Download Factors Affecting the Absorption Properties of Chromophore

Dr Gihan Gawish
1

The
absorption
spectrum
of
a
chromophore is primarily determined by
the chemical structure of the molecule.
A(λ)= e l c
Factors produce changes in max. λ and e
1.
2.
3.
pH effect
Polarity of the solvent or neighboring molecules.
The relative orientation of neighboring
chromophores
Dr Gihan Gawish
2


The pH of the solvent determines the
ionization state of ionizable chromophores.
Absorption spectrum of tyrosine at pH 6 and
13
pH 13
pH 6
4000
Λmax. At pH 6= 274nm
Λmax. At pH 13= 295nm
e
2000
250
270
Λ(nm).
290
310
Dr Gihan Gawish
3


The increase in Λ
max. in the less
polar solvent
Effect of solvent
polarity on the
spectrum of
tyrosine:
e
1. Solvents water (
solid line) and
2. 20% ethylene glycol
(dashed line)
250 260 270 280 290
Λ(nm)
Dr Gihan Gawish
4


Geometric features frequently have strong
effects on Λmax and e.
Spectra of DNA as a double stranded DNA
differs from a single stranded DNA or after
hydrolysis to free nucleuotides.
Dr Gihan Gawish
5
1.
Concentration measurements
According to Beer’s Lambert law we can calculate
concentrations directly from absorbance readings
at specific wavelength if we know the constant (
absorbitivity e).
2.Assay of chemical reactions
 Many chemical reactions can be assayed if
one of the reactants changes in absorbance
during the course of the reaction.
Dr Gihan Gawish
6


Most substances have characteristic spectra
and can be identified thereby
This can be done either by measuring a
complete spectrum or by measuring the
ratio of absorbance at different wavelengths.
Dr Gihan Gawish
7

The Λmax= 260nm of DNA increase if the
DNA
is
heated
through
a
particular
temperature range
Dr Gihan Gawish
8