UNIT 19 OPTICAL METHODS

Structure
19.1 Introduction
Objectives
19.2 Basics of Spectroscopy
The Nature of Electromagnetic Radiation
Spectral Regions
Classification of Spectroscopic Methods
19.3 Absorption Methods
Fundamental Laws of Absorption Methods
Absorbing Species
19.4 Ultraviolet-visible Spectrophotometry
Instrumentation
Some Typical Instruments
Analytical Techniques
Determination of Substances in Waters, Soil and Air
19.5 Emission Methods
Flame Photometry
Atomic Absorption Spectrophotometry
19.6 Summary
19.7 Terminal Questions
19.8 Answers

19.1 INTRODUCTION
In the previous unit, we have studied the electroanalytical methods of analysis.
Another widely used instrumental methods of analysis in the field of environmental
chemistry are optical methods. These methods measure the results of interaction
between electromagnetic radiation and matter. The range of electromagnetic radiation
may vary from X-rays, through visible, to radio waves.

In optical methods of analysis we consider emission, absorption, scattering, or change

in some property of radiation (such as: direction and state of polarization).
Measurement of these effects gives different types of optical methods, which may be
utilized, to identify and determine one or more constituent of the sample.

In modern usage the word spectroscopy is used to characterize important optical

methods where, in general, the study is made of the emission, absorption or scattering
of electromagnetic radiation involving energy changes in nuclei, atoms or molecules.
The absorption of electromagnetic radiation is used in absorption spectrophotometry
and emission used in emission spectophtometry. Whereas scattering results: Raman
spectroscopy, nephelometry and turbidimetry.

Spectroscopy has been found to be an important tool that can quickly solve certain
difficult tasks in chemistry as well as other branches of science. Spectroscopic
methods are used in widely diverse fields, such as, structure elucidation, identification
of compounds and functional groups, qualitative and quantitative analyses,
determination of thermodynamic properties, and pollution analysis. The recent
proliferation of government regulations with respect to atmospheric contaminants has
demanded the development of sensit ive, rapid and specific methods for a variety of
chemical compounds. Absorption Spectrophotometry solves the problem better than
any other single tool. Spectrophotometry involves measuring the extent to which
radiation energy is absorbed by a chemical system as a function of wavelength, as well
as, the measurements at a fixed predetermined wavelength of the radiation.

All spectroscopic methods have in common the interaction of electromagnetic

radiation with the quantized energy states of matter. The emission or absorption of
35
Instrumental Methods radiation of a particular frequency results the transitions between different states of
of Analysis energy. The aim of spectroscopist is to measure the relative amounts of radiant energy
absorbed or emitted at such frequency and to relate these changes with the nature and
amount of various substances.

Especially ultraviolet-visible spectroscopy is still probably the single most frequently

used analytical method for quantitative analysis of trace components in routine work.
Therefore, our main concern here is to deal with ultraviolet-visible absorption methods
and to apply them to pollution studies.

The discussion will also include flame photometry and atomic absorption
spectrophotometry. Flamephotometry although applicable to the determination of a
few elements but is simple.

Objectives
After studying this unit, you will be able to:
• describe electromagnetic radiation,
• classify the spectroscopy methods,
• define and relate various parameters such as wavelength, frequency, wave
number etc. associated with the electromagnetic radiation,
• define Beer-Lambert’s Law,
• describe the different components of a spectrophotometer,
• define absorbing species such as chromophores, auxochromes, etc.
• determine substances in water, waste waters, soil and air samples using
ultraviolet-visible spectrophotometry, and
• describe flame photometry and atomic absorption spectrophotometry.

19.2 BASICS OF SPECTROSCOPY

Before taking up absorption and emission spectrophotometry in detail, let us review
some of the concepts, whic h you may have studied in CHE 1 (Atoms and Molecules),
CHE 5 (Organic Chemistry) and CHE 10 (Spectroscopy) courses of our B.Sc.
Programme.

19.2.1 T he Nature of Electromagnetic Radiation

All optical methods involve the interaction of electromagnetic radiation with matter
following several mechanisms. A brief description of electromagnetic radiation and
their characteristics should be considered first.

The electromagnetic radiation (emr) has dual nature: (i) a stream of discrete particles,
called photons (or quanta), and (ii) a type of energy that is transmitted through space
with enormous velocities, called waves. Various optical phenomena are best
interpreted with the wave nature of emr.

The electromagnetic radiation, in general, may be described as wave motion

characterized by electric and magnetic displacement at right angles to each other and
to the direction of propagation of radiation, as shown in Fig. 19.1

36
 Optical Methods

Electric field
y

Magnetic field x
z

Direction of
propagation

Fig. 19.1: The electric and magnetic Fields of the electromagnetic radiation.

For optical phenomenon only the electric displacement needs to be considered. In

order to characterize many of the properties of emr it is convenient to portray these
waves by such parameters as velocity, frequency, wavelength and wave number.
• Velocity: The velocity of an electromagnetic wave is the rate at which the
wave front moves through a medium. The velocity of all electromagnetic waves
in vacuum is the same (equal to 2.998 x 108 m s-1 (meter per second) and is
denoted by c. However, velocity is dependent upon both the composition of the
medium and the frequency. But in vacuum the velocity of radiation becomes Angstorm unit, A° after
independent of frequency and is at its maximum. A.J. Angstrom, a
• Wavelength: It refers to the distance between (two) adjacent crests or troughs. Swedish Physicist
o
(See Fig. 19.2). It is designated by λ (lambda). The units of wavelength depend A =10 − 10 m
o
upon the region of the spectrum. In ultraviolet and visible region Angstrom ( A ) nm = 10 − 9 m
and nanometer (nm) are widely used.

+
Wavelength

Amplitude, A
Electric field

-
Time of distance

Fig. 19.2: Wavelength (λ) and amplitude (A) associated with electromagnetic radiation.

• Wave number: The number of waves in unit length is referred to as the wave Wave number is often used
by chemist (particularly in
number. The wave number is reciprocal of wavelength. The symbol for wave infrared region as a
number is ν (nu bar). The common unit of wave number is reciprocal frequency unit since
centimetre (cm-1). ν = cν
• Frequency: Frequency is the number of waves (or cycles) passing a point of
space in unit time. Unit of frequency is Hertz, 1Hz = 1 cycle per sec. Frequency
is denoted by ν (nu).

Frequency is determined by the source and remains invariant regardless of the

medium through which the radiation travels.
37
Instrumental Methods
of Analysis These parameters are related among themselves as follows:
1 c
ν= , ?= = cν , c = ??
? ?

Interaction of Radiation with Matter

When electromagnetic radiation comes in contact with atoms or molecules of matter,
there may be an exchange of energy between them. The system may absorb energy
and go from the lower energy state(ground state), E 1, to higher energy state(excited
state), E2. Alternatively, a system initially in the higher energy state E 2 can lose
energy and go to the lower energy state E 1. This transfer of energy is quantized and
energy difference, ∆E , between these two states is given by the following equation.
∆E = E 2 – E1 = hν ..… (19.1)

This equation can be related to λ and ν by following equation

∆E = hc/λ = hc ν

where h is the Plank’s constant and has the value 6.626 × 10 −34 J s (Jules second) ν is
the frequency of electromagnetic radiation which is causing energy changes. The
energy changes are shown in Fig. 19.3.

E2

E1

Fig. 19.3: Energy level diagram; E1, lower energy level, E2 high energy level.

By determining the energy absorbed or emitted, we can know about the energy levels
or transitions present in an atom or a molecule. In other words, these transitions can
be related to the structure of the atoms or the molecules. The energy absorbed or
emitted by matter can be detected by an instrument called spectrophotometer. These
instruments are designed to measure the frequencies or wave number or wavelength of
radiations which are absorbed or emitted by a particular sample on irradiation.

SAQ 1
Give the wave number in cm-1 and the frequency in Hz for radiation of the wavelength
o
4000 A .

…………………………………………………………………………………………
…………………………………………………………………………………………
…………………………………………………………………………………………

19.2.2 Spectral Regions

38
The spectrum of electromagnetic radiation is broken down in several regions Optical Methods
(Fig. 19.4). The limits of these regions are determined on the basis of different
mechanisms involved and different kinds of information achieved from the interaction
of electromagnetic radiation. Table 19.1 provides the different spectral regions with
their approximate wavelength ranges and the types of transition involved.

Ultraviolet Infrared

Radio waves
Vacuum UV

Microwave
Middle IR
Near UV

Near IR
X- rays
- rays

Far IR
Visible

10 nm 100 nm 1000 nm 10 m 100 m 1000 m

(1 m)

Fig 19.4: Representation of electromagnetic spectrum.

Table 19.1: Regions of the electromagnetic spectrum and types of transition

involved.

Region of Electromagnetic Wavelength Type of Transition

Radiation Range (consequential effect)

γ- Rays (Moss Bauer 0.005 – 1.4 A

o Nuclear transition
spectroscopy) (change of nuclear configuration)

X-rays (Diffraction, 0.1 – 100 Å Inner electron transition

Absorption, Emission, (change of electron distribution)
Fluorescence)

Vacuum UV (Absorption) 10 – 180 nm Bonding electrons transition

(change of electron distribution)

Ultraviolet- visible 180 – 780 nm Bonding electrons transition

(Absorption, Emission, (change of electron distribution)
Fluorescence)

Infrared (Absorption) and 0.78 – 300 µm Vibration/Rotation of molecules

Raman (Scattering) (change of configuration)

Microwaves (Absorption) 0.75 – 3.75 nm Rotation of molecules

(change of orientation)

Electron Spin Resonance 3 cm Spin of electrons in a magnetic

(ESR) field (change of electron spin)

Nuclear Magnetic Resonance 0.6 – 10 m Spin of nuclei in a magnetic field

(NMR) (change of nuclear spin)

39
Instrumental Methods You may note that the visible portion of the spectrum to which the human eye is
of Analysis sensitive is a very small part of the emr spectrum.

19.2.3 Classification of Spectroscopic Methods

In the preceding sub-section you have studied that the spectroscopic methods are
based on the energy changes occurred due to the interaction of emr with matter. Thus,
they can be classified on the basis of the energy changes (i.e. nuclear, electronic,
vibration, or rotational etc.) involved in the transition. Another way of classification is
on the basis of the type of the process (i.e., emission, absorption, or scattering)
involved in the transition. Yet, another way to characterize spectroscopic methods is
according to the spectral region of electromagnetic radiation involved. They include:
γ-ray, X-ray, ultraviolet, visible, infrared, microwave, electron spin resonance and
nuclear magnetic resonance methods.

We can choose a suitable spectroscopic method in order to solve the problems of

structure elucidation, quantitative estimation, or measurement of such properties as the
value of dipole moment, equilibrium constant etc.

X-rays usually cause transitions of inner shell (K and L shell) electrons. The most
widespread use of X-rays has been in the field of metallurgy, but X-rays may also be
used to analyse metals, minerals, liquids, glasses and ceramics. They can be used to
determine the crystal structure and to measure the thickness of a very thin layer of tin
plating.

Ultraviolet-visible spectroscopy involves the electronic transitions in atoms and

molecules. It is mainly used for quantitative analysis of substances of different
categories, such as, inorganic, organic and bio-chemicals. The use of this technique is
of wide importance in clinical laboratory and to perform chemical analysis of
environmental samples.

Infrared spectroscopy can be applied to determine the molecular structure.

Identification of compounds can be done by identifying the functional groups.
Quantitative determination is also possible. Raman spectroscopy is another technique,
which utilizes this range of frequency and is used for the same kinds of applications as
infrared (IR) and is complementary to IR spectroscopy.

Nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopic
methods are rather recent techniques but are very useful in structure elucidation
particularly of organic compounds. In these methods the splitting of energy levels
takes place in the influence of an external strong magnetic field.

With these fundamental backgrounds, now we will concentrate our discussion on

absorption method with special emphasis to methods involving ultaviole-visible region
of electromagnetic radiations.

19.3 ABSORPTION METHODS

Absorption Methods in general and using ultraviolet-visible region in particular is
probably the most frequently used analytical technique for the quantitative estimation
of substances in traces. May be a clinical laboratory to analyse the samples of blood or
urine, or an environmental laboratory to know toxic metals in natural waters or waste
waters, or a routine analysis in industries, science laboratories and so forth, this
technique is widely used. In absorption methods involving ultraviolet-visible region,
the sample solution is ideally irradia ted with electromagnetic radiation of single
wavelength (monochromatic radiation), and the amount of absorption at each
wavelength is measured as the wavelength is varied. By plotting the absorbance or
40
percentage transmittance, against the wavelength, an absorption spectrum is obtained. Optical Methods
An instrument called spectrophotometer is used to make the measurement. The plot
of absorbance (A) vs. wavelength (λ) is illustrated in Fig. 19.5. In this figure you can
see that two maxims occur, corresponding to point (i) and (ii), denoting intense
absorption at the corresponding wavelength. We will discuss terms, absorbance and
transmittance in detail in next sub-section. Spectrum shown in Fig. 19.5 is usually
characterized by two parameters.

2.0 100
max =279 nm (a) O
CH-3 C- CH3
Acetone

0 210 230 250 270 290 310

(ii)

Absorbance
max
=255 nm (c) Benzene
vapour
A

1.0 %T 50
(i) 0
230 240 250 260 270
max=255 nm (d) Benzene
in hexane

0
230 240 250 260 270
(i) (ii) A few typical absorption curves
0 0
/nm 300 340 380 420 440 300 340 380 420 440

Fig. 19.5: Presentation of absorption spectral data.

? max value: The value of the wavelength at which maximum absorption occurs is
called wavelength maxima, ? max. Values of ? max for different molecules are different.
For example, ? max for acetone is 279 nm, whereas for benzene, it is 255 nm.
Compounds may have more than one maxima, Fig. 19.5 is showing two ? max values (i)
and (ii).

∈ Value: The extent of absorption for a given concentration of a compound at any

given wavelength is defined by molar absorptivity, which is indicated by ∈ (epsilon)
There is a useful term
value. It is related to the height of the absorption band. We shall define this precisely Transmittance , and is defined
in next sub-section. The parameters, ? max (the position) and ∈ (the extent of as the ratio of radiant power
absorption) are characteristic property of a molecule. These parameters depend on the transmitted through sample to
structure and concentration of the molecules in solution. Therefore, absorption the incident radiant power
spectrum especially in ultraviolet-visible region is extensively used in characterisation T = P/Po
and also in quantitative estimations.
Transmittance is often
In this section we will take up fundamental law s of quantitative analysis relating the expressed in percentage,
amount of radiation absorbed to the concentration of an absorbing substance and
% T = P/P0 × 100
structural requirement of a substance to absorb the Ultraviolet visible region.
while working with solution a
19.3.1 Fundament Laws of Absorption Methods comparison can be made with a
blank, where the ratio of
There are two fundamental laws used in absorption methods. One is Bouguer’s law or transmitted powers through
Lambert’s law, which expresses the relationship between the light absorptive capacity solution and that through blank
(or solvent) is called as
and the thickness of the absorbing medium; and the other is Beer’s law, which transmittance. For this 100% T
expresses the relationship between the light absorptive capacity and the concentration adjustment is made by putting
of a solution. The two laws are combined together to give Beer-Lambert’s law. You the blank in the light path.
will know about these laws in the following discussion.
Note: Transmittance being a
ratio has no unit.
Beer-Lambert’s Law
Reciprocal of transmittance is
known as Opacity. 41

Opacity = 1/T = P0/P

Instrumental Methods Lambert’s Law: Earlier Bouguer and later Lambert gave a mathematical relation based
of Analysis on the transmission of monochromatic light by homogeneous absorbing medium and
stated that, “each unit length of absorbing material through which light passes absorbs
the same fraction of entering light”.

Px Px -d Px
P0 P

dPx
b

Fig. 19.6: Illustration of Lambert’s Law.

In Fig. 19.6, 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, dP x, 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,
dP x ∝ P xdx
or dP x = - k P xdx ..… (19.2)
where k is the proportionality constant and the negative sign indicates that radiant
power decreases with absorption Eq. (19.2) can be rearranged to,
d Px
= − kdx ..… (19.3)
Px

Integrating Eq. 19.3 we get,

P b
dPx
∫
Po
Px
= − k ∫ dx
o

..... (19.4)
P
or ln = −k b
Po

Eq. (19.4) is the mathematical expression for Bouguer-Lambert law or Lambert’s law.

Changing Eq. (19.4) to base 10 logarithms and rearranging we get,

Po k
log = b = k 'b ..… (19.5)
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.

42
Beer’s Law: Beer (1852) modified the Lambert’s law from the point of view of the Optical Methods
effect of concentration of the absorbing species under a constant path length b. He
indicated that, “the decrease in power of a radiant beam of monochromatic radiation is
proportional to the power of the beam multiplied by the change in concentration of
absorbing substance in the path”. The mathematical expression of Beer’s law is
derived in the same manner as the Lambert’s law. Consider a parallel monochromatic
radiation beam 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 is:

dPx ∝ Px dc
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
"
Px

On integration, we get

P c
d Px
∫ Px = − k " ∫ dc
Po o

P
or ln = −k " c
Po
Po
or log = k" c
P ..… (19.6)

It can be shown mathematically that the two Eqs. (19.5) and (19.6) may be combined
to yield:
Po
log = abc ..… (19.7)
P

Where a is a constant (combining two constants k′ and k″) known as absorptivity for
concentration c given in grams per dm 3. It has unit cm−1g −1 dm3.

The term log Po/P is given a special symbol, A , known as “absorbance”. Eq. (19.7)
then is,
A = abc ..… (19.8)

When concentration c is expressed in moles per dm3 (mol dm −3)

A = ∈bc ..… (19.9)

Where∈ (Epsilon) is called molar absorptivity (formerly called the molar extinction
coefficient) and it has unit cm−1 mol-1 dm 3

Eqs. (19.8) and (19.9) are expressions of Beer-Lambert’s law or often referred to as
Beer’s law.

43
Instrumental Methods The Beer-Lambert’s law provides a quantitative relationship to determine the
of Analysis concentration of a solution by measuring the amount of light absorbed by that solution
in a known path length cell.

Deviations from Beer’s Law

The Beer-Lambert’s law (or, as in common practices, simply Beer’s law) given by
equations A = abc, states that a plot of absorbance versus concentration should give a
straight line passing through origin, Fig. 19.7a. However, deviations from this linear
relationship between absorbance A and concentration c may sometimes be
encountered and instead of a straight line a curvature in the plot may be observed. The
upward curvature, curve (b), is known as positive deviation and the downward
curvature, curve(c), as negative deviation.

0 c

Fig. 19.7: Deviations from Beer’s law.

From Eq. (19.8), it is evident that the slope of the absorbance versus concentration
plots will be equal to a b. When both a and b are constant the slope is constant and the
relationship between A and c is linear. However, when any of the two i.e. either a or b
is not constant, there is departure from linearity in the Beer’s law plot. Generally, cell
length is a constant factor and is not involved in deviation. That is, deviations to Aαb,
the Lambert’s law, are not known (if the instrument factors are not changed).

The deviation is then caused due to the variation in absorptivity. Absorptivity, a, is a

function of wavelength and the nature of the substance whose absorbance is being
measured. The nature of the substance depends on a number of variables. The
variation in absorptivity may thus be caused due to several factors, such as: non-
monochromatic radiation, dissociation, association, complex formation,
polymerisation, solvolysis, stability of the absorbing species, pH, photochemical
reaction, reaction time and temperature. Failure of Beer’s law due to these factors is
grouped in apparent deviations since they reflect experimental difficulties rather than
the limitation of Beer’s law itself. These deviations are called apparent since they are
caused by the deviation from the conditions for which the law was derived, and
disappear if the actual conditions are used. The effects of some of these factors are
being discussed in the following paragraphs.

Non-monochromatic Radiation : Beer’s law requires that the radiation be

monochromatic. In usual practice, however, one works with a narrow band of
wavelength and not with a single wavelength. Since absorptivity is a function of
wavelength, the absorptivity, a, at one wavelength λ may not be identical with
absorptivity, a’, at the other wavelength λ’ in the band. Therefore, the relationship
between absorbance and concentration may be non-linear. The deviation will be
greater as the difference between a and a’ becomes greater.
44
 Optical Methods
Association and dissociation : Deviations from Beer’s law arise when an analyte
undergoes dissociation, association, or polymerisation to produce a species with a
different λ max than that of analyte. An example of this behaviour is found with
dichromate and chromate equilibrium, given in equation below:
Cr2 O27 − + H2 O 
→ 2H+ + 2CrO24 −

Obviously the two species dichromate and chromate have different colours and
different spectra with different λmax . The concentrations of C r2 O27 − and C r O24 − be
affected by pH. Cr2 O27 − which is the dominating species at a lower pH will change to A wavelength at which two
or more species in
chromate on dilution with water and the deviation from Beer’s law will be observed. equilibrium with one
another have the same
absorptivity value is called
However, the measurement of absorbance at isobestic point (or is absorptive an isobestic point.
wavelength) at which the two absorbing species in equilibrium have a common value
of absorptivity will not show deviation. Beer’s law then holds, though the
measurements have low sensitivity, even when there is a shift in equilibrium.

Another example of this behaviour is observed with the equilibrium of an acid-base

indicator, where the
HIn ƒ H+ + In−

Two species HIn and the dissociated ion In− have different colours. The colour
changes with change in pH. Therefore, to avoid the deviation effect, we should buffer
the solution before the measurements are made.

Temperature: Although 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 and therefore λ max and absorptivity are changed.

Photochemical reactions: If analyte undergoes a photochemical reaction due to the

effect of radiation the product will be different than the analyte and deviation is
observed.

Real Limitations: The other class of deviations, which may be considered real, rather
that apparent, may be encountered due to the following factors:

Concentration: Beer’s law is applicable to dilute solutions only (that is for

concentration lower than 0.01M). For higher concentrations deviations are caused due
to diminishing the average distance between the species responsible for absorbance in
the solution (i.e., area for capture of photon by the absorbing particles in the solution
is decreased).

Refractive Index: Absorptivity is changed when wavelength is changed. Since

wavelength changes when the medium is changed, that is, the refractive index (µ) of
the medium is changed, absorptivity, therefore, depends on the refractive index of the
solution. Changes in concentration can change the refractive index and therefore
changes in absorptivity resulting deviation from Beer’s law. However, this effect is
very small and is generally well within the experimental errors in spectrophotometry.

Additivity of Absorbance

45
Instrumental Methods According to Beer’s law equation (Eq. 19.9), the absorbance at a given wavelength is
of Analysis proportional to the number of radiant particles, which are effective in absorbing
radiation power. When it is applied for more than one absorbing species, we have,
A = ∈x bcx + ∈y bcy ..… (19.10)

In general,
A = ∑ A i = b ∑ ∈i ci ….. (19.11)

That is, the total absorbance of a solution at a given wavelength is equal to the sum of
the absorbance of the individual components present. This means that the absorbance
is an additive property. This property can be useful in the following ways:
i) To find the contribution of solvent in absorbance measurements, that is the
familiar use of blank.
ii) To find the absorption spectra of unknown chromophore in presence of a known
chromophore by subtraction.
iii) In multiple component analysis, that is, the simultaneous determination of the
concentration of two or more components in a mixture.

SAQ 2
In a photometer at the λmax of a sample using a 2.00 cm cuvettes the value of P o was
85.4 with the solvent and with 1 × 104 M solution of sample, P was 20.3. What is the
molar absorptivity of the sample?
…………………………………………………………………………………………
…………………………………………………………………………………………
…………………………………………………………………………………………

Analysis of Binary Mixtures

The property of additivity of absorbance may be applied to determine the
concentration of two absorbing constituents present in a single solution, provided that
the two constituents have separate wavelength maxima (λ max) and these constituents
do not interact with each other. Consider a solution containing two absorbing
constituents X and Y with wavelength maxima at λ1 and λ2 respectively. In order to
calculate the concentration c x and cy in the mixture, we need to take measurement of
absorbance at two different wavelengths (say λ1 and λ 2) then,
A 1 = (Ax)1 + (Ay )1
= (∈ x)1bc x + (∈y )1bcy ..… (19.12)

and A 2 = (Ax)2 + (Ay )2

= (∈ x)2bc x + (∈y )2bcy ..… (19.13)

Where (Ax)1 is the absorbance contribution due to component X at λ 1; (Ay )1 is the

absorbance contribution due to component Y at λ1; (Ax) 2 is the absorbance
contribution of X at λ2; and (A y)2 is the absorbance contribution of Y at λ 2. Molar
absorptivities (∈) are the respective values with the given suffixes in a manner similar
to that for absorbance. c x and c y are the concentrations of components X and Y in the
mixture being analysed.

Eqs. 19.12 and 19.13 can be solved for c x and cy provided we have the value of the
four constants (molar absorpitivities), (∈x)1, (∈ y)1, (∈ x)2, (∈y )2. The molar

46
absorptivities are determined by making absorbance measurements on pure molar Optical Methods
solution of X and Y at wavelength λ1 and λ 2.

Since, absorptivity is the function of the wavelength and the nature of the substance,
its value will remain constant at a given wavelength for the given component and,
therefore, the values of molar absorptivities calculated for known concentrations from
the absorption spectra can be substituted in Eqs. (19.12) and (19.13) for unknown
concentrations. These equations can be solved algebraically to find the values of cx
and cy in the unknown solution, with knowledge of the components spectra, which
may occur with any of the three possible situations shown in Fig. 19.8.

(a) (b) (c)

A A A

x x y x y
y

1 2 1 2 1 2

Fig. 19.8: Absorption spectra of X and Y with different possibilities.

a) No overlapping b) One -sided overlap c) Double-sided overlap

Fig. 19.8a shows the two spectra with two separate absorption peaks and with no
overlapping. Absorbance of component X is maximum at λ 1 w here component Y does
not absorb and absorbance of component Y is maximum at λ2 where component X
does not absorb. Thus, from Eq. 19.12, A1 is equal to (A x)1 and from Eq. 19.13, A2 is
equal to (A y)2, since (Ay )1 and (Ax)2 are zero. cx and cy can then be calculated directly
from Beer’s law with the molar absorptivities already calculated for known
concentrations. That is, the concentrations of the constituents X and Y are measured
directly at λ1 and λ 2 respectively, without interference.

Fig. 19.8b shows one-sided overlap, that is, at λ 1 there is an overlap of spectrum of Y
on the spectrum of X; but the spectrum of X does not overlap on the spectrum of Y at
λ2. From Eq. 19.12, A 2 is equal to (Ay )2 because (Ax)2 is zero and then (A y )2 is equal to
(∈ y)2bcy gives the direct calculation of cy . Now this value of cy is substituted in Eq.
19.12 to give the value of (Ax)1 and cx is then calculated in simple way.

In Fig. 19.8 c, there is a double -sided overlap that is the spectra of X and Y overlaps
each other. From Eqs. 19.12 and 19.13, we observe that the absorbance of the mixture
of λ1 is A1 which is the sum of (Ax)1 and (A y)1; and the absorbance of the mixture at λ2
is A2 which is the sum of (A x)2 and (Ay )2. The two equations are then solved
algebraically to calculate c x and cy.

19.3.2 Absorbing Species

47
Instrumental Methods Absorption of electromagnetic radiation in the near ultraviolet region (175 – 375 nm)
of Analysis and visible region (375 – 750) results in electronic transition for both organic and
inorganic substances.

Absorption by Organic Species

The absorption of emr by organic compounds is based on the difference in energy
between the ground state and the various excited states (electronic) of the molecule.
Most molecules show only one or two electronic transitions in the visible and near
ultraviolet region and for this only the outermost electrons need to be considered.
Organic molecules have electrons in three kinds of orbital viz: σ (sigma) - bonding, p
(pie)-bonding and n (non)-bonding.

The electronic transitions of these electrons are characterized by their elevation to

excited state antibonding molecular orbitals (π* and σ *). The quantum energies
required for these transitions will be different and will also vary with structure of
organic molecules. The relative energies of the probable transitions are qualitatively
illustrated in Figure 19.9.

Antibonding

Antibonding
Energy

n Nonbonding

Bonding

Bonding

Fig. 19.9: Order of energy for various types of molecular orbital and electronic transitions.

As shown in Fig. 19.9 four types of transition are possible σ → σ *, n → σ*, π → π*

and n → π*. The probable regions of electronic spectrum is regarded as follows:
∆E1 σ → σ* vacuum ultraviolet
∆E2 n → σ* far ultraviolet
∆E3 π → π* ultraviolet
∆E4 n → π* near ultraviolet and visible

Let us discuss these transitions in some detail.

σ → σ * transition: It is obvious from the preceding discussion that relative to other

possible transitions the excitation energy required to induce a σ → σ * transition is
large and corresponds to the absorption peaks in vacuum ultraviolet region (λ < 175
nm). Thus, to excite C – H or C – C single bond electrons in alkanes, radiation of
wavelength lower than 160 nm is required. For example methane shows a peak at 122
nm corresponding to σ → σ* transition.

n → σ * transition: The energies required for n → σ * transitions are lower than σ

→ σ * transitions but larger than π → π* and n → π* type transitions and can be
brought about by the radiation of wavelength range 150 to 250 nm with most
absorption peaks appearing below 200 nm (far ultraviolet range)

48
π → π* transition: The energies required to these transitions are lower and result in Optical Methods
longer wavelength absorption than σ → σ * and n → σ * transitions. The absorption
in hydrocarbons containing double bonds and triple bonds is observed at wavelengths
approaching near ultraviolet regions. Conjugation further increases λmax and is
appreciable 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* transition: In such transitions one of the non-bonding electrons (lone pairs)

may be excited into an empty π* orbital. The energies required to these transitions are
lower than π → π * transitions and result in ultraviolet and visible region. 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.
Chromophores
Among the most obvious characteristics of a chemical compound is its colour. In 1876
Witt related the appearance of colour in molecules due to the existence of certain
chemical groups, termed “chromophores”. Chromophore in Greek means, “colour
bringer”. However, the term chromphore 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 175 and 1000 nm, which is convenient to use
experimentally.

The absorption of emr of a particular frequency results the transitions between states
of different energy. Therefore, the wavelength at which a chromophore shows its
maximum absorbance depends on the type of electronic transition, that is the energy
required to carry out the excitation of a particular kind of electron from the lower
energy state to the possible higher energy state. In the ultraviolet-visible region most
of the absorption bands are due to the excit ation of π-electrons and n-electrons.
Energies required for the transition of π electrons and n-electrons to the π* excited
state bring the absorption peaks into an experimentally convenient spectral region (175
– 1000 nm). Such as carbonyl in aldehydes and ketones give λmax in ultraviolet region
due to the absorption of photons by π or n-electrons. Acetaldehyde has a λmax at 293
nm due to n → π* transition; acetone has λ max at 186 and 280 nm due to n → σ * and
n → π* transitions respectively. Some other examples of chromophores in this region
are: >C=C<, − C ≡ C, − Ν = Ο & −Ν=Ν − and so on.

A listing of common chromophores, along with the types of transitions and

approximate location of their absorption maxima, is given in Table 19.2 although the
peaks are ordinarily broad because of vibration effects, these peaks, however, can
serve as rough guides for the identification of functional groups in organic
compounds.

When two or more chromophores are present in a molecule, absorption depends on

their relative positions of λ max and intensity of absorption varies with the nature of the
solvent. Usually polar solvents tend to shift π to π * transitions to a longer wavelength
(RED SHIFT); an n to π * transitions to a shorter wavelength (BLUE SHIFT).

Table 19.2: Absorption characteristics of some common chromophores.

Chromophore Example λ max Type of Transition
>C = C< C6H13 CH = CH2 177 π →π *

−C ≡ C− C5H11 C ≡ C − CH3 178 π →π *

49
Instrumental Methods >C = Ο CH3CO CH3 186 n → σ*
of Analysis 280 n →π *
CH3CHO 180 n → σ*
293
O n →π *
−C−ΟH CH3COOH 204
O
n →π *

−C−NH2 CH3CONH2 214 n →π *

−N = N− CH3N = NCH3 339

n →π *

−NO 2 CH3NO 2 280

n →π *

−N = Ο C 4H9 NO 300
665 -
n →π *
In conjugated molecules (i.e. containing alternating double bonds) the absorption is
shifted to a longer wavelength due to the fact that the resonance structure results
delocalisation of electrons that is in a conjugated system the electron is less tightly
bound than once in a non-conjugated system.

“Auxochromes” though do not themselves absorb emr but when attached to a

chromophore, alter both the wavelength and the intensity of absorption of the
chromophore. An auxochrome is a saturated (functional) group with non-bonding
.. ..
electrons,
.. which can be donated to the conjugated system. Examples are – OR,
.. – NH2,
– NR 2 ect.

Absorption by Inorganic Compounds

The absorption of emr by inorganic compounds, involving d electrons, f electrons and
the electronic transitions are briefly mentioned below.

The spectra of transition metal ions are due to the involvement of d orbitals in the co-
ordination bonding of these ions with solvent molecules or forming complexes with
specific ligands. The degeneracy of the five d orbitals of the transition metal ions is
removed in complex formation and electronic transitions from the lower energy d
orbitals to higher energy d * orbitals are observed when the emr of proper frequency
(which covers the range from ultraviolet to the near IR region) is absorbed.

The ions of lanthanides and actinides absorb ultaviolet and visible radiation due to the
f to f * transitions. Here, the f electrons being shielded from external influences by
occupied orbitals of higher principal quantum number absorb the radiation of
ultraviolet and visible region in narrow bands.

The absorption of emr involving the charge transfer process may be responsible for the
absorption in many transition metal complexes. This type of transition involves the
electron transfer (via an internal oxidation reduction process) between the two
components of a complex upon the absorption of a photon, where one component of
the complex acts as an electron donor while the other component of the complex acts
as an electron acceptor. Before going further let us try following SAQs.

SAQ 3
Identify the type of absorption π → π *, n → π * or n → σ* among the following
compounds:
O λmax

50
a) CH3CCH3 293 Optical Methods
b) CH3 COOH 294
c) CH3NO2 280
…………………………………………………………………………………………
…………………………………………………………………………………………
…………………………………………………………………………………………

SAQ 4
Arrange following transitions in increasing order
n → π*, π → π*, σ → σ *, n → σ *
…………………………………………………………………………………………
…………………………………………………………………………………………
…………………………………………………………………………………………

19.4 ULTRAVIOLET-VISIBLE SPECTROPHOTOMETRY

Ultraviolet-visible spectrophotometry is a discipline in which the absorption of
ultraviolet (UV) or visible light is used to detect one or more components in a solution
and measure the concentration of these species. The prime advantage of using this
discipline as an instrumental technique is that traces of substances can be determined
in a simple way, which is not possible with classical methods. This technique is one of
the oldest instrumental methods of analysis. In the early stage of development of this
physico-chemical method of analysis, the natural or the artificial white light was used
as light source. The measurements were made with simple instruments and in the
earlier development of the technique the naked eye was used for comparing the colour
intensity of the solution. However, naked eye was replaced by photometers for the
measurements of colour intensity and the instrument thus developed were known as
photometers or colorimeters. Filters were introduced in colorimeters to choose a
spectral band for colour measurement. Later on instrument, which could select a
definite wavelength, were introduced and such instruments are known as
spectrophotometers. Ordinary colorimetric methods do not give accuracy greater than
about 1 percent. For greater accuracy spectrophotometric methods have to be used.

However, both these instrumental techniques have the great advantage of being more
simple and economic.

19.4.1 Components of Instruments for Absorption Measurements

An instrument to be used for measuring intensities of emr requires five basic
components shown in the block diagram in Fig. 19.10.

Radiant Wavelength Sample Detector Signal

Source Selector Indicator

Fig. 19.10: Block diagram showing basic components of an instrument used

for measuring absorption of radiation.

A suitab le radiation source must provide sufficient radiant power over the wavelength
region where absorption is to be measured. The proper wavelength is achieved by a
wavelength selector either by filtering the radiation of the source or by using a
monochromator. Filters are applied in filter photometry and are used mainly in the
visible range whereas the use of a monochromator is applied in spectrophotometry in
the ultraviolet, visible and infrared ranges. The selected wavelength is allowed to pass 51
Instrumental Methods through the solvent or sample placed in the cuvette. The detector measures the
of Analysis intensity of the transmitted radiation and gives a signal, which can be read by a signal
indicator. Now we describe these components in greater detail.

Sources: The usual source of radiation in the ultraviolet region (180-350nm) is

hydrogen or a deuterium discharge lamp operated under low pressure. The important
feature of these lamps is to maintain the discharge to a narrow path with the help of a
mechanical aperture between the cathode and the anode. The use of deuterium in place
of hydrogen enhances brightness of the lamp.

The source of radiation in visible region as well as in near infrared region (325 nm-3
µm) is usually an incandescent lamp with a tungsten wire filament. The coiled wire
filament is enclosed in a sealed bulb of glass filled with an inert gas or vacuum. On
heating the filament by an electric current, radiation is emitted. Incandescent lamps are
rugged and low cost sources, which are adequate to work in the visible and near
ultraviolet region.

Wavelength Selector: The purpose of wavelength selector is to isolate a narrow

range of wavelength from the source. Radiation sources such as tungsten lamps,
hydrogen discharge tubes emit almost continuous radiation over relatively wide ranges
of frequencies. Narrow spectral regions of selected wavelength ranges may be
isolated from a continuous spectrum by the use of filters. However, a monochromator
is required to produce a monochromatic radiation of the desired frequency. A narrow
range can be isolated by either filters or still narrower (monochromatic) can be
achieved by a monochromator equipped with a prism or grating as the dispersing
device.

Filters: An absorption filter is a coloured piece of glass, which absorbs light of some
wavelength to a greater extent than others. We know that white light is made up of
seven different colours (VIBGYOR). An object of a particular colour looks of that
colour because this colour is transmitted and its complementary colour is absorbed. As
a first approximation, the filter should transmit a colour nearly complementary to that
of the sample. Or in other words, the filter should absorb the light of the colour, which
is transmitted by the sample. For example, a blue cobalt glass transmits blue violet
light, but absorbs yellow light. The use of such a filter is illustrated in Figure 19.11.

Yellow light Yellow light absorbed

Violet light Violet light transmitted

Filter

Fig. 19.11: Use of blue cobalt glass filter.

To find the colour of the filter we can take the help of colour wheel (Fig. 19.12).

O
520 nm
Y R
580 nm 700 nm

52
G V
530 nm 420 nm
B
470 nm
 Optical Methods

Fig. 19.12: Colour wheel.

In Fig. 19.12 the colours are shown with their approximate wavelengths. The colours,
which face one another, are said to be complementary to each other. A filter of
complementary colour is most suitable for the measurement. For example, for a red
coloured solution, its complementary, that is, a green coloured filter should be used
which indicates that the λ max of a red coloured solution should lie between 490-525
nm.

The performance of characteristic of a filter is judged by its effective bandwidth,

which is expressed as the wavelength interval at one half of the maximum
transmittance value when the response of a filter is plotted with variation of
wavelength. See Fig.19.13.

Amax

%A

Amax
2
= Effective
band width

Fig. 19.13: Response of a filter.

The narrower the effective bandwidth (∆λ) the better the filter is. Generally ∆λ is of
the order of 30 to 50 nm.

Absorption filters are simple and are totally adequate for many applications in visible
range of emr spectra. 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.

Monochromators: In order to get approximately monochromatic radiation a

dispersing device is to be used. A monochromator, in general consists of an entrance
slit for the heterochromatic radiation from the source, a collimating lens or mirror (to
make the radiation parallel), a prism or grating to disperse the radiation into its
component wavelengths, a lens or mirror to focus the dispersed radiation (more or less
monochromatic), and an exit slit through which the monochromatic radiation is
allowed to pass.
53
Instrumental Methods Grating monochromators are now more widely used than the prism monochromators.
of Analysis A grating is a small piece of metal or glass with numerous parallel and identical
grooves (as many as 10,000 grooves/cm) ruled on it. The grating is ruled with a
diamond knife on the metal piece with numerous precautions. Ruling a high quality
grating is a tedious task. Replica gratings, which are casted by pouring molten plastics
on the original grating, are now used. The replica grating are less expensive and are
not much inferior to the original grating. Advancement in the grating was the
development of concave gratings where the use of lens to focus the radiation is not
required.

Fig. 19.14 represents the optical design of a typical monochromator with grating as the
dispersing device. Light striking the grating is diffracted so that different wavelengths
come off at different angles. Rotating the grating allows radiation of the desired
wavelength to be selected.

Incident
Diffracted beams
Selected beams
wavelength

Slit Grooves

Grating

Fig. 19.14: A typical monochromator-employing grating.

Sample: In ultraviolet-visible spectrophotometery sample is introduced in a cell

called cuvette. The cuvettes must be constructed of a material that does not absorb
radiation in the region of interest. Quartz cells can be used in the range 190nm-84µm.
Silicate glass cells are adequate in the whole visible range (375-950nm), and also a
part of UV region. Cylindrical cuvettes are often used in the interests of economy; but
care should be taken that each cuvette is marked so that its insertion in the cuvette
holder always provides the same incident and emergent surface. The cuvettes are
usually one centimetre in path length.

Detectors : The detectors of most instruments generate a signal, which is linear in

transmittance that is 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 to read out.

A detector is, of course, a transducer, which converts one type of signal to another.
Early instruments used eye or photographic plate as the detector. Most modern
detectors are the photoelectric detector where the intensity of emr (i.e. energy of
photon) is converted into electrical energy causing an electron flow and subsequently,
into a current flow or voltage in the read out circuit.

Human Eye as Detector: In older days identification of colours through naked eye
was one of the major analytical tools. The eye of a common person is quite sensitive to
notice differences in radiant power transmitted through two coloured solutions. It is a
natural photosensitive detector in the visible range. Fig. 19.15 shows average
sensitivity characteristics for the human eye.
54
 Optical Methods

100

Response
50

0 400 500 600 700 nm

Fig. 19.15: Response of an average human eye as a function of wavelength.

The optical nerves carry the signal from retina to the brain through rods and cones.
Comparison of colours and their intensities can be made, approximately, by matching
with a reference.

Photoelectric Detectors: In these detectors the radiant energy is converted into

electrical energy. They are classified as photovoltaic cells and photo emissive
detectors. You will know about them in the following discussion.

Photovoltaic Cell: The photovoltaic cell is used primarily to detect and measure
radiation in the visible region. A typical photovoltaic cell or photo cell (schematically
shown in Fig.19.16) consists of a flat iron or copper electrode (anode) upon which is
deposited a layer of a semi conducting material, such as selenium (or cuprous oxide).
The selenium layer is coated with a transparent film of silver, gold, or some other
metal, which is protected by a transparent plate of glass. A metal ring, which works as
the other terminal (cathode) of the cell, is pressed on the transparent metallic film. The
two terminals are connected to a galvanometer. The whole arrangement is placed in a
plastic case.

Glass Thin layer of silver

Selenium Plastic
Iron case

Fig. 19.16: Schematic of a typical photovoltaic cell.

When the radiation fall upon the cell a current flows through the galvanometer. The
current depends upon the intensity of the photons impinging on the cell. Under proper
conditions the current through photovoltaic cells is proportional to the energy
absorbed per unit of time. Photovoltaic cells require simpler circuitry and no
amplification. In general, they are used in filter photometers.

Photo emissive Detectors: The photo emissive detectors are very sensitive and are
employed to detect even very small variations in the light intensity that is not possible
with a photovoltaic cell. Therefore, in spectrophotometers where the wavelength
resolution using monochromators is the essential requirement, such detectors are made
use of. Two kinds of photo emissive detectors are in use: (i) vacuum phototubes and
(ii) photo multiplier tubes.

55
Instrumental Methods (i) Phototubes: A phototube (Fig. 19.17) consists of two electrodes, a semi
of Analysis cylindrical cathode and a wire anode sealed inside in an evacuated transparent vessel.
The cathode is coated with a photo emissive material, such as potassium or ceasium.
Phototubes with a potassium coated cathode are employed in the range 200-600nm,
and ceasium coat ed cathode are utilized mainly in the 600-1000nm range. The most
sensitive cathodes are bi-alkali types, for example, one is made of potassium, ceasium
and antimony.
Electrons
Wire anode

Photon beam
Cathode

DC amplifier
and readout

90 V DC
Power supply

Fig. 19.17: Schematic diagram of a phototube.

Fig. 19.17 is a diagram of a phototube and its accessory circuit. When photons of
sufficiently high energy hit the cathode, the electrons are dislodged from the photo
emissive material by photoelectric effect and are collected at anode. The number of
electrons ejected from the photosensitive cathode surface is directly proportional to the
radiant power of the beam striking the cathode surface. When a potential is applied
across the electrodes through a dc source, the emitted electrons flow to the wire anode
generate a photocurrent, which depends on the radiant power of the beam and the
applied potential through the dc source. At the saturation potential (about 90 V) across
the two electrodes, essentially all of the electrons emitted by the cathode then become
independent of applied potential and directly proportional to the radiant power of the
beam. Thus, the current flow in the system is related to photon flux coming to the
concave surface of the photosensitive cathode.

(ii) Photo multiplier Tubes: Photo multiplier tubes, which do not require external
amplification proved to be more sensitive and accurate than phototubes. Fig. 19.18
shows the cross-section and electrical circuit of a phtomultiplier tube, which of course,
may be considered as a combination of several phototubes arrang ed in a special
manner. The intermediately dynodes of photo multiplier tubes are covered with a
material which emits several (2 to 5) electrons for each electron being collected on its
surface. The dynodes behave in a manner that the anode of the first stage is the
cathode of the second.

The primary electrons ejected from the first cathode (e1) strike a small area on the first
dynode (e2), which is about 90V more positive than the first cathode (e3). The dynode
(e2) covered with photo emissive material ejects 2 to 5 secondary electrons for each
electron collected on its surface. These secondary electrons strike the second dynode
(e3) and the process multiplies on in each stage. Thus electron amplification occurs on
the dynodes, and for a tube of 9 dynodes the overall amplification factor is between 2 9
and 59.

900 V dc
Several electrons + -
for each
incident electron 90 V Quartz
Numerous envelope
electrons
Quartz 3
5 for each
envelope photon
4 1 9 8 7 6 5 4 3 2 1
6 2 Anode Cathode
8 Grill R Numbered dynodes
7 shown in (a)
Radiation, h v
9 -
56 Anode, -10
7
Photoemissive To readout
+
electrons for Cathode
each Photon

(a) (b)
 Optical Methods

Fig. 19.18: A Photo multiplier tube: a) Cross-section; b) Electrical Circuit.

19.4.2 Some Typical Instruments

The instrumental components discussed in the preceding section have been combined
in various ways to produce a variety of commercial instruments for measurement of
absorption of radiation. In all absorption measurements, the intensity of radiation
transmitted through sample is compared with that of the reference (solvent/blank);
hence relative rather than absolute measurements are made.

In a single beam instrument, the reference and the sample are placed successively in
the path of the monochromatic beam. In a double beam instrument the monochromatic
beam is divided by optical means into two equal intensities. One beam passes through
the reference and the other through the sample. Some advance type of
spectrophotometers are capable of giving direct results of analysis in concentration
units and other information with the help of microprocessors used in automation. For
example, one digital spectrophotometer with programmable statistical calculator can
provide storage of data, calculation of first and second derivative spectra, peak
location, peak area etc. Selection of the instrument is governed by the type of the work
and the cost of the instrument.

A few simple instruments that are typical of ones the student at this stage is likely to
encounter will be described in this section.

Filter Photometers
A relatively inexpensiv e and simple type of instrument is a filter photometer that
works around a set of filters in the visible range. Such instruments are adequate for
many methods especially for absorbing systems with broad absorption bands. There
are two types of filter photometers, namely, single beam and double beam instruments.
The single beam filter photometer will be discussed first.

Single Beam Filter Photometer

It consists of a source of light, S, which is simply a light bulb; a condenser lens, L, to
produce parallel radiation beam; a filter, F, to give the appropriate wavelength band; a
cuvette C; a photovoltaic cell, D, as detector; and a galvanometer as a signal indicator
(See Fig. 19.19).

Variable Solvent
diaphragm cell
to set 100% T
D
S L F %T
50 100
(a) Single- 0
beam C
photometer

Tungsten
lamp Microammeter
Filter Shutter Sample Photocell
Cell

Fig. 19.19: Schematic diagram of a single beam filter photometer.

57
Instrumental Methods
of Analysis The radiation from the light bulb passes through a convex lens fitted in such a way
that its distance from the light bulb is equal to the focal length of the lens. This
arrangement gives the parallel beam of radiation, the parallel beam falls on the filter,
which permits to pass a narrow band of wavelength. The filtered radiation is passed to
the sample cell (cuvette), and the transmitted radiation power is evaluated by a
photovoltaic cell. The function of the photovoltaic cell is to convert the energy of
photons into electrical energy, which moves the galvanometer scale that is calibrated
for percent transmittance or for absorbance.

Working
The following steps are involved in measuring absorbance and transmittance of the
sample solution.
1) Turn the instrument “on”, using the power switch, and wait for about 15 min to
warm up.
2) Insert the proper filter in its place in the instrument.
3) Fill the cuvette with blank (or solvent) and place it in the light path at proper
position.
4) Adjust the pointer of the galvanometer scale at 100% T (or zero A) mark.
5) Remove the solvent/blank from the cuvette, fill it with sample solution and
place in the path at proper position.
6) Read the meter and note the percent transmittance/absorbance.

The single beam filter photometers have the advantages of simple construction, low
cost and simple operation; but have limitations of low accuracy and low sensitivity
because of small galvanometer scale (about 10 cm).

Double Beam Filter Photometers

In a double beam filter photometer the light beam is divided into two parts and two
photocells are used as detectors. The schematic diagram of a double beam filter
photometer is given in Fig. 19.20. S is the Source, which is a tungsten filament lamp.
L is the lens, which is placed at a distance equal to its focal length from the source.
The parallel beam after the lens then falls on the filter F that permits only a narrow
band of wavelength to pass through it. The filtered radiation is allowed to fall on half -
silvered mirror. This divides the original beam into two portions. One beam passes
through the cuvette C1 to fall on photovoltaic cell D1 and the other beam through
cuvette C2 and then falls on identical photovoltaic Cell D2. P 1 and P2 are the two
potentiometers and G is the galvanometer, which is used as the null detector. P 1 is
calibrated in percent transmittance units. K1 and K2 are the two keys.

Filter Shutter Sample cell Photocell

Half-silvered
mirror D2
S
Double-
beam
photometer

Tungsten L F C2
lamp
Solvent C1
cell
100 Null
detector
Reference
D1 C
photocell
%T 50 K2
K1 G
0 P1 P2

Fig. 19.20: Schematic of typical double beam filter photometer.

58
Working Optical Methods
1. Turn the instrument “on” and allow warming up for about 15 min.
2. Place the appropriate filter in its place in the instrument.
3. Fill the cuvettes C1 and C 2 with blank and solvent, respectively and place it in
the light path
4. Put K1 at 100% T value on the scale of potentiometer P 1 and move K2 on P2 so
that the galvanometer G reads zero.
5. Fill the sample solution in the cuvette C 2 and place in the light path.
6. Move K1 on the potentiometer P1 scale so that the galvanometer reads again
zero (without disturbing the position of K2). Note the %T on P1. The double
beam filter photometer has the following advantages over a single beam
instrument:
i) Since the beam of light is divided into two parts and is allowed to fall on
two identical detectors, the fluctuations (if any) of the source will not
disturb the reading, which is noted after balancing the two
potentiometers.
ii) Since the galvanometer is used as a null detector, no current is used in
moving the galvanometer needle and the results are more accurate than
that with a single beam instrument.
iii) Since the reading is noted on a large potentiometer scale, the sensitivity is
high.

Spectrophotometers
Spectrophotometers are more sensitive instruments than filter photometers. A
spectrophotometer is usually a combination of a monochromatic and a photometer.
The light is monochromated by a diffraction grating and slit device. Nevertheless
several designs of spectrophotometers are available, we shall consider here the Bausch
and Lomb spectronic – 20 Spectrophotometer, which is simple to use and is
satisfactorily precise.

The operating features of Bausch and Lomb spectronic – 20 are shown in (Fig. 19.21a)
and the schematic optical arrangement in Fig. 19.21b. It is a single beam
spectrophotometer operating between 340 – 950 nm. With standard phototube the
basic range is 340 to 600 nm, which is extended to 950 nm by adding a red filter and
replacing the standard phototube with the red phototube. The scale of the instrument is
colour-coded to correspond to the operating range of the phototube: black gradations
for the basic 340-600 nm range and red gradation for the 600-950 nm range of the
optional red phototube/filter combinations. Readings are taken directly from the meter
in either absorbance or transmittance mode.

Absorbance
%T scale
scale

Wavelength
Cell selection
compartment

On-off %T Light control

calibration (100%T calibration)

59
Instrumental Methods
of Analysis

Fig. 19.21: a) Bausch and Lomb spectronic – 20 Spectrophotometer;

b) Schematic optical lay out for the Bausch and Lomb Spectronic –20 spectrophotometer.

Working
1. Plug the instrument into a grounded outlet to oper ate on a 230 V AC line.
2. Turn the instrument “on” and allow warming up for about 15 min.
3. Select the wavelength with the wavelength selector knob.
4. Choose matched cuvettes of the appropriate path length (usually 1 cm) for the
analytical method. The cuvettes of the same path length must be used for all
blanks, standards, and samples.
5. Fill one cuvette with blank (or solvent) having sufficient solution to align with
the mark on the cuvette. The solution volume should be enough to cover the
light beam passing through the sample compartment.
6. Open the sample compartment cover and insert the cuvette containing blank
into the sample compartment.
7. Close the sample compartment cover.
8. Set zero absorbance or 100% transmittance on the scale for the blank using the
control knob located on the left side.
9. Remove the blank from the sample compartment.
10. Fill the matched cuvettes with standard solutions and insert in the sample
compartment one after the other. Read and record the absorbance values for
each standard solution.
11. Construct a calibration curve by plotting the absorbance on the y-axis vs the
concentration of each standard solution on the x-axis.
12. Fill the matched cuvette with the sample to be measured and insert in the
sample compartment. Read and record the absorbance value. From the
calibration curve read the concentration corresponding to the absorbance value
of the sample.

19.4.3 Analytical Technique

In this part we will take up general procedures in ultraviolet-visible spectrphtometeric
analysis. In quantitative det ection of a substance, the Beer-Lambert’s Law forms the
basis of the measurement procedure. The amount of light radiation absorbed by a
compound is directly related to the concentration of the compound. Following steps
are involved in general measurement procedure:
1) Preparation of sample to make absorbing species
2) Selection of wavelength
3) Preparation of the calibration plot

Preparation of sample to make absorbing species

60
Samples absorbing in the wavelength range 200 to 800 nm are generally analysed by Optical Methods
ultraviolet-visible method. But it is a common practice; measurements are carried out
in visible range. Sometimes the substance being analysed has its own characteristically
strong absorption in the visible range but more often it may require the addition of a
reagent that form derivative or complex with the necessary high absorptivity.

While choosing the reagent, following points should be considered.

i) The reagent should react selectively with the substance to be determined.
ii) Conditions must be chosen to obtain optimum colour formation if we wish to
detect substance colorimetrically.
iii) Product or complex formed should have high molar absorptivity and should be
stable.

Selection of Wavelength
Quantitative analysis is generally made at λ max. This is because this give rises to
maximum sensitivity in the analysis. λ max is determined by plotting absorption
spectrum.
Spectrum: A plot of absorbance versus wavelength is known as absorption spectrum.
Usually the wavelength is as abscissa and absorbance as the ordinate. All ultraviolet-
visible spectrophotometers have a wavelength scale properly graduated in nanometer
or in Angstrom units.

Proper Wavelength: An absorption spectrum (a plot of A vs λ discussed above) for a

single absorbing species will normally yield a curve having a maximum value of
absorbance at a particular wavelength. This wavelength is designated as λ max. At this
wavelength, the sensitivity is maximum, that is, the change in absorbance per unit
change in the concentration of the absorbing species is a maximum. λ max is the proper
wavelength for determination. However, it should not be located in that portion of the
spectrum where a small change in wavelength causes excessively high change in
absorbance. Under such conditions, λ opt, optimum wavelength, must be used. In
Fig.19.22a the spectrum appears with only one maximum with a suitable curvature
and λ max is also λopt.
A

1 2

max = opt max opt

(a) (b)

Fig. 19.22: Absorption spectra to locate proper wavelength for determination.

61
Instrumental Methods In Fig. 19.22b λmax (λ 1) is in the form of a sharp peak where a small change in
of Analysis wavelength will cause an excessively high change in absorbance, therefore it is not the
proper wavelength for spectrophotometeric determination. Here λ2 with a suitable
curvature, though not λmax , should be selected as proper wavelength (λopt) for
determination.

Preparation of the Calibration Plot

After setting the instrument, with the monochromator providing the optimum
wavelength, a plot is prepared for absorbance values obtained by inserting solutions of
known concentrations in successively increasing order. A plot (See Fig. 19.23a)
obtained for absorbance versus concentration is known as the calibration plot.
Basically, where Beer’s law is obeyed, a plot of absorbance versus concentration will
yield a straight line. A plot of absorbance versus concentration may deviate from a
straight line after certain range of concentration. Such a departure yields a zone of
non-conformity to Beer’s law.
Absorbance

Absorbance

Concentration Concentration

Fig. 19.23: a) Calibration plot; b) Finding the concentration of unknown sample.

For measuring concentration of the unknown solution, it should be adjusted in a

manner to yield concentration ranges where Beer’s law is obeyed. After measuring the
absorbance of the unknown solution, its concentration may directly be read from the
calibration plot Fig. 19.23b.

Concentration of unknown may also be calculated using a ratio method by a

comparison of its absorbance with the absorbance of a solution of known
concentration as:
known × A unknown
cCunknown
cunknown =
A known

19.4.4 Determination of Substances in Water, Soil and Air

Ultraviolet-visible spectrophotometer is most widely used instrumental method,
particularly for routine work at moderately low concentration environmental detection.
62
In metal ions detection in water and soil, ions are converted into coloured species by Optical Methods
treating them with chromophoric reagent such as dithizone or diethyldithiocarbamate.
In table 19.3 we have summarised few metal reagent pairs used in metal ion detection
in water and soil. Inorganic anions and ammonia can also detected by forming
coloured derivative with organic compounds, such as nitrate with xylenol (See Table
19.4). For the detection of gases in air, they must first be absorbed in a selective
reagent and then reacted to give a dyes stuff, which can be measured using
spectrophotometer (Table 19.5), such as a diazo dye for the determination of oxides of
nitrogen.

Organic impurities such as anionic surfactants, cationic surfactants, phenols etc. are
detected by derivatizing or pairing with a coloured dye molecules.

Anionic surfactants: Methylene Blue

Cationic surfactants: Bromophenol Blue
Phenols : 4-Aminoantipyrine

Table 19.3: Reagents for Spectrophotometric determinations of metals.

Metal Reagent Wavelength (nm)

Aluminium Chrome azurol S 545

8-hydroxyquinoline 390
Antimony Iodine 425
Arsenic Diethyldithiocarbamate 515
Bismuth Dithizone 490
Iodide 465
Xylenol Orange 450
Cadmium Cadion 480
Dithizone 520
Chromium Diphenylcarbazide 545
Cobalt Nitrosonaphthol 415
PAR 510
Thiocyanate 620
Copper Diethyldithiocarbamate 436
Dithizone 550
Iron 1,10-phenanthroline 512
Thiocyanate 495
Lead Dithizone 520
PAR 520
Manganese PAN 564
Mercury Dithizone 485
Molybdenum Thiocynate 470
Nickel Dimethylglyoxime 400
Selenium Diaminobenzidine 420
Tellurium Bismuthiol II 330
Vanadium 8-Hydroxyquinoline 550
Zinc Dithizone 538
PAN 560

Table 19.4: Reagents for spectrophotometric determinations of anions

and ammonium.

Anion Reagent Wavelength (nm) 63

Instrumental Methods
of Analysis Ammonium Hypochlorite/phenol 625
Bromide Hypochlorite/Phenol Red 580
Chloride Mercury thiocyanate/Fe(III) 480
Chlorine Orthotolidine 625
Cyanide Chloramine-T/pyridine/barbituric 580
acid
Iodide Bromine/starch 590
Nitrate 3,4-Xylenol phenoldisulphonic acid 410
Nitrite Sulphanilic acid with naphthylamine 520
sulphonic acid
Phosphate Molybdate/vanadate (yellow) 400
Molybdenum Blue 780
Sulphate Barium Chloranilate 530
Sulphide Dimethylaminoaniline/Fe(III) 662

Table 19.5: Reagents for spectrophotometric determ inations of gases.

Gas Reagent Wavelength (nm)

Ozone Iodide/starch 590
Hydrogen sulphide Zinc acetate, then 662
Dimethylaminoaniline/Fe(III)
Oxides of nitrogen Sulphanilic acid with naphthylamine 520
sulphonic acid
Sulphur dioxide Tetrachloromercurate, then 545
Pararosaniline/formaldehyde
Formaldehyde Phenylhydrazine/ferricyanide 515

SAQ 5
Write down the steps of a typical procedure involving a spectrophotometric method of
analysis of the substance.
…………………………………………………………………………………………
…………………………………………………………………………………………
…………………………………………………………………………………………

19.5 EMISSION METHODS

You may be familiar with flame test for sodium, which emits a yellow light, and for
other alkali and alkaline earth metals. Beside this, many other metallic elements, when
subjected to suitable excitation, also emit radiation of characteristic wavelengths.
Under proper control conditions, the intensity of the emitted radiation at some
particular wavelength can also be correlated with the quantity of the element present.
Thus both a quantitative and a qualitative determination can be made using emission
methods. The various analytical methods, which make use of emission spectra, are
characterized by the excitation method used, the nature of the sample (whether solid or
liquid) and the method of detecting and recording the spectra produced. Methods in
this category are Flame emission spectrphotoometry (Flame photometry), inductively
coupled plasma, atomic emission spectrophotometry etc. Out of this flame photometry
is more wid ely used method. This method is used in water and soil analysis for
determining the concentration of alkali and alkaline earth metals such as sodium,
potassium and calcium.
64
 Optical Methods
19.5.1 Flame Photometry
In this technique a flame is used to excite the atoms. When a solution containing an
ion is nebulized through a flame, a series of process occur:
i) the solvent is vaporized leaving particles of salt(s)
ii) the salt is subsequently vaporized and dissociated into atoms
iii) some of the atoms are excited by the flame
iv) the excited atoms emit radiation characteristic of their species

Because of the relatively low energy of the flame, only few elements can be excited.
Therefore, the main application of flame photometry in the quantitative determination
of the alkali and alkaline earth elements at concentrations as low as 0.1 µg/cm3
solution (0.1 ppm). A diagram showing the basic elements of a flame photometer is
given in Fig. 19.24. The basic components are the flame, monochromator, and detector
readout system. The flame is produced with a burner -nebulizer assembly as shown in
Fig19.25. The fuel and oxidant are fed into two separate chambers within the burner
and mix outside the exit orifices. Thus, a turbulent flame is produced. As the oxidant
flows through the sample capillar y a vacuum is produced which draws the solution
into the flame.

A number of fuel gases, such as acetylene, hydrogen or the liquid petroleum gas used
for heating in most laboratories can be used for the flame. The oxidant used is usually
oxygen rather than air. Flames yielding high temperature are capable of exciting more
elements and so are more versatile. List of some common flame gas mixtures are
given in Table 19.5

Table 19.5: Some common flame gas mixtures.

Fuel Oxidant Temperature
Hydrogen Air 2000
Hydrogen Oxygen 2200
Acetylene Air 2000
Acetylene Oxygen 2800
Natural Gas (LPG) Air 1900
Acetylene N2O 2800

The emitted radiation is passed through a monochromator or prism, which separates

the various wavelengths so that the desired regio n can be isolated. A photocell as
detector and some type of amplifier are than used to measure the intensity of the
isolated radiation.

Slits
Lens

Burner Monochromator Readout

nebulizer
Photomultiplier

Fuel Oxidant
supply supply

65
Instrumental Methods Fig.19.24: Basic elements of flame photometer.
of Analysis
The emission spectrum of each metal is different and its intensity depends upon the
concentration of atoms in the flame, the method of excitation used, and the after -
history of the excited atoms. Sodium produces a characteristic yellow emission at 589
nm, lithium a red emission at 671 nm, and calcium a blue emission at 423 nm. Each
also gives a less intense emission at shorter wavelengths. Concentration of these
elements can be measured down to 0.1 µg/cm 3 (or µg/dm 3) or less with some degree of
accuracy, depending upon the sensitivity of the instrument used.

Application of Flame emission spectrophotometry (FES)

In Table19.6 we have listed typical detection limits for the determination of selected
pollutant elements by flame emission spectrometry in nitrous oxide-acetylene flame.
In the present context, FES should be regarded as an inexpensive complementary
techniques to AAS (Atomic Absorption Spectrophotometry).

Table 19.6: Elements that can be determined by Flame emission

spectromotometry.

Element Wavelength Detection Element Wavelength Detection

nm limit nm limit
µg/cm3 µg/cm 3
Ag 328.1 20 In 451.1 2
Al 396.2 5 Mg 285.2 5
Ba 553.6 2 Mn 403.1 5
Ca 422.7 0.1 Mo 390.3 5
Co 345.4 50 Ni 341.5 300
Cr 425.4 5 Pb 405.8 200
Cu 324.7 10 Sr 460.7 0.2
Ga 417.2 10 Tl 377.6 20

19.5 .2 Atomic Absorption Spectrophotometry

Atomic absorption although is an absorption method but it is very similar to flame
emission spectrophotometry. In this technique radiation is absorbed by non excited
atoms in the vapour state. This method has following advantages over flame emission
because:
i) More elements can be quantitatively determined
ii) The spectral interference are decreased
iii) The sensitivity is higher for most elements

Like the flame photometer, an atomic absorption instrument consists of a light source,
flame unit, a prism or grating to disperse and isolate the emission lines, and a detector
with appropriate amplifiers. The light source (Hollow Cathode Lamp) emits a stable
and intense light of a particular wavelength. Each element has characteristic
wavelengths that it will absorb. A light source with wavelength readily absorbed by
the element to be determined is directed through the flame and a measure of its
intensity is made without the sample, and then with the sample introduced into the
flame. The decrease in intensity observed with the sample is a measure of the
concentration of the element.

The amount of radiation absorbed follows Beer -Lambert’s law i.e. it is proportional to
the concentration of the element in the sample. A disadvantage of this method is that a
different light source/hollow cathode lamp has to be used for each element. The
66 advantage of AAS is that it is quite specific for most of the elements. Absorption
depend upon the presence of free unexcited atoms in the flame which ar e generally Optical Methods
available in much greater abundance than the excited atoms.

Flame

Light
source Atomizer
burner Photo
Prism or Grating Slit tube

Sample

Fig. 19.26: Schematic diagram of an atomic absorption spectrophotometer.

Application of AAS in element determinations

Atomic absorption spectrophotometry has been used for the determination of
approximately 70 elements. Application include clinical and biological samples,
forensic materials, foods and beverages, water and effluents, soils, plants, and
fertilizers, iron, steel and various other alloys; minerals, petroleum products,
pharmaceuticals and cosmetics.

A modification to the flame AAS is termed electrothermal atomic absorption

spectrophotometry, which employs a small graphite furnace, which allows analysis for
many heavy metals in further lower ranges i.e. in m icrogram per dm 3 range. Here, the
atomizer burner is replaced by a small cylindrical graphite furnace/tube that can be
programmed through a series of different temperatures. The radiation from the cathode
lamp source passes through the open ends of the horizontal cylinder through a hole in
the side and the temperature programme is initiated. The temperature first rises to just
over 100°C to allow the sample water to evaporate, leaving the metal containing salts
behind. The temperature then increases to several hundred degrees Celsius (upto
3000°C), which volatilizes the cations so they fill the cylindrical space, and the
particular cation to be determined absorbs the characteristics radiation form the
cathode tube. The graphite furnace, as it is called, allows the development of a greater
density of atoms and thus affects greater sensitivity to the atomic absorption
procedure.

Inductively Coupled Plasma (ICP)

ICP method (emission spectroscopy) is a relatively new technique developed in the
1970’s for analys is of trace metals. ICP method uses Plasma Sources for higher-
energy excitation source. The advantage of these more energetic automization sources
is there is lower inter element interference, good spectra can be obtained for many
elements under the same excitation conditions and hence spectra for many elements
can be recorded simultaneously. The disadvantages are high instrument and operating
costs, the need for more skilled operators and often less precision than with atomic
absorption.

In the ICP method, a stream of argon gas flows through three concentric quartz tubes,
which are surrounded by a water -cooled induction coil that is powered by a radio-
frequency generator to form a strong magnetic field. When a spark initiates ionisation
of the argon, the ions with their associated electrons are caused to follow a spiral flow
pattern within the tubes as a result of the magnetic field and heating is the result of
their collisions and resistance to this movement. The result ing temperature is 4000 to
67
Instrumental Methods 8000°C which is two to three times hotter than obtained with the hottest of the
of Analysis combustion flame temperatures. This temperature is sufficient to almost completely
dissociate molecules there by making atomic emission highly efficient. The sample to
be analysed is introduced at the head of the argon flow and into the central tube. The
emissions produced by the elements are focussed through an entrance slit for either a
monochromator or polychrometor, and a portion of the spectrum is isolated for
intensity measurements. The instrument can make measurements in the entire
ultraviolet-visible spectrum form 180 to 900 nm.

SAQ 6
Describe the basic components of Flame emission spectrophotometer.
…………………………………………………………………………………………
…………………………………………………………………………………………
…………………………………………………………………………………………

19.6 SUMMARY
In this Unit, you learnt about the nature and characteristics of electromagnetic
radiation. Various parameters such as frequency, wavelength etc. associated with
electromagnetic radiations were defined and their interrelationship was discussed.
Then we discussed the fundamental law of qualitative analysis, Beer -Lambert’s Law.
Absorbing species responsible for ultraviolet-visible electromagnetic radiations in
organic and inorganic substances were described. This was followed by the functions
of the components of an ultraviolet-visible spectrophotometer. Then wide range of the
application of ultraviolet-visible spectrophotometry in environmental analysis was
highlight. Finally flamephotometry and atomic absorption spectrophotometry were
discussed in brief.

19.7 TERMINAL QUESTIONS

1. Define following terms:
a) absorbance; b) percent transmittance; c) absorptivity;
d) molar absorptivity.

2. List four different cases for deviation from Beer-Lambert’s Law.

3. What are the most frequent electronic transitions in organic molecules observed
during absorption of electromagnetic radiation in ultraviolet-visible region?

4. Define chromophore and auxochrome.

5. What is the main advantage of the double beam photometer over single -beam
photometer?

6. The optimum wavelength used for the analysis of the mixture of o-xylene and
p-xylene are 271 nm and 275 nm. The absorbance of the single components
and the mixtures at these two wavelengths are given below.

Solution 271 nm 275 nm

o-xylene 0.90 0.10

p-xylene 0.34 1.02
68
 mixture 0.47 0.54 Optical Methods

Calculate the various absorptivities and the concentration of o- xylene and

p-xylene. Assume b = 1 cm.

7. Compare flame emission and atomic absorption Spectrophotometry with respect

to instrumentation.

19.8 ANSWERS

Self -Assessment Questions

1 1
1. ν= = = 2.5 × 106 m− 1 = 2.5 × 10
10−44 cm−1
λ 4000 × 10 −10
cc 2.998 × 108
ν = ∈= = 7.495 × 1014 Hz
λ 4000 × 10 −10
Po
2. A = log =∈
∈ bc
P
85.4
= log = ∈ × 2 × 1 × 10−4
20.3
= log 4.207 = ∈ × 2 × 10−4
0.62396
∈=
∈ × 104 = 3.12 × 103 dm3 /mol.cm
mol cm
2
3. a) n → π * ; b) n → π * , c) n → σ* *

4. σ → σ* > n → σ * > π → π * > n → π*

5. i) Preparation of sample to make absorbing species,

ii) Selection of wavelength,
iii) Measurement of absorbance for the reagent blank, standards and sample at
λmax ,
iv) Preparation of calibration curve, and
v) Read the conc. of sample from calibration curve.

6. Flame, monochromator and detector readout system

Terminal Questions
1. Absorbance (A): The logarithm (base 10) of the ratio of incident radiant power
to the radiant power transmitted through sample.
Po ??
A = log or log
P ? 69
Instrumental Methods
of Analysis Percent Transmittance (T)

The ratio of radiant power transmitted through sample to the incident radiant
power is known as transmittance.
P
T =
Po
Where transmittance is expressed in percentage, it is called percent
transmittance
o T = P × 100
o Po

Absorptivity (a):

It is defined as the absorbance of the solution having unit concentration (g dm3)

and unit path length (1 cm). That is
a = A/bc

Molar Absorptivity (ε ):
When the concentration of absorbing substance is taken in mol dm 3, the ratio
A/bc is called as molar absorptivity. Molar absorptivity has Unit dm3 mol- 1cm-1.

2. Use of non-monochromatic radiation, Association and Dissociation of analyte,

change in temperature during experiment, change in analyte due to
photochemical reaction, high concentration of sample etc.

3. σ → σ* > π → π* > n → σ* > n → π * etc.

4. Chromophore: Chromophore has groups, which are responsible for causing

absorption of electromagnetic radiation between 175 and 1000 nm. These
groups have characteristic molar absorptivity and absorb at fairly well defined
wavelengths. Example >C=C<, − C ≡ C, − Ν = Ο & −Ν=Ν − etc.

Auxochromes: They do not themselves absorb emr but when attached to a

chromophore, alter both the wavelength and the intensity of absorption of the
chromophore. Example − OR, − ΝH2, − ΝR 2 etc.

5. i) Since the beam of light is divided into two parts and is allowed to fall on
two identical detectors, the fluctuations (if any) of the source will not
disturb the reading, which is noted after balancing the two
potentiometers.
ii) Since the galvanometer is used as a nu ll detector, no current is used in
moving the galvanometer needle and the results are more accurate than
that with a single beam instrument.
iii) Since the reading is noted on a large potentiometer scale, the sensitivity is
high.

6. For o-xylene (compound X)

70
 0.90 Optical Methods
271 nm (λ1 ) (ax )1 = = 2.25dm −1
dm3 gg−1 cm
3
cm−1−1
0.40
0.10 −1 −1−1
275 nm (λ2 ) (ax ) 2 = = 0.25 dm 3 gg−1 cm
3
cm
0.40
For p − xylene (compound Y)
0.34 −1 −1−1
271 nm (λ1 ) (ay )1 = = 2.0 dm
dm3 g g−1 cm
3
cm
0.17
1.02
275 nm (λ2 ) (ay ) z = = 6.0 dm
dm33 g −g1−1cmcm−1 −1
2 0.17
Therefore, λ1 0.47 = 2.25 cx + 2cy (1)
for λ2 0.54 = 0.25 cx + 6.0 cy (2)

First eliminate cy by multiplying the first equation by 3 and subtracting (2) from
the result. Thus give cx = 0.13 g dm−3

cy can be calculated by putting the value of cx, cy = 0.084 g dm−3

7. See Fig .19.24 and Fig. 19.26. AAS has a light source.

FURTHER READINGS
1. Analytical Chemistry, Gary D. Christin, John Wiley & Sons, Inc.
2. Principal of Instrumental Analysis, Skoog, Holler and Nieman, Saunders
Golden Sunburst Series .

71