Introduction
A spectrophotometer is an instrument used in biochemistry laboratories to measure how much light a substance absorbs. It is widely used in protein estimation, DNA quantification, and enzyme activity studies. Spectrophotometry is based on the principle that different substances absorb light at different wavelengths, and this absorbance can be used to determine their concentration.
Schematic Diagram of a Spectrophotometer
Below is a textual representation of a simple schematic diagram of a spectrophotometer:
Light Source → Collimator → Monochromator → Wavelength Selector → Sample Cuvette → Detector → Readout Display
(Note: In actual document or assignment, this can be replaced with a hand-drawn or digital sketch.)
Basic Components of a Spectrophotometer
Here are the main parts of a spectrophotometer and their functions:
1. Light Source
- Provides the initial beam of light that will pass through the sample.
- Common sources include tungsten lamps (for visible light) and deuterium lamps (for UV light).
2. Collimator (Lens System)
- It directs and focuses the light into a straight beam.
- Ensures that light travels in a uniform direction toward the monochromator.
3. Monochromator
- Separates the white light into individual wavelengths.
- Usually made up of prisms or diffraction gratings.
- Allows only a specific wavelength to pass through to the sample.
4. Wavelength Selector (Slit)
- Ensures only the selected narrow band of light reaches the sample.
- Improves accuracy and sensitivity of the reading.
5. Sample Holder (Cuvette)
- Holds the solution being analyzed.
- Made of glass, quartz, or plastic depending on the wavelength range (quartz is used for UV).
- The path length is usually 1 cm.
6. Detector
- Measures the amount of light that passes through the sample (transmittance).
- Common detectors include photodiodes and photomultiplier tubes.
7. Readout Display (Output Device)
- Shows the absorbance or transmittance value of the sample.
- Can be digital or analog. Modern devices also store data for analysis.
Working Principle in Brief
The spectrophotometer sends light through a sample. If the sample absorbs some of the light, the remaining light reaches the detector. The amount of light absorbed is related to the concentration of the substance in the sample.
Applications of Spectrophotometer
- Measuring concentration of DNA, RNA, and proteins.
- Studying enzyme reactions and rates.
- Analyzing colorimetric reactions.
- Used in quality control in industries like pharmaceuticals and food.
Conclusion
The spectrophotometer is an essential tool in biochemistry labs. Understanding its basic components — from the light source to the detector — helps in using it correctly for accurate and precise results. Drawing and labeling a schematic diagram helps in better understanding of how the instrument works.