Molecular Spectros
Molecular Spectros
Molecular Spectros
UV-VIS
1
PRINCIPLES
2
Molecular Spectroscopy
Molecular Spectroscopy: the interaction of
electromagnetic radiation (light) with matter (organic
compounds). This interaction gives specific structural
information.
3
Principles of molecular spectroscopy:
Electromagnetic radiation
organic light organic relaxation organic
molecule molecule + h
molecule
(ground state) h (excited state) (ground state)
From
http://education.jlab.org
7
Molecular UV-Vis Spectroscopy
Molecular energy levels and absorbance wavelength:
* and p* transitions: high-energy, accessible in vacuum
UV (max <150 nm). Not usually observed in molecular UV-Vis.
n * and p * transitions: non-bonding electrons (lone pairs),
wavelength (max) in the 150-250 nm region.
n p* and p p* transitions: most common transitions observed in
organic molecular UV-Vis, observed in compounds with lone pairs
and multiple bonds with max = 200-600 nm.
10
p-molecular orbitals of butadiene
4: 3 Nodes
0 bonding interactions
3 antibonding interactions
ANTIBONDING MO
3: 2 Nodes
1 bonding interactions
2 antibonding interactions
ANTIBONDING MO
2: 1 Nodes
2 bonding interactions
1 antibonding interactions
BONDING MO
1: 0 Nodes
3 bonding interactions
0 antibonding interactions
BONDING MO
Butadiene
Butadiene
12
Molecular orbitals of conjugated polyenes
A
n
tib
o
n
d
in
g
E
n
rg
e
y
B
o
n
d
in
g
H2C CH2
180 nm 217 nm 258 nm 290 nm
13
Molecules with extended conjugation move toward the visible region
Color of Color
absorbed light observed
violet 400 nm yellow
blue 450 orange
blue-green 500 red
yellow-green 530 red-violet
yellow 550 violet
orange 600 blue-green
red 700 green 14
380 nm 400 nm 450 nm 500 nm 550 nm 600 nm 700 nm 780 nm
15
Many natural pigments have conjugated systems
OH
OH
+
N HO O
OH
N Mg N O
O
N O OH OH OH
HO O
CO2CH3
OH
O
Chlorophyll anthocyanin
-carotene
lycopene 16
N
N Mg N O
N O
CO2CH3
17
18
19
-carotene 20
21
22
Reichardt’s Dyes = solvatochromic dyes
23
24
d-f orbital
charge-transfer (CT) electrons
transition
25
Molecular UV-Vis Spectroscopy and Transition
Metal and Lanthanide/Actinide Complexes
d/f orbitals
– UV-Vis spectra of lanthanides/actinides are particularly sharp, due
to screening of the 4f and 5f orbitals by lower shells.
– Can measure ligand field strength, and transitions between d-
orbitals made non-equivalent by the formation of a complex
36
Quantitative Analysis (Beer’s Law):
1) Widely used for Quantitative Analysis
Characterization
• wide range of applications (organic & inorganic)
• limit of detection 10-4 to 10-5 M (10-6 to 10-7M;
current)
• moderate to high selectivity
• typical accuracy of 1-3% ( can be ~0.1%)
• easy to perform, cheap
2) Strategies
a) absorbing species
• detect both organic and inorganic compounds
containing any of these species
Chromophore: light absorbing portion of a molecule
A=ecl A = absorbance
c = concentration (M, mol/L)
l = sample path length (cm)
e = molar absorptivity (extinction coefficient)
a proportionality constant for a specific
absorbance of a substance
38
Molecular UV-Vis Spectroscopy: Absorption
max is the wavelength(s) of maximum absorption (i.e. the
peak position)
The strength of a UV-Visible absorption is given by the
molar absorption coefficient (e):
e = 8.7 x 1019 P a
where P is the transition probability (0 to 1) –
governed by selection rules and orbital overlap,
and a is the chromophore area in cm2
R² = 0.9926
0.8
0.6
0.4
0.2
0
0 0.01 0.02 0.03
Concentration (M)
b) non- absorbing species
- react with reagent that forms colored product
- can also use for absorbing species to lower limit of detection
- items to consider:
, pH, temperature, ionic strength
- prepare standard curve (match standards and samples as much
possible)
Non-absorbing
reagent Species (colorless)
Complex
(red)
(colorless)
m = y/ x
Instrument Response ( S )
kVs cs kVx cx
b = y-intercept S
Vt Vt
S mVs b
(V s ) 0
S 1Vs cs
Cx
Vs ( S 2 S 1)Vs
Where:
S = signal or instrument response
k = proportionality constant
Vs = volume of standard added
bcs cs = concentration of the standard
cx Vx = volume of the sample aliquot
mVx cx = concentration of the sample
Vt = total volume of diluted solutions
Note: assumes a linear relationship between instrument response and sample
concentration.
c) Analysis of Mixtures
- use two different ’s with different e’s
49
Molecular UV-Visible Spectrophotometers
The traditional UV-
Vis design: double-
beam grating
systems
Sources:
Almost
universal
continuum UV-
Vis source is
the 2H lamp.
Tungsten lamps
used for longer Hamamatsu
(visible) L2D2 lamps
wavelengths.
Advantages:
– Sensitivity
(multiplex)
– Speed
Disadvantages:
– Resolution
4*
2*
3*
2
1 p
1
UV Spectroscopy
log(I0/I) = A
sample
UV-VIS sources I0 I
detector
200 700
, nm
monochromator/
reference
55
UV Spectroscopy
4. As with the dispersive IR, the lamps illuminate the entire band of
UV or visible light; the monochromator (grating or prism)
gradually changes the small bands of radiation sent to the beam
splitter
56
UV Spectroscopy
Diode array
UV-VIS sources
sample
Polychromator
– entrance slit and dispersion device 57
UV Spectroscopy
58
UV Spectroscopy
9. The more non-polar the solvent, the better (this is not always possible)
60
UV Spectroscopy
2. Due to the lack of any fine structure, spectra are rarely shown in
their raw form, rather, the peak maxima are simply reported as a
numerical list of “lamba max” values or max
NH2
max = 206 nm
O O 252
317
376
61
UV Spectroscopy
A is unitless, so the units for e are cm-1 · M-1 and are rarely expressed
5. Since path length and concentration effects can be easily factored out,
absorbance simply becomes proportional to e, and the y-axis is expressed
as e directly or as the logarithm of e
63
UV Spectroscopy
65
Analyte Method λ (nm)
Trace Metals
aluminum react with Eriochrome cyanide R dye at pH6; forms red 535
to pink complex
arsenic reduce to AsH3 using Zn and react with silver 535
diethyldithiocarbamate; forms red complex
cadmium extract into CHCl3 containing dithizone from a sample 518
made basic with NaOH; forms pink to red complex
chromium oxidize to Cr(VI) and react with diphenylcarbazide; 540
forms red-violet product
copper react with neocuprine in neutral to slightly acid solution 457
and extract into CHCl3/CH3OH; forms yellow complex
2+
iron reduce to Fe and react with o-phenanthroline; forms 510
orange-red complex
lead extract into CHCl3 containing dithizone from sample 510
+
made basic with NH3/NH4 buffer; forms cherry red
complex
–
manganese oxidize to MnO4 with persulfate; forms purple solution 525
mercury extract into CHCl3 containing dithizone from acidic 492
sample; forms orange complex
zinc react with zincon at pH 9; forms blue complex 620
66
Inorganic Nonmetals
ammonia reaction with hypochlorite and phenol using a 630
manganous salt catalyst; forms blue indophenol
as product
cyanide react with chloroamine-T to form CNCl and then 578
with a pyridine-barbituric acid; forms a red-blue
dye
fluoride react with red Zr-SPADNS lake; formation of 570
2–
ZrF6 decreases color of the red lake
chlorine react with leuco crystal violet; forms blue product 592
(residual)
–
nitrate react with Cd to form NO2 and then react with 543
sulfanilamide and N-(1-napthyl)-ethylenediamine;
forms red azo dye
68
neocuproine
69
70
Zincon
71
Analysis of Glucose
72
Analysis of Glucose
73
74
Fe-Phenanthroline
75
Cd2+ + Dithizon
76
UV-vis absorption spectrum of (a) dithizone, (b)
complex of dithizone with Cd(II) before adsorption, and
(c) complex of dithizone with Cd(II) after adsorption
77
Analysis of Nitrite
78
79
Analysis of Surfactants
80
81
Organic UV-Vis Spectroscopy
(optional)
82
UV Spectroscopy
III. Chromophores
A. Definition
1. Remember the electrons present in organic molecules are involved
in covalent bonds or lone pairs of electrons on atoms such as O or
N
83
Interpretation of Molecular UV-Visible Spectra
UV-Visible spectra can be
interpreted to help determine
molecular structure, but this
is presently confined to the
analysis of electron behavior
in known compounds.
See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).
Interpretation of UV-Visible Spectra
Other examples:
H2N H3C
– The conjugation of a lone pair on a
enamine shifts the max from 190 nm
CH2 vs. HC CH2
~230 nm ~180 nm
(isolated alkene) to 230 nm. The
nitrogen has an auxochromic effect.
See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).
Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Application of UV/Vis Spectroscopy
Common Problems:
a) Mixtures:
• blank samples often contain multiple absorbing
species.
• the absorbance is the sum of all the individual
absorbencies
A= A1 + A2 +A3 + … = e1bc + e2bc + e3bc …
• substances in both the blank and sample which
absorb can be “blanked out” in both double and
single beam spectrometers.
Applications:
A) Molar Absorptivities (e) in UV-Vis Range:
e = 8.7 x 1019 PA
P – transition probability (ranges from 0.1 to 1, for likely
transitions)
A – cross-section area of target molecule (cm2)
- ~10-15 cm2 for typical organics
- emax = 104 to 105 L/mol-cm
- e < 103 – low intensity (P #0.01)
Name Structure max e
but-1-en-3-yne 219 7,600
If conjugated
- shifts to higher ’s (red shift)
Basic process:
M + h M*
10-8 – 10-9s
M* M + heat
(or fluorescence, light, or phosphorescence)
or
10-8 – 10-9s
M* N
(new species, photochemical reaction)
Note: excited state (M*) is generally short and heat
produced not generally measurable.
Thus, get minimal disturbance of systems (assuming no
photochemical reaction)
2) Absorption occurs with bonding electrons.
-E() required differs with type of bonding electron.
-- UV-Vis absorption gives some information on bonding electrons
(functional groups in a compound.
-Most organic spectra are complex
electronic and vibration transitions superimposed
absorption bands usually broad
detailed theoretical analysis not possible, but semi-quantitative or
qualitative analysis of types of bonds is possible.
effects of solvent & molecular details complicate comparison
-Single bonds usually too high excitation energy for most instruments
(#185 nm)
vacuum UV
most compounds of atmosphere absorb in this range, so difficult to
work with.
usually concerned with functional groups with relatively low
excitation energies (190 ##850 nm).
- Types of electron transitions:
i) , p, n electrons
Example: Formaldehyde
* transition in vacuum UV
n * saturated compounds with non-bonding electrons
~ 150-250 nm
e ~ 100-3000 ( not strong)
n p*, p p* requires unsaturated functional groups (eq. double
bonds) most commonly used, energy good range for UV/Vis
~ 200 - 700 nm
n p* : e ~ 10-100
p p*: e ~ 1000 – 10,000
Absorption Characteristics of Some Common Chromophores
Chromophore Example Solvent max (nm) emax Type of
transition
Alkene C6H13HC CH2 n-Heptane 177 13,000 pp*
Alkyne n-Heptane 178 10,000 pp*
C5H11C C CH3 196 2,000 _
225 160
_
Carbonyl O n-Hexane 186 1,000 n*
280 16 np*
CH3CCH3
O n-Hexane 180 Large
293 12
n*
CH3CH np*
Carboxyl O Ethanol 204 41 np*
CH3COH
Amido O Water 214 60 np*
CH3CNH2
Azo H3CN NCH3 Ethanol 339 5 np*
(a) Vapor
(c)
Aqueous
3) Solvent can also absorb in UV-vis spectrum.
3) Can obtain some functional group information for
certain types of compounds..
- weak band at 280-290 nm that is shifted to shorter ’s
with an increase in polarity (solvent) implies a carbonyl
group.
acetone:
in hexane, max = 279 nm (e = 15)
in water, max = 264.5 nm
- solvent effects due to stabilization or destabilization of
ground or excited states, changing the energy gap.
since most transitions result in an excited state that is
more polar than the ground state
- 260 nm with some fine structure implies an aromatic
ring.
Benzene in heptane
III. Chromophores
B. Organic Chromophores
1. Alkanes – only posses -bonds and no lone pairs of electrons, so
only the high energy * transition is observed in the far UV
C C
106
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
2. Alcohols, ethers, amines and sulfur compounds – in the cases of
simple, aliphatic examples of these compounds the n * is the
most often observed transition; like the alkane * it is most
often at shorter than 200 nm
Note how this transition occurs from the HOMO to the LUMO
C N
*CN
C N
nN sp3 C N anitbonding
orbital
CN C N
107
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
3. Alkenes and Alkynes – in the case of isolated examples of these
compounds the p p* is observed at 175 and 170 nm, respectively
p*
108
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
4. Carbonyls – unsaturated systems incorporating N or O can
undergo n p* transitions (~285 nm) in addition to p p*
109
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
4. Carbonyls – n p* transitions (~285 nm); p p* (188 nm)
p*
It has been
n
C O determined from
spectral studies,
that carbonyl
p O
oxygen more
approximates sp
CO transitions omitted for clarity rather than sp2 !
110
UV Spectroscopy
III.Chromophores
C. Substituent Effects
General – from our brief study of these general
chromophores, only the weak n p* transition occurs
in the routinely observed UV
111
UV Spectroscopy
III. Chromophores
C. Substituent Effects
General – Substituents may have any of four effects on a chromophore
i. Bathochromic shift (red shift) – a shift to longer ; lower energy
Hyperchromic
e Hypsochromic Bathochromic
Hypochromic
200 nm 700 nm
112
UV Spectroscopy
III. Chromophores
C. Substituent Effects
1. Conjugation – most efficient means of bringing about a bathochromic and
hyperchromic shift of an unsaturated chromophore:
H2C max nm e
175 15,000
CH2
217 21,000
258 35,000
465 125,000
-carotene
O
n p* 280 12
p p* 189 900
O n p* 280 27
p p* 213 7,100
113
UV Spectroscopy
III. Chromophores
C. Substituent Effects
1. Conjugation – Alkenes
The observed shifts from conjugation imply that an increase in
conjugation decreases the energy required for electronic excitation
From molecular orbital (MO) theory two atomic p orbitals, f1 and f2 from
two sp2 hybrid carbons combine to form two MOs 1 and 2* in ethylene
2*
f1 f2
1 p
114
UV Spectroscopy
III. Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
When we consider butadiene, we are now mixing 4 p orbitals
giving 4 MOs of an energetically symmetrical distribution
compared to ethylene
4*
2*
3*
2
1 p
1
III. Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
Extending this effect out to longer conjugated systems the energy gap
becomes progressively smaller:
ethylene
butadiene
hexatriene
octatetraene 116
UV Spectroscopy
III. Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
Similarly, the lone pairs of electrons on N, O, S, X can extend
conjugated systems – auxochromes
Here we create 3 MOs – this interaction is not as strong as that of
a conjugated p-system
A
3*
p* 2
Energy
p
nA
1
117
UV Spectroscopy
III.Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
Methyl groups also cause a bathochromic shift, even
though they are devoid of p- or n-electrons
This effect is thought to be through what is termed
“hyperconjugation” or sigma bond resonance
H
C
H
C C H
118
UV Spectroscopy
Next time – We will find that the effect of substituent groups can
be reliably quantified from empirical observation of known
conjugated structures and applied to new systems
s-trans s-cis
120
UV Spectroscopy
Consider:
122
UV Spectroscopy
123
UV Spectroscopy
Allylidenecyclohexane
- acyclic butadiene = 217 nm
one exocyclic C=C + 5 nm
2 alkyl subs. +10 nm
232 nm
Experimental value 237 nm
125
UV Spectroscopy
The increment table is the same as for acyclic butadienes with a couple
additions:
Group Increment
Additional homoannular +39
Where both types of diene
are present, the one with
the longer becomes the
base
126
UV Spectroscopy
C OH C OH
O O
127
UV Spectroscopy
1 exo C=C + 5 nm
234 nm
128
UV Spectroscopy
239 nm
278 nm 129
UV Spectroscopy
130
UV Spectroscopy
p* p*
3*
n n
2
p p
1
O O
134
UV Spectroscopy
Group Increment
6-membered ring or acyclic enone Base 215 nm
5-membered ring parent enone Base 202 nm
Acyclic dienone Base 245 nm
135
UV Spectroscopy
Acid or ester
With a or alkyl groups 208
With a, or , alkyl groups 217
Group value – exocyclic a, double bond +5
Group value – endocyclic a, bond in 5 +5
or 7 membered ring
136
UV Spectroscopy
137
UV Spectroscopy
R
cyclic enone = 215 nm
extended conj. +30 nm
b-ring residue +12 nm
d-ring residue +18 nm
O exocyclic double bond + 5 nm
280 nm
Experimental 280 nm
138
UV Spectroscopy
Eremophilone allo-Eremophilone
139
UV Spectroscopy
p6*
p4* p5*
p2 p3
p1
140
UV Spectroscopy
180 nm
260 nm (allowed)
p2 p3 (forbidden)
A1g
p1
141
UV Spectroscopy
142
UV Spectroscopy
143
UV Spectroscopy
G G G G
144
UV Spectroscopy
G G G G
*
- *
*
*
145
UV Spectroscopy
146
UV Spectroscopy
• For the benzoate ion, the effect of extra n-electrons from the
anion reduces the effect slightly
Primary Secondary
Substituent max e max e
-C(O)OH 230 11,600 273 970
-C(O)O- 224 8,700 268 560
147
UV Spectroscopy
149
UV Spectroscopy
• Consider p-nitroaniline:
O O
H2N N H2N N
O O
150
UV Spectroscopy
O R
G
151
UV Spectroscopy
O R Substituent increment
G o m p
Alkyl or ring residue 3 3 10
-O-Alkyl, -OH, -O-Ring 7 7 25
-O- 11 20 78
G
-Cl 0 0 10
-Br 2 2 15
-NH2 13 13 58
-NHC(O)CH3 20 20 45
-NHCH3 73
-N(CH3)2 20 20 85
152
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• The portion of the EM spectrum from 400-800 is observable
to humans- we (and some other mammals) have the
adaptation of seeing color at the expense of greater detail
V. Visible Spectroscopy
A. Color
1. General
• When white (continuum of ) light passes through, or is reflected
by a surface, those ls that are absorbed are removed from the
transmitted or reflected light respectively
155
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• Organic compounds that are “colored” are typically those with
extensively conjugated systems (typically more than five)
• Consider -carotene
156
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• Likewise:
O
H
N
N
H
O
indigo
V. Visible Spectroscopy
A. Color
1. General
• One of the most common class of colored organic molecules
are the azo dyes:
N N
EWGs EDGs
V. Visible Spectroscopy
A. Color
1. General
• These materials are some of the more familiar
NO2
colors of our “environment”
HO
O3S N N
N H2N N N
N
OH NH2
SO3
159
The colors of M&M’s
Bright Blue Royal Blue
Common Food Uses Common Food Uses
Beverages, dairy products, powders, jellies, confections, Baked goods, cereals, snack foods, ice-cream, confections,
condiments, icing. cherries.
Orange-red Lemon-yellow
Common Food Uses Common Food Uses
Gelatins, puddings, dairy products, confections, beverages, Custards, beverages, ice-cream, confections, preserves,
condiments. cereals.
Orange
Common Food Uses
Cereals, baked goods, snack foods, ice-cream, beverages,
dessert powders, confections
160
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• In the biological sciences these compounds are used as dyes
to selectively stain different tissues or cell structures
HO
O3S N N N N
SO3
161
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• In the chemical sciences these are the acid-base indicators
used for the various pH ranges:
CH3 CH3
H
O3S N N N O3S N N N
CH3 CH3
162
Chemistry 330
Chapter 17
Electronic Spectroscopy
Absorption and Emission
• The absorption
spectrum of
chlorophyll in the
visible region.
• Absorbs in the red
and blue regions,
and that green light
is not absorbed.
The Franck-Condon Principle
• The most intense
vibronic transition is
from the ground
vibrational state to the
vibrational state lying
vertically above it.
• Transitions to other
vibrational levels occur
with lower intensity.
The Q-M Version
• A molecule will
undergo a transition to
the upper vibrational
state when the upper
state wavefunction most
closely resembles the
vibrational wavefunction
of the vibrational ground
state of the lower
electronic state.
Types of Transitions
• Chromophore – a group with a
characteristic optical absorption
• Various types of transition are posssible
– A d-d transition
– Vibronic transitions
– Charge transfer transitions
– p - p* and n - p* transitions
A d-d Transition
• Ligand field theory can be used to
describe the electronic absorption
spectrum of e.g. [Ti(OH2)6]3+ in aqueous
solution.
eg
o
t2g
A Typical Spectrum
• o 20 000 cm-1
for the complex
[Ti(OH2)6]3+ in
aqueous solution.
• Transitions
usually occur in
the visible region
Forbidden Transitions and
Vibronic Spectra
• A d - d transition is
parity-forbidden
because it corresponds
to a g - g transition.
• A vibration of the
molecule can destroy
the inversion symmetry
• The removal of the
centre of symmetry
gives rise to a
vibronically allowed
transition.
Transition Involving p-
electrons
• A C=C double bond
acts as a chromophore.
• One of its important
transitions is the p* p
transition
• The electron is
promoted from a p MO
orbital to the
corresponding
antibonding orbital.
Transition Involving p-
electrons (cont’d)
• A carbonyl (CO)
group acts as a
chromophore
primarily on account
of the excitation of a
nonbonding O lone-
pair electron to an
antibonding CO p
orbital.
Fluorescence vs.
Phosphorescence
• The empirical distinction
between fluorescence
and phosphorescence
• Fluorescence is
extinguished
immediately when the
the exciting source is
removed,
• Phosphorescence
continues with relatively
slowly diminishing
intensity.
Fluorescence
• The sequence of steps
leading to fluorescence.
• The upper vibrational
states undergo
radiationless decay by
giving up energy to the
surroundings.
• Radiative transition then
occurs from the
vibrational ground state
of the upper electronic
state.
Absorption vs. Fluorescence
• An absorption spectrum
(spectrum a) shows a
vibrational structure
characteristic of the
upper state.
• A fluorescence
spectrum (spectrum b)
shows a structure
characteristic of the
lower state.
0,0 bands
Solvent Influences
• The solvent can shift
the fluorescence
spectrum relative to the
absorption spectrum.
• Before fluorescence
occurs, the solvent
molecules relax into a
new arrangement, and
that arrangement is
preserved during the
subsequent radiative
transition.
Phosphorescence
• The sequence of steps
leading to
phosphorescence
• Intersystem crossing -
the switch from a singlet
state to a triplet state
brought about by spin-
orbit coupling.
• The triplet state –
ground state transition
is spin-forbidden.
A Jablonski Diagram for
Naphthalene
• Displays of the
relative positions of
the electronic
energy levels of a
molecule.
• IC - internal
conversion
• ISC - intersystem
crossing.)
Continuum Absorption
• When absorption
occurs to unbound
states of the upper
electronic state, the
molecule dissociates
and the absorption is a
continuum.
• Below the dissociation
limit - normal
vibrational structure.
Dissociative States
• When a dissociative
state crosses a bound
state, molecules
excited to levels near
the crossing may
dissociate.
• This process is called
predissociation, and is
detected in the
spectrum as a loss of
vibrational structure that
resumes at higher
frequencies.
A Three Level Laser
• The transitions involved
in one kind of three-
level laser.
• The pumping pulse
populates the
intermediate state I,
which in turn populates
the laser state A.
• Laser transition is the
stimulated emission A
X.
A Four–Level Laser
• The transitions
involved in a four-
level laser. Because
the laser transition
terminates in an
excited state (A ),
the population
inversion betweeen
A and A' is much
easier to achieve.
The Steps Leading to Laser
Action
• The Boltzmann population of states,
with more atoms in the ground state.
• When the initial state absorbs, the
populations are inverted (the atoms are
pumped to the excited state).
Laser Action
• A cascade of
radiation then
occurs,
• One emitted photon
stimulates another
atom to emit
• The radiation is
coherent (phases in
step).
Q-Switching
• The principle of Q-
switching. The excited
state is populated while
the cavity is
nonresonant.
• The resonance
characteristics are
suddely restored, and
the stimulated emission
emerges in a giant
pulse.
A Mode-Locked Laser
• The output of a
mode-locked laser
consists of a stream
of very narrow
pulses separated by
an interval equal to
the time it takes for
light to make a
round trip inside the
cavity.
The Requirements for Laser
Action
• A summary of the
features needed for
efficient laser action.
The Principles of
Photoelectron Spectroscopy
• An incoming photon
carries an energy
h; an energy Ii is
needed to remove
an electron from an
orbital i, and the
difference appears
as the kinetic energy
of the electron.
The Photoelectron
Spectrometer
• A photoelectron
spectrometer
consists of a source
of ionizing radiation
– a helium discharge
lamp for UPS
– X-ray source for XPS
• Electrostatic
analyser
• Electron detector
The Photoelectron Spectrum
of HBr
• The lowest ionization
energy bands ()
correspond to the
ionization of a Br lone-
pair electron.
• The higher ionization
energy band ()
corresponds to the
ionization of a bonding
electron.
Fall 2005
Chapter 7: UV Spectroscopy
•UV & electronic transition
•Usable ranges & observa
•Selection rules
•Band Structure
•Instrumentation & Spectr
•Beer-Lambert Law
•Application of UV-spec
191
UV Spectroscopy
I. Introduction
A. UV radiation and Electronic Excitations
1. The difference in energy between molecular bonding, non-bonding and
anti-bonding orbitals ranges from 125-650 kJ/mole
Visible
192
UV Spectroscopy
I. Introduction
B. The Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the broad spectrum of
the UV radiation
2. If a particular electronic transition matches the energy of a certain band
of UV, it will be absorbed
3. The remaining UV light passes through the sample and is observed
4. From this residual radiation a spectrum is obtained with “gaps” at these
discrete energies – this is called an absorption spectrum
p*
p*
p
p
193
UV Spectroscopy
I. Introduction
C. Observed electronic transitions
1. The lowest energy transition (and most often obs. by UV) is typically that
of an electron in the Highest Occupied Molecular Orbital (HOMO) to the
Lowest Unoccupied Molecular Orbital (LUMO)
2. For any bond (pair of electrons) in a molecule, the molecular orbitals are
a mixture of the two contributing atomic orbitals; for every bonding
orbital “created” from this mixing (, p), there is a corresponding anti-
bonding orbital of symmetrically higher energy (*, p*)
3. The lowest energy occupied orbitals are typically the ;likewise, the
corresponding anti-bonding * orbital is of the highest energy
5. Unshared pairs lie at the energy of the original atomic orbital, most often
this energy is higher than p or (since no bond is formed, there is no
benefit in energy)
194
UV Spectroscopy
I. Introduction
C. Observed electronic transitions
6. Here is a graphical representation
*
Unoccupied levels
p*
Occupied levels
p
Molecular orbitals
195
UV Spectroscopy
I. Introduction
C. Observed electronic transitions
7. From the molecular orbital diagram, there are several possible electronic
transitions that can occur, each of a different relative energy:
*
* alkanes
p*
p* carbonyls
p p* unsaturated cmpds.
Energy
n
n * O, N, S, halogens
n p* carbonyls
p
196
UV Spectroscopy
I. Introduction
C. Observed electronic transitions
7. Although the UV spectrum extends below 100 nm (high energy), oxygen
in the atmosphere is not transparent below 200 nm
* alkanes 150 nm
p* carbonyls 170 nm
n * O, N, S, halogens 190 nm
n p* carbonyls 300 nm √
197
UV Spectroscopy
I. Introduction
D. Selection Rules
1. Not all transitions that are possible are observed
198
UV Spectroscopy
I. Introduction
E. Band Structure
1. Unlike IR (or later NMR), where there may be upwards of 5 or
more resolvable peaks from which to elucidate structural
information, UV tends to give wide, overlapping bands
199
UV Spectroscopy
I. Introduction
E. Band Structure
5. When these energy levels are superimposed, the effect can be readily
explained – any transition has the possibility of being observed
Disassociation
R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
V2
E1 V1 R1 - Rn
R1 - Rn
Vo
Disassociation
Energy R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
V2
V1 R1 - Rn
R1 - Rn
E0 Vo
200
UV Spectroscopy
log(I0/I) = A
I0 I
sample
UV-VIS sources
200 700
detector
, nm
monochromator/
reference
201
UV Spectroscopy
4. As with the dispersive IR, the lamps illuminate the entire band of
UV or visible light; the monochromator (grating or prism)
gradually changes the small bands of radiation sent to the beam
splitter
202
UV Spectroscopy
Diode array
UV-VIS sources
sample
Polychromator
– entrance slit and dispersion device 203
UV Spectroscopy
3. Only quartz is transparent in the full 200-700 nm range; plastic and glass
are only suitable for visible spectra
204
UV Spectroscopy
6. Since spectra are only obtained up to 200 nm, solvents typically only
need to lack conjugated p systems or carbonyls
205
UV Spectroscopy
9. The more non-polar the solvent, the better (this is not always possible)
206
UV Spectroscopy
2. Due to the lack of any fine structure, spectra are rarely shown in
their raw form, rather, the peak maxima are simply reported as a
numerical list of “lamba max” values or max
NH2
max = 206 nm
O O 252
317
376
207
UV Spectroscopy
A is unitless, so the units for e are cm-1 · M-1 and are rarely expressed
5. Since path length and concentration effects can be easily factored out,
absorbance simply becomes proportional to e, and the y-axis is expressed
as e directly or as the logarithm of e
209
UV Spectroscopy
III. Chromophores
A. Definition
1. Remember the electrons present in organic molecules are involved
in covalent bonds or lone pairs of electrons on atoms such as O or
N
211
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
1. Alkanes – only posses -bonds and no lone pairs of electrons, so
only the high energy * transition is observed in the far UV
C C
212
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
2. Alcohols, ethers, amines and sulfur compounds – in the cases of
simple, aliphatic examples of these compounds the n * is the
most often observed transition; like the alkane * it is most
often at shorter than 200 nm
Note how this transition occurs from the HOMO to the LUMO
C N
*CN
C N
nN sp3 C N anitbonding
orbital
CN C N
213
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
3. Alkenes and Alkynes – in the case of isolated examples of these
compounds the p p* is observed at 175 and 170 nm, respectively
p*
214
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
4. Carbonyls – unsaturated systems incorporating N or O can
undergo n p* transitions (~285 nm) in addition to p p*
215
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
4. Carbonyls – n p* transitions (~285 nm); p p* (188 nm)
p*
It has been
determined from
n
C O
spectral studies, that
carbonyl oxygen more
approximates sp
p O
rather than sp2 !
216
UV Spectroscopy
III.Chromophores
C. Substituent Effects
General – from our brief study of these general
chromophores, only the weak n p* transition occurs
in the routinely observed UV
217
UV Spectroscopy
III. Chromophores
C. Substituent Effects
General – Substituents may have any of four effects on a chromophore
i. Bathochromic shift (red shift) – a shift to longer ; lower energy
Hyperchromic
e
Hypsochromic Bathochromic
Hypochromic
200 nm 700 nm
218
UV Spectroscopy
III. Chromophores
C. Substituent Effects
1. Conjugation – most efficient means of bringing about a bathochromic and
hyperchromic shift of an unsaturated chromophore:
H2C
max nm e
175 15,000
CH2
217 21,000
258 35,000
465 125,000
-carotene
O
n p* 280 12
p p* 189 900
O n p* 280 27
p p* 213 7,100
219
UV Spectroscopy
III. Chromophores
C. Substituent Effects
1. Conjugation – Alkenes
The observed shifts from conjugation imply that an increase in
conjugation decreases the energy required for electronic excitation
From molecular orbital (MO) theory two atomic p orbitals, f1 and f2 from
two sp2 hybrid carbons combine to form two MOs 1 and 2* in ethylene
2 *
f1 f2
1 p
220
UV Spectroscopy
III. Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
When we consider butadiene, we are now mixing 4 p orbitals
giving 4 MOs of an energetically symmetrical distribution
compared to ethylene
4*
2*
3*
2
1 p
1
III. Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
Extending this effect out to longer conjugated systems the energy gap
becomes progressively smaller:
ethylene
butadiene
hexatriene
octatetraene 222
UV Spectroscopy
III. Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
Similarly, the lone pairs of electrons on N, O, S, X can extend
conjugated systems – auxochromes
Here we create 3 MOs – this interaction is not as strong as that of
a conjugated p-system
A
3*
p* 2
Energy
p
nA
1
223
UV Spectroscopy
III. Chromophores
C. Substituent Effects
2. Conjugation – Alkenes
Methyl groups also cause a bathochromic shift, even though they
are devoid of p- or n-electrons
This effect is thought to be through what is termed
“hyperconjugation” or sigma bond resonance
C
H
C C H
224
UV Spectroscopy
Next time – We will find that the effect of substituent groups can be reliably
quantified from empirical observation of known conjugated structures and
applied to new systems
max = 239 nm
225
UV Spectroscopy
s-trans s-cis
226
UV Spectroscopy
227
UV Spectroscopy
Consider:
228
UV Spectroscopy
229
UV Spectroscopy
Allylidenecyclohexane
- acyclic butadiene = 217 nm
one exocyclic C=C + 5 nm
2 alkyl subs. +10 nm
232 nm
Experimental value 237 nm
231
UV Spectroscopy
The increment table is the same as for acyclic butadienes with a couple
additions:
Group Increment
Additional homoannular +39
Where both types of diene
are present, the one with
the longer becomes the
base
232
UV Spectroscopy
C OH C OH
O O
233
UV Spectroscopy
1 exo C=C + 5 nm
234 nm
234
UV Spectroscopy
236
UV Spectroscopy
237
UV Spectroscopy
H
293 nm This is explained by the inductive withdrawal
of electrons by O, N or halogen from the
O carbonyl carbon – this causes the n-electrons
CH3 279 on the carbonyl oxygen to be held more
firmly
O
235
Cl It is important to note this is different from
the auxochromic effect on p p* which
O
214 extends conjugation and causes a
NH2
bathochromic shift
O
204 In most cases, this bathochromic shift is not
O
enough to bring the p p* transition into
O the observed range
204
OH
238
UV Spectroscopy
p* p*
3*
n n
2
p p
1
O O
240
UV Spectroscopy
Group Increment
6-membered ring or acyclic enone Base 215 nm
5-membered ring parent enone Base 202 nm
Acyclic dienone Base 245 nm
241
UV Spectroscopy
Acid or ester
With a or alkyl groups 208
With a, or , alkyl groups 217
Group value – exocyclic a, double bond +5
Group value – endocyclic a, bond in 5 +5
or 7 membered ring
242
UV Spectroscopy
243
UV Spectroscopy
R
cyclic enone = 215 nm
extended conj. +30 nm
-ring residue +12 nm
d-ring residue +18 nm
O exocyclic double bond + 5 nm
280 nm
Experimental 280 nm
244
UV Spectroscopy
Eremophilone allo-Eremophilone
245
UV Spectroscopy
p6*
p4* p5*
p2 p3
p1
246
UV Spectroscopy
E1u
p6*
B1u
200 nm
p4* p5*
(forbidden
) B2u
180 nm
260 nm (allowed)
p2 p3 (forbidden
) A1g
p1
247
UV Spectroscopy
248
UV Spectroscopy
249
UV Spectroscopy
G G G G
250
UV Spectroscopy
G G G G
*
- *
*
*
251
UV Spectroscopy
252
UV Spectroscopy
• For the benzoate ion, the effect of extra n-electrons from the
anion reduces the effect slightly
Primary Secondary
Substituent max e max e
-C(O)OH 230 11,600 273 970
-C(O)O- 224 8,700 268 560
253
UV Spectroscopy
255
UV Spectroscopy
• Consider p-nitroaniline:
O O
H2N N H2N N
O O
256
UV Spectroscopy
O R
G
257
UV Spectroscopy
O R Substituent increment
G o m p
Alkyl or ring residue 3 3 10
-O-Alkyl, -OH, -O-Ring 7 7 25
-O- 11 20 78
G
-Cl 0 0 10
-Br 2 2 15
-NH2 13 13 58
-NHC(O)CH3 20 20 45
-NHCH3 73
-N(CH3)2 20 20 85
258
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• The portion of the EM spectrum from 400-800 is observable
to humans- we (and some other mammals) have the
adaptation of seeing color at the expense of greater detail
V. Visible Spectroscopy
A. Color
1. General
• When white (continuum of ) light passes through, or is reflected
by a surface, those ls that are absorbed are removed from the
transmitted or reflected light respectively
261
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• Organic compounds that are “colored” are typically those with
extensively conjugated systems (typically more than five)
• Consider -carotene
262
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• Likewise:
O
H
N
N
H
O
indigo
V. Visible Spectroscopy
A. Color
1. General
• One of the most common class of colored organic molecules
are the azo dyes:
N N
EWGs EDGs
V. Visible Spectroscopy
A. Color
1. General
• These materials are some of the more familiar
NO2
colors of our “environment”
HO
O3S N N
N H2N N N
N
OH NH2
SO3
265
The colors of M&M’s
Bright Blue Royal Blue
Common Food Uses Common Food Uses
Beverages, dairy products, powders, jellies, confections, Baked goods, cereals, snack foods, ice-cream, confections,
condiments, icing. cherries.
Orange-red Lemon-yellow
Common Food Uses Common Food Uses
Gelatins, puddings, dairy products, confections, beverages, Custards, beverages, ice-cream, confections, preserves,
condiments. cereals.
Orange
Common Food Uses
Cereals, baked goods, snack foods, ice-cream, beverages,
dessert powders, confections
266
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• In the biological sciences these compounds are used as dyes
to selectively stain different tissues or cell structures
O3S N N N N
SO3
267
UV Spectroscopy
V. Visible Spectroscopy
A. Color
1. General
• In the chemical sciences these are the acid-base indicators used for
the various pH ranges:
CH3 CH3
H
O3S N N N O3S N N N
CH3 CH3
268
UV-visible molecular
absorption spectroscopy
Chemistry 243
Transmission and
absorbance and losses
• The reduction in the
intensity of light
transmitted through a
sample can be used to
quantitate the amount
of an unknown material.
P Psample
T
P0 Pblank
P0 Pblank
A log T log log
P Psample
Beer’s Law
• Quantitative P0
relationship between A log e bc
P
absorbance and
e molar absorptivity
concentration of
analyte b pathlength
– See derivation in text c concentration
(Skoog: pages 337-338)
• Absorption is additive Really: A = ebc
for mixtures Beer’s Law is always wavelength-specific
Amixture A1 A2 ... An
Amixture e1bc1 e 2 bc2 ...e n bcn
Limitations and deviations
from Beer’s Law
• Real limitations
– Non-linearities due to intermolecular interactions
• Self aggregation effects and electrolyte effects
• Apparent
– Dynamic dissociation or association of analyte
• Instrumental
– Polychromatic radiation
• Different molar absorptivities at different wavelength leads
to non-linearities in Beer’s LawHow might one avoid?
– Stray radiation
– Mistmatched cells
• Non-zero intercept in calibration curve
How to make a UV-vis absorption measurement
3) Measure %T of sample
• Tungsten filament
– Most common visible and
NIR source
– Blackbody radiator useful
from 350-2500 nm
– Power varies as
(operating voltage)4;
need stable power
supply!
– Tungsten-halogen
sources can operate at
higher temperatures and
give off more UV light.
Light sources for UV-vis, continued2
• LEDs
– 375-1000 nm
– Semi-monochromatic (20-50 nm FWHM)
– “White” LEDs use phosphor to give 400-800 nm
continuum
• Keychain flashlights
• Xenon arc lamps
– Very intense source
– Continuum from 200-1000 nm, peaking at 500 nm
Instrument configurations
• Single-beam
• Double-beam
• Multichannel
Single-beam UV-vis
spectrometers
- Sample
- Dark
- Reference
- Dark
Cary 300 double-dispersing
spectrophotometer
• Why does double dispersion help with extending absorption to
~5.0 absorbance units?
• Two gratings
• Reduced stray light
• 0.00008% or less
• Improved spectral
resolution
• Bandwidth < 4 nm
• If Abs = 5.0, %T = ?
Multichannel UV-vis
spectrometers
• Dispersing optic
(grating or prism) used
to separate different
wavelengths in space.
• Detection with diode
array or CCD
• Fast acquisition of
entire spectrum
Diode array
spectrophotometers
Fairly inexpensive,
but good quality
fiber optic models
available for ~$3000.
• Ocean Optics
• StellarNet
Diode array spectrophotometers
http://www.oceanoptics.com/products/usb4000.asp
89 mm
3.5 inches
250 specta per sec
Reflective dip probes
What is UV-visible absorption
measuring?
• The absorption of a photon generates an electronic
excited state
M + hv M*
• UV-vis energy often matches up with transitions of
bonding electrons
– Often relatively short lifetimes (1-10 nsec)
• Relaxation can occur non-radiatively
M* M + heat
• or by emission of radiation (fluorescence or
phosphorescence)
M* M + hv
Absorption signatures of various
organic functional groups
• Commonly observed transitions are np* or pp*
– Chromophores have unsaturated functional groups
– Rotational and vibrational transitions add detail to spectra
– Single bond excitation energies (n*) are in vacuum UV (
< 185 nm) and have very low molar absorptivities
A
e
bc
e normalized
with respect to
path length and
concentration
Absorption signatures of various organic
functional groups, continued
• Conjugation causes shift to longer wavelength
pp* transitions more 10-100x or more intense than np*
• Nonbonding electrons of heteroatoms in saturated
compounds can give UV absorbance signature.
Note distinct max values
Spectra of inorganic (metal and non-metal)
ions and ionic complexes
• Inorganic anions have broad UV absorption bands from non-
bonding electrons.
• Transition metal ions and complexes absorb visible light upon
excitation between filled and unfilled d-orbitals.
– Dependent upon oxidation state and coordination environment.
Spectra of lanthanide and actinide
ions
• Lanthanide and actinide ions absorptions come from
excitation of 4f and 5f electrons.
– f electrons are shielded from s, p, and d orbitals
and have narrow absorption bands
Charge-transfer complexes
• Electron donor absorbs light and
transfers to acceptor.
– Internal red-ox process
• Typically very large molar
absorptivities (e>10,000)
– Metal-to-ligand charge transfers
(MLCT)
– Ligand-to-metal charge transfer
(LMCT)
http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339
Environmental effects
• The environment that the
analyte is in can have
profound effect on the
observed spectrum
– In the gas phase, rotational
and vibrational fine structure
can be observed given
adequate spectral
bandwidth.
– In solid form or in solution,
molecules cannot rotate as
freely and differences in The visible absorption spectrum of sym-
rotational energy level are tetrazine: I, at room temperature in the vapour;
not observable. II, at 77o K in a 5 : 1 isopentane-
– Solvent molecules can also methylcyclohexane glass, III, in cyclohexane;
lead to a loss of vibrational and IV, in aqueous solution at room
temperature.
detail in the absorbance
spectrum.
J. Chem. Soc., 1959, 1263-1268.
Solvatochromism
• The polarity of solvents can
preferentially stabilize the ground or
excited state leading to different
energy level gaps and thus a solvent-
dependent absorption spectrum.
http://scienceblogs.com/moleculeoftheday/2007/02/reichardts_dye_solvatochromic.php
http://www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/p28_neg_sol-
Solvatochromism, continued
Positive solvatochromism Negative solvatochromism
(red shift) Bathochromic (blue shift) Hypsochromic
http://www.chemie.uni-regensburg.de/Organische_Chemie/Didaktik/Keusch/D-pos_sol-e.htm
http://www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/p28_neg_sol-
e.htm
Qualitative versus quantitative
analysis via UV-vis absorption
• What are the objectives of
qualitative versus quantitative
UV-visible absorption
spectroscopy?
• How might the application guide
slit width selection?
– Large slit width = good sensitivity
but poor resolution
– Small slit width = poor sensitivity
but good resolution
• Qualitative work needs __?? Visible region absorbance spectrum for
cytochrome c with spectral bandwidths of
• Quantitative work needs __?? (1) 20 nm, (2) 10 nm, (3) 5 nm, and (4) 1 nm.
Attributes of UV-visible absorption for
quantitative analysis
1) Applicable to organic and inorganic species
2) Good detection limits: 10-100 mM or better
• Possible need for larger slit widths to
achieve best sensitivities
3) Moderate to high selectivity
4) Accuracy: 1-3% or better
5) Ease and convenience ($$$) of data
acquisition
Considerations for using UV-vis for
quantitative measurements
• Directly monitor absorbing analytes; usually non-destructive
• Can use reagents that react with colorless analyte to generate
measureable species
– Greatly increase molar absorptivity
– Thiocyanate (Fe, Co, Mo), H2O2 (Ti, V, Cr), iodide (Bi, Pd, Te)
• Monitor at wavelength of max absorption, emax at max
– Greatest change in absorbance per unit concentration
– Absorbance least sensitive to a small change in wavelength
• Relaxes requirement on instrument to stringently achieve the
exact same wavelength
• UV-visible absorbance sensitive to environment, pH,
temperature, high electrolyte concentration, interfering
species. Be careful with standards
• Use matched cells.
Calibration and mixture
analysis
• Generate calibration curve (linear) using
external standards
– Must use multiple standards A1 e M bcM e N bcN
1 1
http://www.zeiss.de/c12567bb00549f37/Contents-
Frame/80bd2fe43b50aa3ec125782c00597389
Diffuse Reflectance UV-Visible Spectroscopy
of Solids
Solid powders can be studied using a diffuse reflectance
(DR) accessory either neat or diluted in a non-absorbing
powder
Diffuse Reflectance UV-Visible Spectroscopy
of Solids
Typical diffuse reflectance spectrum of cyanocobalamin
(vitamin B12), diluted to 5% w/w in MgO
100
80
%Reflectance
60
40
20
0
250 300 350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Prediction of UV-Visible Spectra with Quantum
Calculations: Time-dependent DFT
TDDFT: Time-dependent density functional theory currently
provides accurate predictions of UV-visible spectra for
organic molecules
T0
K log 10
T90
A = ebc
200
100
0
CD[mdeg]
-100
-200
1000
800
600
HT[V]
400
200
190 250 300 350
Wavelength [nm]
TDDFT Calculations
TDDFT calculations have largely replaced empirical rules.
Example: (1R)-(+)-10-camphorsulfonic acid (ammonium
salts) and its isomer calculated without solvation:
1R-10-camphorsulfonic acid
2 ammonium salt
1S-10-camphorsulfonic acid
ammonium salt
1
Rvel (10 -40 esu2 cm 2 )
-1
-2
-3
200 220 240 260 280 300 320 340 360 380 400 420 440
Excitation wavelength (nm)
Electronic Circular Dichroism
Variable temperatuer CD spectra of an orally-bioavailable
PTH mimetic peptide, showing conformational changes:
1 H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-
16 Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-
31 Val-(NH2)
h•c
E=
UV Vis
200 400 800 nm
328
329
330
331
But, If the blank absorbance is high, Po will decrease too
much, the response will be slow and the results inaccurate
Large blank
absorbance
scan of substance
10ebc = Po254/P254
8
A = 2.0/(10-10,000x1.0xc + 10-5000x1.0xc)
7
6
A = e bc
5
C A (actual) A(expected)
4
Concentration (M)
The results are the same for more ’s of light. The situation is worse for
greater differences in e’s (side of absorption peak, broad bandpass)
Always need to do calibration curve! Can not assume linearity outside the
range of linearity curve!
b) Chemical Deviations from Beer’s Law:
- Molar absorptivity change in solutions more
concentrated than 0.01M
due to molecular interactions
species.
If solution is buffered, then pH is constant and [HIn] is related to
absorbance.
commonly use the analytical concentration –
concentration of all forms of the species.
But, if unbuffered solution, equilibrium will shift depending on total analyte
concentration
example: if Ka = 10-4 Ka
HIn H+ + In-
Expected
10-5 8.5x10-7 9.2x10-6 0.0924
C
HIn
example: if Ka = 10-4 Ka
HIn H+ + In-
Isosbestic point
At the isosbestic point in spectra:
A = eb([HIn] + [In-])
c) Non-constant b:
- worse for round cuvettes
- use parallel cuvettes to help
B2
P2
P0
B1
P1
Crystal-Field Theory
- In absence of external field d-orbitals are
identical
- Energies of d-orbitals in solution are not
identical
- Absorption involves e- transition between d-
orbitals
- In complex, all orbitals increase in energy where
orbitals along bonding axis are destabilized
UV Spectral
Nomenclature
UV Spectroscopy
I. Introduction
A. UV radiation and Electronic Excitations
1. The difference in energy between molecular bonding, non-bonding and
anti-bonding orbitals ranges from 125-650 kJ/mole
Visible
I. Introduction
B. The Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the broad spectrum of
the UV radiation
2. If a particular electronic transition matches the energy of a certain band
of UV, it will be absorbed
3. The remaining UV light passes through the sample and is observed
4. From this residual radiation a spectrum is obtained with “gaps” at these
discrete energies – this is called an absorption spectrum
p*
p*
p
p
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UV Spectroscopy
I. Introduction
C. Observed electronic transitions
1. The lowest energy transition (and most often obs. by UV) is typically that
of an electron in the Highest Occupied Molecular Orbital (HOMO) to the
Lowest Unoccupied Molecular Orbital (LUMO)
2. For any bond (pair of electrons) in a molecule, the molecular orbitals are
a mixture of the two contributing atomic orbitals; for every bonding
orbital “created” from this mixing (, p), there is a corresponding anti-
bonding orbital of symmetrically higher energy (*, p*)
3. The lowest energy occupied orbitals are typically the ;likewise, the
corresponding anti-bonding * orbital is of the highest energy
5. Unshared pairs lie at the energy of the original atomic orbital, most often
this energy is higher than p or (since no bond is formed, there is no
benefit in energy)
344
UV Spectroscopy
I. Introduction
C. Observed electronic transitions
6. Here is a graphical representation
*
Unoccupied levels
p*
Occupied levels
p
Molecular orbitals
345
UV Spectroscopy
I. Introduction
C. Observed electronic transitions
7. From the molecular orbital diagram, there are several possible
electronic transitions that can occur, each of a different relative
energy:
*
* alkanes
p*
p* carbonyls
p p* unsaturated cmpds.
Energy
n
n * O, N, S, halogens
n p* carbonyls
p
346
UV Spectroscopy
I. Introduction
C. Observed electronic transitions
7. Although the UV spectrum extends below 100 nm (high energy), oxygen
in the atmosphere is not transparent below 200 nm
* alkanes 150 nm
p* carbonyls 170 nm
n * O, N, S, halogens 190 nm
n p* carbonyls 300 nm √
347
UV Spectroscopy
I. Introduction
D. Selection Rules
1. Not all transitions that are possible are observed
348
UV Spectroscopy
I. Introduction
E. Band Structure
1. Unlike IR (or later NMR), where there may be upwards of 5 or
more resolvable peaks from which to elucidate structural
information, UV tends to give wide, overlapping bands
349
UV Spectroscopy
I. Introduction
E. Band Structure
5. When these energy levels are superimposed, the effect can be readily
explained – any transition has the possibility of being observed
Disassociation
R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
V2
V1 R1 - Rn
E1 Vo R1 - Rn
Disassociation
Energy R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
V2
V1 R1 - Rn
R1 - Rn
E0 Vo
350
Molecular UV-Visible Spectroscopy
Molecular UV-Visible spectroscopy is driven by electronic
absorption of UV-Vis radiation
Molecular UV-Visible
spectroscopy can:
– Enable structural analysis
– Detect molecular chromophores
– Analyze light-absorbing properties
(e.g. for photochemistry)
UV-Vis Terminology