UV-Visible Spectroscopy

UV-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.
Types:
 Single beam
 Double beam

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

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