Zoology 6c Explain the principle of spectrophotometer and describe its following components: (i) Radiation sources (IAS 2020/5 Marks), (ii) Phototube (IAS 2020/5 Marks), (iii) Prism (IAS 2020/5 Marks)

Introduction

Spectrophotometry is a technique used in the field of biochemistry and molecular biology to measure the amount of light absorbed by a substance at different wavelengths. This technique is based on the principle that different molecules absorb light at specific wavelengths, allowing for the quantification of substances in a sample. 

Principle of Spectrophotometer

• A spectrophotometer is an analytical instrument that measures the intensity of light at different wavelengths to analyze the composition of substances.
• Basic Principle: It operates on the principle of light absorption, where substances absorb specific wavelengths of light, and the amount of light absorbed is proportional to the concentration of the substance in the sample. This is known as Beer-Lambert Law.

Components of a Spectrophotometer

(i) Radiation Sources

• Purpose: Provide a beam of light that passes through the sample solution. The light source's wavelength range determines the spectrophotometer's operational range.
• Types of Sources:
  
  • Tungsten Lamp: Used for visible light measurements, producing light in the 350-2500 nm range.
  • Deuterium Lamp: Provides ultraviolet light, typically in the 160-400 nm range, suitable for UV spectrophotometry.
  • Xenon Lamp: Offers a broad spectrum of light, useful for applications requiring high-intensity pulses of light.
• Considerations: The choice of light source affects the sensitivity and wavelength range of the spectrophotometer.

(ii) Phototube

• Function: Converts light energy into an electrical signal, allowing for the quantification of the light intensity that passes through the sample.
• Types:
  
  • Photomultiplier Tube (PMT): Highly sensitive and used for detecting low light levels; amplifies the signal through a series of dynodes.
  • Photodiodes: Solid-state devices that convert light into an electric current; used for moderate light levels and offer quicker response times.
• Working Principle: The incident light generates photoelectric emissions within the tube, and the resulting current is proportional to the light intensity.

(iii) Prism

• Role: Disperses incoming light into its constituent wavelengths, allowing the selection of specific wavelengths for measurement.
• Types of Prisms:
  
  • Glass Prism: Commonly used, can disperse light into the visible range.
  • Diffraction Grating: An alternative to prisms, it uses closely spaced slits to separate light into its wavelengths more efficiently.
• Working: As light passes through the prism, it bends at different angles depending on the wavelength, allowing the operator to isolate specific wavelengths for analysis.

Conclusion

The spectrophotometer is a valuable tool in the field of biochemistry and molecular biology, allowing for the accurate measurement of light absorption by substances in a sample. The key components of a spectrophotometer, including the radiation sources, phototube, and prism, work together to provide reliable and precise measurements.