UV-VIS spectroscopy Methods of biophysical chemistry - seminar Jan Novotny novotnyjan@mail.muni.cz Spectroscopic methods - context Complete missing items in the table: Spectral region Observed phenomena Method X-rays UV-VIS IR MW RW Spectroscopic methods - context Complete missing items in the table: Spectral region Observed phenomena Method X-rays UV-VIS IR MW RW transitions of core e valence e molecular vibrations e spin transitions, molecular rotations nuclear spin transitions absorption, diffraction, SAXS absorption, CD, luminescence IR spectroscopy, Raman scattering EPR, rotation spectroscopy NMR J. Novotný □ S1 - = 2/14 Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. J. Novotný □ S> - = Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. The value of extinct coefficient is proportional to transition integral, absorption area of a chromophore and concentration of detected molecule. □ g - = J. Novotný 3/14 Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. O The value of extinct coefficient is proportional to transition integral, absorption area of a chromophore and concentration of detected molecule. O Franck-Condon factor reflects overlap integral of ground and excited states. J. Novotný □ S> - = Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. O The value of extinct coefficient is proportional to transition integral, absorption area of a chromophore and concentration of detected molecule. O Franck-Condon factor reflects overlap integral of ground and excited states. O Electron transitions preserving parity of wavefunction are forbidden J. Novotný □ S> - = Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. O The value of extinct coefficient is proportional to transition integral, absorption area of a chromophore and concentration of detected molecule. O Franck-Condon factor reflects overlap integral of ground and excited states. O Electron transitions preserving parity of wavefunction are forbidden Q Number of normal vibrations modes in N-acetamid is 12. J. Novotný □ S> - = Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. O The value of extinct coefficient is proportional to transition integral, absorption area of a chromophore and concentration of detected molecule. O Franck-Condon factor reflects overlap integral of ground and excited states. O Electron transitions preserving parity of wavefunction are forbidden Q Number of normal vibrations modes in N-acetamid is 12. O Hypochromic shift means transition towards lower wavelength. J. Novotný □ S> - = Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. O The value of extinct coefficient is proportional to transition integral, absorption area of a chromophore and concentration of detected molecule. O Franck-Condon factor reflects overlap integral of ground and excited states. O Electron transitions preserving parity of wavefunction are forbidden Q Number of normal vibrations modes in N-acetamid is 12. O Hypochromic shift means transition towards lower wavelength. O Two maxima: 450nm and 700nm are found in VIS spectrum of chlorophyll. J. Novotný □ S> - = Starting problems: Are the following statements true or false. Explain your decision. O stabilisation of excited states leads to resonance shift towards higher frequency. O The value of extinct coefficient is proportional to transition integral, absorption area of a chromophore and concentration of detected molecule. O Franck-Condon factor reflects overlap integral of ground and excited states. O Electron transitions preserving parity of wavefunction are forbidden Q Number of normal vibrations modes in N-acetamid is 12. O Hypochromic shift means transition towards lower wavelength. O Two maxima: 450nm and 700nm are found in VIS spectrum of chlorophyll. O Measurements of electron transition in gas phase allows to observe fine rotational-vibrational splitting of resonance bands. J. Novotný □ S> - = Exercise 1 Assign the name, biochemical activity and type of transition to the following compounds: J. Novotný 4/14 Exercise 1 Assign the name, biochemical activity and type of transition to the following compounds: xanthophyll - accessory yellow photosyntetic pigment, protection from free radicals, tt-tt* hem - hemo/myoglobin, cytochrome: d-d Exercise 2: Conjugation Which of the isomers is characterized by higher resonance absorption frequency? J. Novotný 5/14 Exercise 2: Conjugation Which of the isomers is characterized by higher resonance absorption frequency? Exercise 3: Effect of pH on absorption spectrum Identify the resonance wavelength and extinct coefficient corresponding to pH < pKa a pH > pKa. Explain your decision. >4i,£i = 270nm, 1450 A2,e2 = 287nm, 2600 Exercise 3: Effect of pH on absorption spectrum Identify the resonance wavelength and extinct coefficient corresponding to pH < pKa a pH > pKa. Explain your decision. AU£! = 270nmf 1450 -OH pH < pKa A2,e2 = 287nm, 2600 -0~ pH > pKa Exercise 4: Effect of solvation Attached absorption spectrum of tyrosine shows the effect of adding of ethylene-glykolu (resulting concentration 15%). Interpret observation using models of electronic levels. I-£_I_I_I_■ ^ 250 260 270 280 290 300 A (nm) Exercise 4: Effect of solvation Attached absorption spectrum of tyrosine shows the effect of adding of ethylene-glykolu (resulting concentration 15%). Interpret observation using models of electronic levels. I-£_I_I_I_■ ^ 250 260 270 280 290 300 A (nm) Bathochromic, Hyperchromic shift. In general, decrease in polarity of environment leads to destabilisation of more polar antibonding 7r* (blueshift n —> 7r*) and lone pair n (redshift n —> 7r*) J. Novotný 7/14 Exercise 5: Retinal The attached scheme shows the structure of vision pigment retinal. Identify HOMO a LUMO orbitals (left column). Try to estimate effect of bond to quaternary nitrogen on value of Xmax. Based on analysis of electron differential density (right column) determine the orientation of transition dipole moment. M. Mohseni, Y. Omar, G. S. Engel, M. B. Plenio: Quantum effects in biology J. Novotný □ S> - = Exercise 6: Circular dichroism In CD spectroscopy several quantities are used to characterize the interaction of polarised light with chiral molecules. Fill in corresponding physical relationship and unit. Optical rotation dispersion Circular dichroism Elipticity /'P....... e\ ••A / E\ 1% s%... A \ / & \ J. Novotný 9/14 Exercise 6: Circular dichroism In CD spectroscopy several quantities are used to characterize the interaction of polarised light with chiral molecules. Fill in corresponding physical relationship and unit. Optical rotation dispersion if = - - np) [rad] Circular dichroism Ae = ei - - ep [M_1cm_1] J. Novotný 9/14 Exercise 7: Lambert-Beer law Molar extinct coefficient of sample at 540 nm is 268 dm3mol_1cm 52.3% of intensity was absorbed upon passing the optical distance mm. What was the concentration of detected compound? Exercise 7: Lambert-Beer law Molar extinct coefficient of sample at 540 nm is 268 dm3mol-1cm-1. 52.3% of intensity was absorbed upon passing the optical distance 7.5 mm. What was the concentration of detected compound? r Řešení A = logA = e.c.l log 'out 1 0.477 £.1 0.321 298,0.75 1.6 mM J. Novotný □ S> - = Exercise 8: Monitoring the reaction using VIS-spectrum The scheme shows series of VIS-spectra of bromthymol blue upon changing the pH. a) What is the name of labelled point and how is the point defined? b) Assign Hind a lnd~ entities to absorption bands. c) Express ratio of conjageted base and acid by means of actual values of A(Ai), A(A2) a their limited counterparts A(Ai)max a A(A2)max- J. Novotný □ S - = Exercise 8: Monitoring the reaction using VIS-spectrum The scheme shows series of VIS-spectra of bromthymol blue upon changing the pH. a) What is the name of labelled point and how is the point defined? b) Assign Hind a lnd~ entities to absorption bands. c) Express ratio of conjageted base and acid by means of actual values of A(Ai), A(A2) a their limited counterparts A(Ai)max a A(A2)max- Řešení 01 u n i_ O l/l -Q < 350 400 450 500 550 Wavelength, nm 600 3í 0 700 a) isosbestic point: e(Hlnd)x* b) H Ind Ai = 430nm, lnd~ X2 [Hlnd] _ A{X1).A{X2)max e(lnd )A* 620nm c) [lnd~] A(X2).A(X1)max J. Novotný □ rS1 ~ = Exercise 9: Transition moment Assume that conjugated system of tt electrons can be approximated as particle in ID- box of infinity potential borders. A) Show relations beteween energy of excitation and the length of the box. B) Derive either analytically or graphically, that the probability of transition n = 1 —>► 2 is non-zero whereas n = 1 —>► 3 is forbidden. Hint: Eigenfunction of free electron in potential box with length L has a form i/jn = Asin^x J. Novotný □ S> - = Exercise 9: Transition moment Assume that conjugated system of tt electrons can be approximated as particle in ID- box of infinity potential borders. A) Show relations beteween energy of excitation and the length of the box. B) Derive either analytically or graphically, that the probability of transition n = 1 —>► 2 is non-zero whereas n = 1 —>► 3 is forbidden. Hint: Eigenfunction of free electron in potential box with length L has a form i/jn = Asin^x Řešení A=y/l/fQLsin^fxdx=Jí h2 ttV fL A2 2=eA2/0L-i-'r-1 sin^xdx = Ml Ml 7t. io sin— 2m L2 7t.2 = eA2 J0L x.x.sin^^xdx = 1 g*u*u=g q sin^x.x.sin^xdx = 0 g*u*g=u sina. sin/3 = ^[cos(a — /?) — cos(a + /?)] f x.cosax = -sinax + -^-cosax -10 ¥1 ¥2 ¥1 ¥2 x - dipol moment __________ ______________ x - dipol m ¥1 — ¥3 — ¥1 >3 oment ---------- J. Novotný 12 / 14 Excercise 10: Frank-Condon factor The ground state of model system is described by vibration wavefunction A/o.e-a(x-x°) . Calculate the Frank-Condon factor into excited state /Vi.e"a(x-X1)2. J. Novotný □ S> - = Futher reading Franz-Xaver Schmid: Biological Macromolecules: UV-visible Spectrophotometry M. Mohseni, Y. Omar, G. S. Engel, M. B. Plenio: Quantum effects in biology P. Atkins, J. de Paula: Physical Chemistry http://wwwxyut.edu.tw/wjchien/BiopolymerSpect/text/absorption.pdf J. Novotný 14 / 14