Spectroscopy
Description
In this book, you will learn the fundamental principles underpinning molecular spectroscopy and the connections between those principles and the design of spectrophotometers.
Spectroscopy, along with chromatography, mass spectrometry, and electrochemistry, is an important and widely-used analytical technique. Applications of spectroscopy include air quality monitoring, compound identification, and the analysis of paintings and culturally important artifacts. This book introduces students to the fundamentals of molecular spectroscopy - including UV-visible, infrared, fluorescence, and Raman spectroscopy - in an approachable and comprehensive way. It goes beyond the basics of the subject and provides a detailed look at the interplay between theory and practice, making it ideal for courses in quantitative analysis, instrumental analysis, and biochemistry, as well as courses focused solely on spectroscopy. It is also a valuable resource for practitioners working in laboratories who regularly perform spectroscopic analyses.
Spectroscopy: Principles and Instrumentation:
* Provides extensive coverage of principles, instrumentation, and applications of molecular spectroscopy
* Facilitates a modular approach to teaching and learning about chemical instrumentation
* Helps students visualize the effects that electromagnetic radiation in different regions of the spectrum has on matter
* Connects the fundamental theory of the effects of electromagnetic radiation on matter to the design and use of spectrophotometers
* Features numerous figures and diagrams to facilitate learning
* Includes several worked examples and companion exercises throughout each chapter so that readers can check their understanding
* Offers numerous problems at the end of each chapter to allow readers to apply what they have learned
* Includes case studies that illustrate how spectroscopy is used in practice, including analyzing works of art, studying the kinetics of enzymatic reactions, detecting explosives, and determining the DNA sequence of the human genome
* Complements Chromatography: Principles and Instrumentation
The book is divided into five chapters that cover the Fundamentals of Spectroscopy, UV-visible Spectroscopy, Fluorescence/Luminescence Spectroscopy, Infrared Spectroscopy, and Raman Spectroscopy. Each chapter details the theory upon which the specific techniques are based, provides ways for readers to visualize the molecular-level effects of electromagnetic radiation on matter, describes the design and components of spectrophotometers, discusses applications of each type of spectroscopy, and includes case studies that illustrate specific applications of spectroscopy.
Each chapter is divided into multiple sections using headings and subheadings, making it easy for readers to work through the book and to find specific information relevant to their interests. Numerous figures, exercises, worked examples, and end-of-chapter problems reinforce important concepts and facilitate learning.
Spectroscopy: Principles and Instrumentation is an excellent text that prepares undergraduate students and practitioners to operate in modern laboratories.
More details
Other editions
Additional editions
Person
MARK F. VITHA is a Windsor Professor of Chemistry at Drake University. He received his Ph.D. from the University of Minnesota. He is the editor of the Chemical Analysis Series (Wiley), the author of Chromatography: Principles and Instrumentation (Wiley 2017), and a co-editor of the books High Throughput Analysis for Food Safety (Wiley, 2014) and Interfaces and Interphases in Analytical Chemistry (ACS, 2011). He has received three teaching awards, including the Levitt Teacher of the Year Award, and has been named a Ronald D. Troyer Research Fellow at Drake University.
Content
ABOUT THE COVER ix
PREFACE xi
1. Fundamentals of Spectroscopy 1
1.1 Properties of Electromagnetic Radiation 1
1.1.1 Speed, c 2
1.1.2 Amplitude, A 2
1.1.3 Frequency, ¿ 3
1.1.4 Wavelength, ¿ 3
1.1.5 Energy, E 3
1.1.6 Wavenumber, 6
1.2 The Electromagnetic Spectrum 7
1.2.1 Radio-Frequency Radiation (10-27 to 10-21 J/photon) 8
1.2.2 Microwave Radiation (10-23 to 10-22 J/photon) 10
1.2.3 Infrared Radiation (10-22 to 10-19 J/photon) 11
1.2.4 Ultraviolet and Visible Radiation (10-19 to 10-18 J/photon) 12
1.2.5 X-Ray Radiation (10-15 to 10-13 J/photon) 13
1.2.6 Alpha, Beta, and Gamma Radiation (10-13 to 10-11 J/photon and Higher) 13
1.3 The Perrin-Jablonski Diagram 15
1.3.1 Timescales of Events 18
1.3.2 Summary of Radiative and Nonradiative Processes 19
1.4 Temperature Effects on Ground and Excited State Populations 19
1.5 More Wave Characteristics 21
1.5.1 Adding Waves Together 21
1.5.2 Diffraction 21
1.5.3 Reflection 25
1.5.4 Refraction 28
1.5.5 Scattering 29
1.5.6 Polarized Radiation 31
1.6 Spectroscopy Applications 34
1.7 Summary 34
Problems 34
References 36
Further Reading 38
2. UV-Visible Spectrophotometry 39
2.1 Theory 40
2.1.1 The Absorption Process 40
2.1.2 The Beer-Lambert Law 43
2.1.3 Solvent Effects on Molar Absorptivity and Spectra 49
2.2 UV-Visible Instrumentation 52
2.2.1 Sources of Visible and Ultraviolet Light 54
2.2.2 Wavelength Selection: Filters 58
2.2.3 Wavelength Selection: Monochromators 61
2.2.4 Monochromator Designs: Putting It All Together 75
2.2.5 Detectors 79
2.3 Spectrophotometer Designs 85
2.3.1 Single-Beam Spectrophotometers 85
2.3.2 Scanning Double-Beam Instruments 89
2.3.3 Photodiode Array Instruments 93
2.4 The Practice of Spectrophotometry 98
2.4.1 Types of Samples That Can Be Analyzed 99
2.4.2 Preparation of Calibration Curves 100
2.4.3 Deviations from Beer's Law 103
2.4.4 Precision: Relative Concentration Error 111
2.4.5 The Desirable Absorbance Range 114
2.5 Applications and Techniques 116
2.5.1 Simultaneous Determinations of Multicomponent Systems 116
2.5.2 Difference Spectroscopy 117
2.5.3 Derivative Spectroscopy 118
2.5.4 Titration Curves 119
2.5.5 Turbidimetry and Nephelometry 121
2.6 A Specific Application of UV-Visible Spectroscopy: Enzyme Kinetics 122
2.6.1 Myeloperoxidase, Immune Responses, Heart Attacks,and Enzyme Kinetics 122
2.6.2 Possible Mechanism for Myeloperoxidase Oxidation of LDL via Tyrosyl Radical Intermediates 123
2.7 Summary 127
Problems 127
References 132
Further Reading 134
3. Molecular Luminescence: Fluorescence, Phosphorescence, and Chemiluminescence 135
3.1 Theory 135
3.1.1 Absorbance Compared to Fluorescence 136
3.1.2 Factors That Affect Fluorescence Intensity 141
3.1.3 Quenching 146
3.1.4 Quantum Yield and Fluorescence Intensity 147
3.1.5 Linearity and Nonlinearity of Fluorescence: Quenching and Self-Absorption 149
3.2 Instrumentation 153
3.2.1 Instrument Design 154
3.2.2 Sources 154
3.2.3 Filters and Monochromators 157
3.2.4 Component Arrangement 158
3.2.5 Fluorometers 158
3.2.6 Spectrofluorometers 159
3.2.7 Cells and Slit Widths 164
3.2.8 Detectors 166
3.3 Practice of Luminescence Spectroscopy 167
3.3.1 Considerations and Options 167
3.3.2 Fluorescence Polarization 168
3.3.3 Time-Resolved Fluorescence Spectroscopy 172
3.4 Fluorescence Microscopy 173
3.4.1 Fluorescence Microscopy Resolution 175
3.4.2 Confocal Fluorescence Microscopy 175
3.5 Phosphorescence and Chemiluminescence 177
3.5.1 Phosphorescence 177
3.5.2 Chemiluminescence 177
3.6 Applications of Fluorescence: Biological Systems and DNA Sequencing 179
3.7 Summary 186
Problems 186
References 190
Further Reading 192
4. Infrared Spectroscopy 193
4.1 Theory 193
4.1.1 Bond Vibrations 196
4.1.2 Other Types of Vibrations 198
4.1.3 Modeling Vibrations: Harmonic and Nonharmonic Oscillators 200
4.1.4 The 3N-6 Rule 207
4.2 FTIR Instruments 209
4.2.1 The Michelson Interferometer and Fourier Transform 210
4.2.2 Components of FTIR Instruments: Sources 224
4.2.3 Components of FTIR Instruments: DTGS and MCT Detectors 226
4.2.4 Sample Handling 227
4.2.5 Reflectance Techniques 231
4.3 Applications of IR Spectroscopy, Including Near-IR and Far-IR 234
4.3.1 Structure Determination with Mid-IR Spectroscopy 235
4.3.2 Gas Analysis 235
4.3.3 Near-Infrared Spectroscopy (NIR) 236
4.3.4 Far-Infrared Spectroscopy (FIR) 245
4.4 Summary 248
Problems 248
References 251
Further Reading 254
5. Raman Spectroscopy 255
5.1 Energy-Level Description 255
5.2 Visualization of Raman Data 258
5.3 Molecular Polarizability 259
5.4 Brief Review of Molecular Vibrations 261
5.5 Classical Theory of Raman Scattering 262
5.6 Polarization of Raman Scattering 265
5.6.1 Depolarization Ratio 266
5.7 Instrumentation and Analysis Methods 266
5.7.1 Filter Instruments 267
5.7.2 Dispersive Spectrometers 270
5.7.3 Fourier Transform Raman Spectrometers 271
5.7.4 Confocal Raman Instruments 271
5.7.5 Light Sources 273
5.8 Quantitative Analysis Methods 274
5.8.1 Calibration Curves 274
5.8.2 Curve Fitting 274
5.8.3 Ordinary Least Squares 275
5.8.4 Classical Least Squares 277
5.8.5 Implicit Analytical Methods 277
5.9 Applications 277
5.9.1 Art and Archeology 277
5.9.2 Pharmaceuticals 278
5.9.3 Forensics 279
5.9.4 Medicine and Biology 279
5.10 Signal Enhancement Techniques 282
5.10.1 Resonance Raman Spectroscopy 283
5.10.2 Surface-Enhanced Raman Spectroscopy 283
5.10.3 Nonlinear Raman Spectroscopy 284
5.11 Summary 286
Problems 286
References 288
Further Reading 289
SOLUTIONS 291
INDEX 315
1
FUNDAMENTALS OF SPECTROSCOPY
All instruments are designed to take advantage of some molecular property or behavior. For example, chromatography is based on the different strength of intermolecular interactions that molecules have with mobile and stationary phases. Electrochemistry is based on the ability of molecules to gain or lose electrons. In this book, we focus on the fact that atoms and molecules absorb and emit electromagnetic radiation (EMR). By measuring the amount and the characteristics of the EMR absorbed and emitted, we can measure the concentration of particular molecules present in a sample or gain structural information about them. You may already be familiar with several of the instrumental methods used to measure the absorption and emission of electromagnetic radiation, such as UV-visible, infrared (IR), and fluorescence spectroscopy.
In order to better understand the fundamental basis of these techniques, in this chapter we examine the properties of electromagnetic radiation and its effects on atoms and molecules. In subsequent chapters, we examine specific spectroscopic techniques. While all the techniques share common features, the specific instruments required and the information we gain from each are quite different and therefore require individual examination.
1.1. PROPERTIES OF ELECTROMAGNETIC RADIATION
Spectroscopic methods ultimately rely on measuring characteristics of electromagnetic radiation, which travels through space as a wave, as shown in Figure 1.1. As the name implies, it has two components, an electric field and a magnetic field, which are at right angles to one another. Figure 1.1 shows only a single wave with its electric field oriented along the x-axis, but in reality, most sources of electromagnetic radiation, like light bulbs and car headlights, emit radiation in which the electric field of the waves are randomly distributed around the x-axis. For now, however, we take a simplified view by focusing on only a single electromagnetic wave.
FIGURE 1.1 Diagram of a single electromagnetic wave propagating through space. The diagram indicates that an electromagnetic wave has both an electric field (E) and magnetic field (B) associated with it and that they are oriented at right angles to each other. It also indicates that the wavelength (?) is the distance the wave travels during one oscillation of the electric and magnetic fields.
Source: Reproduced with permission of Eric Clarke.
All electromagnetic waves have the properties of:
- Speed
- Amplitude
- Frequency
- Wavelength
- Energy
Each of these characteristics is described below.
1.1.1. Speed, c
Electromagnetic radiation in a vacuum travels at 2.998 × 108 m/s, commonly referred to as the speed of light, c. This speed only pertains to light traveling in a vacuum, though, because EMR slows down when it travels through matter such as air and water. We discuss the speed of light when it travels through matter in a later section.
1.1.2. Amplitude, A
The amplitude, A, of a wave is the maximum length of the electric field vector, as shown in Figure 1.2. We seldom consider the amplitude of the wave because detectors are not fast enough to measure the magnitude of the electric field vector. Instead, we measure the radiant power, P, of a beam, which is proportional to the square of the amplitude. Radiant power is the amount of energy transmitted per unit time and is given by Eq. (1.1), where E is the energy of a photon and f is the flux (i.e., the number of photons per unit time) [1]:
(1.1)FIGURE 1.2 A side view of the electric field component of an electromagnetic wave as it propagates from left to right across the page. The amplitude is the displacement along the y-axis and the wavelength is the peak-to-peak distance between a single oscillation of the wave.
Although radiant power is commonly referred to as intensity, I, intensity is strictly defined as the radiant power from a point source per unit solid angle, usually measured in watts per steradian [1, 2].
1.1.3. Frequency, ?
Frequency, ?, is the number of oscillations a wave makes per unit time and is typically measured in hertz, Hz, with units of reciprocal seconds, 1/s or s-1. To visualize the physical meaning of frequency, imagine sitting on a rock out in the ocean with a stopwatch and counting waves that pass the rock. If you count, say, 120 waves in a minute, the frequency is
In other words, two waves pass the rock every second. If more waves pass every second, the frequency is higher, and fewer waves per second are associated with a lower frequency.
The speed and wavelength of electromagnetic radiation change as it passes through different media, but the frequency remains the same. As described below, the frequency of EMR is closely related to the energy of the EMR. Therefore, the frequency is the characteristic that truly differentiates one wave from another.
1.1.4. Wavelength, ?
The wavelength, ?, is the peak-to-peak distance of the wave, as shown in Figure 1.2. Because it is a distance, wavelengths are typically measured in meters. For example, EMR in the visible portion of the electromagnetic spectrum has wavelengths between 380 and 760 nanometers (nm).
1.1.5. Energy, E
As we will see in all of the subsequent chapters, energy is really the fundamental wave characteristic that matters most in terms of the impact electromagnetic radiation has on matter. We often talk about EMR in terms of wavelengths, frequencies, and wavenumbers, but these ultimately relate to energy. In order to understand the energy of radiation, we must consider the wave/particle duality of light. When EMR propagates through space, it is convenient to focus on its wave properties (frequency, wavelength, amplitude). However, when it interacts with matter, it is useful to think of EMR as a discrete particle that contains a fixed amount of energy that can be transferred to an atom or molecule. That discrete particle is called a photon.
The energy that a photon contains is directly related to its frequency and wavelength, as shown in the two relationships in Eqs. (1.2) and (1.3):
(1.2) (1.3)where h is Planck's constant (6.626 × 10-34 Js) and c is the speed of light (2.998 × 108 m/s). These equations are fundamental to the study of spectroscopy. They also clearly show that photons with higher frequencies (i.e., faster oscillations) and shorter wavelengths are higher in energy than those with slower oscillations and longer wavelengths (see Figure 1.3). To help make a mental association between these relationships, consider the two waves shown in Figure 1.3. Imagine trying to draw a wave of each frequency across the entire length of a chalkboard or white board, and that you only have 15 seconds to go from one end of the board to the other. You have the same amount of time in each case because light propagates through space at the same velocity regardless of wavelength. Clearly, the higher frequency wave - the one with little space between peaks and therefore shorter wavelength - will require you to expend a greater amount of energy to fill the board, whereas you can take a leisurely stroll (i.e., exert low energy) along the board when drawing the low-frequency, long-wavelength wave.
FIGURE 1.3 Depiction of the reciprocal relationships between wavelength and frequency. Longer wavelengths are associated with lower frequencies and lower energies, while shorter wavelengths are associated with higher frequencies and higher energies.
From the fact that Eqs. (1.2) and (1.3) are equal to each other, we can see that
(1.4)This relationship has implications for the speed and wavelength of EMR as it travels through different media such as air, water, benzene, etc. As radiation passes through matter, its electric field interacts with the electrons in the matter, slowing its propagation, meaning that the speed of light is different in different media. In a vacuum, the speed is 2.998 × 108 m/s, but in everything else, including air, it is slower. However, as the EMR propagates through matter, its frequency is unaffected, so ? remains the same. In order to maintain the equality in Eq. (1.4), when the speed, c, decreases, then the wavelength must also decrease in order to maintain a constant frequency, ?.
The change in the speed of EMR in matter is measured by the refractive index of a substance, ?, where
(1.5)in which cv is the speed in a vacuum and ci is the speed in the substance of interest. Because EMR is slower in matter than it is in a vacuum, such that ci < cv, refractive indices are greater than 1.00. The velocity of radiation in air is within 1% of the velocity in a vacuum such that using...
