Spectroscopy E-Book

Mark F. Vitha

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 635

Veröffentlichungsjahr: 2018

Beschreibung

Provides students and practitioners with a comprehensive understanding of the theory of spectroscopy and the design and use of spectrophotometers

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.

Leseprobe

Table of Contents

COVER

PREFACE

1 FUNDAMENTALS OF SPECTROSCOPY

1.1. PROPERTIES OF ELECTROMAGNETIC RADIATION

1.2. THE ELECTROMAGNETIC SPECTRUM

1.3. THE PERRIN–JABLONSKI DIAGRAM

1.4. TEMPERATURE EFFECTS ON GROUND AND EXCITED STATE POPULATIONS

1.5. MORE WAVE CHARACTERISTICS

1.6. SPECTROSCOPY APPLICATIONS

1.7. SUMMARY

PROBLEMS

REFERENCES

FURTHER READING

2 UV‐VISIBLE SPECTROPHOTOMETRY

2.1. THEORY

2.2. UV‐VISIBLE INSTRUMENTATION

2.3. SPECTROPHOTOMETER DESIGNS

2.4. THE PRACTICE OF SPECTROPHOTOMETRY

2.5. APPLICATIONS AND TECHNIQUES

2.6. A SPECIFIC APPLICATION OF UV‐VISIBLE SPECTROSCOPY: ENZYME KINETICS

2.7. SUMMARY

PROBLEMS

REFERENCES

FURTHER READING

3 MOLECULAR LUMINESCENCE

3.1. THEORY

3.2. INSTRUMENTATION

3.3. PRACTICE OF LUMINESCENCE SPECTROSCOPY

3.4. FLUORESCENCE MICROSCOPY

3.5. PHOSPHORESCENCE AND CHEMILUMINESCENCE

3.6. APPLICATIONS OF FLUORESCENCE: BIOLOGICAL SYSTEMS AND DNA SEQUENCING

3.7. SUMMARY

PROBLEMS

REFERENCES

FURTHER READING

4 INFRARED SPECTROSCOPY

4.1. THEORY

4.2. FTIR INSTRUMENTS

4.3. APPLICATIONS OF IR SPECTROSCOPY, INCLUDING NEAR‐IR AND FAR‐IR

4.4. SUMMARY

PROBLEMS

REFERENCES

FURTHER READING

5 RAMAN SPECTROSCOPY

5.1. ENERGY‐LEVEL DESCRIPTION

5.2. VISUALIZATION OF RAMAN DATA

5.3. MOLECULAR POLARIZABILITY

5.4. BRIEF REVIEW OF MOLECULAR VIBRATIONS

5.5. CLASSICAL THEORY OF RAMAN SCATTERING

5.6. POLARIZATION OF RAMAN SCATTERING

5.7. INSTRUMENTATION AND ANALYSIS METHODS

5.8. QUANTITATIVE ANALYSIS METHODS

5.9. APPLICATIONS

5.10. SIGNAL ENHANCEMENT TECHNIQUES

5.11. SUMMARY

PROBLEMS

REFERENCES

FURTHER READING

SOLUTIONS

INDEX

END USER LICENSE AGREEMENT

List of Tables

Chapter 01

TABLE 1.1 Refractive Indices of Some Common Materials

TABLE 1.2 Summary of the Approximate Energy, Wavelength, and Frequency Regions of the Electromagnetic Spectrum

TABLE 1.3 Approximate Timescales for Processes Associated with Spectroscopy

Chapter 02

TABLE 2.1 Technical Specifications for the PerkinElmer LAMBDA 1050 UV/Vis/NIR Spectrophotometer

TABLE 2.2 Technical Specifications of the Agilent 8454 Diode Array Spectrometer

TABLE 2.3 Order for Analyzing Samples and Standards

TABLE 2.4 The Effect of the Logarithmic Definition of Absorbance on Ratio of

P

o

and

P

Assuming

P

o

  =  10 000 Photons

Chapter 03

TABLE 3.1 Quantum Yield of Common Fluorescent Molecules

TABLE 3.2 Summary of How Excitation and Emission Spectra Are Collected

Chapter 04

TABLE 4.1 Cutoff Wavenumbers for Optical Materials

TABLE 4.2 Approximate Wavenumber and Wavelength Ranges of UV, Visible, Near‐IR, Mid‐IR, and Far‐IR Electromagnetic Radiation

TABLE 4.3 Approximate Wavenumbers and Wavelengths of the Overtone Bands of C

─

H, O

─

H, N

─

H, and C═O Vibrations

Chapter 05

TABLE 5.1 Qualitative Relationships Between Molecular Properties and Raman Spectral Features

List of Illustrations

Chapter 01

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 (

λ

) is the distance the wave travels during one oscillation of the electric and magnetic fields.

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.

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.

FIGURE 1.4 The regions of the electromagnetic spectrum and the effect that radiation in each region has on matter. Note the labels on the top left and right indicating that the diagram goes from low energy electromagnetic radiation (EMR) on the left to high energy electromagnetic radiation on the right. Also note the reciprocal relationship between energy and wavelength. Long (i.e., high value) wavelength photons on the far left of the diagram have very low energy, and short wavelengths photons (far right on the diagram) have very high energy. The high energy associated with short wavelength photons makes them capable of significant damage to molecules and thus significant negative health effects.

FIGURE 1.5 Depiction of a π–π* transition (left) and the associated energy change, Δ

E

(right). Note that in the ground state, the electrons in the π‐orbital connect both carbon atoms above and below the plane of the bond. The electrons in the π‐bond are thus delocalized and shared between both carbon atoms. In the excited state, the π*‐orbitals, and thus the electrons in them, are localized on a specific carbon atom. Shared electrons like those in the ground state lower the energy of a molecule compared to localized electron density like that seen in the excited state. In order to transition from the ground state to the excited state, the molecule must absorb a photon that has an energy that exactly matches the energy difference (Δ

E

) between the ground and excited states.

FIGURE 1.6 Graphical depiction of approximate ranges within the electromagnetic spectrum. Wavelengths increase and energy decreases from left to right. Vertical displacement has no meaning other than to allow for easier reading.

FIGURE 1.7 The Perrin–Jablonski diagram showing various processes that occur when light is absorbed by a molecule. There is a lot of information contained in this figure, and the reader is encouraged to spend some time to fully understand it as it underpins much of the material in this and subsequent chapters. The arrow at the far left is important to note. It indicates that energy is increasing from bottom to top. Horizontal lines represent the energy of different electronic and vibrational levels of molecules. It should be understood that when a molecule undergoes an electronic transition, while the energy of the molecule increases, the electron that is promoted to a higher energy state does not literally go “up”, but rather is redistributed into a different molecular orbital. Key processes in this diagram include absorption, vibrational relaxation, internal conversion, fluorescence, intersystem crossing (ISC), and phosphorescence, all of which are described in greater detail in the text. The curves shown under the Perrin–Jablonski diagram indicate the spectral features that could be observed for each type of transition.

FIGURE 1.8 A visualization of an electronic rearrangement caused by the absorption of a photon. The Lewis structure of 4‐nitroaniline is shown on the far left. The electron clouds in the middle and right image depict regions of electron density. When 4‐nitroaniline absorbs the energy of a photon, the electrons get redistributed into different orbitals, with the resulting electron distribution being higher in energy than the ground state distribution. Notice that in this case, the electron density shifts away from the amine group toward the nitro group as a result of the electronic transition.

FIGURE 1.9 Three waves (a, b, and c) added together to create a fourth wave (d). Note that each of the waves has a different frequency and amplitude. The amplitudes (

y

‐axis values) at each

x

‐axis value of the first three waves are added to yield the wave in (d). The sum (d) itself has a characteristic pattern. When the process is run in reverse, the single wave in (d) can be deconvolved to yield the frequencies and amplitudes of the three waves that constitute it. This is the basis of Fourier transformation and is depicted in a subsequent figure.

FIGURE 1.10 An illustration of constructive and destructive interference. In both (a) and (b), the top two graphs are added together and create the bottom graph. So the graph on the bottom is the result of adding the

y

‐value of the top two graphs at every point along the

x

‐axis. So in (a), when both curves have a

y

‐value of 1.00 at the same

x

‐value, the result is a

y

‐value of 2.0 at the corresponding

x

‐value. In (b), which represents perfect destructive interference, the value of

y

in the top curve is always offset by the same value in the negative direction in the second curve. The result is zero amplitude at all

x

‐values.

FIGURE 1.11 Depiction of the Fourier transformation during which a single complex signal versus time input (left) gets deconvolved into the constituent frequencies and their associated amplitudes that make up the signal (right). Note that the

y

‐axis scales (i.e., amplitudes) are different for the individual waves on the right. The frequency information contained in spectra provides clues about molecular structure (i.e., qualitative information), and the amplitude (i.e., intensity) provides quantitative information related to concentration.

FIGURE 1.12 Schematic of diffraction of electromagnetic radiation from atoms in a crystal. Constructive interference occurs when the extra distance traveled by rays 2 and 3 is a multiple of the wavelength of incoming radiation.

FIGURE 1.13 Bird’s‐eye view of diffraction. Solid lines represent the crests of waves moving from left to right as they approach and pass through stationary slits. (a) Slits that are much larger than the wavelength of the light cause very little diffraction. (b) Slits that are close to the size of the wavelength passing through them cause significant diffraction.

FIGURE 1.14 Reflection of radiation off of a surface. The angle of incidence (

θ

inc

) equals the angle of reflection (

θ

ref

).

FIGURE 1.15 Diagram of reflection at 90°, which is frequently encountered in spectroscopy, for example, when electromagnetic radiation strikes the surface of a cuvette.

I

o

is the original intensity of the beam,

I

r

is the intensity of the reflected beam, and

I

t

is the intensity of the transmitted beam.

FIGURE 1.16 Diagram of light from a source passing through a quartz cell filled with an aqueous sample.

FIGURE 1.17 Refraction of a beam of light as it transitions from one medium to another, such as the transition from air to water. The degree to which the beam is bent is given by Snell’s law and depends on the refractive indices of the two media. In this diagram,

η

1

<

η

2

.

FIGURE 1.18 Rayleigh scattering creates red sunsets because the light from the sun has to travel further through the atmosphere at sunset than at noon, as seen by comparing the distance labeled “A” (sunset) to the one labeled “C” (noon). At noon, the light passes through the least amount of atmosphere and thus encounters the fewest number of scattering particles. In this case, it is mostly the short wavelength blue light that is scattered. Light with intermediate wavelengths associated with orange, yellow, and green are not scattered much and the result is that the sun looks yellow when we look directly at it, while the sky looks blue due to it having been scattered at all angles by the atmosphere. The light at sunset travels through more atmosphere and thus encounters more scattering particles. Because of the extended path, yellows and greens also get scattered to some extent, creating the reddish orange colors in the sky around the sun that we are accustomed to seeing at sunset.

FIGURE 1.19 Depiction of Raman scattering. As with the Perrin–Jablonski diagram, the increasing energy scale from bottom to top is important. Raman scattering involves the temporary uptake of a photon with some energy, and the subsequent reemission of a photon or either lower (Stokes) or higher (anti‐Stokes) energy. The former leaves the molecule in a higher vibrational state than it was in prior to absorbing the incoming radiation, and the latter results in the molecule being in a lower vibrational state after emission. In Raman scattering, unlike Rayleigh scattering, the energy of the emitted radiation is different than that of the incoming radiation, and therefore also has a different frequency and wavelength because

E

  =  

hυ

  =  

hc

/

λ

.

FIGURE 1.20 An illustration of multiple electromagnetic waves propagating through space, each oriented at a different angle relative to one another. Unpolarized light such as this has electric and magnetic components in planes at all angles.

FIGURE 1.21 (a) A view of a few of the electric field components of unpolarized radiation propagating directly at the viewer. Note that the distribution would be random in all directions around the axis of propagation and varying in amplitude over time for each individual wave. (b) An individual electric field vector broken into

x

‐ and

y

‐components. The same deconstruction into

x

‐ and

y

‐components can be done for all of the waves in part (a).

FIGURE 1.22 Depiction of circularly polarized light. The electric field vector (arrows) rotates around the axis as it propagates through space in the direction indicated by the arrow on the right.

Chapter 02

FIGURE 2.1 A line diagram depicting the energy gap, Δ

E

, between ground and excited state molecular orbitals.

FIGURE 2.2 Energy diagram showing transitions between σ, n, π, π*, and σ* orbitals.

FIGURE 2.3 Representations of the change in electron distribution within orbitals associated with (a) π → π* (bonding to antibonding) and (b) n → π* (nonbonding to antibonding) electronic transitions.

FIGURE 2.4 UV‐visible absorbance spectrum of Reichardt’s dye in 1,2‐dichloroethane.

FIGURE 2.5 Schematic diagram of the change in light intensity due to absorption.

P

o

is the power (intensity) of the light incident on a cell, with path length,

b

, containing a solution of an absorbing species at a concentration,

C

.

P

is the light intensity after absorption and scattering (not depicted in this diagram) occurs.

FIGURE 2.6 Representations of the ground and excited electronic states of

p

‐nitroaniline. (a) Lewis structures: HBAS stands for “hydrogen bond‐accepting solvent.” (b) Molecular orbitals calculated using semiempirical computational methods. (c) Same as (b) but with the molecule rotated so that the electron density above and below the ring can be seen. The molecule is oriented the same way in all three figures, that is, with the amine group on the top and the nitro group on the bottom. Note that the electron density around the ─NH

2

group is much lower in the excited state than in the ground state. This lack of electron density makes the partial positive charge on the hydrogen atoms much greater. This increases their strength of hydrogen bonding with solvents that can accept hydrogen bonds, ultimately lowering the energy of the excited state. The stabilization arising from hydrogen bonding is greater for the excited state than it is for ground state because the partial positive charge on the hydrogen atoms is greater in the excited state than in the ground state. This shrinks the energy difference between the two states in hydrogen bond‐accepting solvents relative to when

p

‐nitroaniline is dissolve in solvents that do not accept hydrogen bonds as depicted in the next figure.

FIGURE 2.7 An energy diagram showing the differential stabilization of the ground and excited states of

p

‐nitroaniline in going from a nonpolar, nonhydrogen‐bonding solvent (e.g., cyclohexane) to a polar, hydrogen bond‐accepting solvent (e.g., methanol) [1, 3, 4].

FIGURE 2.8 The dependence of the spectrum of

p

‐nitroaniline on solvent. The absorbances in each solvent are normalized to the maximum absorbance in the longest‐wavelength band to make the shift in the spectrum easier to observe.

FIGURE 2.9 A simple block diagram of a UV‐visible spectrometer for measuring the absorption of electromagnetic radiation.

FIGURE 2.10 Picture of a deuterium lamp.

FIGURE 2.11 Plot of (a) D

2

, (b) tungsten filament, (c) combined D

2

with tungsten (on the next page), and (d) xenon flash lamp emission (on the next page) as a function of wavelength. Notice in the combined output that some of the distinct spectral features of the deuterium emission, like the sharp emission band at 656.1 and 486.0 nm, are visible above the broad output from the tungsten lamp. Also notice that the intensity between 300 and 650 nm is much lower than at higher and lower wavelengths.

FIGURE 2.12 Schematic of an interference filter and the path of light rays through the filter. Polychromatic radiation strikes surface A from the left, undergoes partial reflection, enters the spacer film made of a dielectric material, and again undergoes partial reflection off of surface B (beam 3). For radiation with a wavelength that is twice the thickness of the spacer film, constructive interference results in the beam being passed and emerging from the right side of the filter (beams 2 and 4). Radiation with wavelengths that are not a multiple of the film thickness experience destructive interference and are therefore not observed on the right side of the filter.

FIGURE 2.13 Spectral transmittance characteristics of two sharp cutoff filters combined to create a notch filter that passes a narrow range of wavelengths.

FIGURE 2.14 Monochromator schematic. Polychromatic light from the source enters the entrance slit, is collimated by the mirror, and is reflected toward the grating. The grating disperses the light into its constituent elements – for clarity, only three wavelengths, labeled

λ

1

,

λ

2

, and

λ

3

, are shown here, but for white light, all the wavelengths in the visible region would be present. The dispersed light is collimated by the second mirror and focused on the plane of the exit slit. The exit slit allows only a narrow range of wavelengths to pass through, while all others are physically blocked. The emerging beam of nearly monochromatic light then passes through the sample where it is absorbed or transmitted.

FIGURE 2.15 (Top) Slit distribution function when the image size and the exit slit are identical. The cone on the right plots the intensity of the light (vertical axis) versus the position of the image from the entrance slit on the exit slit. (Bottom) Another way of visualizing the image from the entrance slit of a narrow band of wavelengths (represented as the hashed rectangle) as it moves across the fixed exit slit.

FIGURE 2.16 Depiction of the linear dispersion provided by a grating as opposed to the nonlinear dispersion typical of a prism.

FIGURE 2.17 Effect of spectral bandwidth on the observed absorption band shapes of cytochrome

c

. Spectral bandwidths are (1) 20 nm, (2) 10 nm, (3) 5 nm, (4) 1 nm, and (5) 0.08 nm (shown in a separate plot for clarity).

FIGURE 2.18 Cross section of a diffraction grating showing the angles of a single groove, which are microscopic on an actual grating.

FIGURE 2.19 The figure depicts the effect of different angles of reflection on the path length traveled by the light. The same angle of incidence,

i

, relative to the grating normal (vertical dashed line) is incident on the blazes. Two different angles of reflection of the diffracted light,

r

1

and

r

2

, are also depicted. The inset shows that there is clearly a difference in the path lengths traveled by light diffracted at the two different angles – the path length for each is highlighted by the bold lines. This difference in path length means that different wavelengths of light are constructively reinforced at different angles, effectively dispersing the incident polychromatic light into its component wavelengths.

FIGURE 2.20 Overlapping orders of spectra from a reflection grating. The overlap results from the fact that light is diffracted according to the equation

mλ

  =  

b

(sin

i

+ sin

r

), where the order, m, is a whole number. As a consequence, the angle at which 700 nm first‐order light appears also has 350 and 233 nm light due to the second‐order (

m

  =  2) and third‐order (

m

  =  3) diffraction. Also notice the scales on the left – they show the overlap in the orders and also clearly demonstrate that higher‐order diffraction has a higher linear dispersion, meaning that the wavelengths are spread out over a greater distance.

FIGURE 2.21 Bird’s‐eye view of the Fastie–Ebert mounting. Electromagnetic radiation enters the entrance slit, strikes the collimating mirror, and is reflected to the grating where it is dispersed into its constituent wavelengths. The dispersed light is reflected off of the collimating mirror and onto the plane of the exit slit. The exit slit allows a narrow band of wavelengths to pass through, while all others are physically blocked by the wall of the monochromator. The Fastie–Ebert mount uses a single collimating mirror, whereas the Czerny–Turner monochromator (Figure 2.22) has two separate collimating mirrors.

FIGURE 2.22 The Czerny–Turner mounting. This arrangement operates in the same manner as the Fastie–Ebert arrangement with the exception of having two distinct concave collimating mirrors rather than a single mirror.

FIGURE 2.23 Littrow mounting with the diffracted beam below the source beam. This arrangement is useful for creating compact spectrophotometers.

FIGURE 2.24 Depiction of total internal reflection in a fiber optic. Electromagnetic radiation traveling in the core of the fiber optic, which has a higher refractive index than the cladding, is totally internally reflected at the core/cladding interface if the angle of interaction is greater than a critical angle. The total internal reflection allows the radiation to propagate down the fiber optic and be carried to a sample or a detector.

FIGURE 2.25 Phototube and its associated circuitry.

FIGURE 2.26 Sensitivity of photoemissive surfaces. S‐1 is Ag─O─Cs, S‐11 is Cs─Sb, S‐17 is also Cs─Sb, S‐20 is (Cs)─Na─K─Sb.

FIGURE 2.27 Bird’s‐eye view of a photomultiplier (PMT). A photon strikes the photoemissive cathode and ejects an electron. The electron is accelerated toward first dynode, which is held at a positive voltage relative to the photocathode. Multiple electrons are ejected from dynode 1 for every electron that strikes it. This cycle of acceleration with subsequent ejection of electrons continues at all of the dynodes until the electrons are collected at the anode.

FIGURE 2.28 Construction of a planar diffused p

–

n junction photodiode. P‐type material has “holes” and n‐type material has “excess” electrons. When a voltage is applied across the p–n junction, holes and electrons separate as depicted in the figure.

FIGURE 2.29 Top view of the optical arrangement of the single‐beam Thermo Scientific

TM

SPECTRONIC

TM

20 visible spectrophotometer. Light from the tungsten lamp is directed through a slit and onto the grating where it is diffracted into its constituent wavelengths. A narrow range of wavelengths selected by the exit slit passes through the test tube that contains the sample where some light may be absorbed, and is ultimately detected by the photodetector.

FIGURE 2.30 Diagram of a double‐beam‐in‐time spectrophotometer (Cary 100 – courtesy of Varian Associates, Inc.). Polychromatic electromagnetic radiation from the UV and visible sources (A) is directed off a mirror (M

1

), through the monochromator entrance slit (S

1

), onto a collimating mirror (M

2

) and onto the grating (G) that disperses it into its constituent wavelengths. The dispersed electromagnetic radiation reflects off the second collimating mirror (M

3

) and is focused onto the plane of the exit slit (S

2

). A narrow band of wavelengths exits the slit and is directed by mirrors onto the chopper wheel (CW

1

). As explained in more detail in the text, depending on whether the open or mirrored portion of the chopper wheel is in place, the light goes through either the reference or the sample cell where it can be absorbed. Whatever light is not absorbed passes through the solution and is ultimately directed onto the detector by mirrors and the second chopper wheel. In this way, independent measurements of

P

to

P

o

are obtained. Taking the ratio yields the transmittance of the sample, which is electronically converted into an absorbance value. The chopper wheel spins continuously so that

P

and

P

o

(and hence absorbance) are constantly updated. While the chopper wheel is spinning, the grating is rotated, which allows different wavelengths to be passed through the reference and sample solutions. In this way, the absorbance is measured at different wavelengths to obtain an entire spectrum without having to manipulate the solutions once the scan is started.

FIGURE 2.31 Diagram of a double‐beam‐in‐space spectrophotometer. Polychromatic light from UV and visible lamps is directed through the monochromator, where it is dispersed into its constituent wavelengths. The beam splitter (half‐mirror) reflects half the light and transmits the other half. The two beams that are created pass through the sample and reference cells simultaneously and proceed to the sample and reference PMTs. The ratio of the two signals is taken and converted into the absorbance.

FIGURE 2.32 Schematic of an Agilent Cary 8454 diode array spectrometer. Polychromatic light from tungsten (visible) and deuterium (UV) lamps passes through the sample and is then dispersed into its constituent wavelengths by the grating. The dispersed light is directed onto a diode array, where individual diodes detect specific wavelengths. An entire spectrum is recorded by first measuring the light intensity striking the diodes when a reference solution is in the cuvette (i.e., measure

P

o

at each wavelength) and then repeating the measurement with the sample solution in the cuvette (i.e., measure

P

).

FIGURE 2.33 General measurement scheme of diode array detection. The linear array of boxes at the top of the figure represents the diode array, and each box within the array represents an individual sensing element (i.e., diode) within the array. Typical diode arrays have hundreds or thousands of diodes. Working your way through the figure from top to bottom explains how an entire spectrum is recorded with a diode array instrument.

FIGURE 2.34 Reaction of dansyl chloride with an amino acid.

FIGURE 2.35 Deviation from Beer’s law: ideal (linear) versus nonideal (curved) behavior of chromate/dichromate system if pH and ionic strength are not controlled.

FIGURE 2.36 Absorbance spectra of phenol red (p

K

a

  =  7.9) as a function of pH. Absorbance maxima are at 433 and 558 nm for the acidic (yellow) and basic (red) forms, respectively. Isosbestic points occur at 338, 367, and 480 nm.

FIGURE 2.37 Effects of narrow (n–n) and wide (w–w) bandpasses. At point (

a

) with a wide bandpass, the range of molar absorptivities is significant as are the associated deviations from Beer’s law. A narrow bandpass has a smaller range of molar absorptivities, but could still produce nonlinearities. Point (

b

) with a narrow bandpass is less prone to deviations because the molar absorptivity is nearly constant. With a wider bandpass, point (

c

), the average molar absorptivity decreases since more curvature, and thus a wider range of molar absorptivities, is incorporated in the range.

FIGURE 2.38 Plot of nonlinear compared to linear Beer’s law behavior. The linear plot, a, is calculated in accord with the equations in this section of the chapter. It is based on two molar absorptivities, both equal to 200 cm

−1

M

−1

, and a path length of 1 cm. Plot b was obtained using the same path length but with two different molar absorptivities (100 and 200 cm

−1

M

−1

). Plot c was obtained using molar absorptivities of 50 and 200 cm

−1

M

−1

. It is clear that wider discrepancies in the molar absorptivities over the wavelength region passed through the sample lead to greater deviations from linearity.

FIGURE 2.39 Relative concentration error, Δ

C

/

C

, in percent, for a constant transmittance error of Δ

T

  =  1%.

FIGURE 2.40 A plot of the absorbance versus percent transmittance data shown in Table 2.4. Note how low the percent transmittance is above an absorbance of 1.5 and how little it changes at higher absorbance values. This makes it difficult to detect small changes in absorbance of highly absorbing samples.

FIGURE 2.41 Simultaneous spectrophotometric analysis of a two‐component system. Selection of analytical wavelengths is indicated by arrows.

FIGURE 2.42 First derivative spectrometry for the quantitative measurement of the intensity of a small band (a) alone and (b) obscured by a broader overlapping band. See text for details.

FIGURE 2.43 Possible shapes of photometric titration curves. See text for details.

FIGURE 2.44 Representation of the structure of human myeloperoxidase from PDB entry 3F9P.

FIGURE 2.45 Rapid‐scan spectra of MPO‐I reduction to MPO‐II by tyrosine. The arrows show the direction of absorbance changes with time. The first, second, and last scans were taken at 2.5, 10, and 100 ms after mixing a solution of 1.0 μm MPO and 50 μM tyrosine with another solution containing 20 μM H

2

O

2

.

FIGURE 2.46 The inset shows a typical trace and curve fit (solid line) of the reaction followed at 456 nm used to determine the pseudo‐first‐order rate constants. Final concentrations were 0.25 μM MPO and 5 μM H

2

O

2

. The second‐order rate constant was calculated from the slope of the plot of the observed pseudo‐first‐order rate constants versus the concentration of tyrosine.

Chapter 03

FIGURE 3.1 (a) The Perrin–Jablonski diagram summarizes a variety of processes related to the interaction of light with molecules. The arrow indicating that energy increases from top to bottom is an important feature of the diagram. In this chapter, we focus on the processes of fluorescence and phosphorescence. However, both of these processes require that the molecule first absorbs light. Other processes detailed in the diagram are explained in the text. Note that the energy involved in absorbance is greater than that in fluorescence, which is greater than that in phosphorescence. (b) Depiction of the shift in wavelengths of absorbance, fluorescence, and phosphorescence spectra.

FIGURE 3.2 (a) Depiction of the change in dipole moment of a solute (shaded gray) upon absorption of a photon and the effect it has on the solvent molecules (open ovals) around the solute. In this case, the excited state dipole is greater than the ground state dipole (i.e.,

μ

e

  >  

μ

g

), as indicated by the width and length of the dipole symbol. Step 1: No change in solvent organization during the absorption process. Note that the dipole moments of the solvent molecules are not oriented optimally around the excited state immediately upon absorption. Step 2: Solvent reorganization in response to the excited state electronic distribution that has a greater dipole moment than does the ground state. Step 3: Fluorescence – note that fluorescence, just like absorption, is so rapid that no solvent reorganization occurs during the event. Step 4: Reorganization of the solvent cage around the ground state dipole. (b) 4‐Nitroaniline is an example of a molecule that has an excited state dipole greater than the ground state. Note that while the ground state is polar, the excited state is much more so. Electron density from the amine nitrogen is transferred toward the nitro group, causing the enhanced excited state dipole.

FIGURE 3.3 The emission spectra above were recorded for the derivative of the bithiophene shown in the figure in solvents with a wide range of polarity [8]. Solvents: toluene (▪), ether (●), tetrahydrofuran (▲), ethyl acetate (▼), chloroform (⧫), dichloromethane (◄), dimethylformamide (►), acetonitrile (), dimethyl sulfoxide (★), and ethanol (). The polarity of the chemical environment of the fluorophore shifts the fluorescence spectrum toward longer wavelengths (lower energies).

FIGURE 3.4 Normalized absorbance (solid line) and emission spectra (dashed line) of quinine sulfate in 0.25 M sulfuric acid. Note that fluorescence occurs at longer wavelength (lower energy), and while the absorbance spectrum reflects both the

S

0

➔

S

2

transition centered around 315 nm and the

S

0

➔

S

1

transition around 345 nm, the fluorescence only occurs from

S

1

due to rapid internal conversion (445 nm peak).

FIGURE 3.5 From left to right: fluorescein, eosin Y, and phenolphthalein.

FIGURE 3.6 From left to right: phenol, anisole, aniline, and the anilinium ion.

FIGURE 3.7 Examples of nonlinear fluorescence calibration curves. These plots depict calibration curves with linear and nonlinear portions (although note that the

x

‐axis in (a) is a log scale). (a) A plot of fluorescence versus concentration for NADH in water. (b) A plot of fluorescence versus absorbance for quinine sulfate in 0.5 M H

2

SO

4

. The

x

‐axis is the absorbance of the solution at the excitation wavelength. Because absorbance is typically linear with concentration, the graph is essentially a plot of fluorescence versus concentration. The solid line with open circle data points is the observed fluorescence. The dotted line is the tangent to the curve as the absorbance approaches zero, showing that the plot is linear until

A

  ≫  0.1.

FIGURE 3.8 (a) A dilute solution of fluorophores (stars) emitting fluorescence (indicated by solid lines). (b) In a more concentrated solution of fluorophores, fluorescence emitted by one molecule can be absorbed by another (indicated by dashed arrows and circled). The second molecule can then nonradiatively relax, diminishing the total fluorescence observed. This phenomenon is more common at higher concentrations because the fluorescent photon is more likely to encounter another fluorophore before exiting the solution.

FIGURE 3.9 An illustration of the inner filter effect. In dilute solutions (a), the intensity of the excitation beam (solid black arrow) remains virtually unaltered as it propagates through the solution. This is important because the intensity of fluorescence is proportional to the intensity of the excitation radiation. As the solution concentration is increased, the fluorescence intensity should increase proportionally, as it does between the solutions depicted in (a) and (b). As the concentration continues to increase (pictures c and d), the inner filter effect attenuates the excitation beam. As a consequence, the fluorescence no longer increases linearly with concentration. In the final picture on the right, the attenuation is so severe that the excitation radiation does not even reach the back of the solution and thus no fluorescence is observed from fluorophores in this part of the solution. Also, keep in mind that as the solution concentration increases, the chances for self‐absorption also increase, further reducing the amount of fluorescence observed.

FIGURE 3.10 Pyrene excimer.

FIGURE 3.11 Excitation and emission spectra of rhodamine B.

FIGURE 3.12 A general schematic of a fluorometer or spectrofluorometer. Electromagnetic radiation from the excitation source passes through a filter or monochromator. The nearly monochromatic light that emerges is then split by a beam splitter. Some of the light is sent to a reference detector in order to correct emission spectra for source intensity variations. The other part of the beam is sent to the sample. Molecules in the sample can absorb the excitation radiation and subsequently emit fluorescence over a range of wavelengths. The fluorescence is passed through the emission filter or monochromator. The nearly monochromatic light that emerges is then detected by the detector. Instruments that use filters are called fluorometers, and those that have monochromators are called spectrofluorometers. Both the excitation and emission monochromator can be scanned to collect excitation or emission spectra.

FIGURE 3.13 A high pressure xenon arc lamp.

FIGURE 3.14 The spectral output of a xenon flash lamp. There is continuous output from 200 to 1000 nm. Spikes in the spectrum are due to specific electronic transitions of xenon atoms.

FIGURE 3.15 A bird’s‐eye view of fluorometer components. Electromagnetic radiation from the source is passed through the excitation filter to select the excitation wavelength. The beam is split by a beam splitter. A portion of the light is directed onto a reference detector to correct for fluctuations in the source intensity. The other portion of the beam is focused onto the sample. Molecules in the sample absorb the excitation radiation and can subsequently fluoresce. The fluorescence is collected at 90° by lenses, passed through the emission filter to select the emission wavelength that is monitored, and is then detected. While fluorescence is collected at 90°, it is worth noting that fluorescent light is emitted in all directions, so only a fraction of the actual light emitted is collected.

FIGURE 3.16 Bird’s‐eye view of the components of a spectrofluorometer. (a) Radiation from the excitation source enters the excitation grating monochromator, which selects the wavelength that is passed to the sample. Fluorescence from the sample is collected at 90° and passed through the emission grating monochromator to select the specific wavelength of fluorescence that is detected by the detector. Emission and excitation spectra are collected by scanning the respective monochromator while keeping the other fixed. (b) This diagram shows a spectrofluorometer with a double emission monochromator, which helps to decrease the stray light that reaches the detector.

FIGURE 3.17 Collecting an excitation spectrum. To collect an excitation spectrum, the grating in the excitation monochromator is rotated in order to scan through the specified wavelength range. The emission monochromator is held constant at a wavelength where it is known that the analyte fluoresces.

FIGURE 3.18 Uncorrected (solid spikey line) and corrected excitation spectra (dashed line) of fluorescein in 0.05 N NaOH compared to its absorbance spectrum (solid smooth line left) and its fluorescence spectrum (solid line right). The spikes in the uncorrected excitation spectrum are caused by variations in the intensity of the lamp at different wavelengths. There are other differences between the corrected and uncorrected spectra that arise from other instrumental components. The result is that even after correcting for lamp intensity variations as a function of wavelength, the corrected spectrum more closely resembles, but is still not identical to the absorbance spectrum.

FIGURE 3.19 Collecting an emission spectrum. To collect an emission spectrum, the grating in the excitation monochromator is held constant at a wavelength where it is known that the analyte absorbs. The grating in the emission monochromator is rotated to scan through the wavelengths emitted by the analyte.

FIGURE 3.20 Normalized fluorescence spectrum of quinine sulfate in 0.25 N H

2

SO

4

, excited at 348 nm.

FIGURE 3.21 Bird’s‐eye view of front‐face vs. 90° collection of fluorescence. (a) In 90° collection with opaque or concentrated samples, the potential for self‐absorption or scattering exists such that light emitted by the excited molecules is lost before it can exit the cuvette and be detected. (b) Because there are fewer molecules between the fluorescent molecules and the front face, some fluorescence can “escape” and be detected before it is absorbed or scattered by other molecules.

FIGURE 3.22 Single photon counting. Note that even though signals “4” and “8” are double in strength compared to most single counts, indicating that two photons arrived simultaneously or nearly so, they are still counted as just a single event.

FIGURE 3.23 Arrangement of components for measuring fluorescence polarization. Polarizers are placed in the path of the excitation beam and the emitted fluorescence. After excitation, molecular rotation can cause the depolarization of fluorescence. Depending on the alignment of the emission polarization, components of the fluorescence that are parallel with or perpendicular to the excitation polarization can be selectively detected. In this figure as shown, the perpendicular component is being measured, and if the emission polarizer is rotated (image that is offset to the right), the parallel component is measured. Note excitation and emission filters or monochromators are still needed to select specific wavelengths but have been omitted from the figure for clarity.

FIGURE 3.24 Emission following no rotation by the fluorescent molecules is polarized parallel to the excitation polarization (a). Emission after rotation is at least partially depolarized and thus has some component that is polarized parallel to the excitation polarization (b).

FIGURE 3.25 Diagrams of an (a) upright and (b) inverted fluorescence microscope. Excitation radiation from the source passes through the excitation filter, strikes a dichroic mirror (beam splitter), and is directed through the objective lens. The objective lens focuses the light onto the specimen. Fluorescence that is subsequently emitted from the specimen is collected by the objective lens, passes through the dichroic mirror and an emission filter, and ultimately strikes the detector or camera. In the upright configuration, the excitation radiation illuminates the specimen from above, and fluorescence is also captured above the specimen. In the inverted configuration, the excitation radiation strikes the specimen from below, and the fluorescence is captured from the underside of the specimen.

FIGURE 3.26 Figure of confocal fluorescence microscopy. (a) Depicts the illumination of the specimen. Electromagnetic radiation from the source (commonly a laser) strikes a dichroic mirror (beam splitter), passes through a lens, and illuminates the sample. (b) This image is similar to (a) except that it adds in the fluorescence caused by the excitation beam. Fluorescence is excited in many planes through the specimen. The fluorescence is collected by the lens and passes through the dichroic mirror toward the detector. Because of the pinholes located in front of the source and detector, only a small spot in a thin plane of the sample is focused on the detector. Light from other planes in the sample, represented by dashed and dotted lines in this figure, is not focused onto the detector and thus is not observed. A 2D image of the in‐focus plane is obtained by scanning the plane, and 3D images are created by layering scans of multiple planes.

FIGURE 3.27 Chemiluminescence produced by the reaction shown in aerated diglyme–acetate buffer, pH  =  5.6, at 25 °C. Time zero is when the reactants are mixed. The molecule in the reaction is an analog of a molecule responsible for blue‐colored bioluminescence emitted by

Cypridina (Vargula)

– a species of ostracod crustacean. The native bioluminescence is produced by a luciferin–luciferase reaction. In this reaction, the analog reacts with triplet oxygen (

3

O

2

), undergoes a series of reactions (not shown), and ultimately creates the product shown while emitting a photon.

FIGURE 3.28 The luminol reaction. The reaction leads to the production of light due to the creation of high energy intermediates that relax back to the ground state via emission.

FIGURE 3.29 Examples of biomolecules with native fluorescence.

FIGURE 3.30 Examples of common fluorescent derivatizing agents.

FIGURE 3.31 Examples of common derivatizing agents for fluorescence microscopy and immunoassays.

FIGURE 3.32 Examples of dyes used to derivatize lipids.

FIGURE 3.33 Examples of DNA bases and fluorescent analogs.

FIGURE 3.34 (a) The first step in the Sanger method of sequencing is to generate fragments of DNA that are radiolabeled (as indicated by the asterisks) and that terminate with dideoxynucleotides. Four separate reaction vessels are used, with one vessel for each of the four different dideoxynucleotides. (b) The ssDNA strands generated in each vessel are separated by slab gel electrophoresis, with a different lane for each reaction vessel. After the separation, the DNA sequence is read from the bottom of the gel to the top.

FIGURE 3.35 (a) Fluorescent dyes used by Smith et al. to derivatize the DNA primers. NBD is 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1‐diazole. (b) The absorbance and emission spectra of fluorescein (solid line), NBD (dotted line), Texas Red (dashed line), and tetramethylrhodamine (dashed/dot line).

FIGURE 3.36 Partial electropherogram from which the DNA sequence of the fragment Smith et al. analyzed was read. (a) The electropherogram collected at the four different wavelengths used to detect the DNA. (b) Electropherogram after adjusting for migration shifts caused by the primers showing how the sequence can be read from the peaks (note the sequence at the bottom of the plot). (c) Enlargement of part of the electropherogram (1000 data points) to make it clearer how the sequence is read from the peaks.

Chapter 04

FIGURE 4.1 A typical IR spectrum taken of methyl salicylate as the analyte. Methyl salicylate is used to make candy “wintergreen” flavored and gives candy a terrific minty smell. Note that the axes are transmittance vs. wavenumber (

in cm

−1

), compared to UV‐visible spectra that are typically plotted as absorbance vs. wavelength (in nm). It is possible, however, to convert between transmittance and absorbance (

A

  =  −log

T

 ) and between wavelength and wavenumber (

λ

  =  1/

).

FIGURE 4.2 Depiction of electronic transitions from the ground state (

S

0

) to the first excited state (

S

1

) state such as those observed in UV‐visible spectroscopy compared to a transition from the lowest vibrational level to the first vibrational level like those probed by IR spectroscopy. Note that energy increases from the bottom to the top of the image. It is clear that vibrational transitions are much lower in energy than electronic transitions.

FIGURE 4.3 A depiction of methyl salicylate (for which the IR spectrum was shown in Figure 4.1). Balls represent atomic nuclei and the gray‐shaded region represents the electron density. Lines representing bonds between atoms have been deliberately omitted to emphasize the fact that Coulombic forces, not sticks or springs, hold the atoms in space relative to one another.

FIGURE 4.4 Starting at the right edge, this curve shows the potential energy as two atoms get increasingly closer to each other. The lowest energy point is referred to as the bond energy. The separation of the two nuclei at this point is known as the bond length. The energy initially decreases as favorable positive/negative interactions between protons located in the nuclei and electrons increase. At very short distances (far left), the energy increases and eventually becomes positive (i.e., unfavorable) as the repulsive forces between the two positive nuclei outweigh the favorable positive/negative interactions. The actual values of bond distance and bond energy change depending on the numbers of protons and electrons in the specific atoms involved.

FIGURE 4.5 Vibration of a carbonyl bond. From top to bottom: atoms close to one another repel each other, and the atoms begin to move apart. As the bond lengthens, restoring forces bring the atoms back together again. The process continues with a frequency that is characteristic of the atoms involved and the strength of the bond.

FIGURE 4.6 Examples of common stretching and bending vibrations. The top two images are stretches, and the middle and bottom images depict bending vibrations. Note that stretching vibrations result in a change in bond lengths, whereas bending vibrations result in changes in bond angles [11].

FIGURE 4.7 Vibrational modes of CO

2

and H

2

O. In all instances except the symmetric stretch of CO

2

(upper middle), the stretching or bending motions result in a change in the dipole of the molecule and are therefore IR‐active.

FIGURE 4.8 Ball and spring model for vibrations. When the system is at rest, it has no potential energy. Displacements of the ball from the rest position due to stretching or compressing the spring are measured relative to the rest position. Displacements in either direction result in the system having potential energy.

FIGURE 4.9 The potential energy versus displacement for two different springs: one with a higher force constant,

k

(solid curve), and one with a lower force constant (dashed line). Note that the spring with the higher force constant has higher potential energy for the same displacement of both springs (follow the dashed vertical line). Similarly, a greater displacement of either spring results in a higher potential energy as indicated by the vertical solid lines.

FIGURE 4.10 Two balls of different mass (

m

1

and

m

2

) attached to a spring. Left: resting or equilibrium position. Middle: spring is stretched. Right: spring is compressed. When let go after being stretched or compressed, the system would begin to vibrate with a characteristic frequency depending on the force constant of the spring and the masses of the two balls.

FIGURE 4.11 Vibrational energy levels within the ground electronic level (

S

0

). In this image, the difference between consecutive energy levels is approximately the same. This approximation does not hold true at high vibrational levels for actual molecules as discussed below, but we use the approximation here for illustrative purposes.

FIGURE 4.12 Depiction of the harmonic (dashed lines) and anharmonic (solid line) oscillator models. Near the potential energy minimum, the harmonic oscillator model approximates anharmonic behavior fairly well. However, when the interatomic distance is very short (far left of the diagram), the potential energy of a bond increases more rapidly than predicted by the harmonic oscillator model because of the repulsion between positively charged nuclei – an effect not present with neutral balls and springs. At long distances between atoms (far right of the diagram), bonds can break, which is also not accounted for in the harmonic oscillator model. Also note that the energy difference between vibrational levels decreases at the higher vibrational levels.

FIGURE 4.13 Translational and rotational motion of molecules on an (a)

x

‐,

y

‐, and

z

‐coordinate system, (b) shows translation of methane on the

x

‐,

y

‐, and

z

‐axes from left to right, and (c) depicts rotations about the

x

‐,

y

‐, and

z

‐axes from left to right.

FIGURE 4.14 Rotation of linear molecules like CO

2

. (a) Coordinate system. (b) Left: rotation about the long axis of the molecule (

x

‐axis in the figure) produces no change in the molecule. Middle and right: rotation about the other axis changes the orientation of the molecule.

FIGURE 4.15 Albert Abraham Michelson, used with permission from AstroLab; the original uploader was Bunzil at English Wikipedia (Public domain), via Wikimedia Commons.

FIGURE 4.16 Bird’s‐eye view of the main components of an interferometer. Infrared radiation emitted by the source strikes the beam splitter, B. Some of the radiation is transmitted through the splitter, strikes the movable mirror, M

2

, and returns to the beam splitter (Path 2). The other part of the radiation is reflected over to the stationary mirror, M

1

, and is reflected back to the beam splitter (Path 1). The waves from M

1

and M

2

recombine at the beam splitter and are directed through the sample compartment and into the detector.

FIGURE 4.17 Constructive and total destructive interference of waves. Constructive interference occurs when waves have the same frequency and are in phase with each other, resulting in a single wave of the same frequency but of greater amplitude. Total destructive interference occurs when waves of the same frequency are 180° out of phase with one another, resulting in the complete cancelation of the waves.

FIGURE 4.18 The interferogram that results from monochromatic radiation passing through an interferometer.

FIGURE 4.19 The superposition of two waves [16]. Top: a wave with wavelength

λ

. Middle: a wave with wavelength 4

λ

. Bottom: the single wave that results from adding the top and middle waves together.

FIGURE 4.20 An interferogram of a broadband source. The center burst occurs at the point of zero path difference (ZPD) [16]. The

y

‐axis is voltage because detectors convert the electromagnetic radiation into an electric signal.

FIGURE 4.21 Depiction of the Fourier transformation of an interferogram (time domain) into an IR spectrum (frequency domain) [16].

FIGURE 4.22 Example of a background spectrum that ultimately gets used as

P

o

to determine the absorbance created by a sample (

A

  =  log

P

o

/

P

) [16]. Signals in the blank mainly arise from CO

2

and water.

FIGURE 4.23 Spectrum with a film of polystyrene in the sample compartment [16]. The ratio of this spectrum to the background spectrum has not yet been taken in this image, but if you visually compare the two, you will see that several signals that are not in the background are present here.

FIGURE 4.24 A spectrum of polystyrene [16] obtained by taking the ratio of the sample spectrum (

P

) to the background spectrum (

P

o

), which were shown in the previous figures, to obtain the transmittance of the polystyrene film.

FIGURE 4.25 The effect of small differences in the starting time of each spectrum. Here, three simulated spectra (dashes) are slightly offset from one another. The solid line shows the average of the three spectra. Notice that in this case, because the spectral features do not perfectly overlap, taking the average distorts and diminishes the actual signal. This emphasizes the importance of having a mechanism for the instrument to start the collection of a new spectrum at exactly the same point in the mirror displacement. Note that the shifts in the spectra have been exaggerated so that the effect is easily seen.

FIGURE 4.26 Spectral output of two Nernst glowers operated under different conditions at approximately 2100 °C. (a) 77.7 W (sensibility 89). (b) 102.5 W (sensibility 108). These curves approximate that of blackbody radiation at a comparable temperature.

FIGURE 4.27 Globar output at four different temperatures. Emittance is the ratio of the output of the source relative to that of a blackbody. In this plot, the ratio is converted to a percent. So in this plot, for example, silicon carbide emits 80% of the radiation at 3 μm and 982 °C that a blackbody would emit.

FIGURE 4.28 A 10 cm gas cell for FTIR analysis. Input and outlet spouts with rotating valves allow gases to be pumped into the cell. Once the cell is full, the valves are close and a spectrum recorded. The gas can then be displaced by another gas. Continuous monitoring of the gas is also possible by continuing to pump the gas of interest through the cell and recording spectra as a function of time.

FIGURE 4.29 A demountable liquid sample cell for FTIR spectroscopy [12]. The cell is assembled by placing the gasket and one cell window between the four screws on the backplate. The spacer is then put in place and a drop of the liquid sample added to it. The second cell window is placed on top of the spacer to sandwich the sample between the two windows. Lastly, the final metal housing piece is slipped over the four screws. The screws are gently tightened to hold the entire assembly in place when it is mounted inside the instrument.

FIGURE 4.30 A fixed path length IR cell [28]. Such cells, unlike those in the previous figure, are not made to be disassembled. They have a spacer of fixed, known thickness that provides the fixed path length. The sample solution is added to the cell by injecting it into the inlet port using a syringe. Once the sample space is full, excess sample comes out from the outlet side. The ports are then plugged and the assembly is mounted in the instrument sample compartment.

FIGURE 4.31 Reflection of radiation off a perfectly smooth surface (specular reflectance) and a rough surface (diffuse reflectance). Solid lines indicate incoming radiation, and dashed lines represent reflected radiation.

FIGURE 4.32 Examples of an (a) integrating sphere and (b) ellipsoidal mirror [27, 29, 30].

FIGURE 4.33 A diffuse reflectance spectrum of an analgesic tablet ground up and diluted to 10% with KBr [16].

FIGURE 4.34 ATR accessories. (a) A single reflection ATR and (b) multiple reflection ATR cell [36]. Note that the depth of penetration of the evanescent waves, which is generally on the order of a few microns, is greatly exaggerated relative to the thickness of the samples in these images.

FIGURE 4.35 Gas sensors based on IR detection [38]. (a) single beam sensor. (b) Double beam in time gas sensor. A rotating chopper is used to create the two beams.

FIGURE 4.36 A depiction of the relative ranges in wavelengths covered by various spectroscopic techniques. As stated in the text, the specific wavelengths associated with each region vary slightly from source to source. Specific definitions are available from governing bodies such as the ISO.

FIGURE 4.37 NIR spectrum of biscuit dough.

FIGURE 4.38 (a) The structures of polyethylene (PE) and polypropylene (PP) [37, 51]. (b) The NIR spectra of different blends of PE and PP. Examining the 100% PE (solid black line) and 100% PP (dotted line) shows that the two materials have different spectra, but determining the percentage of each in the blends is difficult without chemometric methods due to the high degree of overlap [37, 51].

FIGURE 4.39 Figure of a finger oximeter showing that two wavelengths are used to measure the percent saturation of hemoglobin in blood [59].

FIGURE 4.40 Spectrum of hemoglobin and deoxyhemoglobin in a portion of the visible and near‐infrared regions of the spectrum [60].

FIGURE 4.41 A depiction of how the ratio of absorbance at 650–950 nm changes as the percent oxygenation of hemoglobin in blood changes [59].

FIGURE 4.42 Depiction of (a) transmission, (b) reflectance, and (c) multidistance modes of NIR spectroscopy to measure oxygenation changes in different tissue layers [63, 64].

FIGURE 4.43 Structure of (a) acetazolamide and (b) far‐IR spectra of two crystalline forms of it [77].

FIGURE 4.44 Structures of (a) PETN and RDX and (b) their terahertz absorption (extinction) and refractive index spectra. PETN spectra are on the left and RDX spectra are on the right [78].

Chapter 05

FIGURE 5.1 Sir Chandrasekhara Venkata Raman.

FIGURE 5.2 An energy‐level diagram depicting infrared absorption (IR), elastic scattering, Stokes and anti‐Stokes Raman scattering, and fluorescence. Upward straight arrows represent increases in the energy of a molecule caused by incident photons, which are symbolized by horizontal squiggles to the left of upward arrows. Downward straight arrows represent decreases in the energy of a molecule caused by scattering or emission of photons, which are symbolized by the squiggles going off at an angle to the right of the downward lines. The downward squiggly arrow between

v

  =  1 and

v

  =  0 in the excited state,

S

1

, represents nonradiative (NR) decay.

S

0

and

S

1

refer to the ground and first excited electronic states, while the vibrational states are labeled by their corresponding vibrational quantum numbers,

v

. For elastic scattering, the incident and scattered photons have the same energy. Scattering in which the scattered photon has less energy than the incident photon is known as Stokes scattering. Anti‐Stokes scattered photons have more energy than the incident photons.

FIGURE 5.3 Representative Raman spectrum of

N

‐acetyl‐

p

‐aminophenol (Tylenol) with peak assignments highlighting some of the Raman‐active vibrational modes. The structural formula of the molecule is shown in the inset.

FIGURE 5.4 An electromagnetic field or light wave can distort the electron cloud surrounding a molecule.

FIGURE 5.5 Comparison of infrared absorption and Raman spectra of collagen. The absorption spectrum was acquired with a Fourier transform infrared (FT‐IR) spectrometer. Notice the similar but unique and complementary spectral features present in the Raman and FT‐IR spectra.

FIGURE 5.6 Dipole moments and polarizability ellipsoids for the CO

2

symmetric stretch vibration. There is no molecular dipole moment because the two bond dipoles cancel. The vibration is therefore not IR active. The polarizability ellipsoid depicts the magnitude of the polarizability in three dimensions. As explained in the text, the ellipsoids reflect the

inverse

square root of the polarizability, so as the polarizability of the CO

2

molecule decreases as the C═O bonds lengthen, the polarizability ellipsoid increases in size. As the C═O bonds gets shorter during the symmetric stretch, the polarizability increases, which is reflected in a smaller polarizability ellipsoid. As seen above, the size of the ellipsoid changes during vibration around the equilibrium position and the vibration is therefore Raman active.

FIGURE 5.7 Raman‐active modes vibrating at frequency

υ

m

and excited with monochromatic illumination at frequency

υ

will emit light corresponding to elastic (

υ

), Stokes Raman (

υ

−  

υ

m

), and anti‐Stokes Raman scattering (

υ

−  

υ

m

).

FIGURE 5.8 Bird’s‐eye view of a polarization‐sensitive Raman measurement. The excitation light is passed through a polarizer to deliver linearly polarized light to the sample (polarized parallel to the page). The Raman‐scattered light in general has components polarized both parallel and perpendicular to the excitation light. A second polarizer called an analyzer is used to sequentially pass each of these components to the detector.

FIGURE 5.9 Bird’s‐eye view of the optical layout of a scanning Czerny–Turner monochromator.

FIGURE 5.10 Major components of an AOTF.

FIGURE 5.11 Bird’s‐eye view of the optical layout of an axial transmissive spectrometer.

FIGURE 5.12 Basic principle of confocal microscopy. Backscattered light from the focus passes through the confocal pinhole, while light from out‐of‐focus planes is blocked by the pinhole.

FIGURE 5.13 CT tomograms of a human brain from the base of the skull to the top.

FIGURE 5.14 Confocal Raman images of LN‐18 human malignant glioma cells. Each image shows different

spectral

information from the same spatial location (i.e., the same clustering of cells). Different cellular compartments are clearly visible in the panels including (a) the nucleus, (b) actin filaments, and (c) Golgi apparatus. Panel (d) shows a composite image of panels (a)–(c).

FIGURE 5.15 Representative Raman spectrum of bone including the best fit of the phosphate mineral band near 960 cm

−1

.

FIGURE 5.16 Sixteenth‐century German choir book: historiated letter “R”.

FIGURE 5.17 Representative Raman spectra of drug tablets used to treat hypertension (hydrochlorothiazide and verapamil), cardiac arrhythmia (digoxin and verapamil), and other related conditions.

FIGURE 5.18 FTR spectra of the pure alkaloids: (a) heroin, (b) morphine, and (c) codeine. 50 scans, 6 cm

−1

resolution, and incident laser power 200 mW. Scanning time 3 min.

FIGURE 5.19 Raman spectroscopy system for bone diagnostics. Light gray corresponds to the laser illumination path, and dark gray corresponds to the Raman scattering collection path. Abbreviations: ST, shutter; L, lens; MMF, multimode fiber; BP, bandpass filter; BS, dichroic beam splitter; MTS, motorized translation stage; NF, notch filter; FB, fiber bundle; SPEC, spectrograph; HG, holographic grating; CCD, charge‐coupled device.

FIGURE 5.20 Scatterplots of the ultimate torque prediction for each bone sample versus its measured value for the (a) BMD and (b) Raman prediction models. The diagonal line is the perfect prediction line. The closer a data point lies to this line, the more accurate the prediction.

FIGURE 5.21 Comparison of SERS spectrum of blood plasma adsorbed on microwave‐treated Au–PS substrates with conventional Raman spectrum of blood plasma. Note that different spectra have been multiplied by different factors as indicated in the figure for better visualization. The 785 nm laser excitation power is 2.5 mW for SERS measurements, while 80 mW for conventional Raman measurements.

FIGURE 5.22

In vivo

SRS microscopy images of human GBM xenografts. Images are representative of six mice. SRS imaging was carried out via acute cranial window preparation in mice 24 days after implantation of human GBM xenografts. (a) Bright‐field microscopy appears grossly normal, whereas SRS microscopy within the same field of view demonstrates distinctions between tumor‐infiltrated areas and noninfiltrated brain (normal), with a normal brain‐tumor interface (dashed line). (b–d) High‐magnification views (b) within the tumor, (c) at the brain surface, and (d) within normal brain.

FIGURE 5.23 Dipole moments and polarizability ellipsoids for the H

2

O symmetric stretching mode.

Guide

Cover

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