Dr Gihan Gawish 1. The absorption spectrum of a chromophore is primarily determined by the chemical...

1Dr Gihan Gawish

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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