GEC GROUP OF COLLEGE
GWALIOR
ENGINEERING
CHEMISTRY(BE 201)
PREPARED BY:Dr.RITU MENDIRATTA
PROFESSOR
DEPARTMENT OF CHEMISTRY
BE IST YEAR
UNIT - V
INSTRUMENTAL TECHNIQUES IN CHEMICAL ANALYSIS
UNIT - V
INSTRUMENTAL TECHNIQUES IN CHEMICAL ANALYSIS
Absorption Spectroscopy
OR
UV and Visible Spectroscopy
The word spectroscopy is derived from spectrum which means a bend of different
colours formed due to difference in wavelength and skopin means examination or
evaluation. Thus, spectroscopy is the branch of science that deals with the
examination or evaluation of spectrum
Electromagnetic Spectrum (EMS)
The entire range over which the electromagnetic radiation exists is known as
electromagnetic spectrum. This electromagnetic spectrum ranges from very short
wavelengths (including gamma and x-rays) to very long wavelengths (including
microwaves and broadcast radio waves). The following chart displays many of the
important regions of this spectrum.
Fig. 1. The electromagnetic spectrum
According to the fig.1, the major characteristics of various spectrum regions are as
follows:
γ-rays- It lies between 0.2 to 10 nm. They are the shortest waves. They are emitted
by atomic nuclei, involving energy changes of 109 to 1011 joules/gram atom.
x – Rays – it lies between 10 nm to 100 nm. It is emitted or absorbed by movement
of electrons. X- Rays are used for diagnostic purpose.
Visible and ultraviolet region- UV radiation lies between 200 nm to 400 nm.
Visible region lies between 400 nm to 800 nm.
Infrared region- infrared region is further divided in three regions.
1. near infrared region lies between 800 nm to 4000 nm.
2. Middle infrared region lies between 4000 nm to 25000 nm.
3. Far infrared region lies between 25000 nm to 100000 nm.
Microwave region- This region lies between 106 nm to 107 nm. Microwaves are
used in telecommunication.
Radio frequency region- radio frequency region lies between 107 nm to 109 nm.
Absorption Spectroscopy
Absorption spectroscopy refers to spectroscopic techniques that measure the
absorption of radiation, as a function of frequency or wavelength, due to its
interaction with a sample. The sample absorbs energy, i.e., photons, from the
radiating field. The intensity of the absorption varies as a function of frequency,
and this variation is the absorption spectrum. Absorption spectroscopy is
performed across the electromagnetic spectrum.
Electromagnetic radiation(EMR) is absorbed or emitted when the molecule atom or
ion of the sample move from one energy state to another energy and the change in
rotational, vibrational and/or electronic energies are measured.
Theory
Ultraviolet and Visible Spectroscopy Range
The wavelength range of UV radiation is 200 nm- 400 nm. There are mainly two
types of UV region.
1. 200 nm- 400 nm that is called near ultraviolet region.
2. Below 200 nm that is called far ultraviolet region.
The wavelength of visible radiation is 400 nm- 800 nm.
Wavelength in UV and visible region is expressed in nanometers or in angstroms.
Absorption is expressed in terms of wave number (cm-1).
Since the absorption of ultraviolet or visible radiation by a molecule leads transition
among electronic energy levels of the molecule, it is also often called as electronic
spectroscopy. Absorption spectra arise from transition of electron or electrons within
a molecule from a lower electronic energy level to a higher electronic energy level.
Radiation in this region is of sufficient energy to cause electronic transition of outer
valence electrons. Both organic and inorganic species exhibit electronic transitions
in which outermost or bonding electrons are promoted to higher energy levels.
Electronic transitions are associated with vibrational as well as rotational transitions.
A compound appears coloured if it selectively absorbs light in the visible region.
The main function of absorbed energy is to raise the molecule from ground energy
state ( E0 ) to higher excited energy state ( E1 ). The difference is given by:
ΔE= E1- E0 = hv = hc/λ
ΔE depends upon how tightly the electrons are bound in the bonds and accordingly,
absorption will occur in UV or visible range, for example;
If the electrons of a molecule are tightly bound as in compounds containing sigma
bonds (e.g. saturated compounds) no light of region will be absorbed. The light of
UV region will only be absorbed and hence compound appears colourless.
If the electrons of molecule are loosely bound as in unsaturated compound. Such
absorption may occur in visible region and substance will appear as coloured.
Wave length and Color Relationship
For a particular type of light the wavelength for it will have a characteristic value
which will determine the energy of the light. Visible light has wavelengths ranging
from 400 nm (violet) to 780 nm (red). The colors we see with our eyes in our day to
day lives have wavelengths between 400-780 nm. The wavelengths at which
different colors are observed are shown below:
Violet
Indigo
Blue
Green
Yellow
Orange
400-420 nm
420-440 nm
440-490 nm
490-570 nm
570-585 nm
585-620 nm
620-780 nm
Red
Instrumentation of UV or Visible Spectroscopy
Instruments for measuring the absorption of U.V. or visible radiation are made up of
the following components;
1. Sources (UV and visible)
2. Filter or monochromator
3. Sample containers or sample cells
4. Detector
1. Radiation source
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
Various Visible radiation sources are as follows
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbonone lamp
2. Filters or monochromators
All monochromators contain the following component parts;
• An entrance slit
• A collimating lens
• A dispersing device (a prism or a grating)
• A focusing lens
• An exit slit
Polychromatic radiation (radiation of more than one wavelength) enters the
monochromator through the entrance slit. The beam is collimated, and then strikes
the dispersing element at an angle. The beam is split into its component wavelengths
by the grating or prism. By moving the dispersing element or the exit slit, radiation
of only a particular wavelength leaves the monochromator through the exit slit.
3. Sample containers or sample cells
A variety of sample cells available for UV region. The choice of sample cell is
based on
a) the path length, shape, size
b) the transmission characteristics at the desired wavelength
c) the relative expense
The cell holding the sample should be transparent to the wavelength region to be
recorded. Quartz or fused silica cuvettes are required for spectroscopy in the UV
region. Silicate glasses can be used for the manufacture of cuvettes for visible
region. The thickness of the cell is generally 1 cm. cells may be rectangular in shape
or cylindrical with flat ends.
4. Detectors
In order to detect radiation, three types of photosensitive devices are
a. photovoltaic cells or barrier- layer cell
b. phototubes or photoemissive tubes
c. photomultiplier tubes
Types of Electronic Transitions electronic transition level
It was earlier stated that σ, π, and n electrons are present in molecule and can be
excited from the ground state to excited state by the absorption of UV radiation. The
various transitions are n→∏*, ∏→∏*, n→σ*, & σ →σ*
Fig 1: Energy levels of electronic transitions
The energy requirement order for excitation for different transitions is as follows.
n→∏* < ∏→∏* < n→σ* < σ→σ*
n→∏* transition requires lowest energy while σ→σ* requires highest amount of
energy.
1. n→∏* transition
n→π* transition requires lowest energy due to longer wavelength. So they are
forbidden
2. ∏→∏* transition
It is due to the promotion of an electron from a bonding π orbital to an anti-bonding
∏* orbital. Energy requirement is between n→ ∏* and n→σ*. But the extended
conjugation and alkyl substituent shifts the λmax towards longer wavelength
(Bathochromic shift). It is also called K band.
3. n→σ* transition
Saturated compounds with lone pair of electrons undergo n→σ* transition in
addition to σ→σ* transition. Corresponding absorption bands appear at longer
wavelengths in near UV region.
4. σ→σ* transition
These transitions can occur in such compounds in which all the electrons are
involved in single bonds and there are no lone pair of electrons.
Energy required for σ→σ* transition is very large so the absorption band occurs in
the far UV region. So this transition cannot normally be observed.
Beer's and Lambert's Law
Lambert’s law
“When a beam of monochromatic radiation is allowed to pass through a transparent
medium, the rate of decrease of intensity of radiation with thickness of absorbing
medium is directly proportional to the intensity of the incident radiation.”
Mathematically, the Lambert’s law may be expressed as follows.
- dI / dt α I
-dI / dt = KI
. . . . . . . . . .(1)
Where I = intensity of incident light
t = thickness of the medium
K= proportionality constant
By integration of equation (1), and putting I=I0 when t=0,
I0/ It = kt or It= I0 e-kt
Where, I0 = intensity of incident light
It = intensity of transmitted light
k = constant which depends upon wavelength and absorbing medium used.
By changing the above equation from natural log, we get,
It = I0 e-Kt
. . . . . . . . . .(2)
Where K = k/ 2.303
So,
It = I0 e-0.4343 kt
It = I010-Kt
. . . . . . . . . .(3)
Beer’s law
“When a beam of monochromatic radiation is allowed to pass through a transparent
medium, the rate of decrease of intensity of radiation with thickness of absorbing
medium is directly proportional to the intensity of the incident radiation as well as
the concentration of the solution.”
The above sentence is very similar to Lambert’s law. So,
It = I0 e-k' c
It = I0 10-0.4343 k' c
It = I0 10 K' c
. . . . . . . . . .(4)
Where k' and K'= proportionality constants
c = concentration
By combining equation (3) and (4), we get,
It = I0 10 -act
I0 / It = 10 act
Where, K and K' = a or ε
c = concentration
t or b = thickness of the medium
log I0 / It = εbc
. . . . . . . . . .(5)
Where ε = absorptivity, a constant dependent upon the λ of the incident radiation and
nature of absorbing material. The value of ε will depend upon the method of
expression of concentration.
The ratio I0 / It is termed as transmittance T, and the ratio log I0 / It is termed as
absorbance A. formerly, absorbance was termed as optical density D or extinction
coefficient E. the ratio I0 / It is termed as opacity. Thus,
A = log I0 / It
. . . . . . . . . .(6)
From equation (5) and (6),
A = εbc
. . . . . . . . . .(7)
Thus, absorbance is the product of absorptivity, optical path length and the
concentration of the solution.
A = ε /bC
When C=1 mol/dm3
And b=1cm Then
A= ε
Hence, molar absorption coefficient or molar absorptive is the specific absorption
coefficient for a concentration of 1 mole dm-3 and path length of 1 cm. It is also
known as extinction coefficient.
According to equation (7),
A = log I0 / It
Transmittance T is a ratio of intensity of transmitted light to that of the incident
light.
T = I0 / It
The more general equation can be written as follows:
A = log I0 / It = log 1/ T = – log T = abc = εbc
Different shifts in UV-visible Spectroscopy
Bathochromic Shift or Red shift: A shift of an absorption maximum towards
longer wavelength or lower energy. It can occur due to increase in conjugation
(addition of double bond or triple bonds), addition of alkyl substituent in the
molecule etc.
Hypsochromic Shift or Blue Shift: A shift of an absorption maximum towards
shorter wavelength or higher energy. It can occur due to removal of double bond or
triple bonds by saturation dealkylation etc.
Hypochromic Effect: An effect that results in decreased absorption intensity.
Hyperchromic Effect: An effect that results in increased absorption intensity.
CHROMOPHORE
The energy of radiation being absorbed during excitation of electrons from ground
state to excited state primarily depends on the nuclei that hold the electrons together
in a bond. The group of atoms or molecules which are covalently unsaturated
containing electrons responsible for the absorption is called chromophores e.g.:
C=C, C=O, C=N. Most of the simple un-conjugated chromophores give rise to high
energy transitions of little use.
Types of chromophores
1. Chromophores which contains π electrons and undergo ∏→∏* transition
transition. For example, ethylene and acetylene.
2. Chromophores, which contains both π and n electrons and undergo ∏→∏*
transition and n→∏* transition transition. For example, carbonyls and nitriles.
.
AUXOCHROME
The co-ordinatively saturated or unsaturated substituent that themselves do not
absorb ultraviolet radiations but their presence shifts the absorption maximum to
longer wavelength are called auxochromes. The substituents like methyl, hydroxyl,
alkoxy, halogen, amino group etc. are some examples of auxochromes.
Auxochromes are basically colour enhancing group. It has ability to extend the
conjugation of the chromophore by the sharing of the nonbonding electrons and thus
shifts the absorption maximum to longer wavelength.
Applications of Absorption Spectroscopy (UV, Visible)
1. Detection of Impurities
UV absorption spectroscopy is one of the best methods for determination of
impurities in organic molecules. Additional peaks can be observed due to impurities
in the sample and it can be compared with that of standard raw material. By also
measuring the absorbance at specific wavelength, the impurities can be detected.
Benzene appears as a common impurity in cyclohexane. Its presence can be easily
detected by its absorption at 255 nm.
2. Structure elucidation of organic compounds.
UV spectroscopy is useful in the structure elucidation of organic molecules, the
presence or absence of unsaturation, the presence of hetero atoms.
From the location of peaks and combination of peaks, it can be concluded that
whether the compound is saturated or unsaturated, hetero atoms are present or not
etc.
3. Quantitative analysis
UV absorption spectroscopy can be used for the quantitative determination of
compounds that absorb UV radiation. This determination is based on Beer’s law
which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is the length of the cell
that is used in UV spectrophotometer.
4. Qualitative analysis
UVabsorption spectroscopy can characterize those types of compounds which
absorbs UV radiation. Identification is done by comparing the absorption spectrum
with the spectra of known compounds.
UV absorption spectroscopy is generally used for characterizing aromatic
compounds and aromatic olefins.
5. Dissociation constants of acids and bases.
PH = PKa + log [A-] / [HA]
From the above equation, the PKa value can be calculated if the ratio of [A-] / [HA]
is known at a particular PH. and the ratio of [A-] / [HA] can be determined
spectrophotometrically from the graph plotted between absorbance and wavelength
at different PH values.
6. Chemical kinetics
Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is
passed through the reaction cell and the absorbance changes can be observed.
7. Quantitative analysis of pharmaceutical substances
Many drugs are either in the form of raw material or in the form of formulation.
They can be assayed by making a suitable solution of the drug in a solvent and
measuring the absorbance at specific wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength 284
nm.
.