{"product_id":"principles-and-applications-of-fluorescence-spectroscopy-9781405138918","title":"Principles and Applications of Fluorescence Spectroscopy","description":"\u003cb\u003eBook Synopsis\u003c\/b\u003e\u003cbr\u003e* Straightforward overview of absorption and fluorescence shows the student how the phenomenon arises and how it can be used in the course of their research.  * Highly practical approach shows non-specialists how to use the technique to investigate chemical and biochemical problems and generate sophisticated results.\u003cbr\u003e\u003cbr\u003e\u003cb\u003eTable of Contents\u003c\/b\u003e\u003cbr\u003e\u003cp\u003e\u003cb\u003e1 Absorption Spectroscopy Theory 1\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e1.1 Introduction 1\u003c\/p\u003e \u003cp\u003e1.2 Characteristics of an Absorption Spectrum 2\u003c\/p\u003e \u003cp\u003e1.3 Beer–Lambert–Bouguer Law 4\u003c\/p\u003e \u003cp\u003e1.4 Effect of the Environment on Absorption Spectra 6\u003c\/p\u003e \u003cp\u003eReferences 11\u003c\/p\u003e \u003cp\u003e\u003cb\u003e2 Determination of the Calcofluor White Molar Extinction Coefficient Value in the Absence and Presence of \u003ci\u003eα\u003c\/i\u003e\u003c\/b\u003e\u003cb\u003e\u003csub\u003e1\u003c\/sub\u003e-Acid Glycoprotein 13\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e2.1 Introduction 13\u003c\/p\u003e \u003cp\u003e2.2 Biological Material Used 13\u003c\/p\u003e \u003cp\u003e2.2.1 Calcofluor White 13\u003c\/p\u003e \u003cp\u003e2.2.2 \u003ci\u003eα\u003c\/i\u003e\u003csub\u003e1\u003c\/sub\u003e-Acid glycoprotein 13\u003c\/p\u003e \u003cp\u003e2.3 Experiments 16\u003c\/p\u003e \u003cp\u003e2.3.1 Absorption spectrum of Calcofluor free in PBS buffer 16\u003c\/p\u003e \u003cp\u003e2.3.2 Determination of \u003ci\u003eε\u003c\/i\u003e. value of Calcofluor White free in PBS buffer 16\u003c\/p\u003e \u003cp\u003e2.3.3 Determination of Calcofluor White \u003ci\u003eε\u003c\/i\u003e. value in the presence of \u003ci\u003eα\u003c\/i\u003e1-acid glycoprotein 16\u003c\/p\u003e \u003cp\u003e2.4 Solution 17\u003c\/p\u003e \u003cp\u003eReferences 19\u003c\/p\u003e \u003cp\u003e\u003cb\u003e3 Determination of Kinetic Parameters of Lactate Dehydrogenase 21\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e3.1 Objective of the Experiment 21\u003c\/p\u003e \u003cp\u003e3.2 Absorption Spectrum of NADH 21\u003c\/p\u003e \u003cp\u003e3.3 Absorption Spectrum of LDH 22\u003c\/p\u003e \u003cp\u003e3.4 Enzymatic Activity of LDH 22\u003c\/p\u003e \u003cp\u003e3.5 Kinetic Parameters 22\u003c\/p\u003e \u003cp\u003e3.6 Data and Results 22\u003c\/p\u003e \u003cp\u003e3.6.1 Determination of enzyme activity 23\u003c\/p\u003e \u003cp\u003e3.6.2 Determination of kinetic parameters 23\u003c\/p\u003e \u003cp\u003e3.7 Introduction to Kinetics and the Michaelis–Menten Equation 26\u003c\/p\u003e \u003cp\u003e3.7.1 Definitions 26\u003c\/p\u003e \u003cp\u003e3.7.2 Reaction rates 26\u003c\/p\u003e \u003cp\u003eReferences 32\u003c\/p\u003e \u003cp\u003e\u003cb\u003e4 Hydrolysis of \u003ci\u003ep\u003c\/i\u003e-Nitrophenyl-\u003ci\u003eβ\u003c\/i\u003e-D-Galactoside with \u003ci\u003eβ\u003c\/i\u003e-Galactosidase from \u003ci\u003eE. coli \u003c\/i\u003e34\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e4.1 Introduction 34\u003c\/p\u003e \u003cp\u003e4.2 Solutions to be Prepared 35\u003c\/p\u003e \u003cp\u003e4.3 First-day Experiments 35\u003c\/p\u003e \u003cp\u003e4.3.1 Absorption spectrum of PNP 35\u003c\/p\u003e \u003cp\u003e4.3.2 Absorption of PNP as a function of pH 36\u003c\/p\u003e \u003cp\u003e4.3.3 Internal calibration of PNP 37\u003c\/p\u003e \u003cp\u003e4.3.4 Determination of \u003ci\u003eβ\u003c\/i\u003e-galactosidase optimal pH 39\u003c\/p\u003e \u003cp\u003e4.3.5 Determination of \u003ci\u003eβ\u003c\/i\u003e-galactosidase optimal temperature 40\u003c\/p\u003e \u003cp\u003e4.4 Second-day Experiments 40\u003c\/p\u003e \u003cp\u003e4.4.1 Kinetics of \u003ci\u003ep\u003c\/i\u003e-nitrophenyl-\u003ci\u003eβ\u003c\/i\u003e-D-galactoside hydrolysis with \u003ci\u003eβ\u003c\/i\u003e-galactosidase 40\u003c\/p\u003e \u003cp\u003e4.4.2 Determination of the \u003ci\u003eβ\u003c\/i\u003e-galactosidase concentration in the test tube 42\u003c\/p\u003e \u003cp\u003e4.5 Third-day Experiments 44\u003c\/p\u003e \u003cp\u003e4.5.1 Determination of K\u003csub\u003em\u003c\/sub\u003e and V\u003csub\u003emax\u003c\/sub\u003e of \u003ci\u003eβ\u003c\/i\u003e-galactosidase 44\u003c\/p\u003e \u003cp\u003e4.5.2 Inhibiton of hydrolysis kinetics of \u003ci\u003ep\u003c\/i\u003e-nitrophenyl-\u003ci\u003eβ\u003c\/i\u003e-D-galactoside 45\u003c\/p\u003e \u003cp\u003e4.6 Fourth-day Experiments 47\u003c\/p\u003e \u003cp\u003e4.6.1 Effect of guanidine chloride concentration on \u003ci\u003eβ\u003c\/i\u003e-galactosidase activity 47\u003c\/p\u003e \u003cp\u003e4.6.2 OD variation with guanidine chloride 48\u003c\/p\u003e \u003cp\u003e4.6.3 Mathematical derivation of K\u003csub\u003eeq\u003c\/sub\u003e 48\u003c\/p\u003e \u003cp\u003e4.6.4 Definition of the standard Gibbs free energy, \u003ci\u003eΔG\u003c\/i\u003e\u003csup\u003e◦’\u003c\/sup\u003e 51\u003c\/p\u003e \u003cp\u003e4.6.5 Relation between \u003ci\u003eΔG\u003c\/i\u003e\u003csup\u003e◦’\u003c\/sup\u003e and  \u003ci\u003eΔG\u003c\/i\u003e\u003csup\u003e’\u003c\/sup\u003e 51\u003c\/p\u003e \u003cp\u003e4.6.6 Relation between \u003ci\u003eΔG\u003c\/i\u003e\u003csup\u003e◦’\u003c\/sup\u003e and \u003ci\u003eK\u003c\/i\u003e\u003csub\u003eeq\u003c\/sub\u003e 52\u003c\/p\u003e \u003cp\u003e4.6.7 Effect of guanidine chloride on hydrolysis kinetics of \u003ci\u003ep\u003c\/i\u003e-nitrophenyl-\u003ci\u003eβ\u003c\/i\u003e-D-galactoside 56\u003c\/p\u003e \u003cp\u003eReferences 57\u003c\/p\u003e \u003cp\u003e\u003cb\u003e5 Starch Hydrolysis by Amylase 59\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e5.1 Objectives 59\u003c\/p\u003e \u003cp\u003e5.2 Introduction 59\u003c\/p\u003e \u003cp\u003e5.3 Materials 61\u003c\/p\u003e \u003cp\u003e5.4 Procedures and Experiments 61\u003c\/p\u003e \u003cp\u003e5.4.1 Preparation of a 20 g l\u003csup\u003e−1\u003c\/sup\u003estarch solution 61\u003c\/p\u003e \u003cp\u003e5.4.2 Calibration curve for starch concentration 61\u003c\/p\u003e \u003cp\u003e5.4.3 Calibration curve for sugar concentration 63\u003c\/p\u003e \u003cp\u003e5.4.4 Effect of pH 64\u003c\/p\u003e \u003cp\u003e5.4.5 Temperature effect 66\u003c\/p\u003e \u003cp\u003e5.4.6 Effect of heat treatment at 90\u003csup\u003e◦\u003c\/sup\u003eC 69\u003c\/p\u003e \u003cp\u003e5.4.7 Kinetics of starch hydrolysis 70\u003c\/p\u003e \u003cp\u003e5.4.8 Effect of inhibitor (CuCl\u003csub\u003e2\u003c\/sub\u003e) on the amylase activity 73\u003c\/p\u003e \u003cp\u003e5.4.9 Effect of amylase concentration 73\u003c\/p\u003e \u003cp\u003e5.4.10 Complement experiments that can be performed 77\u003c\/p\u003e \u003cp\u003e5.4.11 Notes 77\u003c\/p\u003e \u003cp\u003eReferences 78\u003c\/p\u003e \u003cp\u003e\u003cb\u003e6 Determination of the pK of a Dye 79\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e6.1 Definition of pK 79\u003c\/p\u003e \u003cp\u003e6.2 Spectrophotometric Determination of pK 79\u003c\/p\u003e \u003cp\u003e6.3 Determination of the pK of 4-Methyl-2-Nitrophenol 81\u003c\/p\u003e \u003cp\u003e6.3.1 Experimental procedure 81\u003c\/p\u003e \u003cp\u003e6.3.2 Solution 83\u003c\/p\u003e \u003cp\u003eReferences 87\u003c\/p\u003e \u003cp\u003e\u003cb\u003e7 Fluorescence Spectroscopy Principles 88\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e7.1 Jablonski Diagram or Diagram of Electronic Transitions 88\u003c\/p\u003e \u003cp\u003e7.2 Fluorescence Spectral Properties 91\u003c\/p\u003e \u003cp\u003e7.2.1 General features 91\u003c\/p\u003e \u003cp\u003e7.2.2 Stokes shift 93\u003c\/p\u003e \u003cp\u003e7.2.3 Relationship between the emission spectrum and excitation wavelength 94\u003c\/p\u003e \u003cp\u003e7.2.4 Inner filter effect 95\u003c\/p\u003e \u003cp\u003e7.2.5 Fluorescence excitation spectrum 95\u003c\/p\u003e \u003cp\u003e7.2.6 Mirror–image rule 95\u003c\/p\u003e \u003cp\u003e7.2.7 Fluorescence lifetime 96\u003c\/p\u003e \u003cp\u003e7.2.8 Fluorescence quantum yield 101\u003c\/p\u003e \u003cp\u003e7.2.9 Fluorescence and light diffusion 102\u003c\/p\u003e \u003cp\u003e7.3 Fluorophore Structures and Properties 102\u003c\/p\u003e \u003cp\u003e7.3.1 Aromatic amino acids 104\u003c\/p\u003e \u003cp\u003e7.3.2 Cofactors 108\u003c\/p\u003e \u003cp\u003e7.3.3 Extrinsinc fluorophores 108\u003c\/p\u003e \u003cp\u003e7.4 Polarity and Viscosity Effect on Quantum Yield and Emission Maximum Position 111\u003c\/p\u003e \u003cp\u003eReferences 113\u003c\/p\u003e \u003cp\u003e\u003cb\u003e8 Effect of the Structure and the Environment of a Fluorophore on Its Absorption and Fluorescence Spectra 115\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003eExperiments 115\u003c\/p\u003e \u003cp\u003eQuestions 117\u003c\/p\u003e \u003cp\u003eAnswers 119\u003c\/p\u003e \u003cp\u003eReference 123\u003c\/p\u003e \u003cp\u003e\u003cb\u003e9 Fluorophore Characterization and Importance in Biology 124\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e9.1 Experiment 1. Quantitative Determination of Tryptophan in Proteins in 6 M Guanidine 124\u003c\/p\u003e \u003cp\u003e9.1.1 Introduction 124\u003c\/p\u003e \u003cp\u003e9.1.2 Principle 124\u003c\/p\u003e \u003cp\u003e9.1.3 Experiment 125\u003c\/p\u003e \u003cp\u003e9.1.4 Results obtained with cytochrome b2 core 126\u003c\/p\u003e \u003cp\u003e9.2 Experiment 2. Effect of the Inner Filter Effect on Fluorescence Data 127\u003c\/p\u003e \u003cp\u003e9.2.1 Objective of the experiment 127\u003c\/p\u003e \u003cp\u003e9.2.2 Experiment 127\u003c\/p\u003e \u003cp\u003e9.2.3 Results 128\u003c\/p\u003e \u003cp\u003e9.3 Experiment 3. Theoretical Spectral Resolution of Two Emitting Fluorophores Within a Mixture 130\u003c\/p\u003e \u003cp\u003e9.3.1 Objective of the experiment 130\u003c\/p\u003e \u003cp\u003e9.3.2 Results 132\u003c\/p\u003e \u003cp\u003e9.4 Experiment 4. Determination of Melting Temperature of Triglycerides in Skimmed Milk Using Vitamin A Fluorescence 134\u003c\/p\u003e \u003cp\u003e9.4.1 Introduction 134\u003c\/p\u003e \u003cp\u003e9.4.2 Experiment to conduct 136\u003c\/p\u003e \u003cp\u003e9.4.3 Results 136\u003c\/p\u003e \u003cp\u003eReferences 138\u003c\/p\u003e \u003cp\u003e\u003cb\u003e10 Fluorescence Quenching 139\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e10.1 Introduction 139\u003c\/p\u003e \u003cp\u003e10.2 Collisional Quenching: the Stern–Volmer Relation 140\u003c\/p\u003e \u003cp\u003e10.3 Different Types of Dynamic Quenching 145\u003c\/p\u003e \u003cp\u003e10.4 Static Quenching 147\u003c\/p\u003e \u003cp\u003e10.4.1 Theory 147\u003c\/p\u003e \u003cp\u003e10.5 Thermal Intensity Quenching 154\u003c\/p\u003e \u003cp\u003eReferences 159\u003c\/p\u003e \u003cp\u003e\u003cb\u003e11 Fluorescence Polarization 160\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e11.1 Definition 160\u003c\/p\u003e \u003cp\u003e11.2 Fluorescence Depolarization 162\u003c\/p\u003e \u003cp\u003e11.2.1 Principles and applications 162\u003c\/p\u003e \u003cp\u003e11.3 Fluorescence Anisotropy Decay Time 165\u003c\/p\u003e \u003cp\u003e11.4 Depolarization and Energy Transfer 166\u003c\/p\u003e \u003cp\u003eReferences 167\u003c\/p\u003e \u003cp\u003e\u003cb\u003e12 Interaction Between Ethidium Bromide and DNA 168\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e12.1 Objective of the Experiment 168\u003c\/p\u003e \u003cp\u003e12.2 DNA Extraction from Calf Thymus or Herring Sperm 168\u003c\/p\u003e \u003cp\u003e12.2.1 Destruction of cellular structure 168\u003c\/p\u003e \u003cp\u003e12.2.2 DNA extraction 168\u003c\/p\u003e \u003cp\u003e12.2.3 DNA purification 169\u003c\/p\u003e \u003cp\u003e12.2.4 Absorption spectrum of DNA 169\u003c\/p\u003e \u003cp\u003e12.3 Ethidium Bromide Titration with Herring DNA 169\u003c\/p\u003e \u003cp\u003e12.4 Results Obtained with Herring DNA 170\u003c\/p\u003e \u003cp\u003e12.4.1 Absorption and emission spectra 170\u003c\/p\u003e \u003cp\u003e12.4.2 Analysis and interpretation of the results 173\u003c\/p\u003e \u003cp\u003e12.5 Polarization Measurements 177\u003c\/p\u003e \u003cp\u003e12.6 Results Obtained with Calf Thymus DNA 179\u003c\/p\u003e \u003cp\u003e12.7 Temperature Effect on Fluorescence of the Ethidium Bromide–DNA Complex 180\u003c\/p\u003e \u003cp\u003eReferences 182\u003c\/p\u003e \u003cp\u003e\u003cb\u003e13 \u003ci\u003eLens culinaris \u003c\/i\u003eAgglutinin: Dynamics and Binding Studies 184\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e13.1 Experiment 1. Studies on the Accessibility of I− to a Fluorophore: Quenching of Fluorescein Fluorescence with KI 184\u003c\/p\u003e \u003cp\u003e13.1.1 Objective of the experiment 184\u003c\/p\u003e \u003cp\u003e13.1.2 Experiment 184\u003c\/p\u003e \u003cp\u003e13.1.3 Results 185\u003c\/p\u003e \u003cp\u003e13.2 Experiment 2. Measurement of Rotational Correlation Time of Fluorescein Bound to LCA with Polarization Studies 187\u003c\/p\u003e \u003cp\u003e13.2.1 Objective of the work 187\u003c\/p\u003e \u003cp\u003e13.2.2 Polarization studies as a function of temperature 187\u003c\/p\u003e \u003cp\u003e13.2.3 Polarization studies as a function of sucrose at 20◦C 187\u003c\/p\u003e \u003cp\u003e13.2.4 Results 189\u003c\/p\u003e \u003cp\u003e13.3 Experiment 3. Role of \u003ci\u003eα\u003c\/i\u003e-L-fucose in the Stability of Lectin–Glycoproteins Complexes 190\u003c\/p\u003e \u003cp\u003e13.3.1 Introduction 190\u003c\/p\u003e \u003cp\u003e13.3.2 Binding studies 191\u003c\/p\u003e \u003cp\u003e13.3.3 Results 192\u003c\/p\u003e \u003cp\u003eReferences 196\u003c\/p\u003e \u003cp\u003e\u003cb\u003e14 Förster Energy Transfer 197\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e14.1 Principles and Applications 197\u003c\/p\u003e \u003cp\u003e14.2 Energy-transfer Parameters 202\u003c\/p\u003e \u003cp\u003e14.3 Bioluminescence Resonance Energy Transfer 204\u003c\/p\u003e \u003cp\u003eReferences 208\u003c\/p\u003e \u003cp\u003e\u003cb\u003e15 Binding of TNS on Bovine Serum Albumin at pH 3 and pH 7 210\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e15.1 Objectives 210\u003c\/p\u003e \u003cp\u003e15.2 Experiments 210\u003c\/p\u003e \u003cp\u003e15.2.1 Fluorescence emission spectra of TNS–BSA at pH 3 and 7 210\u003c\/p\u003e \u003cp\u003e15.2.2 Titration of BSA with TNS at pH 3 and 7 210\u003c\/p\u003e \u003cp\u003e15.2.3 Measurement of energy transfer efficiency from Trp residues to TNS 211\u003c\/p\u003e \u003cp\u003e15.2.4 Interaction between free Trp in solution and TNS 211\u003c\/p\u003e \u003cp\u003e15.3 Results 211\u003c\/p\u003e \u003cp\u003e\u003cb\u003e16 Comet Test for Environmental Genotoxicity Evaluation: A Fluorescence Microscopy Application 220\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e16.1 Principle of the Comet Test 220\u003c\/p\u003e \u003cp\u003e16.2 DNA Structure 220\u003c\/p\u003e \u003cp\u003e16.3 DNA Reparation 221\u003c\/p\u003e \u003cp\u003e16.4 Polycyclic Aromatic Hydrocarbons 222\u003c\/p\u003e \u003cp\u003e16.5 Reactive Oxygen Species 223\u003c\/p\u003e \u003cp\u003e16.6 Causes of DNA Damage and Biological Consequences 224\u003c\/p\u003e \u003cp\u003e16.7 Types of DNA Lesions 225\u003c\/p\u003e \u003cp\u003e16.7.1 Induction of abasic sites, AP, apurinic, or apyrimidinic 225\u003c\/p\u003e \u003cp\u003e16.7.2 Base modification 225\u003c\/p\u003e \u003cp\u003e16.7.3 DNA adducts 225\u003c\/p\u003e \u003cp\u003e16.7.4 Simple and double-stranded breaks 225\u003c\/p\u003e \u003cp\u003e16.8 Principle of Fluorescence Microscopy 225\u003c\/p\u003e \u003cp\u003e16.9 Comet Test 227\u003c\/p\u003e \u003cp\u003e16.9.1 Experimental protocol 227\u003c\/p\u003e \u003cp\u003e16.9.2 Nature of damage revealed with the Comet test 227\u003c\/p\u003e \u003cp\u003e16.9.3 Advantages and limits of the method 227\u003c\/p\u003e \u003cp\u003e16.9.4 Result expression 231\u003c\/p\u003e \u003cp\u003eReferences 231\u003c\/p\u003e \u003cp\u003e\u003cb\u003e17 Questions and Exercises 232\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e17.1 Questions 232\u003c\/p\u003e \u003cp\u003e17.1.1 Questions with shorts answers 232\u003c\/p\u003e \u003cp\u003e17.1.2 Find the error 232\u003c\/p\u003e \u003cp\u003e17.1.3 Explain 233\u003c\/p\u003e \u003cp\u003e17.1.4 Exercises 234\u003c\/p\u003e \u003cp\u003e17.2 Solutions 241\u003c\/p\u003e \u003cp\u003e17.2.1 Questions with short answers 241\u003c\/p\u003e \u003cp\u003e17.2.2 Find the error 243\u003c\/p\u003e \u003cp\u003e17.2.3 Explain 243\u003c\/p\u003e \u003cp\u003e17.2.4 Exercises solutions 244\u003c\/p\u003e \u003cp\u003eIndex 253\u003c\/p\u003e","brand":"John Wiley and Sons Ltd","offers":[{"title":"Default Title","offer_id":53187420553559,"sku":"9781405138918","price":67.4,"currency_code":"GBP","in_stock":false}],"url":"https:\/\/bookcurl.com\/products\/principles-and-applications-of-fluorescence-spectroscopy-9781405138918","provider":"Book Curl","version":"1.0","type":"link"}