CHEM 5175: Part 2.5

CHEM 5175: Part 2.5

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

hn 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) hn

hn Excitation Fluorescence Emission Intersystem Crossing hn Emission Triplet Excited State (T1) Phosphorescence Fluorescence Spectroscopy Singlet Excited

State (S1) hn Emission Fluorescence Spin allowed Fast (ns) Organic molecules Triplet Excited State (T1) hn 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)

Kashas Rule: Emission predominantly occurs from the lowest excited state (S0 OR T1) Fluorescence 1920-2013 Kasha Laboratory Building AKA Institute of Molecular Biophysics Kashas Rule: Emission predominantly occurs from the lowest excited state (S0 OR T1) Fluorescence Kashas 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 S1 n n32 n1

n4 S0 n n32 n1 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 Excitation Internal Conversion Fluorescence Non-radiative decay Intersystem Crossing Phosphorescence S0 hn

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 E

Excitation (10-15 s) S0 Intersystem Crossing w/ Heavy atom (< 10-12 s) w/o Heavy atom (> 10-9 s) T1 Fluorescence (10-9 s) Phosphorescence (10-6 s) Emissive Molecules Phosphorescent Fluorescent NH

N N HN Perylene OEP PtOEP Ir(ppy)3 BODIPY Fluorescein Rose Bengal [Ru(bpy)3]2+

I Coumarin Anthracene CH3 Anthracene + ICH3 C60 Fluorometer Source Excitation hn Sample Detector hn

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 5) Emission from the sample enters emission monochromator 3 PMT 4 Sample 5 6 7 Em Grating 8

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 2) Shift excitation grating to desired wavelength (excitation wavelength)

0.4 0.2 3) Light enters sample chamber 0.0 300 400 500 600 Wavelength (nm) 4) Light Hits the Sample 20000

5) Emission from the sample enters emission monochromator 15000 6) Set emission grating 10000 7) Detect emitted light at PMT Intensity (counts) Emission Spectrum 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 Sample 4 5 1 6 5) Emission from the sample enters emission monochromator 6) Detect emitted light at PMT

Em Grating 7) Raster excitation grating Excitation Spectrum Absorption Spectrum 1.0 1.0 0.8 0.8 Absorbance (O.D.) Emission at 650 nm Excitation Spectrum

0.6 0.4 0.2 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 hn Excitation Beam Emission Detector hn 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

Emission Spectrum Ex: 380 nm 15000 10000 5000 0 2d 600 700 800 900 Wavelength (nm) = 2d(sin i + sin r)

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 Butresolution decreases! Exit Slit Entrance Slit Source hn Sample Slit widths Entrance Slit

Source hn 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 hn 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?

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