UV-VIS Molecular Spectrometry

 Ultraviolet–visible (UV-VIS) molecular spectrochemical

methods utilize light in the ultraviolet and visible regions of
the electromagnetic spectrum to analyze laboratory samples
for molecular compounds and complex ions.
 Qualitative analysis (identification of unknowns and detection
of impurities in knowns) is accomplished by comparing
absorption or transmission spectra (molecular fingerprints)
with known spectra.
 Quantitative analysis is accomplished with the use of Beer‟s
law. UV-VIS spectra result from electronic transitions
occurring in the analyte molecules and complex ions.

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UV-VIS Instrumentation
The components of UV-VIS spectrophotometer involve
light source, wavelength selector, sample holder, detector
and readout.
Light Sources
An ideal light source is one that emits an intense
continuous spectrum of light across an entire region of the
spectrum, while also exhibiting a long life. A light source
used frequently for visible light absorption studies is the
tungsten filament source. If an instrument is meant strictly
for visible light studies, then this lamp is the only one
present in the instrument. Such an instrument is often
referred to as a colorimeter.

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• A light source used frequently for ultraviolet absorption
studies is the deuterium lamp. If an instrument use only
deuterium lamp as light source, the instrument is called a UV
spectrophotometer. Deuterium lamp contains deuterium at a
low pressure. Electricity applied to electrodes in the lamp
results in a continuous UV emission due to the presence of the
deuterium. Its wavelength output ranges from 185 nm to
about 375 nm, satisfactory for most UV analyses.
• Xenon arc lamp is a light source that can be used for both
ultraviolet and visible studies. Xenon arc 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.

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Wavelength Selection: to filter out the unwanted
wavelengths and allow only the wavelength of interest to
pass. In reality, there is no single wavelength. 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.

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Absorption Filters
The most inexpensive way to isolate a wavelength band is with
the use of absorption filters. For visible light, such a filter would
consist of colored glass, the color of the glass indicating what
region of the visible spectrum is passed. Thus, if the wavelength
called for in a given method is in the red region of the visible
spectrum, a red colored glass filter is chosen.

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Monochromators
The word “monochromator” is derived from the Latin language, “mono”
meaning “one” and “chromo” meaning “color.” It is a device more
sophisticated than an absorption filter that isolates the narrow band of
wavelengths from visible and ultraviolet sources. A monochromator is
made up of three parts: an entrance slit, a dispersing element, and an
exit slit.
The dispersing element is either a diffraction grating or a prism. A
prism is a three-dimensional triangularly shaped glass or quartz
block. When the light beam strikes one of the three faces of the
prism, the light emerging through another face is dispersed.
A diffraction grating is used more often than a prism. A diffraction
grating is like a highly polished mirror that has a large number of
precisely parallel lines or grooves scribed onto its surface. Light
striking this surface is reflected, diffracted, and dispersed into the
component wavelengths.
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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.
Some spectrophotometers are single-beam instruments, and
some are double-beam instruments. In a double-beam
instrument the light beam emerging from the monochromator
is split into two beams at some point between the
monochromator and the detector.

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Cuvettes used for UV-VIS spectrophotometry must be
transparent to all wavelengths of light for which it is used. If
visible light, colorless plastic and ordinary colorless glass
perfectly suitable.
Glass and plastic are not transparent to light in the ultraviolet
region. For ultraviolet spectrophotometry, the cuvettes must
be made of quartz. If two or more different cuvettes are used
in an analysis, one should be sure that they are matched.
Cuvettes that have scratches or cleaning procedures that may
cause scratching should be avoided. Cotton swabs can be
used for scrubbing, rather than metal-handled brushes. Any
liquid or fingerprints adhering to the outside wall must be
removed with a soft, lint-free cloth or towel.

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Some spectrophotometers are single-beam instruments,
and some are double-beam instruments.
Single-Beam Spectrophotometer: uses one beam of
radiation and passes it through a single cell, generally
less expensive. They are primarily used for studies
performed at a single wavelength, i.e. they are not as
often used to scan through a wavelength region. The
wavelength at which the study is performed can be varied
by adjustment of the monochromator.

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Double beam spectroscopy: divides the radiation into two
beams which are passed through separate cells. In double beam
instruments one of the two cells is filled with either pure solvent
or a blank solution and the second cell is filled with the sample.
Since the readout from the instrument is the difference between
the amount of light absorbed in the two cells, the absorption in
the sample is automatically corrected for absorption occurring in
the blank/ solvent and for changes in intensity of the incident
radiation with wavelength. Double- beam instruments can be
used at fixed wavelength or they can be used to scan through a
wavelength region.

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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.

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Photomultiplier Tube
A photomultiplier tube is a light sensor combined with a signal
amplifier. The light emerging from the sample compartment
strikes the photosensitive surface and the resulting electrical
signal is amplified. The photomultiplier tube consists of a
photocathode, an anode, and a series of dynodes for multiplying
the signal, hence its name. A high voltage is applied between the
photocathode and the anode.

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Photodiodes
Photodiodes make use of the unique properties of semiconductors,
such as silicon. Silicon can be doped with impurities to make it either
electron rich (an n-type semiconductor) or electron poor (a p-type
semiconductor). When an n-type semiconductor is in contact with a
p-type semiconductor, electronic changes occur at the boundary, or
junction. A photodiode is a p–n junction constructed with the top p
layer so thin that it is transparent to light. Light shining through the p
layer creates additional free electrons in the n layer that can diffuse to
the p layer, thus creating an electrical current that depends on the
intensity of the light. This small current is easily amplified and
measured.

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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.

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Electronic excitation of molecular orbital
The energy range required to cause electronic excitations between molecular
orbitals (ΔΕ) is defined as the energy difference between an occupied orbital
(ground state) and an empty (excited state) orbital. The smaller the value of
ΔΕ, the lower the frequency and also the longer the wavelength required to
excite the electron. When the energy of the incoming photon matches ΔΕ, the
radiation is absorbed, and an electron from an occupied level "jumps" from its
ground state to an excited state. Note that this transition could occur from
various occupied to empty levels, (σ to σ*, n to σ*,n to π*,π to π*). But in
reality, 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. In general, this transition will occur
between the highest occupied molecular orbital (HOMO) to the lowest
unoccupied molecular orbital (LUMO).

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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. Vibrational effects broaden absorption peaks in the
ultraviolet and visible regions, which often makes precise
determination of an absorption maximum difficult.

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The Conjugation Effect on λmax
Conjugated compounds are compounds with alternating double
and single bonds. 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.

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The attachment of substituent groups on a basic chromophore
structure changes the position and intensity of an absorption
band of the chromophore. Substituent that change the intensity
of the absorption and possibly the wavelength are called
Auxochromes. Typical auxochromes include methyl, hydroxyl,
alkoxy, halogen, and amino groups. Auxochromes 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
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It is possible to predict the position of absorption maximum in
open chain dienes and six-membered ring compounds. Woodward
put forward rules to predict the positions of absorption maximum
in these compounds which were later modified by Fieser and Scott.
These rules are known as Woodward-Fieser rules and are
applicable to dienes and trienes.
There are essentially four organic molecular systems of interest. The
principal parent chromophore systems are (1) conjugated dienes, (2)
monosubstituted benzene rings, (3) disubstituted benzene rings, and
(4) conjugated carbonyl systems. The method of calculation is to
identify a parent system and assign an absorption maximum. The
parent system is then modified by the presence of other systems
within the molecule. From these modifications, the absorption
maximum of a specific molecular structure can be calculated.
 Conjugated Diene Systems
The parent diene of conjugated diene systems is C=C-C=C. This
system in a hexane solvent absorbs at 217 nm. If the conjugated system
is increased, the wavelength of the absorption maximum is increased
by 30 nm for every double bond extension. The shift due to a
substituent group in the max for any substituted diene can be
calculated by adding the increment to the assigned base value.
Maxima of conjugated dienes nm
Absorption of parent diene system C=C-C=C 217

Shift to longer λ
Double bond extension to diene system 30
Diene system within a ring 36
Exocyclic nature of double bond in conjugated system 5

Each alkyl substituent or ring risidue 5

Auxochrome is
-O-acyl 0
-O-alkyl 6
-S-alkyl 30
-N-alkyl2 60
-Cl, -Br 5
 Example 1: Predict absorption maxima for

Diene 217 nm
Answer Within a ring 36
Alkyl substituent 3x5 = 15
268 nm
Example 2: Predict absorption maxima for

Diene 217 nm
Answer Within a ring 36
Double bond extension 30
Exocyclic double bond 5
Exocyclic double bond is the
double bond that touches the
Alkyl substituent 5x5 = 25
adjacent age of ring 313 nm
Example 3: Predict absorption maxima for

Diene 217 nm
Answer Within a ring 36
Double bond extension 30
Exocyclic double bond 5
Alkyl substituent 3x5 = 15
-OCH3 6
309 nm
Example 4: Predict absorption maxima for

Diene 217 nm
Answer Within a ring 0
Double bond extension 0
Exocyclic double bond 5
Alkyl substituent 3x5 = 15
-OCH3 6
243 nm
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.

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 IR Spectrometry
IR spectrometry differs from UV-VIS spectrophotometry in
the following ways:
1. Absorption of IR light results in vibrational energy
transitions rather than electronic transitions.
2. While liquid solutions are often analyzed in IR work, pure
liquids and undissolved solids, including polymer films,
are also often analyzed, as are gases.
3. In IR work, the containers that hold liquids or liquid
solutions in the path of the light are not called cuvettes.
They are called liquid sampling cells.
4. The liquid sampling cells have extremely short pathlengths
(often fractions of a millimeter), defined by the thickness
of a thin polymer film spacer.
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5. Liquid sampling cells utilize large polished inorganic salt crystals
as windows (cell walls) for the IR light. Analyte solvents must be
water-free organic liquids that do not dissolve the inorganic salt
crystals. Glass and plastic have significant disadvantages and are not
usually used.
6. IR spectra are usually transmission spectra rather than absorption
spectra, and wavenumber, rather than wavelength, is plotted on the x-
axis.
7. IR spectra are characterized by rather sharp absorption bands and
each such band is characteristic of a particular covalent bond in the
sample molecule. Thus, while IR spectra are molecular fingerprints,
as are UV-VIS spectra, they have a greater worth to a qualitative
analysis scheme due to the specificity of the absorption bands.
8. Modern IR spectrometers do not use light dispersion to acquire
spectra. Rather, they utilize a device called an interferometer between
the source and the sample. This design requires a signal processing
circuit that performs a mathematical operation called a Fourier
transformation to obtain the spectra. 30
Principle
Analytical infrared studies are based on the absorption (or
reflection) of the electromagnetic radiation that lies between 1 and
1000µm. This absorption in the infrared region of the
electromagnetic radiation cause vibrational transitions in
molecules. The vibration spectra give information about the
functional groups in molecules. This spectral range is sub-divided
into three smaller areas: the near infrared (near-IR, 1–25µm), the
mid infrared (mid-IR, 25–50µm) and the far infrared (beyond
50µm). Molecules contain bonds of specific spatial orientation and
energy. These bonds are seldom completely rigid and when energy
is supplied, they may bend, distort or stretch. In order to absorb IR
radiation, the dipole moment of the molecule must change during
the vibration. Such vibrations are said to be IR active.

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IR Instrumentation
For mid-IR, NIR and Raman spectrometry, the instrumentation is
different, but the main components of spectrometers are all the same.
The sources used for mid-IR are heated rods, such as a nichrome ribbon
or a „globar‟, which is a rod of silicon carbide. The Opperman source is a
rhodium heater in an alumina tube packed with alumina and zirconium
silicate. When heated to above 1000c, these sources emit energy over a
wide range, resembling a black-body radiator with a maximum intensity
at about 1000 cm. For NIR, tungsten or tungsten halogen lamps are used.
Laser sources such as the Ar+2 laser give strong, sharp lines at 488.0 nm
and 514.5 nm. One disadvantage is that these wavelengths may cause
fluorescence. This is avoided by using NIR laser sources.

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The instruments that utilize the interferometer and Fourier
transformation is known as Fourier transform infrared
spectrometry (FTIR). An interferometer is a device that
creates a pattern of light resulting from the combined
constructive and destructive interference of all component
wavelengths. This combined interference is caused by first
splitting the beam and then directing the two beams through
paths of two different lengths, one of which is fixed and one of
which is variable. When the two beams are recombined at the
same beam splitter (mirrored on both sides), they may be in
phase (when the two paths are of the same length) or out of
phase (when the two paths are not of the same length).

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The advantages of FTIR over the dispersive technique are: 1) it
is faster, making it possible to be incorporated into
chromatography schemes, and 2) the energy reaching the
detector is much greater, thus increasing the sensitivity.
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Sampling
Sampling, in the context of infrared spectrometry, refers to the
method by which a sample is held in the path of the IR light.
Since liquids (including pure liquids and liquid solutions), solids,
and gases can be analyzed by infrared spectrometry, we have
liquid sampling, solid sampling, and gas sampling. In any case, it
is important to point out that glass and plastic are undesirable
materials for the cells. The reason is that glass and plastic are
molecular (covalent) materials and would absorb IR light and
interfere with reading the sample. Inorganic salts, such as NaCl
and KBr, are ionic materials and do not absorb IR light because
ionic bonds cannot undergo vibrational energy transitions.
Covalent bonds can vibrate while ionic bonds cannot.

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Infrared Absorption Spectra
The energy of infrared radiation can excite vibrational and rotational
transitions, but it is insufficient to excite electronic transitions.
Infrared spectra exhibit narrow, closely spaced absorption bands
resulting from transitions among the various vibrational quantum
levels. Variations in rotational levels may also give rise to a series of
peaks for each vibrational state; with liquid or solid samples,
however, rotation is often hindered or prevented, and the effects of
these small energy differences are not detected. Thus, a typical
infrared spectrum for a liquid, consists of a series of vibrational
bands.
For a simple diatomic molecule, this model is easy to visualize:
As a covalent bond oscillates – due to the oscillation of the dipole of the
molecule – a varying electromagnetic field is produced

The greater the dipole moment change through the vibration, the more intense
the EM field that is generated
There are two types of bond vibration:

• Stretch – Vibration or oscillation along the line of the bond

H H
C C
H H

symmetric asymmetric

• Bend – Vibration or oscillation not along the line of the bond

H H H H
C C C C
H H H
H
scissor rock twist wag
in plane out of plane
 Number of Vibrational Frequencies in a
Molecule
• There are 3n-6 possible vibrational modes in
a nonlinear molecule with no symmetry
• There are 3n – 5 possible vibrational modes
for linear molecules.
– Symmetry reduces the number of possible
vibrational modes.
• Water has 3 possible vibrational modes.
• Formaldehyde has 6.
 Characteristic Vibrational Frequencies of
Bonds
• Bonds are not rigid but behave like a spring
with a mass at either end.
– Obey Hooke’s Law: F = -kx
– This gives rise to a characteristic frequency for
the vibration:

1 k

2 reduced _ mass
m1 m2
reduced _ mass 
m1  m2
Molecular vibration induced by IR absorption
• The frequency is affected by
– the masses of the atoms in the bond
– the strength of the bond
• The lower the mass, the higher the vibrational frequency.
Stretching frequencies for bonds to carbon: C-H > C-C > C-N > C-O
• The stronger the bond, the higher the vibrational frequency.
– Stretching frequencies
• C≡C > C=C > C-C
• C≡N > C=N > C-N
• C≡O > C=O > C-O
• C(sp)-H > C(sp2)-H > C(sp3)-H
Absorption Regions
Figure: IR spectrum of octane
Figure: IR spectrum of methylbenzene
Figure: IR spectrum of propanoic acid