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UV-VISIBLE SPECTROMETRY

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 UV-VISIBLE SPECTROMETRY
Introduction
Spectroscopy is the study of the properties of matter through its interaction with
various types of radiation (mainly electromagnetic radiation) of the electromagnetic
spectrum.
Spectrometric Techniques are a large group of analytical methods that are based on
atomic and molecular spectroscopy.
Spectrometry and spectrometric methods refer to the measurement of the intensity of
radiation with a photoelectric transducer or other types of electronic device.
The UV-VIS spectrometry is one of the oldest instrumental techniques of analysis
and is the basis for a number of ideal methods for the determination of micro and
semimicro quantities of analytes in a sample. It concerns with the measurement of the
consequences of interaction of Electromagnetic radiations in the UV and/or visible
region with the absorbing species like, atoms, molecules or ions.

Origen and Characteristics of UV-Visible Spectrum

UV-VIS spectrum results from the interaction of electromagnetic radiation in the UV-
Visible region with molecules, ions or complexes. It forms the basis of analysis of
different substances such as, inorganic, organic and biomolecules. These
determinations find applications in research, industry, clinical laboratories and in the
chemical analysis of environmental samples. It is therefore important to learn about
the origin of the UV-VIS spectrum and its characteristics.

Radiation and energy

Radiation is a form of transmitted energy Electromagnetic radiation is so-named
because it has electric and magnetic fields that simultaneously oscillate in planes
mutually perpendicular to each other and to the direction of propagation through
space Electromagnetic radiation has the dual nature: its exhibits wave properties and
particulate properties.

The nature of light

Light is a form of energy. Energy can be transferred from one point to another point
either by particle motion or by wave motion. Accordingly, different theories on the
nature of light have been proposed. The important theories are as follows:-
1. Particular like theory.
2. Electromagnetic wave theory.
Note\ Electric vector only that can react with matter and exchange the energy.
Wavelength ()[lambda]
is the distance between two consecutive peaks or troughs in a wave the distance
between any two adjacent identical points in a wave. given the notation ()
Wavelength units: length
Angstrom (A) : 1 A = 1x10-10 m
Nanometer (nm): 1 nm=1x10-9 m
Micrometer (m): 1 m = 1x10-6 m

Frequency (f or  [nu]):
Oscillations per sec (Hz) = cycles / s
1 Fresnel= 10 12 Hz
reciprocal of the wavelength (the interval from a given point on one sound wave to
the equivalent point on the next sound wave)
For an oscillating or varying current , frequency is the number of complete cycles per
second in alternating current direction. The standard unit of frequency is the hertz ,
abbreviated Hz.

Wave number(ύ)
Wave number(ύ) is the number of wavelengths that pass a fixed point in one unit of
time (usually per second).
Wave number units: inverse length (often in cm-1), reciprocal of the wavelength

ύ = 1\ ʎ
Particle properties
To describe how electromagnetic radiation interacts with matter, consider the beam
of radiation as a train of photons. The energy of each photon is proportional to the
frequency of radiation given by the relationships.
c =  ,  = c\
C = velocity = 3.0 x 108 m/s in a vacuum
C is independent of  or  in a vacuum
E = h
E (Energy of Radiation)
v (Frequency)
h (Planck’s Constant = 6.626 x 10-34 J  s
E = hc \ ʎ ύ = 1\ ʎ
E α 1\ ʎ

The higher the frequency, the higher the energy of radiation (i.e.) a photon of high
frequency (short wavelength) has higher energy content than one of lower frequency
(longer wavelength).

The nature of electromagnetic radiation and spectral regions

The electromagnetic spectrum is composed of a large range of wavelengths and
frequencies (energies). It varies from the highly energetic gamma rays to the very low
energy radio-waves. The entire range of radiation is commonly referred to as the
electromagnetic spectrum. The major spectral regions of the spectrum are shown in
figure below and the divisions are based on the methods required to generate and
detect various types of radiations.
Note: that spectroscopic methods that employ not only visible but also gamma rays
and x-rays as well as ultraviolet, infrared, microwave and radiofrequency radiation
are often called optical methods despite the fact that the human eye is sensitive to
neither of the later types of radiations. This might be due for the similarities of the
ways of interaction of these types of radiation with matter.

Absorption and Emission of Radiation

Electromagnetic radiation can interact with matter in a number of ways. If the
interaction results in the transfer of energy from a beam of radiant energy to the
matter, it is called "absorption". The reverse process in which a portion of the
internal energy of matter converted into radiant energy is called "emission". In
emission process, species in an excited state can emit photons of characteristic
energies by returning to lower energy states or ground states. Part of the radiation
which passes into matter, instead of being absorbed, may be scattered or reflected or
may be re-emitted at the same wavelength or a different wavelength upon emerging
from the sample. Radiation, which is neither absorbed nor scattered, may undergo
changes in orientation or polarization as it passes through the sample.

Absorption of radiation
Absorption of radiation by matter always involves the loss of energy by the radiation
and a corresponding gain in energy by the atoms or molecules of the medium. The
energy absorbed from radiation appears as increased internal energy, or in increased
irrational and rotational energy of the atoms and molecules of the absorbing medium.
As a general rule, translational energy is not directly increased by absorption of
radiation, although it may be indirectly increased by degradation of electronic energy
or by conversion of rotational or vibration energy to that of translation by
intermolecular collisions.
Atomic absorption
As the name suggests the atomic absorption spectra are the spectral reading obtained
when the electromagnetic radiations are absorbed by the atoms. These spectra are
used for the study of certain atomic reaction where the process require some energy
to activate an atom.
The transition of an electron form lower energy level to higher energy level takes
place by the absorption of photons of certain energy. The magnitude of absorbed
energy must be equal to the difference between those two energy levels

Molecular absorption
Absorption spectra are much more complex than for atomic absorption due to a
large number of possible energy states. The energy of a band in a molecular
absorption spectrum is the sum of three different energy components.
Absorption spectrum of any material is absorbed radiation of different
frequencies, which is determined by the atomic and molecular composition of
the material. Only those radiation of particular frequencies are generally
absorbed which match with the energy difference between two quantum
mechanical states of the molecules. This absorption between two states is
known as an absorption line and a spectrum has many lines.
Interaction of Matter with radiation
The word spectroscopy is used to refer to the broad area of science dealing with the
absorption, emission, or scattering of electromagnetic radiation by molecules, ions,
atoms, or nuclei. Spectroscopic techniques are some of the most widely used
analytical methods in the world today. These techniques are useful in determining
both the identity of unknown substances and their concentration in solution. Different
regions of the electromagnetic spectrum such as infrared, visible, ultraviolet, or, X-
ray radiation can be used to interact with matter
The interaction of radiation with matter can cause redirection of the radiation and/or
transitions between the energy levels of the atoms or molecules.
Absorption: A transition from a lower level to a higher level with transfer of energy
from the radiation field to an absorber, atom, molecule, or solid.
Emission: A transition from a higher level to a lower level with transfer of energy
from the emitter to the radiation field. If no radiation is emitted, the transition from
higher to lower energy levels is called nonradioactive decay.
The data that is obtained from spectroscopy is called a spectrum.
A spectrum is a plot of the intensity of energy detected versus the wavelength (or
mass or momentum or frequency, etc.) of the energy.
A spectrum can be used to obtain information about atomic and molecular energy
levels, molecular geometries, chemical bonds, interactions of molecules, and related
processes. Often, spectra are used to identify the components of a sample (qualitative
analysis).
Ultraviolet- Visible Spectroscopy
Ultraviolet and visible (UV-Vis) absorption spectroscopy is the measurement of the
attenuation of a beam of light after it passes through a sample or after reflection from
a sample surface.

The visible spectrum ranges from 400 nm to about 800 nm. The color we see depends
on wavelength. The color of a substance is determined by which color(s) of light it
absorbs and which color(s) it transmits or reflects (the complementary color(s)).
Color is an important property of a substance. The color of matter is related to its
absorptivity or reflectivity. The human eye sees the complementary color to that
which is absorbed.
Beer – Lambert Law
Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below
shows a beam of monochromatic radiation of radiant power P0, directed at a sample
solution. Absorption takes place and the beam of radiation leaving the sample has
radiant power P.
Sample cell

As the name suggests, these instruments contain a single beam of light. The
same beam is used for reading the absorption of the sample as well as the
reference. The radiation from the source is passed through a filter or a suitable
monochromator to get a band or a monochromatic radiation. It is then passed
through the sample (or the reference) and the transmitted radiation is detected
by the photodetector. The signal so obtained is sent as a read out or is recorded.

Double Beam Spectrometers

Using a spectrophotometer
The specific instructions will differ with other models, but the principles remain.
1. The instrument must have been warm for at least 15 min. prior to use. The
power switch doubles as the zeroing control.
2. Use the wavelength knob to set the desired wavelength. Extreme wavelengths,
in the ultraviolet or infrared ranges, require special filters, light sources, and/or
sample holders (cuvettes).
3. With the sample cover closed, use the zero control to adjust the meter needle to
"0" on the % transmittance scale (with no sample in the instrument the light
path is blocked, so the photometer reads no light at all).
4. Wipe the tube containing the reference solution with a lab wipe and place it into
the sample holder. Close the cover and use the light control knob to set the
meter needle to "0" on the absorbance scale.
5. Remove the reference tube, wipe off the first sample or standard tube, insert it
and close the cover. Read and record the absorbance, not the transmittance.
6. Remove the sample tube, readjust the zero, and recalibrate if necessary before
checking the next sample.
Q \ Why we use a reference solution? Can't you just use a water blank?

Answer\ A proper reference solution contains color reagent plus sample buffer.
The difference between the reference and a sample is that the concentration of
the assayable substance in the reference solution is zero. The reference tube
transmits as much light as is possible with the assay solution you are using. A
sample tube with any concentration of the assayable substance absorbs more
light than the reference, transmitting less light to the photometer. In order to
obtain the best readability and accuracy, the scale is set to read zero absorbance
(100% transmission) with the reference in place. Now you can use the full scale
of the spectrophotometer. If you use a water blank as a reference, you might
find that the assay solution alone absorbs so much light relative to distilled
water that the usable scale is compressed, and the accuracy is very poor.
 REFERENCES
1. principles of instrumental analysis, skoog, 6th edition 2007.
2. Modern Analytical Chemistry, D. Harvey, 2000.
3. Spectrochemical analysis, jams.

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