Principles and Applications of Fluorescence Spectroscopy

✍ Scribed by Jihad Rene Albani

• Publisher: Blackwell Science
• Year: 2007
• Tongue: English
• Leaves: 270
• Edition: 1
• Category: Library

✦ Synopsis

Fluorescence spectroscopy is an important investigational tool in many areas of analytical science, due to its extremely high sensitivity and selectivity. With many uses across a broad range of chemical, biochemical and medical research, it has become an essential investigational technique allowing detailed, real-time observation of the structure and dynamics of intact biological systems with extremely high resolution. It is particularly heavily used in the pharmaceutical industry where it has almost completely replaced radiochemical labelling. Principles and Applications of Fluorescence Spectroscopy gives the student and new user the essential information to help them to understand and use the technique confidently in their research. By integrating the treatment of absorption and fluorescence, the student is shown how fluorescence phenomena arise and how these can be used to probe a range of analytical problems. A key element of the book is the inclusion of practical laboratory experiments that illustrate the fundamental points and applications of the technique.

✦ Table of Contents

Principles and Applications of
Fluorescence Spectroscopy......Page 1
Contents......Page 5
1.1 Introduction......Page 11
1.2 Characteristics of an Absorption Spectrum......Page 12
1.3 Beer-Lambert-Bouguer Law......Page 14
1.4 Effect of the Environment on Absorption Spectra......Page 16
References......Page 21
2.2.2 a1-Acid glycoprotein......Page 23
2.3.3 Determination of CalcofluorWhite e. value in the presence of
a1-acid glycoprotein......Page 26
2.4 Solution......Page 27
References......Page 29
3.2 Absorption Spectrum of NADH......Page 31
3.6 Data and Results......Page 32
3.6.2 Determination of kinetic parameters......Page 33
3.7.2 Reaction rates......Page 36
References......Page 42
4.1 Introduction......Page 44
4.3.1 Absorption spectrum of PNP......Page 45
4.3.2 Absorption of PNP as a function of pH......Page 46
4.3.3 Internal calibration of PNP......Page 47
4.3.4 Determination of B-galactosidase optimal pH......Page 49
4.4.1 Kinetics of p-nitrophenyl-B-D-galactoside hydrolysis with B-galactosidase......Page 50
4.4.2 Determination of the B-galactosidase concentration in the test tube......Page 52
4.5.1 Determination of Km and Vmax of B-galactosidase......Page 54
4.5.2 Inhibiton of hydrolysis kinetics of p-nitrophenyl-B-D-galactoside......Page 55
4.6.1 Effect of guanidine chloride concentration on B-galactosidase activity......Page 57
4.6.3 Mathematical derivation of Keq......Page 58
4.6.5 Relation between G. and G......Page 61
4.6.6 Relation between G. and Keq......Page 62
4.6.7 Effect of guanidine chloride on hydrolysis kinetics of p-nitrophenyl-B-D-galactoside......Page 66
References......Page 67
5.2 Introduction......Page 69
5.4.2 Calibration curve for starch concentration......Page 71
5.4.3 Calibration curve for sugar concentration......Page 73
5.4.4 Effect of pH......Page 74
5.4.5 Temperature effect......Page 76
5.4.6 Effect of heat treatment at 90.C......Page 79
5.4.7 Kinetics of starch hydrolysis......Page 80
5.4.9Effect of amylase concentration......Page 83
5.4.11 Notes......Page 87
References......Page 88
6.2 Spectrophotometric Determination of pK......Page 89
6.3.1 Experimental procedure......Page 91
6.3.2 Solution......Page 93
Reference......Page 97
7.1 Jablonski Diagram or Diagram of Electronic Transitions......Page 98
7.2.1 General features......Page 101
7.2.2 Stokes shift......Page 103
7.2.3 Relationship between the emission spectrum and
excitation wavelength......Page 104
7.2.6 Mirror-image rule......Page 105
7.2.7 Fluorescence lifetime......Page 106
7.2.8 Fluorescence quantum yield......Page 111
7.3 Fluorophore Structures and Properties......Page 112
7.3.1 Aromatic amino acids......Page 114
7.3.3 Extrinsinc fluorophores......Page 118
7.4 Polarity and Viscosity Effect on Quantum Yield and
Emission Maximum Position......Page 121
References......Page 123
Experiments......Page 125
Questions......Page 127
Answers......Page 129
Reference......Page 133
9.1.2 Principle......Page 134
9.1.3 Experiment......Page 135
9.1.4 Results obtained with cytochrome b2 core......Page 136
9.2.2 Experiment......Page 137
9.2.3 Results......Page 138
9.3.1 Objective of the experiment......Page 140
9.3.2 Results......Page 142
9.4.1 Introduction......Page 144
9.4.3 Results......Page 146
References......Page 148
10.1 Introduction......Page 149
10.2 Collisional Quenching: the Ster-Volmer Relation......Page 150
10.3Different Types of Dynamic Quenching......Page 155
10.4.1 Theory......Page 157
10.5 Thermal Intensity Quenching......Page 164
References......Page 169
11.1 Definition......Page 170
11.2.1 Principles and applications......Page 172
11.3 Fluorescence Anisotropy Decay Time......Page 175
11.4 Depolarization and Energy Transfer......Page 176
References......Page 177
12.2.2 DNA extraction......Page 178
12.3 Ethidium Bromide Titration with Herring DNA......Page 179
12.4.1 Absorption and emission spectra......Page 180
12.4.2 Analysis and interpretation of the results......Page 183
12.5 Polarization Measurements......Page 187
12.6 Results Obtained with Calf Thymus DNA......Page 189
12.7 Temperature Effect on Fluorescence of the Ethidium Bromide-DNA Complex......Page 190
References......Page 192
13.1.2 Experiment......Page 194
13.1.3 Results......Page 195
13.2.3 Polarization studies as a function of sucrose at 20.C......Page 197
13.2.4 Results......Page 199
13.3.1 Introduction......Page 200
13.3.2 Binding studies......Page 201
13.3.3 Results......Page 202
References......Page 206
14.1 Principles and Applications......Page 207
14.2 Energy-transfer Parameters......Page 212
14.3 Bioluminescence Resonance Energy Transfer......Page 214
References......Page 218
15.2.2 Titration of BSA with TNS at pH 3 and 7......Page 220
15.3 Results......Page 221
16.2 DNA Structure......Page 230
16.3 DNA Reparation......Page 231
16.4 Polycyclic Aromatic Hydrocarbons......Page 232
16.5 Reactive Oxygen Species......Page 233
16.6 Causes of DNA Damage and Biological Consequences......Page 234
16.8 Principle of Fluorescence Microscopy......Page 235
16.9.3 Advantages and limits of the method......Page 237
References......Page 241
17.1.2 Find the error......Page 242
17.1.3 Explain......Page 243
17.1.4 Exercises......Page 244
17.2.1 Questions with short answers......Page 251
17.2.3 Explain......Page 253
17.2.4 Exercises solutions......Page 254
Index......Page 263
Color plate......Page 267