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Spectrophotometer

Spectrophotometer

It an instrument which is used to measure optical density (O.D), concentration by using light, to determine conc. of solute in a solution

Spectron splits the light into different wavelengths.

On basis of conc. And path length light is absorbed.

Principle

Its principle is based on Beer and Lambert Law which is followings;

Absorbance is directly proportional to the conc.

Or

Absorbance is directly proportional to the path length

Higher the conc. Of solute the higher the absorbance is and increases the path length.

Apparatus

On spectrophotometer 30-mints before use.

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Inner side of spectrophotometer

It has the following parts;

  • Polychromatic light of different wavelengths
  • Slits
  • Dispersion plate
  • Absorber
  • Transmitter
  • Photodetector
  • Tungsten halogen bulb (provide visible light 450-750nm)

 

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Inner side of spectrophotometer

  • Xenon flash lamp (UV + visible light can be adjusted)
  • The bulb is monochromatic it converts light to a single wavelength
  • The cuvette (plastic, glass, quartz)

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  • Calorimeter (read colored reaction and light does not convert to single wavelength)
  • Blank (the medium in which solute is dissolved)
  • Power switch

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  • Controls (absorbance, concentration)

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Uses

It is used for;

Determination of conc. Of solute and microbial cells.

Identification of organic compound (absorption spectrum) absorbance is different at a different wavelength.

Λ max.is the wavelength at which absorbance is maximum

For bacterial cell it is 600-620nm

For proteins it is 540nm

For DNA it is 260-280nm and we use UV light for visualization of DNA (hydrogen bulb)

It is different for every compound as the structure of every compound is different.

Types

It has two types

  1. Double beam spectrophotometer
  2. Single beam spectrophotometer

Double Beam Spectrophotometer

In this light passes through two holes and we can place blank and sample at the same time and take readings.

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