Clinical

Biochemistry 1
Feb.18.2015

Spectrophotometer
Introduction: -
The spectrophotometer is an instrument which measures the amount of light of a specified
wavelength which passes through a medium.
Presence & concentration of dissolved substances is analyzed by passing different types of light
(visible, infrared, or UV) through the sample. Light can either be transmitted or absorbed by
dissolved substances.
Spectrophotometer components: -
1) Light source.
Tungsten lamp: Vis. near IR (320 nm~2500 nm)
Deuterium are lamp: UV (200~400 nm)
Electric discharge lamp + Hg(g) or Xenon:
Globar (silicon carbide rod): IR (5000~200 cm-1)
Laser: intense monochromatic sources.
2) Monochromator consists:
a. Lenses or mirrors: Focus the radiation
b. Entrance and exit slits: Restrict unwanted and control the spectral purity of radiation.
c. Dispersing medium: Separate the l of polychromatic radiation from the source.
3) Cuvate: - It used to hold the sample and let the light pass through the sample.
4) Detector: - Convert radiant energy (photons) into an electrical signal.
Spectrophotometer work:
v The monochromatic light is obtained by allowing the beam of light to pass
through a prism or monochrometer.
v The monochromatic light is directed through a cuvette containing the sample,
and the light that penetrates hits the photoelectric cell.
v The current developed by the photoelectric cell is translated into percent
transmission or absorbance through a galvanometer.
v Then you can read the absorbance on the galvanometer.