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Molecular Spectroscopic
Methods-I

UNIT 2 UV-VISIBLE SPECTROMETRY
Structure
2
...
2

Origin and Characteristics of UV-VIS Spectrum
Origin of UV-VIS spectrum
Characteristics of UV-VIS Spectrum
Absorbing Species

2
...
4

Instrumentation for UV-VIS Spectrometry
Radiation Sources
Wavelength Selectors
Monochromators
Sample Handling
Detectors
Signal Processing and Output Devices

2
...
6

Analytical Applications of UV-Visible Spectrometry
Qualitative Applications
Quantitative Applications
Quantitative Determination Methodology
Simultaneous Determination

2
...
8
2
...
1

Summary
Terminal Questions
Answers

INTRODUCTION

In the previous unit you have learnt about the electromagnetic radiation in terms of its
nature, characteristics and properties
...
Further, the interaction
of radiation with matter may be manifested in terms of its selective absorption or
absorption followed by emission or scattering
...
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
...
Since such measurements need an instrument
called spectrometer the technique is known as UV-VIS spectrometry
...
We will begin by understanding the theory
behind UV-VIS spectrometry in terms of the origin and the features of the UV-VIS
spectrum
...
It will be followed by an account of
the essential components of a UV-VIS spectrometer and their functions
...
In the next unit you will learn about the study of infra- red
spectrometry which concerns the interaction of electromagnetic radiation in the
infrared region with matter
...


2
...
It forms the basis of analysis of
different substances such as, inorganic, organic and biochemicals
...
It is therefore important to learn about
the origin of the UV-VIS spectrum and its characteristics
...


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 would also recall that in molecules, the electronic,
vibrational as well as the rotational energies are quantised
...
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
...
1(a)), constituting what is
called a band spectrum
...
These
collisions and the interactions among the absorbing species and the solvent molecules
cause the energies of the quantum states to spread out
...
Thereby, the spectrum acquires
the shape of a smooth and continuous absorption peak in the solution phase
...
2
...


The absorption of
radiation in the UV-VIS
region of the spectrum
causes the transitions
amongst the electronic
energy levels
...
2
...
2
...
A monochromatic
radiation i
...
, a radiation of a single wavelength is employed at a time
...
The amount of the radiation absorbed at each wavelength is
measured and plotted against the wavelength to obtain the spectrum
...

Have a look again at Fig
...
1 (b)
...
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
...
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 intensity on the other hand is indicative of the probability of the
transition i
...
, whether the transition is allowed or not
...
The relationship between the intensity of
absorption and the concentration is explained in Section 2
...

Analyte: The chemical
species which is to be
determined in the sample
under investigation
...
Therefore, these spectra are extensively used in the
characterisation and in the quantitative estimations of the analyte
...
2
...
The electronic transitions do not occur in all the species when a
radiation in UV region is passed through the sample
...
Let us learn about these species and the nature of the transitions
involved in them
...
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
...
In addition, these have nonbonding electrons on the atoms
like, N,O,S and halogens etc
...
The generalised molecular orbital energy level
diagram and possible transitions for organic compounds is given in Fig
...
2
...
2
...
In most of the
organic compounds, the bonding and sometimes the nonbonding orbitals also are filled
and the antibonding orbitals are empty
...
Let us learn about these two in
the following paragraph
...
For example,
ethylene absorbs at 170 nm
...
The effect of conjugation is quite important in
aromatic molecules
...

n → π ∗ transitions: These transitions are observed in molecules containing lone pairs
or nonbonding electrons
...
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
...
Thus, nitrogen, sulphur and halogens tend to
move absorption to higher wavelengths
...

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
...


33

Molecular Spectroscopic
Methods-I

Fig
...
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
...

Chromophore in Greek means, “colour bearing”
...
A list of some simple chromophoric groups and their light absorption
characteristics is given in Table 2
...

Table 2
...
The ions and the complexes of the
elements of the first two transition series belong to this group and are coloured
...
The complex formation
of these ions with solvent molecules or with other ligands lifts the degeneracy of the
five d orbitals
...

The electronic transitions from the lower energy d orbitals to higher energy d orbitals
are responsible for the observed colour
...

34

The blue colour of the aqueous solutions of copper sulphate and the violet colour of
potassium permanganate are some of the examples
...
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
...
The representative visible spectra of
the ions of transition and inner transition elements are given in Fig
...
4
...
2
...
This happens if one of the species has an
electron donor group and the interacting species has an electron acceptor group
...

This is due to the formation of a complex between the two species
...
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
...
2
...
This has been schematically
illustrated in Fig
...
6
...
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
...
2
...
The intense colour of
such metal chelates is often due to charge transfer transitions
...
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
...
For
example, the alkaloid brucine reacts with nitrate ion in concentrated sulphuric acid to
form a red compound which quickly turns yellow
...
The quantitative determinations are based
on the relationship between the absorption of radiation and concentration of the
analyte
...
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
...
In such a situation a part of
the radiation is reflected, a part is absorbed, and a part is transmitted
...

The transmitted radiation
has information about the
nature and the amount of
the absorbing species
...
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
...
One is Bouguer’s law or Lambert’s
law, which expresses the relationship between the light absorption capacity of the
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
...

Let us discuss these laws one by one
...
3
...
e
...


ii)

The intensity of the transmitted light decreases exponentially when the thickness
of the substance, through which the light is passing, increases linearly
...


Px

Px-dPx

P0

P

dPx
b

b
Fig
...
7: Illustration for establishing the Lambert’s Law

In Fig
...
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
...
1)

where k is the proportionality constant and the negative sign indicates that radiant
power decreases with absorption
...
2
...
2
...


dPx
= − kd x
Px

…(2
...
2
...
3)

Eq
...
3 is the mathematical expression for Bouguer-Lambert law or Lambert’s law
...
303

… (2
...
Lambert’s law
applies to any homogeneous non-scattering medium, regardless of whether it is gas,
liquid, solid, or solution
...
3
...
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
...

Consider a monochromatic radiation beam of intensity Px traversing any thickness of
solution of a single absorbing substance of concentration c
...
We can express it as follows
...
5)
dPx = − k " Px dc
where k″ is the proportionality constant and the negative sign indicates that radiant
power decreases with absorption
...
6)
Px
On integration within the limits of Po to P for intensity and 0 to c for concentration we
get
P

∫
Po

d Px
Px

c

∫

= − k " dc
o

or

P
ln
Po

or

log 10

= − k "c
Po
P

=

k"
c
2
...
7)
Eq
...
7 is the mathematical expression for Beer’s law
...
2
...


log

P
o
= abc
P

… (2
...
The concentration is generally expressed in terms of grams
per dm3 and b in cm
...


UV-Visible
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
...
The modified expression for the Beer-Lambert’s law
becomes,

log

P0
= εbc
P

… (2
...


The term log Po/P is called absorbance and is represented as ‘A’
...
(2
...

Mathematically,
Transmittance, T =

P
P0

Eq
...
10: use ‘a’ if
concentration is
expressed in g/L and path
length in cm and ‘ε’ if the
concentration in mol dm-3
and b in cm
...
11)

It is generally expressed as a percentage and is expressed as,

Percentage transmittance, %T =

P
× 100%
P0

It is related to absorbance as, A = − log T

… (2
...
13)

In typical measurements the radiant power transmitted by a solution is measured and
compared with that observed with solvent (also called a blank)
...


You may note here that
though transmittance and
absorbance are equivalent
expressions which can be
interconverted, it is the
absorbance which is
directly proportional to
the absorbed fraction of
the radiation
...
Let us take some examples illustrating the application of these
expressions
...
0 × 104 cm‒1 mol‒1 dm3
...
0 cm containing 2
...


Solution
As per the Lambert Beer’s law,
Absorbance, A = ε c b
Given:
ε = 2
...
0 × 10‒6 mol dm‒3 and b = 5
...
0 × 104 × 2
...
0 = 0
...
2
∴ A = log 1/T
39

Molecular Spectroscopic
Methods-I

Taking antilog on both sides, we get
l/T = 1
...
63

Example 2
A solution containing 36
...
If
the percentage transmittance is found to be 12, calculate the molar absorptivity of
potassium dichromate
...
0365
= 2
...
12
∴ A = 1/0
...
921
∴ ε = A/cb = 0
...
48 ×10‒4 × 2) = 0
...
96 × 10‒4
∴ε = 1
...
This proportionality is exploited in the determination of
analyte concentration
...
Let us learn about the
possible reasons for the deviation from Beer’s law
...
4 using cuvettes of 2
...
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
...
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
...
85
...

…………………………………………………………………………………………
...

…………………………………………………………………………………………
...

40

2
...
3 Deviations from Beer-Lambert’s Law
As per the Beer’s law discussed above, there is a direct proportionality between the
absorbance and concentration
...
However, this is not always true; there are
certain limitations
...
Many
a times instead of a straight line, a curvature in the plot may be observed as shown in
Fig
...
8
...


UV-Visible
Spectrometry

Fig
...
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
...

However, large amounts of electrolytes may affect the absorption spectrum
qualitatively as well as quantitatively
...
This interaction results
in a deformation of the later, thereby causing a change in its light absorption property
...
For example, the aqueous solution of potassium
dichromate involves the chromate ion-dichromate ion equilibrium as shown below:
2CrO 2 − + 2H +
7

2
Cr2 O 7 − + 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
...
Therefore, the results of the determination of
2
chromate ion ( CrO 7 − ) concentration will depend on the pH
...

In some cases, two absorbing species are in equilibrium and have a common value of
absorptivity at a certain wavelength
...
However, at wavelength of
501 nm, we see that all species have same molar absorptivity (see Fig
...
9)
...
Such a wavelength is known as isosbestic point
...

However, such wavelengths should be avoided for the quantitative work
...
2
...
For example, the colour of cobalt chloride changes from
pink to blue due to the following complex formation
...
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
...

However, most spectrophotometers and all filter photometers, discussed later employ
a finite group of frequencies
...


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
...
Therefore, at higher concentrations
(>10‒3 mol dm‒3) there may be deviation from the law
...
However, changes in temperature
sometimes may shift ionic equilibrium and the absorptivity
...


UV-Visible
Spectrometry

SAQ 4
Why is it important to know about the possible reasons for deviation from
Beer-Lambert’s law?
…………………………………………………………………………………………
...

…………………………………………………………………………………………
...


2
...
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
...
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
...
10
...
2
...

43

Molecular Spectroscopic
Methods-I

2
...
1 Radiation Sources
A spectrophotometric radiation source must provide a stable high energy output over a
broad range of wavelengths
...
The
radiation sources commonly used for the UV and visible range are described below
...
In these, the excitation of the gaseous molecules is brought
about by the passage of electrons through the gas at low pressures
...
The radiant power of the hydrogen lamp is low and these are replaced by
deuterium lamps but it increases the cost of the instrument
...
2
...
The modern instruments use a
tungsten filament lamp as the radiation source
...
This gives radiations in the range of
350-2200 nm
...

Now a days some instruments use tungsten-halogen lamps that contain a small amount
of iodine in the quartz bulb housing the tungsten filament
...


2
...
2 Wavelength Selectors
You would recall that a
radiation consisting of a
single wavelength is
called a monochromatic
radiation
...
This enhances the selectivity and sensitivity of the instrument
...
In many applications we need to continuously vary the wavelength over
a defined range
...
Most modern
instruments use monochromators that employ a prism or diffraction grating as the
dispersing medium
...


Absorption Filters
In low cost instruments catering to measurements in the visible range, coloured glass
44

filters are used to cut off undesirable wavelengths
...
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
...
When white light falls on an object, a part of it
is absorbed and rest is transmitted
...
The absorbed component and the observed colour can
again add up to give white light
...
An object of a particular colour looks of that colour because this colour is
transmitted and its complementary colour is absorbed
...
The colour of the filter
should be the complementary to colour of the solution to be measured
...
e
...
Therefore, for measuring
absorbance of a orange solution you would employ a blue filter
...
2
...


Orange
White light =

Orange

+
Blue

Fig
...
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 find the colour of the filter to be used we can take the help of colour wheel
shown in Fig
...
13, or consult a table of complementary colours given in Table 2
...


Fig
...
13: The colour wheel

45

Molecular Spectroscopic
Methods-I

In a colour wheel, the colours, which face one another, are said to be complementary
to each other
...
In the colour wheel P, S and T refer to the
primary, secondary and tertiary colours, respectively
...
2: Complementary Colours Chart
Wavelength region
eliminated by
absorption (nm)

Colour absorbed
violet
blue
green-blue
blue-green
green
yellow-green
yellow
orange
red

400-450
450-480
480-490
490-500
500-560
560-580
580-600
600-650
650-750

Complementary colour of the
residual light (as seen by the eye)
yellow-green
yellow
orange
red
purple
violet
blue
green –blue
blue –green

Absorption filters are simple and are totally adequate for many applications in visible
range
...
The interference
filters cover a wider range than the absorption filters
...
Such interference filters are available for ultraviolet,
visible and near- infrared region
...
The effective
bandwidths of these filters are narrower than absorption filters
...
4
...
These are of two types; the prism and grating monochromators
...

Glass prisms and lenses
can be used in the visible
region
...


Prism Monochromators
You know that a prism disperses sunlight into seven different colours
...
The radiations of
different colours having different wavelengths are refracted to different extent due to
the difference in the refractive index of glass for different wavelengths
...
2
...


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

46

UV-Visible
Spectrometry

(b)
Fig
...
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
...
2
...
This is then collimated on the prism with the help of a lens
...
The prism is then rotated in a
predetermined way to provide the desired wavelength from the exit slit
...
2
...
The surface
is made reflective by making a thin film of aluminium on it and the etching is done
with the help of a suitably shaped diamond tool
...
2
...


The gratings used for the
ultraviolet and visible
region generally contain
about 1200-1400
grooves/mm
...
2
...
2
...
This is then reflected on the grating which
disperses it
...
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
...

47

Molecular Spectroscopic
Methods-I

Fig
...
16: Schematic diagram of a grating monochromator

2
...
4 Sample Handling
The UV-VIS absorption spectra are usually determined either in vapour phase or in
solution
...
A variety of
quartz cuvettes are available for the spectral determination in the vapour phase
...
For measurements
on solutions in the visible region the cuvettes made of glass can also be used
...
Therefore, most of the spectrophotometers employ quartz cuvettes (Fig 2
...
Usually square cuvettes having
internal path length 1
...
1 mm or 0
...


The eye of a common
person is quite sensitive
to notice differences in
radiant power transmitted
through two coloured
solutions
...

Therefore, early
instruments used eye or
photographic plate as the
detector
...
2
...
It means that it does not absorb in this region
...
Now a
days ‘spectral grade’ solvents are available which have high purity and have negligible
absorption in the region of absorption by the chromophore
...
The
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
...
In double beam spectrometers, the sample and the solvent are scanned
simultaneously (subsection 2
...
2)
...
4
...
The detectors used in most of the
instruments generate a signal, which is linear in transmittance i
...
they respond
linearly to radiant power falling on them
...
There are three types of detectors which are used in modern
spectrophotometers
...


1
...
2
...
These two electrodes are subjected to
high voltage (about 100 V) difference
...
The response of the photoemissive
material is wavelength dependent and different phototubes are available for different
regions of the spectrum
...
2
...


Photomultiplier (PM) Tube

A photomultiplier tube (Fig
...
18 (b)), consists of a series of electrodes, called
dynodes
...
When a radiation falls on the cathode an electron is
emitted from it
...
Here, it releases many more secondary electrons
...
The process continuous upto about 10 stages of amplification
...


3
...
When a UV-VIS radiation falls on the diode, its conductivity increases
significantly
...
Since a large number of diodes can be arranged
together, the intensity at a number of wavelengths can be measured simultaneously
...


2
...
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 output plot between the wavelength and
the intensity of absorption is the resultant of the subtraction process and is
characteristic of the absorbing species
...
But
before proceeding further, try to answer the following SAQ
...

…………………………………………………………………………………………
...

…………………………………………………………………………………………
...


2
...
Broadly
speaking there are three types of spectrometers
...


2
...
1 Single Beam Spectrometers
The instruments using
filters and photoelectric
detectors are called
photometers
...
The same
beam is used for reading the absorption of the sample as well as the reference
...
2
...
The radiation from the source is passed through a filter or a suitable
monochromator to get a band or a monochromatic radiation
...
The signal so obtained is sent as a read out or is recorded
...
2
...
Second, the cuvette is taken out and rinsed and
50

filled with sample solution and the process is repeated
...


UV-Visible
Spectrometry

2
...
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
...
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
...
2
...
There
could be variations depending on the manufacturer, the wavelength regions for which
the instrument is designed, the resolutions required etc
...
2
...
5
...
This allows the radiations of
all the wavelengths to simultaneously fall on the sample
...
The
schematic arrangement of a diode array spectrometer is given in Fig
...
21
...
2
...
These then fall on the array
of the photodiodes arranged side by side
...
The advantage of such
instruments is that a scan of the whole range can be accomplished in a short time
...
2 to answer this question
...


…………………………………………………………………………………………
...

…………………………………………………………………………………………
...
The quantitative determinations are based
on the relationship between the absorption of radiation and concentration of the
analyte
...


2
...
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
...
We will discuss some of
qualitative and quantitative applications of UV-VIS spectrometry
...
6
...

The information obtained from it needs to be supplemented by that from IR, NMR and
mass spectrometries
...

Nonetheless, it can still provide information about the presence or absence and the
nature of the chromophore 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
...
2
...
One of
the spectra happens to be of an azo dye and the other is of permanganate solution
...


Fig
...
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
of a wide array of natural products of plant and animal origin
...
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 details of these
rules and their applications can be obtained from any book on organic spectroscopy
...
The change in the polarity
of the solvent alters the energies of the orbitals
...
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
...


2
...
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 can be used for direct determination of a large number of organic,
inorganic and biochemical species accurately at fairly low concentrations; viz
...
In addition to these, the convenience of conducting the
determination and its reasonable selectivity make it a method of choice for
quantitative determinations
...
It is achieved by
making them react with a reagent that gives a product which absorbs in the region
...
Thus, absorbance measurements
in visible or in UV region, are used in diverse areas
...

1
...


Analytical determination of organic compounds

3
...


Determination of metal-ligand formation constants

5
...
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 such an approach, a series of standard
solutions of the analyte to be determined were prepared in tubes of constant depth and
diameter
...
The concentration of
the test solution was determined from the concentration of the standard solution by
accounting for the dilution factor
...
Today, a given
53

Molecular Spectroscopic
Methods-I

analytical determination can be made in a number of possible ways
...
Let us learn about the methodology of quantitative determinations
...
6
...
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
...
However, for most of the
analytes we have to form a absorbing species by reacting them with a suitable reagent
...

Further, the reagent should be selective towards the analyte and its reaction with the
analyte should be quantitative i
...
100%
...


Ordinarily the absorbance
should be measured at a
wavelength where there is
no interference from other
absorbing species in the
solution
...
At this wavelength the
absorbance is most sensitive to the concentration
...
e
...
This is quite advantageous because even if the
spectrophotometer fails to resolve λ max, the determination does not suffer
...
In such cases we need to identify such a wavelength at
which there is large difference in the absorbance values of pure reagent and the
complex
...
These include solvent polarity, pH, temperature, ionic strength and
interferences from other absorbing species
...


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
dependable method that
the A versus c plot for the
standard analyte is a
straight line
...
However, generally
the value of ε is not known accurately for the species being determined under the
conditions of the determination
...
Therefore in most of the methods, a
calibration curve is obtained by measuring the absorbance values for a series of
standard solutions of the analyte being determined at a fixed wavelength
...
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
...
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
...
23 (a)
...
2
...
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
...
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
...
If the Beer’s law is obeyed,
i
...
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
...
23 (b)
...
2
...
6
...
In order to determine these species we need to find two
wavelengths where molar absorptivity of two species is different
...

This gives a set of simultaneous equations which could be solved for the
concentrations of the individual constituents
...
Consider the
spectra given in Fig
...
24
...
2
...
The molar absorptivity
of spectra 1 and 2 can be found out by taking known amounts of the substance and
recording their absorbance value
...
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
...


C1 =

Aλ1 (ε 2 ) λ2 − Aλ2 (ε 2 ) λ1
(ε 1 ) λ1 (ε 2 ) λ2 − (ε 2 ) λ1 (ε 1 ) λ2
C2 =

Aλ2 − (ε 1 ) λ2 C1
(ε ) λ2

Let us take an example to understand it
...
405 and 0
...
The
absorbance values of pure solutions of K2Cr2O7 (0
...
0002 M) in
similar conditions were as follows:
A (Cr,440 nm) = 0
...
009
A (Mn, 440nm) = 0
...
475
Using the given data determine the concentration of Cr2 O 2− ions and Mn O − ions in
4
7
the mixture
...
374 = εCr, 440 × 1
...
0 × 10‒3
0
...
0 × 1
...
019 = εMn,440 × 1
...
0 × 10‒4
0
...
0 × 2
...
38 × 103

UV-Visible
Spectrometry

For the mixture we can write the simultaneous equations as
A440

2
= εCr, 440[Cr2 O 7− ] + εMn,440[Mn O − ]
4

A545

2
= εCr, 545[Cr2 O 7− ] + εMn,545[Mn O − ]
4

Substituting the values, we get
2
0
...
712 = 9[Cr2 O 2− ] + 2
...
0 × 10‒3 mol dm‒3
[Mn O − ] = 2
...


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

…………………………………………………………………………………………
...

…………………………………………………………………………………………
...
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
...
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
...
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
...
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
...
This law forms the
basis of quantitative determinations of the analyte
...
These need to be considered in
such determinations
...
These are: radiation
source, wavelength selector, sample holder, detector and a signal processing and
output device
...
The wavelength selection is
achieved by absorption filters in low cost instruments for visible region
...
The sample is taken in
quartz cuvettes, though for visible region even glass cells can be used
...

Nowadays, the modern machines employ diode arrays for detection purposes
...

UV-VIS spectrometry can be used to detect one or more components in a solution and
measure the concentration of these species
...
The most important qualitative
application of UV-VIS spectrometry is to detect the presence of unsaturation in the
molecule
...

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
...


2
...

2
...
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
...


4
...
00 microgram of iron (II) in 10 cm3 of solution was measured at
515 nm in 1 cm cuvette
...
0200
...


5
...


6
...


Enlist the essential steps of quantitative determination methodology
...


What is the principle of the photomultiplier tube?

9
...
Explain
...
The molar absorptivities for Ni and Co ions at
500 nm are, 10 and 1000 respectively
...
0 × 103 cm‒1mol‒1dm3
...
091 at 500 nm and 0
...

Calculate the concentrations of Ni2+ and Co2+ in the given mixture
...
9

UV-Visible
Spectrometry

ANSWERS

Self Assessment Questions
1
...


Pо = 85
...
00cm

P = 20
...
4/20
...
624
Since A = є b c; є = A/b
...
624
2
...


є =14000 cm −1 mol −1dm 3
c=?

b = 1cm

A = є cb

A= 0
...
b

c = 0
...
07 × 10 −5 M

4
...

It implies that a plot of concentration versus absorption is a straight line
...
It
therefore becomes important to know about the factors that may cause deviation
so that these could be suitably controlled
...


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
...


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

7
...


Terminal questions
1
...
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
...
Therefore the sample absorbs photons spread over a range of
wavelength and the spectrum acquires the shape of a smooth and continuous
absorption peak
...


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


Some of the factors responsible for the deviation from Beer and Lambert’s law
are as follows
...


Hydrogen Ion Concentration

Temperature

The atomic weight of iron is 56, therefore the molar concentration of the
solution containing 1
...
786 × 10 −6 mol per dm3
...
02
ε = A / b
...
786 mol dm −3 × 1 cm

5
...
12 × 104 dm −3 mol −1 cm −1
A monochromator is a device that can selectively provide radiation of a desired
wavelength out of the range of wavelengths emitted by the source
...
After reflection, it falls on the
grating which causes it to disperse
...
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
...


6
...
As glass absorbs the ultraviolet radiations, these cannot be used for the
purpose
...


The essential steps of the quantitative determinations methodology are,
•
Forming an absorbing species
•
•

Controlling factors influencing absorbance

•

60

Selection of the measurement wavelength

Validation of Beer and Lambert’s law

8
...
The output signal by photoemission from a series of
electrodes called dynodes is much larger than the original signal
...


We know that for simultaneous determination of two absorbing species the
absorbance is measured at two different wavelengths
...
091
A400 nm = 5
...
0 × 103 × [Co 2+] = 0
...
(A)

...
615 – 0
...
160
[ Ni2+] = 0
...
2 × 10 −5 mol dm−3
Substituting it in (A) or (B) and solving we get
[Co 2+] = 9

---