Embed Size (px)
Citation preview
1
Introduction to the Spectroscopy ofDye Molecules
2
Angstroms (10 Å =1 nm)
nλ
2
2 2364.56
2
nnm
n!
" #= $ %&' (
3
4
ΔE = hν = hc/λ
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.
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.
7
σ and π Bonds in Ethylene and inConjugated Systems
C – sp3 hybrid tetrahedral bondingdiamond
C – sp2 hybrid equilateral trianglegraphite
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
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.
10
Particle In A Box
Quantum Treatment
n= 1,2,3,…
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.
12
What 1,3,5 hexatriene looks like …
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)
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.
15
Acid Base Indicators
• Indicators are weak acids or weak bases inwhich the undissociated form has a differentcolor than the dissociated (ionized) form.
16
[ ][ ]
[ ]
[ ][ ]
[ ]
H InK
H In
H InH K
In
+ !
+
!
=
=
H In ↔H+ + In-
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.
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?
19
Energy Diagram
Absorption Fluorescence
Phosphorescence
Intersystem crossing
Internal conversion
λ λ
λ
So
T11
S11
Vibrational levels
Electronic levels
S2 heat
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
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
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
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
Spectrophotometer InstrumentationLet me begin with my favorite movie illustrating how such adevice works … …
Single-Beam Spectrophotometer
Shimadzu BioSpec Mini
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
Spectronic Genesys 20
Spectrofluorimeter
L geometry for optical layout
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.
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.
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.
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.
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.
S0 –> S2
S0 –> S1
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
