Lecture 3: Light absorbance

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1 Lecture 3: Light absorbance Perturbation Response 1

2 Light in Chemistry Light Response 0-3 Absorbance spectrum of benzene 2

3 Absorption Visible Light in Chemistry S 2 S 1 Fluorescence S 0 3

4 Visible light in chemistry Fluorescence microscopy of neurons Super resolution fluorescence microscopy Xiaowei Zhuang Nature Photonics 2009, 3,

5 Infrared light vibration and rotational spectroscopy Near IR ( μm) Mid-IR ( μm) Far-IR ( μm) S 1 S 0 5

6 X-Ray, inner shell electrons 6

7 Summary table Region Wavelength Transition X-ray 10-3 to 10 nm inner shell electrons Ultraviolet 10 to 400 nm electronic transitions Visible 400 to 700 nm outer shell electrons Infrared 0.7 to 1000 mm vibrations and rotations Microwave 0.1 to 100 cm rotations Radio frequency 1 to 1000 m nuclear spins 7

8 Properties of light Electromagnetic radiation, characterized by a frequency, ν (Hz), and wavelength, λ (nm). In vacuum, the speed of light is c = m/s. The energy of a photon, J, E = h ν, ~ Wavenumber, 1 (cm -1 ) Electron volt, 1 ev = 1.60 x J (energy of 1240 nm photon) 8

9 Molecular absorbance spectrum Absorbance spectrum of benzene 9

10 The Jablonski diagram The Jablonski diagram is a good way to understand molecular absorbance, luminescence, and nonradiative relaxation. 10

11 Fluorescence Fluorescence occurs from the ground vibrational level of S 1 to some vibrational level of S 0. As in absorbance, the probability of emission to any vibrational band depends on the overlap between the initial and final wave functions. The potential energy surfaces are similar for many molecules in S 0 and S 1. As a result, the absorbance and emission spectra tend to be mirror images of each other. The Stokes shift: loss of energy between absorbance and emission. 11

12 12

13 There is a noticeable symmetry in the absorption and fluorescence spectra for benzene; both spectra have peaks with similar spacing (90 ± 10 cm -1 ), which equals the vibrational (ringstretch) mode spacing for benzene. The spacing between vibration modes in excited and ground states are similar. 13

14 Fluorescence and phosphorescence The population of excited states decays exponentially, and for molecules in solution, the lifetime is typically a few nanoseconds. Relatively few molecules produce fluorescence, so it is not a universal phenomenon. Many biological molecules can be fluorescently tagged with appropriate derivatizing reagents. If there is a change in the spin of the electron, which is quantum mechanically forbidden, the molecule undergoes phosphorescence. Phosphorescence lifetimes are microsecond to milliseconds. 14

15 Light absorbance The amount of light absorbed by a sample solution is related to its concentration. 15

16 Light absorbance The amount of light absorbed by a sample solution is related to its concentration. Po Sample Pt What is the transmittance : T P t P 0 How is the absorbance defined? A 1 log 10 T 16

17 Absorbance Beer s law: A e b C Light absorbance A is absorbance, e is the molar absorptivity (L mol -1 cm -1 ), b is the path length (cm), C is the concentration (mol L -1 ) Concentration (mm) 17

18 Absorbance Factors affecting absorbance (a) Chemical equilibrium can change e at high concentration by changing its effective concentration. Chromate and dichromate are in equilibrium: 3 2 HCrO 4 - Cr 2 O H 2 O Concentration (M) 18

19 Absorbance Factors affecting absorbance (b) Stray light causes transmittance to be greater than it should be. This phenomenon is important for strongly absorbing samples. If the stray light is 0.1% of P 0, the minimum P t is x P 0, and the maximum absorbance is log 10 (0.001) = 3. A log 10 P0 P Pt P straylight straylight 3 2 LAMBDA 950 UV/Vis/NIR Spectrophotometer Concentration (M) 19

20 Factors affecting absorbance (c) Incident light contains multiple wavelengths. This is similar to the effects of stray light. 20

21 Light absorbance (d) Light scattering from particles in solution. They reduce P t, which increases A. Important at low absorbance. 21

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