Table Of ContentBiotechnology
Introduction
to
Introduction Instrumentation
to
Instrumentation
Life Sciences
in
Life Sciences
in
Instrumentation is central to the study of physiology and genetics in living organisms,
especially at the molecular level. Numerous techniques have been developed to address
this in various biological disciplines, creating a need to understand the physical
principles involved in the operation of research instruments and the parameters required
in using them. Introduction to Instrumentation in Life Sciences fills this need
by addressing different aspects of tools that hold the keys to cutting-edge research and
innovative applications, from basic techniques to advanced instrumentation. The text
describes all topics so even beginners can easily understand the theoretical and 1.0
practical aspects.
90
Comprehensive chapters encompass well-defined methodology that describes the
instruments and their corresponding applications in different scientific fields. The book 80
covers optical and electron microscopy; micrometry, especially in microbial taxonomy;
pH meters and oxygen electrodes; chromatography for separation and purification of 70
products from complex mixtures; spectroscopic and spectrophotometric techniques to
determine structure and function of biomolecules; preparative and analytical centrifuga- 60
tion; electrophoretic techniques; x-ray microanalysis including crystallography;
50
applications of radioactivity, including autoradiography and radioimmunoassays;
and fermentation technology and subsequent separation of products of interest.
40
The book is designed to serve a wide range of students and researchers in diversified
30
fields of life sciences: pharmacy, biotechnology, microbiology, biochemistry, and environ-
mental sciences. It introduces different aspects of basic experimental methods and
20
instrumentation. The book is unique in its broad subject coverage, incorporating funda-
mental techniques as well as applications of modern molecular and proteomic tools that
10
are the basis for state-of-the-art research. The text emphasizes techniques encountered
both in practical classes and in high-throughput environments used in modern industry.
0
As a further aid to students, the authors provide well-illustrated diagrams to explain 200 400 500 600 700 800 900 1000 1100
the principles and theories behind the instruments described.
K14893
Prakash S. Bisen
ISBN: 978-1-4665-1240-5
90000
Anjana Sharma
9 781466 512405
Introduction
to
Instrumentation
Life Sciences
in
Introduction
to
Instrumentation
Life Sciences
in
Prakash S. Bisen
Anjana Sharma
Boca Raton London New York
CRC Press is an imprint of the
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Contents
Preface..............................................................................................................................................xv
About the Book ..............................................................................................................................xvii
Acknowledgments ...........................................................................................................................xix
Chapter 1 Microscopy ...................................................................................................................1
1.1 Introduction .......................................................................................................1
1.2 Magnification, Resolution, and Contrast ...........................................................1
1.3 Light (Bright Field) Microscopy .......................................................................3
1.3.1 Resolution .............................................................................................5
1.3.2 Contrast of a Microscope .....................................................................7
1.3.3 Uses of the Light (Bright Field) Microscope .......................................9
1.3.4 Care of the Microscope ........................................................................9
1.4 Dark Field Microscope ....................................................................................11
1.5 Phase Contrast Microscope .............................................................................12
1.6 Interference Microscope ..................................................................................13
1.7 UV and Fluorescence Microscopes .................................................................13
1.7.1 Uses of UV and Fluorescence Microscopes .......................................15
1.8 Electron Microscopy .......................................................................................15
1.8.1 Operation of TEM ..............................................................................16
1.8.1.1 Parts ....................................................................................16
1.8.1.2 Sample Preparation .............................................................16
1.8.1.3 Operational Problems .........................................................18
1.8.2 Disadvantages of TEM .......................................................................19
1.8.3 Operation of the SEM ........................................................................19
1.8.3.1 Principle ..............................................................................19
1.8.3.2 Parts ....................................................................................19
1.8.3.3 Sample Preparation .............................................................20
1.8.4 Advantages and Disadvantages of SEM over TEM ...........................20
1.9 Tunneling Electron Microscopy ......................................................................20
1.10 Confocal Microscopy ......................................................................................21
1.10.1 Modern Confocal Microscopes ..........................................................22
1.11 Techniques in Microscopy ...............................................................................24
1.11.1 Hanging Drop Technique ...................................................................24
1.11.2 Use of the Hemocytometer .................................................................24
1.11.3 Ocular Meter and Stage Micrometer for Micrometry ........................25
1.12 Electron Microscopy .......................................................................................26
1.12.1 Freeze Etching and Metal Shadowing................................................26
Suggested Reading .....................................................................................................36
Important Links ..........................................................................................................36
Chapter 2 Micrometry ................................................................................................................37
2.1 Introduction .....................................................................................................37
2.2 Structure of an Ocular Micrometer .................................................................37
2.3 Conjugate Image-Forming Focal Planes .........................................................37
v
vi Contents
2.4 Eyepiece Designs .............................................................................................39
2.4.1 Types of Eyepieces .............................................................................39
2.5 Stage Micrometer ............................................................................................40
2.5.1 Counting Chambers Stage Micrometer ..............................................40
2.6 Filar Eyepiece Micrometer ..............................................................................41
2.7 Principle of Filar Eyepiece Micrometer ..........................................................42
2.8 Working ...........................................................................................................42
2.8.1 Components of the Ocular Micrometer..............................................42
Suggested Reading .....................................................................................................44
Important Links ..........................................................................................................44
Chapter 3 Electrochemical Techniques ......................................................................................45
3.1 Introduction .....................................................................................................45
3.2 Structure of a pH Meter ...................................................................................45
3.2.1 Glass Membrane Electrodes ...............................................................45
3.2.2 Reference Electrode ...........................................................................46
3.3 Principles .........................................................................................................47
3.4 Factors Limiting the Accuracy of pH Measurements .....................................48
3.5 Measurement of pH .........................................................................................49
3.6 Working ...........................................................................................................50
3.6.1 Potentiometric Method of pH Measurement ......................................50
3.6.2 Reagents Used in the Potentiometric Method of
pH Measurement ................................................................................50
3.6.2.1 Calibration of the Electrode System against
Standard Buffer Solutions of Known pH ............................50
3.6.2.2 pH 4 Buffer Solution ...........................................................50
3.6.2.3 pH 7 Buffer Solution ...........................................................50
3.6.2.4 pH 9.2 Buffer Solution ........................................................50
3.7 Procedure for Measuring pH Using a pH Meter .............................................50
3.7.1 Modified Glass and Solid-State Membrane Electrodes .....................51
3.7.2 Solid-State Membrane Electrodes ......................................................51
3.7.3 Storage Conditions for Glass Probes ..................................................51
3.8 Cleaning and Troubleshooting of Glass Probes ..............................................52
3.9 Application of pH Measurements ....................................................................53
3.10 Oxygen Electrode ............................................................................................53
3.10.1 Calibration of the Oxygen Electrode ..................................................55
3.10.2 Applications of the Oxygen Electrode ...............................................56
Suggested Reading .....................................................................................................59
Important Links ..........................................................................................................59
Chapter 4 Chromatography .........................................................................................................61
4.1 Introduction .....................................................................................................61
4.2 General Principles ...........................................................................................61
4.3 Column Chromatography ................................................................................62
4.3.1 Chromatography Columns .................................................................62
4.3.2 Stationary Phases ...............................................................................62
4.3.3 Packing of Columns ...........................................................................64
4.3.4 Application of Sample ........................................................................64
4.3.5 Column Development .........................................................................65
4.3.6 Fraction Collection and Analysis .......................................................66
Contents vii
4.4 Paper Chromatography and Thin-Layer
Chromatography ..............................................................................................67
4.4.1 Principles ............................................................................................67
4.4.2 Types of Paper ....................................................................................68
4.4.3 Thin Layer ..........................................................................................68
4.4.4 Sample Application ............................................................................68
4.4.5 Development .......................................................................................68
4.4.6 Component Detection .........................................................................70
4.4.7 High-Performance Thin-Layer Chromatography ..............................70
4.4.8 Advantages of Thin-Layer Chromatography
over Paper Chromatography ...............................................................70
4.5 Sample Collection, Preservation, and Preparation ..........................................71
4.6 Adsorption Chromatography ...........................................................................71
4.6.1 Principle .............................................................................................71
4.6.2 Adsorbents ..........................................................................................72
4.6.3 Solvents ..............................................................................................73
4.7 Partition Chromatography ...............................................................................73
4.7.1 Principles ............................................................................................73
4.7.2 Countercurrent Chromatography .......................................................74
4.8 High-Performance Liquid Chromatography ...................................................74
4.8.1 Principles ............................................................................................74
4.8.2 Column ...............................................................................................74
4.8.3 Column Packing .................................................................................76
4.8.4 Column-Packing Procedure ...............................................................76
4.8.5 Chromatographic Solvent (Mobile Phase) .........................................76
4.8.6 Pumping Systems ...............................................................................76
4.8.7 Detector Systems ................................................................................77
4.8.8 Practical Procedure ............................................................................77
4.9 Fast Protein Liquid Chromatography ..............................................................78
4.9.1 Principles ............................................................................................78
4.9.2 Apparatus and Materials ....................................................................78
4.9.3 Calibrations ........................................................................................81
4.9.4 Maintenance .......................................................................................81
4.9.5 Troubleshooting ..................................................................................82
4.9.6 Advantages .........................................................................................82
4.9.7 Applications ........................................................................................82
4.10 Gas–Liquid Chromatography ..........................................................................85
4.10.1 Principle .............................................................................................85
4.10.2 Solid Support and Stationary Phase ...................................................86
4.10.2.1 Column Packing .................................................................87
4.10.3 Sample Preparation and Application ..................................................87
4.10.4 Carrier Gas .........................................................................................88
4.10.5 Detectors ............................................................................................88
4.10.6 Amplifiers and Recorders ...................................................................89
4.11 Ion-Exchange Chromatography .......................................................................89
4.11.1 Principle .............................................................................................90
4.11.2 Ion-Exchange Materials .....................................................................90
4.12 Exclusion Chromatography .............................................................................91
4.12.1 Principle .............................................................................................91
4.12.2 Materials and Methods .......................................................................92
4.12.3 Applications ........................................................................................94
viii Contents
4.13 Affinity Chromatography ................................................................................94
4.13.1 Principle .............................................................................................94
4.13.2 Materials and Methods .......................................................................95
Suggested Reading ...................................................................................................111
Important Links ........................................................................................................113
Chapter 5 Spectroscopy ............................................................................................................115
5.1 Introduction ...................................................................................................115
5.1.1 Definition and General Principles ....................................................115
5.1.2 Beer–Lambert’s Law ........................................................................117
5.1.3 Mechanics of Measurement .............................................................120
5.2 UV–Visible Spectroscopy ..............................................................................121
5.2.1 Definition ..........................................................................................121
5.2.2 Principle ...........................................................................................121
5.2.3 Instrumentation ................................................................................122
5.2.3.1 Colorimeter .......................................................................122
5.2.3.2 Spectronic 20 Spectrocolorimeter ....................................123
5.2.3.3 Choice of Instruments for Colorimetry ............................123
5.2.3.4 UV–Visible Spectrophotometer ........................................123
5.2.4 Applications ......................................................................................125
5.2.4.1 Concentration Measurement .............................................125
5.2.4.2 Growth Kinetics................................................................126
5.2.4.3 Structural Studies .............................................................126
5.2.4.4 Enzyme Kinetics and Assays ............................................127
5.2.4.5 Difference Spectra ............................................................127
5.2.4.6 Purity and Homogeneity ...................................................127
5.3 IR (Vibrational) Spectroscopy .......................................................................129
5.3.1 Principle ...........................................................................................129
5.3.2 Instrumentation ................................................................................130
5.3.3 Applications ......................................................................................130
5.4 Flame/Atomic Absorption Spectroscopy ......................................................131
5.4.1 Principle ...........................................................................................131
5.4.2 Instrumentation for Emission Flame Spectroscopy .........................132
5.4.3 Instrumentation for Atomic Absorption Spectroscopy ....................132
5.4.4 Applications ......................................................................................133
5.5 Fluorescence Spectroscopy ...........................................................................133
5.5.1 Principle ...........................................................................................133
5.5.2 Instrumentation ................................................................................135
5.5.3 Pre- and Postfilter Effects ................................................................135
5.5.4 Applications ......................................................................................135
5.5.4.1 Concentration Measurement .............................................135
5.5.4.2 Compound Identification/Excitation Spectrum ................136
5.5.4.3 Kinetic and Structural Studies ..........................................136
5.6 ESR Spectroscopy .........................................................................................137
5.6.1 Principles ..........................................................................................137
5.6.2 Instrumentation ................................................................................139
5.6.3 Applications ......................................................................................140
5.7 NMR Spectroscopy .......................................................................................140
5.7.1 Principle ...........................................................................................140
5.7.2 Instrumentation ................................................................................141
