Chapter Four

Ultraviolet and Visible

Molecular Absorption Spectroscopy
Ultraviolet and visible spectrophotometry is the method of choice in
most laboratories concerned with the identification and measurement of
organic and inorganic compounds in a wide range of products and
processes for both research and routine control.
The concentration of an analyte in solution can be determined by
measuring the absorbance, emission, or scattering at some wavelength
and applying the Lambert– Beer Law. Modern spectrophotometers are
quick, accurate and reliable and make only small demands on the time
and skills of the operator. UV/Vis spectrophotometers is routinely used
in the quantitative determination of solutions of transition metal ions,
highly conjugated organic compounds, and biological macromolecules.
Principle: It involves the absorption of electromagnetic radiation by the
substances in the ultraviolet and visible regions of the spectrum. This
will result in changes in the electronic structure of ions and molecules
through the excitations of bonded and non-bonded electrons. In
Ultraviolet and Visible spectrophotometer the amount of absorbed
radiation by molecule is measured.
Absorbing Species
Absorption of ultraviolet and visible radiation by molecules generally
occurs in one or more electronic absorption bands, each of which is
made up of many closely packed but discrete lines.
Absorption by Organic Compounds
Absorption of radiation by organic molecules in the wavelength region
between 180 and 780 nm results from interactions between photons and
electrons that either participate directly in bond formation (and are thus
associated with more than one atom) or are localized about such atoms
as oxygen, sulfur, nitrogen, and the halogens.
The wavelength at which an organic molecule absorbs depends on how
tightly its electrons are bound. The shared electrons in carbon/carbon or
carbon/hydrogen single bonds are so firmly held that their excitation
requires energies corresponding to wavelengths in the vacuum ultraviolet
region below 180 nm. Single-bond spectra have not been widely
exploited for analytical purposes because of the experimental difficulties
of working in this region. These difficulties occur because both quartz
and atmospheric components absorb in this region.
Electrons involved in double and triple bonds of organic molecules are
not as strongly held and are therefore more easily excited by radiation:
thus, species with unsaturated bonds generally exhibit useful absorption
peaks.
Unsaturated organic functional groups that absorb in the ultraviolet or
visible regions are known as chromophores.
Conjugation between two or more chromophores tends to cause shifts in
peak maxima to longer wavelengths.
Saturated organic compounds containing such heteroatoms as oxygen,
nitrogen, sulfur, or halogens have nonbonding electrons that can be
excited by radiation in the 170 - to 250-nm range. Some of these
compounds, such as alcohols and ethers, are common solvents, so their
absorption in this region prevents measuring absorption of analytes
dissolved in these compounds at wavelengths shorter than 180 to 200
nm.
Molecular Orbital: An orbital is a region round a nucleus where there
is a high probability of finding an electron. It is the predicted paths of the
bonding electrons about the nucleus potentially; three types of ground
state orbital may be involved in molecule:
σ(bonding) molecular as in

π (bonding) molecular orbital as in

n (non-bonding) atomic orbital as in

In addition, two types of ant bonding orbital may be involved in the

transition: σ * (sigma star) orbital π * (pi star) orbital. There is no such
thing as an n* anti bonding orbital as the n electrons do not form bonds.
Excitation of electrons between molecular orbital
Excitation only occurs between the two lowest energy transitions, the
outer electron, π -> π* and the n -> π* levels for the energy range of 200-
800 nm associated with UV-Vis radiation.
This transition is from the highest occupied molecular
orbital (HOMO) to the lowest unoccupied molecular
orbital (LUMO).
Chromophores which can be detected by UV/Vis spectrophotometers
always involve a multiple bond (such as C=C, C=O or C=N) and may be
conjugated with other groups to form complex chromophores. A typical
example is the benzene ring which has an absorption peak at 254 nm.
Increasingly complex chromophores move the associated absorption
peak towards longer wavelengths and generally increase the absorption
at the maxima.
Conjugation Effect on λ max
Conjugation:-Conjugated compounds are compounds with alternating
double and single bonds. Compare the molecular orbital levels in a non-
conjugated alkene with the molecular orbitals of a conjugated diene, a
conjugated diene - two π -bonding and two π -antibonding molecular
orbitals compared to one each in the alkene. In other words the greater
the degree of conjugation (number of sp2 hybridized atoms adjacent to
one another), the larger the shift of λmax toward longer wavelength
value. The greater the degree of conjugation the further this shift toward
longer wavelength will be.
For example, the λmax for 1,3-Cyclohexadiene (a four carbon
conjugated system) is 256 nm. The λmax for 1,3,5 Hexatriene (a six
carbon conjugated system i.e. greater conjugation) is 274 nm or 18 nm
longer.
Extending this effect out to longer conjugated systems the energy gap
becomes progressively smaller:

Energy Lower energy =

Longer wavelengths

ethylene
butadiene
hexatriene
octatetraene
The attachment of substituent groups on a basic chromophore structure
changes the position and intensity of an absorption band of the
chromophore. Substituent that increase the intensity of the absorption
and possibly the wavelength are called Auxochromes. Typical
auxochromes include methyl, hydroxyl, alkoxy, halogen, and amino
groups. Other substituent may have any of four kinds of effects on the
absorption.
1. Bathochromic shift ( Red shift ) – a shift to lower
energy or longer wavelength
2. Hypsochromic shift ( blue shift) - a shift to higher
energy or shorter wavelength
3. Hyperchromic effect – an increase in intensity
4. Hypochromic effect – a decrease in intensity
One of the best way to bring about a bathochromic shift is to increase the
extent of conjugation in a double- bonded system. In the presence of
conjugated double bonds the electronic energy levels of a chromophore
move close together. As a result, the energy required to produce a
transition from an occupied electronic energy level to an unoccupied
level decreases and the wavelength of the light absorbed become longer.
Quantification of effect of substituent groups - Woodward-Fieser Rules
which we will apply to three specific chromophores:
Conjugated dienes
Conjugated dienones
Aromatic systems
For acyclic butadiene, two conformers are possible

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

16
 Two possible π  π * transitions can occur for butadiene
   and   


 



175 nm –forb. 175 nm

217 nm 253 nm

s-trans s-cis


The  
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  transition is observed as an intense absorption

17
 The Y2  Y3* 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

18
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
19
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 20
Consider abietic vs. levopimaric acid: find the max

C OH C OH
O O

abietic acid levopimaric acid

21
Be careful with your assignments – three common errors:

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

22
2. Enones : 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 and

gives a very low e, but can routinely be
p observed
For auxochromic substitution on the carbonyl, pronounced
hypsochromic shifts are observed for the n  π* transition (λmax). This is
explained by the inductive withdrawal of electrons by O, N or halogen
from the carbonyl carbon – this causes the n-electrons on the carbonyl
oxygen to be held more firmly. It is important to note this is different
from the auxochromic effect on π  π* which extends conjugation and
causes a bathochromic shift. In most cases, this bathochromic shift is not
enough to bring the π  π* transition into the observed range.

     
 C C C  C C C C C
O O
      
Woodward-Fieser Rules - Enones
 C C C  C C C C C
O O

Group Position 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 and higher 10, 12, 18
-OH and higher 35, 30, 18
-OR  35, 30, 17, 31
-O(C=O)R  6
-Cl  15, 12
-Br  25, 30
-NR2  95
Exocyclic double bond 5
Homocyclic diene component 39
Enones
Woodward-Fieser Rules - Enones
Aldehydes, esters and carboxylic acids have different base
values than ketones

Unsaturated system Base Value

Aldehyde 208
With  or  alkyl groups 220
With  or  alkyl groups 230
With  alkyl groups 242

Acid or ester
With  or  alkyl groups 208
With  or  alkyl groups 217
Group value – exocyclic  double +5
bond
Group value – endocyclic  bond +5
in 5 or 7 membered ring

26
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

27
Some examples – keep in mind these are more complex than
dienes cyclic enone =
215 nm 2 x - alkyl subs.
(2 x 12) O +24 nm

239 nm
Experimental value 238 nm

R
cyclic enone =
215 nm
extended conj.
+30 nm
O -ring residue
+12 nm -ring residue
+18 nm exocyclic
double bond + 5 nm
28
Take home problem – can these two isomers be discerned by
UV-spec
O
O

Eremophilone allo-Eremophilone

29
Aromatic Compounds: 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).
Absorption by Inorganic Species
In general, the ions and complexes of elements in the first two transition
series absorb broad bands of visible radiation in at least one of their
oxidation states and are, as a result, colored. Here, absorption involves
transitions between filled and unfilled d-orbitals with energies that
depend on the ligands bonded to the metal ions. The energy differences
between these d-orbitals (and thus the position of the corresponding
absorption peak) depend on the position of the element in the periodic
table, its oxidation state, and the nature of the ligand bonded to it.
Charge- Transfer Absorption
For quantitative purposes, charge-transfer absorption is particularly
important because molar absorptivities are unusually large (ɛ > 10,000),
which leads to high sensitivity. Many inorganic and organic complexes
exhibit this type of absorption and are therefore called charge-transfer
complexes. A charge-transfer complex consists of an electron-donor
group bonded to an electron acceptor. When this product absorbs
radiation, an electron from the donor is transferred to an orbital that is
largely associated with the acceptor. The excited state is thus the product
of a kind of internal oxidation/reduction process. This behavior differs
from that of an organic chromophore, in which the excited electron is in
a molecular orbital that is shared by two or more atoms. Familiar
examples of charge-transfer complexes include the phenolic complex of
iron (III), the 1,10-phenanthroline complex of iron(II), the iodide
complex of molecular iodine, and the ferro/ferricyanide complex
responsible for the color of Prussian blue.
UV-VIS Instrumentation
Sources
An ideal light source is one that emits an intense continuous spectrum of
light across an entire region of the spectrum, such as the visible region,
while also exhibiting a long life.
visible light = tungsten filament source.
Instrument meant strictly for visible light studies = colorimeter.
Ultraviolet light = deuterium lamp
Instrument meant strictly for ultraviolet studies= UV
spectrophotometer
Instead of having two independently selectable sources, a light source
that can be used for both ultraviolet and visible studies, the xenon arc
lamp, may be present.
Xenon Arc Lamp
This lamp contains xenon at a fairly high pressure and the light is formed
via a discharge across a pair of electrodes. A continuous ultraviolet and
visible emission is emitted due to the presence of the xenon. In some
instruments, the electronic circuitry creates regular pulses of light that
are very intense and therefore more useful. This results in a longer life
for the lamp. The intensity varies with wavelength.
Wavelength Selection
In order to plot the absorption spectrum of a compound or complex ion,
we must be able to carefully control the wavelengths from the broad
spectrum of wavelengths emitted by the source so that we can measure
the absorbance at each wavelength. Additionally, in order to perform
quantitative analysis by Beer’s law, we need to be able to carefully select
the wavelength of maximum absorption, also from this broad spectrum
of wavelengths, in order to plot the proper absorbance at each
concentration. These facts dictate that we must be able to filter out the
unwanted wavelengths and allow only the wavelength of interest to pass.
In the visible region, the spectrum of colors is continuous, meaning that
there is no sharp delineation between green light and blue light, for
example, or where one wavelength ends and the adjacent wavelength
begins. The electromagnetic spectrum is a continuous wavelength band.
Thus wavelength selection in a spectrochemical instrument actually
consists of the selection of a narrow wavelength band from the larger
band. The width of the band that is allowed to pass is called the
bandwidth.
The narrowness of the band that is allowed to pass varies from one
design to another and is called the resolution. Thus a high resolution
(narrow bandwidth) is ideal, although many applications do not require
the best available resolution. The most popular wavelength selector for
Uv - Vis spectrophotometer is monochromator.
Sample Compartment
Following the wavelength selection by the monochromator, the beam
passes on to the sample compartment where the sample solution, held in
the cuvette, is positioned in its path.
Cuvette Selection and Handling
Cuvettes used for UV-VIS spectrophotometry must be transparent to all
wavelengths of light for which it is used. If two or more different
cuvettes are used in an analysis, they should be matched. If the
pathlengths were different, or if the wall of one cuvette reflects more or
less light than another cuvette, then the absorbance measurement could
be different for that reason, and not because the solution concentration is
different. Any liquid or fingerprints adhering to the outside wall must be
removed with a soft, lint-free cloth or towel. In addition to scratches,
fingerprints, water spots, etc., the analyst should be aware that any
foreign particulate matter suspended in the sample or standards would
also be a problem.
Detectors
Photomultiplier tubes or photodiodes (light sensors) are used as detectors
in UV-VIS spectrophotometers. This is the reason UV-VIS instruments
are called spectrophotometers.
Applications: It is the most widely used technique for quantitative
molecular analysis, for this Beer-Lambert law is applied. Sometimes it is
used in conjunction with other techniques such as NMR, IR, etc., in the
identification and structural analysis of organic compounds. For
qualitative analysis it provides valuable information through the
absorption spectrum which is unique for a given compound. The most
use of uv is as a detection device for HPLC.