Optical spectroscopic techniques

Transcription

1 What happens i a saple is illuinated by light? (absorbed light) Optical spectroscopic techniques illuinating light saple transitted light Absorptionspectroscopies: UV-VIS, IR László Seller eitted light Luinescence (Fluorescence and Phosphorescence) spectroscopies scattered light Raan and Rayleigh scattering Raan spectroscopy Static and dynaic light scattering Spectroscopy (Absorption and eission spectroscopy) Why is light absorbed or eitted? Analysis o the wavelength dependence o the transitted or eitted light. Inoration: identiication o atos and olecules, detection o changes in the olecular structure (conoration) deterination o the concentration Jablonski diagra Excited electron and excited vibrational state* Excited electron state Vibrationally excited state.* Ground state S E *only or olecules! (not or atos)

2 E S Why is light absorbed or eitted? UV-VIS IR absorption Raan Fluorescence Phosphorescence T ΔEhhc/λ Absorption spectroscopy (UV-VIS) As a reinder: law o absorption: JJ 0 e -μx where μ(aterial,c,λ) Labert-Beer law: J A lg 0 ε ( λ) cx J spectru: A(λ) easureent: spectrophotoeter (details: see pract. exc.) reerence solution (J 0 ) inoration: identiication (λ ax ), concentration (A) Inrared spectroscopy Molecular vibrations Inrared light: λ800 n - MIR (id-inrared) :,5-50 μ absorption spectroscopy the absorbed inrared radiation excite olecular vibrations very speciic or the structure o the olecule special ethod or detection: FT spectroeter The electrons are light ( e << nucleus ), they can ollow the oveents o the nuclei easily, thereore the oveents o the nuclei are independent o the oveents o the electrons. Classical physical description: the cheical bond is represented by a spring

3 Molecular vibrations: distance o nuclei centre o ass known ro eleentary echanics: π l l centre o ass F Δl l + l l l l F / F / l l + Δl Δl l l + + substituting red + + into Frequency with the reduced ass: requency o the vibration: π is called as reduced ass ( π + ) π red The wavelength: c λ πc In the IR spectroscopy the wavenuber (ν) is used, which is the reciprocal o λ: ν λ πc redukált ν: nuber o waves in a unit length [c - ] Exaple: CO The easured wavenuber: ν 43 c - λ4,67μ 6, Hz 875 N/ C 0-6 kg, O,7 0-6 kg redukált i ν is known, can be calculated i is known, ν can be calculated

4 Classical vs. quantu physics Classical physical Quantu echanical picture picture S center o ass π red resonance with the light with requency ΔEh ΔE Vibrations o the large olecules Molecule consisting o N atos: 3N degree o reedo, 3-3 are the rotations and translations o the whole olecule 3N-6 vibrational degree o reedo (3N-5 or the linear olecules) 3N-6 independent noral vibrations Noral vibrations All the atos vibrate with the sae requency but with dierent aplitude and in dierent direction. Exaple: water Noral vibrations o water syetric stretching asyetric stretching bending These are no vibrations! These are rotations!

5 Typical vibrational requencies (wavenubers) Exaple: Foraldehyde Flavin Benzol

6 Vibrations o the acrooleclues Lipids Coplex global vibrations Localised vibrations, e.g.: CH vibrations o the lipids aid vibrations o proteins (acetaide) Syetric Stretch Asyetric Stretch Twisting Wagging Scissoring Rocking Types o Vibrational Modes. Figure ro Wikipedia

7 Applications phase transitions in lipids protein denaturation Pharaceutical applications synthesis: identiication o the interedate and the end product deterination and justiication o the olecular structure detection o the etabolites quality control (purity) Reark.: Labert-Beer law is valid, deterination o concentration is possible Meersan et al. Biophys J. Identiication o the Molekules ethanol ethanol C 4 H 8 O Exaple: Identiication o olecules Absorbanz Absorbanz Wavenuber Wavenuber Fingerprint Region

8 The technique o the easureent : Fourier transor spectroeter (FTIR) conventional (dispersion) spectroeter onochroator saple detector oving irror standing irror path A path B ényorrás light source Fourier transor spectroeter saple saple intereroeter detector detector book 6.7 book 6.8 Luinescence spectroscopy Which olecues are luorescent? E S T Aino acids (tryptophan, tyrosine, phenylalanine) Fluorescent dyes GFP UV-VIS IR absorption Raan Fluorescence Phosphorescence

9 Measurable quantities in Fluorescence Spectroscopy Aequorea victoria Wavelength o the exciting light Wavelength o the eitted light (luor., phosph.) Tie dependence o the eitted light Polarisation o the eitted light Intensity o the eitted light Schee o the luorescence spectroeter excitation Light source excitation onochroator Eisson onochroator detector (e.g. photoultiplier) saple eission Excitation and eission spectra Excitation spectru Eission at 340 n Eission spectru Excitation at 95 n Phophorescence sp. Exitation at 95 n S E T 6.6 display (coputer) 6.5. Stokes shit Fluorescence Phosphorescence

10 Fluorescence quantu yield(q) The lietie o the excited state E S T k nr k Quantu yield: Q nuber o eitted photons nuber o absorbed photons Q k k + k k probability o the transition with light eission (luoresc.) k nr probability o nonradiating transition dyes, l. arkers Q nr S E where Fro N excited olecules during Δt tie ΔN(k +k nr )NΔt will go back to ground state. ierential equation: dn ( k knr ) N dt + Solution: N τ k + k nr ( k + knr ) t N0e N0 e t τ is the lietie o the excited state ecay o the luorescence intensity Exaple: tryptophan The nuber o eitted photons is proportional with ΔN, i.e. it is proportional also with N, which eans it is decays exponentially with the decay constant o τ. How to easure? -Pulsed illuination (lashlap, or pulse laser) -Photon counting as unction o tie Quantu yield and lie tie can be also deined or phosphorescence, using siilar deinitions. Fluorescence intensity tie Fluorescence intensity tie Typical lieties: τ luor ns τ phosphor μs...s

11 Fluorescence polarization Ligth scattering illuination with polarized light Rayleigh λ scatt λ illu Raan λ scatt λ illu Chandrasekhra Venkata Raan polarization degree o the eitted light is easured The luorescent olecule can rotate between the absorption and the eission dynaic inoration: rotational correlation tie (how ast is the rotational diusion?) Raan scattering: λ scatt λ illu scatt illu E photon,scatt E photon,illu Where is the energy? Excites vibrations o the olecule (cr. IR) very weak (~0-8 ) E S 6.8 IR Raan Raan Spectroeter Raan-Scattering Probe Filter Vibrations are speciic or the olecules corn-starch Monochroator etector Raan Spectroscopy active agent E.g.: active agent content o a tablet -ikroskopie-spektroskopie/raan-spektroskopie-tablette.htl

12 Handheld Raan spectroeter or identiication o aterials Pharaceutical application Rayleigh scattering Size o the particle: a << λ The scattered intensity: 6 a J scatt ~ J 0N 4 λ Inoration: size, contentration (quantity) (e.g. colloids)

13 Meaureent o the Rayleigh scattering i J scatt <<J 0 J scatt is easured (Nepheloetry) Light source saple etector (a) I J scatt J 0 scatt J is easured etector (b) (turbidietry) The sae technique as or the absorption specctrosopy but now J is reduced due to the scattering (and not due to absorption).