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 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

 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 Λmax. At pH 6= 274nm Λmax. At pH 13= 295nm Λmax. At pH 6= 274nm Λmax. At pH 13= 295nm 3

 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 Λ(nm)Λ(nm) 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). 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

 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