21

chapter

Basic Principles of Spectroscopy

Michael H. Penner
Department of Food Science and Technology, Oregon State University,
Corvallis, OR 97331-6602, USA
[email protected]

21.1 Introduction 377 21.3.3 Nuclear Energy Levels in Applied

21.2 Light 377 Magnetic Fields 382
21.2.1 Properties 377 21.4 Energy Level Transitions in Spectroscopy 382
21.2.2 Terminology 378 21.4.1 Absorption of Radiation 382
21.2.3 Interference 378 21.4.2 Emission of Radiation 383
21.3 Energy States of Matter 379 21.5 Summary 384
21.3.1 Quantum Nature of Matter 379 21.6 Study Questions 384
21.3.2 Electronic, Vibrational, and Rotational 21.7 Resource Materials 385
Energy Levels 380

S.S. Nielsen, Food Analysis, Food Science Texts Series, DOI 10.1007/978-1-4419-1478-1_21, 375
c Springer Science+Business Media, LLC 2010
Chapter 21 • Basic Principles of Spectroscopy 377

21.1 INTRODUCTION

Spectroscopy deals with the production, measure-

ment, and interpretation of spectra arising from the
interaction of electromagnetic radiation with mat-
ter. There are many different spectroscopic methods
available for solving a wide range of analytical prob-
lems. The methods differ with respect to the species
to be analyzed (such as molecular or atomic spec-
troscopy), the type of radiation–matter interaction to Representation of plane-polarized electromag-
be monitored (such as absorption, emission, or diffrac- 21-1 netic radiation propagating along the x-axis.
figure
tion), and the region of the electromagnetic spectrum The electric and magnetic fields are in phase,
used in the analysis. Spectroscopic methods are very perpendicular to each other, and to the direc-
tion of propagation.
informative and widely used for both quantitative and
qualitative analyses. Spectroscopic methods based on
the absorption or emission of radiation in the ultra- number of oscillations the wave will make at a given
violet (UV), visible (Vis), infrared (IR), and radio point per second. This is the reciprocal of the period
(nuclear magnetic resonance, NMR) frequency ranges (p) of a wave, which is the time in seconds required for
are most commonly encountered in traditional food successive maxima of the wave to pass a fixed point.
analysis laboratories. Each of these methods is dis- The wavelength (λ) represents the distance between
tinct in that it monitors different types of molecular successive maxima on any given wave. The units used
or atomic transitions. The basis of these transitions is in reporting wavelengths will depend on the region of
explained in the following sections. electromagnetic radiation used in the analysis. Spec-
troscopic data sometimes are reported with respect to
wavenumbers (v̄ ), which are reciprocal wavelengths
21.2 LIGHT in units of cm−1 . Wavenumbers are encountered most
often in IR spectroscopy. The velocity of propagation
21.2.1 Properties (v i ) of an electromagnetic wave, in units of distance
per second, in any given medium “i” can be calculated
Light may be thought of as particles of energy that by taking the product of the frequency of the wave, in
move through space with wavelike properties. This cycles per second, and its wavelength in that particular
image of light suggests that the energy associated with medium.
a ray of light is not distributed continuously through v i = νλi [1]
space along the wave’s associated electric and mag-
netic fields but rather that it is concentrated in dis- where:
crete packets. Light is therefore said to have a dual v i = velocity of propagation in medium i
nature: particulate and wavelike. Phenomena asso- ν = frequency (of associated wave)
ciated with light propagation, such as interference,
λi = wavelength in medium i
diffraction, and refraction, are most easily explained
using the wave theory of electromagnetic radiation. The frequency of an electromagnetic wave is deter-
However, the interaction of light with matter, which mined by the source of the radiation, and it remains
is the basis of absorption and emission spectroscopy, constant as the wave traverses different media. How-
may be best understood in terms of the particulate ever, the velocity of propagation of a wave will vary
nature of light. Light is not unique in possessing slightly depending on the medium through which the
both wavelike and particulate properties. For exam- light is propagated. The wavelength of the radiation
ple, fundamental particles of matter, such as electrons, will change in proportion to changes in wave veloc-
protons, and neutrons, are known to exhibit wavelike ity as defined by Equation [1]. The amplitude of the
behavior. wave (A) represents the magnitude of the electric vec-
The wave properties of electromagnetic radiation tor at the wave maxima. The radiant power (P) and
are described in terms of the wave’s frequency, wave- radiant intensity (I ) of a beam of radiation are propor-
length, and amplitude. A graphical representation of tional to the square of the amplitude of the associated
a plane-polarized electromagnetic wave is given in waves making up that radiation. Figure 21-1 indicates
Fig. 21-1. The wave is plane polarized in that the oscil- that electromagnetic waves are composed of oscillat-
lating electric and magnetic fields making up the wave ing magnetic and electric fields, the two of which are
are each limited to a single plane. The frequency (ν, mutually perpendicular, in phase with each other, and
the lower case Greek letter nu) of a wave is defined as the perpendicular to the direction of wave propagation.
378 Part IV • Spectroscopy

Wavefronts, wave-trains, and rays. [From

21-2 Hugh D. Young, University Physics (8th edn),
figure
p 947, 
c 1992 by Addison-Wesley, Reading,
MA. Courtesy of the publisher.]

As drawn, the waves represent changes in the respec- Interference of identical waves that are (a) in
tive field strengths with time at a fixed location or
21-3 phase, (b) 90◦ out of phase, and (c) 180◦ out of
figure
changes in the respective field strengths over distance phase.
at a fixed time. The electrical and magnetic compo-
nents of the waves are represented as a series of vectors
whose lengths are proportional to the magnitude of another, they result in an instantaneous wave, at the
the respective field. It is the oscillating electric field point of intersection, whose amplitude is the alge-
that is of most significance to spectroscopic phenom- braic sum of the amplitudes of the individual waves
ena such as absorption, transmission, and refraction. at the point of intersection. The law describing this
However, a purely electric field, without its associated wave behavior is known as the principle of super-
magnetic field, is impossible. position. Superposition of sinusoidal waves is illus-
trated in Fig. 21-3. Note that in all cases, the effective
amplitude of the perceived wave at the point in ques-
21.2.2 Terminology tion is the combined effect of each of the waves that
The propagation of electromagnetic waves is often crosses that point at any given instant. In spectroscopy,
described in terms of wavefronts or trains of waves the amplitude of most general interest is that corre-
(Fig. 21-2). A wavefront represents the locus of a set sponding to the magnitude of the resulting electric
of points all of which are in phase. For a point source field intensity. Maximum constructive interference of
of light, a concentric ring that passes through the max- two waves occurs when the waves are completely in
ima of adjacent light rays will represent a wavefront. phase (i.e., the maxima of one wave align with the
The entire ring need not be drawn in all cases, such maxima of the other wave), while maximum destruc-
that wavefronts may represent planes of light in cases tive interference occurs when waves are 180◦ out of
where the observation is sufficiently removed from the phase (the maxima of one wave align with the min-
point source that the curved surface appears planar. ima of the other wave). This concept of interference is
Wavefronts are most typically drawn by connecting fundamental to the interpretation of diffraction data,
maxima, minima, or both for adjacent rays. If max- which represents a specialized segment of qualitative
ima are used for depicting wavefronts, then each of spectroscopy. Interference phenomena also are widely
the wavefronts will be separated by one wavelength. used in the design of spectroscopic instruments that
A train of waves, or wave-train, refers to a series of require the dispersion or selection of radiation, such
wavefronts all of which are in phase, that is, each indi- as those instruments employing grating monochroma-
vidual wave will have a maximum amplitude at the tors or interference filters, as described in Chap. 23.
same location in space. A wave-train also may be rep- Interference phenomena are best rationalized by
resented by a series of light rays. Rays of light are used considering the wavelike nature of light. However,
generally with reference to the corpuscular nature of phenomena such as the absorption and emission of
light, representing the path of photons. A wave-train radiation are more easily understood by considering
would indicate that a series of photons, all in phase, the particulate nature of light. The particles of energy
followed the same path. that move through space with wavelike properties are
called photons. The energy of a photon can be defined
in terms of the frequency of the wave with which it is
21.2.3 Interference associated Equation [2].
Interference is the term used to describe the obser-
vation that when two or more wave-trains cross one E = hν [2]
Chapter 21 • Basic Principles of Spectroscopy 379

where: 21.3 ENERGY STATES OF MATTER

E = energy of a photon 21.3.1 Quantum Nature of Matter
h = Planck’s constant
ν = frequency (of associated wave) The energy content of matter is quantized. Conse-
quently, the potential or internal energy content of
This relationship indicates that the photons making up an atom or molecule does not vary in a continuous
monochromatic light, which is electromagnetic radia- manner but rather in a series of discrete steps. Atoms
tion composed of waves having a single frequency and and molecules, under normal conditions, exist pre-
wavelength, are all of equivalent energy. Furthermore, dominantly in the ground state, which is the state
just as the frequency of a wave is a constant deter- of lowest energy. Ground state atoms and molecules
mined by the radiation source, the energy of associated can gain energy, in which case they will be elevated
photons also will be unchanging. The brightness of to one of their higher energy states, referred to as
a beam of monochromatic light, when expressed in excited states. The quantum nature of atoms and
terms of the particulate nature of light, will be the molecules puts limitations on the energy levels that
product of the photon flux and the energy per pho- are available to these species. Consequently, there will
ton. The photon flux refers to the number of photons be specific “allowed” internal energy levels for each
flowing across a unit area perpendicular to the beam atomic or molecular species. Internal energy levels
per unit time. It follows that to change the bright- not corresponding to an allowed value for that par-
ness of a beam of monochromatic light will require a ticular species are unattainable. The set of available
change in the photon flux. In spectroscopy, the term energy levels for any given atom or molecule will
brightness is generally not used, but rather one refers be distinct for that species. Similarly, the potential
to the radiant power (P) or the radiant intensity (I ) energy spacings between allowed internal energy lev-
of a beam of light. Radiant power and radiant inten- els will be characteristic of a species. Therefore, the
sity often are used synonymously when referring to set of potential energy spacings for a species may be
the amount of radiant energy striking a given area per used qualitatively as a distinct fingerprint. Qualita-
unit time. In terms of International Scientific (SI) Units tive absorption and emission spectroscopy make use
(time, seconds; area, meters; energy, joules), radiant of this phenomenon in that these techniques attempt
power equals the number of joules of radiant energy to determine an unknown compound’s relative energy
impinging on a 1 m2 area of detector per second. The spacings by measuring transitions between allowed
basic interrelationships of light-related properties and energy levels.
a general scheme of the electromagnetic spectrum are
presented in Table 21-1 and Fig. 21-4, respectively.

21-1
table Properties of Light

Symbols/Terms Relationship Frequently Used Units

λ = wavelength λi ν = vi nm (nanometers, 10−9 m)

(vi = c in a vacuum ) Å (Ångstrom units, 10−10 m)
μm (microns, 10−6 m)
mμ (millimicrons, 10−9 m)
ν = frequency Hz (hertz, 1 Hz = 1 oscillation per second)
c = speed of light 2.9979 × 108 m s−1 in vacuum
v̄ = wavenumber = 1/λ cm−1
kK (kilokayser, 1 kK = 1,000 cm−1 )
p = period p = 1/ν s
−2
E = energy E = hν J (1 joule = 1 kg m2 s )
= hc /λ cal (calorie, 1 cal = 4.184 J)
= hc v̄ erg (1 erg = 10−7 J)
eV (1 electron volt = 1.6022 × 10−19 J)
h = Planck’s constant 6.6262 × 10−34 J s
P = radiant power Amount of energy striking a (Joules)(m2 )−1 (s)−1
given unit area per unit time
380 Part IV • Spectroscopy

The electromagnetic spectrum. [From (10), p 3. Courtesy of Milton Roy Company, Rochester, NY, a subsidiary of
21-4 Sundstrand Corporation.]
figure

21.3.2 Electronic, Vibrational, and Rotational Atoms are like molecules in that only specific
Energy Levels energy levels are allowed for atomic electrons. Con-
sequently, an energy level diagram of an atom would
The relative potential energy of an atom or molecule consist of a series of electronic energy levels. In con-
corresponds to the energy difference between the trast to molecules, the electronic energy levels of atoms
energy state in which the species exists and that of the have no corresponding vibrational and rotational lev-
ground state. Figure 21-5 is a partial molecular energy els and, hence, may appear less complicated. Atomic
level diagram depicting potential energy levels for an energy levels correspond to allowed electron shells
organic molecule. The lowest energy state in the figure, (orbits) and corresponding subshells (i.e., 1s, 2s, 2p,
bottom line in bold, represents the ground state. There etc.). The magnitude of the energy difference between
are three electronic energy states depicted, each with the ground state and first excited states for valence
its corresponding vibrational and rotational energy electrons of atoms and bonding electrons of molecules
levels. Each of the electronic states corresponds to a is generally of the same range as the energy content of
given electron orbital. Electrons in different orbitals photons associated with UV and Vis radiation.
are of different potential energy. When an electron The wider lines within each electronic state of
changes orbitals, such as when absorbing or emitting Fig. 21-5 depict the species’ vibrational energy levels.
a photon of appropriate energy, it is termed an elec- The atoms that comprise a molecule are in constant
tronic transition since it is the electron that is changing motion, vibrating in many ways. However, in all cases
energy levels. However, any change in the potential the energy associated with this vibrational motion
energy of an electron will, by necessity, result in a cor- corresponds to defined quantized energy levels. The
responding change in the potential energy of the atom energy differences between neighboring vibrational
or molecule that the electron is associated with. energy levels are much smaller than those between
Chapter 21 • Basic Principles of Spectroscopy 381

Partial molecular energy level diagram depicting three electronic states.

21-5
figure

adjacent electronic energy levels. Therefore, it is com- spacings between rotational energy levels are of the
mon to consider that several vibrational energy levels same magnitude as the energy associated with pho-
are superimposed on each of the molecular electronic tons of microwave radiation. Microwave spectroscopy
energy levels. Energy differences between allowed is not commonly used in food analysis laboratories;
vibrational energy levels are of the same magnitude however, the presence of these different energy levels
as the energy of photons associated with radiation in will impact the spectrum observed in other forms of
the IR region. Vibrational energy levels would not be spectroscopy, as will be discussed later. Similar to the
superimposed on an atomic potential energy level dia- situation of vibrational energy levels, rotational energy
gram since this vibrational motion does not exist in levels are not of consequence to atomic spectroscopy.
a single atom. In this respect, the potential energy In summation, the internal energy of an atom
diagram for an atom is less complex than that for is described in terms of its electronic energy levels,
a molecule, the atomic energy level diagram having while the internal energy of a molecule is dependent
fewer energy levels. on its electronic, vibrational, and rotational energies.
The potential energy of a molecule also is quan- The algebraic form of these statements follows.
tized in terms of the energy associated with the rota-
tion of the molecule about its center of gravity. These Eatom = Eelectronic [3]
rotational energy levels are yet more closely spaced Emolecule = Eelectronic + Evibrational + Erotational [4]
than the corresponding vibrational levels, as depicted
by the narrow lines within each electronic state shown The spectroscopist makes use of the fact that each of
in Fig. 21-5. Hence, it is customary to consider sev- these associated energies is quantized and that dif-
eral rotational energy levels superimposed on each of ferent species will have somewhat different energy
the permitted vibrational energy levels. The energy spacings.
382 Part IV • Spectroscopy

21.3.3 Nuclear Energy Levels in Applied

Magnetic Fields
NMR spectroscopy makes use of yet another type of
quantized energy level. The energy levels of impor-
tance to NMR spectroscopy differ with respect to those
described above in that they are relevant only in the
presence of an applied external magnetic field. The
basis for the observed energy levels may be rational-
ized by considering that the nuclei of some atoms
behave as tiny bar magnets. Hence, when the atoms
are placed in a magnetic field, their nuclear mag-
netic moment will have a preferred orientation, just
as a bar magnet would behave. The NMR-sensitive
nuclei of general relevance to the food analyst have
two permissible orientations. The energy difference
between these allowed orientations depends on the
effective magnetic field strength that the nuclei expe-
rience. The effective magnetic field strength will itself
depend on the strength of the applied magnetic field
and the chemical environment surrounding the nuclei
in question. The applied magnetic field strength will
be set by the spectroscopist, and it is essentially equiv-
alent for each of the nuclei in the applied field. Hence, Absorption spectrum of a 0.005 M benzene in
differences in energy spacings of NMR-sensitive nuclei
21-6 water solution.
figure
will depend solely on the identity of the nucleus
and its environment. In general, the energy spac-
ings between permissible nuclear orientations, under
of photons of that energy, one observes a charac-
usable external magnetic field strengths, are of the
teristic absorption spectrum, the shape of which is
same magnitude as the energy associated with radi-
determined by the relative absorptivity of photons
ation in the radiofrequency range.
of different energy. The absorptivity of a compound
is a wavelength-dependent proportionality constant
that relates the absorbing species concentration to its
21.4 ENERGY LEVEL TRANSITIONS
experimentally measured absorbance under defined
IN SPECTROSCOPY
conditions. A representative absorption spectrum cov-
ering a portion of the UV radiation range is presented
21.4.1 Absorption of Radiation
in Fig. 21-6. The independent variable of an absorp-
The absorption of radiation by an atom or molecule tion spectrum is most commonly expressed in terms
is that process in which energy from a photon of elec- of the wave properties (wavelength, frequency, or
tromagnetic radiation is transferred to the absorbing wavenumbers) of the radiation, as in Fig. 21-6, rather
species. When an atom or molecule absorbs a photon than the energy of the associated photons.
of light, its internal energy increases by an amount Various molecular transitions resulting from the
equivalent to the amount of energy in that particu- absorption of photons of different energy are shown
lar photon. Therefore, in the process of absorption, schematically in Fig. 21-7. The transitions depicted
the species goes from a lower energy state to a more represent those that may be induced by absorption of
excited state. In most cases, the species is in the UV, Vis, IR, and microwave radiation. The figure also
ground state prior to absorption. Since the absorp- includes transitions in which the molecule is excited
tion process may be considered quantitative (i.e., all from the ground state to an exited electronic state with
of the photon’s energy is transferred to the absorb- a simultaneous change in its vibrational or rotational
ing species), the photon being absorbed must have an energy levels. Although not shown in the figure, the
energy content that exactly matches the energy dif- absorption of a photon of appropriate energy also may
ference between the energy levels across which the cause simultaneous changes in electronic, vibrational,
transition occurs. This must be the case due to the and rotational energy levels. The ability of molecules
quantized energy levels of matter, as discussed previ- to have simultaneous transitions between the differ-
ously. Consequently, if one plots photon energy vs. the ent energy levels tends to broaden the peaks in the
relative absorbance of radiation uniquely composed UV–Vis absorption spectrum of molecules relative to
Chapter 21 • Basic Principles of Spectroscopy 383

those peaks observed in the absorption spectrum of associated electronic transitions, are induced by radia-
atoms. This would be expected when one considers tion in the IR region. Independent transitions between
that vibrational and rotational energy levels are absent allowed rotational energy levels also are depicted,
in an atomic energy level diagram. The depicted tran- these resulting from the absorption of photons of
sitions between vibrational energy levels, without microwave radiation. A summary of transitions rele-
vant to atomic and molecular absorption spectroscopy,
including corresponding wavelength regions, is pre-
sented in Table 21-2.

21.4.2 Emission of Radiation

Emission is essentially the reverse of the absorp-
tion process, occurring when energy from an atom
or molecule is released in the form of a photon of
radiation. A molecule raised to an excited state will
typically remain in the excited state for a very short
period of time before relaxing back to the ground state.
There are several relaxation processes through which
an excited molecule may dissipate energy. The most
common relaxation process is for the excited molecule
to dissipate its energy through a series of small steps
brought on by collisions with other molecules. The
energy is thus converted to kinetic energy, the net
result being the dissipation of the energy as heat.
Under normal conditions, the dissipated heat is not
enough to measurably affect the system. In some cases,
molecules excited by the absorption of UV or Vis light
will lose a portion of their excess energy through the
Partial molecular energy level diagram emission of a photon. This emission process is referred
21-7 including electronic, vibrational, and rota- to as either fluorescence or phosphorescence, depend-
figure
tional transitions. ing on the nature of the excited state. In molecular

21-2
table Wavelength Regions, Spectroscopic Methods, and Associated Transitions

Types of Transitions in
Wavelength Wavelength Usual Wavelength Chemical Systems with Similar
Region Limits Type of Spectroscopy Range Energies

Gamma rays 0.01–1 Å Emission <0.1 Å Nuclear proton/neutron

arrangements
X-rays 0.1–10 nm Absorption, emission, 0.1–100 Å Inner-shell electrons
fluorescence, and
diffraction
Ultraviolet 10–380 nm Absorption, emission, and 180–380 nm Outer-shell electrons in
fluorescence atoms, bonding electrons in
molecules
Visible 380–750 nm Absorption, emission, and 380–750 nm Same as ultraviolet
fluorescence
Infrared 0.075–1000 μm Absorption 0.78–300 μm Vibrational position of atoms
in molecular bonds
Microwave 0.1–100 cm Absorption 0.75–3.75 mm Rotational position in
molecules
Electron spin resonance 3 cm Orientation of unpaired
electrons in an applied
magnetic field
Radiowave 1–1000 m Nuclear magnetic resonance 0.6–10 m Orientation of nuclei in an
applied magnetic field
384 Part IV • Spectroscopy

lowest permissible internal energy level. The relax-

ation process that dominates a system will be the one
that minimizes the lifetime of the excited state. Under
normal conditions, the relaxation process is so rapid
that the population of molecules in the ground state is
essentially unchanged.

21.5 SUMMARY

Spectroscopy deals with the interaction of electromag-

netic radiation with matter. Spectrochemical analysis,
a branch of spectroscopy, encompasses a wide range
of techniques used in analytical laboratories for the
qualitative and quantitative analysis of the chemical
composition of foods. Common spectrochemical anal-
ysis methods include UV, Vis, and IR absorption spec-
troscopy; molecular fluorescence spectroscopy; and
NMR spectroscopy. In each of these methods, the ana-
lyst attempts to measure the amount of radiation either
absorbed or emitted by the analyte. All of these meth-
Partial molecular energy level diagram ods make use of the facts that the energy content
21-8 including absorption, vibrational relaxation, of matter is quantized and that photons of radiation
figure
and fluorescence relaxation.
may be absorbed or emitted by matter if the energy
associated with the photon equals the energy differ-
ence for allowed transitions of that given species. The
fluorescence spectroscopy, the photons emitted from above methods differ from each other with respect to
the excited species generally will be of lower energy the radiation wavelengths used in the analysis or the
and longer wavelength than the corresponding pho- molecular vs. atomic nature of the analyte.
tons that were absorbed in the excitation process. The
reason is that, in most cases, only a fraction of the
energy difference between the excited and ground 21.6 STUDY QUESTIONS
states is lost in the emission process. The other fraction
of the excess energy is dissipated as heat during vibra- 1. Which phenomena associated with light are most read-
tional relaxation. This process is depicted in Fig. 21-8, ily explained by considering the wave nature of light?
which illustrates that the excited species undergoes Explain these phenomena based on your understanding
of interference.
vibrational relaxation down to the lowest vibrational
2. Which phenomena associated with light are most readily
energy level within the excited electronic state, and
explained by considering the particulate nature of light?
then undergoes a transition to the ground electronic Explain these phenomena based on your understanding
state through the emission of a photon. The photon of the quantum nature of electromagnetic radiation.
emitted will have an energy that equals the energy 3. What does it mean to say that the energy content of matter
difference between the lowest vibrational level of the is quantized?
excited electronic state and the ground electronic state 4. Molecular absorption of radiation in the UV–Vis range
level it descends to. The fluorescing molecule may results in transitions between what types of energy
descend to any of the vibrational levels within the levels?
ground electronic state. If the fluorescence transition is 5. Molecular absorption of radiation in the IR range results
to an excited vibrational level within the ground elec- in transitions between what types of energy levels?
tronic state, then it will quickly return to the ground 6. Why is an applied magnetic field necessary for NMR
spectroscopy?
state (lowest energy level) via vibrational relaxation.
7. How do the allowed energy levels of molecules differ
In yet other cases, an excited species may be of suffi- from those of atoms? Answer with respect to the energy
cient energy to initiate some type of photochemistry level diagram depicted in Fig. 21-5.
that ultimately leads to a decrease in the system’s 8. In fluorescence spectroscopy, why is the wavelength of
potential energy. In all cases, the relaxation process the emitted radiation longer than the wavelength of the
is driven by the tendency for a species to exist at its radiation used for excitation of the analyte?
Chapter 21 • Basic Principles of Spectroscopy 385

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