Absorption Spectroscopy of Biopolymers Overview

Visible & near-UV regionwavelength (nm) Microwave & radiowave regionfrequency (Hz) Infared regionwavenumber (cm -1 ) Far-UV, x-ray,  -rayenergy (  =h )

Absorption & Emission Rapid process( s)

Absorption & Emission

Radiation-Induced Transition Absorption Stimulated emission Spontaneous emission

UV-Visible Spectroscopy Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state. Ultraviolet/Visible light: wavelengths ( ) between 190 and 800 nm

UV-visible spectrum The two main properties of an absorbance peak are: 1.Absorption wavelength max 2.Absorption intensity A max Housecroft and Sharpe, p. 466

Beer-Lambert Law Beer-Lambert Law : log(I 0 /I) =  bc  = A/c b A =  bc A =  c (when b is 1 cm) I 0 = intensity of incident light I = intensity of transmitted light  = molar absoptivity coefficient in cm 2 mol -1 c = concentration in mol L -1 b = pathlength of absorbing solution in cm -1 A = absorbance = log(I o /I) 0.1 cm ℓ

Beer-Lambert Law AAbsorbance or optical density (OD)  absorptivity; M -1 cm -1 cconcentration; M Ttransmittance

Transmittance, Absorbance, and Cell Pathlength

Deviations from the Beer-Lambert Law The Beer-Lambert law assumes that all molecules contribute to the absorption and that no absorbing molecule is in the shadow of another Low c High c

Sample Concentrations Solution too concentrated Diluted five-fold

UV-visible spectrum of 4-nitroanaline Solvent: Ethanol Concentration: 15.4 mg L -1 Pathlength: 1 cm Molecular mass = 138 Harwood and Claridge, p. 18

UV-visible spectrum of 4-nitroanaline 1.Determine the absorption maxima ( max ) and absorption intensities (A) from the spectrum: max = 227 nm, A 227 = 1.55 max = 375 nm, A 375 = Calculate the concentration of the compound: (1.54 x g L -1 )/(138 g/mol) = 1.12 x mol L -1 3.Determine the molar absorptivity coefficients (  ) from the Beer- Lambert Law:  = A/c ℓ  227 = 1.55/(1.0 cm x 1.12 x mol L -1 ) = 13,900 mol -1 L cm -1  375 = 1.75/(1.0 cm x 1.12 x mol L -1 ) = 15,700 mol -1 L cm -1

Molar absorptivities (  ) Molar absoptivities are very large for strongly absorbing chromophores (  >10,000) and very small if the absorption is weak (  = 10 to 100). The magnitude of  reflects both the size of the chromophore and the probability that light of a given wavelength will be absorbed when it strikes the chromophore. A general equation stating this relationship may be written as follows:  = 0.87 x P x a where P is the transition probability (0 to 1) a is the chromophore area in cm 2 The transition probability depends on a number of factors including where the transition is an “allowed” transition or a “forbidden” transition

UV-visible spectrum of 4-nitroanaline Solvent: Ethanol Concentration: 15.4 mg L -1 Pathlength: 1 cm Molecular mass = 138 Harwood and Claridge, p. 18

UV-visible spectrum of 4-nitroanaline 1.Determine the absorption maxima ( max ) and absorption intensities (A) from the spectrum: max = 227 nm, A 227 = 1.55 max = 375 nm, A 375 = Calculate the concentration of the compound: (1.54 x g L -1 )/(138 g/mol) = 1.12 x mol L -1 3.Determine the molar absorptivity coefficients (  ) from the Beer- Lambert Law:  = A/c ℓ  227 = 1.55/(1.0 cm x 1.12 x mol L -1 ) = 13,900 mol -1 L cm -1  375 = 1.75/(1.0 cm x 1.12 x mol L -1 ) = 15,700 mol -1 L cm -1

UV-visible spectroscopy definitions chromophore Any group of atoms that absorbs light whether or not a color is thereby produced. auxochrome A group which extends the conjugation of a chromophore by sharing of nonbonding electrons. bathochromic shift The shift of absorption to a longer wavelength. hypsochromic shift The shift of absorption to a shorter wavelength. hyperchromic effect An increase in absorption intensity. hypochromic effect A decrease in absorption intensity.

Absorption and Emission of Photons

Absorption and Emission Emission AbsorptionAbsorption: A transition from a lower level to a higher level with transfer of energy from the radiation field to an absorber, atom, molecule, or solid. EmissionEmission: A transition from a higher level to a lower level with transfer of energy from the emitter to the radiation field. If no radiation is emitted, the transition from higher to lower energy levels is called nonradiative decay. Absorption

Singlet and Triplet Excited States

Absorption and emission pathways McGarvey and Gaillard, Basic Photochemistry at

Selection Rules In electronic spectroscopy there are three selection rules which determine whether or not transitions are formally allowed: 1.Spin selection rule:  S = 0 allowed transitions: singlet  singlet or triplet  triplet forbidden transitions: singlet  triplet or triplet  singlet Changes in spin multiplicity are forbidden

Selection rules 2. Laporte selection rule: there must be a change in the parity (symmetry) of the complex Laporte-allowed transitions: g  u Laporte-forbidden transitions: g  g or u  u g stands for gerade – compound with a center of symmetry u stands for ungerade – compound without a center of symmetry 3. Selection rule of  ℓ = ± 1 (ℓ is the azimuthal or orbital quantum number, where ℓ = 0 (s orbital), 1 (p orbital), 2 (d orbital), etc.) allowed transitions: s  p, p  d, d  f, etc. forbidden transitions: s  s, d  d, p  f, etc.

 and  * orbitals

 and  * orbitals

Electronic Transitions:    * McGarvey and Gaillard, Basic Photochemistry at The    * transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”. /Spectrpy/UV-Vis/uvspec.htm#uv2

 * transitions - Triple bonds Organic compounds with -C≡C- or -C≡N groups, or transition metals complexed by C≡N - or C≡O ligands, usually have “low- lying”  * orbitals

Electronic Transitions: n   * McGarvey and Gaillard, Basic Photochemistry at The n-orbitals do not overlap at all well with the  * orbital, so the probability of this excitation is small. The  of the n  * transition is about 10 3 times smaller than  for the  * transition as it is “symmetry forbidden”. /Spectrpy/UV-Vis/uvspec.htm#uv2

Lycopene from Tomatoes

Chlorophyll B-carotene hemoglobin

Quantitative Analysis A plot of absorption versus wavelength is the absorption spectrum

Solutions containing the amino acids tryptophan and tyrosine can be analyzed under alkaline conditions (0.1 M KOH) from their different uv spectra. The extinction coefficients under these conditions at 240 nm and 280 nm are A 10-mg smaple of the protein glucagon is hydrolyzed to its constituent amino acids and diluter to 100 mL in 0.1 M KOH. The absorbance of this solution (1 cm path) was at 240 nm and at 280 nm. Estimate the content of tryptophan and tyrosine in mol (g protein) -1

Isosbestic points Isosbestic wavelength the wavelength at which two or more components have the same extinction coefficient The occurrence of two or more isosbestics in the spectra of a series of solutions of the same total concentration demonstrates the presence of two and only two components absorbing in that spectra region.

Isosbestic points

UV spectrum of BSA UV spectrum of DNA from E. coli

UV Absorption of amino acid

Effect of Secondary structure

Origin of Spectroscopic Changes 1.Change in local charge distribution 2.Change in dielectric constant 3.Change in bonding interaction 4.Change in dynamic coupling between different parts of the molecule

Human EyeRetina Retina Outer segment Light sensitive protein

Rhodopsin is a protein in the membrane of the photoreceptor cell in the retina of the eye. It catalyses the only light sensitive step in vision. The 11-cis-retinal chromophore lies in a pocket of the protein and is isomerised to all- trans retinal when light is absorbed. The isomerisation of retinal leads to a change of the shape of rhodopsin which triggers a cascade of reactions which lead to a nerve impulse which is transmitted to the brain by the optical nerve 1BRD

1BM1