ANALYTICAL CHEMISTRY
`
`GARY D. CHRISTIAN
`
`University of Washington
`
`JOHN WILEY 8: SONS. INC.
`New York
`-
`Chichester
`
`-
`
`Brisbane
`
`v
`
`Singapore
`-
`Toronto
`SENJU EXHIBIT 2279
`
`Page 1 of 66
`‘L _
`
`LUPIN v. SENJU
`
`IPR2015-01105
`
`SENJU EXHIBIT 2279
`LUPIN v. SENJU
`IPR2015-01105
`
`Page 1 of 66
`
`

`
`ACQUISITIONS EDITOR Nedah Rose
`MARKETING MANAGER Catherine Faduska
`PRODUCTION EDITOR Marcia Craig
`DESIGNER Laura Nicholls
`MANUFACTURING MANAGER Inez Pettis
`ILLUSTRATION SUPERVISOR Jamie Perea
`COVER PHOTO Ellen Schusterfl‘he Image Bank
`
`This book was set in It}! 12 Times Roman by The Clarinda Company and printed
`and bound by Hamilton Printing Company. The cover was printed by Phoenix Color Corp.
`
`Recognizing the importance of preserving what has been written. it is a
`policy of John Wiley & Sons. Inc. to have books of enduring value published
`in the United States printed on acid—free paper. and we exert our best
`efforts to that end.
`
`Copyright © 1994. by John Wiley & Sons. Inc.
`
`All rights reserved. Published simultaneously in Canada.
`
`Reproduction or translation of any part of
`this work beyond that permitted by Sections
`107 and 108 of the 1976 United States Copyright
`Act without the permission of the copyright
`owner is unlawful. Requests for permission
`or further information should be addressed to
`the Permissions Department, John Wiley & Sons, Inc.
`
`Ubmry of Congress Cataloging in Publication Data:
`Christian. Gary D.
`Analytical chemistry I Gary D. Christian. — 5th ed.
`p. cm.
`Includes indexes.
`ISBN 0-471-59761-9
`
`1. Chemistry. Aria|ytic—Quantitative.
`QDl0l.2.CS7
`1994
`545—dC20
`
`I. Title.
`
`-
`
`.
`4*.
`
`.v-‘W?’
`
`T-_'-.1".
`
`g.‘
`
`r-. -‘.
`
`':~
`.-I
`"Printed in the United States of America
`1u’9_-_8 7 6 5 4
`
`Pagenniioii S6
`
`93-32933
`CIP
`
`Page 2 of 66
`
`

`
`CHAPTER 14
`
`SPECTROMETRY
`
`Spectrometry, particularly in the visible region of the electromagnetic spectrum,
`is one of the most widely used methods of analysis. It is very widely used in
`clinical chemistry and environmental laboratories because many substances can
`be selectively converted to a colored derivative. The instrumentation is readily
`available and generally fairly easy to operate. In this chapter, we (1) describe the
`absorption of radiation by molecules and its relation to molecular structure; (2)
`make quantitative calculations, relating the amount of radiation absorbed to the
`concentration of an absorbing analyte; and (3) describe the instrumentation re-
`quired for making measurements. Measurements can be made in the infrared,
`visible, and ultraviolet regions of the spectrum. The wavelength region of choice
`will depend upon factors such as availability of instruments, whether the analyte
`is colored or can be converted to a colored derivative, whether it contains func-
`tional groups that absorb in the ultraviolet or infrared regions, and whether other
`absorbing species are present in the solution. Infrared spectrometry is generally
`less suited for quantitative measurements but better suited for qualitative or fin-
`gerprinting information than are ultraviolet (UV) and visible spectrometry. Visible
`spectrometers are generally less expensive and more available than UV spectrom-
`eters.
`We also describe a related technique, fluorescence spectrometry, in which the
`amount of light emitted upon excitation is related to the concentration. This is an
`extremely sensitive analytical technique.
`
`Visible spectrometry is
`probably the most widely
`used analytical technique.
`
`398
`
`Page 3 of 66
`
`Page 3 of 66
`
`

`
`l4.l
`
`INTERACTION OF ELECTROMAGNETIC RADIATION WITH MATTER
`
`399
`
`INTERACTION OF ELECTROMAGNETIC
`14.1
`RADIATION WITH MATTER
`
`In spectrometric methods, the sample solution absorbs electromagnetic radiation
`from an appropriate source. and the amount absorbed is related to the concen-
`tration of the analyte in the solution. A solution containing copper ions is blue
`because it absorbs the complementary color yellow from white light and transmits
`the remaining blue light (see Table 14.] below). The more concentrated the copper
`solution, the more yellow light is absorbed and the deeper the resulting blue color
`of the solution. In a spectrometric method, the amount of this yellow light ab-
`sorbed would be measured and related to the concentration. We can obtain a
`
`better understanding of absorption spectrometry from a consideration of the elec-
`tromagnetic spectrum and how molecules absorb radiation.
`
`Spectrometry is based on
`the absorption of photons
`by the analyte.
`
`The Electromagnetic Spectrum
`
`Electromagnetic radiation, for our purposes, can be considered a form of radiant
`energy that is propagated as a transverse wave. It vibrates perpendicular to the
`direction of propagation and this imparts a wave motion to the radiation, as
`illustrated in Figure 14.1. The wave is described either in terms of its wavelength,
`the distance of one complete cycle, or in terms of the frequency, the number of
`cycles passing a fixed point per unit time. The reciprocal of the wavelength is
`called the wavennmber and is the number of waves in a unit length or distance per
`cycle.
`
`Wavelength, frequency,
`and vvavenumber are
`interrelated.
`
`TABLE 14.1
`
`Colors of Different Wavelength Regions
`
`Wavelength Absorbed, om
`Absorbed Color
`(Complement)
`
`
`Transmitted Color
`
`Violet
`Blue
`Green
`Yellow
`Orange
`Red
`
`Yellow-green
`Yellow
`Violet
`Blue
`Green~blue
`Blue-green
`
`380-450
`450-495
`495-570
`570- 590
`590-620
`620-750
`
`E E
`
`b-
`
`
`
`F: Wavelength —>|
`
`Direction of propagation
`
`FIGURE 14.1 Wave motion of electromagnetic radiation.
`
`Page 4 of 66
`
`Page 4 of 66
`
`

`
`CHAPTER 14 i‘ SPECTROMETRY
`
`The relationship between the wavelength and frequency is
`
`
`
`(14.1)
`
`where A is the wavelength in centimeters (cm),‘ I: is the frequency in reciprocal
`seconds (s"'), or hertz (Hz), and c is the velocity of light (3 X 10"’ cmfs}. The
`wavenumber is represented by 3. in cm“:
`
`
`
`(14.2)
`
`The wavelength of electromagnetic radiation varies from a few angstroms to sev-
`eral meters. The units used to describe the wavelength are as follows:
`
`
`It = angstrom = 10"” meter = 10*‘ centimeter = 10“‘ micrometer
`
`nm = nanometer = 10*‘ meter = 10 angstroms = ‘I0‘3 micrometer
`
`p.m = micrometer = 10"“ meter = 104 angstroms
`
`Wavelengths in the ultravi-
`olet and visible regions are
`on the order of nanome-
`ters. In the infrared region,
`they are micrometers, but
`the reciprocal of wave-
`length is often used {wave-
`numhers, in cm“).
`
`The wavelength unit preferred for the ultraviolet and visible regions of the spec-
`trum is nanometer, while the unit micrometer is preferred for the infrared region?
`In this last case, wavenumbers are often used in place of wavelength, and the unit
`is cm". See below for a definition of the ultraviolet, visible, and infrared regions
`of the spectrum.
`Electromagnetic radiation possesses a certain amount of energy. The energy of
`a unit of radiation, called the photon, is related to the frequency or wavelength by
`
`(14.3)
`
`Shorter wavelengths have
`greater energy. That is why
`ultraviolet radiation from
`
`the sun burns you!
`
`where E is the energy of the photon in ergs and h is Planck’s constant, 6.62 X
`10-“ 1-5. It is apparent, then, that the shorter the wavelength or the greater the
`frequency, the greater the energy.
`As indicated above, the electromagnetic spectrum is arbitrarily broken down
`into different regions according to wavelength. The various regions of the spec-
`
`
`
`'M0re correctly, the units are centimeters per cycle for wavelength and cycles per second for fre-
`quency. but the cycles unit is often assumed. In place of cyclesfs. the unit hertz {Hz} is now commonly
`used.
`
`’Nanometer (nm} is the preferred term over millimicron (mp). the unit used extensively prior to this.
`In the infrared region, micrometer (pm) is the preferred term in place of the previously used term
`micron (pt).
`
`Page 5 of 66
`
`Page 5 of 66
`
`

`
`14.1
`
`INTERACTION OF ELECTROMAGNETIC RADIATION WITH MA'|'l'ER
`
`401
`
`Ultraviolet
`
`infrared
`
`F“”%r“"‘%
`
`
`
`Gammarays
`
`«a
`3-
`E
`x
`
`E
`.9
`';
`L:
`
`We see only a very small
`portion of electromagnetic
`radiation.
`
`Radiowaves
`
`
` Vacuumultraviolet
`
`
`Nearultraviolet Near
`infrared
`
`NaClinfrared
`
`.‘
`lcm ‘i
`.'
`mm
`
`1[.lnn
`to“
`3. 10"‘
`
`J
`lUDnm
`10“
`3- 10”
`
`1000 nrnll pm} ‘llllpm
`10“
`1000
`:3» 10“
`3- 10”
`
`100 urn
`100
`3-. 10‘?
`
`100:] gm
`10
`3x10"
`
`FIGURE 14.2 The electromagnetic spectrum.
`
`truth are shown in Figure 14.2. We will not be concerned with the gamma-ray and
`X-ray regions in this chapter, although these high-energy radiations can be used in
`principle in the same manner as lower-energy radiations. The ultraviolet region
`extends from about 10 to 380 nm, but the most analytically useful region is from
`200 to 380 nm, called the near-ultraviolet region. Below 200 nm, the air absorbs
`appreciably and so the instruments are operated under a vacuum; hence, this
`wavelength region is called the vacuum-ultraviolet region. The visible region is
`actually a very small part of the electromagnetic spectrum, and it is the region of
`wavelengths that can be seen by the human eye, that is, where the light appears
`as a color. The visible region extends from the near-ultraviolet region (380 nm} to
`about T80 nm, The infrared region extends from about 0.78 pm (780 rim) to
`300 nm, but the range from 2.5 to 15 pm is the most frequently used for analysis.
`The 0.8- to 2.5-um range is known as the near-infrared region, the 2.5- to 16-tun
`region as the mid- or NaCl-infrared region, and longer wavelengths as the far-
`infared region. We shall not be concerned with lower~energy radiation (radio or
`microwave) in this chapter. Nuclear magnetic resonance spectroscopy involves
`the interaction of low-energy microwave radiation with the nuclei of atoms.
`
`The Absorption of Radiation
`
`A qualitative picture of the absorption of radiation can be obtained by considering
`the absorption of light in the visible region. We “see“ objects as colored because
`they transmit or reflect only a portion of the light in this region. When polychro-
`matic light (white light), which contains the whole spectrum of wavelengths in the
`visible region, is passed through an object, the object will absorb certain of the
`wavelengths, leaving the unabsorbed wavelengths to be transmitted. These resid-
`ual transmitted wavelengths will be seen as a color. This color is complementary
`to the absorbed colors. In a similar manner, opaque objects will absorb certain
`wavelengths, leaving a residual color to be refiected and “seen."
`Table 14.1 summarizes the approximate colors associated with different wave-
`lengths in the visible spectrum. As an example, a solution of potassium perman-
`ganate absorbs light in the green region of the spectrum with an absorption max-
`imum of 525 nm, and the solution is purple.
`
`Page 6 of 66
`
`The color of an object we
`see is due to the wave-
`
`lengths transmitted or re-
`flected. The other wave-
`
`lengths are absorbed.
`
`Page 6 of 66
`
`

`
`402
`
`CHAPTER 14 I SPECTROMETRY
`
`There are three basic processes by which a molecule can absorb radiation; all
`involve raising the molecule to a higher internal energy level, the increase in
`energy being equal to the energy of the absorbed radiation (hv). The three types
`of internal energy are quantized; that is, they exist at discrete levels. First. the
`molecule rotates about various axes, the energy of rotation being at definite en-
`ergy levels, so the molecule may absorb radiation and be raised to a higher
`rotational energy level, in a rotational transition. Second, the atoms or groups of
`atoms within a molecule vibrate relative to each other, and the energy of this
`vibration occurs at definite quantized levels. The molecule may then absorb a
`discrete amount of energy and be raised to a higher vibrational energy level, in a
`vibrational transition. Third, the electrons of a molecule may be raised to a higher
`electron energy, corresponding to an electronic transition.
`Since each of these internal energy transitions is quantized, they will occur only
`at definite wavelengths corresponding to an energy hv equal to the quantizedjump
`in the internal energy. There are, however, many different possible energy levels
`for each type of transition, and several wavelengths may be absorbed. The tran-
`sitions can be illustrated by an energy level diagram like that in Figure 14.3. The
`relative energy levels of the three transition processes are in the order electronic
`> vibrational > rotational, each being about an order of magnitude different in its
`energy level. Rotational transitions thus can take place at very low energies (long
`wavelengths, that is, the microwave or far-infrared region}, but vibrational tran-
`sitions require higher energies in the near»infrared region, while electronic tran-
`sitions require still higher energies (in the visible and ultraviolet regions).
`Purely rotational transitions can occur in the far-infrared and microwave re-
`gions (ca. I00 pm to 10 cm), where the energy is insufficient to cause vibrational
`
`A molecule absorbs a pho-
`ton by undergoing an en-
`ergy transition exactly
`equal to the energy of the
`photon. The photon must
`have the right energy for
`this quantitized transition.
`
`W
`
`20
`E>-
`E“
`2
`G:
`2l:
`
`E‘
`
`G2c
`
`u
`
`Vibrational
`
`Rotational
`energy levels
`
`energy levels
`J
`
`
`
`A
`
`B
`
`C
`
`a
`a
`l.‘_
`""
`
`E0
`
`FIGURE 14.3 Energy level diagram illustrating energy changes associated with absorption of
`electromagnetic radiation: A, pure rotational changes {far infrared); B, rotationa|—vibrationa|
`changes (near infrared); C, rotational—vibrationa|—electronic transitions (visible and ultraviolet).
`E0 is electronic ground state and E, is first electronic excited state.
`
`Page 7 of 66
`
`Page 7 of 66
`
`

`
`14.1
`
`lNTEFU-‘ACTION OF ELECTROMAGNEHC RADIATION WITH MATTER
`
`403
`
`is usually in its
`or electronic transitions. The molecule, at room temperature,
`lowest electronic energy state, called the ground state (E0). Thus. the pure rota-
`tional transition will occur at the ground-state electronic level (A in Figure l4.3},
`although it is also possible to have an appreciable population of excited states of
`the molecule. When only rotational transitions occur, discrete absorption lines
`will occur in the spectrum, the wavelength of each line corresponding to a par-
`ticular transition. Hence, fundamental information can be obtained about rota-
`tional energy levels of molecules. This region has been of little use analytically,
`however.
`
`As the energy is increased (the wavelength decreased), vibrational transitions
`occur in addition to the rotational transitions, with different combinations of
`vibrational—rotational transitions. Each rotational level of the lowest vibrational
`level can be excited to different rotational levels of the excited vibrational level (B
`
`in Figure 14.3). In addition, there may be several different excited vibrational
`levels, each with a number of rotational levels. This leads to numerous discrete
`transitions. The result is a spectrum of peaks or “envelopes" of unresolved fine
`structure. The wavelengths at which these peaks occur can be related to vibra-
`tional modes within the molecule. These occur in the mid- and far-infrared re-
`
`gions. Some typical infrared spectra are shown in Figure 14.4.
`At still higher energies (visible and ultraviolet wavelengths), different levels of
`electronic transition take place, and rotational and vibrational transitions are su-
`perimposed on these (C in Figure l4.3). This results in an even larger number of
`possible transitions. Although all the transitions occur in quantized steps corre-
`sponding to discrete wavelengths, these individual wavelengths are too numerous
`and too close to resolve into the individual lines or vibrational peaks, and the net
`result is a spectrum of broad bands of absorbed wavelengths. Typical visible and
`ultraviolet spectra are shown in Figure 14.5 and 14.6.
`Not all molecules can absorb in the infrared region. For absorption to occur,
`there must be a change in the dipole moment (polarity) of the molecule. A di-
`atomic molecule must have a permanent dipole (polar covalent bond in which a
`pair of electrons is shared unequally) in order to absorb, but larger molecules do
`not. For example, nitrogen, NEN, cannot exhibit a dipole and will not absorb in
`the infrared region. An unsymmetrical diatomic molecule such as carbon monox-
`ide does have a permanent dipole and hence will absorb. Carbon dioxide,
`O=C=O, does not have a permanent dipole, but by vibration it may exhibit a
`dipole moment. Thus, in the vibration mode O:>C<:O, there is symmetry and no
`dipole moment. But in the mode 0<(=C<:O, there is a dipole moment and the
`molecule can absorb infrared radiation, that is, via an induced dipole. The types
`of absorbing groups and molecules for the infrared and other wavelength regions
`will be discussed below.
`
`Our discussions have been confined to molecules, since nearly all absorbing
`species in solution are molecular in nature. In the case of single atoms (which
`occur in a flame or an electric arc) that do not vibrate or rotate. only electronic
`transitions occur. These occur as sharp lines corresponding to definite transitions
`and will be the subject of discussion in the next chapter.
`The lifetimes of excited states of molecules are rather short, and the molecules
`
`will lose their energy of excitation and drop back down to the ground state.
`However, rather than emitting this energy as a photon of the same wavelength as
`absorbed, most of them will be deactivated by collisional processes in which the
`
`Rotational transitions occur
`
`at very long wavelengths
`(low energy, far infrared}.
`Sharp line spectra are
`recorded.
`
`Vibrational transitions are
`also discrete. But the over-
`
`layed rotational transitions
`result in a "smeared" spec-
`trum of unresolved lines.
`
`Discrete electronic transi-
`tions (visible and ultraviolet
`regions) are superimposed
`on vibrational and rota-
`tional transitions. The
`spectra are even more
`"srneared."
`
`The molecule must un-
`
`dergo a change in dipole
`moment in order to absorb
`infrared radiation.
`
`Single atoms only undergo
`electronic transitions. So
`
`the spectra are sharp lines.
`
`Page 8 of 66
`
`Page 8 of 66
`
`

`
`nl uu. I.I|— I- nu: I.I—
`
`I.rnI.IEiII.l-2-lIII.mfln_§;aw..._.....u...«._aIIII.III...n-u-2v.Eu_.n
`_.§:.iiIn_.
`
`.89.IEIIEIII.
`
`meme:IIBIB
`
`:ul'- k.l.n
`
`.,._fi.,m...§uu
`
`
`
`E.$5....H.Iu_....IEifild...-
`II_=..a_!=H$3:
`
`mi. nu-.
`
`.__:.__.....i....ifl.
`
`CHAPTER 14 I SPECTROMETRY
`
`
`
`
`u.an..§.a..._s...teal..iu.nnis_._._Iil...2_nu_&..EflIu._I!..i_.
`
`a.a.ummus“.-
`
`
`.HiI.-EHIlla»!u..enu“;_he.
`
`.fl.I§fi.unHiullflullSIRE.u
`anInlnslua._.E._n_.-.!_._.._E..__..3....i....!_:_u_a_nn...EI=unnH._a...IniI£lIl=..:.u_25.3nE:-IE......flhnaui
`
`
`
`
`
`....IIIli_._..__.flI__E
`
`ed!“-.aflI!___U....:.un...__.....null.ai3..I-.l:.ua_IiIHI._%........aMfin“H".......“..«......"....u“..___“Eu.Elan._
`
`
`
`.flltflfllllllfllnvflq..___.1
`alfifiilfiliflfir.KinIlflflfi
`_..a.._.ia-.ll.nuu...._I:
`
`Isaac
`
`
`
`EEllzm.3.5:
`
`
`
`ERIE
`I53!
`:5 -=--===i:=:a::::'=
`EEEE
`ufifiiiillii ESE {=3E§IE!EE¥§EEw—iIEEEEE.'
`
`1, l.—DlOxANE
`
`I
`...... -.-.... enun-
`
`GARBON TETRACHLOHIDE
`uumM
`an.
`
`.._._mlIlIIIII
`
`.5HUI!III
`
`
`
`.IIII.I.II_.
`
`lfililpnll-u
`
`9353!!Eu»
`
`flailIi
`kfllillII
`
`u-ch um . a--.. ....
`
`CARBON DISULFIDE
`.... ... ......
`
`.W‘HHlhuflflE;‘RIO«nil&IllI:i
`
`.._n=_...=»I35%»!LI!:5.
`
`.n.
`
`IIo.~fl¥I.I......-
`.is.lIIiiI..nIu-BFIElIl...IIIUH.!1IflIII
`
`cum. 1. r. um (-nun :_...
`
`
`
`IIIIFflI.fi8£E.lrdIIll“-3Eat‘BEHIU
`
`°"——‘—“."=' ma
`NUJOL
`
`.allI
`
`.
`
`.
`
`IKHIKHIIIIIIll
`
`WATER DIST! LLED
`
`IEll'III\—..|JmH
`ana.ni¥...u...=
`
`
`
`II25.!3.5:!.flEflx._
`
`
`
`ate:—§.1\.“._!nIIIO
`
`
`
`Ififlflfl
`
`ucnnl
`
`H3II.-Illufi..-flfllnilia...u::_B:u-_lei-.2
`
`FIGIJIIE 14.4 Typical infrared spectra. (From 26 Frequentiy Used Spectra for the infrared spec-
`troscopist, Standard Spectra-Midget Edition. Copyright © Sadtler Research Laboratories, Inc. Per-
`mission
`’
`for the publication herein of Sadtler Standard Spectra © has been granted, and all rights
`are reserved by Sadtler Research Laboratories, Inc.)
`
`Page 9 of 66
`
`Page 9 of 66
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`
`14.1
`
`INTERACTION OF ELECTROMAGNETIC RADIATION WITH MA'I'|'£ll
`
`405
`
`Absorbance
`
`Ijjj
`400
`500
`600
`?(}D
`Wavelength, nm
`
`FIGURE 14.5 Typical visible absorption spectrum. Tartaric acid reacted with B-naphthol in sul-
`furic acid. 1, Sample; 2, Blank. From G. D. Christian, Talanta, 16 (1969) 255. (Reproduced by
`permission of Pergamon Press, Ltd.)
`
`Absorbanee
`
`.‘.'t
`o_o'._'" .
`220
`200
`210
`
`230
`
`240
`
`250
`
`::::
`:'x'l
`260
`270
`230
`Nanometers
`
`_'.
`':"-'.
`_
`.
`.
`290
`300
`
`':::.
`310
`320
`
`'
`
`330
`
`.
`340
`
`FIGURE 14.6 Typical ultraviolet spectrum. 5-Methoxy—Ei—{p-methoxypheny|)—4—pl1enyI—2(1H)-
`pyridone in methanol. (From Sadt.-‘er Standard Spectra—u.v. Copyright© Sadtler Research Labora-
`tories, Inc., 1963. Permission for the publication herein of Sadtler Standard Spectra® has been
`granted and all rights are reserved by Sadtler Research Laboratories, Inc.)
`
`energy is lost as heat; the heat will be too small to be detected in most cases. This
`is the reason for a solution or a substance being colored. If the light were reemit-
`ted, then it would appear colorless? In some cases, light will be emitted. usually
`at longer wavelengths; we discuss this more under the topic “F|uorescence."
`
`Molecules lose most of the
`energy from absorbing ra-
`diation as heat, via colli-
`sional processes, that is, by
`increasing the kinetic en
`ergy of the collided
`molecules.
`
`“With unidirectional parallel radiation. the solution should still appear colored, however, because the
`emitted light would be emitted as a point source in all directions.
`
`Page 10 of 66
`
`Page 10 of 66
`
`

`
`406
`
`CHAPTER ‘I4 I SPECTROMETRY
`
`14.2 ELECTRONIC SPECTRA AND MOLECULAR STRUCTURE
`
`The electronic transitions that take place in the visible and ultraviolet regions of
`the spectrum are due to the absorption of radiation by specific types of groups,
`bonds, and functional groups within the molecule. The wavelength of absorption
`and the intensity are dependent on the type. The wavelength of absorption is a
`measure of the energy required for the transition. Its intensity is dependent on the
`probability of the transition occurring when the electronic system and the radia-
`tion interact and on the polarity of the excited state.
`
`Kinds of Transitions
`
`Electrons in a molecule can be classified into four different types. (1) Closed-shell
`electrons that are not involved in bonding. These have very high excitation en-
`ergies and do not contribute to absorption in the visible or UV regions. (2) Co-
`valent single-bond electrons (U, or sigma, electrons). These also possess too high
`an excitation energy to contribute to absorption of visible or UV radiation (e.g.,
`single-valence bonds in saturated hydrocarbons, —CH2—CI-I2-——). (3) Paired non-
`bonding outer-shell electrons (n electrons), such as those on N, O, S, and halo-
`gens. These are less tightly held than cr electrons and can be excited by visible or
`UV radiation. (4) Electrons in 'IT (pi) orbitals, for example, in double or triple
`bonds. These are the most readily excited and are responsible for a majority of
`electronic spectra in the visible and UV regions.
`Electrons reside in orbitals. A molecule also possesses normally unoccupied
`orbitals called antibonding orbitals; these correspond to excited-state energy lev-
`els and are either 0* or 11* orbitals. Hence, absorption of radiation results in an
`electronic transition to an antibonding orbital. The most common transitions are
`from 11 or n orbitals to antibonding 11* orbitals, and these are represented by ‘Tr ——>
`-n-* and n —> -n* transitions, indicating a transition to an excited -rr°" state. The
`nonbonding in electron can also be promoted, at very short wavelengths, to an
`antibonding 0* state: :1 —> r:r*. These occur at wavelengths less than 200 nm.
`0
`
`Examples of tr —> 11* and n —> 17* transitions occur in ketones (R—ll——R’).
`
`Representing the electronic transitions by valence bond structures, we can write
`
`11 (double or triple bond)
`and n (outer-shell) elec-
`trons are responsible for
`most UV and visible elec-
`tron transitions.
`
`Excited electrons go into
`antibonding in‘ or cr*J
`orbitals. Most transitions
`above 200 nm are 11 —> 11*
`or n —> 1-r*.
`
`—
`\ +
`\ rs
`=0 -——> C-0
`/
`1: —> 11* transition
`
`/
`
`+
`\ -
`\ r‘\
`=0 ——a~ CEO
`/
`n —> 17* transition
`
`/
`
`Acetone, for example, exhibits a high-intensity 11' —> 11* transition and a low-
`intensity in —r 11* transition in its absorption spectrum. An example of n —) -:r*
`transition occurs in ethers (R—O—R’). Since this occurs below 200 nm, ethers as
`well as thioethers (R——S—R’), disulfides (R—-S—S—R), alkyl amines (R—NH3),
`
`Page 11 of 66
`
`Page 11 of 66
`
`

`
`14.2
`
`ELECTRONIC SPECTRA AND MOLECULAR STRUCTURE
`
`407
`
`and alkyl halides (R—X) are transparent in the visible and UV regions; that is,
`they have no absorption bands in these regions.
`The relative intensity of an absorption band can be represented by its molar
`absorptivity, e, which is really a measure of the probability of the electron tran-
`sition taking place. Molar absorptivity is proportional to the fraction of radiation
`absorbed at a given wavelength and will be described quantitatively below when
`we discuss Beer’s law. For our purposes now, we can simply state that it repre-
`sents the absorbance of radiation passing through a 1 M solution of 1-cm depth,
`where absorbance is —log fraction of radiation transmitted.
`The probability 0f’lI.' —> 11* transitions is greater than for n ——> 1r* transitions, and
`so the intensities of the absorption bands are greater for the former. Molar ab-
`sorptivities at the band maximum for 1r —» 11* transitions are typically 1000 to
`100,000, while for n —> 11* transitions they are less than 1000; e is a direct measure
`of the intensities of the bands.
`
`Absorption by Isolated chromophores
`
`The absorbing groups in a molecule are called chromophores. A molecule con-
`taining a chromophore is called a chromogen. An auxochrome does not itself
`absorb radiation, but, if present in a molecule, it can enhance the absorption by a
`chromophore or shift the wavelength of absorption when attached to the chro-
`mophore. Examples are hydroxyl groups, amino groups, and halogens. These
`possess unshared (n) electrons that can interact with the 11 electrons in the chro-
`mophore (n—'n' conjugation).
`Spectral changes can be classed as follows: (I) bathocliromic shift— absorption
`maximum shifted to longer wavelength, (2) hypsochromic shil‘t—absorption max-
`imum shifted to shorter wavelength, (3) hyperchromism—— an increase in molar
`absorptivity, and (4) hypocl1romism—a decrease in molar absorptivity.
`In principle, the spectrum due to a chromophore is not markedly affected by
`minor structural changes elsewhere in the molecule. For example, acetone,
`
`and 2-butanone,
`
`0
`
`ct-t,—d—cH,
`
`0
`
`cH,iL—cH,cH_,
`
`give spectra similar in shape and intensity. If the alteration is major or is very
`close to the chromophore, then changes can be expected.
`Similarly,
`the spectral effects of two isolated chromophores in a mole-
`cule (separated by at least two single bonds} are, in principle, independent and
`are additive. Hence,
`in the molecule CH3CH3CNS, an absorption maximum
`due to the CNS group occurs at 245 nm with an e of 800.
`In the molecule
`SNCCHZCI-l2CH2CNS, an absorption maximum occurs at 24? nm, with approx-
`imately double the intensity (5 = 2000}. Interaction between chromophores may
`perturb the electronic energy levels and alter the spectrum.
`
`Page 12 of 66
`
`Page 12 of 66
`
`

`
`-.33 -c--..-._‘._._-.. -.._.___..-....-.__. __ __T _
`
`408
`
`CHAPTER 14 I SPECTROMETRY
`
`TABLE 14.2
`
`Electronic Absorption Bands for Representative Chromophores‘
`
`System
`
`—NH3
`—C=C—
`
`:C=0
`
`—CHO
`
`—-N02
`—ONO
`
`—N=N—
`
`Chromophore
`
`Amine
`Ethylene
`
`K‘°"°"°
`
`Aldehyde
`
`Nitro
`Nitrite
`
`A20
`Benzene
`
`Naphthalene
`
`Anth rac ene
`
`hm,
`
`195
`190
`
`30-285
`
`210
`280 — 300
`
`2 l 0
`220-230
`300- 400
`
`285-400
`184
`202
`255
`
`220
`2'?5
`312
`
`252
`375
`
`em,
`
`2,800
`8,000
`
`Iii-(:00
`
`Strong
`l l — 1 8
`
`Strong
`L000-2,000
`10
`
`3—25
`46,700
`6.900
`170
`
`ll2,000
`5 ,600
`l?5
`
`l99,000
`7,900
`
`“From M. M. Willard. L. L. Merritt, and J. A. Dean. Instrumental Methods ofAna!y.rr's, 4th ed. Copyright" I948.
`1951. 1958. I965. by Litton Educational Publishing, Inc.. by pennission of Van Noslrand Reinhold Company.
`
`Table 14.2 lists some common chromophores and their approximate wave-
`lengths of maximum absorption.
`It should be noted that exact wavelengths of an absorption band and the prob-
`ability of absorption (intensity) cannot be calculated, and the analyst always runs
`standards under carefully specified conditions (temperature, solvent, concentra-
`tion, instrument type, etc.). Modern instruments may have databases of standard
`spectra, and standard catalogues of spectra are available for reference.
`
`Absorption by Conjugated Chrornophores
`
`Where multiple (e.g., double. triple} bonds are separated by just one single bond
`each, they are said to be conjugated. The ‘'IT orbitals overlap, which decreases the
`energy gap between adjacent orbitals. The result is a bathochromic shift in the
`absorption spectrum and generally an increase in the intensity. The greater the
`degree of conjugation (i.e., several alternating double, or triple, and single bonds),
`the greater the shift. Conjugation of multiple bonds with nonbonding electrons
`\
`(n—'rr conjugation} also results in spectral changes, for example, C=CH—-N02.
`/
`
`Aromatic compounds are
`good absorbers of UV
`radiation.
`
`Absorption by Aromatic Compounds
`Aromatic systems (containing phenyl or benzene groups) exhibit conjugation. The
`spectra are somewhat different, however, than in other conjugated systems, being
`
`Page 13 of 66
`
`Page 13 of 66
`
`

`
`14.2
`
`ELECTRONIC SPECTRA AND MOLECULAR STRUCTURE
`
`409
`
`more complex. Benzene, © , absorbs strongly at 200 nm (em, = 6900) with a
`weaker band at 230—2'r'0 nm (em, = ITO); see Figure 14.7. The weaker band
`exhibits considerable fine structure, each peak being due to the influence of vi-
`brational sublevels on the electronic transitions.
`As substituted groups are added to the benzene ring, a smoothing of the line
`structure generally results, with a bathochromic shift and an increase in intensity.
`Hydroxy (—OH), methoxy (—OCH3), amino {—NHg). nitro (—-N02), and alde-
`hydic (—CHO) groups, for example, increase the absorption about tenfold; this
`large effect is due to n—1'r conjugation. Halogens and methyl (——CI-13) groups act as
`auxochromes.
`Polynuclear aromatic compounds (fused benzene rings), for example, naphtha-
`
`lene,
`
`, have increased conjugation and so absorb at longer wave.
`
`lengths. Naphthacene (four rings) has an absorption maximum at 470 nm (visible)
`and is yellow, and pentacene (five rings} has an absorption maximum at 5'r'5 nm
`and is blue (see Table 14.1).
`
`In polyphenyl comp0unds,© (®)n©, para-linked molecules (1,6
`positions, as shown) are capable of resonance interactions (conjugation) over the
`entire system, and increased numbers of para-linked rings result in bathochromic
`shifts (e.g., from 250 nm to 320 nm in going from n = 0 to n = 4). In meta—linkecl
`molecules (I ,3 positions), however, such conjugation is not possible and no ap-
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`logI:
`
`220
`
`240
`
`260
`
`280
`
`Wavelength,
`
`|"ll"|"|
`
`0 2
`
`00
`
`FIGURE 14.? Ultraviolet spectrum of benzene.
`
`Page 14 of 66
`
`Page 14 of 66
`
`

`
`410
`
`CHAPTER 14 I SPECTROMETRY
`
`preciable shift occurs up to n = 16. The intensity of absorption increases, how-
`ever, due to the additive effects of the identical chromophores.
`
`Many heterocyclic aromatic compounds, for example, pyridine, (/
`
`\N, absorb
`
`in the UV region, and added substituents will cause spectral changes as for phenyl
`compounds.
`Indicator dyes used for acid—base titrations and redox titrations (Chapters 7’
`and 12) are extensively conjugated systems and therefore absorb in the visible
`region. Loss or addition of a proton or an electron will markedly change the
`electron distribution and hence the color.
`If a compound (organic or inorganic) does not absorb in the ultraviolet or
`visible region,
`it may be possible to prepare a derivative of it that does. For
`example, proteins will form a colored complex with copper(1l) (biuret reagent).
`Metals form highly colored chelates with many of the organic precipitating re-
`agents listed in Table 5.2 in Chapter 5, as well as with others. These may be
`dissolved or extracted (Chapter 16) in an organic solvent such as ethylene chloride
`and the color of the solution measured spectrometrically. The mechanism of
`absorption of radiation by inorganic compounds is described below.
`Spectrometric measurements in the visible region or the ultraviolet region (par-
`ticularly the former) are widely employed in clinical chemistry, frequently by
`forming a derivative or reaction product that is colored and can be related to the
`test substance. For example, creatinine in blood is reacted with picrate ion in
`alkali