You are on page 1of 37

# Circular Dicroism (CD) and Optical Rotational Dispersion

(ORD)
These spectroscopic techniques are suited to determine
chiral structures. In particular in proteins that means helical
and leaf-type structures.
Theory
Light is an electro-magnetic wave and interacts with
matter. Classically speaking the electrons are forced into an
oscillation. A forced oscillation can be modelled as a spring
with an inert mass coupled to a (mechanical) oscillator:
Agitation frequency ν
(ω · 2πν)
natural frequency (eigenfrequency) ν
0
Fig. 1: example for a forced oscillation
After an initial time the oscillation of the sphere reaches a
stationary state in a forced oscillation.
A
R
= amplitude of the forced oscillation of the resonator
A
A
= amplitude of the agitator
ν = frequency of the forced vibration
δ = phase shift (delay) between A
A
and A
R
Fig. 2: phase shift:

1
resonator agitator
0°=360° = 2π
90° = π/2
180° = π
δ
Fig. 3: absorption and phase shift
ν<<ν
0
: the mass follows the agitator almost instantaneously
δ≈0°
ν = ν
0
: resonance δ = - 90°0
ν>>ν
0
: the agitation is so fast that the mass cannot follow,
and the amplitude A
R
becomes almost zero; δ = 180°
There is no energy transfer between agitator and
resonator.
The optical equivalent to the mechanical resonance ν = ν
0
is when the eigenfrequence of the electrons is equal to the
frequency of the incident light, this is an absorption line in a(n
absorption) spectrum. The energy

ν ⋅ ·h E
(1)
is used to raise the energy of the electronic system
correspondingly (excitation). There is also a phase shift due to
the fact that the oscillating electrons act as a Hertzian oscillator
2
180°

δ
A
R
/A
A
ν/ν
0
ν/ν
0
resonance curve
phase shift
from a delay with respect of the excitation since the phase
shift δ is negative. The propagating wave in the matter, now is
a superposition of all secondary waves (Huygens' principle) and
the velocity of the light in matter υ
m
is smaller than the velocity
of light in vacuum c. The quotient is termed refractive index n
of the matter and is correlated with the dielectric constant ε of
the matter:
ε
υ
· ·
m
c
n
(2)
with
ν λ⋅ · c
respectively υ
m
= λ ⋅ ν (3)
n is a function of the wave length λ respectively the frequency
ν. This phenomenon is called dispersion (e. g. of light by a
prism). The dispersion curve decreases usually with λ but in the
vicinity of an absorption band anormal dispersion is observed
and the refractive index increases with the wave length.
⇒ Resonance and phase delay result in absorption and
refraction.
3
ν
λ
A
n
absorption
curve
dispersion
curve
Fig. 4: absorption- and dispersion curve
Although it might look like that, the dispersion curve is not
the first derivative of the absorption curve. Even far away from
the absorption maximum there is always still a wave-length
dependent refractive index (see a quartz prism) even in the
completely transparent region.
In optically anisotropic systems, e. g. some crystals like
calcite, in oriented polymer films or oriented liquids (flow-
birefringence) or solutions with oriented molecules (e. g.:
lyotropic solutions in a shear , electric or magnetic field) also an
anisotropy of physical properties (expansivity, heat
conductivity…) is observed. Correspondingly, refractive index
and absorption become directional.
The extinction coefficient ε (molar or specific) is different
whether the extinction E (eq. 4) is measured parallel (ε
11
) or
orthogonal (ε

) (linear dichroism) to the draw-direction of a(n
uniaxially drawn) film and also n
11
≠ n

. This phenomenon is
called birefringence.
A thin mica plate is birefringent but does not refract an
incident beam significantly so that there is practically no
directional deviation of an incident beam. However, when the
incident beam is linearly polarized and enters the mica plate,
the single beam is splitted-up into two components according to
the different refractive indices n
11
and n

, respectively. If the
mica plate has a thickness of d ≈ 0,038 mm then the two beams
will show a phase delay of λ/4 (λ/4-plate). The superposition of
these two sine-waves phase-shifted against each other by λ/4
creates a circular polarized wave after the λ/4-plate, see fig 5.

Fig. 5: generation of circular polarised light with a λ/4-plate.
4
Is the vector of the incident beam E

rotated by 90° (or π/2) then
the direction of rotation is inverted. Finally, when, as shown in
fig. 8, left, two counter-rotating circular polarized waves (with
phase delay δ = 0!) are superimposed, the result is a linear
polarised wave.
In other words: any linear-polarized wave can be constructed
from two counter-rotating circular polarized waves of the same
phase and amplitude, and likewise any linear polarized wave
can be split-up into the corresponding two circular polarized
waves.
Basics of CD and ORD
An electron exposed to a circular polarized wave is forced
into a corresponding helical movement, fig. 5.
a
In an isotropic
environment it will not experience any difference, however, in a
chiral environment there will be a difference between left-and
right-handed symmetries, see fig. 6.
phenylalanine

left-handed L right-handed D
Fig. 6: chiral structure
a
As in any circular electrical current, a magnetic moment comes
with the electrical dipole moment of the oscillating electrical
charge so that in case of an energy absorption (transition to a
higher energetical state) besides the electrical transition
moment also a magnetical transition moment has to be
considered.
5
D
B
A C

D
B
A C

the sequence of the left circle yields: DABC
the sequence of the right circle yields: DCBA
Hence, when circular polarized light (index L or D, respectively)
passes matter (solid or liquid) containing chiral
b
structures,
there will be two different refractive indices, n
L
and n
D
, and two
different extinction coefficients ε
L
and ε
D
(circular dichroism).
The difference in the refractive indices results in a rotation of
the plane of linear-polarized light, this effect is called optical
rotation dispersion.
The Experiment
Fig. 7: experimental set-up of a CD-experiment. The light-
source LS provides white, unpolarized light that is
monochromatic (a) after it has passed the monochromator
Mono. It is linear polarized (b) after it has passed the polarizer
Polar, and circular polarized (c) after it has passed the λ/4-plate.
This circular polarized light (c) passes the sample cell. During
this passage the left and the right circular polarized parts of the
incident beam are subject to a different fate due to their
interaction with the chiral solution and meet the detector in
their superposition as elliptically polarized light. Finally, the
extinction coefficients ε
L
and ε
D
are determined depending on
the wavelength (after rotating the λ/4-plate accordingly, see
before).
Lambert-Beer's Law gives the extinction E
c
as:
( ) d E
I
I
⋅ ⋅ · ≡

,
_

¸
¸
ρ λ ε
0
ln
(4)
with I
0
the intensity of the incident beam and I the intensity of
the light after the sample, ε (λ) is the extinction coefficient that
is either the specific quantity when the concentrationρ is the
mass density [g/L] the or the molar quantity (mass)
b
Chirality is a symmetry property and comes with the absence of a mirror plane or a
centre of inversion. Therefore, a chiral structure modulates polarized light.
c
Also "absorbance", A. However, the term extinction (may be better: attenuation) usually
also considers effects of luminescence and scattering.
6
LS Mono a Polar b λ/4 c Sample cell
Det
concentration c [mol/L], and d is the length of the sample cell.
Usually, the difference:
∆E(λ) · E
L
(λ) - E
R
(λ) = ρ ⋅d [ε
L
(λ)−ε
R
(λ)] = ρ ⋅d ⋅∆ε(λ) (5)
is determined and the ellipticity θ (molar or specific) is
calculated.
The ellipticity is expressed in terms of ∆ε(λ) and has the
following geometrical explanation, see fig. 8 and fig. 9.
F
ig. 8: linear polarized light as the sum of left and right circular
polarised light of the same intensity without phase delay (left).
When there is a phase delay and a difference in intensity, then
elliptically polarized light results as the sum (right). Given
actual values in CD-spectroscopy, the ellipse would still appear
to be as thin as a single line given the scaling above.
7
Fig. 9: geometrical explanation of the ellipticity θ
Usually the ellipticity is given by:
( )[ ] rad E E
R L r
− ·
4
303 . 2
θ

respectively
( ) [ ] deg
180
4
303 . 2
π
θ
R L d
E E − ·
(6)
d
The difference in extinction is usually very small (10
-2
%…10
-3
%)
but can be determined very accurately. And the results are
given in mdeg (10
-3
deg = 10
-3
°).
The values of θ are frequently
normalized. In protein chemistry this is the mean molar
ellipticity per residue (per amino acid), θ
mrd
.
d
unit of the plane angle, based on the unit circle to give the angle in radian measure. π rad
= 180° ( 180 deg, respectively 180°), 1 rad ≅ 57,295…° Frequently, "rad" is dropped, so
that π = 180°, π/2 = 90°, etc.
8
θ

mass molar residual mean
r
d
mrd
N
M
d c

·
θ
θ
(7)
M = molar mass, c = molar concentration, d = length of the
sample cell, N
r
= number of residues (amino acids). The value
of θ
mrd
is in practice usually of the order of 10
4
.
In general CD-measurements are useful for the following
problems:
• Determining if a protein is folded and gain information
about the secondary and tertiary structure, see fig. 10.
This enables to identify conformational changes during
processes, comparison with mutants or proteins from
different sources
• Study conformational changes under stress such as pH,
Method".
• Interactions with ligands (drugs, other proteins, lipids…)
that change the conformation
• Determination of the influence of the solvent conditions on
the thermal reversible folding/unfolding.
9
Fig. 10: example for a standard curve showing a standard curve
of poly(L-lysine) in α-helical conformation (100%) at pH 10.8, β-
sheet conformation (100%) at pH 11.1, heating to 57°C and re-
cooling, and in random coil conformation (100%) at pH 7. For
analysis of a spectrum that contains all these structures
deconvolution is required.
Fig. 11: influence of trifluoro ethanol (TFE) content on the
conformation of the protein AKQ9.
The protein AKQ9 consists in aqueous solution of 59% random
coil and 41 % helical conformation (per molecule). Addition of
TFE increases the helical content, see fig. 10, until at 40% TFE a
saturation is reached with 71% helical conformation and 29%
coil. The content of the different conformations can be
determined by curve fitting.
10
The secondary structure can be investigated in the far-UV
region (190 nm…250 nm) where signals are caused by a regular
folding of the peptide bond (which is the chromophore), see figs
9 and 10. Although the conformational contribution of a certain
secondary structure can be determined, it is not possible to
specify which particular residues are involved.
The near-UV region (250 nm…350 nm) can serve to
investigate certain aspects of the tertiary structure of proteins.
In this region the chromophores are aromatic amino acids
(phenylalanine 250 nm-270 nm, 270 nm - 290 nm tyrosine, 280
nm - 330 nm tryptophan) and disulphide bridges (broad signals
throughout near UV). All these can be sensitive against changes
of the tertiary protein structure: the presence of significant
near-UV signals can indicate a well-defined folded structure
while the absence of near-UV signals occurs in ill-defined three-
dimensional structures. Although the signal intensity is much
smaller compared with the far-UV, even small changes in
tertiary structure can cause changes in the spectrum (e. g.:
protein-protein interactions, changes of the solvent conditions).
For primary, secondary etc. structures see addendum.
Phe has a small extinction coefficient because of high
symmetry and it is also the least sensitive to alterations in
its environment. Absorption maxima at 254, 256, 262 and
267 nm (vibronic bands).

Tyr has lower symmetry then Phe and therefore has more
intense absorption band. Tyr has absorption maximum at
276 nm and a shoulder at 283 nm. Hydrogen-bonding to
the hydroxyl group leads to a red-shift of up to 4 nm. The
dielectric constant affects the spectrum also.
11

Trp has the most intense absorption band centered at 282
nm. Hydrogen-bonding to the NH can shift the
1
L
a
band by
as much as 12 nm.
Disulfide (S-S) spectra have a broad band at 250 - 300 nm
with no vibronic structure.
12
Fig. 12: small but significant change of the tertiary structure of
a protein due to different amounts of an antibody.
13
Fig. 13: folding and unfolding ("melting") of a protein in three
different buffers.
Optical Rotation Dispersion (ORD) occurs because
chiral substances refract L-respectively R-circular polarized
light in a different way. Generally spoken CD spectroscopy
these days has superseded ORD. The measured value is the
rotation α
λ
of the plane of linear polarized light.
[ ]
λ λ λ
α α ρ α ⋅ ⋅ · ⋅ ⋅ · d c d
(8)
The square brackets indicate here the specific angle of rotation
(per g material) while the dash indicates the molar quantity.
The other symbols are already defined. In the case of polymers
the molar masse of the monomer unit is used so that λ
α
is
independent of the degree of polymerization. In the case of
peptides, proteins and nucleic acids, the already mentioned (eq.
7) "mean residual molar mass"
r
N
M
M ·
(9)
is used. Finally, the "reduced mean residual molar rotation" α′
λ
is obtained:
14
2
3
2
3
2 2
+

·
+

· ′
n d
M
n d c ρ
α α
α
λ λ
λ
(10)
where the term
2
3
2
+ n
accounts for the fact that the system is in
solution and not in vacuum. n is the refractive index of the
solvent.
Summary
• CD has an important role in the structural
determinants of proteins
• However, the effort expended in determining
secondary structure elements is usually not
worth it because it is somewhat unreliable.
• The real power of CD is in the analysis of
structural changes in a protein upon some
perturbation, or in comparison of the structure
of an engineered protein to the parent protein.
• CD is rapid and can be used to analyze a
number of candidate proteins from which
interesting candidates can be selected for more
detailed structural analysis like NMR or X-ray
crystallography.
Investigation of the Secondary Structure of a
Protein
Monitoring λ
222nm
of a protein as a function of temperature or
chemical denaturant yields information about protein stability.
The thermodynamic parameters, ∆G
u
, ∆H
u
, ∆S
u
, T
m
, ∆C
p
can be
determined
Fig. 14
15

,
_

¸
¸

,
_

¸
¸
⋅ + − ∆ −

,
_

¸
¸
− ∆ · ∆
m
m p
m
T
T
T T T c
T
T
H G ln 1
( )
1
1
1
1
]
1

¸

,
_

¸
¸
⋅ ∆ −

− − ∆ + ∆
− +
− ·
RT
T
T
c T
T
H
T T T c H
m
p
m
m p
unfolded
ln
exp 1
1
1 φ
Fig. 15 left: CD of hemoglobin÷, elastase---, and lyosozym…;
Fig. right:CD of various secondary structures from reference: α-
helix (1), antiparallel β-leaf (2), β-leaf (3), coil (4), see also fig.
10
16
1
2
3
4
The goal is to determine the fraction of basis set spectra that
add up to give the CD spectrum of the protein or the other way
around: deconvolute the spectrum into its components.
The absorption should be less than 1.0 (usually < 0.3) for cell
pathlengths of 0.05 to 1 cm in order to maintain reasonable
signal-to-noise ratios and accurate CD measurements.
Protein concentration used is typically 1 mg/mL
Buffer is typically 10 mM phosphate with low salt if any.

Solvent Cut-Off (A=1.0) for
Two Different Cell Pathlengths
Compound 1.0
mm
0.05
mm
H
2
O 182 176
F
6
iPrOH 174.5 163
F
3
EtOH 179.5 170
MeOH 195.5 184
EtOH 196 186
MeCN 185 175
Dioxane 231 202.5
Cyclohexa
ne
180 175
n-Pentane 172 168
The instrument must be calibrated:
typically an aqueous solution of (+)-10-camphorsulfonic acid
(CSA): 1 mg/mL in 1 mm cell: ∆ε = 2.36 at 290.5 nm, ∆E =
1.02⋅10
-3
, ellipticity = 33.5 mdeg. At 192.5 nm ellipticity = 69.6
mdeg. To accurately determine concentration of CSA solution:
E
285nm
= 0.743 in 5 cm cell, ε
285nm
= 34.5 M
-1
cm
-1
.
The protein concentration must be known accurately:
17
ε
190nm
= 8,500 - 11,400 M
-1
cm
-1
per residue (this is not accurate
enough: as you know the ε in the far UV of proteins depends on
the secondary structure).
ε
280nm
(in 6 M GdmCl) = # of Trp residues ⋅ 5,690 M
-1
cm
-1
+ # of Tyr
residues ⋅ 1,280 M
-1
cm
-1
Some results showing also results obtained from X-ray:
Protein Technique
α-
heli
x
H
antiparallel
-β-sheet
A
parallel
-β-
sheet
P
β-
turn
T
othe
r
O
EcoRI
endonucleas
e
X-ray 26 20 8 25 21
EcoRI
endonucleas
e
Deconvolutio
n of CD
spectrum
33 20 5 17 25
calmodulin X-ray 59 3 0 - 41
calmodulin Deconvolutio
n of CD
spectrum
61 2 2 - 35
18
Fig.17: CD-spectrum of thymidylate synthetase is 33% H, 24%
A, 2% P, 21% T, 20% O. Upon binding of FdUMP and 5,10-
methylenetetrahydrofolate CD shows -5% A, -6% T, +8% O
(lower curve). For H, A, P, T and O see table above.
Experimental Conditions
Standard conditions:
Protein Concentration: 0.5 mg/ml
Cell Path Length: 0.5 mm
Stabilizers (Metal ions, etc.): minimum
Buffer Concentration : 5 mM or as low as possible while
maintaining protein stability
The protein concentration might needs to be adjusted to produce the best
data. Changing this has a profound effect on the data, so small increments
or decrements are advised. If that does not produce reasonably good data,
a change in buffer composition may be necessary. It is also a good idea to
check the sample for unexpected aggregation via Dynamic Light
Scattering (DNA repair enzymes are an especially good example of this
behavior). If absorption turns out to be a problem, cells with shorter path
(0.1 mm) and a correspondingly increased protein concentration and
longer scan time might help.
The Increment Method (to Check for Consistency)
Calibration with standards of well-known secondary
structure (e. g. poly(amino acids), see fig. 10, allow a splitting of
the intensive
e
quantities of the measurement into increments
which can be utilized for an approximation of structural
estimates (relative) of secondary structures, in particular when
e
intensive quantity do not depend on the size of a system (e. g.: pressure, density,
temperature…), extensive quantities do (e. g.: volume, mass, entropy, enthalpy…)
19
nm
the structure changes caused by external factors like
temperature or pH.
The extinction E can be written as a sum of increments (in
analogy to thermodynamic quantities like the enthalpy of
formation):
d
c
E
n
i
i i
⋅ ⋅ ·

ε
(11)
This is valid as long as the individual sub-systems i (the
chromophores) do not interact with each other. As long as this
is the case, e. g. the extinction doubles when the concentration
is doubled.
d
x
c
E
E
i
n
i
i
n
i
i
⋅ ⋅ · ·

ε
(12)
x
i
is the molar fraction of the component i.
1 ·
⋅ ≡

n
i
i
i
n
i
i
x
x ε
ε
(13)
For simplification the number of components (or structures) n is
assumed to be n = 2, e. g. component 1 (helix), component 2
(coil). This results for two different wave lengths λ
a
andλ
b
in:
( ) ( )
( ) ( )
( ) ( )
( ) ( )
b b
b b
a a
a a
x and x
λ ε λ ε
λ ε λ ε
λ ε λ ε
λ ε λ ε
2 2
1
2
2 2
1
2

·

·
(14)
Is the same molar fraction x
2
found at more than one wave
length, there is more reliability in the measurements. There
should be as many measurements at different wave lengths as
there are components. The molar fraction can also be
determined with different methods. They should be consistent.
Example:
The molar ellipticity is an intensive variable. If one assumes, for
example, two different structures – an α−helix and a random
20
coil – then the evaluation of the data at only one wave length is
not sufficient. Same is true for 3 structures and evaluation at
only two wave lengths.
If there are interactions between the subsystems that
influence the ellipticity, this can result in misinterpretations.
Again, the only way is to do calculations at more than one
independent methods.
Some more about Problems determining the Secondary
Structure
Far-UV CD for determining protein structure
n -> π* centered around 220 nm
p -> π* centered around 190 nm
n -> π* involves non-bonding electrons of O of the
carbonyl
π -> π* involves the π-electrons of the carbonyl
The intensity and energy of these transitions depends
on φ and ψ (i.e., secondary structure)
In a folded protein the amide is in a continuous array.
For example, the absorption spectrum of poly-L-lysine
in an α-helix, β-sheet, and unordered (random coil)
differ due to long-range order in the amide
chromophore.
21
• Far UV-CD of random coil (RC)
positive at 212 nm (π->π*)
negative at 195 nm (n->π*)
• Far UV-CD of β-sheet
negative at 218 nm (π->π*)
positive at 196 nm (n->π*)
• Far UV-CD of α-helix
exciton coupling of the π->π* transitions leads to
positive (π->π*)
perpendicular
at 192 nm and negative (π-
>π*)
parallel
at 209 nm
negative at 222 nm is red shifted (n->π*)
22
UV-
spectrum
Far UV-CD
spectrum
Figures show deconvolutions of the UV-spectrum (left) and the
CD-spectrum (right).
• Use far-UV CD to determine amounts of secondary
structure in proteins
generate basis sets by determining spectra of pure α-
helix, β-sheet, etc. of synthetic peptides
or deconvoluting CD spectra of proteins with know
structures to generate basis sets of each of secondary
structure
• poly-L-lysine {(Lys)
n
} can adopt 3 different conformations
merely by varying the pH and temperature
random coil at pH 7.0
α-helix at pH 10.8
β-form at pH 11.1 after heating to 52°C and recooling
23
• CD spectrum of unknown protein = f
α
S
α
(λ) + f
β
S
β
(λ) +
f
RC
S
RC
(λ), where S
α
(λ), S
β
(λ), and S
RC
(λ) are derived from
poly-L-lysine basis spectra.
Recently S
α
(λ) was derived from the spectrum of
myoglobin which is 80% α-helix
Recently β-turn has been added to the above equation
{S
T
(λ)}. S
T
(λ) was derived from a combination of L-Pro-
D-Ala, (Ala
2
-Gly
2
)
n
and Pro-Gly-Leu
β-form is now (Lys-Leu)
n
in 0.1 M NaF at pH 7
Random-coil is now (Pro-Lys-Leu-Lys-Leu)
n
in salt free
neutral solution.
The disadvantage of this method is that although these
basis sets are easily determined by direct
measurement, they do not always agree from one lab
to another. In addition, chain length and aggregation
effect the basis set spectra. However, this method is
usually accurate to within 10% for α-helix content.
Techniqu
e
Secondar
y
Structure
carboxypeptida
se
α-
chymotrypsi
n
myoglobi
n
α 23% 8% 68%
X-ray β 18% 22% 0%

RC +
other
59% 70% 32%

α 13% 12% 68%
CD using
(Lys)
n

Basis Sets
β 31% 23% 5%

RC +
other
56% 65% 27%
• If one has at least three proteins of known structure, then
the following equation can be solved for S
i
(λ), where f
i
(λ)
are determined from the X-ray structure:
spectrum of protein with known structure
( )

⋅ ·
rc
i
i i
S f
, , β α
λ
24
usually 5-15 proteins are used to generate basis set
spectra

Disadvantages and problems with this method:
• choice of reference proteins is arbitrary and
effects results
• determination of secondary structure from X-ray
data is subject to error and disagreements among
groups
• secondary structures are NOT ideal in real
proteins (e.g., α-helices can be bent, the spectrum
of a 3
10
-helix is different from α-helix, and β-turn
can be twisted, non-planar, or perpendicular or
parallel β-sheets.
Appendix Proteins
Proteins show a hierarchical structure which is also
known from other systems like liquid crystals:
• primary structure: the linear sequence of
amino acid constitutional units
• secondary structure: the local spatial
arrangement of the chain atoms of a chain
segment without regards to the conformation of
the side group (side chain) or to its relationship
with other segments. This leads to four types of
common secondary structures observed in
proteins, namely: α-helix, turns, β-sheets, see
later
f
, and "other" (sometimes addressed as
"random coils" although these structures are
neither random nor coils in the sense commonly
used in polymer science). The secondary
structure describes the coiling of the chain in
space as it is fixed by hydrogen bonds between
topologically neighbouring –CO- and –NH- groups.
• supersecondary structure: α−helices and/or
β−sheets which are frequently repeated within a
f
α−helix inducing amino acids are: Ala, Arg, Asp, Glu, His, Lys, Met, Phe, Tyr, and Trp,
β−sheets are induced by Cys, Ile, Ser, Thr, and Val.
25
protein. There are four major classifications: a)
proteins containing mostly α−helices, b) proteins
containing mostly β−sheets, c) proteins
containing α−helices and β−sheets in an irregular
sequence, d) proteins with alternating sequences
of α−helices and β−sheets. Some of these
structures can be associated with a particular
biological function. Others may be active only as
part of a larger structure, for example to form a
Ca binding site.
• tertiary structure: the arrangement of all the
atoms of a protein molecule or a subunit of a
protein molecule, regardless to its relationship
with neighbouring subunits or molecules.
• quarternary structure: the arrangement of the
subunits of a protein molecule in space and the
ensemble of its intersubunit contact and
interactions, regardless to the internal geometry
of the subunits. The subunits in a quarternary
structure have to be in non-covalent association.
G. D. Fasman, Circular Dichroism and Conformational Analysis
of Biomolecules, Kleuwer Acad. Publ. (1996) Amsterdam
K. Nakanishi, Circular Dichroism-Principles and Application, John
Wiley & Sons, New York (2000)
D. A. Lighter, J. E. Gurst, Organic Conformational Analysis and
Stereochemistry from Circular Dichroism Spectroscopy, Wiley-
VCh, Weinheim, New York (2000)
For Definitions see in Chemistry and Macromolecular
Science :
Compendium of Chemical Terminology (so-called Gold Book)
http://iupac.org/publications/books/author/mcnaught.html
26
Compendium of Macromolecular Nomenclature and
Terminology (so-called Purple Book):
http://iupac.org/publications/books/author/metanomski.html
PERICYCLIC REACTIONS
CHELETROPIC, DYOTROPIC REACTIONS
FRONTIER MOLECULAR ORBITAL (FMO) THEORY; WOODWARD HOFFMANN
RULES; HUCKEL MOBIUS RULES

Pericyclic reactions are the concerted reactions involving reorganization of electrons which
occur by the way of a single cyclic transition state.

Characteristics of Pericyclic reactions:
* The pericyclic reactions occur in single step and hence there is no intermediate formed during
the reaction.
* The breaking and making of bonds (both σ & π) occur simultaneously in a cyclic transition
state.
* The configuration of the product depends on
1) the configuration of reactants
2) the number of electron pairs undergoing reorganization and
3) the reaction conditions (like thermal or photochemical).

TYPES OF PERICYCLIC REACTIONS
The pericyclic reactions are further classified into following types:
2) Electrocyclic reactions
3) Sigmatropic rearrangements
4) Group transfer reactions
5) Cheletropic reactions
6) Dyotropic rearrangements

Cycloaddition reactions involve the formation of a cyclic product due to addition of two
different π bond containing components, which are joined by newly formed two σ bonds at their
ends at the expense of two π bonds.
It is usually reversible and the backward reaction is also referred to as retro-cycloaddition or a
cycloreversion.
The classic example of cycloaddition is Diels-Alder reaction between a Diene and a Dienophile
27
Note: The direction of curly arrows has no significance here. The movement of electrons can be
shown either clockwise or anti clockwise.
ELECTROCYCLIC REACTIONS
Electrocyclic reactions are intramolecular pericyclic reactions which involve the
rearrangement of π-electrons in an open conjugated system leading to formation of a cyclic
product with a new σ bond at the expense of a π-bond.
However the electrocyclic reactions not only involve ring-closure but also ring opening, which
are referred to as retro-electrocyclic reactions.
E.g. The formation of Cyclohexa-1,3-diene by heating Hexa-1,3,5-triene is an example of ring-
closure electrocyclic reaction.

SIGMATROPIC REARRANGEMENTS
Sigmatropic rearrangements are concerted unimolecular isomerization reactions
characterized by the overall movement of a σ-bond from one position to another with an
accompanying rearrangement of π-electrons of conjugated system so as to accommodate the new
σ-bond.
E.g. The [3,3] Cope rearrangement. The σ-bond undergoing movement is shown as red thick
line.
Note: Though looking like electrocyclic reactions, there is no reduction in the number of π-
bonds in sigmatropic reactions.

GROUP TRANSFER REACTIONS
The concerted transfer of a group from one molecule to another due to concomitant movement
of a σ-bond (from one molecule to another) and formation of a new σ-bond (between two
molecules) at the expense of a π-bond is generally referred to as group transfer pericyclic
reaction.
E.g. The Ene reaction between propene and ethene to give 1-pentene is a classic example of
group transfer reaction.
28
These reactions resemble sigmatropic rearrangements, since a σ-bond moves. However
sigmatropic reactions are unimolecular reactions whereas the group transfer reactions are
bimolecular.
They also resemble cycloadditions, since a new σ-bond is formed at the expense of a π-bond.
However, in group transfer reactions, no ring is formed.

CHELETROPIC REACTIONS
Cheletropic reactions are a special class of cycloadditions or retro-cycloadditions in which the
two σ-bonds are either made or broken to the same atom.
E.g. The reversible addition of sulfur dioxide to 1,3-butadiene is an example of cheletropic
reaction, in which the two new σ-bonds (shown in red) are made to the sulfur atom.

Note: In this reaction, a lone pair on sulfur atom is equivalent a π-bond and is reorganized.
One π-bond and a lone pair are disappeared, whereas two σ-bonds are formed.
Also note that sulfur atom is oxidized from +4 to +6 state.

DYOTROPIC REARRANGEMENTS
The pericyclic reactions which involve concerted intramolecular migration of two σ-bonds
simultaneously are known as dyotropic rearrangements.
However dyotropic reactions can also occur stepwise.
There are two types of dyotropic rearrangements:
Type-I: Two migrating groups interchange their relative positions

Type-II: The σ-bonds are migrated to new bonding sites without any positional interchange
for groups.

COMPARISON OF DIFFERENT TYPES OF PERICYCLIC REACTIONS
All types of pericyclic reactions are concerted and involve cyclic transition state without any
intermediate formed during the reaction. The characteristics which differentiate them from each
other are tabulated below.
S.no
Type of pericyclic
reaction
change in
no. of
σ bonds
change in
no. of
π bonds
1)
reactions
+2 -2
A cyclic product is formed;
may be intermolecular or
intramolecular.
2)
Electrocyclic
reactions
+1 -1 Intramolecular.
3) Sigmatropic 0 0 Intramolecular;
29
reactions
migration of a σ-bond;
rearrangement of π-electrons
4)
Group transfer
reactions
+1 -1
Intermolecular transfer of a group;
migration of a σ-bond from one
molecule to another;
formation of new σ-bond at the expense
of one π-bond.
5) Cheletropic reactions +2
-1 (π-bond)
-1 (lone pair)
A cyclic product is formed;
two σ-bonds are formed to same atom;
A lone pair is disappeared.
6) Dyotropic reactions 0 0 Simultaneous migration of two σ-bonds.

REACTION CONDITIONS FOR PERICYCLIC REACTIONS
It is observed that some of the pericyclic reactions occur only upon heating, whereas the other
are possible only under photochemical conditions.
E.g. The Diels-Alder reaction, a [4+2] cycloaddition occurs under thermochemical conditions
and is not possible under photochemical conditions.
Whereas the following [2+2] cycloaddition is forbidden under thermal conditions. But the
reaction is possible under photochemical conditions.
Even though, most of the times, the final products are thermodynamically stable, there is a
high kinetic barrier due to symmetry considerations under particular conditions to make the
reaction forbidden. However the same symmetry considerations allowed the reaction in different
conditions.
FRONTIER MOLECULAR ORBITAL (FMO) THEORY
Frontier Molecular Orbital (FMO) theory proposed by Kenichi Fukui in 1952, explains whether a
pericyclic reaction is allowed or not under given set of reactions conditions based on interactions
between frontier molecular orbitals (FMOs) like HOMO, LUMO & SOMO.
HOMO = Highly Occupied Molecular Orbital
LUMO = Lowest Unoccupied Molecular Orbital
SOMO = Singly Occupied Molecular Orbital

30
The interaction between one FMO of one molecule with one FMO of another molecule results in
two types of new Molecular Orbitals (MOs) i.e., bonding and antibonding. The bonding orbitals
possess low energy, whereas the antibonding orbitals possess higher energy.
If both of these resulting MOs are filled with electrons, the bonding interaction is cancelled by
the anti bonding interaction. Hence the net result is no bonding between molecules.
However, if only bonding orbitals are filled with electrons, the two molecules attract with each
other.

* Interaction between HOMO & HOMO causes repulsion i.e., no bonding interaction since both
bonding and antibonding MOs are filled with electrons.
* Interaction between HOMO & LUMO causes attraction i.e., bonding interaction, since only the
bonding MO is filled with electrons.
* Interaction between LUMO & LUMO causes neither attraction nor repulsion since all the
resulting MOs are empty.
* Interaction of SOMO with either HOMO or LUMO or another SOMO also causes attraction
between the interacting species.

The effects of interactions between frontier molecular orbitals is summarized in the following
table.
Interacting
Frontier Molecular Orbitals
Type of Interaction
HOMO + HOMO No bonding
HOMO + LUMO Attraction - Bonding
LUMO + LUMO No electrons, null interaction - No bonding
SOMO + HOMO
SOMO + LUMO
SOMO + SOMO
Attraction - Bonding

WOODWARD-HOFFMANN RULES
To predict whether a pericyclic reaction is allowed or not under given condition, Woodward and
Hoffmann proposed following set of rules based onconservation of orbital symmetry concept.
A thermal pericyclic reaction is allowed in the ground state, when the total number of
(4q + 2)s and (4r)a components is odd.
Otherwise, if the total of (4q + 2)s and (4r)a components is even, the pericyclic
reaction is allowed in the excited state i.e., under photochemical conditions.

Number of (4q + 2)s and
(4r)a components
The condition under which the reaction is
allowed
odd Thermal
even Photochemical
Component: A bond(s) or an orbital(s) taking part in the pericyclic reaction as a single unit
can be considered as a component. It can have any number of electrons but may not have
mixtures of π and σ electrons.
E.g.
A double bond is considered as a
π
2 component, since there are two π electrons.
A conjugated diene can be considered as
π
4 component, since there are four π electrons.

31
's' represents suprafacial. A suprafacial component forms new bonds on the same face at its
both ends. In some cases suprafacial is equivalent to "dis-rotation".
'a' represents antarafacial. An antarafacial component forms new bonds on the opposite faces
of its both ends. In some cases antarafacial is equivalent to "con-rotation".
E.g.

π
2s represents a component containing two π electrons and forming new bonds in
suprafacial manner.

π
4a represents a component containing four π electrons and is going to form new bonds in
antarafacial manner.

q & r: These are integers.
(4q + 2)s component: The suprafacial component, which may have either 2 or 6 or 10 or _ _
_ electrons of same type. These numbers are obtained by substituting 'q' by 0 or 1 or 2 or _ _ _.
(4r)a component: The antarafacial component, which may have either 4 or 8 or 12 or _ _ _
electrons of same type. These numbers are obtained by substituting 'r' by 1 or 2 or 3 or _ _ _.
Likewise the meanings of (4q + 2)a & (4r)s can be understood.

Application:
Let us assume the diene and dienophile in Diels-Alder reaction are approaching suprafacially as
shown below.
Since there are 4 π electrons in diene, which is making bonds in suprafacial manner, it is
a (4r)s component.
And the alkene is a (4q + 2)s component, since it has 2 π electrons and is approaching the
diene suprafacially.
i.e, there is one (4q + 2)s component and there are no (4r)a components.
Hence, the total number of (4q + 2)s and (4r)a components = 1 + 0 = 1, an odd number.
Therefore Diels-Alder reaction is thermally allowed in ground state when both the components
are approaching suprafacially. Hence it is termed as
π
4s+
π
Antarafacial addition, for this reaction, is not allowed under thermal conditions. But it is
theoretically allowed under photochemical conditions in the excited state. However, the strain in
the transition state while doing so forbids to do so.
32
Note: The orbitals shown in above diagrams are simple 'p' orbitals and are not the frontier
molecular orbitals. Do not mix descriptions of FMO theory with Woodward-Hoffmann rules.
HUCKEL-MOBIUS RULES BASED ON TOPOLOGY OF AROMATIC TRANSITION STATE
Since application of above Woodward-Hoffmann rules to pericyclic reactions is tedious and
cumbersome, the following simplified rules based on aromatic transition state proposed by
Zimmerman can be used to predict theoretically allowed modes of pericyclic reactions under given
conditions.
These rules are based on the concept of topology of aromatic transition state. The cyclic
transition state with 4n+2π electrons has Huckel topology under thermal conditions and Mobius
topology under photochemical conditions. Hence supra facial interaction between orbitals is allowed
under thermal conditions, whereas antara facial interaction is allowed under photochemical
conditions.
Whereas, the cyclic transition state with 4nπ electrons has Mobius topology under thermal
conditions and Huckel topology under photochemical conditions. Hence antara facial interaction
between orbitals is allowed under thermal conditions, whereas supra facial interaction is allowed
under photochemical conditions.
No. of π electrons Reaction conditions
Type of Aromaticity
in
Transition state
Allowed mode
(4n+2)
A Huckel number
Thermal Huckel Supra (or) Dis
Photochemical Mobius Antara (or) Con
(4n)
A non Huckel number
Thermal Mobius Antara (or) Con
Photochemical Huckel Supra (or) Dis

Remember that even though the pericyclic reactions are allowed theoretically under both the
conditions, most of the times the factors like steric hindrance and strain in the transition state may
forbid the reaction in particular mode, especially the antara facial one.
Inorganic chemistry deals with the chemistry of all the elements, in the periodic table, and
their compounds except that of carbon. It is a broad and complex field which overlaps with other
branches of science and is growing at a rapid pace.
The study of inorganic chemistry includes the extraction or synthesis, chemical properties and
applications of metals, metalloids and non metals as well as their compounds. It involves
interpreting, correlating, and predicting the properties and structures of an enormous range of
materials. We some time lend the physical principles from other areas of science to understand the
structure, characterization and properties of inorganic substances.
The word "inorganic" means non-living. However the study of inorganic chemistry encompasses
the role of inorganic compounds both in material and biological sciences. Some of the important
33
branches of modern inorganic chemistry are: coordination chemistry, bioinorganic chemistry,
organometallics etc.,
You can find various topics from inorganic chemistry from the list given below.
BASIC LEVEL INORGANIC CHEMISTRY
• Periodic table
• Group 14 (carbon family) Introduction
• Silicates
• Carbonates & Bicarbonates
• Coordination chemistry: Jahn-Teller effect
• Isolobal analogy
• Spinel & inverse spinel structures
• Pearson's Hard Soft Acid Base (HSAB) Theory
• Inorganic reaction mechanisms: Trans effect, preparation of cisplatin
• Wilkinson's catalyst
DISCLAIMER
The chemistry study material available on adichemistry web site can be utilized for personal
use only. Since the work on this site is copyrighted and the publication on the web does not imply
that this work becomes general property of public.
You are welcome to browse the site and take print-outs of this study material. However you
are discouraged to store or copy this material in electronic form; or print without prior permission
of the author.
However those who are interested in promoting science like teachers and students are
encouraged to utilize chosen diagrams and material from the site in their lecture presentations and
thesis, provided the source i.e., http://www.adichemistry.com is appropriately acknowledged.
MODERN PERIODIC TABLE
The modern long form of periodic table was constructed by Neils Bohr based on modern
periodic law proposed by Moseley.
LONG FORM OF MODERN PERIODIC TABLE
GROUPS
s-block d-block p-block
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
IA IIA IIIB IVB VB VIB VIIB VIII IB IIB IIIA IVA VA VIA VIIA 0
ns
1
ns
2
np
6
P
E
R
I
O
D
S
1
1H ns
2
ns
2
np
1
ns
2
np
2
ns
2
np
3
ns
2
np
4
ns
2
np
5
2He
2
3Li 4Be 5B 6C 7N 8O 9F 10Ne
3
11Na 12Mg (n-1)d
1-10
ns
1-2
13Al 14Si 15P 16S 17Cl 18Ar
4
19K 20Ca 21Sc 22Ti 23V 24Cr 25Mn 26Fe 27Co 28Ni 29Cu 30Zn 31Ga 32Ge 33As 34Se 35Br 36Kr
5
37Rb 38Sr 39Y 40Zr 41Nb 42Mo 43Tc 44Ru 45Rh 46Pd 47Ag 48Cd 49In 50Sn 51Sb 52Te 53I 54Xe
6
55Cs 56Ba 57-71 72Hf 73Ta 74W 75Re 76Os 77Ir 78Pt 79Au 80Hg 81Tl 82Pb 83Bi 84Po 85At 86Rn
7
87Fr 88Ra
89-
103
104Rf 105Db 106Sg 107Bh 108Hs 109Mt 110Ds 111Rg 112Cn
f-block
Lanthanoids
57La 58Ce 59Pr 60Nd 61Pm 62Sm 63Eu 64Gd 65Tb 66Dy 67Ho 68Er 69Tm 70Yb 71Lu
Actinoids
89Ac 90Th 91Pa 92U 93Np 94Pu 95Am 96Cm 97Bk 98Cf 99Es 100Fm 101Md 102No 103Lr

MODERN PERIODIC LAW
34
The modern periodic law was proposed by Moseley. He found the relation between atomic
numbers (Z) and the frequencies (ν) of X-rays produced when the atoms of different elements are
bombarded with cathode rays. The relation between the square root of frequency (√ν) of highest
energy emission line, called Kα line, with the atomic number, Z was found to be linear.
The mathematical relation can be presented as:
√ν = a(Z-b)
Where a & b are constants, characteristic of elements.
Later on it was clearly established that an element can be characterized by its atomic number,
Z and not by the atomic weight. It was also found that there is a relation between electronic
configuration and properties of elements. The number of electrons in an atom and its electronic
configuration are in turn are related to the atomic number.
Thus the modern periodic law can be stated as:
"The chemical and physical properties of elements are the periodic functions of their
atomic numbers and electronic configurations."
The modern long form of periodic table was constructed based on above law. The following
points are considered while constructing the periodic table.
* The elements in the periodic table are arranged in the increasing order of the atomic number.
* Every row, also called as period, in the periodic table starts with the filling up of
differentiating electron into a new quantum shell.
* The elements in a vertical column called as group should get similar outer electronic
configuration since it is observed that the elements with similar outer electronic configuration show
similar chemical properties.

SALIENT FEATURES OF LONG FORM OF MODERN PERIODIC TABLE
The long form of modern periodic table consists of seven rows called periods and eighteen
columns called groups.
Periods:
* Each period starts with an alkali metal and ends with an inert gas element.
* The first period is a very short period with only two elements i.e., Hydrogen (H) & Helium
(He). In this period, the 1s orbital is being filled up.
1
1H 2He

* The second period starts with Lithium (Li) and ends with Neon (Ne) and contains 8
elements. It is called first short period. In this group, the 2s & 2p orbitals are being filled up.
2
3Li 4Be 5B 6C 7N 8O 9F 10Ne

* The third period also contain 8 elements i.e., from Sodium (Na) to Argon (Ar). It is called
second short period. The 3s & 3p orbitals are being filled up in this period.
3
11Na 12Mg 13Al 14Si 15P 16S 17Cl 18Ar

* The fourth period is the first long period with 18 elements, it starts with Potassium (K) and
ends with Krypton (Kr). It also includes 10 elements belonging to 3d series i.e., from Scandium
(Sc) to Zinc (Zn). In this period, not only 4s & 4p and also the 3d orbitals are being filled up by
electrons.
4
19K 20Ca 21Sc 22Ti 23V 24Cr 25Mn 26Fe 27Co 28Ni 29Cu 30Zn 31Ga 32Ge 33As 34Se 35Br 36Kr
35

* The fifth period is the second long period with 18 elements, it starts with Rubidium (Rb)
and ends with Xenon (Xe). It also includes 10 elements belonging to 4d series i.e. from Yttrium (Y)
to Cadmium (Cd). The 5s & 5p along with 4d orbitals are filled up by electrons.
5
37Rb 38Sr 39Y 40Zr 41Nb 42Mo 43Tc 44Ru 45Rh 46Pd 47Ag 48Cd 49In 50Sn 51Sb 52Te 53I 54Xe

* The sixth period is the longest period with 32 elements. It not only includes 10 elements
belonging to 5d series i.e., Lanthanum (La), Hafnium (Hf) to Mercury (Hg) but also contains 14
elements belonging the 4f series called lanthanides (Cerium (Ce) to Lutetium (Lu)).
In this period, the 6s & 6p along with 4f & 5d orbitals are being filled up.
6
55Cs 56Ba
57-
71
72Hf 73Ta 74W 75Re 76Os 77Ir 78Pt 79Au 80Hg 81Tl 82Pb 83Bi 84Po 85At 86Rn
Lanthanoids
57La 58Ce 59Pr 60Nd 61Pm 62Sm 63Eu 64Gd 65Tb 66Dy 67Ho 68Er 69Tm 70Yb 71Lu

Note: Lanthanum is also considered to be the part of f-block since its properties resemble more
to lanthanoids.
* The seventh period is an incomplete period. It starts with Fr. It also includes the 14
elements belonging to 5f series called actinides (Thorium (Th) to Lawrencium (Lr)).
In this period, the 7s & 5f orbitals are filled up. Since this is an incomplete period, the filling up
of 6d is not yet completed.
7
87Fr 88Ra
89-
103
104Rf 105Db 106Sg 107Bh 108Hs 109Mt 110Ds 111Rg 112Cn
Actinoids
89Ac 90Th 91Pa 92U 93Np 94Pu 95Am 96Cm 97Bk 98Cf 99Es 100Fm 101Md 102No 103Lr
Note: Actinium is also considered to be the part of f-block since its properties resemble more to
actinoids.
* The Lanthanides and actinides are placed below the periodic table separately.

Groups:
* The 18 groups in the periodic table are numbered from 1 to 18 according to IUPAC
convention.
However, according to American convention, these are also denoted by IA , IIA, IIIB, IVB, VB,
VIB, VIIB, VIII (which actually includes 3 groups), IB ,IIB, IIIA, IVA, VA, VIA, VIIA and 0 (zero).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
IA IIA IIIB IVB VB VIB VIIB VIII IB IIB IIIA IVA VA VIA VIIA 0

* The elements of IA to VII A groups i.e., 1, 2, 13, 14, 15, 16 & 17 groups are called
as representative elements.
* The zero group (or 18th group) elements are called as inert gases or noble gases. This
group includes He, Ne, Ar, Kr, Xe and Rn.
* The elements of IIIB to IIB i.e., from groups 3 to 12 are called as transition elements.
* However the Lanthanoids and Actinoids are also considered to be the part of IIIB group (i.e.,
group 3). These are usually called as inner transition elements.
* The elements present in a group show similar physical & chemical properties since they have
similar outer electronic configurations.

36
UV spectroscopy- Absorption of this relatively high-energy light causes electronic
excitation. conjugated pi-electron systems
• Infrared Spectroscopy: Absorption of this lower energy radiation causes vibrational and rotational
excitation of groups of atoms. within the molecule. Because of their characteristic absorptions identification of
functional groups is easily accomplished.
• Nuclear Magnetic Resonance Spectroscopy: Absorption in the low-energy radio-frequency part of the
spectrum causes excitation of nuclear spin states. NMR spectrometers are tuned to certain nuclei (e.g.
1
H,
13
C,
19
F
&
31
P). For a given type of nucleus, high-resolution spectroscopy distinguishes and counts atoms in different
locations in the molecule.
1240692
37