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Inamuddin Editor
Applications
of Ion Exchange
Materials in
Biomedical Industries
Applications of Ion Exchange Materials
in Biomedical Industries
Inamuddin
Editor
Applications of Ion Exchange

Materials in Biomedical
Industries
123
Editor
Inamuddin
Chemistry Department, Faculty of Science
King Abdulaziz University
Jeddah, Saudi Arabia
ISBN 978-3-030-06081-7 ISBN 978-3-030-06082-4 (eBook)

https://doi.org/10.1007/978-3-030-06082-4
Library of Congress Control Number: 2018965911
© Springer Nature Switzerland AG 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
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for any errors or omissions that may have been made. The publisher remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface
Ion exchange chromatography has been the subject of scientific research for the last
three decades. In the beginning, it was primarily a significant process in the field of
analytical chemistry and more especially water softening as well as treatment of
wastewater containing harmful impurities such as heavy metals and dyes using
different types of ion exchange materials. Later on, some of the industrial appli-
cations of ion exchange materials have been explored in areas such as chemical
synthesis concerning dehydration, hydrogenation, alkylation and esterification
reactions and separation and purification of industrial products such as sugar cane
juice, fruit juice, dairy products, beverages. The chromatographic applications of
ion exchange materials thereafter attracted research by healthcare professionals and
were extensively used in biomedical laboratories. The investigation of ion exchange
chromatography in numerous sorts of biomedical applications has been significant
over recent decades. These days, there are various imaginative applications in
material design created to address the issues of the biomedical field.
Ion exchange chromatography stands out among many of the time utilized
separation techniques for the purification of amino acids, proteins, peptides, nucleic
acids, vitamins and urine. This book aims to present the basic principles of ion
exchange chromatography and the continuous improvements in the utilization of
this technique for biomedical and pharmaceutical analyses.
Applications of Ion Exchange Materials in Biomedical Industries will cover
applications of ion exchange materials in biomedical industries. It includes topics
related to the application of ion exchange chromatography in areas such as sepa-
ration and determination and extraction of various compounds like amino acids,
morphine, antibiotics, nucleotides, nucleosides, purine and pyrimidine, penicillin,
streptomycin, and streptothricin, vitamins, sorbitol and citric acid. It provides an
in-depth knowledge of ion exchange materials and their applications suitable for
postgraduate students and researchers as well as industrial research and develop-
ment specialists working in areas such as chemistry, chemical, biomedical and
pharmaceutical sciences and biochemical technology. Additionally, this book
provides an overview of ion exchange columns and operation suitable for engineers
and industrialists. This book is divided into the following ten chapters.
v
vi Preface
Chapter 1 covers modern applications and basic principles of ion exchange

chromatography used in the separation of amino acids.
Chapter 2 pays particular attention to ion exchange chromatography as a robust
tool to improve enzyme immobilization.
Chapter 3 details various methods of determination of urinary morphine. Several
chromatographic and non-chromatographic methods of analysis are discussed in
detail. The role of artificial antibodies, i.e. molecularly imprinted polymers in the
selective detection of morphine, is also discussed. The major focus is given to
communicate the advantages, drawbacks and future feasibility of available proce-
dures reported in the literature.
Chapter 4 discusses the separation of amino acids with the help of different
chromatographic techniques. Additionally, the role of capillary electrophoresis
towards amino acid separation is discussed. The classification of hyphenated
techniques and their roles are also presented in addition to the advantages of
chromatographic techniques over other separation techniques.
Chapter 5 deals with the various chromatographic techniques used in pharma-
ceutical analysis with the help of single-mode and mixed-mode chromatography.
The methods of pretreatment and separation prior to analysis are also discussed.
Chapter 6 explores the study carried out for the adsorption of bovine serum
albumin on Turkish green clay, and some thermodynamic parameters are discussed.
Chapter 7 aims to explore the different ion change chromatographic techniques
for demineralization/deashing of sorbitol.
Chapter 8 intends to explore the methods of separation and purification of nucleo-
tides, nucleosides, purine and pyrimidine bases by ion exchange chromatography.
Chapter 9 overviews the methodologies involved in chromatographic separation
and purification of vitamins B1, B2, B6, C and K1.
Chapter 10 describes the character, composition and origin of coloured sub-
stances. The technological separation processes of coloured substances from both
beet and cane sugar syrups are discussed. The design and set-up of the industrial
system used in colour removal, purification of sugar solutions and a comparison of
ion exchange techniques with other decolorizing methods are also presented.
Jeddah, Saudi Arabia Inamuddin

Contents
1 Separation and Purification of Amino Acids . . . . . . . . . . . . . . . . . . 1

Khush Bakhat Alia, Habibullah Nadeem, Ijaz Rasul, Farrukh Azeem,
Sabir Hussain, Muhammad Hussnain Siddique, Saima Muzammil,
Muhammad Riaz and Shabab Nasir
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Ion Exchange Chromatography in the Separation
of Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Ion Exchange Chromatography of Amino Acids . . . . . . . . . . . . 3
1.4 Ion Exchange Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 Buffer Systems in IEC for Separation of Amino Acids . . . . . . . 5
1.5.1 Sodium Citrate Buffer System . . . . . . . . . . . . . . . . . . . 5
1.5.2 Lithium Citrate Buffer System . . . . . . . . . . . . . . . . . . . 6
1.6 The Relation Between the Concentration of Eluent
and Retention Time of Amino Acids . . . . . . . . . . . . . . . . . . . . 6
1.7 Effect of Temperature on Separation of Amino Acids . . . . . . . . 7
1.8 Effect of pH on Separation of Amino Acids . . . . . . . . . . . . . . . 8
1.9 Effect of the Flow Rate of the Eluting Buffer on the IEC
of Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.10 Regeneration of the Ion Exchange Column . . . . . . . . . . . . . . . . 9
1.11 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2 Ion Exchange Chromatography for Enzyme Immobilization ..... 13
Raissa Pieroni Vaz and Edivaldo Ximenes Ferreira Filho
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2 Enzyme Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2.1 Immobilization Approaches . . . . . . . . . . . . . . . . . . . . . 15
2.3 Ion-Exchange as an Immobilization Tool . . . . . . . . . . . . . . . . . 16
2.4 Enzyme Immobilization Research and Application
by Ion-Exchange in the Laboratory and Industry . . . . . . ..... 18
vii
viii Contents
2.5 Conclusion and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . 22

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3 Determination of Morphine in Urine . . . . . . . . . . . . . . . . . . . . . .. 29
Shrabani De, Raksha Choudhary and Rashmi Madhuri
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1.1 Structural Features of Morphine . . . . . . . . . . . . . . . . . 32
3.1.2 Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1.3 Various Routes of Morphine Administration . . . . . . . . 33
3.1.4 Stay Period of Morphine in the Body . . . . . . . . . . . . . 35
3.2 What Is Drug Abuse? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.2.1 Fatal Dose of Morphine . . . . . . . . . . . . . . . . . . . . . . . 36
3.2.2 Statistics Towards Morphine Addiction . . . . . . . . . . . . 36
3.2.3 Adverse Effect of Morphine . . . . . . . . . . . . . . . . . . . . 36
3.3 Samples Used for Detection of Morphine . . . . . . . . . . . . . . . . . 37
3.3.1 Sample Collection/Preparation Prior to Detection . . . . . 38
3.3.2 Extraction and Derivatization . . . . . . . . . . . . . . . . . . . 38
3.4 Detection of Morphine in Urine . . . . . . . . . . . . . . . . . . . . . . . . 39
3.4.1 Chromatographic Methods . . . . . . . . . . . . . . . . . . . . . 39
3.4.2 Liquid Chromatography (LC) and High-Performance
Liquid Chromatography (HPLC) . . . . . . . . . . . . . . . . . 43
3.4.3 Thin-Layer Chromatography (TLC) . . . . . . . . . . . . . . . 49
3.4.4 Capillary Electrophoresis (CE) . . . . . . . . . . . . . . . . . . 51
3.4.5 Electrochemical Detection . . . . . . . . . . . . . . . . . . . . . . 58
3.4.6 Combination of Molecularly Imprinted Polymer
with Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.4.7 Some Miscellaneous Detection Techniques . . . . . . . . . 62
3.5 Conclusion and Future Scope . . . . . . . . . . . . . . . . . . . . . . . . . 63
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4 Chromatographic Separation of Amino Acids . . . . . . . . . . . . . .... 71
Kartick Chandra Majhi, Paramita Karfa and Rashmi Madhuri
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.1.1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.1.2 Classification of Amino Acids . . . . . . . . . . . . . . . . . . . 75
4.2 Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.2.1 What is Separation? . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.2.2 Why Need to Do Separation of Amino Acids? . . . . . . . 77
4.2.3 What is Chromatography? . . . . . . . . . . . . . . . . . . . . . . 78
4.2.4 Classification of Chromatographic Methods . . . . . . . . . 78
4.2.5 Advantages of Chromatographic Methods Over
Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . .... 79
4.3 Separation of Amino Acids by Gas Chromatography (GC) .... 80
Contents ix
4.4 Liquid Chromatography (LC) . . . . . . . . . . . . . . . . . . . . . . . .. 82

4.4.1 Separation of Amino Acids by High-Performance
Liquid Chromatography (HPLC) . . . . . . . . . . . . . . . .. 84
4.4.2 Advantages of Liquid Chromatography Over the Gas
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 87
4.5 Amino Acid Separation by Countercurrent Chromatography
(CCC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 92
4.6 Separation of Amino Acids by Thin-Layer Chromatography
(TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.6.1 Preparation of Thin Plates . . . . . . . . . . . . . . . . . . . . . . 94
4.6.2 Sample Spotting on the Thin-Layer Plate . . . . . . . . . . . 94
4.6.3 Detection of Amino Acids on the Thin-Layer Plate . . . 95
4.7 Separation of Amino Acids by Capillary
Electrophoresis (CE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.7.1 Various Modes for Capillary Electrophoresis (CE) . . . . 97
4.8 Separation of Amino Acids by the Hyphenated Technique . . . . 98
4.8.1 List of Hyphenated Techniques . . . . . . . . . . . . . . . . . . 100
4.8.2 Separation of Amino Acids Using GC-MS . . . . . . . . . . 101
4.8.3 Separation of Amino Acids by LC-MS . . . . . . . . . . . . 101
4.8.4 Separation of Amino Acids by LC-MS-MS . . . . . . . . . 109
4.8.5 Separation of Amino Acids by CE-MS . . . . . . . . . . . . 109
4.9 Conclusion and Future Scope . . . . . . . . . . . . . . . . . . . . . . . . . 111
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
5 Applications of Ion-Exchange Chromatography
in Pharmaceutical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Sayed M. Derayea and Hytham M. Ahmed
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
5.2 Application of Ion-Exchange Chromatography in Quantitative
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
5.2.1 Single-Mode Ion-Exchange Chromatography . . . . . . . . 120
5.2.2 Analysis of Small Molecules (Organic and
Inorganic Ions) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
5.2.3 Mixed-Mode Chromatography . . . . . . . . . . . . . . . . . . . 125
5.3 Pretreatment and Separation Prior to Analysis . . . . . . . . . . . . . 128
5.3.1 Ionic Solid-Phase Extraction . . . . . . . . . . . . . . . . . . . . 128
5.3.2 Mixed-Mode Ion-Exchange Solid-Phase Extraction . . . 130
5.3.3 Flow Injection Ion-Exchange Preconcentration . . . . . . . 132
5.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
x Contents
6 Thermodynamic Kinetics and Sorption of Bovine Serum

Albumin with Different Clay Materials . . . . . . . . . . . . . . . . . . . . . . 139
Özkan Demirbaş, Mehmet Harbi Çalımlı, Esra Kuyuldar,
İ. Halil Baydilek, Mehmet Salih Nas and Fatih Şen
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
6.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6.3.1 The Effect of Some Specific Physicochemical
Properties BSA onto Adsorption . . . . . . . . . . . . . . . . . 141
6.3.2 Analyses of FTIR, TGA, and SEM Images . . . . . . . . . 143
6.3.3 Kinetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
6.3.4 Thermodynamic Parameters . . . . . . . . . . . . . . . . . . . . 147
6.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
7 Sorbitol Demineralization by Ion Exchange . . . . . . . . . . . . . . . . . . 155
Sabeela Beevi Ummalyma, Haobam Rijeeba, Raveendran Sindhu,
Parameswaran Binod, Ashok Pandey and Edgard Gnansounou
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
7.2 Industrial Application of Sorbitol . . . . . . . . . . . . . . . . . . . . . . . 156
7.3 Importance of Demineralization/Deashing of Sorbitol . . . . . . . . 157
7.4 Role of Ion-Exchange Chromatography . . . . . . . . . . . . . . . . . . 157
7.5 Different Types of Ion Exchangers for Sorbitol
Demineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
7.5.1 Cation-Exchange Chromatography . . . . . . . . . . . . . . . . 159
7.5.2 Anion-Exchange Chromatography . . . . . . . . . . . . . . . . 159
7.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
8 Separation and Purification of Nucleotides, Nucleosides, Purine
and Pyrimidine Bases by Ion Exchange . . . . . . . . . . . . . . . . . . . . . 163
P. Senthil Kumar and P. R. Yaashikaa
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
8.2 Ion-Exchange Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 165
8.2.1 Mechanism of Ion Exchange . . . . . . . . . . . . . . . . . . . . 165
8.2.2 Components of Ion-Exchange Chromatography . . . . . . 166
8.3 Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
8.4 Nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
8.5 Purines and Pyrimidines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
8.6 Column Preparation and Operation . . . . . . . . . . . . . . . . . . . . . 168
8.7 Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
8.8 Impact of Separation Parameters . . . . . . . . . . . . . . . . . . . . . . . 169
8.9 Separation of Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
8.9.1 Fractionation of Nucleotides . . . . . . . . . . . . . . . . . . . . 170
Contents xi
8.9.2 Cation-Exchange Resin . . . . . . . . . . . . . . . . . . . . . . . . 171

8.9.3 Anion-Exchange Materials . . . . . . . . . . . . . . . . . . . . . 171
8.10 Separation of Nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
8.10.1 Purification of Nucleosides . . . . . . . . . . . . . . . . . . . . . 171
8.11 Separation of Purines and Pyrimidines . . . . . . . . . . . . . . . . . . . 172
8.12 Applications of Ion-Exchange Chromatography . . . . . . . . . . . . 173
8.13 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
9 Separation and Purification of Vitamins: Vitamins B1, B2, B6,
C and K1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
P. Senthil Kumar and G. Janet Joshiba
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
9.2 Significance of Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
9.3 Classification of Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
9.3.1 Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . 178
9.3.2 Fat-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . 179
9.4 Sources of Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
9.4.1 B Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
9.4.2 Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
9.4.3 Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
9.5 Vitamin Deficiency Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 180
9.6 B Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
9.6.1 Vitamin B1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
9.6.2 Vitamin B2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
9.6.3 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
9.7 Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
9.8 Vitamin K1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
9.9 Separation and Purification of Vitamin . . . . . . . . . . . . . . . . . . . 183
9.10 Ion-Exchange Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 184
9.11 Mechanism of Ion-Exchange Chromatography . . . . . . . . . . . . . 184
9.12 Separation and Purification of Vitamins B1, B2 and B6 . . . . . . 184
9.13 Separation and Purification of Vitamin C . . . . . . . . . . . . . . . . . 185
9.14 Ion-Exchange Separation and Purification of Vitamin K1 . . . . . 186
9.15 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
10 Colour Removal from Sugar Syrups . . . . . . . . . . . . . . . . . . . . . . . . 189
Svatopluk Henke, Andrea Hinkova and Simona Gillarova
10.1 Colourants in Sugar Solutions . . . . . . . . . . . . . . . . . . . . . . . . . 190
xii Contents
10.1.1 Determination of Colour in Sugar and

Sugar Juices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
10.1.2 Colour Substances in Sugar and Sugar Solutions . . . . . 193
10.1.3 Formation of Beet and Cane Colourants During
the Technological Process . . . . . . . . . . . . . . . . . . . . . . 199
10.1.4 Removal of Colourants from Beet and Cane Sugar
and Sugar Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
10.2 Decolourisation with Ion-Exchange Resins . . . . . . . . . . . . . . . . 201
10.2.1 The Terminology Used in Ion-Exchange
Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
10.2.2 Types of Ion-Exchange Resins . . . . . . . . . . . . . . . . . . 203
10.2.3 Set-up of Industrial Chromatographic Systems
for Colour Removal . . . . . . . . . . . . . . . . . . . . . . . . . . 208
10.2.4 Comparison of Ion-Exchange Technology with Other
Decolourising Techniques . . . . . . . . . . . . . . . . . . . . . . 216
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Chapter 1
Separation and Purification of Amino
Acids
Khush Bakhat Alia, Habibullah Nadeem, Ijaz Rasul, Farrukh Azeem,

Sabir Hussain, Muhammad Hussnain Siddique, Saima Muzammil,
Muhammad Riaz and Shabab Nasir
Abstract The most prevalent type of liquid chromatography is ion exchange chro-
matography. In recent years, the acceptance of ion exchange chromatography has
been increased because of its application in agricultural, pharmaceutical, environ-
mental and biotechnology industries. Amino acids are principal components of pro-
teins and their separation from protein hydrolysate or from a mixture of amino acids,
is always in focus and ion exchange chromatography finds its application in sepa-
ration of amino acids. Separation of amino acids by ion exchange chromatography
has widespread applications because it has high capacity and it is easy to design.
This chapter covers modern applications and basic principles of Ion exchange chro-
matography in the separation of amino acids.
1.1 Introduction
According to the IUPAC, chromatography is a technique for separation of sam-

ple components after distribution between two phases (mobile phase and stationary
phase). While ion exchange chromatography (IEC) is a subset of liquid chromatogra-
phy which allows the separation of polar and ionic molecules depending upon their
K. B. Alia · H. Nadeem (B) · I. Rasul · F. Azeem · M. H. Siddique

Department of Bioinformatics and Biotechnology, Government College University,
Faisalabad, Pakistan
e-mail: habibullah@gcuf.edu.pk
S. Hussain
Department of Environmental Sciences and Engineering, Government College
University, Faisalabad, Pakistan
S. Muzammil
Department of Microbiology, Government College University, Faisalabad, Pakistan
M. Riaz
Department of Food Sciences, University College of Agriculture,
Bahauddin Zakariya University, Multan, Pakistan
S. Nasir
Department of Zoology, Government College University, Faisalabad, Pakistan
© Springer Nature Switzerland AG 2019 1
Inamuddin (ed.), Applications of Ion Exchange Materials in Biomedical Industries,
https://doi.org/10.1007/978-3-030-06082-4_1
2 K. B. Alia et al.
charges. Components of IEC are a separation column, a liquid mobile phase and
a detector which measures the eluted components coming out from the column. In
IEC, separation depends upon ionic interactions between polar analytes, ionic groups
fixed to the chromatographic support and the ions present in eluent [1].
Amino acids are biologically active materials and are found in living cells [2].
Amino acids are building blocks of proteins, and hydrolysis of proteins results in
the production of amino acids. There are 20 amino acids which have been found to
occur in all proteins and for which genetic codons exist. In biological cells, there
are other amino acids which do not occur in proteins but perform specific functions.
Amino acids have two characteristic functional groups, the amino group (–NH2 ) and
the carboxyl group (–COOH). Most amino acids have one amino and one carboxyl
group, but some have more than one of these. The amino acids may be biosynthetic
precursors or metabolic products of protein amino acids. For instance, ornithine and
homoserine are biosynthetic precursors of protein amino acids and decarboxylation of
glutamic acid results in the production of α-aminobutyric acid which is the metabolic
product of protein amino acid.
In biochemical research, techniques for amino acid analysis have gained impor-
tance. From protein hydrolysate, amino acids can be separated for quantitative or
qualitative investigations [2]. The analysis of amino acids can be obtained through
electrophoresis, thin layer chromatography and with more accuracy through auto-
mated ion exchange chromatography [3]. In many cases, carbohydrates and amino
acids may coexist and need to be separated from each other. Free amino acids could
be separated from contaminating material through ion exchange chromatography
[4]. In 1999, chromatographic derivatization-free determination of amino acids was
introduced and amino acids were separated by anion exchange chromatography [5].
1.2 Ion Exchange Chromatography in the Separation

of Amino Acids
Amino acids are structurally important in proteins; therefore, their production has
been increased. The amino acids could be produced by the hydrolysis of proteins
or biosynthesis, but the separation of amino acids from fermentation broths is diffi-
cult [6]. In beverages, medicines and food, amino acids are present and their deter-
mination has a significant role in the assessment of the nutritional worth of food
[7]. In biotechnological, clinical and nutritional fields, the determination of amino
acids is important and many efforts have been made to develop methods to fulfil
the purpose of the separation of amino acids [8]. For analysis of amino acids, pre-
and post-column derivatizations are also in use, but relative complexity, high cost
for maintenance column derivatization, the reagent interference and low stability of
amino acids make pre- and post-column derivatization processes less advantageous
for use [9]. Generally, ion exchange chromatography or crystallization at the isoelec-
tric point is used for separation of amino acids [6], but lack of strong chromophore
1 Separation and Purification of Amino Acids 3
group, high polarity and low volatility makes their separation and detection cumber-
some [10]. The amino acid analysis is significant because it is usually practised in
areas of biochemical investigations.
• In order to investigate the structure and composition of proteins;
• To determine the amino acids in biological tissues and fluids;
• To determine the presence of free amino acids in food articles for the determination
of food values [2].
1.3 Ion Exchange Chromatography of Amino Acids
Small et al. introduced ion chromatography in its modern form in 1975 [11], and it
includes a variety of separation methods like ion exchange, ion exclusion or ion-pair
chromatography. Among these, ion exchange chromatography (IEC) is an important
technique and widely used in order to separate ionic compounds by ion partition
and ion exclusion chromatography [5, 12, 13]. In IEC, the stationary phase has
displaceable oppositely charged ion and mobile phases are generally buffer systems.
Cation and anion exchange processes involved in ion exchange chromatography.
Cation and anion exchangers are packed in glass columns, and the separation depends
on the binding of analytes with positively or negatively charged groups present on
stationary phase [14].
The displacement of analyte ions by co-ions during the separation process is the
distinctive feature of ion exchange chromatography. At the top of the column, the
sample mixture is applied and then allowed to pass through the ion exchange material
present in the column. Eluent flow is then resumed, and eluent fractions from the
column are collected at regular intervals. Ion exchange chromatography has been used
for the analysis of anions and cations, including mono- and oligosaccharides, metal
ions, polyhydroxy compounds, phenols, antibiotics, organic acids, thiols, peptides,
amino acids, amines, alcohols, nucleotides, nucleosides and other polar molecules
[14].
A general separation/purification process by IEC involved the following steps
[15]
(1) Regulate the feedstock composition for mobile phase which is appropriate for
adsorption of the anticipated component.
(2) Interaction of feedstock with ion exchanger to enable adsorption.
(3) Removal of un-retained molecules (contaminants) from the ion exchanger.
(4) Elution of bounded molecules from the ion exchanger.
(5) Re-storing the ion exchanger.
4 K. B. Alia et al.
1.4 Ion Exchange Resins
Nowadays, the synthetic spheroidal shape ion exchange resins are in use. Ion
exchangers with synthetic resin backbone and ion exchangers with polysaccharide
backbone are the two common classes of immobile and commonly used ion exchang-
ers. Ion exchange resins with synthetic resin backbone are usually of polystyrene type,
and these synthetic resin backbones are formed by copolymerization of divinyl-
benzene and styrene. The styrene is replaced with desired functional groups, for
instance weakly acidic (–COOH), strongly acidic (–SO3 H), weakly basic (–NH3 + )
and strongly basic (–NR3 + ) groups [2, 16]. For the synthesis of resins, the concentra-
tion of divinylbenzene is important because cross-links in styrene chains are formed
by divinylbenzene which results in the establishment of the ball-shaped structure. In
addition, the resins have more or less favourable properties depending on the quantity
of cross-linker. Properties of ion exchange resins for purification of amino acids are
given in Table 1.1.
The resin matrix is sulfonated to obtain the strongly acidic cation exchange resin.
The segment which is present inside the skeleton is known as a pore, the charged ions
(like –SO3 − ) are known as linked ions, and oppositely charged ions are known as
exchangeable ions. During the process of ion exchange, ions with opposite charges
in buffer penetrate into the pores of the matrix and replaced with oppositely charged
ions [16]. In IEC, acidic amino acids elute first then hydroxylic, followed by neutral
and finally the basic ones. Resins in neutralized form adsorb neutral amino acids
selectively other than histamine and dicarboxylic acids. To separate almost all types
of amino acids weakly acidic resins are in use [2, 16].
With the increase in the concentration of divinylbenzene, the permeability and
size of particle reduced and cross-linking in styrene chains occur at shorter intervals
and separation power is increased. Resins with less cross-linking or resins (1–4%)
of divinylbenzene have the ability to manage larger molecules. With the decrease in
cross-linking of resins, the permeability increases and equilibrium is maintained more
rapidly. The separation power reduced for certain ions is due to smaller swollen vol-
ume, and along this, the physical stability of resin also decreases. Resins with 8–16%
Table 1.1 Characteristics of polystyrene-type ion exchangers [2]

Name of resin Class Functional group pH
DOWEX 50 Cation exchanger Sulfopropyl 6–10
(strong)
IRC 150 Cation exchanger Carboxymethyl 5–9
(weak)
DOWEX 1 Anion exchanger Quaternary 2–9
DOWEX 2 (strong) ammonium
DOWEX 3 Anion exchanger Diethylaminoethyl 5–9
IR 45 (weak) (DEAE)
of divinylbenzene (low cross-linking) have a small pore size and less permeability,
and this concentration is appropriate for inorganic ions [2].
Smaller particle size increases the exchange rate by reducing the diffusion path
between active groups; therefore, the particle size should be smaller. Shorter columns
are used in order to reduce the separation time; however, the smaller diffusion values
improve the sharpness of amino acid separation. Smaller particle size increases the
mechanical stability which is important during analysis because resin in columns
contracts and expands by continuous change in pH [2]. For the separation of amino
acids with high resolution, it is important to consider the dimension of separating
column. Previously, columns with diameter from 5–9 mm were extensively used,
but currently the columns with 1–2 mm diameter are used. It is desirable to keep the
column diameter as narrow as possible as the separation performance hinges on the
size of ion exchange particles, column diameter and length factor [16].
1.5 Buffer Systems in IEC for Separation of Amino Acids
For the separation of amino acids through IEC, the lithium or sodium buffers have
been used for the elution of separated amino acids [17]. Amino acids obtained in pro-
tein hydrolysate could be separated by three sodium buffer systems. For reasonable
separation between ninhydrin-positive compounds, it is suitable to use four or five
sodium buffer systems because physiological fluids hold 40–50 ninhydrin-positive
compounds. For immediate separation of glutamic acid, aspartic acid, asparagine
and glutamine, lithium buffer system has been most appropriate.
1.5.1 Sodium Citrate Buffer System
Amino acids from protein hydrolysate have been determined by sodium citrate
buffers. NaCl, sodium citrate, sodium azide, boric acid, citric acid, sodium hydroxide
and thiodiglycol are the reagents used in the preparation of sodium citrate buffer.
The first sodium buffer pH 2.95 containing 0.20 M Na and has been used for the
elution of alanine, glycine, proline, aspartic acid, glutamic acid, cysteine, threonine
and serine. As this buffer system has smaller ionic strength, the cysteine is eluted
after glycine and alanine. The increase in temperature or pH decreases the elution
time for cysteine. This buffer has been used for the separation of alanine and glycine
as well as serine and threonine. The second sodium buffer pH 3.50 containing
0.30 M Na has been used for the elution of glutamic acid, aspartic acid, proline,
serine, threonine, cysteine, valine, alanine and glycine. The third sodium buffer
pH 4.25 having 0.40 M Na has been used for the elution of leucine, isoleucine and
methionine. The fourth sodium buffer system pH 7.9 containing 1.12 M Na has
been used for the elution of the amino acids that do not elute by first, second and third
sodium buffer systems, i.e., histidine, arginine, tyrosine, lysine and phenylalanine.
6 K. B. Alia et al.
1.5.2 Lithium Citrate Buffer System
Lithium citrate buffer system has been used to determine the free amino acids from
physiological samples. Citric acid, boric acid, lithium citrate, lithium hydroxide,
lithium chloride, lithium azide and thiodiglycol reagents have been used for the
preparation of lithium citrate buffers.
The first lithium buffer system pH 2.80 containing 0.18 M Li has been used
to elute asparagine, glutamine, serine, threonine, cysteic acid, glutamic acid and
aspartic acid. The elution was carried out at 37–40 °C. Asparagine and glutamine were
sensitive to change in temperature and pH, but glutamic acid was the most sensitive.
The second lithium buffer (pH 3.05 and 0.20 M Li) has been used to elute
glycine, alanine, valine, proline, α-aminobutyric acid, citrulline and α-aminoadipic
acid. Citrulline was sensitive to change in pH and temperature. The third lithium
buffer (0.36 M Li and pH 3.35) system was used for the elution of leucine,
isoleucine, methionine and cystine. The fourth lithium buffer (0.33 M Li and pH
4.05) system was used to elute β-alanine, β-aminobutyric acid, phenylalanine and
tyrosine. The fifth lithium buffer (pH 4.65 and 1.20 M Li) system was used to
elutes histidine, 3-methylhistidine, γ-aminobutyric acid, ornithine, arginine, lysine
and 1-methylhistidine [16].
1.6 The Relation Between the Concentration of Eluent

and Retention Time of Amino Acids
The gradient conditions for elution are divided into three groups; in first group,
hydroxide eluent (NaOH) is used for weakly retained amino acids; in second group,
sodium acetate gradient is used for strongly retained aromatic and acidic amino acid.
In third group, hydroxide concentration is used in order to re-equilibrate the column
for the purpose of next run [9].
In basic conditions, amino acids form anions; therefore, anion exchange chro-
matography is favourable for separation of anions (amino acids). By increasing the
concentration of NaOH from 20 to 120 mM, the retention time of amino acids (except
arginine) decreases because at high alkaline condition, there is arginine lack of nega-
tive charge, so higher concentration of NaOH does not have an effect on its retention
time. By decreasing the concentration of NaOH, separation between glutamine and
thiamine can be improved and the order of elution is also changed by changing the
concentration of NaOH. At a concentration of 8 mM, thiamine separates from glu-
tamine and elute after sucrose, fructose and glucose. This change in elution sequence
is due to the difference in acidic dissociation constants (pK a ). Low concentrations of
NaOH suppressed the ionization of sugars and pK a values of sugar become higher
than 12. On the other hand at low concentrations of NaOH, the ionization of amino
acids having low pK a value is not affected. Therefore, the retention time of amino
acids increases than sugars.
Strongly retained amino acids like tyrosine, histidine, aspartate, phenylalanine,

cysteine and glutamate cannot be eluted from the column at higher concentration if
only NaOH solution is used as eluent. Amino acids which are strongly retained could
be eluted through the strong eluent like sodium acetate (CH3 COONa). Higher con-
centration of CH3 COONa greatly reduces the retention time of dicarboxylic amino
acids than monocarboxylic amino acids. Gradient elution method using NaOH and
CH3 COONa solution has been widely used for amino acids’ separation by anion
exchange chromatography [4].
1.7 Effect of Temperature on Separation of Amino Acids
Change in temperature affects the separation of amino acids in two ways. Tem-
perature affects the amino acids separation by changing their attraction with ion
exchange resin. Change in retention time due to temperature could be reimbursed by
pH. With the increase in temperature, the retention time for leucine, isoleucine and
valine increases; on the other hand with the increase in temperature, the retention
time decreases for methionine and serine. By decreasing the temperature, the sepa-
ration of serine and threonine can be enhanced but at the same time, the separation
of glutamic acid is influenced by the increase in backpressure. Therefore, after the
separation of serine and threonine, it is significant to have a temperature gradient.
In order to decrease the analysis time, increase in temperature (50–70 °C) is rec-
ommended, but this rise in temperature should be after separation of leucine and
isoleucine. With a lithium or sodium buffer system, for the separation of amino acids
like glutamine, 4-hydroxyproline, glutamic acid, aspartic acid, serine, asparagine and
threonine, the optimum temperature has been 37–38 °C. The effect of temperature
on the retention time of amino acids could be explained in a way that anion exchange
chromatography displays an exothermic or endothermic process. In the exothermic
process, the retention time is decreased with the increase in temperature, while in
case of the endothermic process the retention time increases with increase in tem-
perature. The reaction is endothermic or exothermic; it depends upon amino acids
involved in the reaction. The change in temperature could be effectively used for the
separation of two closely eluted amino acids. If two amino acids have elution char-
acteristics identical, their separation could be enhanced by changing the temperature
of the column. For instance, at 25 °C proline and serine have overlapping peaks and
change in concentration of eluent had no significant effect on separation of these
closely eluted amino acids, but at 35 °C, proline and serine separates completely. At
40 °C, methionine and leucine have overlapping peaks, but at 35 °C, good separation
between methionine and leucine was obtained [18].
8 K. B. Alia et al.
1.8 Effect of pH on Separation of Amino Acids
Different polar biological molecules which are ionisable at high pH and have func-
tional groups with pK a values >10 have been separated through anion exchange
chromatography. Anion exchange chromatography has been used for the separation
of basic amino acids. For separation of amino acids, pH of the buffer is important. At
high pH, the peaks representing amino acids appeared earlier. Cysteine is sensitive
amino acids with reference to change in concentration of ions with opposite charge,
temperature and pH. Cysteine has been separated from alanine as it eluted after ala-
nine. With high temperature and pH, the elution time for cysteine decreases. In case
of cystine separation, change in pH is more effective than temperature. The increase
in temperature and pH accelerates the movement of cysteine through the column and
shortens its elution time. For the efficient separation of cysteine, it is important to set
the pH and temperature values in such a way that cysteine just positioned between
valine and alanine [16].
In case of a chromatographic column with negatively charged resins and amino
acids bearing a positive charge at low pH (pH 2.2), the amino acids tend to bind
to the resin. By the increase in pH and ionic strength of buffer used as eluent, amino
acids attain isoelectric point and the force of attraction between amino acids and
resin disappears to facilitate the elution of amino acids from column [2, 16]. The
isoelectric point indicates the pH value at which all molecules present in a solution
have no charge. Therefore, in order to separate amino acids, the conditions can be
adjusted to achieve the isoelectric points of all amino acids at different times. For
instance, at different pH values, the aspartic acid exhibits different charges. At pH
1, the aspartic acid has +1 charge. However, with the increase in pH up to 2.8
(isoelectric point of aspartic acid), the large number of molecules have no charge.
The side chain of aspartic acid is less acidic than the α-carboxylic group, and the
presence of enough H+ ions restricts its ionization. At pH 6.6, the ionization of
carboxylic group present inside chain takes place, and as a result, the molecule
acquired two negative charges and one positive charge, but at pH 11.0, molecule
bears only two negative charges. In case of lysine, side chain has one amino group;
at pH 1.0, lysine has two positive charges, while at pH 5.6 one negative and two
positive charges are present on lysine. However at pH 9.7, lysine possesses one
negative and one positive charge (isoelectric point), but at pH 11, lysine has only
one negative charge [16]. The isoelectric point of different amino acids is given in
Table 1.2.
1.9 Effect of the Flow Rate of the Eluting Buffer on the IEC
of Amino Acids
The flow rate of the eluting buffer is an important factor in the separation of amino
acids by ion exchange chromatography because the flow rate of eluent determines the
Table 1.2 Isoelectric point

Amino acid pI Reference
of different amino acids
Aspartic acid 3.0 [6]
Glutamic acid 3.2 [6]
Cysteine 5.1 [6]
Tryptophan 5.9 [6]
Glycine 6.1 [19]
Alanine 6.0 [19]
Histidine 7.6 [6]
Lysine 9.7 [6]
Arginine 10.8 [6]
l-Serine 5.70 [19]
l-Glutamine 5.70 [19]
time of analysis. If the flow rate of eluent through the column is more than optimal,
then fractions which leave the column become unsymmetrical and lead towards
overlapping in tails and peaks of amino acids. Increase in the flow rate of eluent
leads to higher back pressure, which is undesirable for safety [2, 9]. For successful
separations of amino acids by IEC, a steady flow rate of the buffer is required and this
effect can be achieved through constant pressure. Therefore, most of the analysers
have pumps which are pulse-free and provide an unfluctuating power output. The
limit of pressure provided by the pumps is 1–8 MPa, and pressure is controlled by
software. The flow rate depends upon dimensions of the column, type of resin used
in the column and overall design of instrument [16].
1.10 Regeneration of the Ion Exchange Column
The restoration of the ion exchange column is crucial after the adequate number of
amino acid analysis. From a column in order to remove impurities, sodium or lithium
hydroxide is used which are also used to replace Na+ or Li+ ions that are used during
analysis. For this purpose, the optimum concentration of sodium hydroxide and
lithium hydroxide is 0.4 M and 0.3 M, respectively. If cationic resins are contaminated
with proteins, heavy metals or with other molecules, remove resin from the column,
treat the column resin with one percent EDTA in 2 M HCl, at room temperature for
few hours, and regenerate it by boiling in 6 M HCl for 30 min. After it, allow the resin
to cool at room temperature. Then, dilute it to 3 M HCl, filter it, and wash it with
distilled water. After washing the resins, remove it from the filter and then suspend
the resin in 2 M NaOH/LiOH. Then, dilute it to 0.5 M base by boiling the resin for
some time. Now, the resin is ready to use in the analytical column [16].
10 K. B. Alia et al.
1.11 Conclusion
Ion exchange chromatography is a popular technique for analysis of amino acid. As

IEC is rapid and sensitive than any other available chromatographic method. Along
with this improvement in ion exchange resins and instrumentation, detection has
contributed to make ion exchange chromatography a popular technique for analysis
of amino acids. Ion exchange chromatography promises to continue as an exciting
analytical tool, not only for the analysis of amino acids but for a wide range of other
related compounds.
References
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graphic detectors for the analysis of underivatized amino acids. J Chromatogra A 961(1):9–21
11. Small H, Stevens TS, Bauman WC (1975) Novel ion exchange chromatographic method using
conductimetric detection. Anal Chem 47(11):1801–1809
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Chapter 2
Ion Exchange Chromatography
for Enzyme Immobilization
Raissa Pieroni Vaz and Edivaldo Ximenes Ferreira Filho
Abstract The use of ion-exchange materials for separation and purification of pro-
teins is a widely studied and applied tool since the twentieth century. Following
the same basic principle of charge interactions, new applications for ion-exchange
materials have emerged in recent decades. Enzymatic immobilization technology
is one of the most promising applications in terms of bioconversion processes. The
main current demands of the biotechnology industries that use enzymatic catalysis in
conversion processes are the increase in productivity and reduction of overall costs.
These needs can be met mainly by optimizing the enzymatic properties provided
by enzyme immobilization in several carrier/materials. Enzymatic immobilization
in ion-exchange materials is exceptionally simpler when compared to other immobi-
lization methods. It basically involves electrostatic/ionic interactions of weak nature
between protein and resin. These weak interactions generate minimal conformational
changes, improving enzyme chemical and physical stabilities, and increases speci-
ficity, enzyme selectivity, and catalytic activities. All these advantages make this
application more attractive to industry. However, much research and incentives are
still needed to make this technology more robust, efficient and widespread in several
industrial sectors. This chapter pays particular attention to ion-exchange chromatog-
raphy as a robust tool to improve enzyme immobilization.
2.1 Introduction
The use of enzymes in chemical and biological processes goes back to the beginning
of human history. Consciously or not, the Egyptians used enzymatic catalysis in
bread, wines, and in the production of fermented products [1]. However, only during
the last few decades, the wide applications of biocatalysts have been possible in the
industry owing to the exponential growth of knowledge on both enzyme structure and
mechanism of their action, along with the development of extraction, purification,
and protein engineering techniques for enzymes [1, 2].
R. P. Vaz · E. X. F. Filho (B)

Department of Cell Biology, University of Brasilia, 70910-900 Brasília, Distrito Federal, Brazil
e-mail: eximenes@unb.br
© Springer Nature Switzerland AG 2019 13

Inamuddin (ed.), Applications of Ion Exchange Materials in Biomedical Industries,
https://doi.org/10.1007/978-3-030-06082-4_2
14 R. P. Vaz and E. X. F. Filho
Enzymes are biocatalysts with the enormous potential for application in different
chemical and biological industries [3–5]. Their broad applications are attributed to
high selectivity and specificity, along with easy production [6].
However, these characteristics are possible only under mild environmental con-
ditions, whereas many industrial processes require extreme reaction environments
[4]. In addition, in most industries, productivity is vital for the overall process, both
economically and operationally. Therefore, recovery and recycling procedures are
essential for the use of enzymes in large-scale industrial processes [6–9].
In order to satisfy the current requirements of industrial-scale production with
higher productivity, enzymatic immobilization is one of the possible alternatives.
Enzymatic immobilization allows catalyst recovery and reuse, improve enzyme
chemical and physical stabilities, and increases specificity, enzyme selectivity, and
catalytic activities. It also provides easy product separation and an enhanced resis-
tance to inhibitory agents [3, 10–12].
Several immobilization approaches and different carriers have been developed at
laboratory scale as well as industrial scale. Despite the long history and advantages of
enzymatic immobilization, till 2002, only 20% of industrial processes were reported
to use immobilized enzymes [13]. It is estimated that a wide range of industries
including food, pharmaceutical, biomedical, textile, wastewater treatment, and bio-
fuel production industries are currently using this technology [3]. However, adequate
information on the industrial use of this technology is limited and difficult to obtain,
because it involves intellectual property and market competition.
For the application of immobilized biocatalysts in industry, it is essential to design
efficient, robust, and stable systems [14]. Among the possible approaches for enzy-
matic immobilization, physical absorption (ionic interaction, Van der Waals forces,
hydrophobic interactions, and hydrogen bonds) [15] are considered simple and cost-
effective for industrial use.
2.2 Enzyme Immobilization
Enzyme immobilization, by definition, is the confinement of the free/soluble enzyme

to a phase (matrix, carrier, support, or aggregate) different from its substrate or
product [6, 10, 16].
From a historical point of view, the first scientific article that reported enzyme
immobilization was published in 1916 [17]. This report demonstrated that the inver-
tase had the same catalytic activity as the free enzyme when it was adsorbed on
charcoal and aluminum hydroxide carriers.
It was not until 1967 that the first enzyme immobilization industrial application
was reported. In this case, immobilized aminoacylase on the solid support was used
for amino acid resolution [18]. Since then, immobilization technology has attracted
a great deal of attention, with the increase of publications and patents involving
immobilization of different enzymes from diverse carriers. Consequently, consider-
able advances have been observed in industrial applications of these systems [1].
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Silence reigned, save for the murmur of voices down-stairs—far,
indistinct.
The hall was glorious with indirect rays of the sun. It had wonderful
spaciousness, too. Bonnie May gazed down the broad stairway,
duskily bright and warm and silent, and her expression was quite
blissful. She turned and looked up to the landing above—reached by
a narrower flight of stairs. It seemed splendidly remote, and here the
sunlight fell in a riotous flood.
Her sensations must have been something akin to those of a
mocking-bird that inspects the vernal world in May. She released the
folds of the nightgown and “paraded” to and fro in the hall, looking
back over her shoulder at the train. She had put the garment on
again, after Flora’s advent with the gingham dress, primarily for the
purpose of making the journey from her room to her bath. But there
had been a distinct pleasure in wearing it, too. She thought it made
her look like a fairy queen. She felt the need of a tinsel crown and a
wand with a gilded star at its end.
She was executing a regal turn in the hall when her glance was
attracted upward to some moving object on the landing above.
A most extraordinary ancient man stood there watching her.
Realizing that he had been discovered, he turned in a kind of panic
and disappeared into regions unknown. His mode of locomotion was
quite unusual. If Bonnie May had been familiar with nautical terms
she would have said that he was tacking, as he made his agitated
exit.
As for Bonnie May, she scampered into the bathroom, the flowing
train suddenly gripped in her fingers.
Down-stairs they were listening for her, though they pretended not to
be doing so. They heard her in the bathroom; later they heard
movements in her bedroom. And at last she was descending the
stairs leisurely, a care-free song on her lips.
She invaded the dining-room. Mr. Baron had been lingering over his
coffee. The various parts of the morning paper were all about him.
“Good morning,” was Bonnie May’s greeting. She nodded brightly. “I
hope I’m not intruding?”
“Not at all!” Mr. Baron glanced at her with real friendliness. It had not
occurred to him that her dress was fantastic. What he had noticed
was that her face was positively radiant, and that she spoke as he
imagined a duchess might have done.
“You might like to look at the colored supplement,” he added, fishing
around through the various sections of the paper at his feet.
“I thank you, I’m sure; but isn’t it rather silly?” She added
deferentially: “Is there a theatrical page?”
Mr. Baron coughed slightly, as he always did when he was
disconcerted. “There is, I believe,” he said. He glanced over his
shoulder toward a closed door. “I’m not sure Mrs. Baron would
approve of your looking at the theatrical department on Sunday,” he
added.
“Really! And you don’t think she’d see any harm in looking at the
comic pictures?”
Mr. Baron removed his glasses and wiped them carefully. “She
would probably regard the comic pictures as the lesser of two evils,”
he said.
“Well, I never did like to be a piker. If I’m going into a thing, I like to
go in strong.” She made this statement pleasantly.
A most extraordinary ancient man stood there watching
her.
Mr. Baron put his glasses on somewhat hurriedly and looked hard at
the child. He perceived that she was looking at him frankly and with
a slight constriction at her throat, as was always the case when she
felt she must hold her ground against attack.
“I rather think you’re right,” he said reassuringly. “I’m not sure I know
how to find the theatrical page. Would you mind looking?”
But Flora interrupted here. She entered the room with the air of one
who has blessings to bestow.
“You’re invited to go to Sunday-school with us after a while,” she
informed the guest.
“You’re very kind, I’m sure. What’s it like?”
“Oh, there are children, and music, and—” Flora paused. She wished
to make her statement attractive as well as truthful.
“A kind of spectacle?” suggested the guest.
“Hardly that. But there’s somebody to tell stories. It’s very nice, I
think.”
“It certainly sounds good to me. If they’ve got any good people I
might like to get into it, until I find an opening in my own line.”
Mr. Baron removed his glasses again. “Flora, would you undertake to
tell me what she means?” he inquired.
Miss Baron pinched her lips and looked at him with a kind of ripple of
joy in her eyes. “Isn’t it plain?” she asked. She went out of the room
then, and he heard her laughing somewhere in the distance.
He coughed again and turned to his paper, and so, for the first time
in her life, Bonnie May was in a fair way of going to Sunday-school.
Victor didn’t approve of the idea at all, when it was presently made
known to him. He waylaid his mother in the dining-room at a time
when there was no one else about.
“Why not wait until she can get some things?” he asked.
“Victor,” replied Mrs. Baron, holding her head very high, “you’re
assuming that that extraordinary little creature is going to stay here. I
assure you, she’s not. This may be the only chance she’ll ever have
to place herself in the way of a helpful influence on Sunday. She’s
going to Sunday-school to-day.”
“Governess,” responded Victor, smiling steadily, “if you don’t quit
getting angry with me I mean to sue for separate maintenance. Mark
my words.” After which nothing more was said on the subject.
Victor betook himself to the library, however, and indulged in a
moment of fidgeting. Breakers were ahead—that was certain.
It was forcing things, anyway. He took down his Emerson and turned
to a passage which his mother long ago had pronounced a thing
holding low heathen sentiments. He read:
“And why drag this dead weight of a Sunday-school over the whole
of Christendom? It is beautiful and natural that children should
inquire and maturity should teach, but it is time enough to answer
questions when they are asked. Do not shut up the young people
against their will in a pew and force the children to ask them
questions against their will.”
He could not dismiss from his mind the picture of Bonnie May asking
questions in her elfin yet penetrating way, and he realized that the
answers she would get in that place of ordered forms and
conventions might be very far from satisfactory to one of her
somewhat fearful frankness and honesty.
But suddenly he smiled at the pictures he was drawing in his mind.
“She seems pretty well able to take care of herself,” he concluded.
He came upon the heaped sections of the newspapers he had
examined. That reminded him. The newspapers were not the only
source of information—nor perhaps the most likely source—so far as
his immediate needs were concerned.
No, there was a certain visit he must make that morning.
A little later he emerged from the mansion and stood for an instant
on the steps in the brilliant sunlight. Then he descended the steps
and was gone.
CHAPTER VIII
STILL UNCLAIMED
Baron was on his way to see Thornburg.

On six days and seven nights Thornburg was one of the busiest men
in town. But there was one day in the week when he liked to pose as
a man of leisure. From ten or eleven o’clock on Sunday morning,
and until the latter part of the afternoon, there were few people about
the theatre to disturb him or to claim his attention. And during these
hours it was his practise to lean back in the comfortable chair in his
private office in the theatre and look through old letters and
souvenirs, if there were no callers, or to exchange current gossip or
old reminiscences with the people of his profession who dropped in
to see him. Usually these were managers or agents who happened
to be in town, and sometimes there were veteran players who were
retired, or who were temporarily unemployed. And occasionally there
were politicians who liked to keep on affable terms with the source of
free passes.
When Baron entered the manager’s presence he found that usually
engaged person quite at liberty.
The little office was a place which was not without its fascination to
most people. On the walls there were framed photographs of
Jefferson as Rip Van Winkle, of Booth as Richard III, of Modjeska as
Portia, and of other notable players. In many cases the pictures bore
sprawling autographs across their faces, low enough not to hurt.
Between these authentic ornaments there were fanciful sketches of
dancing girls in extravagant costumes and postures, and a general
ornamentation scheme of masks and foils and armor.
So complacent and open-minded was Thornburg when Baron
appeared that the latter came to a swift, seemingly irrelevant
conclusion.
“Nobody has claimed her! She’s going to stay!” were the words that
formed themselves in Baron’s mind. The dull, monotonous aspects
of the old mansion were to be changed. A new voice, like a melody
rising above droning chords, was to greet his ears at morning and
night. A thing of beauty was to take its place before the background
of dull, long-established things.
No one had come to Thornburg to demand of him the child who had
disappeared from his premises—Baron could read as much in the
manager’s expression. Wonderful! Truly wonderful!
“You haven’t had any word yet?” he began.
Thornburg was used to Baron’s ways. He had a friendly contempt for
the dilettante young man about town and newspaper writer who
could have made a place for himself, as everybody agreed, if he had
chosen to do so, but who indulged himself by following his own ill-
directed bent, merely because he was—well, because he was Baron
—or a Baron.
“Not a word,” he replied, smiling indulgently, as if the matter were
really not at all surprising.
Baron read the other’s thought. “But a child like that!” he exclaimed.
“People are sometimes strange,” said Thornburg. “Now, if she had
been a trained dog, or a cat with an unusual pedigree, or a horse
with power to draw loads—then she would have been hunted up
quick enough. But you see, she’s only a child.”
Baron shook his head. He was rejecting all this as inadequate.
“She’s still with you?” continued the manager.
“Yes. I’m hoping she’ll remain with us.”
“She like it there?”
“Like it?” echoed Baron. He couldn’t answer the question. He
thought of something more pertinent to say. “It means that she will
have a home—if we can keep her.”
Thornburg nodded slowly. “I don’t think anything better could happen
to her than for you to keep her,” he said. “I suppose she’ll get the
kind of care a little girl of her kind needs. If she’s just a waif of the
theatre she probably has a lot to learn about—oh, about life and real
things.”
“Very likely,” Baron agreed. He added: “I was hoping you might throw
some light on the case—as to who she is and where she came
from.”
Thornburg shook his head. “No, I couldn’t,” he said.
“About her coming to the theatre——”
“A woman brought her to the theatre and asked to be admitted. She
belonged to the profession—the woman. We usually pass them in if
there’s any room. There happened to be just one seat left down-
stairs—in the back row—and I told her she could have that. I
supposed she would hold the little girl on her lap. I was provoked
when I saw she had let her wander up into the box where you were.
In fact, I spoke to her about it.”
“And you don’t know who the woman was—even by reputation?”
“Oh, there are thousands of such people—people who are ‘of the
profession.’ Vaudeville people, circus performers, members of little
stock companies, third-rate travelling troupes—they all ask for free
seats.”
Baron reflected. “I suppose,” he said at length, “such people are
often in financial straits?”
“My goodness, yes! Almost always.”
“If she—this actress—had really wanted to find the child, she surely
would have made inquiries here at the theatre before now, wouldn’t
she?”
“It would seem so—certainly.”
“What I’m getting at is this: It looks a good deal like deliberate
desertion, doesn’t it?”
“Yes, I should say so.”
“And that’s what I simply can’t believe,” declared Baron. “Still,” he
added, “under the circumstances, I ought to be justified in not saying
anything—in assuming that I have a right to keep what has come
into my possession?”
“Well, for the time being, certainly. Of course, there may be
developments sooner or later. She must belong to somebody; I
mean, she must have a home somewhere.”
“No, she hasn’t.”
“But of course you can’t be sure of that.”
“I am. She’s my authority.”
“You mean she told you that? It was probably a childish fancy—or a
downright falsehood. You have to take into account all manner of
possible circumstances.”
“I think she told me the truth. She doesn’t seem fanciful, in that way.
She has the most remarkable sort of intelligence—of frankness.”
Thornburg’s eyes brightened with interest. “Has she, really?” he
asked. There was an interval of silence and then the manager
laughed. “It strikes me that you’re an odd sort of a chap, Baron,” he
said. “What was your idea in taking her home—a stray child like
that?”
“I don’t think it was so very remarkable. She wanted to go with me,
for one thing. She seemed quite delighted at the prospect of having
a real home.”
The manager turned this statement over in his mind so long that
Baron supposed he was thinking of something else. He sat, his
hands clasped behind his head, regarding one of the pictures on the
wall, well over Baron’s head. Then he aroused himself abruptly.
“What’s your plan regarding her?” he asked.
“I don’t know that I’ve got that far yet. She’ll have the usual schooling
and the sort of training that is customary. When she’s grown—Well,
it’s hard to look far ahead, where a child like that is concerned. Of
course, if Miss Barry ever turns up.... She would have claims we
couldn’t ignore.”
“Who’s Miss Barry?”
“She’s the woman who brought Bonnie May to the theatre. If you
know of an actress by that name——”
“I don’t.”
“She probably hasn’t very much standing. From what Bonnie May
said I judge she belongs to that vast army we never hear much
about in the cities.”
“It’s like this, Baron,” said the manager, with the air of a man who
hasn’t time for useless speculations, “I’m thinking, and I suppose
you’re thinking, that under the circumstances I ought to assume
some of the responsibility for a waif who was lost on my premises.
I’d want to be fair about it, you know.”
“But I wasn’t thinking anything of the kind,” declared Baron.
Thornburg frowned impatiently. “She’ll be a burden to you, of
course,” he argued. “And there’s clearly my share of the
responsibility——”
“I didn’t say anything about a burden. The word was yours. Of
course I had to take her home with me. Or at least that’s the way I
felt about it. You simply couldn’t turn a child like that over to an
orphan asylum, or to the police. You would as readily think of asking
some grand dame to turn a handspring as to expect Bonnie May to
put on a uniform with a lot of other unclaimed children, and go
through the usual order of childish occupations. Somebody has got
to look after her in a different way: somebody who understands. But I
wouldn’t think of her being a burden any more than I would think of
pigeons or flowers being a burden.”
Again Thornburg laughed. “Still, most people are pretty willing not to
have white elephants thrust upon them.”
Baron regarded him steadily, in silence. There was a sort of threat in
that—or a prophecy. And there was indicated that attitude of mind
which sees no beauty in a generous deed. And these were
reflections which Baron did not care to put into words.
The manager became uncomfortable under that glance. “You see,”
he explained, “I can’t help thinking.... Is it possible that a little
footlight butterfly will be comfortable very long in a home like—in a
home where everything is—is just so?” He flushed a little from the
effort to avoid offensive inferences or words. “Won’t she be
lonesome and out of place after the novelty of the thing passes?”
Baron liked that. It was frank and honest. “I don’t think she’ll be
lonesome,” he declared. “Mother will see that she gets interested in
things: in music, probably, or anything she manifests a taste for.
She’s too bright to feel out of place, if she’s helped in the right way.”
“It might work out all right.” Thornburg nodded. “I’ll tell you,” he
added, “suppose you let me help with the job.”
“Help!” echoed Baron. “You mean——”
“By writing a little check once a month.”
“That won’t be necessary. So far as the expense is concerned that
will scarcely be worth considering.”
“Nonsense! You could use it, if only for extra dresses and trinkets.
I’ve no doubt she’ll want a lot of things.”
That was exactly like a theatrical man’s ideas, Baron thought. But he
couldn’t tell Thornburg that his mother would be sure to oppose
anything that would tend to promote childish vanity, especially in the
case of one who was already inclined to overestimate mere
appearances. The gewgaws of the average petted and spoiled child
would have to give place to simplicity and true childishness. Still, he
didn’t wish to offend Thornburg, whose suggestion had doubtless
been based upon a generous impulse.
“It might be managed,” he said. “We’ll speak of that another time.”
He arose and began to shape a casual exit. “There’s nobody now to
take their places,” he said, indicating the portraits of Jefferson and
Booth and the others.
“Not by a thousand miles,” agreed Thornburg. His thoughts seemed
to have been transferred easily to the players who were gone.
But when Baron emerged from the theatre and lost himself in the
throng which the fine May forenoon had attracted from hotels and
side streets, his face brightened with the joy which he felt he need no
longer conceal.
“She’s ours!” were the words that sang within him. “We’re going to
keep her!”
CHAPTER IX
A DISAPPOINTING PERFORMANCE
Baron looked at his watch twice as he climbed the stairs. Yes, the
family had had time to return from church; but they had not done so.
Mrs. Shepard was busy in the dining-room, but otherwise the house
was unoccupied. Silence reigned in the upper regions.
Thomason, the houseman, was looking impatiently down from the
upper landing; but Thomason didn’t count. He was probably hungry.
Baron realized that he, too, was hungry.
He went into the cheerful sitting-room and looked down upon the
street, and instantly his attitude changed.
There they came! And something was wrong. Oh, plainly, something
was wrong.
Mrs. Baron’s head was held high; she was pale; her lips were
compressed. There was nothing gracious in her carriage. She was
marching.
By her side walked Flora, keeping step with difficulty. She appeared
to be fighting off all realization of her mother’s state.
Mrs. Shepard was no longer present to lend her support to Bonnie
May. The faithful servitor had come home immediately after Sunday-
school to look after the dinner, and the child walked alone, behind
her silent elders. Her whole being radiated defiance. She was
apparently taking in every aspect of the street, but her casual
bearing was obviously studied; the determined effort she was
making was not to be concealed.
Baron hurried down-stairs so that he might meet them in the hall,
and engineer a temporary dispersement. He was affecting a calm
and leisurely demeanor when the door opened and Mrs. Baron,
followed by the others, entered.
There was an ominous silence. Bonnie May caught sight of Baron
and approached him with only a partial concealment of eagerness
and hurry.
Mrs. Baron and Flora ascended the stairs: the former leading the
way sternly; the latter moving upward with wan cheeks and bowed
head.
Baron led the way into the sitting-room, Bonnie May following. He
pretended not to see or to apprehend anything unusual. “Well, what
do you think of Sunday-school?” he began gayly.
“I think it’s fierce!” This took the form of an explosion. “It wouldn’t do
even for one-night stands!”
Baron felt the need of an admonitory attitude. “Bonnie May,” he said,
“you should have discovered that it wasn’t a play. It was something
real. It’s a place where people go to help each other.”
“They certainly need help all right enough.” This with a quite unlovely
jeering laugh.
“I wonder what you mean by that?”
“I suppose I meant the same thing you meant yourself.”
Baron paused, frowning. “I meant,” he explained patiently, “that they
are people who want to be as good as they can, and who want to
give one another encouragement.”
The child was conscious of his wish to be conciliatory. She tried to
restrain herself. “Well,” she asked, “if they want to be good, why
don’t they just be good? What’s the use of worrying about it?”
“I’m afraid it isn’t quite so simple a matter as all that.”
Bonnie May’s wrath arose in spite of herself. She was recalling
certain indignities. “I don’t see anything in it but a bum performance.
Do you know what I think they go there for?”
“That’s what I’m trying to find out.”
“I think they go there to watch each other—to find out something bad
about each other.”
“Bonnie May!”
“I do! And I’ve had pretty near enough, too. You asked me and I told
you. You’re all asking me to do things, and asking me questions; and
then if I don’t agree with you in every way I’m wrong. That may look
all right to you, but it doesn’t to me. If I’ve got to take everything, I
mean to be on my way.”
Baron remained silent a full minute. When he spoke again his voice
was persuasive, gentle. “I’m anxious to understand your difficulties,”
he said. “I’m anxious to have you understand ours. I’m sorry I
criticised you. I’m sure you mean to be fair.”
She looked at him with a light of gratitude in her eyes, a quiver of
emotion passing over her face. She had an intense desire to justify
herself—at least to him.
“Do you know what was the first thing they asked me?”
“Your name, probably.”
“No, Mrs. Shepard told them that. They asked me if I was a good
little girl!”
“But I don’t see any harm in that. Why shouldn’t they have asked
you?”
“You don’t! Do you suppose that I was going to tell them that I was—
or that I wasn’t? What nonsense! Are you ‘a good young man’? How
does a question like that sound?”
Baron pondered. “Well—” he suggested.
“Well, I wouldn’t stand it. I asked her if she was ‘a good old
woman’—and the frowzy old thing stared at me just as ugly! She
walked way down into the parquet without looking back. She’d been
grinning when she asked me. I’ll bet she won’t grin like that very
soon again.”
Baron walked to the window and looked out dully, to gain time.
How extraordinary the child’s attitude was! And yet.... He could
understand that she might have been the only child in the troupe with
which she travelled, and that her older companions, weary of
mimicry and make-believe when their work was done, might have
employed very frank, mature speech toward each other and their
young companion.
He turned away from the window with a sigh. “Won’t you take my
word for it, Bonnie May, that these people mean well, and that one
should speak of them with respect, even if one cannot speak of them
with affection?”
“But they don’t mean well. What’s the good of stalling?” She turned
until her back was toward him, and sat so, her cheek in her hand,
and her whole body eloquent of discouragement.
An instant later she turned toward him with the first evidence of
surrender she had shown. Her chin quivered and her eyes were filled
with misery. “Did you tell the man where I was, so they can come for
me if they want me?” she asked.
Here spoke the child, Baron thought. His resentment fled instantly.
“Truly I did,” he assured her. “I have been doing everything I could
think of to help. I want you to believe that.”
“Oh, I do; but you all put too much on me. I want to go back to where
things are real——”
“Real, child? The theatre, and plays, and make-believe every day?”
“It’s the only thing that’s real. You’d know that if you were an artist. It
means what’s true—that’s what it means. Do you mean to tell me
there’s anything real in all the putting on here in this house—the way
you hide what you mean and what you believe and what you want?
Here’s where the make-believe is: just a mean make-believe that
nothing comes of. The theatre has a make-believe that everybody
understands, and so it really isn’t a make-believe, and something
good and true comes of it.”
Her eyes were flashing. Her hands had been clasped while she
spoke until she came to the final clause. Then she thrust her arms
forward as if she would grasp the good and true thing which came of
the make-believe she had defended.
When Baron spoke again his words came slowly. “Bonnie May,” he
said, “I wish that you and I might try, like good friends, to understand
each other, and not to say or think anything bitter or unkind. Maybe
there will be things I can teach you. I’m sure there are things you can
teach me! And the others ... I honestly believe that when we all get
better acquainted we’ll love one another truly.”
She hung her head pensively a moment, and then, suddenly, she
laughed heartily, ecstatically.
“What is it?” he asked, vaguely troubled.
“I’m thinking it’s certainly a pretty kettle of fish I’ve got into. That’s
all.”
“You know I don’t quite understand that.”
“The Sunday-school, I mean, and your mother, and everything. They
put me in with a lot of children”—this somewhat scornfully—“and a
sort of leading lady asked us riddles—is that what you call them?
One of them was: ‘How long did it take to make the world?’”
“But that wasn’t a riddle.”
“Well, whatever it was; and they caught one Smart Alec. She said,
‘Forty days and forty nights,’ and they all laughed—so you could see
it was just a catch. As if anybody knew! That was the only fun I could
see to the whole performance, and it sounded like Rube fun at that.
One odious little creature looked at my dress a long time. Then she
said: ‘I’ve got a new dress.’ Another looked at me and sniffed, and
sniffed, and sniffed. She wrinkled her nose and lifted her lip every
time she sniffed. It was like a kind of signal. Then she said: ‘My papa
has got a big store, and we’ve got a horse and buggy.’ She sniffed
again and looked just as spiteful! I had to get back at that one. ‘Don’t
cry, little one,’ I said. ‘Wait until it’s a pretty day and I’ll come around
and take you out in my automobile.’”
“But you haven’t any automobile!”
“That,” with great emphasis, “doesn’t make any difference. There’s
no harm in stringing people of a certain kind.”
“Oh, Bonnie May!” cried Baron reproachfully, and with quickly
restored calm he added: “Surely one should tell the truth!”
“Yes, one should, if two would. But you can’t afford to show your
hand to every Bedelia that gets into your troupe. No, you can’t,” she
repeated defiantly, reading the pained look in his eyes.
Baron knew that he should have expressed his disapproval of such a
vagrant philosophy as this; but before he had time to frame a tactful
response the child continued:
“Then the leading lady turned to me, thinking up another question. I
made up my mind to be on hand if I had to sleep in the wings. ‘Why
were Adam and Eve driven out of the garden?’ was mine. I said:
‘Because they couldn’t make good!’ She looked puzzled, and I
patted her on the knee. ‘You can’t put over anything on me,’ I said. I
think I shouted it. That stopped the whole show for a minute, and an
old character man up near the stage got up and said: ‘A little less
noise, please.’ Then your mother came back.” (Baron had
anticipated this detail.) “She had been taking the leading part in a
little sketch up in front.” (Teaching her class, Baron reflected, and
smiled wryly in spite of himself.) “She had got through with her
musical turn, and—well, I don’t want to talk about her. She told me I
must sit still and listen to what the others said. Why? I’d like to know.
I couldn’t agree with her at all. I told her I was a professional and
didn’t expect to pick up anything from a lot of amateurs. And then,”
she added dejectedly, “the trouble began.”
Baron groaned. He had hoped the worst had been told. What in the
world was there to follow?
“Your mother,” resumed Bonnie May, “spoke to the woman who had
been asking questions. She said—so that the children could hear
every word—‘She’s a poor little thing who’s had no bringing up.
She’ll have to learn how to behave.’”
She hung her head in shame at the recollection of this. For the
moment she seemed unwilling to proceed.
“And what happened then?” Baron asked persuasively.
“Oh—I was getting—rattled! She had no right to work in a line like
that.”
“But what did you do?”
“I told her.... You know I am sorry, don’t you?”
“Maybe you’d rather not tell me?”
“You’d better know. I told her that when it came to doing the nasty
stuff I had seen pupils from the dramatic schools that looked like
headliners compared with her.”
Baron stiffened. “Goodness! You couldn’t have said that!”
“Yes, I did. And I didn’t have to wait to hear from any prompter,
either. And she—you know she won’t take anything. The way she
looked! She said she was glad to say she didn’t have any idea what I
was talking about. Just a stall, you know. Oh, these good people!
She called Flora and said I was to be taken into a corner, and that I
was to sit there until we went home. And Flora led me into a corner
and the others looked back as if they were afraid of me. They all
sang after a while—a kind of ensemble affair. Flora held the music
over and invited me to sing. I told her musical turns were not in my
line. She just kept on holding the music for me—honestly, she’s the
dearest thing!—and singing herself. It was a crime, the noise she
made. Isn’t it awful when people try to sing and can’t? As if they had
to. Why do they do it? I felt like screaming to her to stop. But she
looked as if she might be dreaming, and I thought if anybody could
dream in that terrible place it would be a crime to wake them, even if
they did make a noise. They had an intermission, and then a man
down in front delivered a monologue.... Oh, me! Talk about the
moving-picture shows! Why, they’re artistic....”
What, Baron wondered, was one to say to a child who talked in such
a fashion?
Nothing—nothing at all. He groaned. Then, to his great relief, Flora
appeared.
“Dinner is ready,” she said, standing in the doorway. There was a
flush on her cheeks and an odd smile on her lips.
Baron took Bonnie May by the hand—he could not quite understand
the impulse which prompted him to do so—and led her into the
dining-room.
He saw that she bore her face aloft, with a painful effort at
unconcern. He was glad that she was given a place next to him, with
the elder Baron on her right, and Flora across the table from her.
He was dismayed to note that his mother was quite beside herself.
He had expected a certain amount of irritation, of chagrin, but not
this ominous, pallid silence. She avoided her son’s eyes, and this
meant, of course, that her wrath would sooner or later be visited
upon his head.
He sighed with discouragement. He realized sadly that his mother’s
heaviest crosses had always come to her from such trivial causes!
She was oddly childish—just as Bonnie May was strangely
unchildlike. Still, she had all the traditions of propriety, of a rule-made
demeanor, behind her. Strange that she could not have risen to the
difficulty that had confronted her, and emerged from a petty
predicament without so much of loss!
The meal progressed in a constrained silence. Bonnie May
concerned herself with her napkin; she admired the design on the
china; she appeared to appraise the dishes with the care of an
epicure. And at last, unfortunately, she spoke.
“Don’t you think, Mr. Baron”—to the master of the house—“that it is a
pretty custom to converse while at table?”
Mr. Baron coughed. He was keenly aware that something had gone
wrong; he was shrewd enough to surmise that Bonnie May had
offended. But he was in the position of the passenger below decks
who senses an abnormal atmosphere but who is unadvised as to the
nature of the storm.
“I’m afraid I’m not a very reliable hand at small talk,” he said
guardedly. “I think my idea is that you ought to talk when you have
something to say.”
“Very good!” agreed Bonnie May, nodding brightly. She patted her
lips daintily with the corner of her napkin. “Only it seems like

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Applications of Ion Exchange Materials in

Nanophotocatalysis and Environmental Applications

Nanobiomaterials : nanostructured materials for

Materials for Biomedical Engineering Fundamentals and

Applications of Biomedical Engineering in Dentistry

Nanophotocatalysis and Environmental Applications:

Polymers Coatings: Technology and Applications 1st

Biomedical Materials 2nd Edition Roger Narayan (Editor)

Advanced Materials for Sodium Ion Storage 1st Edition

Applications of Ion Exchange

ISBN 978-3-030-06081-7 ISBN 978-3-030-06082-4 (eBook)

Library of Congress Control Number: 2018965911

© Springer Nature Switzerland AG 2019

Chapter 1 covers modern applications and basic principles of ion exchange

Jeddah, Saudi Arabia Inamuddin

1 Separation and Puriﬁcation of Amino Acids . . . . . . . . . . . . . . . . . . 1

2.5 Conclusion and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . 22

4.4 Liquid Chromatography (LC) . . . . . . . . . . . . . . . . . . . . . . . .. 82

6 Thermodynamic Kinetics and Sorption of Bovine Serum

8.9.2 Cation-Exchange Resin . . . . . . . . . . . . . . . . . . . . . . . . 171

10.1.1 Determination of Colour in Sugar and

Khush Bakhat Alia, Habibullah Nadeem, Ijaz Rasul, Farrukh Azeem,

According to the IUPAC, chromatography is a technique for separation of sam-

K. B. Alia · H. Nadeem (B) · I. Rasul · F. Azeem · M. H. Siddique

1.2 Ion Exchange Chromatography in the Separation

1.3 Ion Exchange Chromatography of Amino Acids

1.4 Ion Exchange Resins

Table 1.1 Characteristics of polystyrene-type ion exchangers [2]

1.5 Buffer Systems in IEC for Separation of Amino Acids

1.5.1 Sodium Citrate Buffer System

1.5.2 Lithium Citrate Buffer System

1.6 The Relation Between the Concentration of Eluent

Strongly retained amino acids like tyrosine, histidine, aspartate, phenylalanine,

1.7 Effect of Temperature on Separation of Amino Acids

1.8 Effect of pH on Separation of Amino Acids

Table 1.2 Isoelectric point

1.10 Regeneration of the Ion Exchange Column

Ion exchange chromatography is a popular technique for analysis of amino acid. As

Raissa Pieroni Vaz and Edivaldo Ximenes Ferreira Filho

R. P. Vaz · E. X. F. Filho (B)

© Springer Nature Switzerland AG 2019 13

2.2 Enzyme Immobilization

Enzyme immobilization, by definition, is the confinement of the free/soluble enzyme

Baron was on his way to see Thornburg.

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