Luminescence

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??????????? ?????????????? ?????????????????????
Luminescence Spectroscopy
??.?????? ????????????
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Luminescence Spectroscopy

• Luminescence Spectroscopy
• Molecular Fluorescence Spectroscopy
• Fluorescent Species
• Effect of Concentration Fluorescence Intensity
• Fluorescence Instruments
• Applications of Fluorescence Methods
• Molecular Phosphorescence Spectroscopy
• Chemiluminescence Methods

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Luminescence Spectroscopy

• Luminescence ????????
• Photoluminescence
• ???? emission process ????????????????????????
  ???????????????????????????????????????????
  ??????????? ground state ?????????????????????????
  ??????????? ??? emission process
  ?????????????????? ??????? 10-5 ??????
  ???????????? ???????? ????????? ????
  ????????????? (fluorescence) ?????? emission
  process ??????????????????? ???????????????????
  ???????? ??????????? ???? ?????????????
  (phosphorescence)
• fluorescence ???????????????????????????????
  phosphorescence

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Luminescence Spectroscopy

• Chemiluminescence
• ???? emission process ????????????????????????
  ???????????????????????????? ??????????? ground
  state ????????????????????????????????????
• ????????? ????????????????????????????????????
  ????????????????????????? analyte
  ?????????????????????? (?????????? strong oxidant
  ???? ozone ???? peroxide) ???????????????????????
  ???????????????????????????? ????????? analyte
  ???
• ????????? analyte ??????????????????
  chemiluminescent reaction ??????
  ????????????????????????? chemiluminescence
• ?????????????????????????? molecular
  fluorescence

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Luminescence Spectroscopy

• ???????? luminescence methods ?????????????
• absorption methods
• sensitivity ??????? 1-3 ???? (detection limit
  ??????? 1-3 ????)
• linear concentration range ?????????
• selectivity ? absorption methods
• ??????????? luminescence methods
  ?????????????????????
• ????? chemical systems ?????? luminescence
  ???????

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Molecular Fluorescence Spectroscopy
molecular fluorescence spectroscopy
??????????????????????????????????????????????????
?????????????? ???????? absorption ????
excitation wavelength ????????????????????????????
??????????????????????? emission wavelength ????
?????? (quinine) ????????????????????? 350 nm
??????????????????????????????????????????????????
????? 460 nm
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Molecular Fluorescence Spectroscopy
?????? 1 Excitation ??? emission spectrum
?????????
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(No Transcript)
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Molecular Fluorescence Spectroscopy
molecular fluorescence bands
?????????????????? lines ?????????????????????????
????????????????????????????????????????????
excitation ??????????????????????????????????????
Stokes shift ???????????????????????????????
? vibrational states ?????? ground ??? excited
states ????????????????? absorption spectrum ????
excitation spectrum ??? fluorescence spectrum
?????????????????????????????? mirror images
???????????? (????????? 3) ???????????????????????
?????????????????????????????????????????????
vibrational level 0 ??? E1 ??? vibrational level
0 ??? E0
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Molecular Fluorescence Spectroscopy
?????? 3 Excitation ??? emission spectrum ??????
???????????????? mirror images ???
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Fluorescent Species
????????? 3 ??????????????????????????????????
???????????????????????? ????????????????? ground
state ??????????????????????????????????????
??????????????????????????????????????????????????
???????????????? ????????????????????????????????
????? ?????????????????????????????????????????
radiationless relaxation ?????????????????????????
?? fluorescence emission ????????????????????
????????????? (quantum efficiency)
??????????????????????????????????? ?????????????
???????????????? (quantum yield)
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Fluorescent Species
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Fluorescent Species
? ??????????????????????????????? ????
??????????? (fluorescein) ?? quantum yield
????????? 1 ? ???????????????????????????
quantum yield 0
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Fluorescent Species

• Fluorescence and Structure
• ?????????????????????????????????????? aromatic
  rings
• aliphatic ??? alicyclic carbonyl compounds
  ?????????? ?????????????? ??????? conjugate
  double-bonds ???????????????????
• unsubstituted aromatic hydrocarbons
  ?????????????????????????? ??? quantum efficiency
  ??????????????????? rings ??? degree of
  condensation ?????????

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Fluorescent Species

• heterocyclic compounds ?????????
  ??????????????????? ????
• pyridine furan thiophene
  pyrrole
• ??? fused-ring structures ????? rings ????????
  ????????????????
• quinoline isoquinoline
  indole

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Fluorescent Species

• ??????????? aromatic rings ?????????
• ? ???????????????????????????????????????????
  ?????
• ? ????????????????? fluorescence peaks
• ? ?????????????? fluorescence efficiency
• (?????????? )

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Fluorescent Species
???????? 1 ???????????????? benzene rings
????????????????
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Fluorescent Species
Effect of Structural Rigidity
???????????????? rigid molecule
????????????????????? ??? rigidity
???????????????? nonradiative relaxation
????????????????? relaxation ????????????????
????
? fluorescing dyes ???????????????????????????????
??????????????????????????????
????????????????????????????????? rigidity
?????????
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Fluorescent Species
? fluorene (??????????? rigid ???????????
methylene group ??? benzene rings ??????????) ??
quantum efficiency ????????? 1 ???? biphenyl
(???? benzene rings ??????????????????????????)
?? quantum efficiency ??????? 0.2
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Fluorescent Species
? Zn(8-hydroxyquinoline) complex (???? chelate)
????????????????????????????? (intensity) ???????
8-hydroxyquinoline (???????????????????????????)
??? ???????????????? rigidity ???????
8-hydroxyquinoline
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Fluorescent Species

• Temperature and Solvent Effects
• Quantum efficiency ??????????????????????
  ?????????????????????? ????????????????????
  ??????????????????????????????????????? ?????????
  nonradiative relaxation ??????????
• Quantum efficiency ??????????????????????
  ????????????? (viscosity) ?????????????????
  ??????????????????????????????????????????
  ??????? nonradiative relaxation ??????????

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Effect of Concentration of Fluorescence Intensity
???????? fluorescence radiation (F)
?????????????????????????????? (radiant power)
??? excitation beam ?????????????
F K ?(P0 - P)
(1) P0 ??????????????????????????????
? K ? ???????? ??????????? quantum efficiency
??? fluorescence ???????????????????????? F
?????????????? c ??? fluorescing species ?????
Beers law ????? P/P0 10-?bc
???? P P0 x 10-?bc
(2) ? molar absorptivity of
fluorescing species ?bc absorbance (A)
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Effect of Concentration of Fluorescence Intensity
?????? P ???????? (2) ????????? (1)
F K ? P0 (1 - 10-?bc)
(3) F K ? P0 2.3?bc
(4) ????? ?bc
(A) lt 0.05 2.3?bc ???????????????????????????????
??? ??????? F 2.3K ??bc P0
(5) ??????????????????????????????????? ?????
F K c (6) ?????????? plot
fluorescence power ??????????? ???
?????????????????????? ???????????????????????????
??
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Effect of Concentration of Fluorescence Intensity
????? c ?????????????? A ??????? 0.05 (???? T
???????? 0.9) ??????????????????? F ??? c
???????? (6) ???????????????? ??? F
???????????????????????????????????
????????????? primary absorption
??????????????????????????????????????????????????
???????????????????????????? ???????? (4)
???????????????????? F ???????????????????????????
???????????????????????????? secondary absorption
??????????????????????????????????????????????????
??? analyte ????????????
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Effect of Concentration of Fluorescence Intensity
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Effect of Concentration of Fluorescence Intensity
????????? primary ??? secondary
absorption effects ????????????????????
inner-filter effects ??? ?????????????????????????
??????? sample matrix
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Fluorescence Instruments
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Fluorescence Instruments
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Fluorescence Instruments
?????? 5 ????????????????? fluorometers ???
spectrofluorometers ????????????????????????????
?????????????????????????? UV/visible
spectroscopy fluorescence instrument
????????????? double-beam optics
?????????????????????????? power ??? source
??????????????????????????? ???????????????????
primary excitation filter/monochromator
?????????????????????????????? excitation ??????
emitted radiation ????????????????????????????????
??????????? fluorescence radiation
??????????????????????????????
????????????????????????????????????????
excitation beam ??????????? ????????????????????
???????????
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Fluorescence Instruments
???????????????????????? ?????????????????????????
??????? intensity ??????? emitted radiation
????????? photoelectric detector ???????????
secondary emission filter/ monochromator ???????
fluorescence peak ????????????
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Fluorescence Instruments
fluorescence instrument ?????????????
double-beam optics ??????????????????????????
power ??? source ???????????????????????????
??????????????????? primary excitation
filter/monochromator ?????????????????????????????
? excitation ?????? emitted radiation
???????????????????????????????????????????
fluorescence radiation ???????????????????????????
??? ????????????????????????????????????????
excitation beam ??????????? ?????????????????????
??????????????????????????????????
???????????????????????????????? intensity
??????? emitted radiation ?????????
photoelectric detector ??????????? secondary
emission filter/ monochromator ???????
fluorescence peak ????????????
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Fluorescence Instruments
Radiation Sources fluorescence ??????? source
????? intensity ??????? tungsten ???? hydrogen
lamp ????????? absorption ????????? F 2.303K ?
P0?bc ??????? emitted radiation power (???
sensitivity) ???????????????????? source power
(P0) ???????? absorbance ??? log P0/P
????????????? source power fluorescence
source ????????????????????? ? mercury arc
lamps ? xenon arc lamps ? xenon-mercury
arc lamps ? lasers
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Fluorescence Instruments

• Wavelength Selectors
• ????????????? wavelength selectors
  ??????????? filters ???????? fluorometer
• ??? wavelength selectors ???????????
  monochromators???????? spectrofluorometer
• spectrofluorometer ?????????? filter
  ?????????????????????????? excitation radiation
  ?????? grating monochromator ????????????
  fluorescence radiation

Fluorescence filter
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Fluorescence Instruments
Cells and Cell Compartments
?????????????????????? cylindrical ???
rectangular cells ???????????????????????
????????? cell compartment ??????????????????????
scattered radiation ?????????? detector ????????
baffles ?? cell compartment
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Fluorescence Instruments
Detectors ??????????? photomultiplier (PMT)
????????? fluorescene signal ?? intensity
??? ?????????? amplification factors ???
detectors ??????????????? photomultiplier (PMT)
??????????? diode array detectors ??
spectrofluorometer
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Fluorescence Instruments
fluorometers ??? spectrofluorometers
???????????????????????????????????????? ???????
??????? ???????????? absorption
spectrophotometers ????????? fluorescence
instruments ??????????????? absorption
instruments ??????????????????????????
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Fluorescence Instruments
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Applications of Fluorescence Methods
fluorescence spectroscopy ????????????????????
??????????????????????? (structural analysis)
?????????????????????? (qualitative analysis)
???????????????????????????????????????
fluorescence spectra ???????? ?????????
fluorescence bands ??????????????????????????
(board) ??????????????? fluorescence methods
????????????????????????????????????????????????
absorption methods ???????????????? sensitivity
??? ????????? power ??? fluorescence (F)
????????????????????????? power ??? source (F
2.3K ??bc P0) ???????? sensitivity ???
fluorescence methods ???????????????? P0
??????????? fluorescence signal
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Applications of Fluorescence Methods
?????? absorption methods ??? absorbance
?????????????????????????????? ??????? log P0/P
???????? P0 ??????? P ????????????????????????????
????????????? absorbance ??????????? sensitivity
??????????????????????????????? detector
??????????? P ??? P0 ???????????????? absorbance
??????????????? ????????? fluorescence
methods ?? sensitivity ??????? absorption methods
1-3 ????
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Applications of Fluorescence Methods
???????? (accuracy) ????????????? (precision)
??? fluorescence methods ?????????? absorption
methods ???????????? 2-5 ??? phosphorescence
methods ?? precision ??????? fluorescence
methods fluorescence methods ???????????
inorganic, organic ??? biochemical species
???????????????????????????????????????????
absorption methods ???????????????????????????????
?????????????????? ??????? sensitivity ???????
absorption methods
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Applications of Fluorescence Methods
Methods for Inorganic Species ?? 2 ???? ???
1. Direct Methods ??? analyte ??????????????
complexing agent ???????????? fluorescent complex
??????? fluorescence 2. Indirect Methods
????????????? fluorescence ??????????? quenching
????????????????? analyte ??????????????
fluorescent reagent ???????? quenching
?????????????????????????????? (anions) ???
???????????????????? (dissolved oxygen)
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Applications of Fluorescence Methods
??????????? metal ions transition metals
?????????????????????????? UV/visible ???
transition-metals chelates ????????? fluorescence
????????????? nonradiative relaxation ?????
nontransition-metal ions ?????????????????????
UV/visible ??? nontransition-metal chelates ???
fluorescence ??????? ????????????? metal
ions ?????? fluorescence methods ????????
absorption methods
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Applications of Fluorescence Methods
fluorometric reagents ????????????????????????
?? ???????????????????????????????????? aromatic
structures ????????????????????????????? 2
??????????? ????????????? chelate ??? metal ions
???? ? 8-hydroxyquinoline (???????????????
Al, Be, ??? metal ions ?????) ?
flavanol (??????????????? Zr and Sn) ?
alizarin garnet R (??????????????? Al, F-) ?
benzoin (??????????????? B, Zn, Ge, ??? Si)
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Applications of Fluorescence Methods
???????? 2 Selected Fluorescence Methods for
Inorganic Species
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Applications of Fluorescence Methods
Methods for Organic and Biochemical Species
fluorescence methods ???????????? organic
???biochemical substance ???????? ?????? amino
acids, proteins, coenzymes, vitamins, nucleic
acids, alkaloids, porphyrins, steroids,
flavanoids ??? metabolites ?????????????????????
????????? ?? ????????????????? ???????????????????
? ????????? fluorescence methods ??
sensitivity ??? ???????????????????????? liquid
chromatographic methods, flow analysis methods
??? electrophoresis
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Molecular Phosphorescence Spectroscopy
????????????????????????? delayed emission
???? phosphorescence ??? emission
??????????????????????????????????????????????????
??? fluorescence ??????????????????????
phosphorescence ????????????? 10-4 - 104 s
????????????? phosphorescence ?????????? rigid
solvent structures ?????????? collisional
deactivation ?????????? ?????????????
phosphorescence ????????????????? liquid nitrogen
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Molecular Phosphorescence Spectroscopy
Electron Spins ???????????????????????????????
?? ?????????????????????????????????????
??????????? 2 quantized spin state
???????????????????? ???????????????????????????
???????????????????????????? spin state
??????????????????
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Molecular Phosphorescence Spectroscopy
?? molecular orbital ????? 2 ?????????? spin
??????????????????????????????????? Pauli
Exclusion Principle ???? spin pairing
???????????????????????? ground singlet state
???????????????????????????? spin pairing
???????????????????????? ??????? diamagnetic
?????????????????????????????
?????????????????????????????????????????????????
??? spin ??? excited-state electron
????????????? ground-state electron (spin
pairing) ???????????????????????? excited singlet
state ?????? spin ??? excited-state electron
????????? ground-state electron
???????????????????????? excited triplet state
??? excited triplet state ?????????????????
excited singlet state ???? singlet, doublet
??? triplet ????? spectroscopic multiplicity
consideration ???????????????????????????
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Molecular Phosphorescence Spectroscopy
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Molecular Phosphorescence Spectroscopy
Molecular fluorescence ???????????????
electronic transition ??? excited singlet state
????? ground singlet state transition
????????????????????? ??????? lifetime ???
excited singlet state ??????? (10-5 s
????????????) Molecular phosphorescence
??????????????? electronic transition ??? excited
triplet state ????? ground singlet state
????????? transition ??????????????? electron
spin ?????????????????????????? lifetime ???
excited triplet state ?????????? (10-4 - 104 s)
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Molecular Phosphorescence Spectroscopy
????????? phosphorescence ?? lifetime ???
????????? excited state ?????????????????
nonradiative relaxation ???????????????????
phosphorescence process ??? phosphorescence
intensity ??????????? ???????????????
phosphorescence ?????????????? ???????????? rigid
???? ???? ?????????????????????
???????????????? phosphorescence
????????????????????????????????????
?????????????????????????????????????????????
?????????? molecular cavity
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Molecular Phosphorescence Spectroscopy
phosphorimetry ???????????????? organic ???
biochemical species ????????????? ???? nucleic
acids, amino acids, pyrine, pyrimidine, enzymes,
petroleum hydrocarbons, pesticides
??????????????????????????????????????????????????
fluorometry ???????????????????????????
????????? phosphorescence ?? precision ???????
??????????????? phosphorescence ??? selectivity
???????
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Chemiluminescence Methods
chemiluminescence ????????????????????????????
??????? eletronic excited molecule
?????????????????????????? ground state
chemiluminescence reactions ??????????????????????
? ??????????????????????????????? bioluminescence
???? ???????? ????????????????? ?????????
???????? ??? crustacea chemiluminescence
???????????????????? ??????????????????? external
source ?????? excitation ????????????? wavelength
selector ?????????????????????? reaction vessel
??? photomultiplier tube ????????
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Chemiluminescence Methods
chemiluminescence ??? sensitivity ???
????????? detection limits ???????????? parts per
million (ppm) ??? parts per billion (ppb)
???????????