Applications of Spectroscopic

Techniques

Amandeep Singh

Types of Electromagnetic Radiations

EFFECT OF ELECTROMAGNETIC RADIATION

ON MOLECULES

IR Spectroscopy
Infrared radiation is largely thermal energy.
It induces stronger molecular vibrations in covalent bonds

Specific bonds respond to (absorb) specific frequencies

Applications
1.
2.
3.
4.

Identification of Compounds
Rate of Reactions
Study of Confirmation
Interaction between molecules

Applications
1.

Identification of Compounds

Macromolecules= Large no. of atoms= Large no. of fundamental vibrations

Example: C14H28O
= possess either one ring or one double bond
If double bond involving carbon is present= 1600-1800 cm-1
One lone oxygen atom can exist as carbonyl group or hydroxyl group or as ether
If as hydroxyl group is present= 3100-3500cm-1
Compound showing both these bands, thus there is a double bond involving carbon
is present and the lone oxygen is present as hydroxyl group.

Applications
2.

Rate of Reactions
If substrate possess a hydroxyl group and product do not, then rate of reaction can
be measured by rate of disappearance of O-H stretching vibrations.
Many functional groups in biological macromolecules exchange their protons with
protons of water.

If protein is dissolved in D2O

-N-H + D2O
-O-H + D2O
-S-H + D2O

-N-D + HDO
-O-D + HDO
-S-D + HDO

Atom of mass no. 1 is replaced with atom of mass no. 2 = Frequency of absorption
decreases.

Applications
Study of conformation
Polypeptide repeat units give rise to 9 characteristics infrared absorption bands.
3.

Infrared bands of the peptide linkage

Nuclear Magnetic Resonance

Spectroscopy

Applications
1.
2.
3.
4.
5.
6.
7.
8.
9.

Structural Diagnosis
Study of dynamic characteristics of protein
structure
Complex formation
Biological structures and compartments
Quantitative studies
Thermodynamics studies
Membrane transport
Intact organ studies
Zeugmatography

Applications.
1.

2.

3.

4.

Structural Diagnosis
Structural information which relates to biological functions of antibiotics such as
valinomycin and gramicidin.
Structural information about neurotoxins, cytochromes, hen egg-white lysozyme
and calcium binding proteins.
Study of dynamic characteristics of protein structure
Opening of secondary structure, aromatic side chain rotation etc.
Proteins includes histones, cytochrome b5, prothrombin, plasminogen and
chromogranin A.
Complex formation
Binding of ligand to an enzyme, a drug to DNA, an agonist to a receptor, an
antigen to antibody.
Biological structures & compartments
Confirmation of lipid headgroups of the biological membranes and their
interaction with integral proteins of the membrane.

Applications
5.

6.

7.

8.
9.

Quantitative studies
Concentration of metabolites.
Phosphocreatine concentration in muscles and hexakisphosphatephytic acid in
pith tissues of plants.
Thermodynamic studies
Binding constants, heat and entropy of bindings, pKa values, partition
coefficients etc.
Membrane transport
Alanine and lactate transport in the human erythrocytes by exploiting the
difference in magnetic susceptibility between the inside and outside of the cells.
Intact organ studies
Intact biological specimens as heart, kidney and skeletal muscles.
Zeugmatography
Biological objects such as fruits, nuts and mice have been imaged by n.m.r.
zeugmatography.

ORD & CD Spectroscopy

DIFFERENECES BETWEEN ORD AND CD

OPTICAL ROTATORY DISPERSION
(ORD)

CIRCUALR DICHROISM
(CD)

ORD is If the refractive indices of the

sample for the left and right handed
polarized light are different, when the
components are recombined , the plane
polarized radiation will be rotated through
and angle

Circular dichroism is the differential

absorption of left and right handed
circularly polarized light

ORD spectra are dispersive

CD spectra are absorptive

In ORD the circular polarized light is used

is not converted to elliptical light

In CD the circular polarized lgiht is used

and is converted to elliptical light

ORD graphs are obtained by plotting

specific rotation vs wavelength.

CD graphs are obtained by ploting molar

ellipticity vs wavelength.

Applications

CD/ORD of Proteins

OPTICAL ACTIVITY
L-amino acids &
D-amino acids

Asymmetric distribution of
charges/dipole about a
chromophore;
Tertiary Structure
Ordered
arrangements;
-helix

Example: Poly-L-lysine; Soluble in water at pH 5.7; -amino groups are protonated.

Conformations
Random coil

-helix

-pleated sheets

pH 5.7

pH 11.1

Heat 52C; 15 minutes

Weak +ve CD= 217nm

Weak +ve CD= 191nm

Weak +ve CD= 195nm

Strong ve CD= 197nm

Strong ve CD= 208 & 222 nm

Strong ve CD= 217nm

Applications
Conformational Changes in enzymes
Enzyme= p-hydroxybenzoate hydroxylase
Co-enzyme= NADH or NADPH

On NADH addition, No significant change in CD spectrum

On NADPH addition, significant change will occur

Applications

CD/ORD of Carbohydrates
Give information about nature, extent and position of substitution in carbohydrates.
e.g. Oligosaccharides from blood group substances and human milk.
Analysis of trace amounts of mucopolysacharides such as dermatin sulphate and
heparin sulphate. Both will accumulate in urine of patients with Hurler Syndrome.
Hurler Syndrome

Sanfilippo variant

Heparin sulphate
is accumulated

2 Variants

Hurler variant
Heparin sulphate
+
Dermatin sulphate
are accumulated

Applications

CD/ORD of Nucleic acids

Sugar-phosphate backbone does not absorb significantly above 180 nm.
Therefore, in CD of nucleic acids, most important contributors are: purines and
pyrimidines bases.

1.

Single-double stranded transition of nucleic acids

Denaturation of nucleic acids
Binding of proteins
Difference between B & A forms of DNA
Base stacking
DNA conformation in bound and free state

2.
3.
4.
5.
6.

Thank
s