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Dr Gihan Gawish 1
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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 2 Factors produce changes in max. λ and e 1.pH effect 2.Polarity of the solvent or neighboring molecules. 3.The relative orientation of neighboring chromophores Factors produce changes in max. λ and e 1.pH effect 2.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 pH 6 pH 13 Λ(nm).Λ(nm). e e 2000 4000 250 270 290 310 Λmax. At pH 6= 274nm Λmax. At pH 13= 295nm Λmax. At pH 6= 274nm Λmax. At pH 13= 295nm 3
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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) and 2.20% ethylene glycol (dashed line) Dr Gihan Gawish e e 250 260 270 280 290 Λ(nm)Λ(nm) 4
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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 5
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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). Dr Gihan Gawish 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. 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. 6
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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 7
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The Λmax= 260nm of DNA increase if the DNA is heated through a particular temperature range Dr Gihan Gawish 8
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