INTRODUCTION
AND BASICS OF UV SPECTROSCOPY
UV
spectroscopy is type of absorption spectroscopy in which light of ultra-violet
region (200-400 nm.) is absorbed by the molecule. Absorption of the
ultra-violet radiations results in the excitation of the electrons from the
ground state to higher energy state. The energy of the ultra-violet radiation
that are absorbed is equal to the energy difference between the ground state
and higher energy states (deltaE = hf).
Generally,
the most favoured transition is from the highest occupied molecular orbital
(HOMO) to lowest unoccupied molecular orbital (LUMO). For most of the
molecules, the lowest energy occupied molecular orbital’s are s orbital, which
correspond to sigma bonds. The p orbitals are at somewhat higher energy levels,
the orbital’s (nonbonding orbitals) with unshared paired of electrons lie at
higher energy levels. The unoccupied or anti-bonding orbitals (pie* and sigma*)
are the highest energy occupied orbitals.
In all the
compounds (other than alkanes), the electrons undergo various transitions. Some
of the important transitions with increasing energies are: nonbonding to pie*,
nonbonding to sigma*, pie to pie*, sigma to pie* and sigma to sigma*.
PRINCIPLE OF UV
SPECTROSCOPY
UV spectroscopy obeys the Beer-Lambert law, which states
that: when a beam of monochromatic light is passed through a solution of an
absorbing substance, the rate of decrease of intensity of radiation with
thickness of the absorbing solution is proportional to the incident radiation
as well as the concentration of the solution.
The expression of Beer-Lambert law is-
A = log (I0/I) = Ecl
Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity
From the
Beer-Lambert law it is clear that greater the number of molecules capable of
absorbing light of a given wavelength, the greater the extent of light
absorption. This is the basic principle of UV spectroscopy.
INSTRUMENTATION AND WORKING OF UV
SPECTROSCOPY
UV spectrometers consist of the following parts-
1.
Light Source- Tungsten filament lamps and
Hydrogen-Deuterium lamps are most widely used and suitable light source as they
cover the whole UV region. Tungsten filament lamps are rich in red radiations;
more specifically they emit the radiations of 375 nm, while the intensity of
Hydrogen-Deuterium lamps falls below 375 nm.
2.
Monochromator- Monochromators generally composed of prisms
and slits. The most of the spectrophotometers are double beam
spectrophotometers. The radiation emitted from the primary source is dispersed
with the help of rotating prisms. The various wavelengths of the light source which
are separated by the prism are then selected by the slits such the rotation of
the prism results in a series of continuously increasing wavelength to pass
through the slits for recording purpose. The beam selected by the slit is
monochromatic and further divided into two beams with the help of another
prism.
3.
Sample and reference cells- One of the two
divided beams is passed through the sample solution and second beam is passé
through the reference solution. Both sample and reference solution are contained
in the cells. These cells are made of either silica or quartz. Glass can't be
used for the cells as it also absorbs light in the UV region.
4.
Detector- Generally two photocells serve the
purpose of detector in UV spectroscopy. One of the photocell receives the beam
from sample cell and second detector receives the beam from the reference. The
intensity of the radiation from the reference cell is stronger than the beam of
sample cell. This results in the generation of pulsating or alternating
currents in the photocells.
5.
Amplifier- The alternating current generated in
the photocells is transferred to the amplifier. The amplifier is coupled to a
small servometer. Generally current generated in the photocells is of very low
intensity, the main purpose of amplifier is to amplify the signals many times
so we can get clear and recordable signals.
6.
Recording devices- Most of the time amplifier is
coupled to a pen recorder which is connected to the computer. Computer stores
all the data generated and produces the spectrum of the desired compound.
CONCEPT OF CHROMOPHORE AND AUXOCHROME
IN THE UV SPECTROSCOPY
Chromophore- Chromophore
is defined as any isolated covalently bonded group that shows a characteristic
absorption in the ultraviolet or visible region (200-800 nm). Chromophores can
be divided into two groups-
a) Chromophores which contain p electrons and which undergo
pie to pie* transitions. Ethylenes and acetylenes are the example of such
chromophores.
b) Chromophores which contain both p and nonbonding electrons.
They undergo two types of transitions; pie to pie* and nonbonding to pie*.
Carbonyl, nitriles, azo compounds, nitro compounds etc. are the example of such
chromophores.
Auxochromes- An auxochrome
can be defined as any group which does not itself act as a chromophore but
whose presence brings about a shift of the absorption band towards the longer
wavelength of the spectrum. –OH,-OR,-NH2,-NHR, -SH etc. are the examples of auxochromic
groups.
ABSORPTION AND INTENSITY SHIFTS IN
THE UV SPECTROSCOPY
There are four types of shifts observed in the UV
spectroscopy-
a) Bathochromic effect- This type of shift is also known as
red shift. Bathochromic shift is an effect by virtue of which the absorption
maximum is shifted towards the longer wavelength due to the presence of an
auxochrome or change in solvents.
The nonbonding to pie* transition of carbonyl compounds
observes bathochromic or red shift.
b) Hypsochromic shift- This effect is also known as blue
shift. Hypsochromic shift is an effect by virtue of which absorption maximum is
shifted towards the shorter wavelength. Generally it is caused due to the
removal of conjugation or by changing the polarity of the solvents.
c) Hyperchromic effect- Hyperchromic shift is an effect by
virtue of which absorption maximum increases. The introduction of an auxochrome
in the compound generally results in the hyperchromic effect.
d) Hypochromic effect- Hyperchromic effect is defined as the
effect by virtue of intensity of absorption maximum decreases. Hyperchromic
effect occurs due to the distortion of the geometry of the molecule with an
introduction of new group.
APPLICATIONS OF UV SPECTROSCOPY
1. Detection of
functional groups- UV spectroscopy is used to detect the presence or
absence of chromophore in the compound. This is technique is not useful for the
detection of chromophore in complex compounds. The absence of a band at a
particular band can be seen as an evidence for the absence of a particular
group. If the spectrum of a compound comes out to be transparent above 200 nm
than it confirms the absence of –
a) Conjugation b) A carbonyl group c) Benzene or aromatic
compound d) Bromo or iodo atoms.
2. Detection of extent of conjugation- The
extent of conjugation in the polyenes can be detected with the help of UV
spectroscopy. With the increase in double bonds the absorption shifts towards
the longer wavelength. If the double bond is increased by 8 in the polyenes
then that polyene appears visible to the human eye as the absorption comes in
the visible region.
3. Identification of
an unknown compound- An unknown compound can be identified with the help of
UV spectroscopy. The spectrum of unknown compound is compared with the spectrum
of a reference compound and if both the spectrums coincide then it confirms the
identification of the unknown substance.
4. Determination of
configurations of geometrical isomers- It is observed that cis-alkenes
absorb at different wavelength than the trans-alkenes. The two isomers can be
distinguished with each other when one of the isomers has non-coplanar
structure due to steric hindrances. The cis-isomer suffers distortion and
absorbs at lower wavelength as compared to trans-isomer.
5. Determination of
the purity of a substance- Purity of a substance can also be determined
with the help of UV spectroscopy. The absorption of the sample solution is
compared with the absorption of the reference solution. The intensity of the
absorption can be used for the relative calculation of the purity of the sample
substance.