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Absorbance
molecule returns to the ground or lower energy state via a nonnonradiative transition such as vibration, collision with other molecules, etc. These give off the energy absorbed rather than etc. the emission of light. Some energy is lost through various processes (e.g. nonnonoff. radiative transitions) and then light is given off. The molecule transitions from an excited triplet state to an lower energy singlet state and gives off light. Non-radiative Nontransitions intervene.
Fluorescence
Phosphorescence
M*
M + heat
- Excitation of e- by absorbance of hR. - Re-emission of hv as e- goes to ground state. - Use hR2 for qualitative and quantitative analysis Method Mass detection limit (moles) 10-13 to 10-16 10-15 to 10-17 Concentration Advantages detection limit (molar) 10-5 to 10-8 10-7 to 10-9 Universal Sensitive
UVUV-Vis fluorescence
2.)
Fluorescence ground state to single state and back. Phosphorescence - ground state to triplet state and back.
Fluorescence Phosphorescence
10-5 to 10-8 s
10-4 to 10 s
Forbidden transition: no direct excitation of triplet state because change in multiplicity selection rules.
S1
S0
Resonance fluorescence?
P
STOKES SHIFT
P2
P3
Deactivation Processes:
a) vibrational relaxation: solvent collisions - Pemission > P excitation (Stokes shift) - vibrational relaxation is efficient and goes to lowest vibrational level of electronic state within 10-12s or less. - significantly shorter life-time then electronically excited state - fluorescence occurs from lowest vibrational level of electronic excited state, but can go to higher vibrational state of ground level. b) internal conversion: - crossing of e- to lower electronic state.
c) external conversion: deactivation via collision with solvent (collisional quenching) decrease collision increase fluorescence or phosphorescence decrease temperature and/or increase viscosity decrease concentration of quenching (Q) agent
d) intersystem crossing: spin of electron is reversed - change in multiplicity in molecule occurs (singlet to triplet) - enhanced if vibrational levels overlap - more common if molecule contains heavy atoms (I, Br)
e) Phosphorescence: Deactivation from an triplet electronic state to the ground state producing a photon
J =
JL !
Increase quantum yield by decreasing factors that promote other deactivation processes
Types of Transitions:
- seldom occurs from absorbance less than 250 nm 200 nm => 140 kJ/mol breaks many bonds - fluorescence not seen with W W - typically T* T or T* n
Notation!
kec = external conversion (S1 p S0) kic = internal conversion (S1 p S0) kisc = intersystem crossing (S1 p T1) kpd = predissociation kd = dissociation
Concentration Dependence
F ! K ' ( Po P) P Ibc ! 10 Po F ! Kc
Expanded as Maclaurine series P0 P : Power of the excitation beam absorbed by the system
F } K " Po (2.303Ibc)
H N
Quinoline
indole
fluorene
8-hydroxyquinoline
Application of Fluorescence
- detecting inorganic species by adding a fluorophore
Ion Al3+ Reagent Alizarin garnet R Absorption (nm) 470 Fluorescence (nm) 500 Sensitivity (ppm) 0.007 Interference Be, Co, Cr, Cu, F,NO3-, Ni, PO4-3, Th, Zr Be, Co, Cr, Cu, F,Fe, Ni,PO4-3, Ni,PO4Th, Zr Be, Sb NH3 Mg F-, PO43-, Zr B, Be, Sb, colored ions
F-
470
500
0.001
B4O72
-
OH N O HO OH O
OH N N
HO
O C
OH C H
SO3Na
8-Hydroxyquinoline
flavanol
alizarin garnet R
benzoin
Light sources
high pressure xenon arc lamp lasers filters monchromators photomultipliers CCD cameras quartz cells light tight compartments to minimize stray light
Wavelength selectors
Detectors
Instrumentation
Basic design components similar to UV/Vis spectrofluorometers: observe both excitation & emission spectra.
Extra features for phosphorescence sample cell in cooled Dewar flask with liquid nitrogen delay between excitation and emission
Fluorometers
- simple, rugged, low cost, compact - source beam split into reference and sample beam - reference beam attenuated ~ fluorescence intensity
Spectrofluorometer
- both excitation and emission spectra - two grating monochromators - quantitative analysis
PerkinPerkin-Elmer 204
Chemiluminescence
- chemical reaction yields an electronically excited species that emits light as it returns to ground state. - relatively new, few examples
A+B
C*
C + hR
O2/OH-
+ hR + N2 + H2O
Biological systems
Luciferase (Firefly enzyme)
O O C R N S
1
Luciferase Luciferin + O2
O C R2
R2
Spontaneous
CO2 +
C* R1
Light
Glowing Plants
Luciferase gene cloned into plants
HO
S N
Luciferin (firefly)
HO
Other Applications
Determination of nitrogen monoxide NO + O3 NO2* + NO2* + O2 NO2 + hR (P = 600 2800 nm)