Circular and Linear Dichroism

CIRCULAR DICHROISM AND LINEAR DICHROISM 1
Circular Dichroism References 27
and Linear
Dichroism Circular dichroism (CD) is the difference in absorption,
A, of left and right circularly polarized light, Equation (1):
Alison Rodger CD D A D Al Ar (1)

University of Warwick, Coventry, UK
For randomly oriented systems such as solutions of
molecules, only chiral molecules will show any CD
intensity corresponding to their absorption bands. Chiral
1 Introduction to Circular Dichroism 1 molecules are those molecules that cannot be superposed
1.1 Circularly Polarized Light 2 on their mirror images.(1) Chiral is derived from the Greek
1.2 Normal Absorption Spectroscopy 3 word ceir meaning hand, hence the alternate term for
1.3 Circular Dichroism Spectroscopy 3 chirality, handedness. Two molecules that are mirror
1.4 Units of Circular Dichroism images of each other are often referred to as enantiomers
Spectroscopy 4 and equimolar mixtures of two enatiomers form a racemic
1.5 Optical Activity (Optical Rotation) 4 mixture which has no net CD intensity in solution.
1.6 Enantiomeric Purity and Enan- Linear dichroism (LD) is the difference in absorption
tiomeric Excess 5 of light linearly polarized parallel and perpendicular to an
2 Structure Analysis from Circular orientation axis, Equation (68):
Dichroism 5 LD D Ajj A? (68)
2.1 Introduction 5
2.2 Qualitative Description of Circular
Dichroism and Its Correlation with
Structure for Some Systems 6 1 INTRODUCTION TO CIRCULAR
2.3 Interaction of Radiation with Matter DICHROISM
and the Rosenfeld Equation for
Circular Dichroism 9 CD is the difference in absorption, A, of left and right
2.4 Circular Dichroism of Magnetic circularly polarized light, Equation (1):
Dipole Allowed Transitions:
Why the Octant Rule Works for CD D A D Al Ar .1/
Carbonyls 12
For randomly oriented systems such as solutions of
2.5 Circular Dichroism of Electric
molecules, only chiral molecules will show any CD
Dipole Allowed Transitions 15
intensity corresponding to their absorption bands. Chiral
3 Linear Dichroism 17 molecules are those molecules that cannot be superposed
3.1 Introduction 17 on their mirror images..1/ Chiral is derived from the
3.2 Orientation Parameters 18 Greek word ceir meaning hand, hence the alternate
3.3 Molecular Alignment Techniques 21 term for chirality, handedness. Two molecules that are
3.4 Examples of Linear Dichroism Data 22 mirror images of each other are often referred to as
4 Instrumentation and the Dichroism enantiomers and equimolar mixtures of two enatiomers
Experiment 24 form a racemic mixture which has no net CD intensity in
4.1 Circular Dichrographs solution (Figure 1).
(Spectropolarimeters) 24 In order to understand when chirality is important it
4.2 Design and Implementation of an is helpful to consider the everyday problem of a pair of
Linear Dichroism Experiment 26 shoes, a left foot, and a shoe box. The shoes and the
Acknowledgments 26 foot are chiral and the left foot only fits comfortably
into the left shoe, whereas the achiral shoe box cannot
Abbreviations and Acronyms 26
distinguish between the two shoes. Chirality is important
Related Articles 26 only where two chiral entities (usually molecules) interact.
Further Reading 27 CD depends only on the asymmetry of the molecular
General Circular Dichroism References 27 system and so is uniquely sensitive to this aspect of it.
General Linear Dichroism References 27 As many of the questions we ask about chemical and
Encyclopedia of Analytical Chemistry, Online 2006 John Wiley & Sons, Ltd.
This article is 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a5402
2 ELECTRONIC ABSORPTION AND LUMINESCENCE
Left Right Unoriented

polarized polarized chiral CD spectrum
light light molecule +
CD = +
Wavelength

Achiral molecule Chiral molecule Other enantiomer
Absorbance Absorbance Absorbance

Intensity
Intensity
Intensity
CD
CD
CD
Wavelength Wavelength Wavelength
Figure 1 Schematic illustration of CD.
biological systems relate to the shapes of molecules and B

how this affects their properties, CD is a very useful tool
for studying chiral molecules.
CD is now a routine tool in many laboratories. The k
most common applications are determining whether a
chiral molecule has been synthesized or resolved into
pure enantiomers and probing the structure of biological
macromolecules, in particular determining the a-helical

content of proteins. Wavelength
E
(a)
1.1 Circularly Polarized Light
Although in principle CD can be measured for any kind
of electromagnetic radiation absorbed by a sample, in
k
practice experiments are usually limited to the visible
or the easily accessible ultraviolet (UV) wavelength
range (170 800 nm). We often loosely refer to this
electromagnetic radiation as light. Vibrational circular
dichroism (VCD) using infrared radiation is also used,
but is technically significantly more challenging..2/ We (b)
shall concentrate on the ultraviolet/visible (UV/VIS) CD
Figure 2 Schematic illustration of electromagnetic radiation
as this instrumentation is more readily available and illustrating the electric field, E, the magnetic field, B, and the
significantly more widely used than VCD. propagation direction k. (a) Linearly polarized and (b) right
Electromagnetic radiation, as its name implies, has circularly polarized electromagnetic radiation. Arrows denote
an electric and a magnetic field that oscillate at right direction of E.
angles to one another and to the propagation direction
(Figure 2)..3 7/ Light may be described by a transverse Equation (2).3 7/
wave whose polarization is defined by the direction
hw
of its electric field, E. Electromagnetic radiation also E D hn D D hw .2/
has particle character: if we do an experiment that 2p
would probe wave-like behavior, it is apparent that where h is Plancks constant and w is known as the
light has it, and if we look for particle character we angular frequency. It follows from the wave nature of
find that too. Thus we refer to the wavelength, l, and light that a light beam can be linearly polarized with
frequency, n, of radiation, but acknowledge that if a all photons having their electric field oscillating in the
molecule absorbs energy it absorbs discrete units called same plane (Figure 2). In a circularly polarized light
photons whose energy are given by Plancks relationship, beam the electric field vector retains constant magnitude
DOI: 10.1002/9780470027318.a5402
in time but traces out a helix about the propagation of the intensity of incident, I0 , and transmitted, I, light,
direction. Following the optics convention.6,8/ we take Equation (6):.3 5/
the end of the electric field vector of right circularly
I0
polarized light to form a right-handed helix in space A D log10 .6/
at any instant of time. At each point in space or I
time, the magnetic field, B, is perpendicular to the The Beer Lambert law for the absorption, A, of light by
electric field such that k, the propagation direction, a sample of concentration C is given by Equation (7).3 5/
E, and B form a right-handed system as illustrated in
Figure 2. A D eCl .7/
If we take X to be the direction of propagation of the
light, then unit vectors describing the polarization (by where l is the length of the sample through which the
convention the direction of the electric field component light passes and e is known as the extinction coefficient;
of the light) of two perpendicularly polarized light beams if l is measured in cm and C in M (Dmol dm 3 ), then e
may be written, Equation (3) has units of mol 1 dm3 cm 1 . The Beer Lambert law is
valid as long as the spectrometer can measure I (i.e. the
eO 1 D eO Y D .0, 1, 0/ eO 2 D eO Z D .0, 0, 1/ .3/ concentration is not so large that essentially all photons
are absorbed) and there are no concentration-dependent
If two equal magnitude beams are combined so that one intermolecular interactions.
lags p/2 or a quarter of a wavelength behind the other, In practice, in a collection of molecules, the photons
then the electric field vector will trace out a circle giving absorbed by different molecules will be of slightly
circularly polarized light. The electric field polarization different energies so what we measure is a curve like
vector for right circularly polarized light (as defined the absorbance curves of Figure 1, where the signal
above) may be written:.6 8/ that is plotted is a measure of the probability that a
transition will occur at that energy. Such a plot of the
1 2piX
eO r D p .0, 1, i/ exp iwt .4/ absorbance of light verses l or n is known as an absorption
2 l spectrum.
where i is the square root of 1. The oscillation of the
electric field vector in time is described by the factor 1.3 Circular Dichroism Spectroscopy
exp. iwt/ where w is the angular frequency (2pn) and t
When a molecule absorbs UV and visible light its
is time; the magnitude of the electric field vector remains
electronic distribution is altered. Now consider chiral
constant in time, but its direction rotates about X with
molecules. Because a chiral molecule has no reflec-
frequency w. By taking the real part of Equation (4)
tion plane, any rearrangement of its electrons will
we see that for an observer sitting at a fixed point,
not have one either, so the electrons move in some
say X D 0, the electric field vector of right circularly
kind of helix. As the electric field vectors also trace
polarized light rotates in time about X in a clockwise
out helices (Figure 1) in circularly polarized light, the
manner. At a fixed time, say t D 0, it forms a right-handed
interaction between a chiral molecule and left- and
helix in space. In what follows we use the polarization
right-handed photons will be different. This is the idea
vectors:
behind the definition of CD given in Equation (1). The
1 1 CD version of the Beer Lambert law (Equation 7) is
eO l D p .0, 1, i/ eO r D p .0, 1, i/ .5/
2 2 Equation (8):
for, respectively, left and right circularly polarized light, A D .el er /Cl D .e/Cl .8/
Equation (5).
Thus, a CD spectrum looks similar to an absorbance
spectrum in that it occurs at the same wavelengths
1.2 Normal Absorption Spectroscopy
(Figure 1), but it can be either positive or negative in
Most spectroscopic phenomena arise from the interaction sign and if more than one transition lie close together,
of a molecule with one photon at a time. The molecule cancellation of intensity may occur (Figure 3). The
either absorbs, emits, or scatters the photon. The simplest challenging task is then to relate the helical motions
form of spectroscopy is absorption, where we measure of the electrons to the arrangement of the atoms and
how much light of a given frequency is absorbed by a bonds in space.
collection of molecules. If a molecule absorbs a photon of As noted above, a CD spectrum is only expected for
frequency n, it increases its energy according to Plancks solutions of chiral molecules, though achiral molecules
relationship (Equation 2). Absorbance is defined in terms oriented skew to a light beam in an oriented sample, such
DOI: 10.1002/9780470027318.a5402
between these two is given in Equation (9).6,8/

2
Charge transfer A 100 Eg CD D A.absorbance units/ D Al Ar

1 region 4pq.degrees/ q.millidegrees/
D D .9/
Absorbance
T2g 180 ln 10 32 982

0
A2
The CD analog of the Beer Lambert law (Equation 8)
T1g
then allows conversion to molar absorbance or molar
1
dd region
ellipticity. In this context it is important to be clear
what concentration is being measured. For example
2 A 10 DNA concentrations are often given in terms of bases
or phosphates, but may be in terms of base pairs, whereas
3 protein concentrations are commonly in terms of amino
acid residues and occasionally whole molecules.
(a) 200 300 400 500 600
1.5 Optical Activity (Optical Rotation)

60 E
Optical rotation (OR) is the difference in refractive
40 indices of left and right circularly polarized light upon
Charge transfer (and dd) region passing through the medium, Equation (10):.6/
many transitions overlap
20
pd
0.1 OR D .nl nr / .10/

0 l
the units are radians if l (wavelength) and d (pathlength)
20 have the same units (usually dm are used). (nl nr ) is
called the circular birefringence.
40 A2 OR is usually measured by determining the rotation of
60 In-ligand region
linearly polarized light upon passing through the solution.
If the linearly polarized light is rotated clockwise when
200 300 400 500 600 viewed into the light source the OR is called a positive or
(b) Wavelength/nm right (dextro) OR. OR as a function of l is called optical
rotatory dispersion (ORD). ORD gives an S-shaped curve
Figure 3 CD spectra of 0.1 M -[Co(ethylenediamine)3 ]3C in a centered at the CD maximum (the so-called point of
0.1 cm path length cell and [Ru(1,10-phenanthroline)3 ]2C . Note anomalous ORD since if a transition has zero band width
cancellation of close lying bands. the ORD would go to plus and minus infinity either side of
lmax ). The ORD is also nonzero away from an absorption
as a crystal, will give a signal. The other requirement band, hence aD values (the ORD at the sodium D line,
is that a molecule gives rise to an absorbance signal 589 nm) may be used to characterize the enantiomeric
in the region of interest. If a molecule has no UV/VIS excess of a solution (see below). The long-wavelength side
absorbance spectroscopy then no CD signal will be found. of the ORD curve shows a positive ORD contribution for
Sometimes, as with chiral HPLC (high-performance a positive CD band, but a negative ORD for a negative CD
liquid chromatography) (see below) it is desirable to cause band. ORD is sometimes referred to as optical activity.
a nonabsorbing molecule to have a CD signal. This may Since ORD and CD may be written respectively in
be achieved by derivatizing the analyte molecule with a terms of the dispersive and absorptive parts of the
UV/VIS active chromophore, though care must be taken optical activity tensors, with all other factors the same,
with the chemistry to ensure inversion of configuration or Kramers Kronig.6/ relations may be used to convert one
racemization does not occur. to the other if the complete ORD spectrum or complete
CD spectrum is available. The formalism for this process is
fairly straightforward; however, in practice the problem is
1.4 Units of Circular Dichroism Spectroscopy
that the complete spectrum is not available. It is often
Most CD spectropolarimeters, although they measure possible to perform the conversion on a more limited
differential absorbance, for historical reasons produce a wavelength range with an element of error. Except for sin-
CD spectrum in units of ellipticity, q, in millidegrees, gle wavelength measurements to quantify enantiomeric
versus l, rather than A versus l. The conversion excess, ORD is seldom used now as the more direct
DOI: 10.1002/9780470027318.a5402
relationship between absorbance spectroscopy and CD asymmetry in the sample. It is not much of an over-
facilitates the interpretation of data in structural terms. generalization to say that the only CD studies that have
been interpreted beyond this stage are those where the
1.6 Enantiomeric Purity and Enantiomeric Excess transition being studied is essentially located in an achi-
ral chromophore (or electronic subunit) of the system.
If a CD signal is observed for a solution, then there is Explicit calculations can, of course, be performed for
a net excess of a chiral analyte; however, unless e for intrinsically chiral chromophores (such as CHFClBr),
the compound is known, the enantiomeric purity of the however, this does not usually provide any informa-
sample cannot be determined. Alternative methods that tion except that the input geometry for that molecule
measure the relative amounts of the two enantiomers is correct. By way of contrast, carbonyl compounds con-
directly are chiral HPLC.9/ and NMR (nuclear magnetic taining the achiral chromophore (circled in Figure 4)
resonance) with chiral shift reagents..10/ Simply choosing have been extensively studied using CD spectroscopy
a chiral column or mobile phase additive or chiral and various structure spectra correlation rules have been
shift reagent and then performing an experiment which proposed and applied. It should be noted that any struc-
gives only one peak does not prove that the solution is ture spectrum correlation rule can only be applied with
enantiomerically pure. It must be possible to show that full confidence once its mechanistic origin is understood.
if the mixture is racemic (or any percentage mixture of Given that the chromophores we are interested in are
the two enantiomers) that the experimental method does achiral, the electron redistribution that occurs during
indeed discriminate between the two species. a transition occurs in a plane. This means that either
Enantiomeric purity is often given in terms of the per- the magnetic dipole transition moment, m, (MDTM)
centage of one enantiomer present. Alternatively, we use (by which the transition interacts with the magnetic
enantiomeric excess. Enantiomeric excess relates more field of the radiation) or the electric dipole transition
directly to the CD signal because if there is no enan- moment, , (EDTM) (by which the transition interacts
tiomeric excess then there is no CD signal; conversely the with the electric field of the radiation) vanishes (or
CD observed follows Equation (8) if the solution is enan- they are perpendicular to one another). The m D 0,
tiomerically pure. Enantiomeric excess is usually defined 6D 0 transitions are called electric dipole allowed
as the apparent analyte concentration determined from (EDA), magnetic dipole forbidden (MDF). The m 6D 0,
Equation (8) divided by the total analyte concentration, D 0 transitions are called electric dipole forbidden
often converted to a percentage, Equation (11) (EDF), magnetic dipole allowed (MDA). The theoretical
significance of this division is due to the way CD intensity
Concentration of enantiomer 1
is induced into the two different types of transitions.
EE% D 100 EDA transitions require the induction of a magnetic
component, whereas MDA transitions require an induced
C Concentration of enantiomer 2 electric component. The dependence of the different
.11/ kinds of induced moments on the geometry of the
system is very different, so when we wish to extract
geometric information from CD we must be aware of
2 STRUCTURE ANALYSIS FROM which situation is in effect. The CD spectrum induced
CIRCULAR DICHROISM into EDA transitions is also different if it arises from the
coupling of identical chromophores rather than from the
2.1 Introduction coupling of nonidentical chromophores.
Experimentally the difference between EDA/MDF
The most obvious result to be deduced from the appear-
and EDF/MDA transitions is that the latter have low
ance of a CD spectrum is whether or not there is net
absorbance intensity (what they have is usually gained
from vibronic coupling rather than from the MDTM) so
H3CH2C OC(O)C3H7 the ratio of their CD signal to their absorbance is larger
C CH O than that for EDA/MDF transitions. The dissymmetry
H H
factor, g, Equation (12).6/
H H O
O 4R
gD .12/
cD
Figure 4 Progesterone with the carbonyl chromophores cir-
cled. Note progesterone has one carbonyl conjugated with a where R is the rotational CD strength (Equation 30), c is
double bond so it is not strictly electronically independent of the speed of light, and D is the dipole strength (square
the rest of the molecule. of the EDTM) of the transition summarizes this. The
DOI: 10.1002/9780470027318.a5402
main historical consequence of this is that most of the molecule scaled with their polarizability. This was the
early CD experiments were performed on EDF/MDA first use of what has come to be called the independent
transitions such as the n ! p carbonyl transitions and systems/perturbation (ISP) approach..13 15/
the T1g tris-chelate transitions. To apply the octant rule to hydrocarbon systems,
one simply determines the cartesian coordinates of
each carbon atom (assuming the hydrogens make little
2.2 Qualitative Description of Circular Dichroism and
contribution), evaluates Equation (13) with n D 4 and
Its Correlation with Structure for Some Systems
performs a sum over all carbons in the molecule (or
2.2.1 Carbonyl Circular Dichroism in practice over the net perturbers since atoms located
symmetrically about a plane of the octant system will
Carbonyl chromophores, i.e. carbonyl functional groups give equal magnitude and opposite sign contributions).
that are not conjugated with any other double bond of An example is given in Figure 6. A simple Gaussian type
the molecule, occur in many organic molecules of both curve with sign and relative magnitude determined by
natural and synthetic origin including steroids. That a this sum should then correlate with the observed CD. For
simple reliable empirical rule relating carbonyl molecular nonhydrocarbon systems, the sum must be weighted by
geometry to observed CD spectrum was determined and the polarizabilities of the atoms or chromophores (see
to some extent rationalized in the 1960s was therefore below) of the molecule..16/
extremely convenient. The lowest lying transition of a Although the octant rule has proved to be highly
carbonyl is the n ! p transition which involves rotation successful, there are sufficiently numerous exceptions to
of electron density from the nonbonding oxygen lone pair provide a warning against the blind use of empirical
into the p orbital of the CDO bond (Figures 4 and 5). The rules. Empirical modifications (see Rodger and Rodger
transition, which occurs somewhere between 350 nm and and references therein),.15/ while helpful for a given
240 nm, is weak with an extinction coefficient typically series of compounds, do not provide a global approach.
of 20 50 mol 1 dm3 cm 1 . When it is conjugated with a Consideration of all terms in the ISP expansion (see
double bond as in progesterone (Figure 4) the absorbance below) is really required. The conclusion from this
intensity is increased and any rules to interpret the CD
spectra must be applied with care. The octant rule of
Moffitt et al..11/ is the unifying feature for much of the + x
carbonyl CD work. In its original form, the rule stated
that the CD of the n ! p transition depends on the
positions of other parts of the molecule relative to the
carbonyl group according to Equation (13)
xyz
.13/
rn y
where (x,y,z) is the unit vector along the lines of the

position vector in the right-handed coordinate system
of Figure 5 and r is the distance from the centre of
the carbonyl bond to the chromophore. Hohn and
Weigang.12/ used perturbation theory to give a theoretical
justification of the octant rule and found that n D 4, and
that the relative importance of different parts of the +
z z Above z
y-z plane
y x x>0
+ O Figure 6 Projection onto the x y octant plane of lev-
O O y
Rotate 90o onorgestrel butanoate viewed along the carbonyl bond. All
About z C C + atoms (except the carbonyl oxygen) have coordinates with
C
z < 0. Octant signs are as indicated. The size of the carbon
(a) n * (b) atoms indicates their proximity to the carbonyl group (large
ones are close). The dominant octant rule net perturber lies in
Figure 5 (a) Schematic illustration of the carbonyl n ! p the x < 0, y > 0, z < 0 octant (although more distant atoms lie in
transition illustrating the (x,y,z) coordinate system used for the the x < 0, y < 0, z < 0 octant) Oxygen atoms are shaded. Thus,
octant rule. (b) Octant rule CD signs. Invert the signs illustrated a positive n ! p CD signal is expected from this molecule. In
for the x < 0 region of space. fact e322 nm D 1.5 mol 1 dm3 cm 1 ..45/
DOI: 10.1002/9780470027318.a5402
analysis.15/ is that the octant rule is not valid for polar Charge transfer CD has also been measured mainly for
and charged groups. It also requires the net perturbing tris-chelate complexes with planar aromatic ligands. For
part of the system not to lie on or near one of the such molecules the charge transfer bands often obscure
octant planes. Further, no solvent cavities must be created the d d transitions. The lowest energy EDA charge
by substituents on the molecules and planar zig-zags of transfer transitions for tris-chelate complexes gives a
bonds with the carbonyl bond must be absent for the CD bisignate signal (Eu /A2 ) with the Eu component having
magnitude to be given by the octant rule..18/ the opposite sign from the lowest energy long-axis in-
ligand Eu CD (see above). The sign difference can be used
to assign the polarization of the charge transfer bands.20/
2.2.2 Transition Metal Complexes
whose energy ordering varies from molecule to molecule,
The spectroscopy of transition metal complexes can be in contrast to the situation for the in-ligand transitions.
subdivided into three categories: in-ligand transitions, Interpreting the CD for the MDA d d transitions (CD
which resemble those of the isolated ligands; charge- for MDF/EDF is usually very small) is complicated by
transfer transitions between the ligand and the metal; the fact that the dominant mechanism is determined by
and d d transitions of the chromophore defined by the the symmetry of the d d chromophore. One reliable
central metal ion and its directly ligating atoms. empirical rule has been established over the years for
The in-ligand transitions need only be considered for tris-chelate complexes where transitions of E and A2
aromatic ligands since they are the only ones for which polarization occur as close lying couplets: the E band CD
the CD can be easily measured. This effectively restricts signal is larger than the A2 band signal..19/ The underlying
consideration to tris-chelate (and also cis bis-chelate) reason for this is that if we take the d d chromophore
complexes with planar aromatic ligands. The ligands in to have D3d symmetry (Figure 8) then the Eg band gains
such molecules are usually of C2v symmetry (Figure 7). its CD intensity from a r 4 quadrupolar mechanism (see
The in-ligand CD for these systems can usually be below), whereas the A2 band depends on an r 6 hexade-
accurately predicted by the Kirkwood Kuhn coupled capolar mechanism as discussed below. In reality the d d
oscillator mechanism (see below). As the complexes are chromophore of tris-chelate complexes is not achiral but
based on an octahedral template but themselves have twisted. However, for solution phase work the approxi-
D3 symmetry, the transitions all occur as couplets of A2 mation is satisfactory as the close energy of the A2 and
(z, the three-fold axis, Figure 7) polarized and Eu (x,y) E components of any band means that any intrinsic CD
polarized transitions. The coupling of long axis ligand cancels as it would for the octahedral parent geometry. It
transition moments gives a bisignate CD signal the lower is only for crystal CD that the problem is noted..21/
energy component being the out-of-phase Eu component
that has a negative sign for the enantiomer.19/ and 2.2.3 Macromolecules
conversely for the enantiomer (see below for the the-
The CD of macromolecules.22/ differs from that of small
oretical basis for this conclusion). The A2 component (in
molecules in that the spectroscopy of the component
the absence of coupling with other transitions) is equal in
magnitude and opposite in sign from the Eu component
as illustrated in Figure 3. N
c
N N
C
RC C
B
x Co
x RAC B
Ru x Ru
RB
b N N
RA
A a
A
N
y
N N
y
Figure 7 [Ru(1,10-phenanthroline)3 ]2C , a tris-chelate transi-
tion metal complex of D3 symmetry with C2v symmetry planar
aromatic ligands. The three-fold (z) axis is out of the plane of Figure 8 D3d d d chromophore of [Co(ethylenediamine)3 ]3C ,
the page in the right-hand side of the figure. ethylenediamine D NH2 CH2 CH2 NH2 .
DOI: 10.1002/9780470027318.a5402
parts, such as the amino acids, at least in principle, is and between its N H and the (n 4)th C O; there is a
usually understood and the form of the CD spectra result 1.5 A translation and 100 turn between two consecutive
largely from the arrangement of the units in space. The peptide units, giving 3.6 amino acid residues per turn), the
CD of macromolecules is most commonly used in one chromophores giving rise to the CD are in well-defined
of two ways: (i) to probe changes in the conformation of positions relative to one another so have a well-defined
the macromolecule itself, and (ii) to probe its interaction CD spectrum. The net CD is then the sum of that due to
with small molecules bound to it. the independent structural units. A wide range of fitting
programs for protein CD data are available..24 26/ Some
2.2.4 Proteins work in terms of the spectra of a set of identified structural
At present the main use of CD in the study of proteins is as
an empirical guage of protein structure and conformation.
Proteins are long chains of amino acids, their primary
structure being as illustrated in Figure 9..23/ If the side
30
chain (Ri in Figure 9) is not H, then the ith tetrahedral
carbon is a chiral center and we should expect an
induced CD signal in transitions of the neighboring amide
groups and side chains. If, however, there is free rotation
20
about the bonds of the main chain, the observed amide
n ! p (occurring at 220 nm), p ! p (occurring at
207 190 nm), and n ! s (occurring as a shoulder at
175 nm) CD signals will be small since they will be an
/mol1dm3 cm1
average of different geometry factors (see carbonyl CD 10

above and coupled oscillator CD below). Thus a truly
random or denatured protein will have only a small CD
signal at energies accessible to most CD machines. The
most stable conformation of a protein under physiological
conditions, however, is not random but composed of well-
defined structural units that give macrochiral units with 200 220 240
significant CD intensities.
The basis of CD structural analysis of proteins is
that the CD spectrum from 250 nm downwards is due
largely to transitions of the main backbone chain, so it is 10
independent of the nature of the side chains (this is not
always true if there are a large number of well-ordered
aromatic side chains or prosthetic groups such as a heme
present). In any well-defined structural motif (such as the
a-helix where the nth peptide unit forms hydrogen bonds 20
between its C O and the N H of the (n C 4)th peptide Wavelength/nm
Figure 10 Typical standard protein motif CD spectra,.26,43/
Ri +1 a-helix ( , average of poly(g-methyl-L-glutamate) in F6 i-PrOH
O and poly(L-alanine) as a film), antiparallel b-sheet (- - - -,
tert-butyloxycarbonyl (BOC) (L-alanine)7 OMe as a film),
parallel b-sheet ( , BOC (L-valine)7 OMe as a film),
Free Free poly(L-proline) II-type helix ( , systemin in 10 mM phosphate
C
rotation rotation buffer at 5 C, pH D 7.43/ ), and random coil ( , collagen at
C 45 C in 0.01 M phosphate buffer, pH D 3.5). The CD charac-
teristics of a-helices are a negative signal with separate maxima
N of similar magnitude at 222 nm (the n ! p transition) and
208 nm (part of the p ! p transition that is polarized parallel
Peptide to the helix axis); and a positive signal at 195 nm (part of the
bond
Ri p ! p transition that is polarized perpendicular to the helix
axis). The CD characteristics of b-sheets are a negative band at
about 220 nm and a positive band of near 200 nm and the CD
Figure 9 Protein primary structure. Ri denotes the side chain characteristics of the poly(L-proline) II-type helix are a positive
of the ith amino acid. band at 225 nm and a negative band at about 200 nm.
DOI: 10.1002/9780470027318.a5402
motifs, others use a basis set of real protein spectra where 5

the percentage of different structural units is known. The
approach of Manalavan and Johnson,.25/ where a large
basis set of such proteins is used and a subset of these 0
randomly chosen until a good fit is obtained probably
gives the best estimate of the full range of structural motifs

present in proteins. Almost all approaches will give a good
a-helix content estimate as its profile is the most distinct 5
and also largest in magnitude as illustrated in Figure 10.
2.2.5 DNA 10
CD experiments performed on DNAs generally fall into

one of two categories: those used to probe the DNA and (a) 220 240 260 280 300
those used to probe a drug (or other molecule) bound 10
to the DNA. DNA can be viewed as a more-or-less
vertical spiral staircase where the steps are made up of
pairs of planar aromatic molecules, called base pairs, and CD/millidegrees 5
the sides are chains of alternating phosphate groups and
ribose sugars..21/ The source of the chiral structure in
DNA is the sugar backbone which imposes the twisted 0
structure onto the whole molecule. Usually the twist is
right-handed, though it appears that left-handed Z-DNA
is intrinsically not much less stable than the right-handed 5
forms..27/ The CD of a DNA molecule in the 180 300 nm
region is due to the skewed orientation of transitions in the
bases. Thus, if the DNA is untwisted there will be less CD 10
190 210 230 250 270 290 310
and if it is twisted the other way the relative geometries of
the coupling transition moments (see below) will change (b) Wavelength/nm
and the CD signal may even invert (as it does for Z-DNA
relative to much of the B-DNA spectrum, Figure 11). Figure 11 CD spectra of (a) , B-form calf thymus DNA
(pH D 6.8, 5 mM NaCl, 1 mM sodium cacodylate); ,
B-form poly[d(G C)]2 (pH D 6.8, 5 mM NaCl, 1 mM sodium
2.2.6 Induced Circular Dichroism of a Molecule cacodylate), and - -, Z-form poly[d(G C)]2 (pH D 6.8, 5 mM
NaCl, 1 mM sodium cacodylate, 50 M [Co(NH3 )6 ]6C ) (b) 372
Bound to DNA nucleotide A-form mRNA (pH D 7)..44/ The CD characteristics
of these nucleic acid polymorphs are as follows. B-form: a
If an achiral molecule is bound to DNA, its transitions gain
positive band centered at 275 nm, a negative band at 240 nm,
an induced circular dichroism (ICD) due to perturbation with the zero being around 258 nm. At 220 nm the CD signal
by the chiral DNA. The sign and magnitude of this ICD is either becomes positive or less negative; a small negative peak
crucially dependent on the binding geometry, so in princi- is then followed by a large positive peak from 180 190 nm.
ple should yield information about that binding geometry. A-form: a positive CD band centered at approximately 260 nm
that is larger than the corresponding B-form band, a fairly
At its crudest level the appearance of the CD signal indi-
intense sharp negative band at 210 nm and a very intense positive
cates that the drug molecule is bound. If the binding is in band at 190 nm. The 250 230 nm region is also usually fairly
a single binding mode or a constant proportion of mixed flat though not necessarily zero. Z-form: a negative CD band
modes then the ICD signal will be proportional to the con- at 290 nm, a positive band at approximately 260 nm, a large
centration of bound drug and a titration series of data can negative CD signal in the 195 200 nm region, and the CD
passes through zero between 180 and 185 nm.
be used to determine the binding constant of the drug to
DNA..8,28/ An example is given in Table 1 and Figure 12.
by Equation (14).3 5,7/
2.3 Interaction of Radiation with Matter and the Hint D E mB C higher order multipole terms .14/
Rosenfeld Equation for Circular Dichroism
where is the electric dipole moment of the molecule
In classical mechanics, the energy of the interaction, and m is its magnetic dipole moment. and m are
Hint , between a molecule and the electric, E, and determined by summing over the electron distribution
magnetic, B, fields of electromagnetic radiation is given of the molecule, Equation (15):.3 5,7/
DOI: 10.1002/9780470027318.a5402
Table 1 Induced CD signals at 215 nm (DNA signal where e is the (negative) unit charge on an electron, ri
subtracted out) determined for calf thymus DNA with denotes the position vector of the ith electron, pi is the
additions of [Co(NH3 )6 ]3C
momentum vector for the ith electron, and me is the mass
Total ICD215 D r Concentration of of an electron.
[[Co(NH3 )6 ]3C ](M) (mdeg) bound ligand, Lb (M) When we are interested in spectroscopic phenomena
such as absorbance, CD, and LD (see below), we have to
15 1.2 11.4
move beyond classical mechanics to quantum mechanics
30 2.13 19.8
50 2.98 27.7 since the transitions we study involve discrete amounts
100 3.74 34.7 of energy, i.e. the energy is quantized. Thus, instead of
an expression for the classical energy of the interaction
Path length 1 cm, 100 mM in 10 mM NaCl, 1 mM sodium cacodylate,
pH D 7.
between the radiation and the molecule, we need the
interaction Hamiltonian operator..3 5,7/ This follows from
70 Equation (14) by converting and m to operators and the
y = 9.3 + 4.3x
interaction energy to the Hamiltonian operator. Curved
60
hats are used in what follows to denote operators,
50 Equation (16):
40
y
A int D AE
H A B C higher order terms
m .16/
30
When we compare the relative strengths of different
20
transitions experimentally we measure the intensity of
10
0 2 4 6 8 10 12 14
the transition which is the area under the absorption
(a) x band. The related theoretical quantity is the probability
per unit time, P, that a transition will occur from the
0.005 initial state, jii, (which is usually the ground state) to the
y = 0.0063 0.099x final state, jf i:.3 5,7/
0.004
2
0.003 P.jii ! jf i/ D k0 jhf jA
H int jiij
r
0.002
D k0 hf jA
H int jii hf jA
H int jii
D k0 hijA
H int jf ihf jA
H int jii .17/
0.001
0.000 where the integral is over molecular position coordinates,

0.01 0.02 0.03 0.04 0.05 0.06 and k0 is a constant. Equation (17) is a form of the Fermi
(b) r /If Golden Rule..3 5,7/ It is written in terms of bra ket
notation where the bra h0j state is the complex conjugate
Figure
12 (a)j Induced
. spectroscopy method plot.8,28/ where of the ket j0i. When a bra and a ket are coupled
xD
Lktot Ltot
.rk rj / and y D Lktot
j
Ltot /.rk rj / for ri together either directly or either side of an operator,
rk rj
such as A in Equation (18) below, then we imply that
the ICD signal for the ith spectrum, and Litot the ligand an integral over all space is being performed. Round
concentration for that spectrum. The intercept equals a,
where a relates the bound ligand concentration to the ICD brackets j/ in what follows denote unperturbed states
r: Lb D ar, and n is the number of DNA bases per bound of chromophores A and C (see below), and pointed
ligand. The slope is Stot /a where Stot is the total binding site ones ji denote the states of the whole system. Upon
concentration (i.e. the DNA concentration divided by n). From substituting the expression for HA int into Equation (17) we
the graph: a D 9.3 1 M mdeg 1 ; n D 16 2. (b) Scatchard
have Equation (18)
plot.8,28/ using value of a determined from (a). r is binding
ratio (concentration of ligand bound divided by DNA base
concentration), If is the concentration of free ligand. The slope P.jii A jf ibO g
! jf i/ D kfhijAjf iOe C hijm
is K, for K the equilibrium binding constant. The binding O
constant is therefore approximately 100 000 M 1 . fhf jAjiiOe C hf jm
A jiibg .18/
X where eO is the unit vector along E, bO is the unit vector along

De .ri / B, the asterisk denotes the complex conjugate, jii D hij,
i and the magnitudes of the electric and magnetic fields are
e X absorbed into the constant k. hf jAjii is the EDTM from
mD fri pi g .15/
2m e i state i to state f , and hf jm
A jii the corresponding MDTM.
DOI: 10.1002/9780470027318.a5402
It follows that,.8/ Equation (19) a laboratory-fixed axis system about which the molecule
knows nothing unless we tell it. In the absence of any
P.jii ! jf i/ D k[fi eO if eO C mfi bO mif b]
O .I/ molecular orientation the LD in Equation (23) vanishes
C k[if eO mfi bO C fi eO mif b]
O .II/ .19/ because all molecular axes are equally likely to coincide
with Z so, Equation (24)
where if D i!f D hf jAjii and so on (note inversion of if if
order of states in the notation). hjZ j2 i D hjY j2 i D 13 jif j2 .24/
2.3.1 Interaction of a Molecule with Linearly Polarized 2.3.2 Interaction of a Molecule with Circularly Polarized
Light: Absorbance and Linear Dichroism Light: Circular Dichroism
If no external magnetic field is present (except that due When the radiation is circularly polarized, CD becomes
to the radiation) we can choose jii and jf i to be real possible. As long as there is no magnetic field (apart from
wavefunctions; thus fi D if but mfi D mif since m A is that of the radiation) present, we can again choose to use
imaginary. The polarization vectors for E, and hence B, real wavefunctions so fi D if and mfi D mif . To evalu-
are also real for linearly polarized light. It follows that ate Equation (1) we substitute first the expression for left
the two parts of term (II) in Equation (19) are equal circularly polarized light into Equation (19) then subtract
in magnitude but opposite in sign, so term II is zero. from it the result of substituting the expression for right
As magnetic terms are invariably small compared with circularly polarized light. We also make use of the equality
electric ones, we can ignore the second term within term eO r D eO l , and note that,.6 8/ Equations (25) and (26)
(I) and therefore write the probability of the transition
occurring from jii to jf i with light polarized parallel to bO D kOe .25/
the electric field (the absorbance, Ae , of the transition)
so
to be:
bO l D p1 .1, 0, 0/ .0, 1, i/ D p1 .0, i, 1/ D bO r .26/
Ae .jii ! jf i/ D kjif eO j2 .20/ 2 2
Thus.8/
For the usual situation where the system is a collection of
randomly oriented molecules, the interaction averages to CD D Al Ar
1
Equation (20) and becomes Equation (21):.3 5/
3 .fi eO l if eO l C mfi bO l mif bO l /
Dk
k if 2 .fi eO l if eO l C mfi bO l mif bO l / .I/
Aiso D A D j j .21/
3 .if eO l mfi bO l C fi eO l mif bO l /
Ck .27/
where Aiso is the isotropic absorbance. LD, which .if eO l mfi bO l C fi eO l mif bO l / .II/
forms the subject matter of the next section, is the
difference in absorption of two in-phase linearly polar- Because fi eO l if eO l D fi eO l if eO l and so on (the wavefunc-
ized light beams propagating in the same direction tions are real), the term (I) vanishes. Upon rearranging
with perpendicular polarizations. Take the .jj/ direc- term (II) so that the EDTMs are all from jf i to jii and
tion to be (0,0,1) and the perpendicular direction to the magnetic moments are the reverse, Equation (27)
be (0,1,0). Then Equation (22) follows from Equa- becomes Equation (28).8/
tion (20)
CD D Al Ar D 2k[fi eO l mif bO l fi eO l mif bO l ] .28/
if if
LDmolecule D .Ajj A? /molecule D kfjZ j2 jY j2 g .22/
Substitution of explicit forms for the electric and magnetic
if
where Y
is the Y component of the jii to jf i transition field (Equations 5 and 25) polarizations for the circularly
dipole moment, and so on. For a collection of N polarized radiation, gives Equation (29).8/
molecules we sum over the LD for each molecule, if if
Equation (23) CD D 2ik[fiY mY C fiZ mZ ]
X if
D 2kIm[fiY mY C fiZ mZ ]
if
.29/
LDtotal D .Ajj A? /molecule
molecules where Im denotes the imaginary part of.
D
if
NkfhjZ j2 i
if
hjY j2 ig .23/ CD experiments are usually performed on collections
of randomly oriented molecules. In that case the lab-
where hi denotes average. The net LD thus depends on the oratory (radiation) defined axis system does not relate
method and extent of sample orientation since fX, Y, Zg is in any fixed way to a molecular axis system, so what
DOI: 10.1002/9780470027318.a5402
is measured is a rotational average of Equation (29) in A C

Equation (30a):
qa qc
CD D 43 khijjf Q D 43 kIm[fi mif ]
Q ihf jmjii .30a/
ra rc
The CD for the transition from state jii to state jf i is thus
given by the Rosenfeld equation (Equation 30b):.29/
RAC
CD D Im[fi mif ] .30b/
A origin C origin
Recall that fi is the EDTM from state jf i to state jii.
If we limit our consideration to achiral molecules but
Figure 13 A and C chromophores.
impose an external magnetic field, then term (II) of
Equation (27) vanishes, but term (I) does not (the states
are complex rather than real). Further, we ignore the origin of chromophore A to the origin of chromophore C
terms that involve squares of MDTMs as they will be much (Figure 13).
smaller than any EDTM terms. The magnetic circular The electric dipole moment operator, , Q where is used
dichroism (MCD) may then be written as Equation (31) here to indicate an operator for the whole system, may be
if if if if written in terms of A and C operators as, Equation (33)
MCD D k[.fiY ifiZ /.Y C iZ / .fiY C ifiZ /.Y C iZ /]
( )
if if X X
D 2ik.fiY Z fiZ Y / .31/ Q D e A A C Ar a / C
.R A C C Ar c /
.R .33/
a c
We shall not deal further with MCD in this article. See
Rodger and Norden.8,31/ for more details. where RA A D RA is the position operator for the A origin in
the global coordinate system, and Ar a D ra is the position
2.4 Circular Dichroism of Magnetic Dipole Allowed operator within A for particle a (Figure 13). We shall
Transitions: Why the Octant Rule Works for be considering only transition moments, so since RA is
Carbonyls constant it follows that.8/
( )
In this section and the next we shall be working within X X
the chromophoric or independent systems/perturbation Q D e .Ar a / C .Ar c / D AA CAC .34/
a c
(ISP) approach to CD theory,.8,12,13,15/ and will limit our
consideration to transitions in achiral chromophores, A, where AA is the electric dipole moment operator for A
that acquire CD intensity by coupling to transitions in and operates only on wavefunctions of A.
other chromophores C. C need not itself be chiral but Similarly it follows from Equation (15) that the mag-
it must be positioned so that fA C Cg forms a chiral netic dipole moment operator for the whole system is,.8/
system. A chromophore is strictly a part of a system Equation (35)
whose wavefunctions have no overlap with the rest of
the system; electronic wavefunctions in a chromophore m
Q DmMACm MC
therefore have no electron exchange with the rest of the ( )
e X X
system. In practice, a chromophore is usually identified D A A C Ar a /Apa C
.R .RA C C Ar c /Apc
for a given electronic transition if that transition seems 2me a c
to be more or less dependent on only the identity of a e X e X
subset of the molecule (such as the carbonyl functional D AA
R Apa C mAAC AC
R Apc C m AC
2me a
2me c
group).
e e
We also need to be able to write an equation for D A AApA C m
R AAC A Ap C m
R AC .35/
the interaction between A and C. Since there is no 2me 2me C C
electron exchange between A and C (by definition), their where m O A is the total magnetic moment for chromophore
interaction, V, is purely coulombic, Equation (32) A,ApA is the linear momentum operator for chromophore
X qa qc A, mA A is the intrinsic magnetic dipole moment operator
VD .32/
jRAC C rc ra j within A, and so on. Thus the total magnetic moment for
a,c
A includes both the intrinsic magnetic moment operator
where qa is the charge of a particle in A located at the within the chromophore and any magnetic moment due
end of the vector, ra , that begins at the A origin, similarly to the linear motion of its charges about the system
qc and rc , and RAC D RC RA is the vector from the origin.
DOI: 10.1002/9780470027318.a5402
To evaluate the Rosenfeld equation (Equation 30b) for the interacting system is, Equation (39)
two interacting chromophores we need to know 01 and
m01 for the whole system, i.e. for the j0i ! j1i transition, A DH
H A CCV
A ACH .39/
which is the j0/ ! j1/ transition on A in the presence of The wavefunctions for the combined system could be
any perturbation due to C. We write the wavefunctions determined using the Equation (32) expression for V and
for the A/C system when their interaction is switched off the variation principle. We shall use perturbation theory
as the product wavefunctions ja/jc/ D jac/ where ja/ is a to give expressions for the wavefunctions and later assume
wavefunction of A and jc/ is a wavefunction of C. The A that A and C are far enough apart to expand V as a
function is always written first even in the ket form, thus, double Taylor series expansion to give terms dependent
Equation (36) on monopoles, dipoles, quadrupoles and so on of A and
C. This approach has the disadvantage of giving different
fja/jc/g D fjac/g D .aj.cj D .acj .36/ final equations for degenerate and nondegenerate systems
(and ignores near degenerate systems); however, it has
A operators only operate on A wavefunctions, similarly C the advantage of enabling us to write equations that can
operators only operate on C wavefunctions. For example, be used to relate system geometry to CD signal.
Equation (37) The perturbed wavefunctions jii D j00i and jf i D j10i
0 for the MDTM case where the perturbation comes
.a0 c0 jAA jac/ D .a0 jAA ja/.c0 jc/ D A
aa
dcc0 .37/ from EDTMs are determined from nondegenerate per-
turbation theory to first order in V Equations (40)
where dcc0 D 0 unless c0 D c.
and (41):.3 5,32/
We are considering the situation when the j0/ ! j1/
transition of A is MDA and EDF so m01 A 6D 0 but A D 0.
10 .1cjVj00/
j00i D j00/ j1c/ .40/
An EDF transition of an achiral chromophore, A, must ec C ea
borrow some electric character in order to have a CD .ocjVj10/
signal. If we also assume that transition moments give j10i D j10/ j0c/ .41/
ec ea
significantly stronger effects than permanent moments,
then the dynamic coupling model for the CD of MDA from which it follows that, Equation (42)
transitions follows..12/ Historically much more effort has
been put into what is known as the static coupling 10 D h00jj10i
Q D h00jAA CAC j10i
or one electron mechanism (since one electron on A is .00jVj1c/
D .00jAA j10/ .1cjAC j10/
assumed to be the only one to move) where the molecular ec C ea
framework provides a static perturbation via permanent .0cjVj10/
moments. This mechanism certainly contributes to the .00jAC j0c/
ec ea
net observed CD, however, its contribution is almost
always smaller than that due to the dynamic coupling .00jVj1c/ 0c 2ec V 1c c0
.0cjVj10/ c0
D A AA D
mechanism. More details about the static coupling model ea C ec C ec ea C
e2c e2a C
may be found in references in Schellman..31/ .42/
To determine the wavefunctions of the interacting A/C since the A and C wavefunction are real. Similarly,
system we first consider an A/C system where A and C do Equation (43)
not interact. The Hamiltonian, H A , of the noninteracting
system is the sum, Equation (38) m01 D h10jmj00i
Q D h10jm
A ACm
A C j00i
.1cjVj00/
D .10jm
A A j00/ .10jm
A C j1c/
A DH
H AC
A ACH .38/ ea C ec
.10jVj0c/
where H A A is the Hamiltonian of isolated A, and similarly .0cjm
A C j00/
A C . This Hamiltonian is separable and the total energy ec ea
H
of a state jac/ is the energy of ja/ in A plus that of jc/ in .1cjVj00/ c0 .10jVj0c/ 0c
D m01
A mC mC
C. So, for example, j01/ means A is in its ground state ea C ec ec ea
and C is in state j1/. The energy of the j01/ state of the 2ea V 1c 0c
combined system is therefore 0 C e D e (taking the zero D m01
A m .43/
e2c e2a C
point of energy to be when both A and C are in their
ground states). Substitution into the Rosenfeld equation (Equation 30b)
When the electrostatic interaction, V (Equation 32), is gives the so-called dynamic coupling CD expressions to
switched on between A and C then the Hamiltonian of first order in V,.14,33/ Equation (44)
DOI: 10.1002/9780470027318.a5402

2ec V 1c c0 01 Unless fi, jg equals fx, yg or fy, xg the A-moment product
R D Im m .44/ (first square brackets in Equation 48) vanishes. Thus the
ec e2a C A
2
CD strength for MDA z-polarized transitions of C2v
The above equations are for systems composed of A plus
chromophores such as the n ! p transition of carbonyls
one C. If there are two or more Cs then a sum over Cs is
(Figure 4) is, Equation (49):.15/
introduced.
In order to apply Equation (44) we need to have explicit
forms for V 1c in terms of electric transition moments of 6 01 10
ec .0c
C /z
R.C2v , z/ D Im .QA /xy .mA /z
A and C. It is now convenient to use a general form for R4AC .e2c e2a /
the terms in the expansion of V using i, j, k D fx, y, zg and c0 b b
the Kronecker delta, di,j , which is zero unless i D j. The .C /x .RAC /y C .c0 C /y .RAC /x
b b b .49/
dipole dipole term in the expansion of V is.20/ given in 5.c0
C /k .RAC /x .RAC /y .RAC /k
Equation (45)
The octant rule follows from Equation (49) when we
V 1c .A C / D V 10,0c D .0cjVj10/
reduced our consideration to the term that has an isotropic
A C
10 0c b AC 10 0c R
3R b polarizability component (the octant rule term), i.e. set
A C AC
D
R3AC k D z and ignore the first two terms in the final brackets.
As noted above, there are occasions when the octant rule
1 b AC /i .R
b AC /j ]
D .10 /i .0c
C /j [dij 3.R is not appropriate because other terms in Equation (49)
R3AC A (or even higher order terms in the V expansion) become
.45/ dominant.
where a sum over all repeated indices is implied. When Equation (49) after appropriate symmetry averag-
substituted into Equation (44) the dipole dipole term of ing also leads to the CD expressions for degener-
V leads to a CD expression containing a .01 10
A /i .mA /k term.
ately x,y-polarized transitions of molecules with D3d
This moment product vanishes except for chromophores A (Figures 7 and 8), since the A moment product of
of Cs symmetry (i.e. those with only one reflection plane Equation (49) does not vanish for these polarizations
and no rotation axes) and x- and y- polarized transitions under this symmetry. Take z to be the three-fold rota-
of C2v chromophores. An MDA transition in such a tion axis and x to align with one of the two-fold
chromophore is also EDA so the CD will probably be rotation axes of A. By symmetry, the only nonvan-
dominated by the coupled-oscillator CD of the EDTM ishing A moment products are those in Equation (50)
which is dependent on .RAC / 2 (see below). so the E-polarized tris chelate transition metal complex
For magnetically z-polarized transitions in A chro- CD is:.20/
mophores of C2v symmetry such as the n ! p transition

of carbonyls (see above) the quadrupole dipole term of x 3ec
V is the first nonvanishing one, Equation (46):.14/ R D3d , D Im 4
y 4RAC .e2c e2a /
3 10
V 1c .QA C / D [.Q10 0c
A /ij .C /k ] .QA /xx .m01 A /x .Q10A /yy .mA /x
01

2R4AC

2.Q10 A /xy .mA /y
01

b AC /k C dik .R
dij .R b AC /i
b AC /j C djk .R

2. c0
/ . 0c
/ . Rb / 5. c0
/

b AC /i .R
b AC /j .Rb AC /k
C x C x AC x C k

5.R b b b

0c
. / . R / . R / . R /
.46/

C x AC x AC x AC k

c0 0c b c0

where QA A is the electric quadrupole moment operator
4. / x . / y . R AC / y C 5. / k

C
b
C
b b
C

AC k
0c
of A. It is a second rank tensor whose components are,
. C / x . R AC y / . R AC y/ . R /

Equation (47)
c0

X

2.C /y .C /y .RAC /x C 10.C /k
c0 0c b

A A /ij D e
.Q .ri rj / .47/ .0c / x . b AC /x .R
R b AC /y .R b AC /k .50/
C

a

4 [ .Q 10
/yz .m01 /x .Q10 /xz .m01 /y ]

(cf. Equation 15 for notation). The quadrupole dipole

A A A A

c0 0c b c0

CD term is thus given by Equation (48)
. / .
C y C z / . R AC x/ C 5. C k/

0c

.C /x .RAC /y .RAC /z .RAC /k
0c b b b

3 ec .c0
C /l .C /k

RD Im [.Q 10
/ .m 01
/ ]
c0 0c b
.C /z .C /x .RAC /y 5.C /k c0
R4AC A ij A l
.e2c e2a /

b b b
.C /y .RAC /x .RAC /z .RAC /k
0c
b

dij .RAC /k C dik .Rb AC /j C djk .R b AC /i

C .c0 b

.48/
/ .
C x C y
0c
/ . R /
AC z . c0
C y/

b AC /i .R
5.R b AC /j .R
b AC /k 0c b
.C /x .RAC /z
DOI: 10.1002/9780470027318.a5402
The isotropic polarizability part of Equation (50) is given j10 i D p1 fj10/ j01/g .52/
2
in Equation (51)
where the j10C i state takes the upper sign of the
15ec aC .ea / n
R.D3d /x,y D Im[.Q10 01
A /xx .mA /x
or terms in this equation and those that follow. As
4R4AC .e2c e2a / above, we use real normalized wavefunctions. If we also
.Q10 01
2.Q10 01 assume that permanent moments are much smaller than
A /yy .mA /x A /xy .mA /y ]
o transition moments, then the energies of these two states
.R b AC /2
b AC /x [.R b AC /2 ]
3.R .51/ are, Equation (53).3 7,32/
x y
Equation (51) is the basis of the above discussion of Eg e D h10 jA A C Vj10 i

HA C H
polarized d d transitions of tris-chelate metal complexes. D 12 f.10j .01jgjH A C C Vjfj10/ j01/g
A ACH
When A has higher than two-fold rotational symmetry,
then the quadrupole dipole term of Equation (50) is zero D 12 f.1jA
H A j1/ C .0jA
H C j0/ .10jVj01/
for transitions polarized along the high symmetry rotation C .0jA
H A j0/ C .1jA
H C j1/ .01jVj10/g
axes (e.g. for A2 polarized transitions of tris-chelates).
Since the octupole term in the V expansion also vanishes D e0 C e1 V 10,01 D e V 11 .53/
under these circumstances,.20/ the term in the V expansion
for A2 polarized transitions is the hexadecapole dipole where, for example, Equation (54)
6
term which has an RAC dependence so is smaller than the
4 V ba,dc D .acjVjbd/
Eg RAC term; hence the dominant E band rule discussed
above. C
ba dc
D .A b AC ba dc R
3R b
A C AC
3
C higher order terms/ RAC .54/
2.5 Circular Dichroism of Electric Dipole Allowed
Transitions Because all wavefunctions are real, when one state on
For EDA transitions, the independent systems approach each of A and C is the ground state we further simplify
leads to mechanisms that are usually referred to as the notation as indicated in the last line of Equation (53).
coupled-oscillator models. Because we choose to use V 11 is much smaller than e so the two new perturbed
perturbation theory to give expressions for wavefunctions states are close in energy.
(as discussed above), we need to consider identical and The EDTMs for the transitions from the two excited
nonidentical fA,Cg systems separately. states of Equation (52) to the ground state j00/ are,
Equation (55):
2.5.1 Identical A and C: the Degenerate Coupled h00jj10
Q i D p1 f.00jA CAC [j10/ j01/]g
2 A
Oscillator Model
Most transitions for which we measure UV/VIS absorb- D p1 f10
2 A C10 g .55/
ance spectra are EDA. If an EDA transition, with EDTM
The j0/ ! j1/ transitions in both A and C are MDF,
A01
and transition energy e, of an achiral chromophore,
and so have no intrinsic magnetic moment. We there-
A, has a measurable CD spectrum, then it must have
fore set mAADm A C D 0 in Equation (35). The momenta
borrowed some magnetic character to give a net
MDTM terms are more conveniently dealt with when
helical electron rearrangement. The degenerate coupled-
re-expressed in terms of EDTMs for the appropriate
oscillator model describes the situation where A and C
transition exploiting a very useful relationship,.32/ Equa-
are identical and the magnetic character comes from the
tion (56)
coupling of A 01
with the same energy EDTM in C, C01 .
C must be oriented skew relative to A
01 01
for a CD signal ime jk
to be observed near e. C induces a magnetic effect in A
01 .kjApC jj/ D .ek ej /C .56/
e h
since, although it is a linear motion of charge within C,
when viewed from A it moves (circles) around As origin from which follows Equation (57)
01
(A simultaneously induces a magnetic component into jk e
the C transition C01 ).
mC D .kjm
Q C jj/ D RCApC jj/
.kjA
2me
In this case, where A and C are identical, by i jk
symmetry (or degenerate perturbation theory) the two D RC .ek ej /C .57/
2 h
states that result from the mixing of j10/ (A in j1/
and C in the ground state) and j01/ are given by This is the magnetic moment at the origin (0,0,0) created
jk
Equation (52).3 5,32/ by the tangential EDA transition moment C . The
DOI: 10.1002/9780470027318.a5402
MDTMs for the return transition from to the ground state 2.5.2 Different A and C: Nondegenerate Coupled
to the perturbed excited states are then, Equation (58) Oscillator Circular Dichroism
ie When A and C are not identical then the wavefunc-

.10 jmj00/
Q D p RA A
01
RC C01 .58/ tions of the A/C system follow from nondegenerate
8 h perturbation theory..3 5,32/ We may write Equations (62)
and (63)
Substituting Equations (55) and (58) into the Rosenfeld
equation (Equation 30b) gives the CD strengths of bands .1cjVj00/
centered at eC and e (Equation 53) arising from the j00i D j00/ j1c/ .62/
ec C ea
coupling of degenerate j0/ ! j1/ transitions on A and C
.0cjVj10/
to be,.8/ Equation (59) j10i D j10/ j0c/ .63/
ec ea
e 01 01
R01
D C A .RC RA / The EDTM of the perturbed j10i ! j00i transition is
4 h given by Equation (64)
e 01 01
D C A RAC .59/
4 h 10 D h00jj10i
Q D h00jAA CAC j10i
where the C transition is not necessarily higher in energy .0cjVj10/

D .00jAA j10/ .00jAC j0c/
than the transition. ec ea
The physical interpretation and applications of Equa- .00jVj1c/ 2ec V 1c c0
tion (59) for solving geometric and spectroscopic prob- .1cjAC j10/ D A 10
.64/
ec C ea e2c e2a C
lems is most easily seen by choosing the coordinate system Similarly,.8/ Equation (65)
illustrated in Figure 14 in which case Equation (59) may

be written, Equation (60): 01 e 01 2ea V 1c 0c
m D h10jmj00i D
Q RA pA RC pC
2me e2c e2a
e2 RAC
R D sin a sin g sin t .60/ i 2ea ec V 1c
4 h D ea RA A
01
R
C C
0c
.65/
2 h e2c e2a
and the energies of the two bands are, Equation (61)
since the transition has no intrinsic magnetic moment
within A as in the case of the degenerate coupled oscillator
2 .sin a sin g cos t C 2 cos a cos g/ model.
e D e .61/
R3AC The CD strength is therefore (Equation 31).8/
where is the magnitude of A and C . ea ec V 1c

R.j00i ! j10i/ D
The expected CD spectrum for two degenerate chro- h.e2c e2a /
mophores as a function of t is illustrated in Figure 15. 10
fA RC C0c C Cc0 RA A
01
g
Because independent systems theory is identical to the
theory of exciton particles, these degenerate couplets are ea ec V 1c 0c 01
D f RAC g .66/
often referred to as exciton CD spectra. h.e2c e2a / C A
CAR right-handed CAR left-handed

R+ > 0 R>0
z
z z
a c c
a y a
y
RAC
x
A A
C C
A C
c
Figure 14 Diagram illustrating the geometry and coordinates for an A/C system. Note that t is the angle taken in the anticlockwise
direction between the projections of the EDTMs onto the y z plane when the observer is looking down the x-axis. 0 < t < 180
if c a RAC > 0 (i.e. if the three vectors form a right-handed parallelepiped), and 180 < t < 360 if c a RAC < 0. CAR
right-handed summarizes a mnemonic for the situation where c , a , and RAC form a right-handed axis system with 0 < t < 180 .
DOI: 10.1002/9780470027318.a5402
Again using the coordinate system defined in Figure 14

we may write Equation (67)
ea ec .a c /2
R.ea / D fsin a sin g cos t C 2 cos a cos gg
h.e2c e2a /R2AC
sin a sin g sin t .67/
which is usually easier to implement than Equation (66).
CD
0
3 LINEAR DICHROISM
3.1 Introduction
LD is the difference in absorption of light linearly
Wavelength
polarized parallel and perpendicular to an orientation
axis, Equation (68): (a) Energy
a
c

LD D Ajj A? .68/ c z
a

LD is related to CD in that both require the difference RAC y
between the absorbances of different polarized light A
beams to be measured and CD spectropolarimeters can A C C
be adapted to produce the required alternating beams of
polarized light for LD. However, LD measurements are +

0 < < 90 90 < < 180
performed on systems that are either intrinsically oriented
+
R+
or are oriented during the experiment (Figure 16). The R
general LD references given below may be consulted
for more details on the definitions and equations that

CD
follow. Rodger and Norden.8/ use the formalism adopted 0 +

+
here.
The equations for LD were given in Equations (22)
and (23) in the context of the interaction of radiation R R
with matter. In order to understand LD more fully,

consider what happens when a molecule absorbs a photon: (b)
absorption can be pictorially viewed as either the electric
+
field or the magnetic field (or both) of the radiation o o
o
180 < < 270
o
270 < < 360 R
pushing the electron density from a starting arrangement
to a higher energy final one. The direction of net linear R
displacement of charge is known as the polarization of the

transition. The polarization and intensity of a transition
CD
0
are characterized by the EDTM (see Equations 18 and
+ +
19) which may be regarded as the antenna by which the
molecule absorbs light. Each transition thus has its own
antenna and the maximum probability of absorbing light R+ R+
is obtained when the antenna and the electric field of the
light are parallel.
Now imagine a linearly polarized light beam (Figure 16) (c) Wavelength Wavelength
and a sample of molecules all oriented in exactly the
same way. If the normal absorption is first measured with Figure 15 Exciton CD spectra for coupling short-axis polar-
ized transitions. (a) Illustrates characteristic form that results
the light polarized so that it is parallel to the direction
from cancellation of overlapping bands; - - - -, higher energy
of orientation of the sample and then measured when component; , lower energy component; , observed
the light is polarized perpendicular to this direction, the CD spectrum. (b) Illustrates the geometry of the system.
difference between these two spectra is the LD spectrum. (c) Shows the net spectra as a function of t (assuming Gaussian
The two extreme situations are: band shapes for the two transitions).
DOI: 10.1002/9780470027318.a5402
II Polarized Polarized Oriented

light light molecule
+ LD spectrum
LD = +

Wavelength
Figure 16 Schematic illustration of LD.
,,,,,, ,,,,,,
1. If the polarization of the transition that is being ,,,,,,
,,,,,, ,,,,,,
,,,,,,
,,,,,, ,,,,,,
probed is perfectly parallel to the orientation direc- Ez
tion, then Equation (69) follows k
Ey
LD D Ajj A? D Ajj > 0 .69/
2. If the polarization of the transition that is being

,,,,,,
,,,,,, ,,,,,,
,,,,,,
probed is perpendicular to the orientation direction, (a) ,,,,,,
,,,,,, ,,,,,,
,,,,,,
then Equation (70) follows
E
LD D Ajj A? D A? < 0 .70/
For intermediate polarizations, the LD is between these y polarized

LD
cases. Thus, the polarization for a given transition can

be determined from its LD spectrum if the orienta- 10
z polarized
tion of the molecule is known; conversely, LD can be
used as a probe of molecular orientation if the polar-
ization of a transition moment within the molecule is
known. In practice, one type of experiment is often per- 220 260 300 340 380
formed, followed by the other, using different orientation (b) Wavelength/nm
methods.
The two extreme cases of Equations (69) and (70) are Figure 17 (a) Stretched film orientation showing propagation
both illustrated in the simplified anthracene spectrum of direction, k, and electric field polarization, E, of the elec-
tromagnetic radiation, and (b) LD spectrum of long (z) and
Figure 17..8/ The spectrum is simplified in two ways: short (y) axis polarized transitions of an idealized (see text for
assumptions) anthracene sample.
1. perfect orientation of the molecules has been
assumed, and
2. the long wavelength band has been assumed to be of parameter S can be used. Equation (71) is then valid
pure short-axis polarization. LD Ajj A? 3
LDr D D D S.3 cos2 a 1/ .71/
In reality perfect orientation is never achieved and A A 2
there is a significant long-axis polarized component at LDr is the so-called reduced LD, S may be viewed as
320 nm due to coupling with the 250 nm band. a scaling factor defining the efficiency of macroscopic
orientation, and a is an angle that specifies the orienta-
3.2 Orientation Parameters tion of the transition moment that is responsible for the
absorption of light at the particular wavelength (if several
A variety of different orientation methods for LD spec- transitions absorb at the same wavelength an average is
troscopy are outlined below. In practice, with the possible obtained) with respect to the orientation axis (Figure 19).
exception of a crystalline environment, the orientation is S D 1 for perfect orientation and S D 0 for random (i.e.
never perfect and we need to use the concept of an ori- no) orientation. In Equation (71) A is the absorption of
entation distribution. In many cases, either the averaging the LD sample under isotropic (unoriented) conditions so
inherent in the orientation method or the nature of the Ajj , A? , and A all have Cl as a common factor, hence when
sample means that (at least) local uniaxial orientation using LDr it is not necessary to know the sample concen-
can be assumed (Figure 18) and the simple orientation tration or path length. If the sample is macroscopically as
DOI: 10.1002/9780470027318.a5402
Uniaxial Biaxial (Figure 19). q is used instead of z, to which the same def-
orientation orientation
inition is given below, as a reminder that Equation (73)
only holds for rod-like molecules in uniaxial samples. It
Circle
is important to note that the averages of (cos2 angles)
Ellipse
are appearing in the above equations. These are not the
same as the (cos2 ) of (average angles), though in practice
we usually have to ignore what can be a considerable
difference.
More generally, instead of a single orientation parame-
ter we need to have three since in some LD experiments,
including rectangular squeezing of a gel or when the light
is incident radially upon a cylindrical couette flow cell
(Figure 20), the system behaves as if the orientation were
biaxial (Figure 18) for which the constant probability
contour for a given molecular axis traces out an ellipse.
(Note: flow-oriented DNA, see below, is uniaxially
Figure 18 Uniaxial and biaxial orientation. Uniaxial orienta-
tion requires all orientations on a cone about the orientation oriented due to the helical nature of the molecule.) A
axis to be statistically equally probable. This may be due to laboratory (macroscopic) axis system fX, Y, Zg is defined
static or dynamic factors. first, such that X is the direction of propagation of the inci-
dent radiation and Z is the macroscopic direction of ori-
Z, orientation direction entation. Y then completes the right-handed axis system.
The more general definition of LD is then, Equation (74)
z
y z
LD D AZ AY .74/
x
(cf. Equation 23). Similarly, we write Equation (75)
x
Y AZ AY
y
r
LD D 3 D 3.hO 2Z i hO 2Y i/ .75/
AX C AY C AZ
When studying LD it is also convenient to define the
Radiation X molecular axis system, fx, y, zg. We take z to be the
molecular orientation axis, i.e. the axis in the molecule
Molecule which has maximum value shown by Equation (76) of
hcos2 zi .76/
where z is the angle between z and Z. x is the axis
Figure 19 Macroscopic and molecular axes systems and angles perpendicular to z that has the smallest value shown by
relating them.
Equation (77) of
well as molecularly uniaxial, such as molecules oriented in hcos2 xi .77/
a polymer film drawn in one direction or polar molecules
in an electric field, there is a simple relation between Ajj , where x is the angle between x and Z. y is similarly the
A? , and A which makes it unneccessary to measure all angle between y and Z. z is often, but not always, the
three quantities, Equation (72): highest order symmetry determined axis.
The general definitions of the orientation parameters
A D 13 .Ajj C 2A? / .72/ are given by Equation (78)
When the orientation method used in an LD experiment
Sxx Sxy Sxz
provides a uniaxial sample (Figure 18) and the sample S D Syx Syy Syz
molecules are also characterized by a unique axis around
Szx Szy Szz
which all orientations are equally probable (so the
molecule is rod-like), we may then write, Equation (73): 1 2
2 .3hcos xi 1/ 0 0
S D 12 .3hcos2 qi 1
1/ .73/ D 0 2
.3hcos2 yi 1/ 0
1
where q is the angle between the macroscopic orientation 0 0 2
.3hcos2 zi 1/
direction Z and the molecular orientation axis z .78/
DOI: 10.1002/9780470027318.a5402
Inner
Outer cylinder
cylinder
Moving wall 0.5 mm
annular
gap
Incident Emergent
radiation radiation
Y
Rotation axis
Rotating quartz
cylinder
Z
Sample solution
X
Macroscopic
orientation
To PMT axis
Incident radiation linearly

Quartz window polarized in XY plane
in outer cylinder (as shown) or XZ plane
Figure 20 Flow orientation in a coaxial (couette) flow cell with radial incident light. The total light path is typically 1 mm.
PMT D Photomultiplier tube.
Since we defined z to be the most oriented molecular axis, also, Equation (82)
and x to be the least oriented Equation (79) follows: 0 0
Sx x Sx0 y0 Sx0 z0
Szz Syy Sxx .79/ S0 D Sy0 x0 Sy0 y0 Sy0 z0
Sz0 x0 Sz0 y0 Sz0 z0
Further, the trace of the orientation tensor is zero so that,
1
Equation (80): 2
.3hcos2 x0 i 3hcos x0 cos y0 i 3hcos x0 cos z0 i
1/
Sxx C Syy C Szz D 0 .80/ 0

3hcos x0 cos y0 i 1 .3hcos2 y0 i 3hcos y 0
cos z i
D 2
1/
For rod-like molecules Sxx D Syy and S for the uniaxial
0 0 0 0 1 2 0
case is Szz . 3hcos x cos z i 3hcos y cos z i 2 .3hcos z i
In solving any particular problem, the positions of 1/
fx, y, zg are seldom known, so it is usually convenient first .82/
to choose a temporary molecular axis system fx0 , y0 , z0 g For planar molecules in uniaxially oriented systems,
which probably bears no relationship to the orientation x0 D x so x0 D x. It follows that,.8,34/
axis system but is convenient for our analysis (see example
below). Analogously to the definitions we used with LDr D 3.Sy0 y0 sin2 a0 C Sz0 z0 cos2 a0 C Sy0 z0 sin a0 cos a0 /
the true molecular axis system, the orientation of the .83/
macroscopic orientation axis Z in the new coordinate where a0 is the angle between the transition dipole
system may then be written, Equation (81), as the moment and axis z0 . The three orientation parameters
vector in Equation (83) are three unknowns, so three different
transitions of known polarization are needed to determine
.cos x0 , cos y0 , cos z0 /x0 ,y0 ,z0 .81/ them. The position of z is almost determined from
DOI: 10.1002/9780470027318.a5402
Equation (84).8,34/ is polarizable) may be achieved by an electric field

between two parallel plates..37/ Electric orientation is
Sy0 z0 conceptually simpler than flow orientation, and the data
tan.2b/ D .84/
Sz0 z0 Sy0 y0 can be fairly easily extrapolated to infinite orientation
where b is the angle between z0 and z. Either chemical which contrasts with the situation for flow orientation.
intuition or another experiment such as fluorescence Heating effects may be circumvented by using pulsed
polarization anisotropy.35/ is required to determine on field techniques, which additionally enable the study
which side of z0 , z lies. It is often apparent upon of relaxation phenomena. However, the pulsed nature
inspection. of the field generally requires each electric dichroism
experiment to be performed at a fixed wavelength or
else a diode array system to be used; it is also usual to
3.3 Molecular Alignment Techniques measure Ajj and A (the unoriented absorbance) rather
In addition to the requirement of linearly polarized light than A? (as the orientation is uniaxial, only two of the
for LD, LD experiments are made technically demanding three absorbances need be determined independently as
because of the need to orient the sample. The orientation discussed above).
method used for this depends on the sample. Long
polymers may be oriented by shear flow whereas small 3.3.4 Squeezed Gel Orientation
molecules require a stronger orienting force. A selection The method of orientation that has proved most successful
of orientation methods is described below. More details for membrane proteins, particularly photosynthetic ones,
may be found in the general LD references. is to embed the protein in a gel (usually polyacrylamide)
and then physically squeeze the gel either unidirectionally
3.3.1 Stretched Polymers or bidirectionally (Figure 21)..28/ The main shortcomings
Small molecules can often be absorbed into polymer films, of the gel orientation technique are its limitation to
usually polyethylene or poly(vinyl alcohol) (both can be wavelengths longer than 250 nm, and the limited dynamic
used well into the UV even down to 200 nm) depending on range of deformation.
the polarity of the analyte. When the film is mechanically
stretched, either before or after the small molecules are 3.3.5 Migrative Orientation
added, the absorbed molecules align their long axes While migrating in an electrophoresis gel, DNA is
preferentially along the stretch direction (Figure 17). macroscopically oriented and gives an LD signal.
There has been no evidence that a solute is better oriented
when introduced into the film before stretching compared 3.3.6 Magnetic Field Orientation
with it being added after stretching; with poly(vinyl
Magnetic fields may be used to orient molecules in much
alcohol); however, the film is usually cast containing the
the same way as electric fields, however, the effect is small
analyte so it cannot be added after stretching.
and this method is not widely used unless the particles are
large (such as chloroplasts) and carry substantial magnetic
3.3.2 Flow Orientation dipole moments.
Long polymers may be oriented by the viscous drag
caused when a solution is flowed between narrow walls..36/
Depending on the cell design, the light is then propagated
either along the flow direction or perpendicular to it. Gel Gel
This technique is commonly used for LD studies of DNA
(the DNA needs to be at least of the order of 1000
base pairs in length to get significant orientation). The Unidirectional
most successful flow cell has proved to be a cylindrical
couette flow cell where the solution containing the DNA
is subjected to a constant gradient over the annular gap
between two coaxial cylinders one of which is rotating
Gel Gel
(Figure 20).
3.3.3 Electric Field Orientation Bidirectional

Effective uniaxial orientation for polar or polarizable
molecules (including DNA since its ionic environment Figure 21 Principles of biaxial and uniaxial squeezing of gels.
DOI: 10.1002/9780470027318.a5402
3.3.7 Crystalline Samples 1.5

If the absorbance is not too large for the spectrometer to
measure, crystals may be used for LD studies.
1.0
Absorbance
3.3.8 Liquid Crystal Orientation
The molecules within a liquid crystal are oriented
0.5
with respect to one another and by sandwiching the
liquid crystal between two quartz plates, macroscopic
orientation may be achieved..38/ The sample must be
tilted with respect to the light beam to get an LD signal. 0.0
Molecules dissolved in a liquid crystal may also have a (a) 200 250 300
preferential orientation.
0.000
3.3.9 Orientation by Evaporation, for Example onto
Quartz
0.002
This method may be appropriate for some samples,
particularly planar aromatic molecules that readily adsorb
onto quartz. The quartz plate has to be tilted for LD LD
0.004
measurement.
3.4 Examples of Linear Dichroism Data 0.006

(b) 200 250 300
3.4.1 DNA
0.00
The absorption, and hence LD, spectra of nucleic acids
in the easily accessible UV region of the spectrum (down
to 180 190 nm) are dominated by the p ! p transitions
of the purine and pyrimidine bases which, by symmetry,
LDr
are all polarized in the plane of the bases. If DNA 0.01

is in the idealized B-DNA form with the DNA bases
perpendicular to the helix axis.39/ then a flow oriented
DNA sample (Figure 20) should have its LDr across the
whole spectrum constant and negative. In practice, there
is some variation in magnitude (Figure 22) which indicates 0.02
200 250 300
that the bases are tilted.
Normal B-DNA, which according to the classical fiber
(c) Wavelength/nm
structure has qX D 2.1 and qY D 4.0 (Figure 23, though
Figure 22 Absorbance (a), LD (b), and LDr (c) spectra of
fiber B-DNA has 10 base pairs per turn of the helix rather B-form and Z-form poly[d(G C)]2 (60 M in 10 mM NaCl,
than the 10.4 10.6 of solution DNA), should display 1 mM cacodylate buffer, pH D 7). The Z-form is induced
only small variations in LDr in the wavelength region by adding 40 M [Co(NH3 )]3C . The DNA has been flow
230 300 nm with LDr /S D 1.47 0.02. A-form DNA, oriented. As all p ! p transitions of the bases are polarized
approximately perpendicular to the helix axis, the LD signals
with qX D 19.3 and qY D 3.2 , is, however, expected
are negative. , B-DNA; - -, Z-DNA.
to exhibit a sloping LDr , with LDr /S varying from about
1.2 at 230 nm to about 1.4 at 300 nm, depending on
polarizations, intensities, and bandwidths. An effective
the sequence of the DNA..40/ Z-DNA is longer and stiffer
average value of 84 for a at 260 nm is often assumed for
and so is expected to have a significantly larger LD signal
B-DNA based on the fiber diffraction structure and an
than the same sequence of B-DNA as shown in Figure 22.
estimate of contributions from different transitions to the
LD data has been analyzed to determine the average
total LD..42/
values for base roll, tilt, twist, and slide for the
different DNA bases in solution B-DNA; a value of
a D 73 is appropriate for purines, whereas a D 65 3.4.2 Linear Dichroism of DNA-bound Ligands
for pyrimidines..40/ These results are dependent on Once the orientation of DNA itself is understood a next
the accuracy of the assignment of base transition step is to use LD to probe the binding and orientation
DOI: 10.1002/9780470027318.a5402
9-hydroxyellipticine aligns with the long axis of the

base-pair pocket and has a slightly smaller LDr signal
than the long axis of the molecule which is perpen-
dicular to the pocket and also more perpendicular to
the helix axis than the base pairs (Figure 25).
Z
3. In the minor groove: this mode is frequently
adopted by aromatic molecules containing internu-
clear bonds with some rotational freedom. For exam-
ple, long molecules such as netropsin, distamycin,
Hoechst 33258, and 40 ,6-diamidino-2-phenylindole
Y
(Figure 24), which can fit snugly into the minor groove
with the molecule following the curvature of the
Y
groove but are too large to fit into an intercalation
X site, favor this mode of binding. The long axis of
X
HO
NH2 CH3
H
N+ N+
C2H5 NH
CH3
(a) (b)
H2N + NH2
Figure 23 Geometry parameters for DNA as used in the text.
N
H
H O
on DNA of small ligand molecules and even proteins. N
A key feature of such LD studies is that if there is
no specific binding, then there will be no LD of the H N
ligand transitions. Conversely, the presence of an LD N CH3
O
signal from an absorption band of the putative ligand
H
immediately implies that it is bound. Although LD cannot N N
tell us where a ligand binds to DNA, it can be used to CH3
O
determine the orientation of the ligand on the DNA (if H2N
+
the ligands transition moments have been assigned). This (c) NH2
often provides significant clues as to the binding mode. A
ligand may bind to DNA in a number of different types of
N
site, as well as with sequence and orientation preferences. HO
Broadly speaking a ligand may bind in the following ways. N N
H
N
1. Externally bound to the phosphate backbone: this H
is usually orientationally fairly nonspecific and the CH3
N
induced LD (and CD) is correspondingly small. N+
(d) H
2. Intercalated between DNA bases: this mode requires
planar aromatic molecules and the DNA to unwind
and open up a slot between adjacent base-pairs so NH2
+
the ligand may be sandwiched between them. The H2N NH NH2
DNA is stiffer and locally better oriented near the +
(e) NH2
intercalation site, so the LDr of an intercalator is
usually slightly larger (more negative) at 260 nm than
Figure 24 The intercalators (a) ethidium bromide and
that of the average DNA base. If the bases are tilted, (b) 9-hydroxyellipticine, and some minor groove binders:
then the intercalator follows that tilt and this may be (c) netropsin, (d) Hoechst 33258, and (e) 40 ,6-diamidino-
apparent in the LDr . For example, the short axis of 2-phenylindole.
DOI: 10.1002/9780470027318.a5402
0.000 films, because the polarization of vibrations are often

known simply from their position in the spectrum. For
example, riboflavin has three infrared bond stretching
vibrations..35/ For convenience we choose the primed
LD/OD
axis system as illustrated in Figure 26. The values

0.004
of the LDr for each transition are also given in
Figure 26.
For the three transitions for which the data is given in
Figure 26, Equation (83) leads to Equation (85)
0.008
C0.070 D 3.Sz0 z0 /
(a) 200 300 400 500
C0.580 D 3.0.030Sz0 z0 C 0.970Sy0 y0 0.174Sy0 z0 /
0.012
ct-DNA C0.110 D 3.0.250Sz0 z0 C 0.750Sy0 y0 0.433Sy0 z0 / .85/
0
20
0.008 40 From which Equation (86) follows
60
80
LD/OD
100 Sz0 z0 D 0.024 Sy0 y0 D 0.256 Sy0 z0 D 0.373 .86/

0.004
Thus, b (Equation 84) either equals 61 or 151 . By
inspection of Figure 26, we conclude that b D 61 is
0.000
correct and the dashed line of Figure 26 is the molecular
orientation axis z.
0.004
200 300 400 500 600 700
(b) Wavelength/nm
4 INSTRUMENTATION AND THE
Figure 25 Some examples of LD spectra for DNA bound lig- DICHROISM EXPERIMENT
ands. (a) 9-Hydroxyellipticine (1 10 M in 2.5 M increments,
signal increases with ligand concentration) with 100 M calf 4.1 Circular Dichrographs (Spectropolarimeters)
thymus DNA in NaCl (20 mm) and phosphate buffer (1 mM,
pH 7);.41,45,46/ (b) trans-bis-(4-N-methylpyridiniumyl)diphenyl- The essential features of a CD spectropolarimeter are
porphyrin (5 M) with 40 M calf thymus DNA in phosphate a source of (more-or-less) monochromatic left and right
buffer (1 mM, pH 7) and varying NaCl concentrations (see figure
circularly polarized light and a means of detecting the
insert) showing the change in binding orientation as a function
of ionic strength. Signal increases with ligand concentration. difference in absorbance of the two polarizations of light.
The normal method of achieving these requirements,
such molecules is oriented at about 45 to the helix because the CD of molecules is generally quite weak com-
axis and the short axis polarized transition perpen- pared with the total absorbance signal, is to implement a
dicular to it (approximately parallel to the base polarization phase-modulation technique. A photoelastic
transitions). This mode is selective (or even spe- modulator (PEM) (in older instruments a Pockels cell)
cific) for adenine thymine-rich DNAs as the guanine produces alternatively right and left circularly polarized
amino group sterically hinders the minor groove of = 0
guanine-containing sequences. z LDr = +0.07
4. In the major groove: this type of binding is found for
O 1
several regulatory proteins and there is enough space
H3C N H
to accommodate most smaller ligands in a variety of N 2
= 100
orientations. H3C N O LDr = +0.58
CH2 3 = 120
Some examples of LD spectra for DNA bound ligands HOHC r
are given in Figure 25. LD = +0.11
CHOH
HOHC
CH2OH
3.4.3 Determination of the Molecular Orientation Axis of
a Molecule in a Stretched Film Figure 26 Riboflavin indicating the temporary axis system used
in the calculation and the polarizations and LDr values for the
Infrared LD is particularly useful for determining ori- three transitions used in the text. Data are from Matsuoka and
entations, particularly of small molecules in stretched Norden..34/
DOI: 10.1002/9780470027318.a5402
light. The light intensity is constant, but upon passage equal magnitude vertical and horizontal components.
through a sample exhibiting CD an intensity fluctua- The PEM exhibits birefringence so that .nZ nY / 6D 0,
tion (corresponding to the different absorptions of left i.e. the refractive indices nZ and nY for vertical and
and right circularly polarized light) that is in phase with horizontal polarizations are different. During passage
the modulator frequency appears. The unabsorbed pho- through the PEM the phase difference between the
tons hit a photomultiplier tube which produces a current vertical and horizontal light beam components will
whose magnitude depends on the number of incident amount to, Equation (87):
photons.
2pD 2pd.nZ nY /
The light source in most CD instruments is a xenon arc dD D .87/
lamp for UV and visible CD measurements. This means l l
that the instrument has most sensitivity in the 300 400 nm where d is the thickness of the PEM, l is the wavelength
region of the spectrum. The fact that a lamp is getting of the light, and D is the path difference between the
old becomes apparent by decreased light intensity in the light components. At a certain value of .nZ nY /, d will
200 nm and 700 nm regions of the spectrum. The optics be equal to p/2 and D equal to l/4, making the so-
of a typical CD machine are illustrated in Figure 27. A called quarter wave plate required to produce circularly
series of mirrors and prisms and slits are used to produce polarized light.
collimated monochromatic radiation (in reality it is not An ac (alternating current) voltage is applied to the
monochromatic but has a well-defined wavelength range, crystal part, causing the whole PEM assembly to oscillate
typically of 0.5 2 nm). This light is then linearly polarized (typically at 50 kHz). By adjusting the voltage amplitude
and subsequently circularly polarized. The conversion of so that the birefringence amplitude corresponds to the
linearly polarized light into circularly polarized light is quarter wave condition at each l, the time dependence
achieved by the PEM. of .nZ nY / as it oscillates between CjnZ nY j and
The PEM consists of a piece of crystal quartz jnZ nY j gives light that is alternately left and right
mechanically coupled (glued) to a piece of isotropic circularly polarized. The polarized light thus produced
(silica) quartz, the light passing through the latter. then passes through the sample compartment (and
The quartz plate is oriented so that light polarized the sample) and what is not absorbed is detected by
(say) vertically travels through it at one speed and the photomultiplier tube. If the sample has no CD
light polarized horizontally travels more slowly. If the the photomultiplier current will be a constant, direct
linearly polarized light is incident on the plate at a current (dc) with a magnitude determined by the normal
45 angle, then it may be considered as split into absorbance of the sample. If the sample exhibits CD
M0 M1
Xe
lamp
M2
1
MP
P1
M3
2
M4
P2
PO PO PEM
Lens
3
M5
Slit assembly
Figure 27 Schematic diagram of the optics of a CD spectropolarimeter. M denotes mirror, P denotes prism, and PO denotes
polarizer. As illustrated the monochromator prisms (P1 and P2) are also the polarizers; to improve the polarization further a series
of tilted quartz plates are inserted in the light path preceding the PEM. Sample and photomultiplier tube are attached after the
PEM.
DOI: 10.1002/9780470027318.a5402
the photomultiplier current will also show an oscillating ACKNOWLEDGMENTS

component (ac). The CD is obtained as the ratio between
the ac and dc components. Its sign is determined Professor Curtis Johnsons contribution to this article is
from the phase of the ac component using a lock-in gratefully acknowledged.
amplifier that has the ac voltage of the PEM as a time
reference.
With both DNA and protein CD experiments it
ABBREVIATIONS AND ACRONYMS
is important to think carefully about the buffer
to be used. CD instruments are single beam spec-
trometers and so there is no automatic compensa- ac Alternating Current
tion mechanism for buffer absorbance as there is CD Circular Dichroism
for normal absorbance spectroscopy where the buffer dc Direct Current
can be placed in a reference cuvette. Many CD EDA Electric Dipole Allowed
experiments fail or give nonsense results as all the EDF Electric Dipole Forbidden
light is absorbed by the nonchiral parts of the EDTM Electric Dipole Transition Moment
solution. HPLC High-performance Liquid Chromatography
ICD Induced Circular Dichroism
ISP Independent Systems/Perturbation
4.2 Design and Implementation of an Linear LD Linear Dichroism
Dichroism Experiment MCD Magnetic Circular Dichroism
The components of an LD experiment are a source MDA Magnetic Dipole Allowed
of linearly polarized light, a means of detecting how MDF Magnetic Dipole Forbidden
much light is absorbed, a method of orienting the sample MDTM Magnetic Dipole Transition Moment
(as discussed above), and a way to change the relative NMR Nuclear Magnetic Resonance
orientations of sample and light beam. There are two main OR Optical Rotation
methods for measuring LD spectra. The one requiring ORD Optical Rotatory Dispersion
less specialized equipment is the two-spectra method. PEM Photoelastic Modulator
Using a double beam spectrophotometer equipped with UV Ultraviolet
a polarizer (e.g. of Glan type) which is not sensitive to UV/VIS Ultraviolet/Visible
beam divergence, the sample is oriented parallel to the VCD Vibrational Circular Dichroism
polarization direction of the polarizer to obtain Ajj . It is
then oriented perpendicular to the polarization direction
of the polarizer to obtain A? . (Alternatively, the polarizer RELATED ARTICLES
may be rotated, in which case any effect of internal
polarization of the light by the optics of the spectrometer Biomedical Spectroscopy (Volume 1)
must be considered.) Photodynamic Therapy
The other method, the differential method, is much
easier to implement and a wider range of sample Biomolecules Analysis (Volume 1)
orientation techniques may be used. However, much Biomolecules Analysis: Introduction Circular Dichro-
more sophisticated instrumentation is required as the ism in Analysis of Biomolecules Vibrational Optical
phase-modulation technique of a circular dichrometer is Activity of Pharmaceuticals and Biomolecules
used, either by supplementing the latter with a quarter
wave device (e.g. a Fresnel rhomb or an Oxley prism Clinical Chemistry (Volume 2)
to achieve achromacy.3 7/ ) or by increasing the driving Ultraviolet/Visible Light Absorption Spectrophotometry
voltage of the PEM and doubling the beat frequency of the in Clinical Chemistry
lock-in amplifier. A factor of two increase in the voltage
across the quarter wave PEM that turns linearly polarized Forensic Science (Volume 5)
light into alternating left and right circularly polarized Chiroptical Spectroscopy in Drug Analysis
light for CD measurements, turns the PEM into a half-
wave plate that produces alternating pulses of orthogonal Nucleic Acids Structure and Mapping (Volume 6)
beams of linearly polarized light for LD spectroscopy. The Nucleic Acids Structure and Mapping: Introduction
differential method has the advantage of being extremely DNA Probes DNA Structures of Biological Relevance,
sensitive, needing only very low sample orientation or Studies of Unusual Sequences PNA and Its Applications
concentration. RNA Tertiary Structure
DOI: 10.1002/9780470027318.a5402
Pharmaceuticals and Drugs (Volume 8) General Linear Dichroism References

Pharmaceuticals and Drugs: Introduction Chiral Purity
J. Michl, E.W. Thulstrup, Spectroscopy with Polarized Light,
in Drug Analysis Proteins and Peptides Purification VCH, New York, 1986.
in Pharmaceuticals Analysis Steroid Analysis Vibra- B. Norden, Applications of Linear Dichroism Spectroscopy,
tional Spectroscopy in Drug Discovery, Development and Appl. Spectrosc. Rev., 14, 157 248 (1978).
Production B. Norden, C. Elvingson, M. Jonsson, B. Akerman, Micro-
scopic Behaviour of DNA During Electrophoresis: Elec-
Electronic Absorption and Luminescence (Volume 12) trophoretic Orientation, Quart. Rev. Biophys., 24, 103 164
Electronic Absorption and Luminescence: Introduction (1991).
Detectors, Absorption and Luminescence Ultraviolet B. Norden, M. Kubista, T. Kuruscev, Linear Dichroism Spec-
and Visible Molecular Absorption and Fluorescence Data troscopy of Nucleic Acids, Q. Rev. Biophys., 25, 51 170
Analysis (1992).
A. Rodger, B. Norden, Circular Dichroism and Linear Dichro-
Infrared Spectroscopy (Volume 12) ism, Oxford University Press, Oxford, 1997.
Infrared Spectroscopy: Introduction Infrared J. Schellman, H.P. Jensen, Optical Spectroscopy of Oriented
Reflection Absorption Spectroscopy Interpretation of Molecules, Chem. Rev., 87, 1359 1399 (1987).
Infrared Spectra, A Practical Approach Theory of A. Rodger, Linear dichroism, Methods Enzymol., 226, 232 258
Infrared Spectroscopy (1993).
Liquid Chromatography (Volume 13)

Liquid Chromatography: Introduction Biopolymer REFERENCES
Chromatography Chiral Separations by High-perfor-
mance Liquid Chromatography Column Theory and 1. R.E. Allen, (ed.) The Concise Oxford Dictionary, Claren-
Resolution in Liquid Chromatography Normal-phase don Press, Oxford, 1990.
Liquid Chromatography Reversed Phase Liquid Chro- 2. P.J. Stephens, M.A. Lowe, Ann. Rev. Phys. Chem., 36,
matography 213 241 (1985).
3. P.W. Atkins, Molecular Quantum Mechanics, Oxford
Nuclear Magnetic Resonance and Electron Spin Reso- University Press, Oxford, 1983.
nance Spectroscopy (Volume 13) 4. P.W. Atkins, Physical Chemistry, 4th edition, Oxford
Chemical Shifts in Nuclear Magnetic Resonance University Press, Oxford, 1991.
5. J.M. Hollas, Modern Spectroscopy, 2nd edition, John
Wiley and Sons, Chichester, 1992.
6. L.D. Barron, Molecular Light Scattering and Optical
FURTHER READING
Activity, Cambridge University Press, Cambridge, 1982.
7. D.P. Craig, T. Thirunamachandran, Molecular Quantum
General Circular Dichroism References Electrodynamics, An Introduction to Radiation Molecule
L.D. Barron, Molecular Light Scattering and Optical Activity, Interaction, Academic Press, London, 1984.
Cambridge University Press, Cambridge, 1982. 8. A. Rodger, B. Norden, Circular Dichroism and Linear
D.P. Craig, T. Thirunamachandran, Molecular Quantum Elec- Dichroism, Oxford University Press, Oxford, 1997.
trodynamics: An Introduction to Radiation Molecule Interac- 9. S. Allenmark, Chromatographic Enantiomer Separation,
tion, Academic Press, London, 1984. 2nd edition, Ellis Horwood Limited, Chichester, 1991.
N. Harada, K. Nakanishi, Circular Dichroic Spectroscopy: Exci- 10. W.H. Pirkle, A. Tsipouras, 3,5-Dinitrobenzoyl Amino-
ton Coupling in Organic Stereochemistry, University Science acid Esters Broadly Applicable Chiral Solvating Agents
Books, California, 1983. for NMR Determination of Enantiomeric Purity, Tetra-
S.F. Mason, Molecular Optical Activity and the Chiral Discrimi- hedron Lett., 26, 3989 3992 (1985).
nation, Cambridge University Press, Cambridge, 1982. 11. W. Moffitt, R.B. Woodward, A. Moscowitz, W. Klyne,
J. Michl, E.W. Thulstrup, Spectroscopy with Polarized Light, C. Djerassi, Structure and the Optical Rotatory Dis-
VCH, New York, 1986. persion of Saturated Ketones, J. Am. Chem. Soc., 83,
K. Nakanishi, N. Berova, R.W. Woody, (eds.) Circular Dichro- 4013 4018 (1961).
ism: Principles and Applications, VCH, New York, 1994. 12. E.G. Hohn, O.E. Weigang, Electron Correlation Models
F.S. Richardson, Theory of Optical Activity in the Ligand-field for Optical Activity, J. Chem. Phys., 48, 1127 1137
Transitions of Chiral Transition Metal Complexes, Chem. (1968).
Rev., 79, 17 36 (1979). 13. F.S. Richardson, Theory of Optical Activity in the
A. Rodger, B. Norden, Circular Dichroism and Linear Dichro- Ligand-field Transitions of Chiral Transition Metal Com-
ism Oxford University Press, Oxford, 1997. plexes, Chem. Rev., 79, 17 36 (1979).
DOI: 10.1002/9780470027318.a5402
14. P.E. Schipper, A. Rodger, Symmetry Rules for the 30. A. Rodger, B. Norden, A Coupled-oscillator Type App-
Determination of the Intercalation Geometry of Host/ roach to Magnetic Circular Dichroism, Enantiomer, 3,
Guest Systems Using Circular Dichroism: A Symmetry 409 421 (1998).
Adapted Coupled-Oscillator Model, J. Am. Chem. Soc., 31. J.A. Schellman, Symmetry Rules for Optical Rotation,
105, 4541 4550 (1983). Acc. Chem. Res., 1, 144 151 (1968).
15. A. Rodger, P.M. Rodger, The Circular Dichroism of 32. H. Eyring, J. Walter, G.E. Kimball, Quantum Chemistry,
the Carbonyl n-p Transition: An Independent Sys- John Wiley and Sons, New York, 1944.
tems/Perturbation Approach, J. Am. Chem. Soc., 110, 33. J.G. Kirkwood, On the Theory of Optical Rotatory
2361 2368 (1988). Power, J. Chem. Phys., 5, 479 491 (1937).
16. A. Rodger, M. Moloney, n-p Circular Dichroism of 34. Y. Matsuoka, B. Norden, Linear Dichroism Studies of
Planar Zig-Zag Carbonyl Compounds, J. Chem. Soc., Nucleic Acid Bases in Stretched Poly(vinyl alcohol)
Perkin II, 919 925 (1991). Film. Molecular Orientation and Electronic Transition
17. A. Toumadje, W.C. Johnson, Jr, Systemin has the Char- Moment Directions, J. Phys. Chem., 86, 1378 1386
acteristics of a Poly(L-proline)-II Type Helix, J. Am. (1982).
Chem. Soc., 117, 7023 7024 (1995). 35. J.R. Lacowitz, Principles of Fluorescence Spectroscopy,
18. D.A. Lightner, T.D. Bouman, W.M.D. Wijekoon, Plenum Press, New York, 1983.
A. Hansen, Mechanism of Ketone n ! p Optical Activ- 36. A. Wada, Dichroic Spectra of Biopolymers Oriented by
ity. Experimental and Computed Chiroptical Properties Flow, Appl. Spectros. Rev., 6, 1 30 (1972).
of 4-axial and 4-equatorial alkyladamantan-2-ones, J. 37. E. Fredericq, C. Houssier, Electric Dichroism and Electric
Am. Chem. Soc., 108, 4484 4497 (1986). Birefringence, Clarendon Press, Oxford, 1973.
19. B. Bosnich, The Application of Exciton Theory to 38. B. Norden, G. Lindblom, I. Jonas, Linear Dichroism Sp-
the Determination of the Absolute Configurations of ectroscopy as a Tool for Studying Molecular Orientation
Inorganic Complexes, Acc. Chem. Res., 2, 266 273 in Model Membrane Systems, J. Phys. Chem., 81,
(1969). 2086 2093 (1977).
20. P.E. Schipper, A. Rodger, Generalized Selection Rules 39. R.R. Sinden, DNA Structure and Function, Academic
for Circular Dichroism: A Symmetry Adapted Perturba- Press Ltd., San Diego, 1994.
tion Model for Magnetic Dipole Allowed Transitions, 40. P.-J Chou, W.C. Johnson, Jr, Base Inclinations in Natural
Chem. Phys., 109, 173 193 (1986) and references therein. and Synthetic DNAs, J. Am. Chem. Soc., 115, 1205 1214
21. J.A. Schellman, H.P. Jensen, Optical Spectroscopy of (1993) and references therein.
Oriented Molecules, Chem. Rev., 87, 1359 1399 (1987). 41. A. Rodger, H.C. Latham, P. Wormell, A. Parkinson,
22. L. Stryer, Biochemistry, 3rd edition, W.H. Freeman and M. Ismail, K.J. Sanders, DNA-drug Systems: how
Company, New York, 1988. Circular Dichroism Data is Complemented by other
23. K. Nakanishi, N. Berova, R.W. Woody, (eds.) Circular Spectroscopic Techniques, Enantiomer, 3, 393 408
Dichroism: Principles and Applications, VCH, New York, (1998).
1994. 42. Y. Matsuoka, B. Norden, Linear Dichroism Studies of
24. W.C. Johnson, Analyzing Protein Circular Dichroism Nucleic Acids. II Calculation of Reduced Dichroism
Spectra for Accurate Secondary Structures, Proteins: Curves of A- and B-form DNA, Biopolymers, 21,
Struct. Funct. Genet., 7, 307 312 (1999). 2433 2452 (1982).
25. P. Manavalan, W.C. Johnson, Variable Selection Method 43. W.C. Johnson, Jr, Circular Dichroism and Its Empirical
Improves the Prediction of Protein Secondary Structure Application to Biopolymers, Methods Biochem. Anal.,
Using a Simple Matrix Multiplication, Anal. Biochem., 31, 61 163 (1985).
167, 76 85 (1987). 44. S.F. Newbury, J.A. McClellan, A. Rodger, Spectroscopic
26. W.C. Johnson, Jr, Secondary Structure of Proteins and Thermodynamic Studies of Conformational Changes
Through Circular Dichroism Spectroscopy, Ann. Rev. in Long, Natural mRNA Molecules, Anal. Commun., 33,
Biophys. Biophys. Chem., 17, 145 166 (1988). 117 122 (1996).
27. H. Votavova, R. Matlova, J. Sponar, B Z Transition of 45. K.J. Sanders, Spectroscopic Investigations of Interactions
Synthetic Oligonucleotides Containing Non-Z Forming of Chiral Molecules in Biological Systems, PhD thesis,
Elements, J. Biomol. Struct. Dyn., 12, 163 172 (1994). University of Warwick, 1998.
28. A. Rodger, Linear Dichroism, Methods Enzymol., 226, 46. M.A. Ismail, DNA Ligand Interactions: A Biophysical
232 258 (1993). Study of 9-Hydroxyellipticine, Hoechst 33258 and a Meso-
29. L. Rosenfeld, Quantenmechanische Theorie Der Natur- substituted Porphyrin Derivative Binding to DNA, PhD
lichen Optischen Aktivitat Von Flussigkeiten Und thesis, University of Warwick, 1998.
Gasen, Z. Phys., 52, 161 174 (1928).
DOI: 10.1002/9780470027318.a5402

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CIRCULAR DICHROISM AND LINEAR DICHROISM 1

Circular Dichroism References 27

Alison Rodger CD D A D Al Ar (1)

Left Right Unoriented

Achiral molecule Chiral molecule Other enantiomer

Absorbance Absorbance Absorbance

Figure 1 Schematic illustration of CD.

biological systems relate to the shapes of molecules and B

between these two is given in Equation (9).6,8/

Charge transfer A 100 Eg CD D A.absorbance units/ D Al Ar

T2g 180 ln 10 32 982

1.5 Optical Activity (Optical Rotation)

0.1 OR D .nl nr / .10/

where (x,y,z) is the unit vector along the lines of the

average of different geometry factors (see carbonyl CD 10

motifs, others use a basis set of real protein spectra where 5

CD experiments performed on DNAs generally fall into

0.000 where the integral is over molecular position coordinates,

X where eO is the unit vector along E, bO is the unit vector along

is measured is a rotational average of Equation (29) in A C

Equation (51) is the basis of the above discussion of Eg e D h10 jA A C Vj10 i

ie  When A and C are not identical then the wavefunc-

where the C transition is not necessarily higher in energy .0cjVj10/

where is the magnitude of A and C . ea ec V 1c

CAR right-handed CAR left-handed

Again using the coordinate system defined in Figure 14

which is usually easier to implement than Equation (66).

follow. Rodger and Norden.8/ use the formalism adopted 0 +

II Polarized Polarized Oriented

2. If the polarization of the transition that is being

For intermediate polarizations, the LD is between these y polarized

cases. Thus, the polarization for a given transition can

Incident radiation linearly

Equation (84).8,34/ is polarizable) may be achieved by an electric field

3.3.3 Electric Field Orientation Bidirectional

3.3.7 Crystalline Samples 1.5

3.4 Examples of Linear Dichroism Data 0.006

are all polarized in the plane of the bases. If DNA 0.01

9-hydroxyellipticine aligns with the long axis of the

0.000 films, because the polarization of vibrations are often

axis system as illustrated in Figure 26. The values

100 Sz0 z0 D 0.024 Sy0 y0 D 0.256 Sy0 z0 D 0.373 .86/

the photomultiplier current will also show an oscillating ACKNOWLEDGMENTS

Pharmaceuticals and Drugs (Volume 8) General Linear Dichroism References

Liquid Chromatography (Volume 13)

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ie When A and C are not identical then the wavefunc-