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1Dr Gihan Gawish
2
The absorption spectrum of a chromophore is primarily determined by the chemical structure of the molecule.
A(λ)= e l c
Dr Gihan Gawish
Factors affecting the absorption of chromophore
Factors produce changes in max. λ and e 1. pH effect2. Polarity of the solvent or neighboring molecules.3. The relative orientation of neighboring
chromophores
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The pH of the solvent determines the ionization state of ionizable chromophores.
Absorption spectrum of tyrosine at pH 6 and 13
Dr Gihan Gawish
1. pH affect
pH 6pH 6 pH 13pH 13
Λ(nm).Λ(nm).
ee
20002000
40004000
250250 270270 290290 310310
Λmax. At pH 6= 274nmΛmax. At pH 13= 295nmΛmax. At pH 6= 274nmΛmax. At pH 13= 295nm
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The increase in Λ max. in the less polar solvent
Effect of solvent polarity on the spectrum of tyrosine: 1. Solvents water
( solid line) and2. 20% ethylene
glycol (dashed line)
Dr Gihan Gawish
2.Polarity effect
ee
250250 260260 270270 280280 290290
Λ(nm)Λ(nm)
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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
3.Orientation effect
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1. Concentration measurementsAccording to Beer’s Lambert law we can calculate
concentrations directly from absorbance readings at
specific wavelength if we know the constant
( absorbitivity e).
Dr Gihan Gawish
Application of Absorption Spectroscopy
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.
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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
3. Identification of substances by spectral measurement
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The Λmax= 260nm of DNA increase if
the DNA is heated through a particular
temperature range
Dr Gihan Gawish
4. The helix coil transition of double stranded DNA: denaturation& renaturation
