Introduction:

 UV-Visible spectroscopy is a mature and well-established analytical technique used extensively in many industry sectors including Environmental Analysis, Pharmaceutical Testing, Food and Beverage Production etc.

Spectroscopy is the measurement and interpretation of electromagnetic radiation absorbed or emitted when the molecules or atoms or ions of a sample moves from one energy state to another energy state.

UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400 nm) is absorbed by the molecule which results in the excitation of the electrons from the ground state to higher energy state.

Principle:

UV-Visible Spectroscopy is based on the Lambert-Beer principle which states that the absorbance of a solution (A) is directly proportional to its path length (l) and its concentration (c) when the wavelength of the incidence light remains fixed.

Basically, spectroscopy is related to the interaction of light with matter.

As light is absorbed by matter, the result is an increase in the energy content of the atoms or molecules.

When ultraviolet radiations are absorbed, this results in the excitation of the electrons from the ground state towards a higher energy state.

Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb energy in the form of ultraviolet light to excite these electrons to higher anti-bonding molecular orbitals.

The more easily excited the electrons, the longer the wavelength of light it can absorb.

The absorption of ultraviolet light by a chemical compound will produce a distinct spectrum which aids in the identification of the compound.

If there is no absorption of the light passing through the solution, the transmittance is 100%.

Apparatus:

The UV-Visible Spectrophotometer is the analytical instrument used for the UV-Vis spectroscopic analysis. Spectrophotometers are available in different configurations however most can be categorized into either single beam, split beam or double beam types depending on the design of their optical system. Such types of instrument comprise the following components in their constructions:

1. Light Source :

• Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region.
• Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.

2. Monochromator :

• Monochromators generally is composed of prisms and slits.
• Most of the spectrophotometers are double beam spectrophotometers.
• The radiation emitted from the primary source is dispersed with the help of rotating prisms.
• The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose.

3. Cell Compartment :

• One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution.
• Both sample and reference solution are contained in the cells.
• These cells are made of either silica or quartz. Glass can’t be used for the cells as it also absorbs light in the UV region.

4. Detector :

• Generally two photocells serve the purpose of detector in UV spectroscopy.
• One of the photocell receives the beam from sample cell and second detector receives the beam from the reference.
• The intensity of the radiation from the reference cell is stronger than the beam of sample cell.

5. Signal Processing System :

• Most of the time amplifier is coupled to a pen recorder which is connected to the computer.
• Computer stores all the data generated and produces the spectrum of the desired compound.

This absorption spectroscopy uses electromagnetic radiation between 190 and 800 nm and is divided into two regions

1. UV (190–400 nm)- Deuterium lamp

2. Visible (400–900 nm) – Tungsten lamp

Because the absorption of UV or visible radiation by a molecule leads to transition among electronic energy levels of the molecule, it is also often called electronic spectroscopy.

UV-Visible Analysis is Suitable For,

  1. Analytes that can be dissolved in solvents like water, ethanol and hexane.

  2. The analyte need to absorb UV or Visible light.

  3. With UV /Vis we can do quantitative measurements a single analyte in solution(or more than one analytes om solution provided that do not interfere with each other).

Not Suitable For,

1. Analytes that have a photochemical reaction at the wavelength range of interest.

2. Partially dissolved, unclear or colloidal samples.

The UV-Visible spectrum shows the absorbance of one or more sample component in the cuvette when we scan through various wavelengths in the UV/Vis region of the electromagnetic spectrum.

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