Unit 2

QUALITATIVE ANALYSIS BY UV
AND VISIBLE SPECTROCOPY
 Electronic Spectroscopy
Ultraviolet (UV) and visible (VIS) spectroscopy
This is the earliest method of molecular
spectroscopy.
A phenomenon of interaction of molecules
with ultraviolet and visible lights.
Absorption of photon results in electronic
transition of a molecule, and electrons are
promoted from ground state to higher
electronic states.
Instrumentation of UV-Visible spectrophotometer

The instrument used to record the spectra of

molecules is called a spectrometer. The
sophisticated double beam recording UV-Visible
spectrophotometer covers the entire wavelength
range of 190 -800 nm. The basic components are
1. Source of radiation
2. Monochromator
3. Sample cell
4. Detector
5. Display/ Recorder
Fig. 2.1 The block diagram of ultraviolet and visible
spectrophotometer
 Ultraviolet and visible spectrophotometer
1. Radiation source: Hydrogen discharge lamp or deuterium lamp is
used as UV radiation source. For visible light, tungsten filament
lamp is used.
2. Monochromator: It disperses the polychromatic radiation from
the source to a narrow range of wavelength. For UV and visible
light, quartz prism or a grating is used. Two types of prisms,
namely 60o Cornu quartz prism and 30o Littro prisms are
employed. For visible light, a glass prism can be used.
3. Sample holder (cells or cuvettes): Sample containers should be
transparent to UV and visible radiation. Cuvettes made of quartz
are used for both UV and Visible region, whereas for visible
light, glass cuvettes are used. Standard path length of these
cuvettes is usually 1 cm.
4. Sector mirror: The monochromatic beam of radiation is split into
two parallel beams by the sector mirrors which pass through the
sample and reference cells and reach the detector.
 Ultraviolet and visible spectrophotometer
5. Solvents for UV region: Electronic absorption spectra are
usually recorded for solutions. Solvent used should absorb in
the same region as the solute. Solvents used in the UV and
visible region are water, methyl alcohol, ethyl alcohol,
chloroform, hexane, etc. 95% ethyl alcohol is the most
widely used solvent in UV region since it is a polar solvent,
cheaper and transparent up to 210 nm.
6. Detectors: Photovoltaic cells or photo emissive cells or the
more sensitive photomultiplier tubes are used to convert the
incident photons into electric current.
7. Display/Recorder: The wavelength drive of the recorder and
display unit are synchronized so that the detector signal
converted into the transmittance or absorbance units is
recorded as a function of wavelength of the incident beam of
radiation.
 Working Principle
In UV-visible spectrometer, a beam of light is split into two
equal halves.
One half of the beam (sample beam) is directed towards the
sample cell containing the solution of the compound being
analyzed and the other half (reference beam) through the
reference cell that contains only the solvent.
The instrument is so designed that it can compare the
intensities of both the beams at each wavelength of the
region 190-800 nm.
If the compound absorbs light at a particular wavelength, the
intensity of the sample beam, I will be less than the intensity
of the reference beam Io. An output graph, which is a plot of
the wavelength (λ) versus the absorbance (A) at each
wavelength obtained, is known as absorption spectrum.
 Characteristics of UV and Visible spectra
1. λmax value is the wavelength at which absorption
maximum occurs and is different for different
molecules.
2. ε value (molar absorptivity) for a given
concentration of the compound is related to the
height of the absorption band.
The λmax and ε value depend upon the concentration
and structure of the molecule and therefore used in
characterization and in quantitative estimation of a
compound. Unsaturated groups having n or 𝜋
electrons are essentially responsible for absorption
and these fragments are known as chromophores.
 Characteristics of UV and Visible spectra

Simple chromophores such as C=C, C≡C, C≡N,

N=N, C=O undergo n-π*transitions in the short
wavelength regions of UV light.
Saturated groups containing hetero atoms which
modify the absorption of the chromophores are
called auxochromes - e.g. -OH, -Cl,-OR, NR2, etc.
UV visible spectrum of benzene in hexane is shown
below.
Types of electronic transitions
Three types of electrons are involved in organic molecules
a) σ-electrons: Electrons forming sigma bond.
b) π–electrons: Electrons responsible for double and triple
bond.
c) n-electrons: These are unshared or non-bonded electrons.
Fig.2.1. Electronic absorption spectrum of a solution of benzene in hexane
(λmax = 225 nm)
 Applications of UV-Visible Spectrophotometry
a) UV-Visible spectra aids in the identification of unknown organic
samples.

Observation Possible conclusion

Molecules contain only σ bonds or lone pairs or
isolated double bonds. E.g. CH2=CH2 (λmax = 180 nm)

Presence of conjugated double bonds is indicated by

Absorption an increase in λmax
below 200 nm
E.g. butadiene (CH2 = CH-CH = CH2) absorbs at 210
nm. Long chain conjugated molecules such as
polyenes, carotenes, etc absorb in the visible region
with very highε value.
 Observation Possible conclusion
Strong absorption Presence of aromatic ring. E.g. benzene (λmax=
between 200 and 250 nm)
250 nm (ε=1000)
Weak absorption Carbonyl compound (containing C=O)
near 300 nm

(b) Purity check: ε value is used in the identification of the

substance. The magnitude of ε value depends on the chemical nature
of the absorbing substance and the wavelength of incident light
(λ).The purity of the sample can be checked by comparing the ε
values of the test sample with the standard sample. Deviations show
the presence of impurity or adulteration in the test sample.
 Applications of UV-Visible Spectrophotometry

2. Quantitative analysis -Many organic compounds and inorganic

complexes may be determined by direct absorbance
measurement values using the Beer Lambert’s law.

A plot of Absorbance (A) vs. C the concentration gives a linear plot.

3. Determination of dissociation constants of weak acids and bases
from the change in absorption spectra with pH.
4. Study of kinetics of chemical reactions.
5. Study of electronic structure of molecules such as vitamins,
detecting steric hindrance, etc.
 Applications of UV-Visible Spectrophotometry
1. Qualitative analysis: For characterizing aromatic
compound, conjugated dienes and dyes by comparing the
spectra (Hartley’s Rule).
2. Detection of impurities: It is one of the best method for
detecting impurities in organic solvents. Example
benzene is the most common impurity in cyclohexane.
Benzene can be detected by its absorption band at 255 nm.
3. Quantitative analysis: Determination of unknown
concentrations. Basis is Beer-Lamberts law.
4. Study kinetics of chemical reaction: Fix the wavelength
of ether reactant or product and measure absorbance at
different time intervals. Plot absorbance vs time. Slope
contains information about rate constant.
 Lambert’s law
When a monochromatic beam of radiation passes
through an absorbing medium, the intensity of the
transmitted radiation decreases exponentially with
the thickness of the absorbing medium. The law is
expressed as
It = Io10 –kx
It and Io are the intensities of the transmitted and
incident beams of radiations, x is the thickness of
the absorbing medium and k is a constant.
 Beer’s law

When a monochromatic beam of radiation passes

through an absorbing medium, the intensity of the
transmitted radiation decreases exponentially
with the concentration of the absorbing substance.
The law is expressed as
It = Io10 – k’C
where C is the molar concentration of the
absorbing substance and k’ is another constant
 Beer Lamberts Law
When a monochromatic light or radiation is
passed through a transparent absorbing medium,
the absorbance (decrease in the intensity of
radiation) is proportional to product of
concentration and thickness the absorbing
medium.

A= ε c t

[ε is molar absorptivity, c is concentration and t

is thickness of solution.
 Beer Lamberts Law Derivation
Consider a parallel beam of monochromatic
electromagnetic radiation of intensity I o, is passed
through an absorbing solution of thickness x and
concentration c .
 Beer Lamberts Law Derivation
The rate of decrease in intensity –d I of radiation with thickness of
solution dx is proportional to intensity of incident light I at that
point, concentration of solution and dx.
 Beer Lamberts Law

According to beer lamberts law absorbance increases when

one increases concentrations and/or path length (solution
thickness).
 Limitations of Beer –Lambert’s law
Beer-Lambert’s law is strictly valid only in
dilute solutions. For dilute solutions, a linear
relationship is exhibited by a plot of absorbance
(A) as a function of concentration of the
absorbing substance (C), as shown in Fig.
 Limitations of Beer –Lambert’s law
i. Real deviations occur at higher concentration of the
absorbing species. At higher concentrations (>10 -
3
M), there is a change in the refractive index of the
solution.
ii. Chemical deviations occur when there is more than
one absorbing species present in the solution. When
the absorbing molecules associate or dissociate in the
solution, there is a change in the number of
absorbing species.
iii. Instrumental deviation occurs due to changes in
absorptivity of the species as a function of
instrumental bandwidth.
 UV-Visible Spectroscopy
Ultraviolet visible spectroscopy or UV-vis
spectroscopy involves the measurement of absorption
or transmittance of energy in the ultraviolet and visible
regions of the electromagnetic spectrum. It is primarily
used to measure the multiple chemical bonding in
organic compounds and colour of transition metals.
A diagram of the components of a typical spectrometer
are shown in the following diagram. The functioning of
this instrument is relatively straightforward.
UV-Visible Spectroscopy
 UV-Visible Spectroscopy working principle

A beam of light from a visible and/or UV light source

(colored red) is separated into its component
wavelengths by a prism or diffraction grating.
Each monochromatic (single wavelength) beam in turn
is split into two equal intensity beams by a half-
mirrored device.
One beam, the sample beam (colored magenta), passes
through a small transparent container (cuvette)
containing a solution of the compound being studied in
a transparent solvent.
 Working principle
The other beam, the reference (colored blue), passes
through an identical cuvette containing only the solvent.
The intensities of these light beams are then measured by
electronic detectors and compared.
The intensity of the reference beam, which should have
suffered little or no light absorption, is defined as I0. The
intensity of the sample beam is defined as I. Over a short
period of time, the spectrometer automatically scans all the
component wavelengths in the manner described. The
ultraviolet (UV) region scanned is normally from 200 to
400 nm, and the visible portion is from 400 to 800 nm.
 WOODWARD- FIESER RULES

Woodward–Fieser rules are several sets of

empirically derived rules which attempt to predict
the wavelength of the absorption maximum (λmax)
in an ultraviolet-visible spectrum of a given
compound.
Inputs used in the calculation are based on the type
of chromophores present, the auxochromes
(substituents on the chromophores) present
and the solvent used.
 WOODWARD- FIESER RULES

According to Woodward’s rules the λmax of the

molecule can be calculated using a formula:
λmax= Base value + Σ Substituent Contributions +
Σ Other Contributions
Woodward-Fieser rules can be used to calculate
the λmax of
1. Conjugated Dienes and Polyenes
2. Conjugated Carbonyl Compounds
 Steps involved in calculation of λmax
Each type of diene or triene or α, β-unsaturated aldehydes or
ketones have a certain fixed value where absorption takes place-
Base value
The contribution made by various alkyl substituents or ring residues
or double bond extension conjugation or polar groups such as Br, Cl
etc are added to base value to obtain the λmax for a particular
compound.
• λmax = Base value + ΣSubstituent contribution + ΣOther
contribution
There are three sets of rules
• 1. Woodword – Fieser rule for conjugated dienes and polyenes
• 2. Woodword – Fieser rule for carbonyl compounds
• 3. Woodword – Fieser rule for aromatic compounds
 Wood ward Fieser rule for conjugated
diene and polyenes
A. Homo annular diene : cyclic diene having
conjugated double bond in same ring.
B. Hetero annular diene : cyclic diene having
conjugated double bond in different ring.
C. Endo cyclic double bond : double bond
present in a ring.
D. Exocyclic double bond : double bond in which
one of the double bond atom is a part of a ring.
E. Double bond extending : when more double
bond are present other than conjugated.
 α, β UNSATURATED CARBONYL COMPOUNDS OR
KETONES
1. Base value:
a) Acyclic α, β unsaturated ketones = 214 nm
b)6 membered cyclic α, β unsaturated ketones = 215 nm
c) 5 membered cyclic α, β unsaturated ketones = 202 nm
d)α, β unsaturated aldehydes = 210 nm
e) α, β unsaturated carboxylic acids & esters = 195 nm
2. Alkyl substituent or Ring residue in α position = 10 nm
3. Alkyl substituent or Ring residue in β position = 12 nm
4. Alkyl substituent or Ring residue in γ and higher positions = 18
nm
5. Double bond extending conjugation = 30 nm
 α, β UNSATURATED CARBONYL COMPOUNDS
OR KETONES
7. Homodiene compound = 39 nm
8. Polar groups:
a) –OH in α position = 35 nm –OH in β position = 30 nm
–OH in δ position = 50 nm
b) –OAc in α, β, γ, δ positions = 6 nm
c) –OMe in α position = 35 nm
–OMe in β position = 30 nm
–OMe in γ position = 17 nm
–OMe in δ position = 31 nm
d) –Cl in α position = 15 nm
–Cl in β position = 12 nm
e) –Br in α position = 25 nm
–Br in β position = 30 nm
f) –NR2 in β position = 95 nm
 AROMATIC COMPOUNDS

1) Base value: for

a) ArCOR = 246 nm
b) ArCHO = 250 nm
c) ArCO2H = 230 nm
d) ArCO2R = 230 nm
2) Alkyl group or ring residue in ortho and meta
position = 3 nm
3) Alkyl group or ring residue in para position =10
nm
 Aromatic Compounds
4) Polar groups:
a) –OH, –OCH3, –O Alkyl in o, m position = 7 nm
b) –OH, –OCH3, –O Alkyl p position = 25 nm
c) –O (oxonium) in o position = 11 nm
d) –O (oxonium) in m position = 20 nm
e) –O (oxonium) in p position = 78 nm
f) –Cl in o, m position = 0 nm
g) –Cl in p position = 10 nm
h) –Br in o, m position = 2 nm
i) –Br in p position = 15 nm
j) –NH2 in o, m position = 13 nm
k) –NH2 in p position = 58 nm
l) –NHCOCH3 in o, m position = 20 nm
m) –NHCOCH3 in p position = 45 nm
n) –NHCH3 in p position = 73 nm
o) –N(CH3)2 in o, m position = 20 nm
p) –N(CH3)2 in p position = 85 nm
 Significant applications of UV spectroscopy to
organic chemistry
 Extent of conjugation
 Distinction between conjugated and Non-conjugated compounds
 Detection of a chromophore in an unknown compound
 Identification of a chromophore (functional group)
 Study of Geometric Isomerism
 Effect of Geometric Isomerism and Steric Effects
 Study of Tautomerism
 Study of Structural features in different solvents
 As an analytical tool
 Effect of S-Cis (cisoid) and S-Trans (transoid) conformations
 Effect of Alkyl Substitution and Ring residues
 Effect of Exocyclic double bonds
 Extent of conjugation
The longer the conjugation, longer the
absorption maximum λmax
Isomeric conjugated dienes, conjugated trienes,
conjugated tetraenes etc. show appropriate
increase in λmax
Sufficient conjugation shifts the absorption
maximum λmax to visible region and the
compound will be coloured. Eg. Lycopene-
compound with 11 conjugated bonds gives red
colour to tomatoes
 Distinction between conjugated and Non-
conjugated compounds
 Conjugated compounds show λmax at longer
wavelength whereas non conjugated
compounds shows λmax at shorter wavelength
 UV spectroscopy can differentiate conjugated
dienes from non-conjugated dienes, conjugated
dienes from conjugated trienes, α,β-unsaturated
ketones from their β,γ-analogs, etc
UV-Vis spectrum of Lycopene
 Woodward-Fieser Rule for conjugated dienes
or polyenes
1. Acyclic dienes
An acyclic diene exists mostly in s-trans (transoid)
conformation as shown in the case of butadiene. As acyclic
diene can rotate about their single bond to give either
cisoid or transoid, therefore they exist in trans-form due to
the high stability of the later. When this diene becomes a
part of a ring system as in 1,3 cyclohexadiene, it is forced
into a cisoid configuration. The wavelength of absorption is
also shifted to longer wavelength and intensity is lowered
in comparison with acyclic dienes. The absorption maxima
for 1,3 cyclohexadiene is 256 nm, whereas 1,3-butadiene
shows λmax at 217 nm.
Acyclic dienes

cyclic dienes
 Cyclic dienes
Cyclic dienes can be classified into two classes depending upon
whether the double bonds are in the same ring or in different rings.
a) Homo-annular conjugated dienes: Both conjugated double
bonds are in the same ring and are having s-cis (cisoid)
configuration. The s-cis configuration causes strain which raises the
energy of ground state, while the energy of the excited state is
relatively unchanged. Thus the transition energy is lowered resulting
absorption at longer wavelength.
b) Hetero-annular dienes: Conjugated double bonds are present
in different rings and have Strans configuration.
The homo-annular diene resembles with 1,3 cyclohexadiene, whereas
hetero-annular diene with butadiene.
Endocyclic double bond: Double bond/s is
present inside the ring.

Exocyclic double bond: Double bond/s

projecting outside the ring is called as exocyclic
double bonds.
Exocyclic double bond
In example II, the double bond present within ring B is
exocyclic to ring A, whereas endocyclic to ring B.
In example III, double bond present in ring A is exocyclic to
ring B, similarly double bond present in ring B is exocyclic to
ring A.
In example IV, both double bonds are present within ring B
making them endocyclic to ring B, while both the double
bonds are exocyclic to ring A.
In example V, the double bond present in the ring B is
exocyclic to ring A and ring C. Similarly the double bond
present in the ring A is exocyclic to ring B.
In example VI, the double bond is exocyclic to two different
rings at a time. In such cases, the contribution would be 2
times.
Nickel (II) ion detection
Nickel (II) ion detection
 Terms describing UV absorptions
1. Chromophores: functional groups that give
electronic transitions.
2. Auxochromes: substituents with unshared pair e's
like OH, NH, SH ..., when attached to π
3. chromophore they generally move the absorption
max. to longer λ.
4. Bathochromic shift: shift to longer λ, also called red
shift.
5. Hysochromic shift: shift to shorter λ, also called blue
shift.
6. Hyperchromism: increase in ε of a band.
7. Hypochromism: decrease in ε of a band.
UV absorptions
UV absorption of Isoprene
 Introduction to nitrite
Nitrite is one of the pollutants found in the atmosphere and
natural water and is an important intermediate in biological
nitrogen cycle.
Traces of nitrite and nitrate in drinking water may lead to
mathemeglobmenia in infants and with long term exposure is a
possible cancer risk.
Various instrumental methods such as polarography,
voltammetry, fluorimetry, biamperometry and flow
injection spectrophotometry have been used for nitrite
determination.
Nitrite is determined spectrophotometrically based on diazo
coupling reaction extraction of the azo dye into suitable
organic solvent provides a much lower detection limit and
improved sensitivity
 Determination to nitrite
This method is based on the reactions involving
sulfanilic acid with methyl anthranilate as the coupling
agents.
It is based on their reduction to nitrite in the presence of
Zn/NaCl.
The produced nitrite is subsequently diazotized with
sulfanilic acid then coupled with methyl anthranilate to
form an azo dye which is measured at 493 nm.
The method is optimized for acidity, amount of reagents
required and tolerance amount of other ions.
 Determination to nitrite
The range of linearity for sulfanilic acid-methyl
anthranilate couple was found to be 0.2-8.0 µg/mL of
nitrite with molar absorptivity be 1.03x10 4 Lmol-1cm-1
and sandell’s sensitivity 4.5x10-3 µg cm-2 .
The detection limit and quantitation limit of the nitrite
determination are found to be 0.93 µg/mL and 2.82
µg/mL respectively.
This method has been successfully applied to the
determination of trace amounts of nitrite and nitrate in
water, soil and pharmaceutical preparations
Determination to nitrite
Fig.. (a) Absorption spectra of azo dye; sulfanilic acid -methyl
anthranilate couple and (b) reagent blank vs distilled water
Fig. Adherance to beer’s law for the determination of nitrite
using methyl anthranilate as a reagent
 Photometric titration
A photometric titration curve is a plot of absorbance,
corrected for volume changes, as a function of the volume
of titrant. If the conditions are properly chosen the curve
will consists of two linear regions of different slopes. One
region occurs at the start of the titration and the other is
well after the equivalence point. The end point is the
volume of titrant corresponding to the point where the two
linear regions intersect. A few examples of photometric
titration curves are shown in Figure 9.5.1
where A indicates the analyte, P indicates the product
and T indicates the titration for the reaction A + T --> P.
Photometric titration