Molecular analysis using UV/Visible spectroscopy

//Molecular analysis using UV/Visible spectroscopy

Molecular analysis using UV/Visible spectroscopy

Spectroscopy
Spectroscopy deals with production, measurement and interpretation of spectra arising from the interaction of electromagnetic radiation with matter. It helps in the qualitative or quantitative study of matter. It was experimentally observed that when light impinges on solutions or crystals of molecules, at least two distinct processes occur- Light scattering and Light Absorption. The interaction of radiation with matter depends upon both radiation properties and structural parts of the materials involved. This interaction between matter and radiation leads to a variety of spectra.

Types of spectra

Emission spectrum: When atoms or molecules are subjected to intense heat or an electric discharge, they absorb energy and get excited. During this excitation, electrons get shifted from ground state to the excited state. Since the lifetime of these electrons is short, they return to lower state and the energy so absorbed is released as light energy. When it is passed through a prism, it gives rise to an emission spectrum.
The experimental setup used to obtain an emission spectrum for hydrogen with the wavelengths of the 4 visible emission lines noted.

Absorption spectrum: When an electromagnetic radiation is passed through a solution, some of it is absorbed, some is reflected and the remainder is transmitted. When this transmitted light is passed through a prism and absorption spectra is obtained. For example, an object that absorbs blue, green and yellow light will appear red when viewed under white light.
Absorption spectra can be used to identify elements present in a gas or liquid. This method is used in deducing the presence of elements in stars and other gaseous objects which cannot be measured directly.

UV/Visible Spectroscopy
The range of UV region in electromagnetic spectrum is 100-400 nm and that of visible region is 400-800 nm. The UV region can be further divided into two regions– far UV (100-200nm) and near UV (200-400nm). UV and visible absorption spectroscopy measures the absorption of a beam of light after it has passed through a sample or after reflection from sample surface.
Principle
Whenever UV or visible radiation is passed through the solution of an organic compound, it is either absorbed or transmitted depending on the wavelength and the molecular structure of the compound. As a result of absorption, an electron gets excited from the ground state to the excited state. During absorption, some molecules of compound collide with the photons of radiant energy and only those will result in absorption of energy in which energy of photon exactly matches the difference in energy of ground and excited states of molecule. If E0 and E1 are the energies of ground state and excited state of a molecule respectively and ΔE is the difference in energy between two states, then
ΔE = (E1 – E0) = hv = hc/ λ
Absorption of energy in UV or visible region occurs due to transitions between electronic energy levels within the molecule. Thus UV/ Visible spectroscopy is called electronic spectroscopy.

The absorption of radiant energy is based on two laws:

  1. Beer’s Law
  2. Lambert’s Law

Beer’s Law states that amount of energy absorbed is directly proportional to the concentration of absorbing solute or number of absorbing molecule present in a medium.
A = log (Io/I) = ε c
In this formula, A is absorbance, Io is intensity of incident light, I is intensity of light transmitted through sample, ε is absorption coefficient/absorptivity, c is concentration of absorbing material in sample.

Lambert’s Law states that fraction of light absorbed by a homogenous medium is independent of the intensity of light and light absorption is directly proportional to path length of light.
A = log (Io/I) = ε l where, l is path length. Combining these two laws, we get Beer-Lambert Law which is expressed as A = log (Io/I) = ε c l

Spectrophotometer

Spectrophotometer is an instrument used to measure the absorbance of light by a sample. This instrument works on the principle of Beer-Lambert Law. In this instrument, wavelength of light to be passed through sample is selected using prisms or gratings and absorption of light by the unknown sample is measured. Following are the components of spectrophotometer
Radiation Source For visible range, tungsten lamp with a wavelength of 340-800 nm is used as a radiation source and for UV range high pressure hydrogen or deuterium electrical discharge lamps with wavelength between 200-340 nm are used. Light source should emit a strong bond of radiation over the entire wavelength range for which instrument is designed. Power of radiation should be sufficient for adequate detector response.

Monochromator

Monochromatic light is a light of a specific single wavelength. Monochromators are used to select specific wavelength of light in spectroscopic assay. Modern instruments use a prism or more often a diffraction grating to produce desired wavelength.

By |2018-04-02T09:40:39+00:00April 2nd, 2018|Molecular Biology|Comments Off on Molecular analysis using UV/Visible spectroscopy

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