Skip to main content

UV-Visible Spectroscopy


Spectroscopy is an analytical technique based on the interaction of the electromagnetic radiation with matter.

Electromagnetic radiation (E.M.R.) – Electromagnetic radiation is a form of energy which is dual in nature.
 Wave nature - frequency, wave-length and velocity.
 Particulate nature – individual photon, discrete packet of energy.

The energy carried by an E.M.R. or a photon is directly proportional to the frequency, i.e.
E = hv =hc/ = hv' c
Where h is Planck’s constant h=6.626x10-34J×s

Interaction of electromagnetic radiation with Matter

The electromagnetic radiation interacts with matter because electrons and molecules in materials are polarizable.

Different types of interactions:

• Absorption
• Reflection
• Transmission
• Scattering
• Refraction

Each interaction can disclose certain properties of the matter – When applying E.M.R. of different frequency different type information can be obtained.

The energy, E, associated with the molecular bands:

E = Eelectronic + Evibrational + Erotational

Electromagnetic radiation is absorbed when the energy of photon corresponds to difference in energy between two states. Conjugated systems form the basis of chromophores, which are light-absorbing parts of a molecule. In conjugated pi-system, the electrons jump between extended pi-orbitals created by a series of alternating single and double bonds, usually in aromatic systems.

Different Spectroscopic methods:

•Absorption spectrophotometric methods: ultraviolet-visible (UV-VIS), infrared (IR).
•Emission spectrophotometric methods: Fluorimetry (Fl).
•Light scattering: turbidimetry, nephelometry.

Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a
molecule causing the promotion of an electron from a ground electronic state to an excited electronic state.
• Ultraviolet/Visible light: wavelengths (l) between 190 and 800 nm

Beer-Lambert Law:

The law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.

A=-log 10(Io/I) = E c l, A = 2 - log10 %T

A: Absorbance or optical density (OD)
E : Absorptivity; M-1 cm-1
C : concentration; M
T : transmittance

Thus the absorbance depends upon the concentration, path length and molar extinction coefficient.
The absorption spectrum can be generated by using a UV-Visible Spectrophotometer in UV and visible region of the electromagnetic spectrum, as the molecules undergo electronic transitions. Molecular electronic transitions take place when valence electrons in a molecule are excited from one energy level to a higher energy level.

Following are some typical electronic transitions:
 σ → σ*
 π → π*
 n → σ*
 n → π*
Bands associated with electronic transitions:
 R band - Radicle like
 B band - Benzoic
 E band - Ethylenic
 K band - Conjugated (Konjugated)
UV Spectrophotometer:
The basic parts of a spectrophotometer are:
 Light source- Tungsten (300-2500 nm), Deuterium (190-400 nm)
 Sample holder,
 Monochromator (diffraction grating)
 Detector- photodiode or CCD.
 Single beam
 Double beam

 Photometric: For quantitative analysis
 Spectrum: For both qualitative and quantitative
 Can be used as a detector in LC.

 Control of wavelength
 Control of absorbance
 Limit of stray light
 Resolution power
 Base line flatness


Popular posts from this blog

Made in Japan

Made in Japan
Japan – the island nation is famous for many things. The first developed country in Asia despite adverse geographical conditions, the oldest empire existing on earth, the land of rising sun, Japanese tea ceremony Sado, Sushi the national dish, the great Samurai warriors, the gigantic Sumo wrestlers and above  all “Made in Japan” robots. Its technology had made it an iconic nation.
I was amazed to see many marvels of science and technology during my recent visit to Tokyo Science Center. What impressed me most was apart from being a research and training center for healthcare professionals it offers many novel modes of training like using realistic organ models for endoscopic surgery and cardiovascular disease treatment in an effort to introduce its cutting edge medical devices and technological advancement to the whole world enabling the healthcare professionals to enhance their knowledge and skills in treating patients across the globe for unmet medical needs making it a…