introduction to the spectroscopy of dye...
TRANSCRIPT
![Page 1: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/1.jpg)
1
Introduction to the Spectroscopy ofDye Molecules
![Page 2: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/2.jpg)
2
Angstroms (10 Å =1 nm)
nλ
2
2 2364.56
2
nnm
n!
" #= $ %&' (
![Page 3: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/3.jpg)
3
![Page 4: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/4.jpg)
4
ΔE = hν = hc/λ
![Page 5: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/5.jpg)
5
Ephoton = hν = Eupper state - Elower state
True for atoms, true for molecules
To interpret the color of an object wemust know the array of possibleenergy levels for its molecules.
Visible absorptive coloration arises when visiblephotons are absorbed and excite moleculesfrom their ground or lowest-energy electronicstate to a higher-energy electronic state.
Transitions between electronic states areresponsible for the majority of the colors we seein the natural world.
![Page 6: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/6.jpg)
6
Q: Why are most substances colorless or white?
A: Most molecular substances are colorlessbecause the spacing between the highestoccupied electronic energy level and the lowestunoccupied level typically is larger than theenergy of any photon in the visible range.
![Page 7: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/7.jpg)
7
σ and π Bonds in Ethylene and inConjugated Systems
C – sp3 hybrid tetrahedral bondingdiamond
C – sp2 hybrid equilateral trianglegraphite
![Page 8: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/8.jpg)
8
Polyenes
Organic molecules that contain alternating single anddouble bonds are said to be conjugated. The simplestexample is butadiene (C4H6), whose structure is shownbelow.
If we examine the p-bonding arrangement in butadienewe can imagine each carbon atom with a p-orbitaloverlapping in a side-on fashion, with each p-orbitalcontaining 1 electron.
BIG CONCEPT: delocalized electrons
![Page 9: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/9.jpg)
9
One example is β-carotene, the pigment that makes carrotsorange and makes butter yellow.
In such molecules the electrons in the π-bonds can beconsidered to be delocalized over all the atoms of theconjugated chain, and to a crude approximation theelectrons can be thought of as moving freely along thelength of the carbon skeleton.
The color of β -carotene arises from an absorption in thevisible spectrum with λmax at 450 nm.
![Page 10: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/10.jpg)
10
Particle In A Box
Quantum Treatment
n= 1,2,3,…
![Page 11: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/11.jpg)
11
Reconsideration of Butadiene
Each π orbital can holdtwo electrons, one spinup, the other spin down.In 1,3 butadiene, thereare 4 π electrons, so thefirst two π MOs are filled.
![Page 12: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/12.jpg)
12
What 1,3,5 hexatriene looks like …
![Page 13: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/13.jpg)
13
So How Well Does This CrudeTheory Work?
A
B
C
Assume L = (2k+2)b,where: k = number of doublebonds along the chain b = 139 pm (C-Clength in benzene)
![Page 14: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/14.jpg)
14
L (pm) Theory Experiment
A 556 328 nm 523 nmB 834 453 nm 605 nmC 1112 580 nm 706 nm
The Result:
The simple one-dimensional particle-in-the-boxmodel does not match the experimental resultsexactly, but it does show the same trend ofdecreasing energy (longer wavelength) as the"box" gets larger.
![Page 15: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/15.jpg)
15
Acid Base Indicators
• Indicators are weak acids or weak bases inwhich the undissociated form has a differentcolor than the dissociated (ionized) form.
![Page 16: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/16.jpg)
16
[ ][ ]
[ ]
[ ][ ]
[ ]
H InK
H In
H InH K
In
+ !
+
!
=
=
H In ↔H+ + In-
![Page 17: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/17.jpg)
17
Phenothalein
colorless magenta
Under acidic conditions, the equilibrium is to the left, andthe concentration of the anions is too low for the magentacolor to be observed.
Under alkaline conditions, the equilibrium is to the right, andthe concentration of the anion becomes sufficient for themagenta color to be observed.
![Page 18: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/18.jpg)
18
After a molecule is excited(1) it is more reactive than before it absorbed light(2) it takes time for the molecule to emit
light and return to the starting molecule (ground state).
During this time, various things can happen:(1) solvent rearrangement(2) rotation of the molecule(3) reaction (quenching)(4) loss of energy to a neighboring molecule
(energy transfer) in a process that is distance related
What Can Light Tell us About aMolecule and its Environment?
![Page 19: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/19.jpg)
19
Energy Diagram
Absorption Fluorescence
Phosphorescence
Intersystem crossing
Internal conversion
λ λ
λ
So
T11
S11
Vibrational levels
Electronic levels
S2 heat
![Page 20: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/20.jpg)
20
Energy DiagramRates of Relaxation
Absorption Fluorescence
So
T11
S11
Electronic levels
S2 heat
10-15 s 10-9 sec 10-6 to 1 s
10-12 s
10-9 s
10-13 s
Phosphorescence
![Page 21: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/21.jpg)
21
Naturally Fluorescent Chromophores
tryptophantyrosinephenylalanine
H2N
CC
CH2
OH
O
H
H2N
CC
CH2
OH
O
H
OH
H2N
CC
H2C
OH
O
H
HN
1La
1Lb
Amino acids Cofactors
N
H
N
NH
N O
O
3HC
3HC
N
C
NH2
O
O
OP
O
N
O
OHP
OO
OHHO
OH OH
HO
HO
N
N
N
NH2
Flavin, FMN
NADH
porphyrin
![Page 22: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/22.jpg)
22
Local Electric Fields CauseSpectral Shifts
Gas phase
µ
µ*
solvent
Solvent can affect the groundstate and excited statemolecules causing spectralshifts
Example: H bonding totryptophan. Changes itsabsorption by about 10 nmChanges its emission byabout 60 nm
![Page 23: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/23.jpg)
23
Solvent Relaxes AroundExcited State Trp
NH2CHC
CH2
HO
O
NH
Ground state dipole moment: 2.1D
Excited state dipole moment: 5.4 D
More polar in the excited state
H
O
H H
O
H
H
O
H
Fluorescence spectral shifts are sensitiveindicators of conformational changes
![Page 24: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/24.jpg)
![Page 25: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/25.jpg)
Spectrophotometer InstrumentationLet me begin with my favorite movie illustrating how such adevice works … …
![Page 26: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/26.jpg)
Single-Beam Spectrophotometer
![Page 27: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/27.jpg)
Shimadzu BioSpec Mini
![Page 28: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/28.jpg)
Hardware Specifications
Spectral Bandwidth 5 nm
Wavelength Range 190 – 1100 nm
Wavelength Accuracy ±1 nm
Recording Range -399 to 399% transmittance
Photometric Accuracy ±0.005 abs at 1.0 abs;
±0.003 abs at 0.5 abs
![Page 29: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/29.jpg)
Spectronic Genesys 20
![Page 30: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/30.jpg)
![Page 31: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/31.jpg)
Spectrofluorimeter
L geometry for optical layout
![Page 32: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/32.jpg)
![Page 33: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/33.jpg)
![Page 34: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/34.jpg)
Stage 2 : Excited-State Lifetime
The excited state exists for a finite time (typically 1–10 x 10-9 seconds). During thistime, the fluorophore undergoes conformational changes and is also subject to amultitude of possible interactions with its molecular environment.
These processes have two important consequences.
First, the energy of S1' is partially dissipated, yielding a relaxed singlet excited state(S1) from which fluorescence emission originates.
Second, not all the molecules initially excited by absorption (Stage 1) return to theground state (S0) by fluorescence emission. Other processes such as collisionalquenching, fluorescence energy transfer, and intersystem crossing (see below) mayalso depopulate S1.
The fluorescence quantum yield, which is the ratio of the number of fluorescencephotons emitted (Stage 3) to the number of photons absorbed (Stage 1), is ameasure of the relative extent to which these processes occur.
![Page 35: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/35.jpg)
The entire fluorescence process is cyclical.Unless the fluorophore is irreversibly destroyedin the excited state (an important phenomenonknown as photobleaching), the samefluorophore can be repeatedly excited anddetected. This concept is key to single-molecule spectroscopy.
For polyatomic molecules in solution, thediscrete electronic transitions represented byhvEX and hvEM in the Jablonski diagram arereplaced by rather broad energy spectra calledthe fluorescence excitation spectrum and thefluorescence emission spectrum, respectively.
![Page 36: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/36.jpg)
The bandwidths of these spectra are parametersof particular importance for applications in whichtwo or more different fluorophores aresimultaneously detected. With few exceptions, thefluorescence excitation spectrum of a singlefluorophore species in dilute solution is identical toits absorption spectrum.
Under the same conditions, the fluorescenceemission spectrum is independent of theexcitation wavelength, owing to the partialdissipation of excitation energy during the excited-state lifetime.
![Page 37: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/37.jpg)
Stage 3 : Fluorescence Emission
A photon of energy hvEM is emitted, returningthe fluorophore to its ground state S0. Owingto energy dissipation during the excited-statelifetime, the energy of this photon is lower,and therefore of longer wavelength, than theexcitation photon hvEX.
The difference in energy or wavelengthrepresented by
(hvEX–hvEM)
is called the Stokes shift.
![Page 38: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/38.jpg)
The Stokes shift is fundamental to thesensitivity of fluorescence techniques becauseit allows emission photons to be detectedagainst a low background, isolated fromexcitation photons.
In contrast, absorption spectrophotometryrequires measurement of transmitted lightrelative to high incident light levels at thesame wavelength.
![Page 39: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/39.jpg)
![Page 40: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/40.jpg)
S0 –> S2
S0 –> S1
![Page 41: Introduction to the Spectroscopy of Dye Moleculesweb.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf · Dye Molecules. 2 Angstroms (10 Å =1 nm) n ... in the natural world](https://reader031.vdocuments.site/reader031/viewer/2022030420/5aa7708e7f8b9a294b8c1083/html5/thumbnails/41.jpg)
41
The Fluorescence Quantum Yield
The fluorescence quantum yield (ΦF) is the ratio of photonsabsorbed to photons emitted through fluorescence.
In other words the quantum yield gives the probability of theexcited state being deactivated by fluorescence rather than byanother, nonradiative mechanism.
0 ≤ ΦF ≤ 1