Absorption Electromagnetic waves and interactions; spectroscopy - PDF document

4/11/2016 Content PÉCSI TUDOMÁNYEGYETEM ÁLTALÁNOS ORVOSTUDOMÁNYI KAR Light; www.aok.pte.hu Absorption Electromagnetic waves and interactions; spectroscopy Definition of absorption; Absorption measurements; (Miklós Nyitrai; 2 nd of March 2016) Applications. www.aok.pte.hu Reminder Reminder increasing energy far red red orange yellow green kék violet UV 760nm 647nm 586nm 535nm 492nm 422nm 390nm What is the correlation between frequency, energy and wavelength? The light is electromagnetic radiation. www.aok.pte.hu www.aok.pte.hu Spektroscopy Absorption in diluted solutions - transmittance - absorption www.aok.pte.hu www.aok.pte.hu 1

4/11/2016 Transmittance Absorption or I light absoprtion I 0 detector light substance source T = I / I 0 Usually given in % www.aok.pte.hu www.aok.pte.hu Similar to the radioactive decay Absorption low! I 0 Sample I = I 0 exp (-kx) N = N 0 exp (- λ t) or Exponential light intensity or I = I 0 e -kx function!!! N = N 0 e - λ t I distance www.aok.pte.hu www.aok.pte.hu A different presentation What are the bases? I 0 sample Exponential The Born-Oppenheimer approximation; too! Nuclear vs. Electron motions. intensity I ln(distance) www.aok.pte.hu www.aok.pte.hu 2

4/11/2016 The splitting of the energy levels Example: water molecule E sum = E elect + E vibr + E rot The changes can be then written as: ∆ E sum = ∆ E elect + ∆ E vibr + ∆ E rot http://www1.lsbu.ac.uk/water/vibrat.html www.aok.pte.hu www.aok.pte.hu Energy scheme: band spectra The energy scale The magnitude of the energy types: ∆ E elect ~ 1,000 * ∆ E vibr ~ 1,000,000 * ∆ E rot www.aok.pte.hu www.aok.pte.hu Spin multiplicity within the molecules Interpretation of absorption The number of possible quantum states (n) of a Excited ed state system based on the spin quantum number S: Vibrational relaxation (10 -12 s) S 1 M = 2S+ S+1 S 1 → T 1 : intersystem crossing (10 -10 – 10 -8 s) T 1 excitation (10 -15 s) excited singlet Szingulett → S=0; M = 1 state excited triplet T 1 → S 0 (10 -3 – 10 -1 s ) S 0 – S 1 state Triplett → S=1; M = 3 h ν 5 4 3 2 vibrational levels S 0 1 0 singlet ground Ground state state www.aok.pte.hu www.aok.pte.hu 3

4/11/2016 Distance dependence The definition of absorption I 0 Aim: sample intensity - easy understanding I - measurable - additive distance Note: transmittance (T = I / I 0 ) is not additive. A solution of 50% I = I 0 10 - ε ( λ ) c x transmittance is mixed with a solution of 60% transmittance (keeping the concentrations) will never give a solution of 110% Why ε ( λ ) and not only ε ? transmittance. Define parameters! www.aok.pte.hu www.aok.pte.hu Why ε ( λ ) and not only ε ? The definition of absoprtion I 0 I absorption substance OD = A = - log (I / I 0 ) = ε ( λ ) c x λ (nm) I = I 0 10 - ε ( λ ) c x „optical density” ε depends on λ ! www.aok.pte.hu www.aok.pte.hu How to measure absorption? Absorption measurements A scheme of a photometer. light source monochromator sample detector photometry = absorption spectroscopy www.aok.pte.hu www.aok.pte.hu 4

4/11/2016 Spektrophotometer Main componen ents: How does the prism work? 1. Ligh ght source ce • UV lamp (~180-350nm): Deuterium lamp • Visible source (~350-800nm): Wolfram 2. Monochromat ator: to select wavelength. 3. Sample holder er: e.g. a cuvette 4. photodet etekt ektor: PMT, diode… 5. Other componen ents: lenses, filters, slits. www.aok.pte.hu www.aok.pte.hu The wavelength dependence of the How does the prism work? refraction index n λ λ www.aok.pte.hu www.aok.pte.hu The wavelength dependence of the Photometers refraction index Why we use reference? Monochromatic light White light red yellow purple www.aok.pte.hu www.aok.pte.hu 5

4/11/2016 The absorption of proteins The absorption of proteins www.aok.pte.hu www.aok.pte.hu Applications: protein The absorption of proteins concentration Measured absorption: A Extinkciós coefficient: ε Usual units for ε : M -1 cm -1 , or (mg/ml) -1 cm -1 If A = 0.55 and ε = 1.1 (mg/ml) -1 cm -1 c = (A/ ε ) in mg/ml; c = 0.5 mg/ml www.aok.pte.hu www.aok.pte.hu What is the advantage of Application: gel electrophoresis additivity? (example) 1.0 0.8 absorption fluorescent probe 0.6 S protein 0.4 0.2 0.0 λ (nm) 0 5 10 15 20 [mDia3-FH2] ( µ M) www.aok.pte.hu www.aok.pte.hu 6

4/11/2016 Summary Thank you! - interpretation of absorption; - definition of absorption; - absorption measurements; - applications. www.aok.pte.hu www.aok.pte.hu 7