Principle of UV Visible Spectroscopy: Methods & Applications

The principle of UV-visible spectroscopy is based on the production of spectrum by the absorption of visible light by the chemical compounds. According to the UV-visible spectroscopy, all the molecules have both bonding and non-bonding electrons.

• UV Visible spectroscopy can absorb ultraviolet radiations or visible light to excite the electrons.
• The electrons are present in higher molecular orbitals(anti-bonding). 
• The excitation of electrons takes place from lower energy ground state to higher energy states.
• Excitation takes place due to the absorption of ultraviolet radiation (or visible light). 
• The electronic transitions is of four types: σ–σ* transitions > n–σ* transitions > π–π* transitions > n–π* transitions.
• UV Visible spectroscopy is applicable for all organic, inorganic, and coordination compounds. 
• The spectrum formed during the spectroscopy is used to identify the compound. 
• Detection of impurities and medical laboratory tests are the main applications of this type of spectroscopy.​

Key Terms: Principle of UV-visible Spectroscopy, UV-Visible Spectroscopy, Spectrum, Electrons, Ultraviolet Radiation, Molecular Orbitals, Electromagnetic Radiation, Spectroscopy, Wavelength

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What is UV-Visible Spectroscopy?

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UV Visible Spectroscopy or UV Vis spectroscopy is generally defined as the interaction of ultraviolet radiation (or visible light) with matter. 

• The electronic spectrum is formed due to the electromagnetic radiation in the UV Visible region. 
• It measures the amount of light which is absorbed by a chemical substance.
• UVA, UVB, and UVC are three main types of UV rays.
• The residual radiation will produce a spectrum with a given between it called the absorption spectrum.
• Electrons in the occupied orbitals move towards the unoccupied orbitals.
• It is used for the characterization of dyes and the structural interpretation of compounds.

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Principle of UV-Visible Spectroscopy

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The principle of UV-visible spectroscopy is a form of quantitative technique that is used to measure the amount of light absorbed by a substance. When light rays fall on a substance, a part of the light is absorbed.

• The principle of UV-visible spectroscopy is based on the interaction between light and matter.
• A certain amount of light is reflected back to the surface.
• The wavelength of light is known as the absorption maximum.
• The amount of radiation is equal to the difference between the incident and transmitted radiation.
• Transmittance is the ratio of incident radiation and transmitted radiation.
• It also indicates the amount of light that passes through the sample.
• The principle of UV-visible spectroscopy is mathematically represented by:

Transmittance, T = I/ Io

Absorbance, A = log10(Io /I) = log10 (1/T) = - log10 (T)

• where Io is incident radiation and I is transmitted radiation

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Important terms of UV-Visible Spectroscopy

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Some important terms of UV-Visible Spectroscopy are as follows:

Spectroscopy

The field of science which involves the study of the interaction of electromagnetic radiation with matter is called spectroscopy.

Electromagnetic Radiations

Electromagnetic radiations are radiations associated with electric and magnetic fields.

Electromagnetic Spectrum

The arrangement of electromagnetic radiations in the increasing order of their frequency or decreasing order of their wavelength is called the electromagnetic spectrum.

UV Visible Spectrophotometer

UV Visible Spectrophotometer is an instrument working on the principle of UV Visible spectroscopy. It measures the intensity of the transmitted light through a sample chemical compound.

• UV -Visible spectrometer uses a reference of the incident source of light for comparison with measurements of transmitted light.

UV-visible spectroscopy 

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Beer-Lambert’s Law

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Beer-Lambert’s law states that when monochromatic light is incident on a homogenous medium, then the intensity of the transmitted light decreases with the increase in the concentration of the absorbing solution.

• It increases with the increase in the thickness of the medium.
• Beer-Lambert’s law is one of the major absorption laws and corresponds to the absorption of light by molecules.
• It determines that absorption of radiation increases with an increase in the number of the absorbing molecules.

According to Beer Lamert’s Law, the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length. The efficiency is determined by molar absorptivity.

• An increase in the absorption of radiation will increase the absorption of molecules.
• Shorter wavelengths can easily pass through the smaller aperture.
• It can be mathematically represented as:

A = εLc 

• Where, A is the amount of light which is absorbed by the sample
• ε is the molar absorptivity 
• L is the distance covered by the light within the solution
• c is the concentration of the absorbing radiation

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Instrumentation of UV Visible Spectroscopy

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The instrumentation used to carry out UV-visible spectroscopy is carried out with the help of a UV-visible spectrophotometer. It consists of a monochromator, data recorder, light source and a detector.

• The light source is used as an illumination source to identify different wavelengths.
• Monochromator will identify the wavelength that will pass through the solution of the sample.
• Detector will measure the intensity of light that will pass through the sample.
• Data recorder will determine the absorbance or transmission of light.

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Applications of UV Visible Spectroscopy

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The UV Visible spectroscopy has a wide range of applications in the field of science and day-to-day life. Some of them are as follows:

• UV Visible spectroscopy is used for the characterization of dyes and colourants.
• It is used in medical labs for tests done in determining blood sugar and cholesterol.
• The test is used for the detection of aromatic compounds.
• It is also used for the structural interpretation and detection of impurities of organic molecules.
• UV Visible spectrophotometer is used as a detector for High-Performance Liquid Chromatography (HPLC).

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Advantages of UV Visible Spectroscopy 

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Various advantages of UV Visible Spectroscopy are as follows:

• The method can be reused any number of times.
• The process is simple and easy, for which no training is required.
• Setup for the experiment can be easily performed.
• The result can be achieved in a short span of time.

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Things to Remember

• The principle of UV Visible spectroscopy will have all the molecules having both bonding and non-bonding electrons.
• It can absorb ultraviolet radiations or visible light to excite the electrons.
• UV-visible spectroscopy is generally defined as the interaction of ultraviolet radiation (or visible light) with matter.
• Beer-Lambert’s law determines that the intensity of the transmitted light decreases with the increase in the concentration of the absorbing solution.
• The UV Visible spectroscopy is applicable to all organic, inorganic, and coordination compounds.
• It is used to identify the concentration of samples containing RNA and DNA molecules.