Analytical techniques in biochemistry and molecular biology

Analytical Techniques in Biochemistry
and Molecular Biology



Rajan Katoch

Analytical Techniques
in Biochemistry
and Molecular Biology


Rajan Katoch
Biochemistry Laboratory
Department of Crop Improvement
CSKHPKV
Palampur, HP
India


ISBN 978-1-4419-9784-5
e-ISBN 978-1-4419-9785-2
DOI 10.1007/978-1-4419-9785-2
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011931968
# Springer Science+Business Media, LLC 2011

All rights reserved. This work may not be translated or copied in whole or in part without the written
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Foreword

Biochemistry is one of the fundamental subjects in life sciences. Biological systems
of virtually all sorts can be controlled in ways not thought possible as recently as
a decade ago. Different disciplines are now being translated through common
language of biochemistry. The twenty-first century is going to be an era of Plant
Biology with emphasis on Biochemistry and Biotechnology. These disciplines
are still expected to answer some of the most urgent questions in the discipline
of life sciences. Basic knowledge has been linked, wherever appropriate, to the
applicability of that knowledge for understanding various physiological processes.
Inter-relatedness and regulations of biological processes are emphasized all
through. In fact, this is a work of trans-disciplinary synthesis where excellence
and relevance are sought to be combined. For successful researchers, it is necessary
to acquire new skills and knowledge in the fields of biochemistry. Therefore, there
was a dire need for such a book which would provide various biochemical and
biotechnological procedures which are in frequent use in modern research.
Dr. Rajan Katoch must be congratulated for producing what I consider a truly
monumental and unique work, thereby rendering a most valuable service to plant
sciences and in particular to biochemistry. The book is targeted to all students of
plant sciences. Scientists and researchers are to be benefitted by this book for
intelligent and enlightened teaching and research work. I hope that the present

book “Techniques in biochemistry and Molecular Biology” will equip students and
teachers alike with the present day concept of understanding of biochemistry.
Dr. S.K. Sharma
Vice-Chancellor

v



Preface

In the recent years we have seen a remarkable increase of interest in biochemical
and molecular methods for the elucidation of structural and functional relationship
among different physiological processes. Science and its application to biochemistry today are facing the greatest opportunities. Detailed mechanistic knowledge
resulting from the application of biochemical methods combined with numerous
interdisciplinary techniques has aided the understanding of biological processes.
Now a variety of new, faster and sensitive methods have enabled us for the
examination of vital processes at biochemical and molecular level. The present
day developments in the field of biochemistry and biotechnology have been made
possible by isolation and purification of numerous enzymes, understanding nucleic
acid metabolism, by refinement of existing techniques and the development of new
ones. Biochemistry can and does contribute to the understanding and solutions of
problems involved in many of the more specialized aspects of plant biology.
The aim of bringing out this book was not to produce a comprehensive text, but a
general and wherever necessary, simplified methods for postgraduate students and
researchers who have recourse to use various techniques during their research
programmes. The book is mainly written for postgraduate students, researchers,
lecturers and scientists in biochemistry and biotechnology. The methods described
are also important components of courses in microbiology, genetics, plant physiology, etc. The text is designed, therefore, to bring researchers of different disciplines
to a level of competence in biochemistry and molecular biology. I believe that this

book can help students and research workers in these diverse fields by providing
them with ready source of biochemical information directly applicable to plant
sciences.
The book has been divided into 15 different chapters covering different aspects.
The first three chapters of the book deal with fundamental aspects which are
necessary for conducting any biological experimentation. The later part of the
book deals with various advanced techniques in biochemistry. Chapter 1 introduces
the readers to the concept of preparation of different solutions which are indispensible part of any experiment. Chapter 2 presents important fundamentals of

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Preface

expression of concentration of solutions. Buffers are important for any enzymatic
work and the concept of buffers and their preparation have been described in the
Chap. 3. Chapter 4 introduces the readers to different techniques used during
biochemical analysis. Carbohydrates and their estimations have been described in
Chap. 5. Chapters 6 and 7 deal with the estimation of lipids, amino acids and
proteins. Protein purification techniques, cell disruption techniques, estimation of
enzymes and isozyme analysis have been described in Chaps. 8–11. Chapter 12
concentrates on various chromatographic separation procedures. Chapter 13 deals
exclusively with the nutritional and anti-nutritional evaluation of different food
stuffs, whereas the methodology for the nutritional evaluation of forages has been
described in Chap. 14. Different biotechnological procedures which are in vogue in
modern research have been described in Chap. 15. Thus, every effort has been made
to cover the different topics which are used in biochemistry and related disciplines.
I hope that the reader, after having studied the concepts and methodology will find

himself quantified to go to the laboratory and start investigating process applicable
to his field.
I am indebted to my family for untiring support that helped me in bringing out
this manuscript.
Palampur, HP, India

Rajan Katoch


Contents

1

Preparation of Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1
Types of Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.1 Standard Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.2 Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.3 Saturated Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.4 Solution of Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.5 General Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
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3
4
4
5

2

Expression of Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1
Molarity (M) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2
Molality (m) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3
Normality (N). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4
Mass Concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5
Mass Fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6
Mass Percent % (w/w). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7
Percentage by Volume or % (v/v) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8
Parts per Million (ppm). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8.1 Preparation of 1,000 ppm Solutions. . . . . . . . . . . . . . . . . . . . . . . . .
2.9
Parts per Billion (ppb) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.10 Preparation of 10À1–10À10 M Solutions from 1 M Stock
Solution by Serial Dilutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.11 Conversion of Molarities into ppm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.12 Conversion of ppm to Parts per Hundred (%)
(ppm  10À6  100). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.13 Preparation of One Molar (1 M) Solutions
of Concentrated Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.14 Formula to Calculate the Volume of Stock Solution

Required to Prepare Solution of Desired Normality . . . . . . . . . . . . . . . .
2.15 Formula to Calculate Volume of Stock Solution
Required to Prepare Solution of Desired Percentage . . . . . . . . . . . . . . .

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2.16

Formula to Calculate the Dilution Factor Used
in Spectroscopic Estimations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of Standard Solution of Acids and Bases . . . . . . . . . . . . . .
2.17.1 Normal Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Percentage Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.18.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.18.2 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ratio Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17
17
18
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24
25
25
26

3

Buffers and Their Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1
Selection of Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2
Acids and Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3
Concept of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4

Buffer System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5
Preparation of Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6
Preparation of Some Commonly Used Buffers . . . . . . . . . . . . . . . . . . . . .

29
29
30
30
33
34
34

4

Techniques in Biochemical Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1
Spectrophotometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2
Measurement of pH with Glass Electrode . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1
Types of Chromatographic Techniques . . . . . . . . . . . . . . . . . . .
4.4
Centrifugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5
Ultracentrifugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6

Isotopic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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41
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5

Carbohydrate Estimations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1
Classification of Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2
Qualitative Tests for Carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.1
Molisch’s Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.2
Anthrone Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.3
Iodine Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.4
Barfoed’s Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.5
Seliwanoff’s Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.6
Fehling’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.7
Benedict’s Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.8
Picric Acid Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.9
Mucic Acid Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.10 Bial’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.11 Quantitative Determination of Carbohydrates . . . . . . . . . . . . .

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2.17
2.18

2.19
2.20

Contents

6

7

Estimation of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1
Extraction, Separation and Estimation of Lipids from Oil Seed. . .
6.2
Determination of Saponification Value of Fats and Oils . . . . . . . . . .
6.3
Determination of Acid Value of Fats and Oils. . . . . . . . . . . . . . . . . . . . .
6.4
Determination of Iodine Number of Fat. . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5
Solubility Test for Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.6
Acrolein Test for Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.7
Qualitative Test for the Presence of Fatty Acids
by Titrimetric Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.8
Test for Unsaturation of Fatty Acids in Lipid Sample . . . . . . . . . . . . .
6.9
Separation of Different Lipid Fractions by Thin Layer
Chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.10 Separation and Identification of Lipids by Column
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.11 Estimation of Fatty Acids by Gas–Liquid Chromatography . . . . . . .
Qualitative and Quantitative Estimations of Amino
Acids and Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1
Qualitative Tests for Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2
Nitrogen Estimation by Micro-Kjeldahl Method (FAO 1970). . . . .
7.3
Sample Preparation for Amino Acid Estimation . . . . . . . . . . . . . . . . . . .
7.4
Estimation of Total Free Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5
Estimation of Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.6
Lysine Estimation in Grains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.7
Estimation of Lysine in Cereal Grains . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.8
Estimation of Methionine in Food Grains. . . . . . . . . . . . . . . . . . . . . . . . . .
7.9
Estimation of Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.10 In Vitro Protein Digestibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.11 Protein Efficiency and Net Protein Ratios . . . . . . . . . . . . . . . . . . . . . . . . .
7.12 Determination of Net Protein Utilization, Digestibility
and Biological Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.13 Protein Estimation by Lowry’s Method . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.14 Polycrylamide-Sodium Dodecyl Sulphate Slab Gel
Electrophoresis (SDS-PAGE) of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . .
7.15 Fluorography of Polyacrylamide Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.16 Quantification of Protein in Polyacrylamide Gels . . . . . . . . . . . . . . . . .

7.17 Protein Electrophoresis Using Starch Gel . . . . . . . . . . . . . . . . . . . . . . . . . .
7.18 Isoelectric Focusing of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.19 Western Blotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.20 Production of Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.21 Immunodiffusion in Agarose Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.22 Enzyme-Linked Immunosorbant Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.23 Preparations of S-30 Extract for Protein Synthesis In Vitro . . . . . . .
7.24 In Vitro Translation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.25 Ammonium Sulphate Fractionation of Proteins. . . . . . . . . . . . . . . . . . . .

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7.26

Methods for Determining Amino Acid Sequences of Protein . . . .

7.26.1 Identification of the NH2-Terminal Amino Acid . . . . . . . .
7.26.2 Identification of the COOH-Terminal Amino Acid . . . . . .
7.26.3 Cleavage of Protein into Smaller Units . . . . . . . . . . . . . . . . . .

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144
144
146

8

Protein Purification Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1
Protein Extraction Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2
Ammonium Sulphate Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3
Ion-Exchange Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.1 Selection of Ion-Exchange Matrix . . . . . . . . . . . . . . . . . . . . . . . .
8.4
Gel Filtration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5
Characterization of Proteins by SDS-PAGE . . . . . . . . . . . . . . . . . . . . . .
8.6
Protein Analysis by Reversed Phase HPLC. . . . . . . . . . . . . . . . . . . . . . .
8.6.1 Separation of Proteins on C-18 Column . . . . . . . . . . . . . . . . . .

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151

153
153
157
161
165
168

9

Cell Disruption and Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.1
Homogenization Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2
Cell Disruption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3
Fractionation of Tissue Homogenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4
Isolation of Mitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.5
Isolation of Chloroplasts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169
169
169
170
170
173

10

Enzymes in Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1 Main Steps During Enzyme Purification . . . . . . . . . . . . . . . . . . . . . . . . . .
10.2 Preparation of Acetone Powder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3 Estimation of Activities of Some Important Enzymes . . . . . . . . . . . .
10.3.1 Malate Dehydrogenase (L-Malate: NAD+
Oxidoreductase EC 1.1.1.37) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.2 Glutamate Dehydrogenase (L-Glutamate: NAD
Oxidoreductase (Deaminating) EC 1.4.1.2)
(L-Glutamate: NADP Oxidoreductase (Deaminating)
EC 1.4.1.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.3 Glutamate Synthase (L-Glutamate: NADP+
Oxidoreductase (Transaminating) EC 1.4.1.13). . . . . . . . . .
10.3.4 Ascorbic Acid Oxidase (L-Ascorbate: Oxygen
Oxidoreductase EC 1.10.3.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.5 Catalase (Hydrogen Peroxide: Hydrogen Peroxide
Oxidoreductase EC 1.11.1.6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.6
Nitrate Reductase (NADH: Nitrate Oxidoreductase
EC 1.6.6.1 (NADH-Dependent)) (NAD(P)H:
Nitrate Oxidoreductase EC 1.6.6.2
(NAD(P)H-dependent)) (NADPH: Nitrate
Oxidoreductase EC 1.6.6.3 (NADPH-Dependent)) . . . . .

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175
177
177
177

178

180
181
182

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Contents

xiii

10.3.7
10.3.8
10.3.9

10.3.10
10.3.11
10.3.12
10.3.13
10.3.14
10.3.15
10.3.16

10.3.17

10.3.18
10.3.19
10.3.20
10.3.21

10.3.22
10.3.23
10.3.24
10.3.25
10.3.26

Nitrite Reductase (NAD(P)H: Nitrite
Oxidoreductase EC 1.6.6.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peroxidase (Donor: H2O2 Oxidoreductase
E.C. 1.11.1.17) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polyphenol Oxidase (Monophenol,
Dihydroxyphenylalanine: Oxygen Oxidoreductase
EC 1.14.18.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Indole Acetic Acid Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lipase (Triacylglycerol Acylhydrolase EC 3.1.1.3) . . . .
Acetylcholine Esterase (Acetylcholine Hydrolase
EC 3.1.1.7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pectin Methylesterase (Pectin Pectyl Hydrolase
EC 3.1.1.11) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aspartate Aminotransferase (Glutamate:
Oxaloacetate Aminotransferase EC 2.6.1.1) . . . . . . . . . . . .
Alanine Aminotransferase (Glutamate:
Pyruvate Aminotransferase EC 2.6.1.2) . . . . . . . . . . . . . . . .
Phosphatases (Orthophosphoric-Monoester
Phosphohydrolase, Alkaline Medium 3.1.3.1)
(Orthophosphoric-Monoester Phosphohydrolase,
Acid Medium 3.1.3.2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Amylases (a-1,4 Glucan 4-Glucanohydrolase
EC 3.2.1.1) and (a-1,4 Glucan Maltohydrolase
EC 3.2.1.2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cellulases (1,4-(1,3:1,4): b-D-Glucan
4-glucanohydrolase EC 3.2.1.4). . . . . . . . . . . . . . . . . . . . . . . . .
Polygalacturonase Activity (Poly 1,4-a-DGalacturonide Glycanohydrolase EC 3.2.1.15) . . . . . . . . .
Estimation of Papain (Papainase EC 3.4.22.2) . . . . . . . . .
Phosphoenol Pyruvate Carboxylase
(Orthophosphate: Oxaloacetate Carboxylase
Phosphorylative EC 4.1.1.31) . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ribulose Biphosphate Carboxylase (3-Phospho
D-Glycerate Carboxylyase EC 4.1.1.39) . . . . . . . . . . . . . . . .
Phenylalanine Ammonia Lyase (L-Phenylalanine
Ammonia Lyase EC 4.3.1.5) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glutamine Synthetase (L-Glutamate: Ammonia
Ligase EC 6.3.1.2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Asparagine Synthetase (L-Aspartate; Ammonia
Ligase EC 6.3.1.4). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lactate Dehydrogenase (L-Lactate: NAD
Oxidoreductase, EC 1.1.1.27) . . . . . . . . . . . . . . . . . . . . . . . . . . .

185
186

188
190
191
193
195
196
198

199

201
203
205
208

210
211
212
213
215
216


xiv

Contents

10.3.27
10.3.28
10.3.29

Glutamate–Oxaloacetate Transaminase. . . . . . . . . . . . . . . . . 218
Glutamate–Pyruvate Transaminase . . . . . . . . . . . . . . . . . . . . . 221
Phosphatases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

11

Isoenzyme Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.1 General Methodology for Isozyme Analysis . . . . . . . . . . . . . . . . . . . . . .

11.2 Isozyme Analysis of Esterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3 Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4 Polyphenol Oxidase (PPO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.5 Acid Phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.6 Glutamine Synthetase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.7 Phosphoenol Pyruvate Carboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.8 Glutamate Dehydrogenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.9 Indolacetic Acid Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.10 Malate Dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

227
228
228
229
229
229
230
230
230
231
231

12

Chromatographic Separations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1 Separation and Identification of Amino Acids
by Descending Paper Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12.1.4 Observations and Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1.5 Note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.2 Separation and Identification of Amino Acids
by Ascending Paper Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.2.1 Reagents and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.2.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.3 Separation and Identification of the Amino Acids
by Two-Dimensional Paper Chromatography. . . . . . . . . . . . . . . . . . . . .
12.3.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.3.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.3.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4 Identification of Lipids by TLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.2 Reagents and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5 Separation of Pigments by Adsorption
Column Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5.2 Materials and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6 Separation and Identification of Sugars by Adsorption TLC . . . . . .
12.6.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233
233
233
233
234
235
235

236
236
236
237
237
237
238
238
238
239
239
240
240
240
240
241
241


Contents

12.7

12.8

12.9

12.10

13

xv

12.6.2 Reagents and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6.4 Note. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Separation of Amino Acids by Ion Exchange Column
Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.7.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.7.2 Materials and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.7.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concentration of Dilute Protein Solutions
with Sephadex G-25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.8.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.8.2 Materials and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.8.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concentration of Dilute Protein by Column
Chromatography using Sephadex G-25 . . . . . . . . . . . . . . . . . . . . . . . . . .
12.9.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.9.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.9.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of Molecular Weight of Protein
by Gel Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.10.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.10.2 Materials and Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.10.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Methods for Nutritional Quality Evaluation of Food Materials . . . . .
13.1
Determination of Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2
Estimation of Protein Content by Micro-Kjeldahl
Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3
Estimation of Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.4
Estimation of Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5
Estimation of Starch. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.6
Estimation of Amylose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.7
Estimation of Gluten in Wheat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.8
Estimation of Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.9
Protein Fractionation in Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.10 Estimation of Crude Fibre. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.11 Estimation of Dietary Fibre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.12 Estimation of Oil Content in Oilseeds . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.13 Estimation of Fatty Acids by Gas–Liquid
Chromatography (GLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.14 Determination of Lipase and Lipoxygenase Activity . . . . . . . . . . . .
13.15 Estimation of Carotenoid Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.16 Estimation of Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.17 Estimation of Chlorophylls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

242
242
243

244
244
244
244
245
245
245
246
247
247
247
247
248
248
248
249
251
251
253
255
256
257
258
259
260
262
264
265
266
268

270
272
276
277


xvi

Contents

13.18
13.19
13.20
13.21
13.22
13.23

Estimation of Curcumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Thiamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biological Evaluation of Protein Quality by Rat Feeding
Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of In Vitro Protein Digestibility
in Food Grains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Blood Glucose and Cholesterol . . . . . . . . . . . . . . . . . . . .
Estimation of High Density Lipoprotein (HDL),
Low Density Lipoprotein (LDL) and Very Low
Density Lipoprotein (VLDL) Cholesterol in Serum . . . . . . . . . . . . . .

Estimation of Phenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chlorogenic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Capsaicin in Chillies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Glucosinolates Content in Oil Seeds . . . . . . . . . . . . . .
Determination of Polyphenols in Pulse Grains . . . . . . . . . . . . . . . . . . .
Estimation of Aldehydes in Food Stuffs . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of ODAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Assessment of Rancidity of Oil and Fats . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of Phytin Phosphorus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Trypsin Inhibitor in Legumes. . . . . . . . . . . . . . . . . . . . . .
Estimation of Cyanogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Indoleactic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Ethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297
299
300
301
303
305
306
309
310
311
312
315
317
319

320
321

Nutritional Evaluation of Forages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.1
Preparation of Plant Extract for Analysis . . . . . . . . . . . . . . . . . . . . . . . . .
14.2
Proximate Analysis of Forages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3
Estimation of Dry Matter (DM). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.1 DM in Bulk Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.2 Dry Matter in Laboratory Samples . . . . . . . . . . . . . . . . . . . . .
14.3.3 DM in Silage, Haylage and Molasses . . . . . . . . . . . . . . . . . .
14.4
Crude Protein (CP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5
Determination of True Protein and Non-Protein Nitrogen . . . . . . .
14.6
Estimation of Ammonia Nitrogen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.7
Estimation of Urea Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.8
Estimation of Ether Extract (Crude Fat) . . . . . . . . . . . . . . . . . . . . . . . . . .
14.8.1 Preparation of Thimble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.9
Determination of Crude Fibre (CF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.10 Estimation of Total Ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.11 Estimation of Acid Insoluble Ash. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323

323
324
327
327
328
329
330
332
333
334
335
335
336
338
339

13.24
13.25
13.26

13.27
13.28
13.29
13.30
13.31
13.32
13.33
13.34
13.35
13.36

13.37
13.38
13.39
13.40
13.41
14

279
279
282
283
284
287
292
294


Contents

14.12
14.13
14.14

Nitrogen-Free Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of Free Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Fibre Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.14.1 Estimation of Neutral-Detergent Fibre
(Determination of Cell Contents
and Cell Wall Constituents) . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.14.2 Determination of Acid-Detergent Fibre. . . . . . . . . . . . . . .

Determination of Acid-Detergent Lignin . . . . . . . . . . . . . . . . . . . . . . . . .
Permanganate Lignin, Cellulose and Silica . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Non-Starch Polysaccharides . . . . . . . . . . . . . . . . . . . . . . .
14.17.1 Determination of Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of Digestibility by In Vitro
and In Sacco Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.18.1 In Vitro Dry Matter Digestibility . . . . . . . . . . . . . . . . . . . . .
14.18.2 Modified Method of In Vitro Dry
Matter Digestibility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantification of Tanins in Foliage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method for Extraction of Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Measurement of Total Phenolics and Tannins
Using Folin–Ciocalteu Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of Condensed Tannins (Proanthocyanidins) . . . . . .
Gallotannin Determination by Rhodanine Assay . . . . . . . . . . . . . . . . .
Gallotannin Determination Using HPLC . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of Protein-Precipitable Phenolics . . . . . . . . . . . . . . . . .
Protein-Binding Capacity by Filter Paper Assay . . . . . . . . . . . . . . . . .
Characterization of Phenolic Compounds by Thin Layer
Chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Nitrates and Nitrites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Total Glucosinolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Oxalic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Trypsin Inhibitor in Forages . . . . . . . . . . . . . . . . . . . . . . .
Estimation of Cyanogenic Glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.32.1 Qualitative Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.32.2 Titrimetric Method for Quantitative Test . . . . . . . . . . . . .
Qualitative Estimation of Ricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

341

341
342

Techniques in Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.1 Polymerase Chain Reaction (PCR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.2 Isolation of Plant DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.3 Isolation of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.4 Quantitative Estimation of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.5 Quantitative Estimation of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.6 Southern Blot Analysis of Plant DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.7 Isolation of Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

377
377
380
383
385
386
387
394

14.15
14.16
14.17
14.18

14.19
14.20
14.21
14.22

14.23
14.24
14.25
14.26
14.27
14.28
14.29
14.30
14.31
14.32

14.33
15

xvii

343
344
346
347
350
350
351
352
353
355
356
357
358
359

361
362
364
365
368
370
371
372
373
373
373
375


xviii

Contents

15.8
15.9
15.10
15.11
15.12
15.13
15.14
15.15

Rapid Isolation of Plasmid DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miniprep of Plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cultivation of Lambda (l) Phage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Extraction Lambda DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Restriction (Digestion) of DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DNA Electrophoresis in Agarose Gel. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recovery of DNA Fragments from Agarose Gels . . . . . . . . . . . . . . . .
Isolation of mRNA by Affinity Chromatography. . . . . . . . . . . . . . . . .

397
399
401
403
405
407
409
410

Appendix A

Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417

Appendix B

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419

Appendix C

Reference Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431

Chapter 1

Preparation of Solutions

The techniques in biochemistry generally involve the use of solutions. These
solutions have different uses, e.g. extraction of biomolecules from tissues and
their separation, quantitative estimation, purification and characterization of different compounds. Different kinds of solutions are integral part of molecular biology
techniques. Therefore, it is important that one must be fully familiar with the proper
preparation of solutions. A solution is homogeneous mixture of two or more nonreacting substances and has uniform properties such as chemical composition,
density, refractive index, etc. However, its composition can be varied within certain
fixed limits. A solution which is made up of two components is called binary
solution. The dissolved substance in solution is called the solute and the medium
in which it is dissolved is known as the solvent. The solute in a solution is always
present in a smaller quantity than the solvent. The most commonly used solutions in
biochemical work are of solid–liquid and liquid–liquid type.
Composition of a solution can be expressed in two ways: quantity and concentration. Quantity is the amount of any substance (solute) present in a solution/
solvent irrespective of the amount/volume of the solvent/solution. In contrast,
concentration refers to the quantity of the solute present in an exact or a specific
amount of solvent or that of solution. For example, a solution containing 4 g NaOH
in a volume of 100 mL of a solution has a total quantity of 4 g NaOH. When this
amount is expressed per unit volume such as 4 g/100 mL of the solution, then it
is termed as 4% (concentration) solution.

1.1
1.1.1

Types of Solutions
Standard Solutions

A standard solution is one that contains a precisely known concentration of solute.
We express the concentration in terms of molality, molarity and normality. Sometimes
the concentrations are also expressed as % solution and in ppm (parts per million)
R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,
DOI 10.1007/978-1-4419-9785-2_1, # Springer Science+Business Media, LLC 2011

1


2

1

Preparation of Solutions

Molality of standard solution (m): The molality of solution is defined as the number
of moles of solute dissolved in 1,000 g of the solvent. We can make 1 molal solution
by weighing out precisely 1 mole of the substance and then dissolving it in 1,000 g
solvent. A 0.5 molal solution contains one half mole of solute per 1,000 g of
solvent. A 2 M solution has 2 moles of solute in 1,000 g of solvent. This term has
no relevance with the volume.
Molarity of standard solution (M): The molarity of a solution is defined as the
number of moles of solute present per litre of the solution. The standard solution of
1 M concentration contains 1 mole of solute per litre of solution. The gram-formula
weight of NaCl is 58.5 g. Weigh out this quantity of NaCI and dissolve it in water
and make its volume to 1 L in a volumetric flask of 1,000 mL capacity. This will be
called 1 M solution of NaCI. If we use only one half mole i.e. 29.25 g in 1 L of
solution, our solution would be 0.5 M. Using 2 moles or 117 g in 1 L of solution
makes a 2 M solution.
Normality of standard solution (N): The normality of solution is defined as the

number of gram equivalent of substance present/litre of the solution. 1 N solution
contains 1 GEW of substance per litre of solution. In the case of acids it is the
number of gram equivalent of H+ present in 1 L of the solution. Thus, 1 N acid
solution contains 1 GEW of H+ per litre of solution. To prepare a litre of 0.1 N HCl
(the mol. wt. of HCl is 36.5 g) only 0.1 gEq of HCl per litre of solution is required.
Hence, use 3.65 g of HCl diluted to 1 L. But this is 3.65 g of the anhydrous
hydrogen chloride in one 1 L of solution, not 3.65 g of the concentrated
hydrochloric acid in bottle of the laboratory. How may we determine the volume
of concentrated hydrochloric acid to be measured out which contain 3.64 g of
hydrogen chloride? This may be found very easily as the assay information is
printed on the manufacture’s label on the bottle of concentration HCl.
The concentrated acid is generally 37.23% hydrogen chloride by weight and has
a specific gravity of 1.19; it means 1 mL of conc. HCl weighs 1.19 g of which
37.23% is hydrogen chloride.
1 mL conc. HCl contains: 0.3723 Â 1.19 g ¼ 0.443 g of hydrogen chloride.
Thus, the volume of solution needed to provide 3.65 g of HCl is 3.65/
0.443 g/mL ¼ 8.43 mL of conc. HCl.
If 8.43 mL of conc. HCl is made to 1 L with distilled water with earlier
mentioned information, it will make 0.1 N HCl.
We know that 1 mole (98 g) of H2SO4 contains 2 gEq of H+ and is a dibasic acid.
Therefore, 1 N H2SO4 contains (98/2) or 49 g of H2SO4 per litre of solution. A 5 N
solution contains 245 g (49 Â 5) of H2SO4 per litre, and 0.01 N H2SO4 contains
0.49 g (49/100) H2SO4 per litre of solution. Concentrated sulphuric acid is usually
95–98% and has a specific gravity of about 1.84. (for details see appendix. Reagent 1).
Percentage of standard solution (%): Parts of solute dissolved to form hundred
parts of solution is termed as % solution. One gram of any compound dissolved in to
form 100 mL of solution is termed as 1% solution and 2 g of NaCI dissolved in
water to make to a final volume of 100 mL is a 2% solution.

1.1 Types of Solutions

3

Standard solution of known concentration in parts per million (ppm): Parts of
solute dissolved to form million parts of solution is termed as a ppm solution. In
a simple way 1 g dissolved in. 10,00,000 g or in other words 1 mg in 1 L makes
1 ppm solution.
Standard solution of hydrated salts: Crystalline salts containing water of hydration
must be given special consideration in making up standard solutions. For example,
crystalline cupric sulphate has the empirical formula CuSO4∙5H2O, a 1 M CuSO4
solution. However, the formula weight of this hydrated salt is 249.5. This fact has to
be taken into consideration when weighing out moles or gram equivalent of such
hydrated salts i.e. 249.9 g of salt is to be weighed for making 1 M solution 1 L
instead of 159.5 g.
One millimolar or 1 mM ¼ 1=1; 000 M ¼ 0:001 M ¼ 1 Â 10À3 M
One micromolar or 1 mM ¼ 1=10; 00; 000 M ¼ 1 Â 10À6 M
Where N1 and V1 are normality of a solution and N2 and V2 are normality and
volume of the solution to be prepared.

1.1.2

Stock Solution

Stock solution of a substance is the one having a concentration many folds higher
than that actually required in the experiment. Stock solutions are prepared for the
substances that are to be used frequently and are stable at higher concentration for
several days and can be used after appropriate dilution just before use. It is sometimes convenient to weigh out a relatively large amount of the compound and
prepare a stock solution of that compound from which small amounts can be
withdrawn at convenience and added to solution of other components. The use of

a stock solution thus cuts down on the amount of pipetting and at the same time
reduces variability between a number of similar incubation mixtures, assay mixture
etc. A particular volume of solution containing a desired concentration of a substance has to be prepared by using its stock solution. A simple mathematical
equation can be used to calculate the volume of the stock solution needed to prepare
a solution of required volume containing desired concentration of the compound:
N1 V1 ¼ N2 V2
Where
N1 ¼ concentration of the solution to b prepared
V1 ¼ volume of the solution to be prepared
N2 ¼ concentration of the stock solution
V2 ¼ volume of the stock solution.


4

1

Preparation of Solutions

For example, for preparing 200 mL of a solution containing 15 mM Tris from a
stock of 80 mM Tris, the volume of the stock solution which should be used can be
calculated as:
V2 ¼

N1 V1 10 Â 200
¼
¼ 25 mL
N2
80

Therefore, by taking 25 mL of 80 mM stock solution of Tris and making its final
volume to 200 mL (by adding water or other solutions) a solution containing 10 mM
Tris is obtained.

1.1.3

Saturated Solution

Solubility of any substance in a particular medium varies with temperature. When a
solution contains the solute in an amount in excess of that which can completely be
dissolved at a given temperature and the solute in solution is in equilibrium with the
excess of undissolved solute, the solution is said to be saturated.

1.1.4

Solution of Acids

The commonly used acids like hydrochloric acid and sulphuric acid are not pure
and their effective strength is low which is to be taken into account while making
their solutions of desired concentrations. The following formula can be applied to
compute the volume of desired concentrations. The following formula can be
applied to calculate the volume of the commercially available concentrated acid
required for preparing a solution of required normality:
V1 ¼

Eq: wt: of acid  V2  normality  100
1; 000  specific gravity  purity ð%Þ

Where
V1 ¼ required volume of the concentrated acid

V2 ¼ total volume of the acid solution of desired normality to be prepared.
The use of above formula could be explained by considering the following
example.
Prepare 1,000 mL of 1 N sulphuric acid from concentrated acid (purity, 96%;
specific gravity, 1.84; Mol. wt., 98).
Eq. wt. of sulphuric acid ¼ 49 (since sulphuric acid is disprotic).
Putting various values in the above equation, we get
V1 ¼

49 Â 1; 000 Â 1 Â 100
¼ 27:7 mL
100 Â 1:84 Â 96


1.1 Types of Solutions

5

Thus, take 27.7 mL of concentrated sulphuric acid and dilute it to 1,000 mL with
water to get 1 N solution of sulphuric acid.

1.1.5

General Precautions

1.1.5.1

Cleaning of Glassware

1. Ordinary glassware should be thoroughly cleaned with washing soda or any

detergent followed by washing with ordinary tap water and rinsing with distilled
water. Care should be taken to remove previous markings on the glassware, if
any while cleaning.
2. Cleaning of oil flasks, used while estimating ether extract, should be done by
slight boiling with dilute alkali (NaOH) followed by same procedure adopted for
cleaning ordinary glassware. Care, however, should be taken not to use any
brush for cleaning inside the flask to avoid scratch formations.
3. Graduated glassware may be cleaned by initially keeping in chromic acid
solution (dissolve about 60 g potassium dichromate in 300 mL tap water by
thorough stirring and boiling, to which around 450 mL of commercial sulphuric
acid is added slowly after cooling) kept in cylindrical jar for reasonable time
followed by washing and cleaning as per ordinary glassware. Discard chromic
solution when it develops green colour.

1.1.5.2

Drying of Glassware

1. Ordinary glassware can be dried by keeping in hot air oven at low temperatures.
2. Graduated glassware (pipettes, burettes, measuring cylinders, volumetric flasks
etc.) should never be dried in hot air oven as high temperature would change the
volume for which they are graduated for.

1.1.5.3

Other Laboratory Precautions

1. Laboratory floor, working tables and water sinks should be kept neat and clean
and it should be well ventilated and provided with an exhaust fan to remove
unwanted gases, fumes and smoke.

2. One should work fully protected in laboratory by wearing white drill aprons and
shoes.
3. Store chemicals and glassware in alphabetical order in well protected cupboards.
4. Reagents should be properly labelled with date of preparation before placing
them on shelf.
5. Systematic breakage record should be maintained.


6

1

Preparation of Solutions

6. Always use acid and alkali gloves while handling strong acids and alkalies.
7. While working in Kjeldahl digestion room, use fume protecting face mask to
avoid inhalation of highly irritating sulphur dioxide fumes.
8. Distilled water bottles should be kept tightly corked to avoid absorption of
atmospheric gases.
9. Always add acid to water slowly from the sides of the container near the sink.
10. While opening liquor ammonia bottles, especially during summer season, cool
it for some time in a freezer to avoid sudden spurt of ammonia gas accumulated
in the bottles.
11. Set the balance and check the oscillation of pans before using analytical
balances. After use the pan and platform of the balance should be cleaned
with a camel hair brush, in case of any spillage of chemical, sample etc. during
weighing.
12. Proper record of usage of special equipment should be made in log book meant
for it showing date, time and condition of the equipment.
13. Fire extinguishers should be provided in each laboratory.

14. Always work in company during odd hours for the fear of fire and electrical
accidents.
15. All used filter papers and other materials should be deposited in water baskets.
16. Smoking in laboratory premises should be restricted to avoid catching accidental firing by highly inflammable chemicals.
17. All observations should be recorded at least in duplicate.
18. Never pipette strong acids and alkalies with mouth. Always use adopter or
rubber bulb or bulb pipette.
19. Never blow the solution left at the tip of the pipette and delivery of the reagent
drawn into pipette should be uniform giving appropriate time, varying from 10
to 30 s. for quantities of 2–50 mL.
20. Mouth should be washed quickly with water or weak solution of washing soda
during accidental sucking of acids.
21. Acid and alkali spillage on working tables, floor and clothes should be thoroughly washed with water after suitable neutralising with either weak alkali in
case of acid and weak acid in case of alkali.
22. Use always glass distilled water while analysing minerals.
23. Commercial sulphuric acid should only be used for digestion of samples for
nitrogen/protein estimation.
24. No need to use distilled water while making up the volume of digested sample
for protein estimation and during the analysis of crude fibre.
25. Always use self prepared reagents and indicators.
26. Consider lower meniscus for clear colourless and upper meniscus for
coloured solutions while recording observations with the help of measuring
glassware.
27. During cooling samples in a desiccator, the lid should be displaced to leave a
small space, which can be closed after complete cooling.