Molecular Spectros

Molecular Spectroscopy
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.
Methods of structure determination

• Ultraviolet-visible (UV-Vis) Spectroscopy
• Nuclear Magnetic Resonances (NMR) Spectroscopy
• Infrared (IR) Spectroscopy
• Mass (MS) spectrometry (not really spectroscopy)
3
Principles of molecular spectroscopy:
Electromagnetic radiation
organic light organic relaxation organic
molecule molecule + h
molecule
(ground state) h (excited state) (ground state)
Electromagnetic radiation has the properties of a particle

(photon) and a wave.
= distance of one wave

 = frequency: waves per unit time (sec-1, Hz)
c = speed of light (3.0 x 108 m • sec-1)
h = Plank’s constant (6.63 x 10-34 J • sec) 4
Electronic Spectroscopy
 Spectroscopy of the electrons surrounding an atom or a
molecule: electron energy-level transitions
Atoms: electrons are Molecules: electrons are in
in hydrogen-like molecular orbitals (HOMO,
orbitals (s, p, d, f) LUMO, …)
From
http://education.jlab.org
(The Bohr model for nitrogen) (The LUMO of benzene)

Principles of molecular spectroscopy:
Quantized Energy Levels
molecules have discrete energy levels

(no continuum between levels)
A molecule absorbs electromagnetic radiation when

the energy of photon corresponds to the difference in
energy between two states 6
organic light organic relaxation organic
molecule molecule + h
molecule
(ground state) h (excited state) (ground state)
UV-Vis: valence electron transitions

- gives information about p-bonds and conjugated systems
Infrared: molecular vibrations (stretches, bends)

- identify functional groups
Radiowaves: nuclear spin in a magnetic field (NMR)

- gives a map of the H and C framework
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.
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Molecular UV-Vis Spectroscopy: Transitions
 Major classes of electron transitions
– HOMO: highest occupied molecular orbital

– LUMO: lowest unoccupied molecular orbital
– Types of electron transitions:
(1) , p and n electrons (mostly organics)
(2) d and f electrons (inorganics/organometallics)
(3) charge-transfer (CT) electrons
, p and n electrons transition
10
p-molecular orbitals of butadiene
4: 3 Nodes
0 bonding interactions
3 antibonding interactions
ANTIBONDING MO
3: 2 Nodes
ANTIBONDING MO
2: 1 Nodes
BONDING MO
1: 0 Nodes
BONDING MO
y2 is the Highest Occupied Molecular Orbital (HOMO)

y3 is the Lowest Unoccupied Molecular Orbital (LUMO)
11
UV-Vis light causes electrons in lower energy molecular orbitals
to be promoted to higher energy molecular orbitals.
HOMO LUMO
Chromophore: light absorbing portion of a molecule
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
380 nm 400 nm 450 nm 500 nm 550 nm 600 nm 700 nm 780 nm
violet-indigo blue green yellow orange red
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
violet-indigo blue green yellow orange red
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
 Charge transfer (CT) – occurs when electron-donor and

electron-acceptor properties are in the same complex –
electron transfer occurs as an “excitation step”
– MLCT (metal-to-ligand charge transfer)
– LMCT (ligand-to-metal charge transfer)
– Ex: tri(bipyridyl)iron(II), which is red – an electron is exicted from
the d-orbital of the metal into a p* orbital on the ligand
27
28
29
30
31
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iii) Charge Transfer Complexes
 Important analytically because of large e (> 10,000)
 Absorption of radiation involves transfer of e- from the donor to
orbital associated with acceptor
- excited state is product of pseudo oxidation/reduction process
 Many inorganic complexes of electron donor (usually organic) &
electron acceptor (usually metal)
- examples: Iron III thiocyanate
Iron II phenanthroline
(colorless) (deep red color)

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35
QUANTITATIVE ASPECT
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
Beer’s Law: There is a linear relationship between

absorbance and concentration
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
 Molar absorption coefficient (e) then gives A via the Beer-

Lambert Law:
A = ebc
Quantitative UV-Visible Spectroscopy
 UV-visible spectra can be used for direct quantitative
analysis with appropriate calibration
Ezetimibe calibration plot

1.2
y = 36.891x + 0.0814
1
Absorbace at 231 nm
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)
When all the protein is bound to Fe3+,

no further increase in absorbance.
As Fe3+ continues to bind protein

red color and absorbance increases.
Standard Addition Method (spiking the sample)
- used for analytes in a complex matrix where interferences in the UV/Vis
for the analyte will occur: i.e. blood, sediment, human serum, etc..
- Method:
(1) Prepare several identical aliquots, Vx, of the unknown sample.
(2) Add a variable volume, Vs, of a standard solution of known

concentration, cs, to each unknown aliquot.
Note: This method assumes a linear relationship between instrument

response and sample concentration.
(3) Dilute each solution to an equal volume, Vt.
(4) Make instrumental measurements of each sample to get an

instrument response, IR.
(5) Calculate unknown concentration, cx, from the following equation.
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
A1 = e1MbcM + e1NbcN (1)
A2 = e2MbcM + e2NbcN (2)
Note: need to solve simultaneous equations

d) Photometric titration
- can measure titration with UV-vis spectroscopy.
- requires the analyte (A), titrant (T) or titration product (P) absorbs radiation
Example 7: Given:
Absorbance (1.00 cm cell)
Species 475 nm 700 nm
A (7.50x10-5 M) 0.155 0.755
B (4.25x10-5 M) 0.702 0.091
Calculate the concentrations of A and B in solutions that

yielded an absorbance of 0.439 at 475 nm and 1.025 at 700 nm
in a 2.50-cm cell.
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INSTRUMENTATION
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.
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1

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52
Molecular UV-Visible Spectrophotometers
 Diode array detectors can acquire all UV-Visible
wavelengths at once.
 Advantages:
– Sensitivity
(multiplex)
– Speed
 Disadvantages:
– Resolution
Figure from Skoog, et al., Chapter 13

Spring 2014
 4*
 2*
 3*
2
1 p
1
UV Spectroscopy
II. Instrumentation and Spectra

A. Instrumentation
1. The construction of a traditional UV-VIS spectrometer is very similar to an
IR, as similar functions – sample handling, irradiation, detection and
output are required
2. Here is a simple schematic that covers most modern UV spectrometers:
log(I0/I) = A
sample
UV-VIS sources I0 I
detector
200 700
, nm
monochromator/
reference
beam splitter optics I0 I0
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UV Spectroscopy

A. Instrumentation
3. Two sources are required to scan the entire UV-VIS band:
• Deuterium lamp – covers the UV – 200-330
• Tungsten lamp – covers 330-700
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
5. The beam splitter sends a separate band to a cell containing the

sample solution and a reference solution
6. The detector measures the difference between the transmitted

light through the sample (I) vs. the incident light (I0) and sends
this information to the recorder
56
UV Spectroscopy

A. Instrumentation
7. As with dispersive IR, time is required to cover the entire UV-VIS band
due to the mechanism of changing wavelengths
8. A recent improvement is the diode-array spectrophotometer - here a

prism (dispersion device) breaks apart the full spectrum transmitted
through the sample
9. Each individual band of UV is detected by a individual diodes on a silicon

wafer simultaneously – the obvious limitation is the size of the diode, so
some loss of resolution over traditional instruments is observed
Diode array
UV-VIS sources
sample
Polychromator
– entrance slit and dispersion device 57
UV Spectroscopy

B. Instrumentation – Sample Handling
1. Virtually all UV spectra are recorded solution-phase
2. Cells can be made of plastic, glass or quartz
3. Only quartz is transparent in the full 200-700 nm range; plastic

and glass are only suitable for visible spectra
4. Concentration (we will cover shortly) is empirically determined
A typical sample cell (commonly called a cuvet):
58
UV Spectroscopy

5. Solvents must be transparent in the region to be observed; the
wavelength where a solvent is no longer transparent is referred to
as the cutoff
6. Since spectra are only obtained up to 200 nm, solvents typically

only need to lack conjugated p systems or carbonyls
Common solvents and cutoffs:

acetonitrile 190
chloroform 240
cyclohexane 195
1,4-dioxane 215
95% ethanol 205
n-hexane 201
methanol 205
isooctane 195
water 190 59
UV Spectroscopy

7. Additionally solvents must preserve the fine structure (where it is actually
observed in UV!) where possible
8. H-bonding further complicates the effect of vibrational and rotational

energy levels on electronic transitions, dipole-dipole interacts less so
9. The more non-polar the solvent, the better (this is not always possible)
60
UV Spectroscopy

C. The Spectrum
1. The x-axis of the spectrum is in wavelength; 200-350 nm for UV,
200-700 for UV-VIS determinations
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

C. The Spectrum
1. The y-axis of the spectrum is in absorbance, A
2. From the spectrometers point of view, absorbance is the inverse

of transmittance: A = log10 (I0/I)
3. From an experimental point of view, three other considerations

must be made:
i. a longer path length, l through the sample will cause
more UV light to be absorbed – linear effect
ii. the greater the concentration, c of the sample, the

more UV light will be absorbed – linear effect
iii. some electronic transitions are more effective at the

absorption of photon than others – molar absorptivity, e
this may vary by orders of magnitude…
62
UV Spectroscopy

C. The Spectrum
4. These effects are combined into the Beer-Lambert Law: A = e c l
i. for most UV spectrometers, l would remain constant (standard cells

are typically 1 cm in path length)
ii. concentration is typically varied depending on the strength of

absorption observed or expected – typically dilute – sub .001 M
iii. molar absorptivities vary by orders of magnitude:

• values of 104-106 are termed high intensity absorptions
• values of 103-104 are termed low intensity absorptions
• values of 0 to 103 are the absorptions of forbidden transitions
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

D. Practical application of UV spectroscopy
1. UV was the first organic spectral method, however, it is rarely
used as a primary method for structure determination
2. It is most useful in combination with NMR and IR data to elucidate

unique electronic features that may be ambiguous in those
methods
3. It can be used to assay (via max and molar absorptivity) the

proper irradiation wavelengths for photochemical experiments, or
the design of UV resistant paints and coatings
4. The most ubiquitous use of UV is as a detection device for HPLC;

since UV is utilized for solution phase samples vs. a reference
solvent this is easily incorporated into LC design
UV is to HPLC what mass spectrometry (MS) will be to GC

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QANTITATIVE ANALYSIS
APPLICATIONS
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
phosphate react with ammonium molybdate and then reduce 690

with SnCl2; forms molybdenum blue
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Organics
phenol react with 4-aminoantipyrine and 460
K3Fe(CN)6; forms yellow antipyrine
dye
anionic react with cationic methylene blue dye 652

surfactant and extract into CHCl3; forms blue ion
pair
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neocuproine
Eriochrome cyanide R dye silver diethyldithiocarbamate
69
70
Zincon
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Analysis of Glucose
• Measure the concentration of glucose by

detecting the reducing end of the monosaccharide.
• This group converts the oxidized form of 3,5-
dinitrosalicylic acid, DNS, to reduced form which
absorbs at 540nm.
• Amount of reduced DNS proportional to
amount of glucose.
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Analysis of Glucose
73
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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
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Analysis of Surfactants
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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
2. Since similar functional groups will have electrons capable of

discrete classes of transitions, the characteristic energy of these
energies is more representative of the functional group than the
electrons themselves
3. A functional group capable of having characteristic electronic

transitions is called a chromophore (color loving)
4. Structural or electronic changes in the chromophore can be

quantified and used to predict shifts in the observed electronic
transitions
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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.
 Information from other

techniques (NMR, MS, IR) is
usually far more useful for
structural analysis
 However, UV-Vis evidence

should not be ignored!
Figure from Skoog, et al., Chapter 14
Calculation of Molar Absorption Coefficient
 The molar absorption coefficient (e) for each absorbance
in a UV spectrum is calculated as follows:
– e, Molar Abs Coeff (AU mol-1 cm-1) = A x mwt / mass x pathlength
 Solvent “cutoffs” for UV-visible work:

Solvent UV Cutoff (nm)
Acetonitrile (UV grade) 190
Acetone 330
Dimethylsulfoxide 268
Chloroform (1% ethanol) 245
Heptane 200
Hexane (UV grade) 195
Methanol 205
2-Propanol 205
Tetrahydrofuran (UV grade) 212
Water 190
Burdick and Jackson High Purity Solvent Guide, 1990

Interpretation of UV-Visible Spectra
 Although UV-Visible spectra are no longer frequently used
for structural analysis, it is helpful to be aware of well-
developed interpretive rules.
 Examples:
– Woodward-Fieser rules for max dienes and polyenes
– Extended Woodward rules for unsaturated ketones
– Substituted benzenes (max base value = 203.5 nm)
Substituent (X) Increment (nm)

X -CH3 3.0
-Cl 6.0
-OH 7.0
-NH2 26.5
-CHO 46.0
-NO2 65.0
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.
 Why does increasing conjugation cause bathochromic shifts

(to longer wavelengths)?
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
cyclohex-2-enone 225 10,300
toluene 206 7,000
3,4-dimethylpent-3-en-2-one 246 5,300

- For Compounds with Multiple Chromophores:
 If greater then one single bond apart
- e are additive
-  constant
CH3CH2CH2CH=CH2 max= 184 emax = ~10,000
CH2=CHCH2CH2CH=CH2 max=185 emax = ~20,000
 If conjugated
- shifts to higher ’s (red shift)
H2C=CHCH=CH2 max=217 emax = ~21,000

Example 6: The equilibrium constant for the conjugate acid-
base pair
e = 8.04x103 K = 8.00x10 -5 e = 0.755x10 3
HIn + H2O H3O+ + In-
Calculate the absorbance at 430 nm for an indicator

concentration of 3.00x10-4 M
Applications:
B) Absorbing Species in UV/Vis:
1) Electronic transitions involving organic compounds,
inorganic compounds, complexes, etc.
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
Sigma () – single bond electron
Low energy bonding orbital High energy anti-bonding orbital

Pi (p) – double bond electron
Low energy bonding orbital High energy anti-bonding orbital
Non-bonding electrons (n): don’t take part in any bonds,

neutral energy level.
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 pp*
Alkyne n-Heptane 178 10,000 pp*
C5H11C C CH3 196 2,000 _
225 160
_
Carbonyl O n-Hexane 186 1,000 n*
280 16 np*
CH3CCH3
O n-Hexane 180 Large
293 12
n*
CH3CH np*
Carboxyl O Ethanol 204 41 np*
CH3COH
Amido O Water 214 60 np*
CH3CNH2
Azo H3CN NCH3 Ethanol 339 5 np*
Nitro CH3NO2 Isooctane 280 22 np*

Nitroso C4H9NO Ethyl ether 300 100 _
665 20 np*
Nitrate C2H5ONO2 Dioxane 270 12 np*
Other Examples of Some
Common Chromophores
Magnitude of  depends on:
- charge on metal ion
- position in periodic table
- ligand field strength :
I- < Br- < Cl- < F- < OH- < C2O42- ~ H2O < SCN- < NH3
< ethylenediamine < o-phenanthroline < NO2- < CN-
 increases with increasing field strength, so wavelength decreases
C) Qualitative Analysis:
1) Limited since few resolved peaks
- unambiguous identification not usually possible.
2) Solvent can affect position and shape of curve.
- polar solvents broaden out peaks, eliminates fine structure.
Loss of fine structure for acetaldehyde
when transfer to solvent from gas phase
Also need to consider

absorbance of solvent.
2) Solvent can affect position and shape of curve.
- polar solvents broaden out peaks, eliminates fine structure.
(a) Vapor
Loss of fine structure for 1,2,4,5-

tetrazine as solvent polarity
(b) Hexane increases
solution
(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
More complex ring systems

shift to higher ’s (red shift)
similar to conjugated alkenes
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
This transition is destructive to the molecule, causing cleavage of

the -bond
C C
*
 C C
106
UV Spectroscopy
III. 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
3. Alkenes and Alkynes – in the case of isolated examples of these
compounds the p  p* is observed at 175 and 170 nm, respectively
Even though this transition is of lower energy than   *, it is still in

the far UV – however, the transition energy is sensitive to substitution
p*
108
UV Spectroscopy
III. Chromophores
4. Carbonyls – unsaturated systems incorporating N or O can
undergo n  p* transitions (~285 nm) in addition to p  p*
Despite the fact this transition is forbidden by the selection rules

(e = 15), it is the most often observed and studied transition for
carbonyls
This transition is also sensitive to substituents on the carbonyl
Similar to alkenes and alkynes, non-substituted carbonyls undergo

the p  p* transition in the vacuum UV (188 nm, e = 900);
sensitive to substitution effects
109
UV Spectroscopy
III. 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
The attachment of substituent groups (other than H)

can shift the energy of the transition
Substituents that increase the intensity and often

wavelength of an absorption are called auxochromes
Common auxochromes include alkyl, hydroxyl, alkoxy

and amino groups and the halogens
111
UV Spectroscopy
III. Chromophores
General – Substituents may have any of four effects on a chromophore
i. Bathochromic shift (red shift) – a shift to longer ; lower energy
ii. Hypsochromic shift (blue shift) – shift to shorter ; higher energy
iii. Hyperchromic effect – an increase in intensity
iv. Hypochromic effect – a decrease in intensity
Hyperchromic
e Hypsochromic Bathochromic
Hypochromic
200 nm 700 nm
112
UV Spectroscopy
III. Chromophores
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
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
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
E for the HOMO  LUMO transition is reduced

115
UV Spectroscopy
III. Chromophores
Extending this effect out to longer conjugated systems the energy gap
becomes progressively smaller:
Energy Lower energy =

Longer wavelenghts
ethylene
butadiene
hexatriene
octatetraene 116
UV Spectroscopy
III. Chromophores
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
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
This quantification is referred to as the Woodward-Fieser

Rules which we will apply to three specific chromophores:
1. Conjugated dienes
2. Conjugated dienones
3. Aromatic systems
max = 239 nm 119

UV Spectroscopy
IV. Structure Determination

A. Dienes
1. General Features
For acyclic butadiene, two conformers are possible – s-cis and s-
trans
s-trans s-cis
The s-cis conformer is at an overall higher potential energy than

the s-trans; therefore the HOMO electrons of the conjugated
system have less of a jump to the LUMO – lower energy, longer
wavelength
120
UV Spectroscopy

A. Dienes
1. General Features
Two possible p  p* transitions can occur for butadiene 2  3*and 2
 4*
4*
175 nm –forb. 175 nm
3*
217 nm 253 nm
2
s-trans s-cis
1
The 2  4* transition is not typically observed:

• The energy of this transition places it outside the region
typically observed – 175 nm
• For the more favorable s-trans conformation, this transition is

forbidden
The 2  3* transition is observed as an intense absorption
121
UV Spectroscopy

A. Dienes
1. General Features
The 2  3* transition is observed as an intense absorption (e =
20,000+) based at 217 nm within the observed region of the UV
While this band is insensitive to solvent (as would be expected) it is

subject to the bathochromic and hyperchromic effects of alkyl
substituents as well as further conjugation
Consider:
max = 217 253 220 227 227

256 263 nm
122
UV Spectroscopy

A. Dienes
2. Woodward-Fieser Rules
Woodward and the Fiesers performed extensive studies of terpene
and steroidal alkenes and noted similar substituents and structural
features would predictably lead to an empirical prediction of the
wavelength for the lowest energy p  p* electronic transition
This work was distilled by Scott in 1964 into an extensive treatise

on the Woodward-Fieser rules in combination with comprehensive
tables and examples – (A.I. Scott, Interpretation of the Ultraviolet
Spectra of Natural Products, Pergamon, NY, 1964)
A more modern interpretation was compiled by Rao in 1975 –

(C.N.R. Rao, Ultraviolet and Visible Spectroscopy, 3rd Ed.,
Butterworths, London, 1975)
123
UV Spectroscopy

A. Dienes
2. Woodward-Fieser Rules - Dienes
The rules begin with a base value for max of the chromophore being
observed:
acyclic butadiene = 217 nm
The incremental contribution of substituents is added to this base value

from the group tables:
Group Increment
Extended conjugation +30
Each exo-cyclic C=C +5
Alkyl +5
-OCOCH3 +0
-OR +6
-SR +30
-Cl, -Br +5
-NR2 +60 124
UV Spectroscopy

A. Dienes
For example:
Isoprene - acyclic butadiene = 217 nm

one alkyl subs. + 5 nm
222 nm
Experimental value 220 nm
Allylidenecyclohexane
- acyclic butadiene = 217 nm
one exocyclic C=C + 5 nm
2 alkyl subs. +10 nm
232 nm
125
UV Spectroscopy

A. Dienes
3. Woodward-Fieser Rules – Cyclic Dienes
There are two major types of cyclic dienes, with two different base values
Heteroannular (transoid): Homoannular (cisoid):
e = 5,000 – 15,000 e = 12,000-28,000

base max = 214 base max = 253
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

A. Dienes
In the pre-NMR era of organic spectral determination, the power
of the method for discerning isomers is readily apparent
Consider abietic vs. levopimaric acid:
C OH C OH
O O
abietic acid levopimaric acid
127
UV Spectroscopy

A. Dienes
For example:
1,2,3,7,8,8a-hexahydro-8a-methylnaphthalene
heteroannular diene = 214 nm
3 alkyl subs. (3 x 5) +15 nm
1 exo C=C + 5 nm
234 nm
128
UV Spectroscopy

A. Dienes

1 exo C=C + 5 nm
C OH
O
239 nm
homoannular diene = 253 nm

1 exo C=C + 5 nm
C OH
O
278 nm 129
UV Spectroscopy

A. Dienes
Be careful with your assignments – three common errors:
R
This compound has three exocyclic

double bonds; the indicated bond is
exocyclic to two rings
This is not a heteroannular diene; you

would use the base value for an acyclic
diene
Likewise, this is not a homooannular

diene; you would use the base value for
an acyclic diene
130
UV Spectroscopy

B. Enones
1. General Features
Carbonyls, as we have discussed have two primary
electronic transitions:
p* Remember, the p  p*
transition is allowed and gives
a high e, but lies outside the
n
routine range of UV
observation
p
The n  p* transition is
forbidden and gives a very low
e, but can routinely be
observed
131
UV Spectroscopy

B. Enones
1. General Features
For auxochromic substitution on the carbonyl, pronounced
hypsochromic
O
shifts are observed for the n  p* transition (max):
H
293 nm This is explained by the inductive
withdrawal of electrons by O, N or
O
halogen from the carbonyl carbon – this
279
causes the n-electrons on the carbonyl
CH3
O oxygen to be held more firmly

Cl 235
It is important to note this is different
O
from the auxochromic effect on p  p*
214
NH2
which extends conjugation and causes a
O bathochromic shift
O
204
In most cases, this bathochromic shift is
O
204 not enough to bring the p  p*
OH transition into the observed range 132
UV Spectroscopy

B. Enones
1. General Features
Conversely, if the C=O system is conjugated both the n  p* and
p  p* bands are bathochromically shifted
Here, several effects must be noted:

i. the effect is more pronounced for p  p*
ii. if the conjugated chain is long enough, the much

higher intensity p  p* band will overlap and drown
out the n  p* band
iii. the shift of the n  p* transition is not as predictable
For these reasons, empirical Woodward-Fieser rules for

conjugated enones are for the higher intensity, allowed p  p*
transition
133
UV Spectroscopy

B. Enones
1. General Features
These effects are apparent from the MO diagram for a conjugated
enone:  4*
p* p*
 3*
n n
2
p p
1
O O
134
UV Spectroscopy
IV. Structure Determination  a d g  a

B. Enones  C C C d C C C C C
2. Woodward-Fieser Rules - Enones O O
Group Increment
6-membered ring or acyclic enone Base 215 nm
5-membered ring parent enone Base 202 nm
Acyclic dienone Base 245 nm
Double bond extending conjugation 30

Alkyl group or ring residue a,,gand higher 10, 12, 18
-OH a,,gand higher 35, 30, 18
-OR a,,g,d 35, 30, 17, 31
-O(C=O)R a,,d 6
-Cl a, 15, 12
-Br a, 25, 30
-NR2  95
Exocyclic double bond 5
Homocyclic diene component 39
135
UV Spectroscopy

B. Enones
2. Woodward-Fieser Rules - Enones
Aldehydes, esters and carboxylic acids have different base values
than ketones
Unsaturated system Base Value
Aldehyde 208
With a or  alkyl groups 220
With a, or , alkyl groups 230
With a,, alkyl groups 242
Acid or ester
Group value – exocyclic a, double bond +5
Group value – endocyclic a, bond in 5 +5
or 7 membered ring
136
UV Spectroscopy

B. Enones
Unlike conjugated alkenes, solvent does have an effect on max
These effects are also described by the Woodward-Fieser rules

Solvent correction Increment
Water +8
Ethanol, methanol 0
Chloroform -1
Dioxane -5
Ether -7
Hydrocarbon -11
137
UV Spectroscopy

B. Enones
Some examples – keep in mind these are more complex than dienes
cyclic enone = 215 nm
O 2 x - alkyl subs. (2 x 12) +24 nm
239 nm
R
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

B. Enones
Take home problem – can these two isomers be discerned by UV-
spec O
O
Eremophilone allo-Eremophilone
Problem Set 1: (text) – 1,2,3a,b,c,d,e,f,j, 4, 5, 6 (1st, 2nd and 5th

pairs), 8a, b, c
Problem Set 2: outside problems/key -Tuesday
139
UV Spectroscopy

C. Aromatic Compounds
1. General Features
Although aromatic rings are among the most widely studied and observed
chromophores, the absorptions that arise from the various electronic
transitions are complex
On first inspection, benzene has six p-MOs, 3 filled p, 3 unfilled p*
p6*
p4* p5*
p2 p3
p1
140
UV Spectroscopy

1. General Features
One would expect there to be four possible HOMO-LUMO p  p*
transitions at observable wavelengths (conjugation)
Due to symmetry concerns and selection rules, the actual

transition energy states of benzene are illustrated at the right:
E1u
p6*
B1u
200 nm
p4* p5*
(forbidden)
B2u
180 nm
260 nm (allowed)
p2 p3 (forbidden)
A1g
p1
141
UV Spectroscopy

1. General Features
The allowed transition (e = 47,000) is not in the routine range of UV obs.
at 180 nm, and is referred to as the primary band
The forbidden transition (e = 7400) is observed if substituent effects shift

it into the obs. region; this is referred to as the second primary band
At 260 nm is another forbidden

transition (e = 230), referred to
as the secondary band.
This transition is fleetingly allowed

due to the disruption of symmetry
by the vibrational energy states,
the overlap of which is observed
in what is called fine structure
142
UV Spectroscopy

1. General Features
Substitution, auxochromic, conjugation and solvent
effects can cause shifts in wavelength and intensity of
aromatic systems similar to dienes and enones
However, these shifts are difficult to predict – the

formulation of empirical rules is for the most part is not
efficient (there are more exceptions than rules)
There are some general qualitative observations that

can be made by classifying substituent groups --
143
UV Spectroscopy

2. Substituent Effects
a. Substituents with Unshared Electrons
• If the group attached to the ring bears n electrons, they can
induce a shift in the primary and secondary absorption bands
• Non-bonding electrons extend the p-system through

resonance – lowering the energy of transition p  p*
• More available n-pairs of electrons give greater shifts
G G G G
144
UV Spectroscopy

• The presence of n-electrons gives the possibility of n  p*
transitions
• If this occurs, the electron now removed from G, becomes an

extra electron in the anti-bonding p* orbital of the ring
• This state is referred to as a charge-transfer excited state
G G G G
*
- *
*
*
145
UV Spectroscopy

• pH can change the nature of the substituent group
• deprotonation of oxygen gives more available n-pairs,
lowering transition energy
• protonation of nitrogen eliminates the n-pair,
raising transition energy
Primary Secondary
Substituent max e max e
-H 203.5 7,400 254 204
-OH 211 6,200 270 1,450
-O- 235 9,400 287 2,600
-NH2 230 8,600 280 1,430
-NH3+ 203 7,500 254 169
-C(O)OH 230 11,600 273 970
-C(O)O- 224 8,700 268 560
146
UV Spectroscopy

b. Substituents Capable of p-conjugation
• When the substituent is a p-chromophore, it can interact with
the benzene p-system
• With benzoic acids, this causes an appreciable shift in the

primary and secondary bands
• For the benzoate ion, the effect of extra n-electrons from the
anion reduces the effect slightly
Primary Secondary
-C(O)OH 230 11,600 273 970
-C(O)O- 224 8,700 268 560
147
UV Spectroscopy

c. Electron-donating and electron-withdrawing
effects
• No matter what electronic influence a group
exerts, the presence shifts the primary
absorption band to longer 
• Electron-withdrawing groups exert no

influence on the position of the secondary
absorption band
• Electron-donating groups increase the  and e

of the secondary absorption band
148
UV Spectroscopy

c. Electron-donating and electron-withdrawing effects
Primary Secondary
-H 203.5 7,400 254 204
Electron withdrawing Electron donating
-CH3 207 7,000 261 225

-Cl 210 7,400 264 190
-Br 210 7,900 261 192
-OH 211 6,200 270 1,450
-OCH3 217 6,400 269 1,480
-NH2 230 8,600 280 1,430
-CN 224 13,000 271 1,000
C(O)OH 230 11,600 273 970
-C(O)H 250 11,400
-C(O)CH3 224 9,800
-NO2 269 7,800
149
UV Spectroscopy

d. Di-substituted and multiple group effects
• With di-substituted aromatics, it is necessary to consider both
groups
• If both groups are electron donating or withdrawing, the

effect is similar to the effect of the stronger of the two groups
as if it were a mono-substituted ring
• If one group is electron withdrawing and one group electron

donating and they are para- to one another, the magnitude of
the shift is greater than the sum of both the group effects
• Consider p-nitroaniline:
O O
H2N N H2N N
O O
150
UV Spectroscopy

• If the two electonically dissimilar groups are ortho- or meta-
to one another, the effect is usually the sum of the two
individual effects (meta- no resonance; ortho-steric hind.)
• For the case of substituted benzoyl derivatives, an empirical

correlation of structure with observed max has been
developed
• This is slightly less accurate than the Woodward-Fieser rules,

but can usually predict within an error of 5 nm
O R
G
151
UV Spectroscopy

Parent Chromophore max
R = alkyl or ring residue 246
R=H 250
R = OH or O-Alkyl 230
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

d. Polynuclear aromatics
• When the number of fused aromatic rings increases, the  for
the primary and secondary bands also increase
• For heteroaromatic systems spectra become complex with the

addition of the n  p* transition and ring size effects and are
unique to each case
153
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
400 500 600 700 800

, nm
Violet 400-420
Indigo 420-440
Blue 440-490
Green 490-570
Yellow 570-585
Orange 585-620
Red 620-780
154
UV 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
• What is “seen” is the complimentary colors (those that are not

absorbed)
• This is the origin of the “color wheel”
155
UV Spectroscopy
A. Color
1. General
• Organic compounds that are “colored” are typically those with
extensively conjugated systems (typically more than five)
• Consider -carotene
-carotene, max = 455 nm
max is at 455 – in the far blue region

of the spectrum – this is absorbed
The remaining light has the

complementary color of orange
156
UV Spectroscopy
A. Color
1. General
• Likewise:
lycopene, max = 474 nm
O
H
N
N
H
O
indigo
max for lycopene is at 474 – in the near blue

region of the spectrum – this is absorbed, the
compliment is now red
max for indigo is at 602 – in the orange region

of the spectrum – this is absorbed, the
compliment is now indigo! 157
UV Spectroscopy
A. Color
1. General
• One of the most common class of colored organic molecules
are the azo dyes:
N N
EWGs EDGs
From our discussion of di-subsituted aromatic

chromophores, the effect of opposite groups is
greater than the sum of the individual effects – more
so on this heavily conjugated system
Coincidentally, it is necessary for these to be

opposite for the original synthetic preparation!
158
UV 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
Fast Brown Sunset Yellow (Food Yellow 3)

Para Red
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
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
A. Color
1. General
• In the biological sciences these compounds are used as dyes
to selectively stain different tissues or cell structures
• Biebrich Scarlet - Used with picric acid/aniline blue for

staining collagen, recticulum, muscle, and plasma. Luna's
method for erythrocytes & eosinophil granules. Guard's
method for sex chromatin and nuclear chromatin.
HO
O3S N N N N
SO3
161
UV Spectroscopy
A. Color
1. General
• In the chemical sciences these are the acid-base indicators
used for the various pH ranges:
• Remember the effects of pH on aromatic substituents

Methyl Orange
CH3 CH3
H
O3S N N N O3S N N N
CH3 CH3
Yellow, pH > 4.4 Red, pH < 3.2
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
CHMBD 449 – Organic Spectral Analysis
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
2. This energy corresponds to EM radiation in the ultraviolet (UV) region,

100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum
3. For comparison, recall the EM spectrum:
g-rays X-rays UV IR Microwave Radio
Visible
4. Using IR we observed vibrational transitions with energies of 8-40 kJ/mol

at wavelengths of 2500-15,000 nm
5. For purposes of our discussion, we will refer to UV and VIS spectroscopy

as UV
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
4. p-orbitals are of somewhat higher energy, and their complementary anti-

bonding orbital somewhat lower in energy than *.
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
6. Here is a graphical representation
*
Unoccupied levels
p*
Atomic orbital Atomic orbital

Energy n
Occupied levels
p

Molecular orbitals
195
UV Spectroscopy
I. Introduction
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
7. Although the UV spectrum extends below 100 nm (high energy), oxygen
in the atmosphere is not transparent below 200 nm
8. Special equipment to study vacuum or far UV is required
9. Routine organic UV spectra are typically collected from 200-700 nm
10. This limits the transitions that can be observed:
 * alkanes 150 nm
 p* carbonyls 170 nm
p p* unsaturated cmpds. 180 nm √ - if conjugated!
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
2. For an electron to transition, certain quantum mechanical

constraints apply – these are called “selection rules”
3. For example, an electron cannot change its spin quantum number

during a transition – these are “forbidden”
Other examples include:
• the number of electrons that can be excited at one time
• symmetry properties of the molecule
• symmetry of the electronic states
4. To further complicate matters, “forbidden” transitions are

sometimes observed (albeit at low intensity) due to other factors
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
2. It would seem that since the electronic energy levels of a pure

sample of molecules would be quantized, fine, discrete bands
would be observed – for atomic spectra, this is the case
3. In molecules, when a bulk sample of molecules is observed, not

all bonds (read – pairs of electrons) are in the same vibrational or
rotational energy states
4. This effect will impact the wavelength at which a transition is

observed – very similar to the effect of H-bonding on the O-H
vibrational energy levels in neat samples
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

A. Instrumentation
1. The construction of a traditional UV-VIS spectrometer is very similar to an
IR, as similar functions – sample handling, irradiation, detection and
output are required
2. Here is a simple schematic that covers most modern UV spectrometers:
log(I0/I) = A
I0 I
sample
UV-VIS sources
200 700
detector
, nm
monochromator/
reference
beam splitter optics I0 I0
201
UV Spectroscopy

A. Instrumentation
3. Two sources are required to scan the entire UV-VIS band:
• Deuterium lamp – covers the UV – 200-330
• Tungsten lamp – covers 330-700
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
5. The beam splitter sends a separate band to a cell containing the

sample solution and a reference solution
6. The detector measures the difference between the transmitted

light through the sample (I) vs. the incident light (I0) and sends
this information to the recorder
202
UV Spectroscopy

A. Instrumentation
7. As with dispersive IR, time is required to cover the entire UV-VIS band
due to the mechanism of changing wavelengths
8. A recent improvement is the diode-array spectrophotometer - here a

prism (dispersion device) breaks apart the full spectrum transmitted
through the sample
9. Each individual band of UV is detected by a individual diodes on a silicon

wafer simultaneously – the obvious limitation is the size of the diode, so
some loss of resolution over traditional instruments is observed
Diode array
UV-VIS sources
sample
Polychromator
– entrance slit and dispersion device 203
UV Spectroscopy

1. Virtually all UV spectra are recorded solution-phase
2. Cells can be made of plastic, glass or quartz
3. Only quartz is transparent in the full 200-700 nm range; plastic and glass
are only suitable for visible spectra
4. Concentration (we will cover shortly) is empirically determined
A typical sample cell (commonly called a cuvet):
204
UV Spectroscopy

5. Solvents must be transparent in the region to be observed; the
wavelength where a solvent is no longer transparent is referred to as the
cutoff
6. Since spectra are only obtained up to 200 nm, solvents typically only
need to lack conjugated p systems or carbonyls
Common solvents and cutoffs:

acetonitrile 190
chloroform 240
cyclohexane 195
1,4-dioxane 215
95% ethanol 205
n-hexane 201
methanol 205
isooctane 195
water 190
205
UV Spectroscopy

7. Additionally solvents must preserve the fine structure (where it is actually
observed in UV!) where possible
8. H-bonding further complicates the effect of vibrational and rotational

energy levels on electronic transitions, dipole-dipole interacts less so
9. The more non-polar the solvent, the better (this is not always possible)
206
UV Spectroscopy

C. The Spectrum
1. The x-axis of the spectrum is in wavelength; 200-350 nm for UV,
200-700 for UV-VIS determinations
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

C. The Spectrum
1. The y-axis of the spectrum is in absorbance, A
2. From the spectrometers point of view, absorbance is the inverse

of transmittance: A = log10 (I0/I)
3. From an experimental point of view, three other considerations

must be made:
i. a longer path length, l through the sample will cause
more UV light to be absorbed – linear effect
ii. the greater the concentration, c of the sample, the

more UV light will be absorbed – linear effect
iii. some electronic transitions are more effective at the

absorption of photon than others – molar absorptivity, e
this may vary by orders of magnitude…
208
UV Spectroscopy

C. The Spectrum
4. These effects are combined into the Beer-Lambert Law: A = e c l
i. for most UV spectrometers, l would remain constant (standard cells

are typically 1 cm in path length)
ii. concentration is typically varied depending on the strength of

absorption observed or expected – typically dilute – sub .001 M
iii. molar absorptivities vary by orders of magnitude:

• values of 104-106 104-106 are termed high intensity
absorptions
• values of 103-104 are termed low intensity absorptions
• values of 0 to 103 are the absorptions of forbidden transitions
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

D. Practical application of UV spectroscopy
1. UV was the first organic spectral method, however, it is rarely
used as a primary method for structure determination
2. It is most useful in combination with NMR and IR data to elucidate

unique electronic features that may be ambiguous in those
methods
3. It can be used to assay (via max and molar absorptivity) the

proper irradiation wavelengths for photochemical experiments, or
the design of UV resistant paints and coatings
4. The most ubiquitous use of UV is as a detection device for HPLC;

since UV is utilized for solution phase samples vs. a reference
solvent this is easily incorporated into LC design
UV is to HPLC what mass spectrometry (MS) will be to GC

210
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
2. Since similar functional groups will have electrons capable of

discrete classes of transitions, the characteristic energy of these
energies is more representative of the functional group than the
electrons themselves
3. A functional group capable of having characteristic electronic

transitions is called a chromophore (color loving)
4. Structural or electronic changes in the chromophore can be

quantified and used to predict shifts in the observed electronic
transitions
211
UV Spectroscopy
III. Chromophores
1. Alkanes – only posses -bonds and no lone pairs of electrons, so
only the high energy   * transition is observed in the far UV
This transition is destructive to the molecule, causing cleavage of

the -bond
C C
*
 C C
212
UV Spectroscopy
III. 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
3. Alkenes and Alkynes – in the case of isolated examples of these
compounds the p  p* is observed at 175 and 170 nm, respectively
Even though this transition is of lower energy than   *, it is still in

the far UV – however, the transition energy is sensitive to substitution
p*
214
UV Spectroscopy
III. Chromophores
4. Carbonyls – unsaturated systems incorporating N or O can
undergo n  p* transitions (~285 nm) in addition to p  p*
Despite the fact this transition is forbidden by the selection rules

(e = 15), it is the most often observed and studied transition for
carbonyls
This transition is also sensitive to substituents on the carbonyl
Similar to alkenes and alkynes, non-substituted carbonyls undergo

the p  p* transition in the vacuum UV (188 nm, e = 900);
sensitive to substitution effects
215
UV Spectroscopy
III. 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 !
CO transitions omitted for clarity
216
UV Spectroscopy
III.Chromophores
General – from our brief study of these general
chromophores, only the weak n  p* transition occurs
in the routinely observed UV
The attachment of substituent groups (other than H)

can shift the energy of the transition
Substituents that increase the intensity and often

wavelength of an absorption are called auxochromes
Common auxochromes include alkyl, hydroxyl, alkoxy

and amino groups and the halogens
217
UV Spectroscopy
III. Chromophores
General – Substituents may have any of four effects on a chromophore
i. Bathochromic shift (red shift) – a shift to longer ; lower energy
ii. Hypsochromic shift (blue shift) – shift to shorter ; higher energy
iii. Hyperchromic effect – an increase in intensity
iv. Hypochromic effect – a decrease in intensity
Hyperchromic
e
Hypsochromic Bathochromic
Hypochromic
200 nm 700 nm
218
UV Spectroscopy
III. Chromophores
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
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
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
E for the HOMO  LUMO transition is reduced

221
UV Spectroscopy
III. Chromophores
Extending this effect out to longer conjugated systems the energy gap
becomes progressively smaller:
Energy Lower energy =

Longer wavelengths
ethylene
butadiene
hexatriene
octatetraene 222
UV Spectroscopy
III. Chromophores
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
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
This quantification is referred to as the Woodward-Fieser Rules which we

will apply to three specific chromophores:
1. Conjugated dienes
2. Conjugated dienones
3. Aromatic systems
max = 239 nm
225
UV Spectroscopy

A. Dienes
1. General Features
For acyclic butadiene, two conformers are possible – s-cis and s-
trans
s-trans s-cis
The s-cis conformer is at an overall higher potential energy than

the s-trans; therefore the HOMO electrons of the conjugated
system have less of a jump to the LUMO – lower energy, longer
wavelength
226
UV Spectroscopy

A. Dienes
1. General Features
Two possible p  p* transitions can occur for butadiene 2  3*and 2
 4*
4 *
175 nm –forb. 175 nm
3 *
217 nm 253 nm
s-trans  2 s-cis
1
The 2  4* transition is not typically observed:

• The energy of this transition places it outside the region
typically observed – 175 nm
• For the more favorable s-trans conformation, this transition is

forbidden
The 2  3* transition is observed as an intense absorption
227
UV Spectroscopy

A. Dienes
1. General Features
The 2  3* transition is observed as an intense absorption (e =
20,000+) based at 217 nm within the observed region of the UV
While this band is insensitive to solvent (as would be expected) it is

subject to the bathochromic and hyperchromic effects of alkyl
substituents as well as further conjugation
Consider:
max = 217 253 220 227 227 256 263 nm
228
UV Spectroscopy

A. Dienes
2. Woodward-Fieser Rules
Woodward and the Fiesers performed extensive studies of terpene
and steroidal alkenes and noted similar substituents and structural
features would predictably lead to an empirical prediction of the
wavelength for the lowest energy p  p* electronic transition
This work was distilled by Scott in 1964 into an extensive treatise

on the Woodward-Fieser rules in combination with comprehensive
tables and examples – (A.I. Scott, Interpretation of the Ultraviolet
Spectra of Natural Products, Pergamon, NY, 1964)
A more modern interpretation was compiled by Rao in 1975 –

(C.N.R. Rao, Ultraviolet and Visible Spectroscopy, 3rd Ed.,
Butterworths, London, 1975)
229
UV Spectroscopy

A. Dienes
The rules begin with a base value for max of the chromophore being
observed:
acyclic butadiene = 217 nm
The incremental contribution of substituents is added to this base value

from the group tables:
Group Increment
Extended conjugation +30
Each exo-cyclic C=C +5
Alkyl +5
-OCOCH3 +0
-OR +6
-SR +30
-Cl, -Br +5
-NR2 +60
230
UV Spectroscopy

A. Dienes
For example:
Isoprene - acyclic butadiene = 217 nm

one alkyl subs. + 5 nm
222 nm
Allylidenecyclohexane
- acyclic butadiene = 217 nm
one exocyclic C=C + 5 nm
2 alkyl subs. +10 nm
232 nm
231
UV Spectroscopy

A. Dienes
There are two major types of cyclic dienes, with two different base values
Heteroannular (transoid): Homoannular (cisoid):
e = 5,000 – 15,000 e = 12,000-28,000

base max = 214 base max = 253
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

A. Dienes
In the pre-NMR era of organic spectral determination, the power
of the method for discerning isomers is readily apparent
Consider abietic vs. levopimaric acid:
C OH C OH
O O
abietic acid levopimaric acid
233
UV Spectroscopy

A. Dienes
For example:
1,2,3,7,8,8a-hexahydro-8a-methylnaphthalene
1 exo C=C + 5 nm
234 nm
234
UV Spectroscopy

A. Dienes

1 exo C=C + 5 nm
C OH
O
239 nm
homoannular diene = 253 nm

1 exo C=C + 5 nm
C OH
O
278 nm
235
UV Spectroscopy

A. Dienes
Be careful with your assignments – three common errors:
R
This compound has three exocyclic

double bonds; the indicated bond is
exocyclic to two rings
This is not a heteroannular diene; you

would use the base value for an acyclic
diene
Likewise, this is not a homooannular

diene; you would use the base value for
an acyclic diene
236
UV Spectroscopy

B. Enones
1. General Features
Carbonyls, as we have discussed have two primary electronic
transitions:
p* Remember, the p  p* transition is

allowed and gives a high e, but lies
outside the routine range of UV
n observation
The n  p* transition is forbidden

p and gives a very low e, but can
routinely be observed
237
UV Spectroscopy

B. Enones
1. General Features
For auxochromic substitution on the carbonyl, pronounced hypsochromic
shifts are observed for the n  p* transition (max):
O
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

B. Enones
1. General Features
Conversely, if the C=O system is conjugated both the n  p* and
p  p* bands are bathochromically shifted
Here, several effects must be noted:

i. the effect is more pronounced for p  p*
ii. if the conjugated chain is long enough, the much

higher intensity p  p* band will overlap and drown
out the n  p* band
iii. the shift of the n  p* transition is not as predictable
For these reasons, empirical Woodward-Fieser rules for

conjugated enones are for the higher intensity, allowed p  p*
transition
239
UV Spectroscopy

B. Enones
1. General Features
These effects are apparent from the MO diagram for a conjugated
enone:  4*
p* p*
 3*
n n
2
p p
1
O O
240
UV Spectroscopy
IV. Structure Determination  a d g  a

B. Enones  C C C d C C C C C
2. Woodward-Fieser Rules - Enones O O
Group Increment
6-membered ring or acyclic enone Base 215 nm
5-membered ring parent enone Base 202 nm
Acyclic dienone Base 245 nm
Double bond extending conjugation 30

Alkyl group or ring residue a,,gand higher 10, 12, 18
-OH a,,gand higher 35, 30, 18
-OR a,,g,d 35, 30, 17, 31
-O(C=O)R a,,d 6
-Cl a, 15, 12
-Br a, 25, 30
-NR2  95
Exocyclic double bond 5
Homocyclic diene component 39
241
UV Spectroscopy

B. Enones
Aldehydes, esters and carboxylic acids have different base values
than ketones
Unsaturated system Base Value
Aldehyde 208
With a,, alkyl groups 242
Acid or ester
Group value – exocyclic a, double bond +5
Group value – endocyclic a, bond in 5 +5
or 7 membered ring
242
UV Spectroscopy

B. Enones
Unlike conjugated alkenes, solvent does have an effect on max
These effects are also described by the Woodward-Fieser rules

Solvent correction Increment
Water +8
Ethanol, methanol 0
Chloroform -1
Dioxane -5
Ether -7
Hydrocarbon -11
243
UV Spectroscopy

B. Enones
Some examples – keep in mind these are more complex than dienes
O 2 x - alkyl subs. (2 x 12) +24 nm
239 nm
R
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

B. Enones
Take home problem – can these two isomers be discerned by UV-
spec O
O
Eremophilone allo-Eremophilone
Problem Set 1: (text) – 1,2,3a,b,c,d,e,f,j, 4, 5, 6 (1st, 2nd and 5th

pairs), 8a, b, c
Problem Set 2: outside problems/key -Tuesday
245
UV Spectroscopy

1. General Features
Although aromatic rings are among the most widely studied and observed
chromophores, the absorptions that arise from the various electronic
transitions are complex
On first inspection, benzene has six p-MOs, 3 filled p, 3 unfilled p*
p6*
p4* p5*
p2 p3
p1
246
UV Spectroscopy

1. General Features
One would expect there to be four possible HOMO-LUMO p  p*
transitions at observable wavelengths (conjugation)
Due to symmetry concerns and selection rules, the actual transition

energy states of benzene are illustrated at the right:
E1u
p6*
B1u
200 nm
p4* p5*
(forbidden
) B2u
180 nm
260 nm (allowed)
p2 p3 (forbidden
) A1g
p1
247
UV Spectroscopy

1. General Features
The allowed transition (e = 47,000) is not in the routine range of UV obs.
at 180 nm, and is referred to as the primary band
The forbidden transition (e = 7400) is observed if substituent effects shift

it into the obs. region; this is referred to as the second primary band
At 260 nm is another forbidden

transition (e = 230), referred to
as the secondary band.
This transition is fleetingly allowed

due to the disruption of symmetry
by the vibrational energy states,
the overlap of which is observed
in what is called fine structure
248
UV Spectroscopy

1. General Features
Substitution, auxochromic, conjugation and solvent
effects can cause shifts in wavelength and intensity of
aromatic systems similar to dienes and enones
However, these shifts are difficult to predict – the

formulation of empirical rules is for the most part is not
efficient (there are more exceptions than rules)
There are some general qualitative observations that

can be made by classifying substituent groups --
249
UV Spectroscopy

• If the group attached to the ring bears n electrons, they can
induce a shift in the primary and secondary absorption bands
• Non-bonding electrons extend the p-system through

resonance – lowering the energy of transition p  p*
• More available n-pairs of electrons give greater shifts
G G G G
250
UV Spectroscopy

• The presence of n-electrons gives the possibility of n  p*
transitions
• If this occurs, the electron now removed from G, becomes an

extra electron in the anti-bonding p* orbital of the ring
• This state is referred to as a charge-transfer excited state
G G G G
*
- *
*
*
251
UV Spectroscopy

• pH can change the nature of the substituent group
• deprotonation of oxygen gives more available n-pairs,
lowering transition energy
• protonation of nitrogen eliminates the n-pair,
raising transition energy
Primary Secondary
-H 203.5 7,400 254 204
-OH 211 6,200 270 1,450
-O- 235 9,400 287 2,600
-NH2 230 8,600 280 1,430
-NH3+ 203 7,500 254 169
-C(O)OH 230 11,600 273 970
-C(O)O- 224 8,700 268 560
252
UV Spectroscopy

b. Substituents Capable of p-conjugation
• When the substituent is a p-chromophore, it can interact with
the benzene p-system
• With benzoic acids, this causes an appreciable shift in the

primary and secondary bands
• For the benzoate ion, the effect of extra n-electrons from the
anion reduces the effect slightly
Primary Secondary
-C(O)OH 230 11,600 273 970
-C(O)O- 224 8,700 268 560
253
UV Spectroscopy

c. Electron-donating and electron-withdrawing
effects
• No matter what electronic influence a group
exerts, the presence shifts the primary
absorption band to longer 
• Electron-withdrawing groups exert no

influence on the position of the secondary
absorption band
• Electron-donating groups increase the  and e

of the secondary absorption band
254
UV Spectroscopy

c. Electron-donating and electron-withdrawing effects
Primary Secondary
Electron donating
-H 203.5 7,400 254 204

-CH3 207 7,000 261 225
-Cl 210 7,400 264 190
-Br 210 7,900 261 192
-OH 211 6,200 270 1,450
-OCH3 217 6,400 269 1,480
-NH2 230 8,600 280 1,430
Electron withdrawing
-CN 224 13,000 271 1,000

C(O)OH 230 11,600 273 970
-C(O)H 250 11,400
-C(O)CH3 224 9,800
-NO2 269 7,800
255
UV Spectroscopy

• With di-substituted aromatics, it is necessary to consider both
groups
• If both groups are electron donating or withdrawing, the

effect is similar to the effect of the stronger of the two groups
as if it were a mono-substituted ring
• If one group is electron withdrawing and one group electron

donating and they are para- to one another, the magnitude of
the shift is greater than the sum of both the group effects
• Consider p-nitroaniline:
O O
H2N N H2N N
O O
256
UV Spectroscopy

• If the two electonically dissimilar groups are ortho- or meta-
to one another, the effect is usually the sum of the two
individual effects (meta- no resonance; ortho-steric hind.)
• For the case of substituted benzoyl derivatives, an empirical

correlation of structure with observed max has been
developed
• This is slightly less accurate than the Woodward-Fieser rules,

but can usually predict within an error of 5 nm
O R
G
257
UV Spectroscopy

Parent Chromophore max
R = alkyl or ring residue 246
R=H 250
R = OH or O-Alkyl 230
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

d. Polynuclear aromatics
• When the number of fused aromatic rings increases, the  for
the primary and secondary bands also increase
• For heteroaromatic systems spectra become complex with the

addition of the n  p* transition and ring size effects and are
unique to each case
259
UV 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
400 500 600 700 800

, nm
Violet 400-420
Indigo 420-440
Blue 440-490
Green 490-570
Yellow 570-585
Orange 585-620
Red 620-780
260
UV 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
• What is “seen” is the complimentary colors (those that are not

absorbed)
• This is the origin of the “color wheel”
261
UV Spectroscopy
A. Color
1. General
• Organic compounds that are “colored” are typically those with
extensively conjugated systems (typically more than five)
• Consider -carotene
-carotene, max = 455 nm
max is at 455 – in the far blue region

of the spectrum – this is absorbed
The remaining light has the

complementary color of orange
262
UV Spectroscopy
A. Color
1. General
• Likewise:
lycopene, max = 474 nm
O
H
N
N
H
O
indigo
max for lycopene is at 474 – in the near blue

region of the spectrum – this is absorbed, the
compliment is now red
max for indigo is at 602 – in the orange region

of the spectrum – this is absorbed, the
compliment is now indigo! 263
UV Spectroscopy
A. Color
1. General
• One of the most common class of colored organic molecules
are the azo dyes:
N N
EWGs EDGs
From our discussion of di-subsituted aromatic

chromophores, the effect of opposite groups is
greater than the sum of the individual effects – more
so on this heavily conjugated system
Coincidentally, it is necessary for these to be

opposite for the original synthetic preparation!
264
UV 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
Fast Brown Sunset Yellow (Food Yellow 3)

Para Red
265
The colors of M&M’s
Bright Blue Royal Blue
Beverages, dairy products, powders, jellies, confections, Baked goods, cereals, snack foods, ice-cream, confections,
condiments, icing. cherries.
Orange-red Lemon-yellow
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
A. Color
1. General
• In the biological sciences these compounds are used as dyes
to selectively stain different tissues or cell structures
• Biebrich Scarlet - Used with picric acid/aniline blue for

staining collagen, recticulum, muscle, and plasma. Luna's
method for erythrocytes & eosinophil granules. Guard's
method for sex chromatin and nuclear chromatin.
HO
O3S N N N N
SO3
267
UV Spectroscopy
A. Color
1. General
• In the chemical sciences these are the acid-base indicators used for
the various pH ranges:
• Remember the effects of pH on aromatic substituents

Methyl Orange
CH3 CH3
H
O3S N N N O3S N N N
CH3 CH3
Yellow, pH > 4.4 Red, pH < 3.2
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 = ebc
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
1) Make a 0%T (dark current) measurement
2) Make a 100%T (blank) measurement
3) Measure %T of sample
4) Determine %T ratio and thus the

absorbance value
Instrumental noise
• Precision of measurement is limited by
instrumental noise sources
Use proper slit widths
• Resolution improves with narrower slit
width, but power decreases as square
of slit width.
– 10-fold narrower slit gives 100x less
radiant power
• General rule: Use the widest slit that
gives required resolution.
Light sources for UV-vis
• Deuterium lamp
– Most common UV
source
– Arc between oxide-
coated filament and
metal electrode
– Low voltage and low
pressure of D2
– Aperture gives 1-1.5
mm spot
– Continuum from
190-400 nm,
emission lines >400nm
Light sources for UV-vis, continued
• 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
Skoog, Fig. 13-1
Good light throughput, but

what if the source
power fluctuates?
Double-beam in time UV vis
spectrometers
• Beam is split in two, but measured by
same detector
“in time” because
the beam appears in
2 places over one
cycle in time
- Sample
- Reference
- Sample
- Reference
What if the source

power fluctuates?
Skoog, Fig. 13-13
Double-beam in space UV-vis
spectrometers
• Beam is split into two paths and measured by
matched detectors
– Difficult to find perfectly matched detectors
“in space” because Continuous

two beams are always Reference
present in space
What if the source

Continuous
power fluctuates? Sample
Cary 100 double beam
spectrometer
- 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 np* or pp*
– 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
 pp* transitions more 10-100x or more intense than np*
• 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.
acetone isopropanol ethanol
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
Resonance structures of 4,4'-

bis(dimethylamino)fuchsone
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 A1  e M  bcM  e N bcN
1 1
• Standards hopefully match sample A2  e M  bcM  e N bcN

2 2
matrix
• Matrix matching is hard—consider using
standard addition.
• Mixtures are additive
– Need to monitor at as many wavelengths
as components to be analyzed.
– Requirement of solving multiple
equations with multiple unknowns.
Color Analysis with Visible Spectra
 The visible region of a UV-Visible spectrum can be
decomposed into a color analysis (typically three numbers)
by simple calculations
– Involves multiplying the visible portion of the spectrum by color
functions and then taking the total area of the spectrum as a
single number
– Tristimulus values, which mimic the eye, are generally used and
then other values are determined from these algebraically
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
J. Mol. Struct. 2010, 984, 246–261,

Plane (or Linearly) Polarized Light
 If the electric vector of an EM wave points in the same

direction as that of the wave propagating through a
medium, the light is said to be linearly polarized
T0
K  log 10
T90
Figure from Sears, et al., “University Physics”, 7th Ed., 1988

Polarimetry and Optical Rotation
 A polarimeter measures the
angle of rotation of linearly
polarized monochromatic light
as it passes through a sample
– Source: sodium arc lamp (589
nm), now commonly replaced
with a yellow LED
– Two polarizers before and after
the sample. One is fixed and
the other is rotated to find the
maximum light transmitted, and
the rotation is recorded.
– Result is a single number, e.g.
-10.02, the specific rotation
– What happens when we vary
the wavelength?
Optical Rotation and ORD
 The rotation of plane polarized light by molecules:
Eliel et al., “Stereochemistry of Organic Compounds”, p. 997.

R. P Feynman, et al., “The Feynman Lectures on Physics”, 1963, Addison-Wesley. p. 33-6
Optical Rotatory Dispersion (ORD)
 The measurement of specific rotation as a function of

wavelength, in the absence of absorption, is monotonic
(and governed by the Fresnel equation)
 In the vicinity of an absorption, one obtains “anomalous

dispersion”
UV-Visible Circular Dichroism
 UV-visible or electronic circular dichroism (ECD or just

CD) is the study of differential absorption of polarized UV-
Visible radiation by chiral molecules.
 CD measures the difference between LCPL and RCPL
 Beer’s law for CD:
A = ebc
 Where e = (eLPCL - eRPCL)

e is the molar absorptivity (cm-1 M-1)
A is absorption
See Eliel, et al. Stereochemistry of Organic Compounds, pg. 1003.
Circularly-Polarized UV-Visible Radiation
 Circularly-polarized UV-visible radiation is made by mixing
two orthogonal electric field components 90 degrees out
of phase.
 In practice, a quartz crystal is subjected to mechanical
stress and (via the piezoelectric effect) causes circular
polarization of the light
Animation from http://www.bip.bham.ac.uk/osmart/bcm201_cd/cd_movie/index.html

UV-Visible Circular Dichroism
 A typical UV-Visible CD spectrometer, the Jasco J-715
Electronic Circular Dichroism
 CD spectra of (1S)-(+)-10-camphorsulfonic acid and (1R)-
(+)-10-camphorsulfonic acid (ammonium salts) in H2O
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:
TDDFT ECD B3LYP/6-311+G(2d,p) 50-50

3
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)
 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)
Anal. Chem. 2012, 84, 4357-4372, http://dx.doi.org/10.1021/ac203478r

 ECD has extensive applications to structural analysis in
proteins, antibodies, and other biopolymers
N. Sreerama and R. W. Woody, Meth. Enzymology, 2004, 383, 318-351.

 Different protein conformations give rise to different spectra
 CD spectra are numerically fitted to extract conformational
population
N. Sreerama and R. W. Woody, Meth. Enzymology, 2004, 383, 318-351.

Hyphenated Circular Dichroism Experiments
Example: Related
atropoisomeric
compounds
studied in
stopped-flow LC-
CD experiments
T. J. Edkins and D. R. Bobbitt, Anal. Chem., 2001, 73, 488A-496A

G. Bringmann, et al., Anal. Chem., 1999, 71, 2678-2686.
The Cotton Effect
 The Cotton effect:
– An extrema in the ECD spectrum
– Or, a zero-crossing in the ORD spectrum
Other Notes on Electronic Circular Dichroism
 Background signals – UV absorbance that does not
depend on the polarization constitutes the background
(Dynamic Reserve).
 DR = A/A = e/e = e/(eLPCL - eRPCL)
e is the molar absorptivity (cm-1 M-1)
A is absorption
 DR values of 2 x104 are possible
 Electronic background suppression is almost always
used instead of optical background suppression
(technical design issues)
Elliptically Polarized Light
 Combining left and right circularly polarized waves of
unequal amplitudes = elliptically polarized light
 Basis of ellipsometry – a surface analysis method used to

study:
– Layer/film thickness
– Optical constants (refractive index and extinction coefficient)
– Surface roughness
– Composition
– Optical anisotropy
Further Reading
Optional:
J. Cazes, Ed. Ewing’s Analytical Instrumentation Handbook, 3rd Edition, 2005, Marcel
Dekker, Chapters 5 and 6.
D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of Instrumental Analysis, 6th

Edition, 2006, Brooks-Cole, Chapters 13 and 14.
D. H. Williams and I. Fleming, “Spectroscopic Methods in Organic Chemistry”, McGraw-

Hill (1966).
D. A. Lightner and J. E. Gurst, “Organic Conformational Analysis and Stereochemistry

from Circular Dichroism Spectroscopy,” Wiley-VCH, 2000.
Molecular UV-Vis Spectroscopy: Quantum Theory
 UV-Visible spectra and the states involved in electronic
transitions can be calculated with theories ranging from Huckel
to ab initio/DFT.
 Example: p  p* transitions responsible for ethylene UV
absorption at ~170 nm calculated with ZINDO semi-empirical
excited-states methods (Gaussian 03W):
HOMO pu bonding molecular orbital LUMO pg antibonding molecular orbital
Instrument noise:
noise - short term baseline fluctuations, which decrease the
precision of the analysis
-can not measure A precisely
- the various sources of noise each cause some uncertainty in the
absorbance measurement and can be treated as individual
standard deviations.
1) 0%T noise:
- noise when light beam is blocked
- seldom important
- typically " 0.01%T
2) Readout Precision:
- especially with a meter
- typically " 0.5%T  1-3% error in concentration
3) Shot Noise:
- occurs when e- transfers across a junction (like the space between
cathode & anode in PMT).
- causes random fluctuations in current since individual e- arrive at
random times
- increases with increase current (%T). Especially bad above 95%T.
4) Flicker Noise:
- noise from the lamp due to intensity changes
-important at high transmittances.
5) Cell positioning uncertainty:
- not really noise, but affects precision
- minor imperfections, scratches or dirt change %T
- may be the major cause of imprecision
Category Characterized by Typical Sources Likely to be Important in

Case I Limited Readout resolution Inexpensive photometers and
S T = k1 spectrophotometers having small meter
scales
Heat detector Johnson noise IR and near-IR spectrophotometers and
photometers
Dark current and amplifier noise Regions where source intensity and detector
are low
Case II Photon detector shot noise High-quality UV-visible spectrophotometers
ST = k2 rT2 +T
Case III Cell positioning uncertainties High-quality UV-visible and IR

ST = k3T spectrophotometers
Source flicker Inexpensive photometers and
spectrophotometers
Taken together, these noise sources
indicate that the intermediate absorbance
and transmittance ranges should be used.
- at low %T, 0%T, noise and readout precision

are important
- at high %T, shot and flicker noise are large.
Keep A in range of 0.1 – 1.5 absorbance units (80 -3%T)

Quantum: the energy of a photon
c
E = h =

h•c
E=

E a  E a 1  a 1
g-rays x-rays UV Vis IR microwaves radiowaves
10-10 10-8 10-6 10-5 10-4 10-2 1 108  (cm)
short Wavelength () long

high Frequency () low
high Energy (E) low
327
Ultraviolet-Visible (UV-Vis) Spectroscopy
UV Vis
 200 400 800 nm
Recall bonding of a p-bond
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
If blank absorbance too high:

- dilute the sample
- use a different  where the analyte absorbs more
relative to the interference.
- use a different method of separation
b) Instrumental Deviations from Beer’s Law:
- stray light (already discussed).
- polychromatic light (more then a single )
 since all instruments have a finite bandpass, a range of
’s are sent through the sample.

 e may be different for each 
Deviation’s from
Beer’s law at high
concentration
Illustration of Deviation from Beer’s law:
Let us say that exactly 2 wavelengths of light were

entering the sample
 = 254 nm e254 =10,000
 = 255 nm e255 = 5,000
let Po = 1 at both ’s
What happens to the Beer’s law plot as c increases?
A = A254 + A255
A = log Po/P (total) = log (Po254 + Po255)/(P254 + P255)
At the individual ’s:
A254 = e254bc = log Po254/P254
10ebc = Po254/P254
P254 = Po/10ebc = Po10-ebc

For both together:
A = log (Po254 + Po255)/(Po25410-e bc
254 + Po25510-e bc)
255
Since Po254 = Po255 =1:
A = 2.0/(Po25410-e bc + Po25510-e bc)
254 255
8
A = 2.0/(10-10,000x1.0xc + 10-5000x1.0xc)
7
6
A = e bc
5
C A (actual) A(expected)
4
10-6M 0.0075 0.0075

3 Negative Deviation
10-5M 0.074 0.075 2
10-4M 0.068 0.75 1
10-3M 5.3 7.5 0

2 10-4 4 10-4 6 10-4 8 10-4 1 10-3
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
 Beer’s law assumes species are independent
 electrolytes may also cause this problem
 e is also affected by the index of refraction
- association, dissociation, precipitation or reaction of

analyte
phenolphthalein:
Ka
HIn H+ + In-
Red,  =600nm colorless
 c in Beer’s law is the concentration of the absorbing
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-
CHIn [HIn] [In-] [HIn]/[In-]
Expected
10-5 8.5x10-7 9.2x10-6 0.0924
10-4 3.8x10-5 6.2x10-5 0.613 Actual
10-3 7.3x10-4 2.7x10-4 2.70
C
HIn
“Apparent” deviation since can be accounted for by chemical equilibrium

But, if unbuffered solution, equilibrium will shift depending on total analyte
concentration
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
A = log10 Po/P = ebc

ii) d/f electrons (transition metal ions)
 Lanthanide and actinide series
- electronic transition of 4f & 5f electrons
- generally sharp, well-defined bands not affected by
associated ligands
 1st and 2nd transition metal series
- electronic transition of 3d & 4d electrons
- broad peaks
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
2. This energy corresponds to EM radiation in the ultraviolet (UV) region,

100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum
3. For comparison, recall the EM spectrum:
g-rays X-rays UV IR Microwave Radio
Visible
4. Using IR we observed vibrational transitions with energies of 8-40 kJ/mol

at wavelengths of 2500-15,000 nm
5. For purposes of our discussion, we will refer to UV and VIS spectroscopy

as UV
342
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
343
UV Spectroscopy
I. Introduction
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
4. p-orbitals are of somewhat higher energy, and their complementary anti-

bonding orbital somewhat lower in energy than *.
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
6. Here is a graphical representation
*
Unoccupied levels
p*
Atomic orbital Atomic orbital

Energy n
Occupied levels
p

Molecular orbitals
345
UV Spectroscopy
I. Introduction
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
7. Although the UV spectrum extends below 100 nm (high energy), oxygen
in the atmosphere is not transparent below 200 nm
8. Special equipment to study vacuum or far UV is required
9. Routine organic UV spectra are typically collected from 200-700 nm
10. This limits the transitions that can be observed:
 * alkanes 150 nm
 p* carbonyls 170 nm
p p* unsaturated cmpds. 180 nm √ - if conjugated!
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
2. For an electron to transition, certain quantum mechanical

constraints apply – these are called “selection rules”
3. For example, an electron cannot change its spin quantum number

during a transition – these are “forbidden”
Other examples include:
• the number of electrons that can be excited at one time
• symmetry properties of the molecule
• symmetry of the electronic states
4. To further complicate matters, “forbidden” transitions are

sometimes observed (albeit at low intensity) due to other factors
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
2. It would seem that since the electronic energy levels of a pure

sample of molecules would be quantized, fine, discrete bands
would be observed – for atomic spectra, this is the case
3. In molecules, when a bulk sample of molecules is observed, not

all bonds (read – pairs of electrons) are in the same vibrational or
rotational energy states
4. This effect will impact the wavelength at which a transition is

observed – very similar to the effect of H-bonding on the O-H
vibrational energy levels in neat samples
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)
 Basic UV-Vis spectrophotometers acquire data in the 190-

800 nm range and can be designed as “flow” systems.
Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
Molecular UV-Vis Spectroscopy: Terminology
 UV-Vis Terminology
– Chromophore: a UV-Visible absorbing functional group

– Bathochromic shift (red shift): to longer wavelengths
– Auxochrome: a substituent on a chromophore that
causes a red shift
– Hypsochromic shift (blue shift): to shorter wavelengths
– Hyperchromic shift: to greater absorbance
– Hypochromic shift: to lesser absorbance

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Molecular Spectroscopy

Methods of structure determination

Electromagnetic radiation has the properties of a particle

= distance of one wave

(The Bohr model for nitrogen) (The LUMO of benzene)

molecules have discrete energy levels

A molecule absorbs electromagnetic radiation when

UV-Vis: valence electron transitions

Infrared: molecular vibrations (stretches, bends)

Radiowaves: nuclear spin in a magnetic field (NMR)

Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

 Major classes of electron transitions

– HOMO: highest occupied molecular orbital

y2 is the Highest Occupied Molecular Orbital (HOMO)

380 nm 400 nm 450 nm 500 nm 550 nm 600 nm 700 nm 780 nm

violet-indigo blue green yellow orange red

violet-indigo blue green yellow orange red

 Charge transfer (CT) – occurs when electron-donor and

(colorless) (deep red color)

Beer’s Law: There is a linear relationship between

 Molar absorption coefficient (e) then gives A via the Beer-

Ezetimibe calibration plot

When all the protein is bound to Fe3+,

As Fe3+ continues to bind protein

(2) Add a variable volume, Vs, of a standard solution of known

Note: This method assumes a linear relationship between instrument

(4) Make instrumental measurements of each sample to get an

A1 = e1MbcM + e1NbcN (1)

A2 = e2MbcM + e2NbcN (2)

Note: need to solve simultaneous equations

Calculate the concentrations of A and B in solutions that

Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1

Figure from Skoog, et al., Chapter 13

II. Instrumentation and Spectra

2. Here is a simple schematic that covers most modern UV spectrometers:

beam splitter optics I0 I0

II. Instrumentation and Spectra

5. The beam splitter sends a separate band to a cell containing the

6. The detector measures the difference between the transmitted

II. Instrumentation and Spectra

8. A recent improvement is the diode-array spectrophotometer - here a

9. Each individual band of UV is detected by a individual diodes on a silicon

II. Instrumentation and Spectra

2. Cells can be made of plastic, glass or quartz

3. Only quartz is transparent in the full 200-700 nm range; plastic

4. Concentration (we will cover shortly) is empirically determined

A typical sample cell (commonly called a cuvet):

II. Instrumentation and Spectra

6. Since spectra are only obtained up to 200 nm, solvents typically

Common solvents and cutoffs:

II. Instrumentation and Spectra

8. H-bonding further complicates the effect of vibrational and rotational

II. Instrumentation and Spectra

II. Instrumentation and Spectra

2. From the spectrometers point of view, absorbance is the inverse

3. From an experimental point of view, three other considerations

ii. the greater the concentration, c of the sample, the