Model Answer
0 min readIntroduction
A spectrophotometer is a scientific instrument used to measure the intensity of light as a function of wavelength. It is a cornerstone tool in various scientific disciplines, including biochemistry, molecular biology, and analytical chemistry, enabling quantitative analysis of substances. The instrument operates based on the principle that different substances absorb light at different wavelengths, and the amount of light absorbed is proportional to the concentration of the substance. Understanding its components and their functions is vital for accurate data interpretation and experimental design. This answer will detail the principle behind spectrophotometry and describe the function of its key components: radiation sources, phototubes, and prisms.
Principle of Spectrophotometry
The fundamental principle governing spectrophotometry is the Beer-Lambert Law. This law states that the absorbance of a solution is directly proportional to the concentration of the analyte and the path length of the light beam through the solution. Mathematically, it is expressed as:
A = εbc
Where:
- A = Absorbance
- ε = Molar absorptivity (a constant specific to the substance and wavelength)
- b = Path length (the width of the cuvette holding the sample)
- c = Concentration
Spectrophotometers measure the amount of light that passes through a sample (transmittance) and calculates the amount of light absorbed. By comparing the absorbance of a sample to a known standard, the concentration of the analyte can be determined.
Components of a Spectrophotometer
1. Radiation Sources
The radiation source provides the initial beam of light for the spectrophotometer. The type of source used depends on the wavelength range being measured. Common radiation sources include:
- Tungsten-Halogen Lamp: Used for visible and near-infrared (320-2500 nm) regions. These lamps provide a continuous spectrum of light.
- Deuterium Lamp: Used for ultraviolet (190-400 nm) regions. Deuterium lamps emit a continuous spectrum in the UV range.
- LEDs (Light Emitting Diodes): Increasingly used as sources due to their stability, long lifespan, and narrow bandwidth. They can be tailored to specific wavelengths.
The emitted light from the source is often polychromatic (containing multiple wavelengths) and needs to be filtered to select the desired wavelength for analysis.
2. Prism (or Diffraction Grating)
The prism (or more commonly, a diffraction grating in modern spectrophotometers) is responsible for separating the polychromatic light emitted by the source into its constituent wavelengths.
- Prism: A prism refracts light, separating it into its spectrum based on the principle of dispersion – different wavelengths are bent at different angles.
- Diffraction Grating: A diffraction grating consists of a surface with closely spaced parallel grooves. When light strikes the grating, it is diffracted, and constructive interference creates a spectrum. Diffraction gratings offer better wavelength separation and are more commonly used in modern spectrophotometers.
A monochromator, which includes the prism/grating and slits, selects a narrow band of wavelengths to pass through the sample. The narrower the bandwidth, the higher the resolution of the spectrophotometer.
3. Phototube (or Photodiode)
The phototube (or more commonly, a photodiode) is the detector that measures the intensity of the light that has passed through the sample.
- Phototube: A vacuum tube containing a photosensitive material that emits electrons when struck by light. The number of emitted electrons is proportional to the light intensity.
- Photodiode: A semiconductor device that generates a current when exposed to light. Photodiodes are more compact, sensitive, and have a faster response time than phototubes.
The current generated by the photodiode is amplified and converted into a digital signal, which is then displayed as absorbance or transmittance on the spectrophotometer’s readout. Modern spectrophotometers often use photomultiplier tubes (PMTs) for even greater sensitivity, especially in the UV range.
| Component | Function | Technology |
|---|---|---|
| Radiation Source | Provides initial light beam | Tungsten-Halogen, Deuterium, LEDs |
| Prism/Diffraction Grating | Separates light into wavelengths | Refraction (Prism), Diffraction (Grating) |
| Phototube/Photodiode | Detects light intensity | Photoemission, Semiconductor |
Conclusion
In conclusion, the spectrophotometer is a powerful analytical tool based on the Beer-Lambert Law. Its functionality relies on the coordinated operation of its key components: the radiation source, the prism (or diffraction grating), and the phototube (or photodiode). Each component plays a crucial role in generating, selecting, and detecting light, enabling accurate quantitative analysis of substances. Advancements in technology continue to refine spectrophotometers, increasing their sensitivity, resolution, and versatility for a wide range of applications in scientific research and industry.
Answer Length
This is a comprehensive model answer for learning purposes and may exceed the word limit. In the exam, always adhere to the prescribed word count.