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
2!
natural frequency eigenfrequency!
#
$ig. %" e&ample for a forced oscillation
After an initial time the oscillation of the sphere reaches a
stationary state in a forced oscillation.
A
'
( amplitude of the forced oscillation of the resonator
A
A
( amplitude of the agitator
( frequency of the forced vibration
( phase shift delay! between A
A
and A
'
$ig. )" phase shift"

%
resonator agitator
#*(+,#* ( )
unit radius
-#* ( /)
%.#* (

$ig. +" absorption and phase shift
<<
0
" the mass follows the agitator almost instantaneously
0*
(
0
" resonance ( - -#*0
>>
0
" the agitation is so fast that the mass cannot follow/
and the amplitude A
'
becomes almost 0ero1 ( %.#*
There is no energy transfer between agitator and
resonator.
The optical equivalent to the mechanical resonance (
0
is when the eigenfrequence of the electrons is equal to the
frequency of the incident light/ this is an absorption line in an
absorption! spectrum. The energy

h E
%!
is used to raise the energy of the electronic system
correspondingly e&citation!. There is also a phase shift due to
the fact that the oscillating electrons act as a 2ert0ian oscillator
emitting secondary radiation. This secondary radiation su3ers
)
%.#*
#*

A
'
4A
A
/
0
/
0
resonance curve
phase shift
from a delay with respect of the e&citation since the phase
shift is negative. The propagating wave in the matter/ now is
a superposition of all secondary waves 2uygens5 principle! and
the velocity of the light in matter
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 of
the matter"

m
c
n
)!
with
c
respectively
m
( +!
n is a function of the wave length respectively the frequency
. This phenomenon is called dispersion e. g. of light by a
prism!. The dispersion curve decreases usually with but in the
vicinity of an absorption band anormal dispersion is observed
and the refractive inde& increases with the wave length.
Resonance and phase delay result in absorption and
refraction.
+

A
n
absorption
curve
dispersion
curve
$ig. 6" absorption- and dispersion curve
Although it might look like that/ the dispersion curve is not
the 7rst derivative of the absorption curve. 8ven far away from
the absorption ma&imum there is always still a wave-length
dependent refractive inde& see a quart0 prism! even in the
completely transparent region.
In optically anisotropic systems/ e. g. some crystals like
calcite/ in oriented polymer 7lms or oriented liquids 9ow-
birefringence! or solutions with oriented molecules e. g."
lyotropic solutions in a shear / electric or magnetic 7eld! also an
anisotropy of physical properties e&pansivity/ heat
conductivity:! is observed. Correspondingly/ refractive inde&
and absorption become directional.
The e&tinction coe;cient molar or speci7c! is di3erent
whether the e&tinction E eq. 6! is measured parallel
1 1
! or
orthogonal

! linear dichroism! to the draw-direction of an

unia&ially drawn! 7lm and also n
1 1
n

. This phenomenon is
called birefringence.
A thin mica plate is birefringent but does not refract an
incident beam signi7cantly so that there is practically no
directional deviation of an incident beam. 2owever/ when the
incident beam is linearly polari0ed and enters the mica plate/
the single beam is splitted-up into two components according to
the di3erent refractive indices n
1 1
and n

/ respectively. If the
mica plate has a thickness of d #/#+. mm then the two beams
will show a phase delay of 46 46-plate!. The superposition of
these two sine-waves phase-shifted against each other by 46
creates a circular polari0ed wave after the 46-plate/ see 7g <.

$ig. <" generation of circular polarised light with a 46-plate.
6
Is the vector of the incident beam E

rotated by -#* or 4)! then

the direction of rotation is inverted. $inally/ when/ as shown in
7g. ./ left/ two counter-rotating circular polari0ed waves with
phase delay ( #=! are superimposed/ the result is a linear
polarised wave.
In other words" any linear-polari0ed wave can be constructed
from two counter-rotating circular polari0ed waves of the same
phase and amplitude/ and likewise any linear polari0ed wave
can be split-up into the corresponding two circular polari0ed
waves.
Basics of CD and ORD
An electron e&posed to a circular polari0ed wave is forced
into a corresponding helical movement/ 7g. <.
a
In an isotropic
environment it will not e&perience any di3erence/ however/ in a
chiral environment there will be a di3erence between left-and
right-handed symmetries/ see 7g. ,.
phenylalanine


left-handed L right-handed >
$ig. ," 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.
<
>
?
A C

>
?
A C

the sequence of the left circle yields" >A?C
the sequence of the right circle yields" >C?A
2ence/ when circular polari0ed light inde& L or >/ respectively!
passes matter solid or liquid! containing chiral
b
structures/
there will be two di3erent refractive indices/ n
L
and n
>
/ and two
di3erent e&tinction coe;cients
L
and
>
circular dichroism!.
The di3erence in the refractive indices results in a rotation of
the plane of linear-polari0ed light/ this e3ect is called optical
rotation dispersion.
The Experiment
$ig. @" e&perimental set-up of a CDexperiment. The light-
source LA provides white/ unpolari0ed light that is
monochromatic a! after it has passed the monochromator
Bono. It is linear polari0ed b! after it has passed the polari0er
Colar/ and circular polari0ed c! after it has passed the /4-plate.
This circular polari0ed light c! passes the sample cell. >uring
this passage the left and the right circular polari0ed parts of the
incident beam are subDect to a di3erent fate due to their
interaction with the chiral solution and meet the detector in
their superposition as elliptically polari!ed light. $inally/ the
e&tinction coe;cients
L
and
>
are determined depending on
the wavelength after rotating the /4-plate accordingly/ see
before!.
Lambert-?eer5s Law gives the extinction E
c
as"
( ) d E
I
I

,
_

0
ln
6!
with I
#
the intensity of the incident beam and I the intensity of
the light after the sample/ ! is the e&tinction coe;cient that
is either the speci"c quantity when the concentration is the
mass density Eg4LF 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 polari0ed light.
c
Also GabsorbanceG/ A. 2owever/ the term e&tinction may be better" attenuation! usually
also considers e3ects of luminescence and scattering.
,
LA Bono a Colar b /4 c Aample cell
>et
concentration c Emol4LF/ and d is the length of the sample cell.
Hsually/ the di3erence"
E() E
L
() - E
'
() ( d E
L
()
'
()F ( d () <!
is determined and the ellipticity molar or speci7c! is
calculated.
The ellipticity is e&pressed in terms of () and has the
following geometrical e&planation/ see 7g. . and 7g. -.
$
ig. ." linear polari0ed light as the sum of left and right circular
polarised light of the same intensity without phase delay left!.
Ihen there is a phase delay and a di3erence in intensity/ then
elliptically polari0ed light results as the sum right!. Jiven
actual values in C>-spectroscopy/ the ellipse would still appear
to be as thin as a single line given the scaling above.
@
$ig. -" geometrical e&planation of the ellipticity
Hsually the ellipticity is given by"
( )[ ] rad E E
R L r

4
303 . 2

respectively
( ) [ ] deg
180
4
303 . 2

R L d
E E
,!
d
The di3erence in e&tinction is usually very small %#
-)
K:%#
-+
K!
but can be determined very accurately. And the results are
given in mdeg %#
-+
deg ( %#
-+
*!.
The values of are frequently
normali0ed. In protein chemistry this is the mean molar
ellipticity per residue per amino acid!/
mrd
.
d
rad better" radian because the unit GradG is the non-unit of absorbed radiation! is the
unit of the plane angle/ based on the unit circle to give the angle in radian measure. rad
( %.#* %.# deg/ respectively %.#*!/ % rad <@/)-<:* $requently/ GradG is dropped/ so
that ( %.#*/ /2 ( -#*/ etc.
.

mass molar residual mean

r
d
mrd
N
M
d c

@!
M ( molar mass/ c ( molar concentration/ d ( length of the
sample cell/ N
r
( number of residues amino acids!. The value
of
mrd
is in practice usually of the order of %#
6
.
In general C>-measurements are useful for the following
problems"
>etermining if a protein is folded and gain information
about the secondary and tertiary structure/ see 7g. %#.
This enables to identify conformational changes during
processes/ comparison with mutants or proteins from
di3erent sources
Atudy conformational changes under stress such as p2/
heat/ denaturants etc/ see 7g. %%/ see also GThe Increment
BethodG.
Interactions with ligands drugs/ other proteins/ lipids:!
that change the conformation
>etermination of the in9uence of the solvent conditions on
the thermal reversible folding4unfolding.
-
$ig. %#" e&ample for a standard curve showing a standard curve
of polyL-lysine! in -helical conformation %##K! at p2 %#../ -
sheet conformation %##K! at p2 %%.%/ heating to <@*C and re-
cooling/ and in random coil conformation %##K! at p2 @. $or
analysis of a spectrum that contains all these structures
deconvolution is required.
$ig. %%" in9uence of tri9uoro ethanol T$8! content on the
conformation of the protein ALM-.
The protein ALM- consists in aqueous solution of <-K random
coil and 6% K helical conformation per molecule!. Addition of
T$8 increases the helical content/ see 7g. %#/ until at 6#K T$8 a
saturation is reached with @%K helical conformation and )-K
coil. The content of the di3erent conformations can be
determined by curve 7tting.
%#
The secondary structure can be investigated in the far-HN
region %-# nm:)<# nm! where signals are caused by a regular
folding of the peptide bond which is the chromophore!/ see 7gs
- and %#. 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-HN region )<# nm:+<# nm! can serve to
investigate certain aspects of the tertiary structure of proteins.
In this region the chromophores are aromatic amino acids
phenylalanine )<# nm-)@# nm/ )@# nm - )-# nm tyrosine/ ).#
nm - ++# nm tryptophan! and disulphide bridges broad signals
throughout near HN!. All these can be sensitive against changes
of the tertiary protein structure" the presence of signi7cant
near-HN signals can indicate a well-de7ned folded structure
while the absence of near-HN signals occurs in ill-de7ned three-
dimensional structures. Although the signal intensity is much
smaller compared with the far-HN/ even small changes in
tertiary structure can cause changes in the spectrum e. g."
protein-protein interactions/ changes of the solvent conditions!.
$or primary/ secondary etc. structures see addendum.
Che has a small e&tinction coe;cient because of high
symmetry and it is also the least sensitive to alterations in
its environment. Absorption ma&ima at )<6/ )<,/ ),) and
),@ nm vibronic bands!.

Tyr has lower symmetry then Che and therefore has more
intense absorption band. Tyr has absorption ma&imum at
)@, nm and a shoulder at ).+ nm. 2ydrogen-bonding to
the hydro&yl group leads to a red-shift of up to 6 nm. The
dielectric constant a3ects the spectrum also.
%%

Trp has the most intense absorption band centered at ).)
nm. 2ydrogen-bonding to the O2 can shift the
%
L
a
band by
as much as %) nm.
>isul7de A-A! spectra have a broad band at )<# - +## nm
with no vibronic structure.
%)
$ig. %)" small but signi7cant change of the tertiary structure of
a protein due to di3erent amounts of an antibody.
%+
$ig. %+" folding and unfolding GmeltingG! of a protein in three
di3erent bu3ers.
Optical Rotation Dispersion P'>! occurs because
chiral substances refract L-respectively '-circular polari0ed
light in a di3erent way. Jenerally spoken C> spectroscopy these
days has superseded P'>. The measured value is the rotation

of the plane of linear polari0ed light.

[ ]

d c d
.!
The square brackets indicate here the speci7c angle of rotation
per g material! while the dash indicates the molar quantity. The
other symbols are already de7ned. In the case of polymers the
molar masse of the monomer unit is used so that

is
independent of the degree of polymeri0ation. In the case of
peptides/ proteins and nucleic acids/ the already mentioned eq.
@! Gmean residual molar massG
r
N
M
M
-!
is used. $inally/ the Greduced mean residual molar rotationG

is obtained"
%6
2
3
2
3
2 2
+

n d
M
n d c

%#!
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 inde& of the
solvent.
#ummary
C> has an important role in the structural
determinants of proteins
2owever/ the e3ort e&pended in determining
secondary structure elements is usually not
worth it because it is somewhat unreliable.
The real power of C> 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.
C> is rapid and can be used to analy0e a
number of candidate proteins from which
interesting candidates can be selected for more
detailed structural analysis like OB' or Q-ray
crystallography.
$nvestigation of the #econdary #tructure of a
%rotein
Bonitoring
)))nm
of a protein as a function of temperature or
chemical denaturant yields information about protein stability.
The thermodynamic parameters/ J
u
/ 2
u
/ A
u
/ T
m
/ C
p
can be
determined
$ig. %6
%<

,
_

,
_

,
_

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
$ig. %< left" C> of hemoglobin/ elastase---/ and lyoso0ym:1
$ig. right"C> of various secondary structures from reference" -
heli& %!/ antiparallel -leaf )!/ -leaf +!/ coil 6!/ see also 7g.
%#
%,
%
)
+
6
The goal is to determine the fraction of basis set spectra that
add up to give the C> spectrum of the protein or the other way
around" deconvolute the spectrum into its components.
The absorption should be less than %.# usually R #.+! for cell
pathlengths of #.#< to % cm in order to maintain reasonable
signal-to-noise ratios and accurate C> measurements.
Crotein concentration used is typically % mg4mL
?u3er is typically %# mB phosphate with low salt if any.

Solvent Cut-Of (A=1.0) for
Two Dferent Cell !"t#len$t#%
Compound %.#
mm
#.#<
mm
2
)
P %.) %@,
$
,
iCrP2 %@6.< %,+
$
+
8tP2 %@-.< %@#
BeP2 %-<.< %.6
8tP2 %-, %.,
BeCO %.< %@<
>io&ane )+% )#).<
Cyclohe&a
ne
%.# %@<
n-Centane %@) %,.
The instrument must be calibrated"
typically an aqueous solution of S!-%#-camphorsulfonic acid
CAA!" % mg4mL in % mm cell" ( ).+, at )-#.< nm/ 8 (
%.#)%#
-+
/ ellipticity ( ++.< mdeg. At %-).< nm ellipticity ( ,-.,
mdeg. To accurately determine concentration of CAA solution"
8
).<nm
( #.@6+ in < cm cell/
).<nm
( +6.< B
-%
cm
-%
.
The protein concentration must be known accurately"
%@

%-#nm
( ./<## - %%/6## B
-%
cm
-%
per residue this is not accurate
enough" as you know the in the far HN of proteins depends on
the secondary structure!.

&'(nm
(in ) * +dmCl) , - of Trp residues ./)0( *
1
cm
1
2 - of Tyr
residues 1/&'( *
1
cm
1
#ome results sho3ing also results obtained from 4ray5
Crotein Technique
-
heli
&
2
antiparallel
--sheet
A
parallel
--
sheet
C
-
turn
T
othe
r
P
Eco'I
endonucleas
e
Q-ray ), )# . )< )%
Eco'I
endonucleas
e
>econvolutio
n of C>
spectrum
++ )# < %@ )<
calmodulin Q-ray <- + # - 6%
calmodulin >econvolutio
n of C>
spectrum
,% ) ) - +<
$ig. %,"cadmodulin and Eco'I endonuc
%.
$ig.%@" C>-spectrum of thymidylate synthetase is ++K 2/ )6K
A/ )K C/ )%K T/ )#K P. Hpon binding of $dHBC and </%#-
methylenetetrahydrofolate C> shows -<K A/ -,K T/ S.K P
lower curve!. $or 2/ A/ C/ T and P see table above.
6DDE7D8*
Experimental Conditions
Atandard conditions"
!roten Concentr"ton" #.< mg4ml
Cell !"t# &en$t#" #.< mm
St"'l(er% (Met"l on%) etc.)* minimum
+ufer Concentr"ton * < mB or as low as possible while
maintaining protein stability
The protein concentration might needs to be adDusted to produce the best
data. Changing this has a profound e3ect on the data/ so small increments
or decrements are advised. If that does not produce reasonably good data/
a change in bu3er composition may be necessary. It is also a good idea to
check the sample for une&pected aggregation via >ynamic Light
Acattering >OA repair en0ymes are an especially good e&ample of this
behavior!. If absorption turns out to be a problem/ cells with shorter path
#.% mm! and a correspondingly increased protein concentration and
longer scan time might help.
The $ncrement *ethod (to Chec9 for Consistency)
Calibration with standards of well-known secondary
structure e. g. polyamino acids!/ see 7g. %#/ allow a splitting of
the intensive
e
quantities of the measurement into increments
which can be utili0ed for an appro&imation of structural
estimates relative! of secondary structures/ in particular when
e
intensive quantity do not depend on the si0e of a system e. g." pressure/ density/
temperature:!/ e&tensive quantities do e. g." volume/ mass/ entropy/ enthalpy:!
%-
nm
the structure changes caused by e&ternal factors like
temperature or p2.
The e&tinction 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

%%!
This is valid as long as the individual sub-systems the
chromophores! do not interact with each other. As long as this is
the case/ e. g. the e&tinction doubles when the concentration is
doubled.
d
x
c
E
E
i
n
i
i
n
i
i

%)!
,

is the molar fraction of the component .
1

n
i
i
i
n
i
i
x
x

%+!
$or simpli7cation the number of components or structures! n is
assumed to be n ( )/ e. g. component % heli&!/ component )
coil!. This results for two di3erent wave lengths
a
and
b
in"
( ) ( )
( ) ( )
( ) ( )
( ) ( )
b b
b b
a a
a a
x and x




2 2
1
2
2 2
1
2

%6!
Is the same molar fraction &
)
found at more than one wave
length/ there is more reliability in the measurements. There
should be as many measurements at di3erent wave lengths as
there are components. The molar fraction can also be
determined with di3erent methods. They should be consistent.
8&ample"
The molar ellipticity is an intensive variable. If one assumes/ for
e&ample/ two di3erent structures T an heli& and a random coil
)#
T then the evaluation of the data at only one wave length is not
su;cient. Aame is true for + structures and evaluation at only
two wave lengths.
If there are interactions between the subsystems that
in9uence the ellipticity/ this can result in misinterpretations.
Again/ the only way is to do calculations at more than one
independent methods.
#ome more about %roblems determining the #econdary
#tructure
$ar-HN C> for determining protein structure
n : ; centered around &&( nm
p : ; centered around 10( nm
n : ; involves nonbonding electrons of O of the
carbonyl
: ; involves the electrons of the carbonyl
The intensity and energy of these transitions depends
on and (i<e</ secondary structure)
In a folded protein the amide is in a continuous array.
=or example/ the absorption spectrum of poly>lysine
in an helix/ sheet/ and unordered (random coil)
di?er due to longrange order in the amide
chromophore<
)%
$ar HN-C> of random coil 'C!
positive at &1& nm (:;)
negative at 10. nm (n:;)
$ar HN-C> of -sheet
negative at &1' nm (:;)
positive at 10) nm (n:;)
$ar HN-C> of -heli&
exciton coupling of the :; transitions leads to
positive (:;)
perpendicular
at 10& nm and negative (
:;)
parallel
at &(0 nm
negative at &&& nm is red shifted (n:;)
))
HN-
spectrum
$ar HN-C>
spectrum
$igures show deconvolutions of the HN-spectrum left! and the
C>-spectrum right!.
Hse far-HN C> to determine amounts of secondary
structure in proteins
generate basis sets by determining spectra of pure
helix/ sheet/ etc< of synthetic peptides
or deconvoluting CD spectra of proteins 3ith 9no3
structures to generate basis sets of each of secondary
structure
poly-L-lysine ULys!
n
V can adopt + di3erent conformations
merely by varying the p2 and temperature
random coil at p@ A<(
helix at p@ 1(<'
form at p@ 11<1 after heating to .&BC and recooling
)+
C> spectrum of unknown protein ( f

! S f

! S
f
'C
A
'C
!/ where A

!/ A

!/ and A
'C
! are derived from
poly-L-lysine basis spectra.
Recently #

() 3as derived from the spectrum of

myoglobin 3hich is '(C helix
Recently turn has been added to the above eDuation
E#
T
()F< #
T
() 3as derived from a combination of >%ro
D6la/ (6la
&
+ly
&
)
n
and %ro+ly>eu
form is no3 (>ys>eu)
n
in (<1 * 7a= at p@ A
Randomcoil is no3 (%ro>ys>eu>ys>eu)
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 al3ays agree from one lab to
another< $n addition/ chain length and aggregation
e?ect the basis set spectra< @o3ever/ this method is
usually accurate to 3ithin 1(C for helix content<
TechniDu
e
#econdar
y
#tructure
carboxypeptida
se

chymotrypsi
n
myoglobi
n
)+K .K ,.K
Q-ray %.K ))K #K
'C S other <-K @#K +)K

%+K %)K ,.K
C> using
Lys!
n

?asis Aets
+%K )+K <K
'C S other <,K ,<K )@K
If one has at least three proteins of known structure/ then
the following equation can be solved for A
i
!/ where f
i
!
are determined from the Q-ray structure"
spectrum of protein 3ith 9no3n structure
( )

rc
i
i i
S f
, ,

usually .1. proteins are used to generate basis set

spectra
)6

Disadvantages and problems 3ith this method5
choice of reference proteins is arbitrary and
e?ects results
determination of secondary structure from 4ray
data is subGect to error and disagreements among
groups
secondary structures are 7OT ideal in real
proteins (e<g</ helices can be bent/ the spectrum
of a H
1(
helix is di?erent from helix/ and turn
can be t3isted/ nonplanar/ or perpendicular or
parallel sheets<
6ppendix %roteins
Croteins 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" -heli&/ turns/ -sheets/ see
later
f
/ and GotherG sometimes addressed as
Grandom coilsG 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 7&ed by hydrogen bonds between
topologically neighbouring TCP- and TO2- groups.
supersecondary structure" helices and4or
sheets which are frequently repeated within a
protein. There are four maDor classi7cations" a!
proteins containing mostly helices/ b! proteins
f
heli& inducing amino acids are" Ala/ Arg/ Asp/ Jlu/ 2is/ Lys/ Bet/ Che/ Tyr/ and Trp/
sheets are induced by Cys/ Ile/ Aer/ Thr/ and Nal.
)<
containing mostly sheets/ c! proteins
containing helices and sheets in an irregular
sequence/ d! proteins with alternating sequences
of helices and sheets. Aome of these
structures can be associated with a particular
biological function. Pthers may be active only as
part of a larger structure/ for e&ample 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.
Duarternary 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.
=urther Reading
J. >. $asman/ Circular >ichroism and Conformational Analysis of
?iomolecules/ Lleuwer Acad. Cubl. %--,! Amsterdam
L. Oakanishi/ Circular >ichroism-Crinciples and Application/ Wohn
Iiley X Aons/ Oew York )###!
>. A. Lighter/ W. 8. Jurst/ Prganic Conformational Analysis and
Atereochemistry from Circular >ichroism Apectroscopy/ Iiley-
NCh/ Ieinheim/ Oew York )###!
=or De"nitions see in Chemistry and *acromolecular
#cience 5
Compendium of Chemical Terminology so-called Jold ?ook!
http"44iupac.org4publications4books4author4mcnaught.html
Compendium of Bacromolecular Oomenclature and Terminology
so-called Curple ?ook!"
http"44iupac.org4publications4books4author4metanomski.html
),
)@