Circular Dichroism: Principle (Part A) - e-PG Pathshala

Biochemistry Biochemical Techniques

Circular Dichroism: Principle (Part A)

Description of Module

Subject Name Biochemistry

Paper Name 12 Biochemical Techniques

Module Name/Title 32 Circular Dichroism: Principle (Part A)

Paper : 12 Biochemcial Techniques Module : 32 Circular Dichroism: Principle (Part A)



Principal Investigator Dr. Sunil Kumar Khare, Professor, Department of Chemistry, IIT-Delhi

Paper Coordinator Dr.Y.S. Rajput, Emeritus Scientist, Animal Biochemistry Division, National Dairy Research Institute, Karnal

Content Writer Dr. Jai K. Kaushik, Principal Scientist Animal Biotechnology Centre National Dairy Research Institute, Karnal

Content Reviewer :

Circular Dichroism: Principle (PART A)

1. Objectives

1.1 To understand the concepts of optical activity, linear and circular polarization

1.2 To understand the principle of optical rotatory dispersion and circular dichroism



Circular Dichroism: Principle

1.0 Introduction

Optical rotatory dispersion (ORD) has been used to explore the conformations of

asymmetric molecules since very early days.ORD and circular dichroism (CD) are closely

related techniques and are very simple and quick for studying the structure and

conformation of macromolecules like nucleic acids and proteins. A protein may assume a

random or extended chain like structure, or folded structure constituted of helices, sheets,

turns, loops and coils. The secondary structural elements fold into tertiary structure and

subsequently to quaternary structure in some cases. Similarly, nucleic acids also possess

various polymorphic structures depending upon their sequence and environment. The

double stranded DNA may assume A-form, B-form, or Z-form or even higher order

structures like triplex and quadruplexes. On the other hand, single stranded DNA or RNA

may fold in to structures like hair pins, stem-loops, triplex, G-guadruplex or random coil

forms. The conformation of biological macromolecules is highly dynamic and may

change with physiological and environmental conditions. The conformation of proteins

and nucleic acids depends on the external factors like solution pH, ionic strength,

temperature and solvent nature.CD can be used to determine the secondary structural

content of proteins. CD has also been used to study tertiary interactions in proteins as well

as protein-solvent, protein-cofactors, and protein-protein or protein-drug/ligand

interactions. Nucleic acids also show specific CD spectrum for various forms and can be

used to identify them in solution under defined conditions. CD is also very handy in

understanding the DNA-protein and DNA-drug/ligand interactions. In general,

conformational changes in proteins or nucleic acids because of molecular binding,

association/dissociation or induced by solution condition (solvents, co-solutes, pH or

temperature) can be conveniently studied by CD spectroscopy. Commercially available

CD machines have very high sensitivity and can detect very minor changes in

macromolecular conformations.



2.0 Molecular asymmetry

Molecular asymmetry is the hallmark of optically active molecules. Therefore, it is

important to understand what makes an object or molecule structural asymmetric. The

asymmetry could be related to shape, structure, conformation or configuration of the

molecules. When we talk about small molecules, it is usually configurational asymmetry

while in case of macromolecules like (bio) polymers it is usually conformational

asymmetry. To simply the concept of molecular symmetry, we can consider the shape of

various objects we confront in our daily life. Objects which look identical when kept in

any position like a sphere has perfect symmetry, while other objects like cubes, discs,

cones, cylinders, hexagons and parallelograms, which over a particular rotation look

identical to the original object also possess some kind of symmetry defined by extent of

rotation or translation. Mirror images of such objects can be perfectly superimposed over

the original object. On the other hand, mirror images of some objects cannot be

superimposed on the original one and are known as asymmetric objects. Examples of

asymmetric objects are screws that have directional motion, likewise impeller and spiral

are also asymmetric objects. Some examples with explanation of their shape symmetry

are shown in Figure 1.

Like the symmetry of the shape of objects, if we investigate the structure and

configuration of molecules, they may also be classified either as symmetrical or

asymmetrical (Figure 2). Molecule whose mirror image can be superimposed on the

original molecule is known as symmetrical molecule, e.g. methane, tetrachloromethane

and benzene are examples of symmetrical molecules. On the other hand, mirror image of

molecules like 1-bromo-1-chloroethane and pyruvic acid cannot be superimposed on the

original molecules as shown in Figure 2. Among the amino acids, the simplest amino acid

glycine is also symmetric, while all other naturally occurring amino acids are asymmetric.

Glycine has two hydrogens at alpha carbon, while in case of alanine and all other amino

acids one of the hydrogen is substituted by a carbon chain (R-group) making all the four

groups around the central carbon (alpha carbon) dissimilar and hence are asymmetric

centres, also known as chiral centre. In case of 1-bromo-1-chloroethane the central carbon

is bonded to one chlorine, one bromine, one hydrogen and one methyl (R-group), a

constellation that renders the central carbon atom as an asymmetric centre. Similarly, the



pyruvic acid is also asymmetric because all the four groups around the central carbon are

dissimilar.

Figure 1: Images of symmetric and asymmetric objects. The objects shown on left side

are symmetric and their mirror image can be exactly superimposed on them. Images on

right side are inherently asymmetric and contained handedness. A nail without threads

constitute a symmetric shape while a screw is always asymmetric with threads running in

only one particular direction. Propellers, screws, seashells and rearview side mirrors of a

car have particular handedness. Rearview mirror inside the car is usually symmetric.

Rearview side mirrors placed on the door of the car have handedness. Left side mirror

cannot be used on right side and vice-versa. Seashells have either left handed curvature or

right handed curvature. Some species make only left handed while other only right

handed, while some other may produce both type of shells with preference for one type.

The above examples indicate that it is rather a particular centre which has asymmetric

constellation of groups around it that make it an asymmetric molecule, although other

centre could be symmetric in the same molecule, e.g. the central carbon in pyruvic acid

(Figure 2) is asymmetric because it makes bonds with four different groups, viz.,

hydrogen, hydroxyl, methyl and carboxyl groups; while the carbon in methyl group is

symmetric because it makes bonds with three equivalent hydrogens and a carbon.



Figure 2: Symmetric and asymmetric (chiral) molecules.

3.0 Optical activity

Optical properties of matter depends upon the chromophores present in the constituting

molecules. The discussion on interaction between light and matter is beyond the scope of

the current chapter and should be read elsewhere;however, the optical activity which is

related to asymmetry or chirality of the molecules will be explained here. Optical activity

is related to the rotation of the planepolarized light by a medium constituted of chiral

molecules. It may be noted that plane polarized light is same as linearly polarized light.

The emergent plane polarized light is seen by the observer approaching toward him/her

rotatingeither clockwise, i.e. in a right handed direction (dextro), or anticlockwise, i.e. left

handeddirection (laevo/levo). The dextrorotatory or laevorotatory molecules are denoted

by d- or l- for the respective rotationby prefixing the molecule name.The d- and l-

prefixes that are related to optical activity and direction of rotation can also be denoted by



(+) and (-) signs, respectively. The d- and l- are strictly related to only optical activity and

should not be confused with the D and L denotations that are also used as prefixes to

molecule name. The D and L is a system of writing the relative configuration or

stereochemistry(with respect to glyceraldehyde) of chiral molecules and has nothing to do

with optical activity. It is quite possible that a molecule with prefix D may rotate the

plane polarized light in anticlockwise direction, i.e. it can be l- or (-) in term of optical

activity.

If an optically active molecule rotates a plane polarized light by a certain angle (α), the

mirror image configurationof the molecule, which is called enantiomer (enantio means

opposite),would rotate the same light by angle (–α). The mirror image of the chiral

molecule isnon-superimposable and rotates light with equal magnitude in opposite

direction.The extent of rotation (angle α) is proportional to the concentration of the

medium and length of the cuvet containing the medium. Only optically active molecules

exhibit this property in pure form and the presence of equal ratio of d- and l- enantiomers

in a solution would generate clockwise and anticlockwise rotation, resulting in

cancellation of the rotations. Such mixtures are called racemic mixture. However, unequal

concentrations of d- and l- enantiomers should result in net rotation in one direction

depending upon the excess population of one of the enantiomer over the other.

A peculiar configuration or structure that results into chirality of the molecules is also

responsible for the rotation of the polarized lights. The asymmetric distribution of

electronic cloud of the chiral molecules rotate the plane of polarized light in a particular

direction irrespective of the orientation of the molecules in the solution. Thus all the

molecules present in the solution cause a net rotation of the plane of polarized light

emerging from the medium. In fact,components of the non-polarized lightare also rotated

individually by such medium, but the change in the orientation cannot be measured as

both the incident and emergent lights oscillate in all the planes. The rotation of the plane

of polarization is measurableonly if we use incident light oscillating in only one

direction/plane.

The optical activity is prominently exhibited when we use other types of polarized lights

known as circularly polarized light, which can either be right handed circularly polarized



light (RCPL) or left handed circularly polarized light (LCPL) as shown in Figure 3. The

optically active medium interact differentially with the RCPL and LCPL because of

different refractive indices (birefringent medium) and absorbance of the two lights in an

optically active medium. It can be emphasized that optically active medium show

birefringence. Also, the refractive index of the medium varies as a function of light

wavelength in an optically active medium, a phenomenon called optical rotatory

dispersion (ORD).

Figure 3: The schematic diagrams of ORD and CD phenomenon observed for an

optically active medium. Panel i. shows the superimposed incident LCPL (EL vector) and

RCPL (ER vector) components resulting in a linearly polarized light with electric vector

(ER+EL) oscillating linearly (vector shown in blue colour). Panel ii. shows that RCPL

experiences higher refractive index (n> 1.0) as well as absorption (> 0) while LCPL is

neither polarized nor absorbed in this medium (shown for the sake of clarity, in real both

components could be polarized and absorbed to different extent). This results in the

change of polarization of the emergent elliptical light by an angle α with respect to the

incident light. The ratio of the minor axis (b) to the major axis (a) of the ellipse is a

tangent of the angle , that is what we know asellipticity ( = tan-1(b/a) and is a

measurable property in a CD experiment.



Cotton while investigating the optical properties of potassium chromium tartarate solution

noted that for an optically active medium the extinction coefficient of LCPL and RCPL

were different. Here it is important to note that this happens only in the absorption band

of only optically active medium. Optically inactive material or mixtures of optically

active material (enantiomers) in equal ratio do not shown differential absorption of LCPL

and RCPL components. This also means that the electronic system or electronic

transitions responsible for the absorption of light is also responsible for the differential

absorption of polarized light by the optically active medium. If the chiral centre is

responsible for the absorbance by the medium, the absorbance of RCPL and LCPL

components will be unequal resulting into the phenomenon of circular dichroism (CD). In

other words, the optically active medium will exhibit different extinction coefficients (R

L) for the RCPL and LCPL. When equal amount of RCPL and LCPL is introduced in

the optically active medium, differential absorption of the light results in elliptically

polarized emerging light rather than the circularly polarized light. In fact, ORD and CD

are related optical properties having common structural basis and hence one can be

derived from another, if known.

It has also been observed that the molar rotation increases with decrease in wavelength

for one isomer of an optically active medium and this is known as positive cotton effect.

You can see curves in Figure 4. As wavelength decreases, optical rotation which is shown

by curve in red color also increases and reaches to maxima and suddenly starts decreasing

and reaches to zero, which is also the point of inflection, and then it becomes negative.

The optical rotation keep decreasing and reaches to maximal magnitude with negative

value. From where it start increasing toward zero.You can see that at wavelength where

CD band has a peak (see the curve shown in blue color), ORD is zero. The sudden change

in the angle of rotation when wavelength approaches peak of CD band is the hall mark of

optically active media and is known as cotton effect. It is important to note that a medium

exhibit Cotton effect only in its own absorption band, and also if the electronic system

responsible for the absorption band contributes to the optical activity. An optically



inactive medium will not show ORD passing through zero in the middle of the ORD

curve.

Figure 4:The cotton effect in the absorption region of an optically active medium.

There are two kinds of cotton effects, positive cotton effect if molar rotation increases with

decrease in wavelength. In positive Cotton effect, the optical molar rotation peak is at a

higher wavelength than its trough, whereas in negative Cotton effect, opposite is true. The

panel A in Figure 4shows positive cotton effect, while panel B shows negative cotton

effect.The enantiomers show cotton effect which are mirror image of each other. The above

two cases are for two enantiomers.You can also see that absorptive CD band is restricted to a

very narrow zone of wavelength, while ORD goes away from absorption band.This could

have several ramification in the experimental setup and gathering amount of information. CD

band is located where medium have normal absorption band and hence describe the



electronic system more accurately.CD signals are more sensitive and precise due to small

windows of absorbance, while ORD is more extended and dispersive taper down in a shallow

manner from the centre of absorbance peak as you can see in Figure 4. On the other hand, the

ORD spectrum is extended well beyond absorption band and that may provide additional

information in the wavelength zone where CD measurement is difficult either due to too

week signal or in region of high absorbance e.g. in a wavelength region below 190 nm. LCPL

and RCPL will always experience different refractive index at all wavelengths and hence

ORD can be also helpful in the region of very small or no CD absorbance but where ORD

signal is measurable.

In part B, I shall introduce you with the various types of polarization and how are they

produced, their interaction with optically active media resulting in to the phenomena of ORD

and CD.

Circular Dichroism: Principle (Part A) - e-PG Pathshala

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