]. Thus, even if the scale-up is carried out follow-
ing a well-thought-out methodology, it is still very likely that the peak width
and shape may differ from that obtained at lab scale. Thus, the fraction collec-
tion strategy must be revisited at preparative scale based on column perfor-
mance at that scale. The critical monitoring device, e.g., the UV detector,
should be placed as close to the fraction collector as possible to ensure good
representation of the process stream. Also, it is good to have a fraction collector
that can collect fractions based on several process parameters, such as UV ab-
sorbance, conductivity, time, volume, first or second derivative of the signal, etc.

9. Media Availability
Although selectivity for a separation may be the primary criterion for selection
of a resin for analytical separation, several other factors need to be considered
before a resin is selected for preparative separation. These include the avail-
ability of large quantities of the resin, cost, continuity of supply, batch-to-
batch consistency, column lifetime, and support documentation to aid in regu-
latory filing [44].

10. Costing
The cost of the feedstock is generally not given adequate consideration when
the process optimization is carried out at bench scale. However, as the process
is scaled up, the process should be modeled and raw material and facility costs
must be examined. Resin costs usually account for the biggest contribution
to the raw material costs. Thus, although a certain resin may offer the best
selectivity at bench scale, it may be too expensive, under linear scale-up, at
process scale [8].

11. Sample Pretreatment

Often cells or inclusion bodies undergo a rupture step by homogenization or
some other mechanism prior to a chromatography step with the purpose of
capturing the product from the cell culture or fermentation broth. In these
situations the process stream is replete with lipids, nucleic acids, proteins, and

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 other macromolecular contaminants. These impurities affect the passage of
the product through the column and have a very significant effect on column
performance. Because most cell-rupture operations are more effective at large
scale than at bench scale, pretreatment of the process stream at large scale
to remove all the interfering contaminants may become crucial to ensuring
satisfactory and robust column performance [8].

12. Scale-Down
Although our focus is on scale-up, it is worth mentioning that some issues
related to validation are often addressed by scaling down. Thus, using a small-
scale study for viral clearance is often faster, safer, and cheaper while being at
least as accurate as larger scale studies [45]. Many of the scale-up approaches
discussed above apply, mutatis mutandis, to scale-down.

B. Other Modes of Operation

Although isocratic and gradient elution are the most common modes of opera-
tion and displacement chromatography has a small but significant role, there
are other modes of interest at large scale. In this context, frontal chromatogra-
phy has always played an important role under the guide of “adsorption steps”
in a variety of applications, especially in the chemical industries. The process
of feed introduction in isocratic, gradient, and displacement runs is nothing
more than frontal chromatography, so it is clearly an important part of the
run. Here, expanded bed chromatography and simulated moving bed (SMB)
chromatography are discussed briefly. Though the basic scale-up rules men-
tioned above are applicable to both these techniques, there are some unique
considerations that are worth highlighting.

Expanded Bed Chromatography

An expanded bed consists of specially designed packing particles that are flu-
idized in a column with controlled flow distribution so as to provide a large
number of plates (high mass transfer) with minimal back-mixing. Thus, al-
though expanded bed chromatography (EBC) offers more plates than batch
adsorption, it also allows better utilization of binding capacity of the adsorbent
[46].
Scale-up in EBC is a little more complicated than in other modes of
chromatography because of the additional requirements of maintaining the
stability of the expanded bed. The flow distribution, flow velocity, composi-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 tion, and other physical/chemical properties of the feed and the particle distri-
bution and the physical/chemical stability of the adsorbent can all have a sig-
nificant effect on the bed stability. Conditions optimized for high productivity
may not be able to provide long-term stability, so a process optimized at small
scale should be carefully evaluated with these two objectives in mind [47].

Simulated Moving Bed Chromatography

Simulated moving bed chromatography (SMB) is a continuous chromato-
graphic process in which a mobile phase and the sample components are in-
jected into and withdrawn from a ring of chromatography columns at points
that are rotating between the columns during the process. SMB is slowly be-
coming the technique of choice for performing efficient enantiomeric separa-
tions [48].
The key to successful SMB operation is proper selection of the operating
flow rates and the valve-switching times of the feed and eluant streams [49].
Pumps typically used in this technique provide control of flow rates of better
than 1%. These are important both for stability of the different zones and for
achieving satisfactory separation. Just as in other modes of chromatography,
the efficiency of an SMB separation is negatively impacted by extracolumn
volume, which in this case consists of the volume of the recycling pump, flow
meter, pressure sensor, and valves. Antia (Chapter 5) describes a simple and
effective approach to the design and control of SMB separations.

C. Practical Considerations for Modes of Interaction

As with modes of operation, there are many practical issues peculiar to each
mode of interaction. The general features of each mode are discussed briefly
in this section. Again, the relevance of these issues in the design of practical
separations will be highlighted by the design choices made in the case studies
described in the following chapters.

1. Ion-Exchange Chromatography
Separation in ion-exchange chromatography (IEC) takes place because of dif-
ferential ionic interactions between the charged ligands on the stationary phase
and the charged sample components in the feed in the presence of aqueous
buffer solution. Elution is then performed with increasing salt concentration
by altering the pH of the mobile phase, resulting in weakening of the ionic
forces, with the components eluting in the order of increasing binding strength
with the stationary phase.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Ion-exchange chromatography is the most widely used mode of chroma-
tography for protein separation. This is due to the high dynamic capacities
and low relative costs of the IEC resins, simple buffers used, high usable
flow rates, and the robustness, scalability, and ease of operation of most IEC
methods. Most IEC resins have large volumetric capacities, with operation
limited only by the total concentration of the product and contaminants in
the feed. Scale-up in IEC can be performed following the generic guidelines
mentioned above, and it is the technique of choice as an early processing step
due to the large qualities of material that have to be handled [50,51].
Watler et al. (Chap. 4) and Levison (Chap. 6) provide a clear and com-
prehensive discussion of the use of IEC in preparative separations.

2. Hydrophobic Interaction Chromatography

Separation in hydrophobic interaction chromatography (HIC) is based on dif-
ferences in hydrophobicity of the different sample components. At high salt
concentrations, the solubility of the hydrophobic product is reduced and the
hydrophobic side chains of the sample component associate with the hy-
drophobic ligands on the stationary phase [52]. Elution can be performed by
reducing the polarity of the mobile phase in a continuous or stepwise manner.
Because of the differences in the mechanisms of separation of HIC and IEC,
HIC is frequently used to process the eluant stream from an IEC column.
Scale-up in HIC can be performed by following the generic guidelines
outlined above [53–55]. However, there are several considerations peculiar
to HIC that must be kept in mind. Denaturation of the product molecule may
occur at high salt concentrations during the separation process; the mobile
phase may be too viscous and thus impair the accuracy and reproducibility of
gradient formation, apart from limiting the usable bed height; and temperature
variations may change column performance considerably.

3. Size Exclusion Chromatography

The separation in size exclusion chromatography (SEC) is due to differences
in the sizes and shapes of the sample components as they are carried by the
mobile phase through the three-dimensional porous structure of the stationary
phase. In most cases the maximum bed height and diameter that can be used
are limited by the physical stability of the gel. This is resolved by using a
series of smaller columns instead of a single long column during scale-up.
Silica and agarose matrices are most commonly used in SEC and often contain
negatively charged moieties at the surface that may cause adsorption of posi-
tively charged proteins and the exclusion of negatively charged proteins at
low ionic concentrations.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 In SEC, although the separation time increases linearly with column
length as in other forms of chromatography, the resolution increases only as
the square root of the length. Also, the resolution of the sample components
is more sensitive to the width of the sample band than the flow velocity. One
of the suggested strategies for SEC optimization and scale-up is to select the
bed height and linear flow velocity to obtain the desired cycle time and satis-
factory resolution [56,57]. Next, the width of the sample zone is varied to
fine-tune resolution. Finally, the column diameter is changed to meet the pro-
ductivity requirements for the column.
Feed volume in SEC normally varies between 1% and 5% of the total
column volume to obtain satisfactory resolution for most large-scale separa-
tions and up to 30% for desalting. Resins used in SEC have low binding capac-
ities and are often compressible, so the separations are generally performed
at very low flow velocities, and all the issues mentioned earlier in Section
IV.A.1 apply. Therefore, SEC is not considered by most an ideal technique
for scale-up except for desalting or buffer exchange or as a final polishing step
when the volumes and sample quantities are small enough (as in purification of
human serum albumin).

4. Reversed-Phase Chromatography
Separation in reversed-phase chromatography (RPC) is based on the differ-
ences in the hydrophobicities of the different sample components. The hy-
drophobic groups on the surface of the sample molecules bind with the very
hydrophobic stationary phase particles in the presence of an apolar mobile
phase. Elution occurs when a mobile phase is used with increasing concentra-
tion of an organic phase such as acetonitrile, methanol, or isopropanol. The
components elute in the order of their hydrophobicity, and a separation is
achieved.
Reversed-phase chromatography is very popular in the separation of small
molecules. Its limited use in protein separation, particularly at large scale, is due
to the various issues that are associated with the use of the organic solvents in
the purification process. These include protein denaturation and/or unfolding,
waste handling, and the need for special explosion-proof handling due to solvent
volatility. Scale-up in RPC follows the guidelines outlined above.

5. Affinity Chromatography
The separation of a target molecule from a mixture of species in affinity chro-
matography (AC) takes place by virtue of specific and reversible binding with
a ligand that is immobilized on the matrix. This technique offers short process

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 times and high specificity and resolution and is particularly useful when the
target is present in very small quantities in the complex mixture. The primary
issues in this technique are that the ligands may be expensive and unstable
and may leach from the matrix and be present in the product. Fouling and
regeneration of the ligand upon use may also offer challenges, so AC is gener-
ally used as the final polishing step in the purification scheme [58].
Scale-up in AC is carried out following the generic guidelines outlined
in the previous section [59–62]. The bed height is kept constant, and column
productivity is increased by increasing column diameter. However, resins used
in AC are often compressible, so all the issues mentioned earlier in Section
IV.A.1 apply and should be considered during scale-up.
Another approach to scaling up affinity separations is to increase the
capacity of the adsorbent. This may be accomplished by increasing the concen-
tration of the binding ligand that is coupled to the support particles. However,
the binding capacity does not increase linearly with the ligand concentration
and depends on the characteristics of the ligand as well as those of the binding
product. This route for scale-up is therefore sensitive to the problems of leach-
ing of ligands and fouling of resins. In addition, there is a partical limit on
the maximum attainable ligand concentration on the resin surface. Thus, the
final decision should depend on the comparison of economics and issues asso-
ciated with the two approaches highlighted above.

6. Metal (Chelate) Chromatography

The separation and operation principles in metal (chelate) chromatography
(MC) are very similar to those in affinity chromatography. Separation is based
on interactions between the affinity tail on the sample component (such as
dihistidyl tag) with the complexed heavy metals such as zinc and nickel on
the stationary phase [9]. MC can be as efficient as affinity chromatography,
particularly in the removal of endotoxins.
Scale-up issues for metal chromatography are very similar to those of
affinity chromatography. The resins are generally custom-made and are rela-
tively very expensive. The affinity tail often needs to be clipped before the
end of the purification process. Care must be taken to avoid denaturation of
protein feedstocks by the metals used in MC.

V. CONCLUSIONS

In this chapter we have provided an overview of the basic principles and prac-
tice of scale-up in the preparative chromatography. Modes of interaction and

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 modes of operation were defined to clarify the options available upon scale-
up. Because it is necessary to optimize a bench scale separation before attempting
to scale it up, attention was first focused on method development at bench scale.
Thermodynamic issues (resin screening) and kinetic issues (determination of op-
erating conditions) were decoupled. A simple but generally applicable approach
to resin screening was described. After a physically motivated introduction to
scale-up, a straightforward calculation based on pore diffusion as the controlling
kinetic contribution was presented and should suffice for many realistic separa-
tions. More detailed analyses of the fundamental chromatographic processes,
modeling approaches to important modes of interaction and operation, and indus-
trial case studies are given in the subsequent chapters.

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Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Exploring the Variety of Random
Documents with Different Content
The Swift Construction Company never had so much bother with a
contract in its life. When those fellows want some more machinery,
I’d say, let them apply to some one else. We’re fed up with them.”
“Right you are,” agreed Tom. “Just what part of Texas did you
ship it to?”
“Copperhead,” answered Ned. “That’s right in the heart of a
newly developed oil region.”
“Copperhead,” repeated Tom. “Sounds like a snake.”
“There’ll be some snakes there when the Hankinshaw crowd get
on the job,” affirmed Ned.
“I think we’ll make that our destination,” said Tom thoughtfully.
“Why?” asked Ned.
“Well,” said Tom, “we’ve got to pick out some place to begin, and
that’s probably as good as any other. Then, too, I have some
curiosity to see what Hankinshaw and his brother snakes are doing.”
“Whom they’re doing, you mean,” suggested Ned.
“I suppose that would be more correct,” said Tom with a smile.
“What I’m thinking of especially is that old blind man that Mr. Damon
told us about. You remember, don’t you, that he said that the
Hankinshaw crowd were out to swindle him out of his property?
Perhaps we can put a spoke in their wheel.”
Ned groaned.
“Great Scott, Tom!” he exclaimed. “Can’t you stick to business
and stop looking for trouble?”
 CHAPTER XV
OFF FOR THE OIL FIELDS

Tom grinned at the earnestness in Ned’s tone.

“Don’t be worried, old boy,” he said. “We’ll give all the attention to
business that it needs. But if on the side we can at the same time
help an old fellow along, why shouldn’t we? But that’s a matter that
we can settle when we come to it. All we have to do now is to get
ready for the trip.”
Before he left for Texas, Tom went up again to the hospital to get
news of the young aviator.
“Oh, he’s getting along,” replied Dr. Sherwood to Tom’s question.
“That is, physically. His leg has mended, though he still has to use a
cane. But his head isn’t clear yet. He can’t talk intelligently. As a
matter of fact, perhaps he never will.”
“You don’t mean that he may be insane for the rest of his life!”
exclaimed Tom, genuinely shocked.
“That’s among the possibilities,” affirmed the doctor. “Though
considering his youth and vitality—he’s a strong young fellow—the
chances are that he’ll recover. Still, no one can tell. You can go out
and take a look at him yourself if you like. He’s sunning himself on a
bench in the garden.”
Tom went out and seated himself on the bench beside the invalid.
He had a good look at him for the first time under anything like
normal conditions. The young aviator had evidently been sinewy and
stalwart when in health, judging from his frame, though now thin from
his long illness. His face must have been a pleasant one, though
marred now by the vacant look in his eyes.
Tom tried to get in conversation with him.
“How are you, Hillobie?” he asked, using the name at a venture.
The boy looked at him without any sign of interest and made no
reply. Tom tried again several times but fruitlessly, and at last had to
give it up. He left the hospital in a depressed mood, wondering if,
after all, he had done the young man a real service in saving his life.
Of what use was life without reason?
The Swift Construction Company hurried things along, and in
about a week Tom was ready for the start. The young inventor had
decided to take Koku with him, as his great strength and loyal
devotion might prove invaluable. The giant was in great glee and
grinned from ear to ear when apprised of Tom’s decision, but Rad
was thoroughly disgusted.
“To hev dat big chunk o’ beef clutterin’ up the plane!” he snorted.
“Doan know whut you must be t’inkin’ uv, Marse Tom.”
“But don’t you see, Rad,” said Tom soothingly, trying to keep a
serious face, “that I wouldn’t dare go off and leave this place alone
without you to take care of things? How could I have an easy minute
unless I could keep saying to myself: ‘Everything’s all right at home.
Rad’s on the job?’ ”
The old negro bridled up proudly.
“Guess yo’s right, Marse Tom,” he chuckled, all his resentment
vanishing. “Didn’t see it dat way befo’. Takes brains to run dis yere
house. An’ when it cums to brains, dat big grampus Koku ain’t dere.
Nussah, he jes’ ain’t dere.”
So peace was reëstablished between Tom’s faithful retainers,
each of whom thought the other had special reason to envy him.
At last all was ready. Tom had had a long and tender interview
with Mary, the final directions had been given for the running of the
works during the absence of Tom and Ned, and one bright morning,
with many of Tom’s friends and all the workmen assembled to bid the
voyagers Godspeed, the Winged Arrow rose like a huge bird from
the grounds of the plant, soared high in air to the altitude of two
thousand feet, and turned her nose toward the oil fields of Texas.
Tom had figured that, barring accidents and with ordinary good
luck, he would be able to reach his destination on the following day.
This was asking a good deal of the Winged Arrow, but the result
justified Tom’s confidence. The engines ran like a dream, the
weather was superb, neither fog nor storm intervened, and on the
afternoon of the following day those in the airship sighted the town of
which they were in search.
 It lay beneath them sprawling in the heat of the Texas sun, one of
the hastily built pioneer towns that spring up like magic in the wake
of an oil strike. It was a mere collection of wooden shacks that
looked as though little more than a breath would be required to blow
them down. But they sufficed for the immediate needs of the hardy
adventurers who were seeking a quick road to fortune. Many had
found it, many more hoped to find it. The man “stony broke” in the
morning might be a millionaire before night.
Tom circled about, looking for a likely landing place. This was not
a matter of much difficulty, for there was a host of open spaces on
the outskirts of the town. He soon found one suited to his purpose,
and the Winged Arrow came down as softly as a swan.
Scarcely had she stopped before the passengers were out on the
ground, stretching their cramped legs on Texas soil. It was a
delightful change after their long confinement in the plane. The
warmth, too, was congenial, contrasting as it did so strongly with the
chill of the upper air. They were in high spirits over the successful
termination of the flight. Tom and Ned laughed aloud, and Koku was
one broad grin.
“So far, so good,” remarked Tom, patting the Winged Arrow
proudly. “You certainly are the goods, old girl. Now for the town and a
hot meal. You’ll have to stay here and mind the plane, Koku, but we’ll
see that you get yours later.”
In a few minutes they were in the town. It was their first
experience in such surroundings, and they looked about them
curiously.
There was no pretence at order or regularity in the shacks that
served as dwellings and business houses. It seemed as though they
had been built wherever the traveler had dropped his pack. There
was one main street, a long, straggling, crooked thoroughfare, from
which a number of smaller streets branched off here and there at
irregular intervals. The houses were of the rudest description. Two or
three men and one day would have sufficed to build most of them.
Many of them were of the one-story type with one or two rooms and
earth floors. Others, more pretentious, had two stories, the lower part
occupied for business purposes and the upper floor as a residence.
Let the mere tail-end of a Texas norther come along and they would
all have been leveled to the ground like a pack of cards.
Most of the “business” houses were saloons and dance halls.
The prohibition law was largely a dead letter as far as Copperhead
was concerned. From almost every door the young men passed
came the rattle of dice and the clink of bottle against glass, the
wheeze of an old accordion or raucous jazz music from a
phonograph.
Through the main street passed an almost endless column of
wide-wheeled trucks with tugging horses straining in the harness, the
trucks themselves loaded with iron casings, and, some of them, with
red flags at the back, carrying enough nitro-glycerine to blow the
town sky-high in the event of a collision. Weaving in and out of these
were dusty automobiles, mule carts driven by negroes, “buggies,”
every kind of conveyance, some of them looking as though they
dated back to Revolutionary times. Other vehicles were parked in
rows about saloons, on the front porches of which loungers sat in
tipped-back chairs.
And derricks! There were derricks everywhere, some of them in
the town itself, in back yards where the precious fluid had been
discovered. Some of the buildings were plastered with oil that had
spattered against them in a black flood when a strike had been
made. And all about the town for as far as the eye could reach rose
a multitude of derricks in a perfect forest, towering, some of them, to
the height of eighty or a hundred feet.
Through the roughly dressed multitudes that thronged the
principal street, Tom and Ned threaded their way. Airplanes had
become common in that locality, and no one paid any especial
attention to the aviator suits in which the youths were clad. There
was little choice as to restaurants. None of them was good, and it
was only a question of which was the least bad. Even this could not
be determined at a glance, and the lads finally entered one that
seemed to be at least no worse than the others.
Nor was there much choice as to food. The rough-looking waiter
in a dirty apron told them they could have corned beef and cabbage
or ham and eggs. They ordered the latter, which soon made their
appearance, accompanied by cups of weak, muddy coffee. Then,
while they ate, they looked curiously about them.
The restaurant, like all others in the town, was only an adjunct to
a saloon, and the sale of drinks was much more profitable than that
of food. Before the bar a long line of the thirsty stood.
Suddenly Ned nudged Tom.
“Look who’s here,” he whispered.
 CHAPTER XVI

ON THE TRAIL OF FORTUNE

Tom looked in the direction indicated, and among the men

standing at the bar saw Hankinshaw. The mottled face was even
redder than usual.
“Can’t seem to avoid that fellow,” remarked Tom disgustedly.
“He’s a regular jinx.”
Either Hankinshaw had not seen them or he had not recognized
them in their aviators’ suits, for he paid no attention to them. He took
one drink more and then lurched unsteadily out of the place.
Tom and Ned finished their meal and went to the shack that
served as post-office and telegraph station. Tom sent off messages
to his father and Mary, announcing their safe arrival, and then made
some inquiries of the telegraph operator, a bright young college boy
who was “fingering the key” to earn some extra money during his
vacation.
“Is the town of Goby near here?” asked Tom.
“No town of that name in this section,” was the answer. “But
there’s a Goby farm owned by a man of that name about four miles
from here.”
“I guess that’s the place I have in mind,” said Tom, “though from
what a friend of mine told me I thought it was the name of a town. Is
the owner of it a blind man?”
“Yes,” was the answer. “A nice old fellow he is, too, and he has a
daughter that’s a perfect peach.”
“Tell me about him, will you?”
“I understand that he’s a Northerner who came down to this part
of the country to regain his health. Since he came here he’s gone
blind. I imagine he’s had rather hard sledding to get along on his half
section of land. That is, before the oil craze began. As far as I can
learn, his property is right in the midst of the oil region, and he’ll
probably be able to sell it at a good price. That is, if he doesn’t get
cheated out of it. Some of these oil prospectors are a pretty slick lot.
They’d steal the penny off a dead man’s eyes, and they’d rob a blind
man as quickly as they’d take a drink.”
“I suppose so,” remarked Tom. “You say that this farm is about
four miles from here. Could you give us more exact directions?”
The obliging operator could and did, and Tom and Ned hurried
back to their plane, taking with them two quarts of coffee and a
double portion of ham and eggs and rolls that they had had put up
for the faithful Koku.
While the giant feasted, Tom took his bearings, and as soon as
Koku had finished they climbed into the Winged Arrow and turned it
in the direction of the Goby farm. To make a mile a minute was
nothing to the powerful plane, and in less than five minutes it was
hovering over a homestead which answered to the description that
the operator had given. It was in slightly rolling country with several
hills in the vicinity, differing from the unrelieved flat plain on which
Copperhead stood. It was a pleasant place and seemed like an oasis
amid the throng of derricks that reared themselves on every side.
The house itself was a substantial two-story structure with a
sloping roof. There were white curtains at the windows and a perfect
riot of flowers at the front and around two sides of the building. The
whiteness and the daintiness of the curtains seemed to show the
presence of a feminine hand.
Tom made a landing about three hundred feet from the house. A
movement among the curtains showed that the roar of the engine
had attracted attention from at least one of the inmates. Leaving
Koku with the plane, Tom and Ned made their way to the house.
Tom knocked and the door was promptly opened by one of the
most charming girls imaginable. She was slightly above medium
height, had a perfect figure, beautifully formed features, and wavy
chestnut hair and limpid brown eyes. It was evident that the
enthusiastic young telegraph operator had not erred when he had
described her.
To Tom, with his mind and heart full of Mary, she was simply a
very pretty girl. To Ned, she seemed a heavenly vision, the sweetest
thing he had ever seen.
“Does Mr. Goby live here?” asked Tom, removing his cap.
 “Yes,” was the reply, in a soft, musical voice that completed Ned’s
undoing. “I am his daughter. Won’t you walk in?”
“My name is Swift,” said Tom, as they accepted her invitation,
“and this is my friend, Mr. Newton. We came to see your father on a
little business matter.”
“If you will sit down,” she said, as she ushered them into the living
room and indicated chairs, “I will call him.”
She vanished, followed by Ned’s eyes. Tom, looking at his friend,
saw him staring at the door through which she had disappeared.
“Hard hit, old boy?” he bantered. “Come out of your trance.”
Ned glared at him, but before he could frame a suitable retort the
young girl came back, accompanied by a man whom she introduced
as her father. Then she excused herself, flushing a trifle as she
caught the all too evident admiration in Ned’s eyes.
Mr. Goby was a medium-built man somewhat over fifty, with a
kindly and intelligent face. He wore a pair of colored spectacles,
evidently to cover his loss of sight. The young men took to him at
once.
After a few preliminary remarks, Tom launched into the object of
their visit. He told him that he and a few of his friends had decided to
go into an oil venture, and were looking about for a likely spot to
commence operations. They had selected the Copperhead district
and had noted that his farm was right in the center of the producing
field. Did Mr. Goby care to sell or lease all or part of his property? If
he did, Tom thought they might make a deal, as he and his
associates were prepared to offer perfectly fair terms.
The blind man listened attentively, though, for a time, the boys
could not tell whether interestedly or not.
“Well,” said he, “it seems highly probable that oil abounds on this
farm of mine. All my friends have told me so, and the fact that rich
strikes have been made all around it seems to confirm the
probability. Then, too, I’ve had a number of offers from speculators
and prospectors. One group in particular have been especially
pressing. A man named Thompson with two of his friends have
seemed very anxious to make some arrangement. But—” here Mr.
Goby hesitated for a moment—“perhaps it isn’t fair to say so, but
there’s something about them that doesn’t seem to ring quite true.
What they offer sounds fair enough, but, somehow, I don’t quite feel
as though I could trust them. My daughter, Carol, who is pretty
shrewd, feels the same way about it.”
“I see,” murmured Tom, nodding.
“You see,” continued Mr. Goby, after some further talk concerning
the offer Tom was prepared to make, “I’m at a disadvantage on
account of my blindness. So I have to leave most of my business
affairs in the hands of an old friend of mine, Judge Wilson, of the
district court. I’ve referred this Mr. Thompson and his partners to the
judge, but for some reason or other they seem reluctant to deal with
him. They say that I’m the owner of the property, and they’d rather
deal direct with me than through my attorney. But how can I sign
papers that I haven’t read? To be sure, my daughter could read them
to me, but even then I’m not versed in legal matters, and there might
be some clause in the contract that would seem perfectly innocent
and yet be used to rob me. This farm is all I have in the world, and I
have to look out for the future of my daughter.”
 CHAPTER XVII

CLOSING THE DEAL

“You are perfectly right, Mr. Goby,” declared Tom. “Men who
refuse to submit a contract to a lawyer are to be distrusted on
general principles. No honest man would object. As far as myself
and my associates are concerned, we are ready, with your approval,
to submit our proposition to Judge Wilson and have him draw up the
papers.”
“That sounds fair,” replied the blind man. “If you like, I’ll have my
daughter telephone to the judge and ask him to come over to-
morrow. By the way, where are you stopping in town?” Mr. Goby had
taken a liking to the boys, both so frank and friendly.
“Why, the fact is,” answered Tom, “we haven’t made any
arrangements yet. We just reached here to-day and came right over.
We’ll have to fix that up when we go back.”
“You don’t need to do anything in a rush,” said Mr. Goby heartily.
“We have plenty of room here, and maybe we could let you stay with
us, especially if we come to a deal to work the farm for oil. You might
stay to-night, if you care to.” And so, a little later, with Carol’s
consent, it was arranged.
“Seem to be mighty nice people, Ned,” remarked Tom, after the
young inventor and his chum had been shown to a room where they
might wash and make themselves otherwise presentable.
“You are right, Tom; and I hope we come to a satisfactory
arrangement with them.”
“So do I.”
“It would be great to strike something big down here, wouldn’t it?”
“Well, we mustn’t let our imagination run away with us. We’ll have
to take what comes.”
They had an excellent supper, prepared by Carol with the
assistance of an old colored mammy, and a very delightful evening,
spent chiefly by Tom in conversation with Mr. Goby, whom he found
to be well informed and an entertaining talker.
 Ned had developed a sudden interest in flowers, and was very
anxious to have Carol show him her garden. She was not unwilling,
for this handsome young man who seemed to have dropped down
on them from the skies was not an unwelcome visitor.
“Carol’s a beautiful name,” murmured Ned later that night, as he
and Tom were getting ready for bed in the comfortable room to which
they had been shown.
Tom stopped in his work of unlacing a shoe and stared at him.
“Sounds like the singing of birds,” mused Ned dreamily.
“For the love of Pete!” cried Tom, “what’s the matter with you?”
“Oh,” said Ned in some confusion, “did I say anything? Guess I
must have been thinking out loud.”
The next day Judge Wilson came over to the farm. He was a
keen, cultivated man of high standing in the legal profession.
“Swift,” he repeated, when he was introduced to Tom. “That’s a
famous name. Any relation to the inventor, Tom Swift?”
Tom flushed with embarrassment.
“A slight relation,” put in Ned, with a laugh. “In fact, he’s the man
himself.”
“But you’re only a boy, lad!” exclaimed the judge, in wonderment.
“Old enough to have a number of good inventions to his credit,”
affirmed Ned.
“I’m amazed!” cried the judge, when finally convinced that the boy
before him was the noted inventor, Tom Swift. “Well, well, this is
indeed an honor! I’ve heard a lot about your wonderful inventions—
who hasn’t?—but I never expected to have the pleasure of shaking
you by the hand.”
As a matter of fact, the recognition stood Tom in good stead. It
simplified matters immensely. His standing was established at once,
and the tedious delay otherwise necessary in looking up his
references was obviated.
They were deep in the discussion of terms, when Ned, who
happened to be facing the window, saw an automobile coming up
the road. It stopped at the gate and three men got out.
Ned gave a low whistle of surprise as he recognized them, and
Tom and the judge looked up inquiringly.
“Hankinshaw and his partners,” explained Ned.
 “Friends of yours?” asked the judge.
“No,” replied Ned. “We’ve known them chiefly in a business way.
We——”
Further explanations were prevented by a knock on the door.
Carol opened it and ushered the three men into the room.
Blank surprise showed in the faces of all of them when they
caught sight of Tom and Ned, who had risen on their entrance. The
blank looks were quickly succeeded by looks of intense vexation.
Thompson and Bragden, as the more diplomatic of the trio, banished
these promptly, but Hankinshaw’s brows remained drawn together in
a forbidding scowl.
“This is an unexpected pleasure,” said Thompson suavely, as the
visitors seated themselves. “Who would have thought that you were
down in this part of the country? On a little pleasure trip, I suppose?”
“More business than pleasure,” answered Tom coolly.
“Looking for contracts to make some more oil-well machinery?”
asked Bragden.
“No,” returned Tom. “Though if any came our way we might
consider them. We’re going to do a little digging on our own
account.”
“In this neighborhood?” asked Thompson, looking with alarm at
the papers that lay on the table near Judge Wilson’s elbow.
“Yes,” replied Tom, who was getting a little impatient at this cross-
examination. “Right on this farm, if Mr. Goby and I can come to
terms.”
“Cutting in under us, eh?” snarled Hankinshaw. “Poaching on our
preserves.”
“That remark is quite uncalled for,” remarked Judge Wilson,
entering the conversation for the first time since the introduction.
“Why do you use the phrase ‘our preserves’? These gentlemen have
no option or claim of any kind on the property, have they, Mr. Goby?”
he continued, turning to the blind man.
“Not at all,” replied the owner of the farm. “They have discussed
the matter with me several times, but no agreement has been
reached.”
“No written agreement perhaps,” broke in Thompson. “But I
certainly thought that we had reached a verbal agreement, or at least
a practical understanding the last time we were over here.”
“That’s what I thought,” said Bragden, backing up his partner.
“Sure we did,” growled Hankinshaw.
“Nothing of the kind,” returned Mr. Goby indignantly. “That is
wholly your own assumption. I distinctly told you then, as I had told
you before, that you would have to take the matter up with Judge
Wilson and that I would do nothing without his approval.”
The judge looked at the three men keenly.
“I have always been within easy reach,” he remarked. “May I ask
why you have not brought the matter to my attention?”
“Our plan has always been to save expense and delay by dealing
directly with the owners of property,” replied Thompson.
“Even when that owner happens to be a blind man?” asked the
judge, with a tinge of sarcasm in his tone.
“His daughter could read the papers to him,” replied Thompson
defensively.
“A blind man and an inexperienced young girl,” mused Judge
Wilson, and before the contempt expressed, Thompson and
Bragden winced, while Hankinshaw glared.
“Do you give me authority to deal with these gentlemen, Mr.
Goby?” asked the judge.
“Absolutely,” returned the blind man. “Whatever you say or do will
be wholly satisfactory to me.”
“That being the case, gentlemen,” said the judge, turning to the
three partners, “I think we will not detain you any longer. You are
doubtless busy men and have many things to attend to.”
It was a clear case of dismissal. Thompson fumed white with
anger, as he and Bragden rose from their chairs.
“You may regret this,” said Thompson threateningly, moving
toward the door.
“Possibly,” replied Judge Wilson indifferently, turning toward his
papers.
“You bet you will,” bullied Hankinshaw, who remained obstinately
planted in his chair.
Tom sprang to his feet.
“Miss Goby,” he said, “would you mind stepping from the room for
a moment?”
 The young girl vanished through a door at the back.
Tom went to the front door and threw it open.
“Just to save Mr. Hankinshaw the trouble,” he remarked.
“I’ll go when I get ready,” snarled Hankinshaw, who was fighting
mad at the collapse of his scheme. “I’ll——”
He stopped short as the gigantic form of Koku blocked the door.
“Come in, Koku,” said Tom. “By the way, Hankinshaw, you
remember Koku, don’t you? You met him the night that you couldn’t
sleep. He’s a genial sort of fellow, and——”
But Hankinshaw at the sight of Koku had risen from his chair with
alacrity and followed his partners from the room.
When they had gone, Tom and Ned and the judge got down to
business, and it was not long before they had settled on terms.
Tom had agreed previously with Ned and Mr. Damon that they
would go into the oil venture as partners with equal investments and
equal profits or losses. And the terms that were made with Mr. Goby
were not only fair, but generous. He was to receive a large lump sum
at once for the privilege accorded Tom and his partners of drilling on
his farm. If the venture failed, he would still have the farm and a
large sum of money. If oil were struck, he was to have a good share
of the profits. So that either way he would win.
With the contract signed, Tom and Ned set to work. Through the
aid of Judge Wilson, they were able to secure the services of
experienced and reliable drillers. Much of their material had already
arrived, and other necessaries were secured from the owners of
abandoned wells in the vicinity.
In a surprisingly short time, a derrick was rigged, the machinery
installed, and all was ready for the venture.
“Now,” said Tom, on the morning they started work, as he patted
lovingly his new patent drill, “don’t fall down on me. Show me what
you can do.”
While he was busy with these preparations, Tom had not come
across any of the Hankinshaw crowd, but from various sources he
heard that they were furious at their failure to get hold of the Goby
farm and that they were making dire threats of getting even. But he
was too busy to pay any attention to these. He felt perfectly confident
of his ability to take care of himself no matter what they might do.
Not so Mr. Damon.
“They’ll be after us, Tom,” said the eccentric man, one day, and
his manner showed his nervousness.
“You bet they’ll be after us,” put in Ned. “Especially if we strike
oil.”
“We’ll keep our eyes trimmed for them,” answered the young
inventor. “For Hankinshaw especially,” he added soberly. He had
seen a look in that rascal’s eyes that proved the unscrupulous fellow
was becoming desperate.
 CHAPTER XVIII

A TEST OF COURAGE

At first the well went down rapidly. The earth was soft and
sandy near the surface, and with even an ordinary drilling outfit
progress would have been fairly rapid. But Tom’s newly perfected
drill fairly ate its way through the soil, “like a gimlet going through a
nice soft piece of cheese,” as Ned expressed it. They were all
delighted with the performance of the new invention, and promised
themselves an early and successful strike.
But this rapid progress did not keep up long. After the first
hundred feet or so, the ground became harder, and they often
encountered rocks that slowed up even Tom’s marvelous drill. It kept
hammering away, though, and gradually thrust through obstacles
that would have splintered and shattered any ordinary well-boring
outfit.
Deeper and deeper grew the hole, and heavier and heavier grew
the pipe as it was sunk through the earth’s crust. The big derrick
creaked and groaned, and they had to stop drilling for several days
while they added massive beams to the structure to reënforce it.
Then drilling was resumed, but as the shaft sank deeper and deeper,
and still with no sign of oil, one member after another of the party
began to get discouraged. At first they would hardly admit this, even
to themselves, but at last the facts had to be faced.
Mr. Damon had arrived a few days before the strengthening of
the derrick. At first he had been his usual bright and voluble self, but
as day followed day even his good spirits died away, and at length
he put in words what the others had been thinking for some time
past.
“Bless my oil cups, Tom, I’m the last man in the world to want to
discourage you, but it begins to look to me as though there wasn’t a
drop of oil on this whole farm—except what they burn in the lamps at
night.”
 “I must admit that it begins to look that way, as you say,” Tom
replied. “But don’t forget that more than once oil has been struck at
greater depths than we’ve penetrated so far. Why, we’re not down a
thousand feet yet, and the famous ‘spouter’ well didn’t break until
they’d gone down to nearly fourteen hundred. Besides, we’ve struck
a softer stratum of earth now, and the old drill is beginning to bite
through in fine style once more.”
“That new drill of yours has done wonders, and if you let it go I
think it would reach China eventually, but even then it might not
strike an oil deposit. Why, bless my good, muscular right arm, if you
go down much farther, you’ll have to strengthen your derrick again. A
thousand feet of iron pipe weighs something, let me tell you.”
“Well, if the derrick breaks, we’ll build a new one,” returned Tom,
doggedly. “I’ve got a hunch that there’s oil under this farm, and I
want pretty good proof that there isn’t before I give up looking for it.
Besides, it isn’t only ourselves that we’ve got to think of. Can’t you
imagine how disappointed Mr. Goby and his daughter would be if we
had to admit failure?”
“Yes, and then there’s the Hankinshaw gang, too,” chimed in
Ned. “They’d have the laugh on us good and plenty if we went to all
this trouble and then didn’t get anything after all. We’d just be saving
them the expense of doing the work themselves.”
“Very true. But you’ve got to look at this from a business
viewpoint,” came from Mr. Damon. “Every ten feet you go deeper
now will cost you many times more than the same distance did at
first, and if the chances seem all against you, it’s better policy to take
your losses and get out while you’ve got something left. That blessed
hunch of yours, Tom, may prove to be a very expensive one before
you’re through.”
“That’s very true, Mr. Damon. But remember that it hasn’t cost us
nearly as much to drill this hole as it would if we had reached the
same depth with the ordinary drilling equipment. I think we’d better
add a little more bracing to the derrick and drill through another
hundred feet or so. If we don’t strike oil here, I want to feel that we
did our best, anyway. There may be oil within ten feet of the drill
point right now.”
 Tom’s confidence and eagerness were infectious, and while Mr.
Damon still shook his head doubtfully and blessed everything he
could think of, it was finally decided to “carry on” a little while longer.
Ned, while still unconvinced, did not advance any further arguments
against a continuation of the drilling, as he knew how bitterly
disappointed Carol would be if they failed in the undertaking.
Day followed day at the scene of the drilling, and still there was
the same heartbreaking lack of success. Deeper and deeper went
the drill, faster now, but still with no result. Finally their supply of pipe
ran out, and it was almost a week before they could get more—a
week during which Tom paced restlessly about the confines of the
farm, counting the minutes until they could resume operations. The
time was not entirely wasted, however, as they added some heavy
shoring to the derrick, together with some new crossbeams to
support those that were bending and splitting under the tremendous
strain.
In drilling for oil, as the drill bores a hole through the earth’s crust,
lengths of wrought-iron pipe are lowered into the hole to keep the
earth from caving in and filling the shaft. When one length of pipe,
usually twelve to fifteen feet, is all the way in, another length is
coupled on to it, and this in turn is sunk as the drill goes deeper.
Now, the entire weight of this pipe is supported by a wooden—or, in
some cases, steel—framework, which is erected over the boring.
One length of four-inch pipe is not such a trifling weight, and when
dozens of these lengths are coupled together, their combined weight
becomes enormous. Quite often the pipe or its supports will break,
and then the whole length drops down into the hole and has to be
fished out again before operations can be resumed. This is often a
very difficult job, and may hold up progress for many days. In the
feverish rush to get the shaft sunk, derricks are often overloaded
until they fall under the strain, often badly injuring or killing the
workmen, and in any event causing delay and expense.
Tom and his friends had guarded as far as possible against these
accidents, and so far had had no trouble in that direction. But with
every length of pipe that was added to that already in the hole, the
chance of an accident grew greater.
 However, Tom, with characteristic grit, had determined to see the
enterprise through to a finish, and the others of the party, seeing that
he was not to be dissuaded, concealed as far as possible their own
despondency as to the outcome.
“Bless my suspenders, Tom Swift, you look as though you had
lost your last friend!” exclaimed Mr. Damon, one day. “The world
won’t come to an end just because we’ve happened to run out of
pipe. We’ll have more in a few days, and in the meantime you ought
to be getting a rest instead of pacing up and down like a wild animal
in its cage. You’ll make yourself sick if you don’t look out—bless my
pill box, but you will!”
“I’ll get well fast enough when we strike oil,” Tom assured him.
“When we strike oil! Bless my timepiece! What about now? Now,
Tom Swift?”
Tom laughed, but merely reiterated that he would be well enough
when the oil began to flow.
“That’s all right. But in the meantime, why not be sociable?” came
from Ned, as he linked his arm with that of his friend “You take hold
of him on the other side, Mr. Damon, and we’ll trot him up to the
farmhouse and give him a good home-cooked meal. In other words,
we’ll feed the brute.”
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