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Fluorescence Fluorescent corals.

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Presentation on theme: "Fluorescence Fluorescent corals."— Presentation transcript:

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

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