1. Fluorescence
Fluorescent corals

3. Jablonski Diagram

4. Fluorescence
From v = 0 down
Absorption
From v = 0 up
So expect the emission and absorption spectra to overlap here
Mostly don’t – because of changes of energy due to solvent interactions

6. Fluorescence is always at a longer
wavelength than absorption, because of the loss of vibrational energy
This is known as Stoke’s shift

7. Mirror image rule (Kasha’s Rule)
The shape of an emission spectrum of a simple molecule is the mirror image of the absorption spectrum.
The vibrational levels have approximately the same space in the ground state and the first excited level because the shape of the molecule does not really change.

11. Excited Singlet state – paired electrons but one is excited
No splitting of electronic energy levels occurs when the molecule is in a magnetic field
Triplet state – unpaired electrons –the
spins of the two electrons are parallel
Paramagnetic
Triplet because of spectroscopic
multiplicity

12. Intersystem Crossing
When the lowest vibrational state of S1 has the same energy as an upper vibrational level of the triplet state.
Lifetime of a triplet state is long – there are lots of chances for loss of energy in collisions
We do not see phosphorescence in liquids at room temperature

13. What molecules fluoresce?
Rigid, coplanar (reduces collisional dectivation)
Conjugated
Have to freeze to get phosphorescence or put on filter paper
Use micelles, cyclodextrin
All reduce collisional deactivation

14. Note the difference in rigidity

15. Applications

16. Advantages of Fluorescence over Absorption
Greater selectivity and freedom from spectral interferences
Fewer species which luminesce
Can vary the absorption (excitation) and emission wavelengths
Lower LOD than Absorption for same compound
F is linear with conc over 3-4 orders of magnitude

17. Lower LOD than Absorption for same compound
Fluorescence is read directly by detector
Absorption is a ratio
F is linear with conc over 3-4 orders of magnitude (extending to lower conc range)

18. Lysergic acid diethylamide
50 µg is active
Plasma or urine
Make basic
Extract with 98:2
n-heptane:isoamyl alcohol
Excitation: 335 nm
Emission: 435 nm

19. Phosphorescence Radiative relaxation from T1 to G
Is forbidden – so has long lifetime
10-6 – 10 sec
To make a glow-in-the-dark toy, what you want is a phosphor that is energized by normal light and that has a very long persistence. Two phosphors that have these properties are Zinc Sulfide and Strontium Aluminate. Strontium Aluminate is newer -- it's what you see in the "super" glow-in-the-dark toys. It has a much longer persistence than Zinc Sulfide does. The phosphor is mixed into a plastic and molded to make most glow-in-the-dark stuff.

20. Phosphorescence Occurs in solids Which may be frozen solvents
Reduces the number of collisions
Paramagnetic species increase the likelihood of intersystem crossing
So reduce fluorescence and phosphorescence

21. Shape of Emission spectrum
Does not change with excitation wavelength
BUT the intensity changes
The most fluorescence will occur when a lot of light is absorbed
Can find an excitation λ by running an absorption spectrum
Use this to find λemmax and then λexmax

22. Quantum Yield = Φ
ΦF = number of fluorescence quanta emitted divided by number of quanta absorbed to a singlet excited state
Φ F = ratio of photons emitted to photons absorbed

24. Quenching

25. Xenon arc lamp
Laser
High power is more important than stability
A reference system is added to measure the stability

26. Xenon Arc Lamp Unstable
Some portion of initial light goes to reference detector to ratio with F signal to compensate for changes in lamp intensity
Sometimes a fluorescent standard of rhodamine is included
May have to restrict intensity of light to minimize sample decomposition (photobleaching)

27. Sources of UV produce ozone. Fan disperses this and cools lamp.
Ozone is toxic but also absorbs certain wavelengths
Detector at right angles to lamp
Two wavelength selectors
Slits: narrow for high resolution
Wider(5-10 nm) to give greater sensitivity

29. L = k[P0 – P] P = P0 10-abc (Beer’s law)
= k[P0 – P0 10-abc]
= kP0[1 – 10-abc]
Note: L is proportional to P0

30. 10-abc = e-(2.3abc)
Expand in a series:

31. Calibration using Raman peak for water
Raman peak maxima of water at various Exc λ’s
Excitation wavelength/nm
Raman emission/nm
200
212
250
272
300
337
350
397
400
463
450
530
500
602

32. Excitation wavelength/nm
Positions of the Raman bands of various solvents when excited at selected wavelengths
Solvents
Excitation wavelength/nm
Water
350
418
469
511
Acetonitrile
340
406
457
504
Cyclohexane
344
409
458
499
Chloroform
346
411
461
502

33. Excitation wavelengths for quinine
450
450
Excitation wavelengths for quinine
Excitation
(Em at 450 nm)
Absorbance

35. 0.05 M H2SO4 Ex at 250 nm

36. Excitation spectrum with emission at 450 nm