Transcript Fluorescence Spectroscopy

CHM 5175: Part 2.5
Fluorescence Spectroscopy
Source
Detector
hn
Sample
Ken Hanson
MWF 9:00 – 9:50 am
Office Hours MWF 10:00-11:00
1
Fluorescence Spectroscopy
• First observed from quinine by Sir J. F. W. Herschel in 1845
Filter
Church Window
400nm SP filter
Yellow glass of wine
400 nm LP filter
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Quinine
Solution
(tonic water)
Observe
Blue emission
Herschel concluded that “a species in the
solution exert its peculiar power on the
incident light and disperses the blue light.”
Fluorescence Spectroscopy
Measuring the light given off by an electronically excited state.
Ground State
(S0)
Singlet Excited
State (S1)
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Excitation
Fluorescence
Emission
Intersystem
Crossing
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Emission
Triplet Excited
State (T1)
Phosphorescence
Fluorescence Spectroscopy
Singlet Excited
State (S1)
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Emission
Fluorescence
Spin allowed
Fast (ns)
Organic molecules
Triplet Excited
State (T1)
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Emission
Phosphorescence
Spin “forbidden”
slow (ms to s)
Transition metal complexes
Jablonski Diagram
S2
S1
T2
Energy
T1
S0
Excitation
Internal Conversion
Fluorescence
Non-radiative decay
Intersystem Crossing
Phosphorescence
5
Fluorescence
S2
2
V4
1
V3
V2
V1
Vo
S1
Energy
3
V4
V3
S0
V2
V1
Vo
Geometry
1) Excitation
-Very fast (< 10-15 s)
-No structure change
2) Internal Conversion
-Fast (10-12 s)
-Structure change
3) Fluorescence
-”Slow” (10-9 s)
- No structure change
Fluorescence
Sprinter (7 m/s)
S2
S1
Absorption
Snail (0.005 m/s)
n3
n2
n1
n3
n2
n1
IC
Internal Conversion (sprinter)
“always” wins!
Fluorescence
S0
Internal Conversion (1012 s-1)
S2 Fluorescence (109 s-1)
Kasha’s Rule:
Emission predominantly
occurs from the lowest
excited state (S0 OR T1)
Fluorescence
1920-2013
Kasha Laboratory Building
AKA Institute of Molecular Biophysics
Kasha’s Rule:
Emission predominantly
occurs from the lowest
excited state (S0 OR T1)
Fluorescence
Kasha’s Rule:
Emission predominantly
occurs from the lowest
excited state (S0 OR T1)
S1
Blue
Higher E
Red
Lower E
S0
Internal
Conversion
Eabsorption > Eemission
Emission is red-shifted (bathochromic)
relative to absorption
Absorption is blue-shifted (hypsochromic)
relative to emission
Mirror Image Rule
• Vibrational levels in the excited states and ground
states are similar
• An absorption spectrum reflects the vibrational
levels of the electronically excited state
v’=5
v’=4
v’=3
v’=2
v’=1
v’=0
• An emission spectrum reflects the vibrational
levels of the electronic ground state
S1
• Fluorescence emission spectrum is mirror image
of absorption spectrum
v=5
v=4
v=3
v=2
v=1
v=0
S0
Mirror Image Rule
n4
n
S1 nn32
1
n4
n
3
n
S0 n21
Mirror Image Rule
fluorescein
Anthracene
ethidium bromide
Stokes Shift
Stokes Shift:
Difference in energy/wavelength
between absorption max and
emission max.
S1
S0
Internal
Conversion
Sensitivity to local environment:
Solvent polarity
Temperature
Hydrogen bonding
Solvent Dependence
Stokes Shift:
Difference in energy/wavelength
between absorption max and
emission max.
4-dimethylamino-4'-nitrostilbene (DNS)
Solvatochromism
Solvatochromism
Jablonski Diagram
S2
S1
T2
Energy
T1
S0
Excitation
Internal Conversion
Fluorescence
Non-radiative decay
Intersystem Crossing
Phosphorescence
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Intersystem
Crossing
Singlet Excited
State (S1)
Emission
Triplet Excited
State (T1)
Ground
State (S0)
Phosphorescence
S2
T2
V4
2
2) Internal Conversion
-Fast (10-12 s)
-Structure change
V3
V2
V1
Vo
S1
3
V4
2
1
E
4
V3
S0
V2
V1
Vo
V4
2
Geometry
V3
V2
V1
Vo
1) Excitation
-Very fast (10-15 s)
-No structure change
T1
3) Intersystem Crossing
-Fast (10-12 s)
-No Structure change
4) Phosphorescence
-”Slow” (10-6 s)
- No structure change
Emission
Rates:
Lifetime:
Dl:
O2 sensitive:
Fluorescence
Phosphorescence
Fast (10-9s-1)
nanoseconds
<100 nm
no
Slow (10-6 – 0.1 s-1)
>microseonds
>100 nm
Yes
Fluorescence vs Phosphorescence
S2
Internal Conversion
(10-12 s)
S1
Intersystem Crossing
w/ Heavy atom (< 10-12 s)
w/o Heavy atom (> 10-9 s)
E
Excitation
(10-15 s)
S0
T1
Fluorescence
(10-9 s)
Phosphorescence
(10-6 s)
Emissive Molecules
Phosphorescent
Fluorescent
Perylene
OEP
PtOEP
Ir(ppy)3
BODIPY
Fluorescein
Rose Bengal
[Ru(bpy)3]2+
Coumarin
Anthracene
Anthracene + ICH3
C60
Fluorometer
Source
Excitation hn
Sample
Detector
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Emission
Variables
Excitation Wavelength
Excitation Intensity
Emission Wavelength
Filters
Fluorometer
3
1
2
Components
1) Light source
2) Monochrometer
3) Sample
4) Detector
5) Filters
6) Slits
7) Polarizers
2
4
Fluorometer: Simple Diagram
Xenon Lamp
Grating
Mirrors
Excitation
Monochromator
Two light sources =
Two monochromators!
1 for excitation
1 for emission
Emission
Monochromator
PMT
Sample
Grating
Fluorometer: Medium Diagram
Grating
Mirror
Lens
Sample
Mirror
Fluorometer: Hard Mode
Grating
Mirrors
Mirror
Grating
Fluorometer: Hard Mode 2
450 W Xe
300 nm blaze
1200 g/mm
exit slit
iris
shutter
NIR:
9170-75=950-1700 nm
1000 nm blaze
600 g/mm grating
polarizer
slit
r
V
V
V
UV-VIS:
R928 = 250-850nm
500 nm blaze
1200 g/mm grating
Horiba JY Fluoromax-4
Horiba JY Fluoromax-4
MAC Lab
(Materials Characterization)
Dr. Bert van de Burgt
CSL 116
Measuring Emission Spectra
Procedure
1) White light source on
Ex Grating
Xenon Lamp
1
2) Shift excitation grating to desired
wavelength (excitation wavelength)
Excitation
Monochromator
3) Light enters sample chamber
2
Emission
Monochromator
4) Light Hits the Sample
3
PMT
7
4
Sample
5
6
Em Grating
8
5) Emission from the sample enters
emission monochromator
6) Set emission grating
7) Detect emitted light at PMT
8) Raster emission grating
Measuring Emission Spectra
Absorption Spectrum
Absorbance (O.D.)
1.0
Procedure
0.8
1) White light source on
0.6
0.2
2) Shift excitation grating to desired
wavelength (excitation wavelength)
0.0
3) Light enters sample chamber
0.4
300
400
500
600
4) Light Hits the Sample
Wavelength (nm)
5) Emission from the sample enters
emission monochromator
Emission Spectrum
Intensity (counts)
20000
15000
6) Set emission grating
10000
7) Detect emitted light at PMT
8) Raster emission grating
5000
0
600
700
800
900
Wavelength (nm)
Excitation at 450 nm
Emission from 550 – 900 nm
Excitation Spectrum
Absorbance (O.D.)
1.0
0.8
S3
n3
n2
n1
S2
n3
n2
n1 IC
S1
n3
n2
n1
S3
0.6
0.4
S1
S2
0.2
0.0
300
400
500
Wavelength (nm)
600
Absorption
Fluorescence emission spectrum is the same
regardless of the excitation wavelength!
S0
Fluorescence
Absorbance
Excitation Spectrum
Fluorescence emission spectrum is the same
regardless of the excitation wavelength!
S3
n3
n2
n1
S2
n3
n2
n1 IC
S1
n3
n2
n1
Absorption
But intensity changes!
S0
Fluorescence
Excitation Spectrum
Absorbance
Monitor emission
(Fixed l)
Scan Through Excitation l
Measuring Excitation Spectra
Procedure
1) Shift emission grating to desired
wavelength (monitor emission max)
Ex Grating
Xenon Lamp
3
Excitation
Monochromator
2
7
2) Shift excitation grating to stating
wavelength
3) Light source on
Emission
Monochromator
4) Light Hits the Sample
PMT
6
Sample
4
5
1
5) Emission from the sample enters
emission monochromator
6) Detect emitted light at PMT
Em Grating
7) Raster excitation grating
Excitation Spectrum
Absorption Spectrum
Excitation Spectrum
1.0
Absorbance (O.D.)
Emission at 650 nm
1.0
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
0.0
0.0
300
400
500
600
Excitation Wavelength (nm)
300
400
500
Wavelength (nm)
If emitting from a single species:
Excitation spectrum should match absorption spectrum!
600
Fluorometer
3
1
2
Components
1) Light source
2) Monochrometer
3) Sample
4) Detector
5) Filters
6) Slits
7) Polarizers
2
4
Samples
Solutions
Powders
Thin Films
Crystals
Solution Fluorescence
Top View
Source
Excitation
Sample
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Excitation
Beam
Emission
Detector
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Emission
non-emitting molecules
filter effect
“self”-absorption
Filter Effect
Anthracene
For Fluorescent Samples:
Absorbance < 1.0
Solid Samples
Thin Films/Solids
Intensity (counts)
20000
Emission Spectrum
Ex: 380 nm
15000
10000
5000
0
600
700
800
900
Wavelength (nm)
Source
Detector
Intensity (counts)
20000
15000
10000
5000
0
600
700
800
Wavelength (nm)
Sample
Real emission spectrum +
Second Order
900
Solid Samples
Thin Films/Solids
Intensity (counts)
20000
2d
Emission Spectrum
Ex: 380 nm
15000
10000
5000
0
600
700
800
900
Wavelength (nm)
λ = 2d(sin θi + sin θr)
Detector at 760 nm sees 380 nm light!
Source
Detector
Intensity (counts)
20000
15000
10000
5000
0
600
700
800
Wavelength (nm)
Sample
Real emission spectrum +
Second Order
900
Filters
Filters
Band Pass Filter
Fluorometer
3
1
2
Components
1) Light source
2) Monochrometer
3) Sample
4) Detector
5) Filters
6) Slits
7) Polarizers
2
4
Fluorometer: Slits
Entrance Slit
Exit Slit
Mirrors
Fluorometer: Slits
Entrance Slit
Slit widths
Wider Slits:
More light hitting sample
More emission
More light hitting the detector
More signal
Greater signal-to-noise
But…resolution decreases!
Exit Slit
Entrance Slit
Source
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Sample
Slit widths
Entrance Slit
Source
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Sample
Small Slit
Large Slit
bandpass (nm) =
slit width (mm) x dispersion (nm mm-1)
1.0
Intensity
0.8
for a 4.25 nm mm-1 grating
0.6
0.4
0.2
460
480
500
520
Wavelength (nm)
540
Excitation Slit widths
Single Component:
Absorbance
Wider slit:
Larger bandwidth
Intensity increase
No emission spectra change
Excitation Slit widths
Wider slit:
Larger bandwidth
Intensity increase
Emission ratio changes (1:2)
-small slit less of dye 2
-large slits more of dye 2
Absorbance (a.u.)
Multi Component :
Dye 1
Dye 2
1.5
1.0
0.5
0.0
400
500
600
Wavelength (nm)
700
Emission Slit widths
Wider slit:
Larger bandwidth
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More light hitting the detector
More signal
Grating
Sample
Lower Resolution
Exit Slit
Detector
doubled slits = intensity2
570 nm emission
Small Slit (0.5 mm)
summing 569-571 nm
(2.125 nm bandwidth)
Large Slit (2.0 mm)
summing 566-574 nm
(8.5 nm bandwidth)
Nyquist Rule: scanning increment should be greater than 1/2 slit widths
Ex: For 8 nm bandwidth set emission acquisition to 4 nm per step.
Emission Intensity
Emission Intensity
Emission Slit widths
Always report your slit widths (in nm)!
Fluorometer
3
1
2
Components
1) Light source
2) Monochrometer
3) Sample
4) Detector
5) Filters
6) Slits
7) Polarizers
2
4
Fluorometer: Polarizer
Mirrors
Polarizer
Polarizer
Fluorescence Anisotropy
Absorption is polarized
Fluorescence is also polarized
Absorption Probablity
Fluorescence Anisotropy
Detector
End View
Unpolarized
Light
Fluorescence Anisotropy
Detector
End View
Unpolarized
Light
Fluorescence Anisotropy
Detector
End View
End View
Unpolarized
Light
Unpolarized
Light
Fluorescence Anisotropy
Polarizer
End View
Polarized
Light
Detector
Fluorescence Anisotropy
Polarizer
End View
Polarized
Light
Detector
Fluorescence Anisotropy
Polarizer
End View
Detector
End View
I^
Polarized
Light
Slightly
Polarized
Light
I||
Fluorescence Anisotropy
Sample
I||
Detector
I^
Polarized Excitation
r = anisotropy factor
I|| and I^ are the intensities of the
observed parallel and perpendicular
components
Fluorescence Anisotropy
r = anisotropy factor
I|| and I^ are the intensities of the
observed parallel and perpendicular
components
Monitor Binding
Reaction Kinetics
Other Sampling Accessories
Spatial Imaging
Integrating Sphere
Cryostat
Microplate Reader
Potential Complications
With Sample
• Solvent Impurities
-run a blank
• Raman Bands
• Concentration to high
-A>1
- Self-absorption
• Scatter (2nd order or spikes)
With the Instrument
• Stray light
• Slit Widths
• Signal/Noise
Fluorescence Spectroscopy End
Any Questions?