UV AND VISIBLE SPECTROPHOTOMETRY

Principle
1. Beer lambert’s law
When a beam of monochromatic radiation is allowed to pass through a homogenous light
absorbing medium, the intensity of the light coming out (transmitted light) decreases
exponentially with the increase in the concentration of absorbing medium and the path length
of the medium through which the light passes.

E = Ɛʎcd
E - Extinction
Ɛʎ - Molar extinction coefficient
c – Concentration
d - Path length
2. Absorption spectra

(i) Absorption spectrum or absolute absorption spectrum is shown as a

plot of light absorbed by that compound against wavelength.

(ii) Such a plot for a coloured compound has one or more absorption

(extinction) peaks in the visible region of the spectrum

(iii) Absorption spectra in the UV (200-400) and visible regions are

due to energy transition of both bonding and non-bonding outer
electrons of the molecule

(iv) Usually delocalised electrons are involved such as π bonding

electrons of c-c double bond [c ═ c] and lone pairs of nitrogen and
oxygen

(v) Since at room temperature most of the electrons in a molecule are at ground state, spectra
in this region will give information about this state and the next higher state

(vi) As the wave length of the light absorbed are determined by the actual occurring

 Specific absorption peaks may be recorded

 Related to known molecular structure

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 1

3. Chromaphore

Chromophore is a molecule or a part of a molecule that can be excited by absorption (or)

Chromophore is a part of the molecule which independently gives rise to distinct part of an
absorption spectrum.

4 Bathochromic Shift

Conjugation of double bond (ie, alternating) single and double bond (-c ═ c - c ═ c-) lower
the energy required for transition and hence causes an increase in the wave length at which a
chromophore absorbs. This is referred it as an Bathochromic shift

5 Hypsochromic Shift

A decrease in conjugation (eg: protonation)of an aromatic nitrogen increase the energy

required for transition and hence cause a decrease in wave length at which a chromopore
absorbs this is referred to as hypsochromic shift

6 Hyperchromic Effect

Hyperchromic effect refers to an increase in extinction

7 Hypochromic Effect

Hypochromic effect refers is a decrease in extinction (OD)

INSTRUMENTATION

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 2

1. Source of Light

 Tungsten lamp for visible region

 Hydrogen/Deuterium lamp for UV region

2. Monochromator

Monochromator is an optical system which produces a parallel beam of monochromtic

radiation form a multi wave length source of radiation if is usually based on

 refraction by a prism
 diffraction by grating

Prisms are made up of

 glass for visible region

 quartz/silica for uv region
3. Cuvette

(i) Material under study is normally dissolved in a suitable solvent and taken in an optically
transparent cell called cuvette

(ii) Cuvettes must be constructed of such a material which will not absorb in the region in
which the contents in the cuvettes absorb

 glass cuvettes are used for visible region

 quartz/silica cuvettes are used for UV region

(iii) If the solvent used is volatile/corrosive, stoppered cuvettes are used

(iv) Scratched/contaminated cuvettes reflect and absorb radiation, resulting in inaccurate

measurement

(v) To obtain valid data the contents of the cuvettes must be homogenous

(vi) Formation of bubbles or turbidity inside the cuvettes will lead to completely error result

(vii) The cuvettes commonly used for accurate work will have - optical path length of 1cm
and require 2.5 - 3 cm of sample for accurate reading.

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 3

4. Photocells

Photocell converts quanta of radiation (light energy) to electrical energy which may be -
amplified, detected and recorded.

In photo emission type, photons impinging on a metal surface in vacuum cause emission, of
electrons (in proportion to the intensity of radiation). The emitted electrons are attracted by a
+ ve electrode hence current flow which cause a potential difference across a resistor
incorporated in the system. By electronically amplifying the potential and balancing it against
a potentiometer calibrated directly to extinction units. Photomultiplier tube is more sensitive
than photocell.

5. Slit

To obtain very reliable data, the narrowest possible slit width should be used. Zero extinction
is usually obtained by adjusting the slit width.

APPLICATION:-

1. Qualitative Analysis
UV and visible spectra may be used to
 identify number of compound in pure state and in biological preparation
 Indicate chemical structure and intermediate occurring in a system.

2. Quantitative Analysis

 Protein, nucleic acid and other biological compound may be measured semi
quantitatively using UV and visible spectrophotometry
 Correction for impurities may than be applied by measuring the extinction at ʎ where
the impurities absorb more than sample.

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 4

3. Enzyme Assays and Kinetic Analysis

The determination of the activity of an enzyme is based upon the rate of utilization of
substrate or formation of product.

Enzyme
SUBSTRATE PRODUCT
Many substrate and products absorb light in visible and UV region and so the change in
absorbance can be used as the basic of enzyme assays if

 the substrate and product do not absorb at same ʎ [wave length]

 Beer-lambert’s law is obeyed.

Enzyme assay using UV and visible spectrophotometer are based on

 inter conversion of NAD (P)+ and NDA(P)H+H+

 Coupled reaction.
 using artificial substrate
 Using synthetic dyes.
 Conversion of product in to coloured derivative.

Based on the inter conversation of NAD (P) + and NAD (P) H+H+

A large number of assay are based on inter conversion of NAD (P) + and NAD (P) H+H+ both
oxidation and reduced form of these two nucleotides absorb light

eg. NAD+ NAD (P) H+H+

Oxidised form (260 nm) reduced form (340 nm)
Oxidised form absorb at 260nm & reduced form absorb at 340nm

Based on coupled reaction

Enzyme which do not involve the inter conversion of NAD (P)+ may be assayed by using
coupled reaction. In such reaction the enzyme to be assayed in linked to enzyme utilizing
NAD+/NAD (P)+system.

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 5

Example

Assay of phosphor fructo kinase (PFK)

Phosphofrutokinase can be linked via aldolase to glyceraldehyde 3-(P) reaction the assay
mixture should contain fru-6-(P) ATP, mg2+aldolase, gly-3-(P) Dehydrogenase and inorganic
phosphate in excess, so the rate of NADD+H+ production, ie, increase in absorption at 340nm
would be determination only by the concentration phosphor fructokinase in the known
volume of test preparation added to assay mixture

Use of artificial Substrate

The scope of visible spectro phometric assays is extended by use of artificial substrate and by
production of coloured derivative of substrate (or) product

Eg: assay of α-glucosidase (maltase)

Using synthetic dyes

An extension of this approach is the use of synthetic dyes for the study of oxidoreductases
oxidised and reduced form of these dyes have different colour

Eg :-


Tetraolium dyes


2,6 dichloro phenol indo phenol,


Methlylene blue
Methyl viologen
 Benzene viologen

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 6

By converting the product in to a coloured derivative

Product containing certain functional group can be converted in to coloured derivative

Eg:-
Orange dinitro-phenyl hydrazine derivatives of aldehyde and ketones
Assay of isocitrate lyase is based on this reaction

Isocitrate dehydrogenase

Isocitrate Succinate + Glyoxalate

Dinitro phenyl hydrazine

Glyoxalate dinitro-phenyl hydrazine [Orange]

PROTEIN STRUCTURAL STUDIES

The spectrum of the chromophore depends upon the polarity of the environment

SOLVENT PERTURBATION:-

The determination of whether an amino acid is external or internal by measuring the spectra
of a protein in a polar and nonpolar solvent is called the solvent perturbation method. Using
this method, the position of a particular amino acid in a protein can be located.

Perturbation solvent commonly used is solution of dioxane, glycrol, manitol, sucrose or

polyethylene glycol.

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 7

ROLE OF TYROSINE RESIDUE IN STRUCTURAL STUDIES OF PROTEIN:-

 The effect of pH, temperature and ionic strength on protein denaturation may be
studied if unfolding protein exposes tyrosine residues to the external environment
 If a protein –protein interaction (OR) -binding of ligand to protein involves tyrosine
residues it may be studied.
 Eg:- binding of substrate/inhibitor to the active site of an enzyme

NUCLEIC ACID STRUCTURAL STUDIES:-

Effect of pH temp and ionic strength on secondary structure of DNA may be studied

Hyperchromism of DNA:-

The extinction at 260nm of DsDNA in solution increase when it is related through its TM
(transition temp) due to denaturation of helix therefore unstacking of base leads to
hyperchromism

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 8

Hypochromism of DNA

On cooling the denatured DNA (SSDNA) is annealed to Ds DNA and hence the extinction at
260nm decrease due to stacking of based in DNA

Solvent perturbation of nucleic acid spectra can be clone by replacement of 50% of normal
water with Double distilled water it changes the spectral components due to unpaired
nucleotide. So the effect of 50% double distilled water on the spectrum of RNA allow the
estimation of the fraction of unpaired based

eg:- in t-RNA

ACTION SPECTRA

Action spectra is shown as a plot of physiological Parameters (non-extinction) against

wavelength

Eg: Plotting the rate of O2 evolution by green plant tissue against the wavelength of light
used to irradiate the system.

Mr. V. Magendira Mani., Asst. Professor., IC., VNB. Page 9