UV- Visible Spectroscopy

        Fig: UV- Visible Spectrophotometer

Content

• Introduction
• Instrumentation
• Working Principle
• Operating Process
• Application

Introduction:

 Spectroscopy is  the interaction of  electromagnetic radiation with matter. When light is absorbed by matter, this results an increase in the energy of the atoms or molecules.

Ø In UV-Vis spectroscopy, the UV-Vis light is passed through a sample and the transmittance(T) of light by a sample is measured.

Ø  Most organic molecules with or without functional groups are transparent in the portions of the electromagnetic spectrum that we call the ultraviolet (UV) and visible (VIS) regions.

Ø  Wavelength in this region ranges from 200 nm to 800 nm (for UV 200 nm – 400 nm and for Visible region 400 nm-800 nm)

 Instrumentation: 

Light source

Ø  Tungsten filament lamps emit the radiations of 375 nm.

Ø  The intensity of Hydrogen-Deuterium lamps falls below 375 nm.

Monochromator

Ø  Generally monochromators are Composed of prisms and slits.

Ø  The radiation emitted from the light source is dispersed with the help of rotating prism.

Ø  The various wavelengths of the light source which are separated by the prism

     are then selected by the slits for recording purpose.

Ø  The beam selected by the slit is monochromatic and further divided into two beams.

Cuvette

Ø  One of the two divided beams is passed through the sample cuvette and second beam is passed through the reference cuvette.

Ø  Cuvettes are made of either silica or quartz.

Detector

Ø  Two photocells serve the purpose of detector in UV spectroscopy. (Photocell is a resistor that changes resistance depending on the amount of light incident on it).One of the photocell receives the beam from sample cell and the other receives the beam from the reference

Amplifier

Ø  The main purpose of amplifier is to amplify the signals transferred from photocells many times so we can get clear and recordable signals.

Recorder

Ø  Computer stores all the data generated and produces the spectrum of the desired compound.

Principle of UV-Visible Spectrophotometer: 

• Ø  The principle of UV-Visible spectroscopy is based on the absorption of ultraviolet light by chemical compounds, which results in a distinct spectra.
• Ø  The greater the number of molecules capable of absorbing light of a given wavelength, the greater the extent of light absorption. From these the following expression can be formulated which is known as Beer-Lamberts law.

            Beer-Lamberts law can be expressed as-

        

                                 A= log (I₀/I) = εcl

            Where,

         A= Absorbance, I₀ = Intensity of incident light, I = Intensity of transmitted light, ε = Molar             extinction coefficient , C = Concentration , l = length of the sample cell (cm)

• Ø  When a monochromatic light fall on a sample, a portion of the light got absorbed by the sample and this results excitation of electrons from the ground state to a higher energy state.
• Ø  The absorbed energy must be equal to the energy difference between the excited state and the ground state.
• Ø  The most probable transition is from the highest occupied molecular orbital(HOMO) to the lowest unoccupied molecular orbital(LUMO).
• Ø  There are four possible types of transitions (π–π*, n–π*, σ–σ*, and n–σ*), and they can be ordered as follows: σ–σ* > n–σ* > π–π* > n–π*

• The most common transitions that fall in the UV-Vis range are π-π* and n-π*.
• π-orbitals arise due to double bonds and n-orbitals are for non-bonding electrons.
•   π*are anti-bonding π-orbitals.
• Ø  The best UV-Vis absorption is by molecules that contain double bonds.
• Ø  π-orbitals adjacent to each other that are connected called conjugation, typically increases absorption.
• σ-σ* transitions, associated with single bonds are higher energy and fall in the deep UV (range 100nm to 200nm), so they are less useful for routine use

Some examples of possible transitions fall in the UV-Vis range are,

(i) σ–σ* transition

(ii) n–σ* transition 

(iii) π–π* transitions

(iv) n–π* transitions

Operating process: 

Applications: 

1. Detection of impurities
2. Detection of functional groups
3. Qualitative and quantitative determination of compounds
4. To study Kinetics of reaction
5. Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds.
6. UV spectrophotometer may be used as a detector for HPLC.