Molecular Spectroscopic

Methods-I UNIT 2 UV-VISIBLE SPECTROMETRY

Structure
2.1 Introduction
Objectives
2.2 Origin and Characteristics of UV-VIS Spectrum
Origin of UV-VIS spectrum
Characteristics of UV-VIS Spectrum
Absorbing Species
2.3 Principle of UV-VIS Spectrometry
Lambert’s Law
Beer’s Law
Deviations from Beer-Lambert’s Law
2.4 Instrumentation for UV-VIS Spectrometry
Radiation Sources
Wavelength Selectors
Monochromators
Sample Handling
Detectors
Signal Processing and Output Devices
2.5 Types of UV-Visible Spectrometers
Single Beam Spectrometers
Double Beam Spectrometers
Photodiode Array Spectrometer
2.6 Analytical Applications of UV-Visible Spectrometry
Qualitative Applications
Quantitative Applications
Quantitative Determination Methodology
Simultaneous Determination
2.7 Summary
2.8 Terminal Questions
2.9 Answers

2.1 INTRODUCTION
In the previous unit you have learnt about the electromagnetic radiation in terms of its
nature, characteristics and properties. You also learnt that the interaction of radiation
with matter brings about transitions among some of the quantised energy levels of the
matter and study of such an interaction is called spectroscopy. Further, the interaction
of radiation with matter may be manifested in terms of its selective absorption or
absorption followed by emission or scattering.

In this unit you would learn about the ultraviolet-visible (UV-VIS) spectrometry. This
concerns the measurement of the consequences of interaction of EM radiations in the
UV and/or visible region with the absorbing species like, atoms, molecules or ions. In
such determinations the extent to which radiation energy is absorbed by a chemical
system as a function of wavelength, as well as, the absorption at a fixed predetermined
wavelength of the radiation is measured. Since such measurements need an instrument
called spectrometer the technique is known as UV-VIS spectrometry.

The UV-VIS spectrometry is one of the oldest instrumental techniques of analysis and
is the basis for a number of ideal methods for the determination of micro and semi-
micro quantities of analytes in a sample. We will begin by understanding the theory
behind UV-VIS spectrometry in terms of the origin and the features of the UV-VIS
spectrum. Then we will discuss the fundamental principles that form the basis of the
analytical applications of UV-VIS spectrometry. It will be followed by an account of
the essential components of a UV-VIS spectrometer and their functions. Thereafter,

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we will discuss the qualitative and quantitative applications of UV-VIS spectrometry UV-Visible
in diverse areas. In the next unit you will learn about the study of infra- red Spectrometry
spectrometry which concerns the interaction of electromagnetic radiation in the
infrared region with matter.

Objectives
After studying this unit, you should be able to:
• explain the origin and the characteristics of UV-VIS spectrum,
• outline different types of species absorbing in the UV-VIS region,
• explain the Lambert’s and Beer’s laws,
• explain the factors leading to deviation from Beer-Lambert’s law,
• describe the components of UV-VIS instruments,
• state the principles of different types of UV-VIS instruments,
• elaborate the methodology of quantitative applications of UV-VIS spectrometry,
and
• state different applications of UV-VIS spectrometry.

2.2 ORIGIN AND CHARACTERISTICS OF UV-VIS

SPECTRUM
UV-VIS spectrum results from the interaction of electromagnetic radiation in the UV-
VIS region with molecules, ions or complexes. It forms the basis of analysis of
different substances such as, inorganic, organic and biochemicals. These
determinations find applications in research, industry, clinical laboratories and in the
chemical analysis of environmental samples. It is therefore important to learn about
the origin of the UV-VIS spectrum and its characteristics. Let us try to understand the
origin of the UV-VIS spectrum.

2.2.1 Origin of UV-VIS Spectrum

The absorption of radiation in the UV-VIS region of the spectrum is dependent on the
electronic structure of the absorbing species like, atoms, molecules, ions or complexes.
You have learnt in Unit 1 that the absorption spectrum of atomic species consists of a
number of narrow lines that arise as a consequence of the transition amongst the
atomic energy levels. You would also recall that in molecules, the electronic,
vibrational as well as the rotational energies are quantised. A given electronic energy
level has a number of vibrational energy levels in it and each of the vibrational
energy level has a number of rotational energy levels in it. When a photon of a given
wavelength interacts with the molecule it may cause a transition amongst the
electronic energy levels if its energy matches with the difference in the energies of
these levels.
The absorption of
In the course of such transitions, for the sample in gaseous or vapour phase, the radiation in the UV-VIS
spectrum consists of a number of closely spaced lines (Fig 2.1(a)), constituting what is region of the spectrum
called a band spectrum. However, in the solution phase, the absorbing species are causes the transitions
surrounded by solvent molecules and undergo constant collisions with them. These amongst the electronic
collisions and the interactions among the absorbing species and the solvent molecules energy levels.
cause the energies of the quantum states to spread out. As a consequence, the sample
absorbs photons spread over a range of wavelength. Thereby, the spectrum acquires
the shape of a smooth and continuous absorption peak in the solution phase. A typical
UV-VIS spectrum in the solution phase is depicted in Fig. 2.1 (b).

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Molecular Spectroscopic
Methods-I

Fig. 2.1: Representative UV spectrum: a) in vapour phase and b) in solution phase

2.2.2 Characteristics of UV-VIS Spectrum

In order to obtain a UV-VIS spectrum the sample is ideally irradiated with the
electromagnetic radiation varied over a range of wavelength. A monochromatic
radiation i.e., a radiation of a single wavelength is employed at a time. This process is
called scanning. The amount of the radiation absorbed at each wavelength is
measured and plotted against the wavelength to obtain the spectrum. Thus, a typical
UV spectrum is a plot of wavelength or frequency versus the intensity of absorption.

Have a look again at Fig. 2.1 (b). The abscissa (x-axis) indicates the wavelengths
absorbed and therefore, is marked in wavelength though sometimes frequency may
also be used. The ordinate (y-axis) on the other hand represents the intensity of
absorption and is generally represented in terms of absorbance (explained in subsec.
2.2.3). The UV spectra of substances are characterised by two major parameters,
namely, the position of the maximum of the absorption band called λmax, and the
intensity of the bands. The λmax refers to the wavelength of the most absorbed
radiation and is a measure of the difference in the electronic energy levels involved in
the transition. The intensity on the other hand is indicative of the probability of the
transition i.e., whether the transition is allowed or not. It is also is a measure of the
concentration of the absorbing species. The relationship between the intensity of
absorption and the concentration is explained in Section 2.3.

Analyte: The chemical

The characteristics of UV-VIS spectrum depend on the structure and concentration of
species which is to be the absorbing species in solution. Therefore, these spectra are extensively used in the
determined in the sample characterisation and in the quantitative estimations of the analyte.
under investigation.
2.2.3 Absorbing Species
As discussed above, the UV-VIS spectra arise due to electronic transitions in the
absorbing species. The electronic transitions do not occur in all the species when a
radiation in UV region is passed through the sample. Generally three types of species
show the mentioned transitions, these are organic, inorganic and complexes formed by
change transfer. Let us learn about these species and the nature of the transitions
involved in them.

Organic Molecules
The molecular absorptions in the UV-VIS region depend on the electronic structure of
the molecule. The wavelength of the radiation absorbed by an organic molecule is
determined by the difference in energy between the ground state and the various
excited electronic states of the molecule. You would recall from your earlier

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knowledge of bonding in organic molecules that the constituent atoms are bonded UV-Visible
through σ and π bonds. In addition, these have nonbonding electrons on the atoms Spectrometry
like, N,O,S and halogens etc. There are a number of transitions possible involving the
bonding and the nonbonding electrons. The generalised molecular orbital energy level
diagram and possible transitions for organic compounds is given in Fig. 2.2.

Fig. 2.2: Generalised molecular orbital energy level diagram and possible transitions for
organic compounds

As a rule, the transitions occur from the highest occupied molecular orbital (HOMO)
to the lowest unoccupied molecular orbital (LUMO) in a molecule. In most of the
organic compounds, the bonding and sometimes the nonbonding orbitals also are filled
and the antibonding orbitals are empty. Of the six possible transitions indicated in the
figure, only the two of the lowest energy ones ( n → π ∗ and π → π ∗ ) can be achieved
by the energies available in the 200 to 800 nm region. Let us learn about these two in
the following paragraph.

π → π ∗ transitions: These transitions are observed in molecules containing

π = electrons and occur at wavelengths approaching near ultraviolet regions The
hydrocarbons containing double and triple bonds are typical examples. For example,
ethylene absorbs at 170 nm. The conjugation of unsaturation further increases λ max;
but-1, 3-diene absorbs at 217 nm. The effect of conjugation is quite important in
aromatic molecules. For example single ring aromatics absorb in the vicinity of
250 nm, naphthalene in the vicinity of 300 nm and anthracene in the vicinity of
360 nm.

n → π ∗ transitions: These transitions are observed in molecules containing lone pairs

or nonbonding electrons. In such transitions one of the nonbonding electrons may be
excited into an empty π ∗ orbital. The energies required for these transitions are lower
than that for π → π ∗ transitions and result in the absorption in the ultraviolet and
visible region. The presence of atoms or groups containing n = electrons, can cause
remarkable changes in the spectrum. Thus, nitrogen, sulphur and halogens tend to
move absorption to higher wavelengths.

The π → π ∗ transitions are generally intense while the n → π ∗ transitions are weak.
For example, acetone, exhibits a high intensity π → π ∗ transition at 195 nm and a low
intensity n → π ∗ transition at 274 nm in its absorption spectrum (Fig 2.3).

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Molecular Spectroscopic
Methods-I

Fig. 2.3: UV spectrum of acetone showing the π → π ∗ and n → π ∗ transitions

Thus, only those molecules are likely to absorb light in the 200 to 800 nm region
which contain π-electrons and may also have atoms with non-bonding electrons. Such
groups that absorb light in the UV-VIS region are referred to as chromophores.
Chromophore in Greek means, “colour bearing”. However, the term chromophore is
now not limited to colours only but is used in a general way for groups which are
responsible for causing an absorption of electromagnetic radiation between about 175
to 800 nm. A list of some simple chromophoric groups and their light absorption
characteristics is given in Table 2.1.

Table 2.1: Absorption characteristics of some common chromophoric groups

Chromophore Example λmax Type of Transition

>C = C<
< C6H13 CH = CH2 177 π →π*

−C ≡ C−
− C5H11 C ≡ C − CH3 178 π →π*

>C = Ο CH3CO CH3 186 n → σ*

280 n→π*

CH3CHO 180 n → σ*
293 n→π*

−COΟ
ΟH CH3COOH 204 n→π*

−CONH2 CH3CONH2 214 n→π*

−N = N−
− CH3N = NCH3 339 n→π*

−NO2 CH3NO2 280 n→π*

−N = Ο C4H9 NO 300 -
665 n→π*

Inorganic Species
A large number of inorganic salts containing atoms with electrons in d-orbitals give
weak absorption bands in the visible range. The ions and the complexes of the
elements of the first two transition series belong to this group and are coloured. The
colour of these species is due to transitions amongst d-orbitals. The complex formation
of these ions with solvent molecules or with other ligands lifts the degeneracy of the
five d orbitals. As a consequence, these split into groups having different energies.
The electronic transitions from the lower energy d orbitals to higher energy d orbitals
are responsible for the observed colour. These transitions are called d-d transitions.

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The blue colour of the aqueous solutions of copper sulphate and the violet colour of UV-Visible
potassium permanganate are some of the examples. Spectrometry

The ions of the lanthanides and actinides are also coloured, however, these involve
f-f transitions. The nature of spectrum in case of these ions is different because the
f-electrons are relatively less affected by external influences due to shielding by the
occupied orbitals of higher principal quantum number. These ions absorb the radiation
of ultraviolet and visible region in narrow bands. The representative visible spectra of
the ions of transition and inner transition elements are given in Fig. 2.4.

Fig. 2.4: Representative absorption spectra for the a) transition and b) inner
transition element ions

Charge Transfer Complexes

Many a times a given compound that is transparent in the UV region starts absorbing
after interacting with another species. This happens if one of the species has an
electron donor group and the interacting species has an electron acceptor group.
When the two species bind to each other, the resulting species is intensely coloured.
This is due to the formation of a complex between the two species. Such a complex is
called charge transfer complex. For example, the blood red color of the complex ion,
thiocyanatoiron (III) ion, Fe (SCN) 2+ is due to the formation of a charge transfer
complex.

Fig. 2.5: The charge transfer spectrum of the complex ion, Fe (SCN)2+
The radiation absorbed by this product or complex causes the transfer of an electron
from an orbital on the donor to an orbital of the acceptor. This has been schematically
illustrated in Fig. 2.6. The transitions in the donor and the acceptor species
individually are of high energy and absorb outside the UV-VIS region. However, in
the complex, the energies of the orbitals are such that the HOMO to LUMO transition
is of much lower energy and falls in the visible region.

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Molecular Spectroscopic
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Fig. 2.6: Schematic diagram showing the disposition of the molecular orbitals of the
donor, acceptor and the charge transfer complex species
A large number of chelating agents are used for the spot tests. The intense colour of
such metal chelates is often due to charge transfer transitions. They simply involve the
movement of electrons from the metal ion to the ligand, or vice-versa. In the complex
between thiocyanate (SCN ̶ ) and the Fe (III) ions, the absorption of photon transfers
an electron from the thiocyanate ion to Fe (III) ion. Besides metal ions, non-metals
and their ions also form coloured substances which produce characteristic spectra. For
example, the alkaloid brucine reacts with nitrate ion in concentrated sulphuric acid to
form a red compound which quickly turns yellow.

It was mentioned earlier that UV-VIS spectrometry can be used for qualitative and
quantitative determinations of the analytes. The quantitative determinations are based
on the relationship between the absorption of radiation and concentration of the
analyte. Let us learn about the principle of spectrometric determinations. Before that
why don’t you assess your understanding by answering the following self assessment
question?

SAQ 1
List different types of species that may absorb in the UV-VIS region of the
spectrum and indicate the type of transitions involved in them.
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...

2.3 PRINCIPLE OF UV-VIS SPECTROMETRY

In a typical absorption spectral measurement a monochromatic radiation is made to
fall on a sample taken in suitable container called cuvette. In such a situation a part of
the radiation is reflected, a part is absorbed, and a part is transmitted. The intensity of
original radiation, Po is equal to the sum of the intensities of reflected (Pr), absorbed
(Pa) and transmitted (Pt ) radiation.
The transmitted radiation Po = Pr + Pa + Pt
has information about the
nature and the amount of The effect of reflection can be compensated by passing equal intensities of beams
the absorbing species. through the solution and through the solvent contained in the same or similar container
and comparing the transmitted radiations. We can then write the above equation as
follows:
Po = Pa + Pt
The intensity of the transmitted light is measured and is found to depend on the
thickness of the absorbing medium and the concentration, besides the intensity of the
incident radiation. This dependence forms the basis of spectrometric determinations
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and is given in terms of two fundamental laws. One is Bouguer’s law or Lambert’s UV-Visible
law, which expresses the relationship between the light absorption capacity of the Spectrometry
sample and the thickness of the absorbing medium; and the other is Beer’s law, which
expresses the relationship between the light absorptive capacity of the sample and its
concentration. The two laws are combined together to give Beer-Lambert’s law.
Let us discuss these laws one by one.

2.3.1 Lambert’s Law

Lambert (1760) and Bouguer independently studied the decrease in the intensity of
radiation when it passes through a substance and made the following observations:
i) The amount of monochromatic light absorbed by a substance is proportional to
the intensity of the incident light i.e. the ratio of the intensity of the transmitted
and incident light is constant.
ii) The intensity of the transmitted light decreases exponentially when the thickness
of the substance, through which the light is passing, increases linearly.

These observations, called Lambert’s law can be translated into a mathematical

expression as described below.

Px Px-dPx
P0 P

dPx
b
b
Fig. 2.7: Illustration for establishing the Lambert’s Law
In Fig. 2.7, if Po represents the radiant power of incident light and P represents the
radiant power of transmitted light after passing through a slab of thickness b, consider
a small slab of thickness dx, then the change in power (dPx), is proportional to the
power of incident light (Px) multiplied by the change in thickness (dx) of the slab
through which the light is passed. That is,

dPx ∝ Pxdx
or dPx = ‒ k Pxdx … (2.1)

where k is the proportionality constant and the negative sign indicates that radiant
power decreases with absorption. Eq. 2.1 can be rearranged to Eq. 2.2.

dPx
= − kd x …(2.2)
Px

Integrating Eq. 2.2 within the limits of Po to P for intensity and 0 to b for the thickness
we get,

p b
dPx
∫ Px
p0
= − k ∫0 dx

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Molecular Spectroscopic P
Methods-I ln = − kb … (2.3)
Po

Eq. 2.3 is the mathematical expression for Bouguer-Lambert law or Lambert’s law.
Changing this equation to base 10 logarithms and rearranging we get,

Po k
log = b = k 'b … (2.4)
P 2.303

Note that the ratio P/Po has been inverted to remove the negative sign. Lambert’s law
applies to any homogeneous non-scattering medium, regardless of whether it is gas,
liquid, solid, or solution.

2.3.2 Beer’s Law

In 1852, Beer and Bernard independently studied the dependence of intensity of
transmitted light on the concentration of the solution. It was found that the relation
between intensity of the transmitted light and concentration was exactly the same as
found by Lambert for the intensity of the transmitted light and the thickness of the
absorbing medium. Mathematically, the Beer’s observations can be expressed as
follows.

Consider a monochromatic radiation beam of intensity Px traversing any thickness of

solution of a single absorbing substance of concentration c. If c is changed by a small
amount dc to c + dc, the change in transmitted power dPx is proportional to the
incident intensity Px and dc. We can express it as follows.

dPx ∝ Px dc
…(2.5)
dPx = − k " Px dc
where k″ is the proportionality constant and the negative sign indicates that radiant
power decreases with absorption. This equation can be rearranged to:

dPx
= − k " dc …(2.6)
Px
On integration within the limits of Po to P for intensity and 0 to c for concentration we
get

P c
d Px
∫
Po
Px
= − k " dc ∫
o

P
or ln = − k "c
Po
Po k"
or log 10 = c
P 2.303
… (2.7)
Eq. 2.7 is the mathematical expression for Beer’s law. The Lambert’s and Beer’s laws
are combined and are expressed as per Eq. 2.8.

Po
log = abc
P
… (2.8)
In this expression, ‘a’ is a constant (combining two constants k′, k″ and the numerical
factor) and is called absorptivity (earlier called extinction coefficient) whose value
depends on unit of concentration used and is a function of wavelength of the

38
monochromatic light used. The concentration is generally expressed in terms of grams UV-Visible
per dm3 and b in cm. Therefore, it has units of cm−1g−1 dm3. Spectrometry

However, if the concentration is expressed as mol dm‒3 and b in cm then it is called

molar absorptivity (earlier called molar extinction coefficient) and expressed as ε. Its
units are cm−1 mol−1dm3. The modified expression for the Beer-Lambert’s law
becomes,

P0
log = εbc … (2.9)
P
Absorbance is defined as
The term log Po/P is called absorbance and is represented as ‘A’. the logarithm of the ratio
of the intensities of the
P0 incident and transmitted
A = log radiations.
P

The expressions for Beer-Lambert’s law then becomes,

P0 Eq. 2.10: use ‘a’ if

log = A = abc or εbc ...(2.10) concentration is
P expressed in g/L and path
length in cm and ‘ε’ if the
The absorbance, A is related to another important term called transmittance which is concentration in mol dm-3
defined as the fraction of the incident radiation transmitted by the absorbing medium. and b in cm.
Mathematically,
P
Transmittance, T = … (2.11)
P0
It is generally expressed as a percentage and is expressed as,
P You may note here that
Percentage transmittance, %T = × 100% … (2.12) though transmittance and
P0 absorbance are equivalent
expressions which can be
It is related to absorbance as, A = − log T … (2.13) interconverted, it is the
absorbance which is
directly proportional to
In typical measurements the radiant power transmitted by a solution is measured and the absorbed fraction of
compared with that observed with solvent (also called a blank). The ratio of the radiation.
transmitted powers through solution and that through blank or solvent is called as
transmittance.

It is desirable that you get acquainted with the expressions of the Beer-Lambert’s law
and learn to use them. Let us take some examples illustrating the application of these
expressions.

Example 1
The molar absorptivity of a substance is 2.0 × 104 cm‒1 mol‒1 dm3. Calculate the
transmittance through a cuvette of path length 5.0 cm containing 2.0 × 10‒6 mol dm‒3
solution of the substance.

Solution
As per the Lambert Beer’s law,
Absorbance, A = ε c b
Given:
ε = 2.0 × 104 cm‒1 mol‒1 dm3, c = 2.0 × 10‒6 mol dm‒3 and b = 5.0 cm
Substituting the values we get,
A = 2.0 × 104 × 2.0 10‒6 × 5.0 = 0.2
=> log l/T = 0.2 ∴ A = log 1/T
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Molecular Spectroscopic Taking antilog on both sides, we get
Methods-I
l/T = 1.585
∴T = 0.63

Example 2
A solution containing 36.5 mg of potassium dichromate per 500 cm3 was taken in a
cuvette having a path length of 2 cm and its transmittance was measured at 455 nm. If
the percentage transmittance is found to be 12, calculate the molar absorptivity of
potassium dichromate.

Solution
The molarity of given potassium dichromate solution can be calculated as

1000 × 0.0365
= = 2.48 × 10 − 4 mol dm −3
500 × 294
(Mm of potassium dichromate = 294 g mol‒1)

12% transmittance means T = 0.12

∴ A = 1/0.12 = 0.921
∴ ε = A/cb = 0.921 / (2.48 ×10‒4 × 2) = 0.921/4.96 × 10‒4
∴ε = 1.86 × 103 cm‒ 1 mol‒1 dm3
From the Lambert-Beer’s law expression given above, absorbance ( A = log P0 / P ) is
a direct measure of the concentration of the analyte if the thickness of the absorbing
medium is kept constant. This proportionality is exploited in the determination of
analyte concentration. However, some factors challenge the validity of the Beer’s law
and need to be controlled to take up quantitative determinations. Let us learn about the
possible reasons for the deviation from Beer’s law. Why don’t you answer the
following SAQs before proceeding further?

SAQ 2
In a spectrophotometer set at the λmax of a sample the value of P0 (with the solvent)
was found to be 85.4 using cuvettes of 2.00 cm path length. The value of P for a
solution of the sample having a concentration of 1 × 104 M was measured in the same
cuvette and was found to be 20.3. Calculate the molar absorptivity of the sample.
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...

SAQ 3
A substance has a molar absorptivity of 14,000 cm-1 mol-1 dm3 at the wavelength of its
maximum absorption. Calculate the concentration of the substance whose solution in a
cuvette of path length 1 cm has an absorbance of 0.85.
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...

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2.3.3 Deviations from Beer-Lambert’s Law UV-Visible
Spectrometry
As per the Beer’s law discussed above, there is a direct proportionality between the
absorbance and concentration. A plot of absorbance versus concentration is expected
to be a straight line passing through origin. However, this is not always true; there are
certain limitations. The law does not hold for all species under every condition. Many
a times instead of a straight line, a curvature in the plot may be observed as shown in
Fig. 2.8. The upward curvature, curve (a), is known as positive deviation and the
downward curvature, curve(c), as negative deviation.

Fig. 2.8: Beer-Lambert law plots; the curvatures show deviations from the law
Some of the factors responsible for the deviation from Beer’s law are as follows.

Presence of Electrolytes
The presence of small amounts of colourless electrolytes which do not react
chemically with the coloured components does not affect the light absorption as a rule.
However, large amounts of electrolytes may affect the absorption spectrum
qualitatively as well as quantitatively. This is due to the physical interaction between
the ions of the electrolyte and the coloured ions or molecules. This interaction results
in a deformation of the later, thereby causing a change in its light absorption property.

Hydrogen Ion Concentration

There are a number of substances whose ionic state in solution is greatly influenced by
the presence of hydrogen ions. For example, the aqueous solution of potassium
dichromate involves the chromate ion-dichromate ion equilibrium as shown below:

2CrO 27 − + 2H +
Cr2 O 72− + H 2 O

Chromate ion Dichromate ion
( λ max,375 nm) ( λ max 350,450 nm)

The chromate ion has a single λ max at 375 nm whereas dichromate ion has two peaks in
the spectra; λmax at 350 and 450 nm. The position of equilibrium depends on the pH of
the solution and yellow colour of solution changes to orange on increasing the
concentration of hydrogen ions. Therefore, the results of the determination of
chromate ion ( CrO 72 − ) concentration will depend on the pH. Thus, it is imperative that
substances, whose colour is influenced by change in hydrogen ion concentration, must
be studied under the condition of same pH.

In some cases, two absorbing species are in equilibrium and have a common value of
absorptivity at a certain wavelength. For example, in case of bromothymol blue the

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Molecular Spectroscopic absorption spectra at different pH values are different. However, at wavelength of
Methods-I
501 nm, we see that all species have same molar absorptivity (see Fig. 2.9). Therefore,
no matter to what extent does one species change into the other, there is no change in
the total absorption. Such a wavelength is known as isosbestic point. At this
wavelength the Beer’s law holds, though the measurements have low sensitivity.
However, such wavelengths should be avoided for the quantitative work.

Fig. 2.9: The absorption spectra for bromothymol blue at different pH values showing
the isosbestic point at 501 nm

Complexation, Association or Dissociation

Some salts have a tendency to form complexes whose colours are different from those
of the simple compounds. For example, the colour of cobalt chloride changes from
pink to blue due to the following complex formation.

2 CoCl2
Co[CoCl4]
pink blue

The degree of complex formation increases with increase in concentration, therefore,

Beer’s law does not hold at high concentrations. Similar discrepancies are found when
the absorbing solute dissociates or associates in solution because the nature of the
species in solution depends on the concentration.

Non-monochromatic Nature of the Radiation

In order for the Beer’s law to hold, it is necessary that monochromatic light is used.
However, most spectrophotometers and all filter photometers, discussed later employ
a finite group of frequencies. The wider the bandwidth of radiation passed by the filter
or other dispersing devices, the greater will be the apparent deviation of a system from
adherence to Beer’s law.

Concentration of the Analyte

As per Beer and Lambert’s law, the plot of absorbance versus the concentration of
absorbing substance should be a straight line when ε and b are constant. The path
length can always be held constant but there are some factors which affect ε and it is
found that at high concentration, ε is not constant. Therefore, at higher concentrations
(>10‒3 mol dm‒3) there may be deviation from the law.

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Temperature UV-Visible
Spectrometry
The temperature is not considered as an important factor since ordinarily the
measurements are made at a constant temperature. However, changes in temperature
sometimes may shift ionic equilibrium and the absorptivity. For example, the colour of
acidic ferric chloride solution changes from yellow to reddish brown on heating due to
change in λmax and absorptivity.

SAQ 4

Why is it important to know about the possible reasons for deviation from
Beer-Lambert’s law?
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...

2.4 INSTRUMENTATION FOR UV-VIS

SPECTROMETRY
Today a wide range of instruments are available for making molecular absorption
measurements in the UV-visible range. These vary from simple and inexpensive
machines for routine work to highly sophisticated devices that can be used for
specialised work and of course the routine jobs also. However, the basic components
of these instruments remain the same. The five essential components of UV-VIS
instruments are as follows
• A stable radiation source
• Wavelength selector
• Sample holder
• Radiation detector or transducer , and
• Signal processing and output device

The general layout of the essential components in a simple absorption instrument is

given in Fig. 2.10.

Fig. 2.10: General layout of the essential components in a simple absorption instrument

Let us learn about these components one by one.

43
Molecular Spectroscopic 2.4.1 Radiation Sources
Methods-I
A spectrophotometric radiation source must provide a stable high energy output over a
broad range of wavelengths. There is no inexpensive source available that may
provide stable output over the entire UV-visible range (190 nm to 780 nm). The
radiation sources commonly used for the UV and visible range are described below.

Sources for UV Region

For measurements in the UV region, electric discharge sources like hydrogen or a
deuterium lamp are used. In these, the excitation of the gaseous molecules is brought
about by the passage of electrons through the gas at low pressures. A hydrogen lamp is
commonly used in the spectrophotometers and gives light in the wavelength region of
160-375 nm. The radiant power of the hydrogen lamp is low and these are replaced by
deuterium lamps but it increases the cost of the instrument.

Fig. 2.11: Radiation sources: a) deuterium lamp for UV range b) tungsten lamp for
visible range
Sources for Visible Region
In the beginning of development of instruments for colorimetric work, sunlight was
used as source for measurements in the visible range. The modern instruments use a
tungsten filament lamp as the radiation source. This consists of a thin, coiled tungsten
wire that is sealed in an evacuated glass bulb. This gives radiations in the range of
350-2200 nm. As the output depends on the voltage, the tungsten lamp is energised by
a 6 or 12 volt storage battery or by the output of a constant voltage transformer.

Now a days some instruments use tungsten-halogen lamps that contain a small amount
of iodine in the quartz bulb housing the tungsten filament. The presence of iodine
extends the output wavelength range of the lamp from 240 -2500 nm.

2.4.2 Wavelength Selectors

You would recall that a In spectrophotometric measurements we need to use a narrow band of wavelengths of
radiation consisting of a light. This enhances the selectivity and sensitivity of the instrument. Less expensive
single wavelength is instruments use a filter to isolate the radiant energy and provide a broad band of the
called a monochromatic wavelengths. In many applications we need to continuously vary the wavelength over
radiation. a defined range. This can be achieved by using monochromators. Most modern
instruments use monochromators that employ a prism or diffraction grating as the
dispersing medium. The selection and use of fillers and monochromators is described
below.

Absorption Filters
In low cost instruments catering to measurements in the visible range, coloured glass

44
filters are used to cut off undesirable wavelengths. A typical filter is a coloured piece UV-Visible
of glass, which absorbs light of certain wavelength and allows that of the other to pass Spectrometry
through. You know that white light is made up of seven different colours; the acronym
is VIBGYOR for violet, indigo, blue, green, yellow, orange and red. These seven
colours add up to give white light back. When white light falls on an object, a part of it
is absorbed and rest is transmitted. These transmitted components add up to give the
observed colour of the object. The absorbed component and the observed colour can
again add up to give white light. These are therefore called as complementary to each
other. An object of a particular colour looks of that colour because this colour is
transmitted and its complementary colour is absorbed.

In order to select a suitable filter for a measurement of absorbance of a solution, we

need to have an understanding of complementary colours. The colour of the filter
should be the complementary to colour of the solution to be measured. If a solution
appears orange, this implies that orange light is not being absorbed but that the
complement to orange i.e. blue is being absorbed. Therefore, for measuring
absorbance of a orange solution you would employ a blue filter. The use of such a
filter is illustrated in Fig. 2.12.

Orange
Orange
White light = +
Blue

Fig. 2.12: Use of an absorption filter for the measurement of absorbance

Since these filters work by absorbing part of the radiation incident on them therefore
they do not provide monochromatic light. In order to get a narrow band of
wavelengths we can use certain filters which actually contain two glass filters; one
filter absorbs strongly above a certain wavelength, while the second absorb strongly
below a certain wavelength.

In order to find the colour of the filter to be used we can take the help of colour wheel
shown in Fig. 2.13, or consult a table of complementary colours given in Table 2.2.

Fig. 2.13: The colour wheel

45
Molecular Spectroscopic In a colour wheel, the colours, which face one another, are said to be complementary
Methods-I
to each other. In order to measure a red coloured solution, its complementary, that is, a
green coloured filter should be used. In the colour wheel P, S and T refer to the
primary, secondary and tertiary colours, respectively.

Table 2.2: Complementary Colours Chart

Wavelength region
eliminated by Complementary colour of the
Colour absorbed
absorption (nm) residual light (as seen by the eye)

400-450 violet yellow-green

450-480 blue yellow
480-490 green-blue orange
490-500 blue-green red
500-560 green purple
560-580 yellow-green violet
580-600 yellow blue
600-650 orange green –blue
650-750 red blue –green
Absorption filters are simple and are totally adequate for many applications in visible
range. However, for extended ranges we need interference filters. The interference
filters cover a wider range than the absorption filters. Interference filters are
essentially composed of two transparent parallel films of silver, which are so close as
to produce interference effects. Such interference filters are available for ultraviolet,
visible and near- infrared region. The performance characteristics of interference
filters are significantly superior to those of absorption (coloured) filters. The effective
bandwidths of these filters are narrower than absorption filters.

2.4.3 Monochromators
As mentioned above, monochromators are devices that can selectively provide
radiation of a desired wavelength out of the range of wavelengths emitted by the
source. These are of two types; the prism and grating monochromators. These are
described in the following paragraphs.
Glass prisms and lenses
can be used in the visible Prism Monochromators
region. However, since
glass absorbs ultraviolet You know that a prism disperses sunlight into seven different colours. This occurs due
light, a quartz or fused to the refraction of the light when it passes through the prism. The radiations of
silica is a better choice for different colours having different wavelengths are refracted to different extent due to
the material of the prism the difference in the refractive index of glass for different wavelengths. Shorter
because it can be used in wavelengths are refracted more than longer wavelengths as depicted in Fig. 2.14 (a).
both the regions.

(a)
Fig. 2.14: a) Dispersion of radiation by prism

46
 UV-Visible
Spectrometry

(b)
Fig. 2.14: b) Schematic diagram of the prism monochromator
If a prism is rotated, different wavelengths of the radiation, coming out after refracting
through it, can be made to pass through the exit slit. In a prism monochromator,
shown in Fig. 2.14 (b), a fine beam of the light from the source is obtained by passing
through an entrance slit. This is then collimated on the prism with the help of a lens.
The refracted beams are then focused on an exit slit. The prism is then rotated in a
predetermined way to provide the desired wavelength from the exit slit.

Grating Monochromators
A grating is made by cutting or etching a series of closely spaced parallel grooves on
the smooth reflective surface of a solid material as shown in Fig. 2.14 (a). The surface The gratings used for the
ultraviolet and visible
is made reflective by making a thin film of aluminium on it and the etching is done
region generally contain
with the help of a suitably shaped diamond tool. The intensity of radiation reflected by about 1200-1400
a grating varies with the wavelength, the wavelength of maximum intensity being grooves/mm.
dependent on the angle from which the radiation is reflected from the surface of the
line of the grating as shown in Fig. 2.15.

Fig. 2.15: Diffraction of Radiation by a grating

In grating monochromator (Fig. 2.16), a fine beam of the light from the source falls on
a concave mirror through an entrance slit. This is then reflected on the grating which
disperses it. The dispersed radiation is then directed to an exit slit. The range of
wavelengths isolated by the monochromator is determined by the extent of dispersion
by the grating and the width of the exit slit. Rotation of the grating in a predetermined
way can be used to obtain the desired wavelength from the exit slit.

47
Molecular Spectroscopic
Methods-I

Fig. 2.16: Schematic diagram of a grating monochromator

2.4.4 Sample Handling

The UV-VIS absorption spectra are usually determined either in vapour phase or in
solution. In order to take the UV spectrum of the analyte it is taken in a cell called a
cuvette which is transparent to the wavelength of light passing through it. A variety of
quartz cuvettes are available for the spectral determination in the vapour phase. These
are of varying path lengths and are equipped with inlet and outlets. For measurements
on solutions in the visible region the cuvettes made of glass can also be used.
However, since glass absorbs the ultraviolet radiations, these cannot be used in the UV
region. Therefore, most of the spectrophotometers employ quartz cuvettes (Fig 2.17),
as these can be used for both visible and UV region. Usually square cuvettes having
internal path length 1.0 cm are used, though cuvettes of much smaller path lengths say
of 0.1 mm or 0.05 mm are also available.

The eye of a common

person is quite sensitive Fig. 2.17: Quartz cuvettes for measurements in solution and in vapour phase
to notice differences in
radiant power transmitted The sample whose spectrum is to be measured is dissolved in a solvent that is
through two coloured transparent in the UV region. It means that it does not absorb in this region. Hexane,
solutions. We can say that 95% ethanol, methanol and 1,4 dioxane are commonly employed as solvents. Now a
eye is a natural days ‘spectral grade’ solvents are available which have high purity and have negligible
photosensitive detector in absorption in the region of absorption by the chromophore.
the visible range.
Therefore, early
In a typical measurement of a UV spectrum, the solution of the sample is taken in a
instruments used eye or
photographic plate as the suitable cuvette and the spectrum is run in the desired range of the wavelengths. The
detector. absorption by the solvent, if any, is compensated by running the spectrum for the
solvent alone in the same or identical cuvette and subtracting it from the spectrum of
the solution. This gives the spectrum only due to the absorption species under

48
investigation. In double beam spectrometers, the sample and the solvent are scanned UV-Visible
simultaneously (subsection 2.5.2). Spectrometry

2.4.5 Detectors
The detectors are used to convert a light signal to an electrical signal which can be
suitably measured and transformed into an output. The detectors used in most of the
instruments generate a signal, which is linear in transmittance i.e. they respond
linearly to radiant power falling on them. The transmittance values can be changed
logarithmically into absorbance units by an electrical or mechanical arrangement in
the signal read out device. There are three types of detectors which are used in modern
spectrophotometers. These are described in the following paragraphs.

1. Phototube
A phototube consists of a photoemissive cathode and an anode in an evacuated tube
with a quartz window as shown in Fig. 2.18 (a). These two electrodes are subjected to
high voltage (about 100 V) difference. When a photon enters the tube and strikes the
cathode, an electron is ejected and is attracted to the anode resulting in a flow of
current which can be amplified and measured. The response of the photoemissive
material is wavelength dependent and different phototubes are available for different
regions of the spectrum.

(a) (b)

Fig. 2.18: Detectors of UV-VIS radiation; a) Phototube and b) Photomultiplier tube

2. Photomultiplier (PM) Tube

A photomultiplier tube (Fig. 2.18 (b)), consists of a series of electrodes, called
dynodes. The voltage of successive electrodes is maintained 50 to 90 volt more
positive than the previous one. When a radiation falls on the cathode an electron is
emitted from it. This is accelerated towards the next photoemissive electrode by the
potential difference between the two. Here, it releases many more secondary electrons.
These, in turn are accelerated to the next electrode where each secondary electron
releases more electrons. The process continuous upto about 10 stages of amplification.
The final output of the photomultiplier tube gives a much larger signal than the
photocell.

3. Diode Array Detector

These detectors employ a large number of silicon diodes arranged side by side on a
single chip. When a UV-VIS radiation falls on the diode, its conductivity increases
significantly. This increase in conductivity is proportional to the intensity of the
radiation and can be readily measured. Since a large number of diodes can be arranged
together, the intensity at a number of wavelengths can be measured simultaneously.

49
Molecular Spectroscopic Though the photodiode array is not as sensitive as the photomultiplier tube, the
Methods-I
possibility of being able to measure a large number of wavelengths makes it a detector
of choice in the modern fast instruments.

2.4.6 Signal Processing and Output Devices

The electrical signal from the transducer is suitably amplified or processed before it is
sent to the recorder to give an output. The subtraction of the solvent spectrum from
that of the solution is done electronically. The output plot between the wavelength and
the intensity of absorption is the resultant of the subtraction process and is
characteristic of the absorbing species.

Having learnt about different components of UV-VIS instruments and their

importance; you are now equipped to learn about the types of instruments used. But
before proceeding further, try to answer the following SAQ.

SAQ 5
In what way a diode array detector is better than a photomultiplier tube?
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...

2.5 TYPES OF UV-VISIBLE SPECTROMETERS

Having learnt the basics of the different components of UV-Visible spectrometers let
us now see how these individual components are arranged in a spectrometer. Broadly
speaking there are three types of spectrometers. Let us learn about their set ups.

2.5.1 Single Beam Spectrometers

The instruments using As the name suggests, these instruments contain a single beam of light. The same
filters and photoelectric beam is used for reading the absorption of the sample as well as the reference. The
detectors are called schematic diagram of a typical single beam UV- Visible spectrometer is given in
photometers. Fig. 2.19. The radiation from the source is passed through a filter or a suitable
monochromator to get a band or a monochromatic radiation. It is then passed through
the sample (or the reference) and the transmitted radiation is detected by the
photodetector. The signal so obtained is sent as a read out or is recorded.

Fig. 2.19: Schematic diagram for a single beam UV-VIS spectrometer

Typically, two operations have to be performed – first, the cuvette is filled with the
reference solution and the absorbance reading at a given wavelength or the spectrum
over the desired range is recorded. Second, the cuvette is taken out and rinsed and

50
filled with sample solution and the process is repeated. The spectrum of the sample is UV-Visible
obtained by subtracting the spectrum of the reference from that of the sample solution. Spectrometry

2.5.2 Double Beam Spectrometers

In a double beam spectrometer, the radiation coming from the monochromator is split
into two beams with the help of a beam splitter. These are passed simultaneously
through the reference and the sample cell. The transmitted radiations are detected by
the detectors and the difference in the signal at all the wavelengths is suitably
amplified and sent for the output.

The general arrangement of a double beam spectrometer is shown in Fig. 2.20. There
could be variations depending on the manufacturer, the wavelength regions for which
the instrument is designed, the resolutions required etc.

Fig. 2.20: Schematic diagram for a double beam spectrometer

2.5.3 Photodiode Array Spectrometer

In a photodiode array instrument, also called a multi-channel instrument, the radiation
output from the source is focused directly on the sample. This allows the radiations of
all the wavelengths to simultaneously fall on the sample. The radiation coming out of
the sample after absorption (if any) is then made to fall on a reflection grating. The
schematic arrangement of a diode array spectrometer is given in Fig. 2.21.

Fig. 2.21: Schematic diagram for a photodiode array spectrometer

The grating disperses all the wavelengths simultaneously. These then fall on the array
of the photodiodes arranged side by side. In this way the intensities of all the
radiations in the range of the spectrum are measured in one go. The advantage of such
instruments is that a scan of the whole range can be accomplished in a short time.

SAQ 6
What colour absorption filter should be used to measure each of the following
absorptions? (You may use Table 2.2 to answer this question.)
a) Iron salicylate, λ max = 530 nm

51
Molecular Spectroscopic b) Ti (H2 O2), λ max = 410 nm
Methods-I
c) Glucose (o-toluidine), λ max = 625 nm.
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
It was mentioned earlier that UV-VIS spectrometry can be used for qualitative and
quantitative determinations of the analyte. The quantitative determinations are based
on the relationship between the absorption of radiation and concentration of the
analyte. Let us learn about this relationship and other applications.

2.6 ANALYTICAL APPLICATIONS OF UV-VISIBLE

SPECTROMETRY
UV-VIS spectrometry provides a technique that may be used to detect one or more
components in a solution and measure the concentration of these species. The primary
advantage of this technique is that even traces of substances can be determined in a
simple way which is not possible with classical analytical methods like gravimetric
and volumetric procedures. In addition, UV spectrometry is also used for obtaining
structural information of substances; particularly the organic compounds and it may as
well help in establishing the identity of a molecule. We will discuss some of
qualitative and quantitative applications of UV-VIS spectrometry.

2.6.1 Qualitative Applications

In terms of qualitative analysis of the analyte, the UV-VIS spectrometry is of a
secondary importance for the identification and the determination of structural details.
The information obtained from it needs to be supplemented by that from IR, NMR and
mass spectrometries. This is because of simple and broad nature of the spectra.
Nonetheless, it can still provide information about the presence or absence and the
nature of the chromophore in the molecule. For example, the presence of one or more
signals in the 200-400 nm region of the spectrum is a clear indication of the presence
of unsaturation in the molecule.

Though lack of fine structure in the UV-visible spectrum does not allow unambiguous
identification of the molecule, yet sometimes the spectrum can be quite useful in
distinguishing two molecules having similar colour. For example, Fig. 2.22 gives the
visible spectra of two purple coloured species of distinctly different structures. One of
the spectra happens to be of an azo dye and the other is of permanganate solution. The
shapes of the absorption curves help in distinguishing; the smooth curve with a single
maximum is for the dye.

Fig. 2.22: Absorption spectra of two distinctly different compounds but with similar
colour

52
Historically speaking, the UV spectroscopy has played an important role in the study UV-Visible
of a wide array of natural products of plant and animal origin. UV spectrometry was Spectrometry
used by Woodward to assign chemical structures of closely related compounds;
differing in only the location of a carbonyl group or a carbon-carbon double bond. In
fact extensive studies of the UV spectra of a large number of molecules led to the
establishment of empirical rules for calculating the λ max for organic molecules. The
rules proposed by Fieser, Woodward and Scott could be used to predict to a reasonable
degree of accuracy the λmax of unsaturated organic compounds. The details of these
rules and their applications can be obtained from any book on organic spectroscopy.

The changes in spectra due to the changes in the pH of the solution or the solvent can
provide useful information about the nature of the analyte. The change in the polarity
of the solvent alters the energies of the orbitals. This leads to change in the absorption
maxima. On increasing the polarity of the solvent, the n → π ∗ transitions are shifted
to lower wavelengths while the π → π ∗ transitions are shifted to longer wavelengths.
This helps in the identification and distinction of two closely associated molecules.

2.6.2 Quantitative Applications

As discussed above, UV-VIS spectrometry has limited applications in qualitative
analysis of the analytes; however, it is probably the most useful tool available for
quantitative determinations in diverse areas. It is due its versatility, accuracy and
sensitivity. It can be used for direct determination of a large number of organic,
inorganic and biochemical species accurately at fairly low concentrations; viz., 10‒4 to
10‒5 or even lower. In addition to these, the convenience of conducting the
determination and its reasonable selectivity make it a method of choice for
quantitative determinations.

Another significant feature of this technique is that it may be used for the quantitative
determination of analyte which do not absorb in the UV-VIS region. It is achieved by
making them react with a reagent that gives a product which absorbs in the region.
The same technique can be used to undertake quantitative determination of all such
species which have an absorption in the region but are present in system which contain
other constituents that also absorb in the same region. Thus, absorbance measurements
in visible or in UV region, are used in diverse areas. Some of the common ones are
related to the following quantitative aspects of solution chemistry.
1. Analytical determination of metals and non-metals
2. Analytical determination of organic compounds
3. Determination of dissociation constants of organic acids and dyes
4. Determination of metal-ligand formation constants
5. Determination of kinetic stability of complexes
The basis for the first two aspects of quantitative analysis lies in the Beer and
Lambert’s law discussed earlier. It involves comparing the extent of absorption of
radiant energy at a particular wavelength by a solution of the test material with a series
of standard solutions. In the earlier times, visual comparators were used for such
determinations which were carried out with natural light (white light) and employed
human eye to judge the colour intensity. In such an approach, a series of standard
solutions of the analyte to be determined were prepared in tubes of constant depth and
diameter. The sample solution was transformed to a duplicate tube and diluted to the
extent that its colour matched with one of the standard solutions. The concentration of
the test solution was determined from the concentration of the standard solution by
accounting for the dilution factor.

The rest three aspects of solution chemistry require measurements of the UV-visible
spectrum of the analyte as a function of pH or ligand concentration etc. Today, a given

53
Molecular Spectroscopic analytical determination can be made in a number of possible ways. These methods
Methods-I
depend on the nature of the species being determined and follow a kind of
methodology. Let us learn about the methodology of quantitative determinations.

2.6.3 Quantitative Determination Methodology

The methodology followed for the quantitative determinations have certain essential
steps. These are,
• Forming an absorbing species
• Selection of the measurement wavelength
• Controlling factors influencing absorbance
• Validation of Beer and Lambert’s law
Let us briefly discuss these one by one.

Forming an Absorbing Species

There are only a few analytes that have a strong absorption in the UV or Visible
region and can be subjected to direct determinations. However, for most of the
analytes we have to form a absorbing species by reacting them with a suitable reagent.
In any case, the absorbance of the solution should be stable and should not change
with minor variations in the pH, temperature and the ionic strength of the solution.
Further, the reagent should be selective towards the analyte and its reaction with the
analyte should be quantitative i.e. 100%.

Selection of the Measurement Wavelength

In the absence of interfering substances, the measurements are made at the wavelength
The wavelength chosen
of the maxima ( λ max) of the largest peak in the spectrum. At this wavelength the
for quantitative work is
called the analytical absorbance is most sensitive to the concentration. Further, as in many cases there is
wavelength. broadening of peak so within certain interval of wavelength near the λ max , there is no
appreciable change in the value of absorbance i.e. the measurement is not very
sensitive to the wavelength. This is quite advantageous because even if the
Ordinarily the absorbance spectrophotometer fails to resolve λ max, the determination does not suffer.
should be measured at a
wavelength where there is However, in systems where the reagent, metal and products all absorb light, it may so
no interference from other happen that at λ max there is not much of difference in the absorbance value for pure
absorbing species in the reagent and the metal complex. In such cases we need to identify such a wavelength at
solution. which there is large difference in the absorbance values of pure reagent and the
complex.

Controlling Factors Influencing Absorbance

As discussed earlier, a number of factors can affect the absorption spectrum of the
analyte. These include solvent polarity, pH, temperature, ionic strength and
interferences from other absorbing species. It is therefore, essential for a reliable
quantitative determination that the conditions of the determination are so chosen that
there is minimal effect of these factors.

Validation of Beer and Lambert’s law

Let us rewrite the expression for the Beer and Lambert’s law as

It is essential for a log Po / P = A= abc or εbc

dependable method that
According to this expression, if we know the values of a (or ε) and b then we can
the A versus c plot for the
standard analyte is a
determine the concentration directly from the absorbance value. However, generally
straight line. the value of ε is not known accurately for the species being determined under the
conditions of the determination. Quite often even the validity of the Beer and

54
Lambert’s law expression is questionable. Therefore in most of the methods, a UV-Visible
calibration curve is obtained by measuring the absorbance values for a series of Spectrometry
standard solutions of the analyte being determined at a fixed wavelength. These
solutions should be under similar solution conditions and in the range of the
concentration of the analyte. For the law to be valid, the plot of absorbance, A versus
the concentration, c for the standard solutions should be a straight line passing through
origin.

Such a curve is known as calibration curve and is handy in determination of

concentration of unknown solutions from a measurement of absorbance value. For
example, the unknown concentration of a solution of iron can be determined by using
a calibration curve obtained by plotting the absorbance values of a series of standard
solutions of iron measured at 562 nm depicted in Fig. 2.23 (a).

Fig. 2.23: Calibration curve: a) Standard solution method

Sometimes, it may so happen that all the constituents in the sample may not be known;
so a standard solution with the same chemical composition cannot be prepared. In
such a case, a method called standard addition method is used which eliminates any
error arising from molar absorptivities (ε) being different in the standard solution from
that in the sample solution. In this method known amounts of the standard is added to
identical aliquots of the sample and the absorbance is measured. The first reading is
the absorbance of sample alone and the second reading is absorbance of sample
containing analyte plus, a known amount of analyte and so on. The readings so
obtained are then plotted to obtain the calibration curve. If the Beer’s law is obeyed,
i.e. a straight line is obtained; the unknown concentration of the solution can then be
obtained by the extrapolation of the calibration curve as shown in Fig. 2.23 (b).

Fig. 2.23: Calibration curve: b) Standard addition method

2.6.4 Simultaneous Determination

Sometimes we come across a situation wherein the analyte contains two species which
have overlapping spectra. In order to determine these species we need to find two
wavelengths where molar absorptivity of two species is different. In such a case,

55
Molecular Spectroscopic measurements are made on the solution of the analytes at two different wavelengths.
Methods-I
This gives a set of simultaneous equations which could be solved for the
concentrations of the individual constituents. For best results it is desirable to select
two such wavelengths where the ratio of molar absorptivities is largest. Consider the
spectra given in Fig. 2.24.

Fig. 2.24: Schematic UV spectra of analyte having two species with overlapping spectra
Let us select the wavelengths λ1 and λ 2 on these two spectra. The molar absorptivity
of spectra 1 and 2 can be found out by taking known amounts of the substance and
recording their absorbance value. Let these values be (ε 1 ) λ1 , (ε 2 ) λ1 , (ε 1 ) λ2 , and
(ε 2 ) λ2 . If the absorbance of the solution at the wavelength λ1 and λ2 are Aλ1 and Aλ2
respectively then we can write, for 1 cm path,
Aλ1 = C1 (ε 1 ) λ1 + C 2 (ε 2 ) λ1
Aλ 2 = C1 (ε 1 ) λ2 + C2 (ε 2 ) λ 2
The above two simultaneous equations could be solved for C1 and C2. The expressions
are found to be.
Aλ1 (ε 2 ) λ2 − Aλ2 (ε 2 ) λ1
C1 =
(ε 1 ) λ1 (ε 2 ) λ2 − (ε 2 ) λ1 (ε 1 ) λ2

Aλ2 − (ε 1 ) λ2 C1
C2 =
(ε ) λ2
Let us take an example to understand it.

Example
The absorbance values of a mixture of K2Cr2O7 and KMnO4 at 440 nm and 545 nm
using a cell of 1 cm path length were found to be 0.405 and 0.712 respectively. The
absorbance values of pure solutions of K2Cr2O7 (0.001 M) and KMnO4 (0.0002 M) in
similar conditions were as follows:

A (Cr,440 nm) = 0.374, A (Cr, 545 nm) = 0.009

A (Mn, 440nm) = 0.019, A (Mn, 545 nm) = 0.475
Using the given data determine the concentration of Cr2 O 27− ions and Mn O −4 ions in
the mixture.

Solution: Using the given absorbance values for the pure solutions, we can calculate
the molar absorptivities as for Cr and Mn at 440 and 545 nm as follows:

0.374 = εCr, 440 × 1.0 × 1.0 × 10‒3 => εCr, 440 = 374
0.009 = εCr, 545 × 1.0 × 1.0 × 10‒ 3 => εCr, 545 = 9

56
 0.019 = εMn,440 × 1.0 × 2.0 × 10‒4 => εMn,440 = 95 UV-Visible
0.475 = εMn,545 × 1.0 × 2.0 × 10‒4 => εMn,545 = 2.38 × 103 Spectrometry

For the mixture we can write the simultaneous equations as

A440 = εCr, 440[Cr2 O 72− ] + εMn,440[Mn O −4 ]

A545 = εCr, 545[Cr2 O 72− ] + εMn,545[Mn O −4 ]

Substituting the values, we get

0.405 = 374 [Cr2 O 72− ] + 95 [Mn O −4 ]
0.712 = 9[Cr2 O 27− ] + 2.38 × 103[Mn O −4 ]
Solving simultaneous equations, we get
[Cr2 O 72− ] = 1.0 × 10‒3 mol dm‒3
[Mn O −4 ] = 2.95 × 10‒4 mol dm‒3

Let us conclude our section on applications by leaving the following SAQ for you to
solve.

SAQ 7
What is the advantage of the standard addition technique as compared to a calibration
curve method?
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...
…………………………………………………………………………………………...

2.7 SUMMARY
UV-VIS spectrometry is an instrumental technique that provides ideal methods for the
determination of micro and semi-micro quantities of analytes in a sample. The UV-
VIS spectrum arises due to the transitions amongst the electronic energy levels of the
absorbing species. For the sample in gaseous phase, the spectrum consists of a number
of closely spaced lines whereas in the solution phase, the spectrum has the shape of a
smooth and continuous absorption peak. This is due to the collisions and the
interactions among the absorbing species and the solvent molecules. A typical UV
spectrum is a plot of wavelength versus the intensity of absorption and is characterized
by two major parameters-the position of the maximum of the absorption band (λmax)
and the intensity.

There are three types of species that absorb in the UV-VIS region. These are organic
molecules, inorganic salts containing the ions of the transition and inner transition
elements and the species that involve formation of charge transfer complexes. The
relationship of the absorption with the concentration and the path length of the
absorbing sample is expressed in terms of Beer-Lambert’s law. This law forms the
basis of quantitative determinations of the analyte. A number of factors like pH,
concentration of electrolytes, wavelength, the possibility of association and
dissociation etc may cause deviations from the law. These need to be considered in
such determinations.

57
Molecular Spectroscopic There are five essential components of UV-visible instruments. These are: radiation
Methods-I
source, wavelength selector, sample holder, detector and a signal processing and
output device. For UV region a deuterium lamp is used as the source whereas for
visible range a tungsten filament lamp is employed. The wavelength selection is
achieved by absorption filters in low cost instruments for visible region. In the modern
instruments this is done by using suitable monochromators. The sample is taken in
quartz cuvettes, though for visible region even glass cells can be used. The detection
of the transmitted radiation is done generally by a phototube or a photomultiplier tube.
Nowadays, the modern machines employ diode arrays for detection purposes. These
components are assembled into single beam, double beam and diode array
spectrometers.

UV-VIS spectrometry can be used to detect one or more components in a solution and
measure the concentration of these species. In addition, it may also used for obtaining
structural information of substances; particularly the organic compounds and it may as
well help in establishing the identity of a molecule. The most important qualitative
application of UV-VIS spectrometry is to detect the presence of unsaturation in the
molecule. In addition, the changes in spectra due to the changes in the pH of the
solution or the solvent can provide useful information about the nature of the analyte.
On the other hand, due to its versatility, accuracy and sensitivity UV-VIS
spectrometry can be used for direct determination of a large number of organic,
inorganic and biochemical species accurately at fairly low concentrations of the order
of 10‒4 to 10‒5 M or even lower. The quantitative determinations follow a general
methodology involving four steps as, identifying or forming an absorbing species,
selection of the measurement wavelength, controlling factors that influence
absorbance and validation of Beer and Lambert’s law.

2.8 TERMINAL QUESTIONS

1. The UV-VIS spectra in the solution phase have band structure whereas in the
vapour phase we get sharp lines. Explain.

2. The d-d transitions are forbidden transitions so most of the transition metal ions
produce very weak bands in aqueous solution with various ligands. How do you
account for the intense blood red colour produced in a solution of Fe (III) ion
when the ligand like thiocyanate is added?

3. Outline the factors that may cause deviations from Beer and Lambert’s law.

4. The absorbance of a solution of ferrous 1, 10-phenanthroline complex

containing 1.00 microgram of iron (II) in 10 cm3 of solution was measured at
515 nm in 1 cm cuvette. It was found to be 0.0200. Calculate the molar
absorptivity of the complex.

5. What are monochromators? Describe the functioning of a grating

monochromator.

6. Why do we need to take UV spectra in quartz cuvettes?

7. Enlist the essential steps of quantitative determination methodology.

8. What is the principle of the photomultiplier tube?

9. A mixture of Ni2+ and Co2+ is analyzed by forming the salicyaldoxime

complexes in chloroform solvent. The molar absorptivities for Ni and Co ions at
500 nm are, 10 and 1000 respectively. However, at 400 nm both molar
absorptivities are 5.0 × 103 cm‒1mol‒1dm3. The absorbances were recorded using
1 cm cuvette and were found to be 0.091 at 500 nm and 0.615at 400 nm.
Calculate the concentrations of Ni2+ and Co2+ in the given mixture.

58
 UV-Visible
2.9 ANSWERS Spectrometry

Self Assessment Questions

1. Different types of species absorbing in the UV-VIS region and type of transition
occurring in them are as follows

Species Type of Transition

Organic molecules n→π or π → π*
*

Inorganic molecules d-d or f-f transitions

Charge Transfer complexes Donor HOMO to acceptor LUMO

2. Pо = 85.4 b = 2.00cm

P = 20.3 c = 1 × 10 −4 M є =?

A = log Pо/P = A = log 85.4/20.3 = 0.624

Since A = є b c; є = A/b.c

0.624
Substantially the value ε =
2.0 cm ×1 ×10 − 4 M

=> 3120 cm −1 mol −1dm 3

3. є =14000 cm −1 mol −1dm 3

c=? b = 1cm A= 0.85
A = є cb c = A/ є.b
c = 0.85/14000 × b
= 6.07 × 10 −5 M
4. According to Beer’s law there is a direct proportionality between the absorption
and concentration of the absorbing species taken in a cuvette of unit path length.
It implies that a plot of concentration versus absorption is a straight line. In
order to use this property for quantitative determination of the concentration of
the absorbing species there should not be any deviation from the linearity. It
therefore becomes important to know about the factors that may cause deviation
so that these could be suitably controlled.

5. A diode array detector is better than a photomultiplier tube as it allows

simultaneous determination of the intensity of a number of electromagnetic
radiations of different wavelengths.

6. i) purple
ii) yellow-green
iii) green-blue

7. The standard addition method allows the determination of the concentration of

the species in such solution also in which the other constituents are not fully
known.

Terminal questions
1. The UV-VIS spectra arise as a consequence of the interaction of the radiation

59
Molecular Spectroscopic with absorbing species causing a transition amongst their electronic energy
Methods-I
levels. In case of gaseous samples, the absorbing species the spectrum consists
of a number of closely spaced discreet lines that resemble a band and the
resulting spectrum is called a band spectrum.
In the solution phase the energies of the quantum states of the molecules are
somewhat spread out due to constant collisions with the surrounding solvent
molecules. Therefore the sample absorbs photons spread over a range of
wavelength and the spectrum acquires the shape of a smooth and continuous
absorption peak.

2. The intense red colour produced on addition of thiocyanate ion to Fe(III) ions is
due to the charge transfer complex formed between them.

3. Some of the factors responsible for the deviation from Beer and Lambert’s law
are as follows.
• Presence of Electrolytes
• Hydrogen Ion Concentration
• Complexation, Association or Dissociation
• Non monochromatic nature of the radiation
• Concentration of the Analyte
• Temperature
4. The atomic weight of iron is 56, therefore the molar concentration of the
solution containing 1.00 microgram of iron (II) present in 10 mL of solution will
be

1× 10 −6 g 1000mL
× = 1.786 × 10 −6 mol per dm3.
56 10mL
0.02
ε = A / b.c ⇒
1.786 mol dm −3 × 1 cm

= 1.12 × 104 dm −3 mol −1 cm −1

5. A monochromator is a device that can selectively provide radiation of a desired
wavelength out of the range of wavelengths emitted by the source. In grating
monochromator, a fine beam of the light from the radiation source is made to
fall on a concave mirror through an entrance slit. After reflection, it falls on the
grating which causes it to disperse. The dispersed radiation is then emerges out
of an exit slit. The range of wavelengths isolated by the monochromator is
determined by the extent of dispersion by the grating and the width of the exit
slit. Rotation of the grating in a predetermined way used to obtain the desired
wavelength from the exit slit.

6. In order to take the UV absorption spectra the analyte is taken in a quartz

cuvette because it is transparent to the electromagnetic radiations of the UV
range. As glass absorbs the ultraviolet radiations, these cannot be used for the
purpose.

7. The essential steps of the quantitative determinations methodology are,

• Forming an absorbing species
• Selection of the measurement wavelength
• Controlling factors influencing absorbance
• Validation of Beer and Lambert’s law

60
8. A photomultiplier tube is based on the principle of successive increase in the UV-Visible
number of electrons. The output signal by photoemission from a series of Spectrometry
electrodes called dynodes is much larger than the original signal.

9. We know that for simultaneous determination of two absorbing species the

absorbance is measured at two different wavelengths. For measurements in a
cuvette of 1 cm path length, the absorbances, concentrations and the extinction
coefficients are related as:
Aλ1 = C1 (ε 1 ) λ1 + C 2 (ε 2 ) λ1
Aλ2 = C1 (ε1 )λ 2 + C2 (ε 2 )λ2
With given data we can write as:
A500 nm = 10 × [Ni2+] + 1000 × [Co 2+] = 0.091 ... (A)
A400 nm = 5.0 × 103 × [ Ni2+] + 5.0 × 103 × [Co 2+] = 0.615 ... (B)

Multiplying (A) by 5 and subtracting from (B) we get,

4950 [ Ni2+] = 0.615 – 0.455 = 0.160

[ Ni2+] = 0.160 / 4950 = 3.2 × 10 −5 mol dm−3

Substituting it in (A) or (B) and solving we get

[Co 2+] = 9.1 × 10 −5 mol dm−3

61