1/2/2014 U.V. Spectroscopy: What is Spectroscopy? Contd….

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U.V. Spectroscopy: Applications
What is Spectroscopy?
Perspective
Dr. Poonam Piplani
Professor (Pharm. Chemistry)
University Institute of Pharmaceutical
Sciences
Panjab University, Chandigarh
The Electromagnetic
Spectrum
•
From the viewpoint of an organic chemist the 4 most
frequently used spectroscopic investigations are:
•
Infrared (IR) spectroscopy measures the bond
vibration frequencies in a molecule and is used to
determine the functional groups.
Mass spectrometry (MS) fragments the molecule and
measures the masses.
Nuclear magnetic resonance (NMR) spectroscopy in
which
source
produces
radiation
in
the
radiofrequency region.
Ultraviolet (UV) spectroscopy in which the
instruments use a source which can produce
radiation of wavelength between 200-800 nm.
•
n= c /l
• The word spectroscopy implies the use
of electromagnetic spectrum to gain
information about organic molecules.
• Ultraviolet spectroscopy incultates that
the information will be obtained from a
specific region of the electromagnetic
spectrum called the ultraviolet region
(190 to 400 nm U.V. Region and 400 to
800 nm Visible Region) .
Ultraviolet spectroscopy
Types of Spectroscopy
•
E = hn
Contd….
• Sunlight is composed of many
components which can be separated
by passing through some medium
(prism). Such a separation of the
components
constitutes
the
formation of a spectrum. This study
of interaction of the components of
light with matter is known as
Spectroscopy.
•
• Ultraviolet radiation is the part of the electromagnetic
spectrum which bridges the gap between the longest
wavelength x-rays and shorter wavelength visible
light.
• This UV region is divided into into far UV (10- 200
nm) and near UV or quartz (200-380 nm) regions.
Far UV radiation is absorbed by air because of the
moisture and also because of electronic transitions in
atmospheric O2, N2 and CO2. One must use vacuum
apparatus to study far UV radiation. That is why this
region is known vacuum region.
• Quartz optics are therefore used to study in the near
UV region. Glass absorbs UV radiation in this region.
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Contd…..
Contd…..
• The total energy of the molecule is the sum total
of its binding or electronic, vibrational and
rotational energy.
• Energy absorbed in the UV region produces
changes in the electronic energy of the molecule
resulting from the transitions of valency electrons
in the molecule.
• In the case of molecules, electronic transitions
occur at the molecule orbital levels. Such
transitions require the amount of energy
possessed by the photons in the UV region and
become the chief area of interest in UV
spectroscopy.
Electronic Transitions
In the ground state both the  e- are in the bonding 
orbital and have opposite spins whereas the antibonding
* orbital is empty. When the molecule absorbs photons
of correct energy, one of these e- is promoted to
antibonding * molecular orbital. This state of the
molecule is referred to as the excited state.
Similar transitions can take place for s and n elctrons
also. For radiation to cause electronic excitation it
must be in the UV region of electromagnetic spectrum.
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The valence electrons are the only ones whose energies permit them to
be excited by near UV/visible radiation.
Characteristic groups may be recognized in the complex structures.
Ultraviolet (UV) Spectroscopy – The Instrumentation
s* (anti-bonding)
* (anti-bonding)
Four types of transitions
*
n (non-bonding)
n*
 (bonding)
The recorder
assem bly
The absorption
results from the
conjugated
“enone
enone”” portion
of the two
compounds.
 *
n *
s (bonding)
s  s* transition in vacuum UV
n  s* saturated compounds with non-bonding electrons
n ~ 150-250 nm
e ~ 100-3000 ( not strong)
n  *,   * requires unsaturated functional groups (eq. double bonds)
most commonly used, energy good range for UV/Vis
n ~ 200 - 700 nm
n  * : e ~ 10-100
  *: e ~ 1000 – 10,000
The spectrom eter itself – this houses the lam ps,
m irrors, prism s and detector. The spectrom eter
splits the beam of radiation into tw o and passes
one through a sam ple and one through a
reference solution (that is alw ays m ade up of the
solvent in w hich you have dissolved the sam ple).
The detector m easures the difference between
the sam ple and reference readings and
com m unicates this to the recorder.
The sam ples are dissolved in a solvent w hich is transparent to UV light and put into sam ple cells
called cuvettes. The cells them selves also have to be transparent to UV light and are accurately
m ade in all dim ensions. They are norm ally designed to allow the radiation to pass through the
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sam ple over a distance of 1cm .
Double-beam UV
Spectrophotometer
U.V. Spectrum
Applications of U.V. Spectroscopy:
UV spectrum is a plot of absorption intensity
(ordinate) versus wavelength (abscissa). The
absorption intensity is plotted either as Є or
log Є and is reported as Єmax meaning that
this is the maximum molar extinction
coefficient.
The wavelength of absorption is usually
expressed as λmax, meaning that this is the
maximum absorption.
1. QUALITATIVE ANALYSIS
 UV
absorption 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.
Optical system of a double-beam spectrophotometer
COMPARISON OF U.V. SPECTRA OF
BENZENE WITH PHENOL
U.V. SPECTRA OF PARACETAMOL (PCM)
2. 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.
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3. Structure elucidation of organic compounds.
4. QUANTITATIVE ANALYSIS

UV absorption spectroscopy can be used for the quantitative determination of
compounds that absorb UV radiation. This determination is based on Lambert

Beer’s law which is as follows.
UV spectroscopy is useful in the structure elucidation of
5. CHEMICAL KINETICS

Kinetics of reaction can also be studied using
UV spectroscopy. UV radiation is passed through the
organic molecules, the presence or absence of unsaturation
reaction cell and the absorbance changes can be

and the presence of hetero atoms.
observed.
Where :

I0 -Intensity of incident light
From the location of peaks and combination of peaks, it can
I -Intensity of transmitted light
be concluded that whether the compound is saturated or
ε -is extinction co-efficient,
unsaturated, hetero atoms are present or not etc.
c- is concentration, and
b- is the length of the cell that is used in UV spectrophotometer.
7. QUANTITATIVE ANALYSIS OF PHARMACEUTICAL
8. EXAMINATION
OF
POLYNUCLEAR HYDROCARBONS
SUBSTANCES
 Many
drugs (raw material or formulation ) e.g.

Diazepam, Chloramphenicol etc., can be assayed by
NAPHTHALENE
DIPHENYL
Benzene and polynuclear hydrocarbons have characteristic spectra
in ultraviolet and visible region. Thus identification of poly
nuclear hydrocarbons
making a suitable solution of the drug in a solvent and
can be made by comparison with the
spectra of known poly nuclear compounds.
measuring the absorbance at a specific wavelength.

Polynuclear hydrocarbons are the hydrocarbon molecules with
two or more closed rings; examples are naphthalene, C10H8, with

two benzene rings side by side, or diphenyl, (C6H5)2, with two
bond-connected benzene rings. Also known as polycyclic
hydrocarbon.
9. MOLECULAR WEIGHT DETERMINATION
DERIVATIVE

The concentration of the solution in gm moles per litre can
be calculated by using the following formula.

Molecular
weight
of
compounds
can
be
measured
spectrophotometrically by preparing the suitable derivatives of
SPECTROSCOPY
It shows better resolution of the overlapping bands than fundame ntal
spectrum and may pe rmit the actual dete rmination of the l max of the
individual bands.
In de rivative spectroscopy, DA/Dl is plotted against the wavelength. It
is excellent for dete rmination of multi components in a sample, if they
can be resolved.
these compounds.

For example, to determine the molecular weight of an amine it
is converted to amine picrate. Then optical density of a known
concentration of amine picrate solution is measured at λmax 380
nm.
 "c"
can be calculated using above equation, the
weight
"w"
of
amine
picrate
is
known.
From "c" and "w", molecular weight of amine picrate can
be calculated.
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SOLID STATE U.V. SPECTROSCOPY
It is specifically designed to measure the
reflectance or transmittance of solids,
liquids, powders, or other small objects
that can fit in the transmittance or
reflectance ports.
Loading a standard or sample into
the sample holder.
SOLID STATE U.V. SPECTROSCOPY:
THE INSTRUMENTATION
Basic components of the accessory
include the integrating sphere, transfer
optics and detector pre amplification
module.
Optical Setup
APPLICATIONS
Measuring reflectance of a solid sample.
TACKLES DIFFICULT LIQUID SAMPLES WITH EASE
• Turbid and cloudy liquids and suspensions, including DNA,
proteins and blood are handled with ease, which can make highly
accurate measurements up to 6 Absorbance units.
• Choice of cell and autosamplers speed and simplify multi-sample
analyses.
Specular
Diffuse
Depending on the equipment used, it is possible to take separate
measurements for specular reflectance, diffuse reflectance or
overall reflectance.
CONTD……
• A range of simple fixed and variable-angle
solid sample holders allow transmittance
measurements of materials such as glass,
polymer films and filters.
• A range of single and multiple-cell accessories ensures precise
and rapid thermostatting for biological studies such as enzyme
kinetics, DNA melt and general kinetics measurements in
chemistry.
• Huge choice of cells and sample holders allows virtually any
liquid and solid sample to be measured. Options include longpath cells, test-tube holders, flow-through cells and micro and
semi-micro cells
• For
relative
and
absolute
reflectance
measurements on mirrors, optics and thin-film
coatings. Used in Paint industry to determine the
quality of the paint.
CONCLUSION
 To
conclude, UV-Visible spectrometers
have been in general use for the last 35
years and over this period have become
the most important analytical instrument
in the modern day laboratory.
 In many applications other techniques
could be employed but none rival UVVisible spectrometry for its simplicity,
versatility, speed, accuracy and costeffectiveness.
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