Spectrophotometer – Principle, Components, Applications, Advantages & Limitations

Table of Contents

What is a Spectrophotometer?

A spectrophotometer is a scientific instrument used to measure how much light a sample absorbs.
It is mainly used to determine the concentration of solutes in a solution.
The instrument passes a beam of light through the sample placed in a cuvette, and measures the amount of light absorbed.
Absorption depends on the type of solute, its concentration, and the wavelength of light used.

In simple words: A spectrophotometer helps us know how much of a substance is present in a solution by measuring light absorption.

History: The first commercial spectrophotometer, the Beckman DU, was invented in 1940 by Arnold J. Beckman and his colleagues.

Principle of Spectrophotometer

The working principle is based on the absorption of light at specific wavelengths.

Light from a source is passed through a monochromator (prism or grating) that splits it into different wavelengths.
Selected wavelengths fall on the sample solution in the cuvette.
The sample absorbs part of the light depending on its properties and concentration.
The remaining transmitted light reaches a detector, which converts it into an electrical signal.
The signal is displayed as a reading or absorption spectrum.

The spectrophotometer follows Beer–Lambert’s Law:

A=ε⋅c⋅l

Where:

A = Absorbance
ε = Molar absorptivity (constant)
c = Concentration of solution
l = Path length of light through the sample

Key Point: The higher the concentration of a solute, the more light it absorbs.

**Figure:** Principle of Spectrophotometer

Components of Spectrophotometer

A spectrophotometer is made of several key components:

1. Light Source

Provides the initial beam of radiation.
Types:
- Tungsten lamps → visible range
- Deuterium/ Hydrogen lamps → UV range
- Xenon lamps → wide spectrum

2. Monochromator

Splits polychromatic light into individual wavelengths.
Two main types:
- Prism → disperses light based on refraction. (e.g., Cornu Quartz Prism, Littrow Prism).
- Diffraction Grating → disperses light using interference; widely used for UV, Visible, and IR regions.

3. Sample Holder (Cuvette)

Small transparent container that holds the sample.
Glass cuvettes → for visible light.
Quartz cuvettes → for UV light (since glass absorbs UV).

4. Detector

Detects transmitted light and converts it into an electrical signal.
Works on photoelectric effect.
Examples: Photodiodes, Photomultiplier tubes.

5. Readout System

Displays absorbance or transmittance.
May include amplifiers, potentiometers, recorders, or digital displays.
Modern spectrophotometers use computer-based data systems for graphical output.

Working of Spectrophotometer

Calibration → The spectrophotometer is first calibrated using a blank solution (usually solvent without solute).
Sample Placement → The sample solution is placed in a cuvette.
Light Selection → A monochromator selects the required wavelength of light.
Absorption Measurement → Light passes through the sample, and the detector measures transmitted intensity.
Calculation → The device converts signals into absorbance values, which are proportional to solute concentration.
Data Output → Absorbance is displayed numerically or graphically as an absorption spectrum.

Types of Spectrophotometers

UV-Visible Spectrophotometer
- Works in 200–800 nm range.
- Common in biological and chemical labs.
Infrared (IR) Spectrophotometer
- Measures absorption in infrared region.
- Used in structural analysis of molecules.
Fluorescence Spectrophotometer
- Measures emission of light by molecules after excitation.
- Very sensitive; used for DNA, proteins, and trace detection.
Atomic Absorption Spectrophotometer (AAS)
- Used for measuring metal ions in samples.
Double-Beam Spectrophotometer
- Splits light into two beams (one passes through sample, the other through reference).
- Provides more accuracy by compensating fluctuations.

Applications of Spectrophotometer

Spectrophotometry has wide applications in biology, medicine, chemistry, and industry:

Quantitative Analysis
- Measure solute concentration in solutions (proteins, nucleic acids, enzymes).
Quality Control
- Detect impurities in chemicals, food, and pharmaceuticals.
Structural Analysis
- Identify functional groups in organic molecules.
Clinical & Medical Use
- Respiratory gas analysis in hospitals.
- Blood and urine sample testing.
Environmental Monitoring
- Measure dissolved oxygen in water bodies.
- Check pollution levels.
Molecular Biology
- Protein characterization.
- DNA/RNA quantification using UV absorbance at 260 nm.
Pharmaceutical & Industrial Applications
- Drug testing and formulation.
- Food color and preservative testing.

Advantages of Spectrophotometer

Provides highly accurate and precise results.
Can analyze very small sample volumes.
Useful for both qualitative and quantitative analysis.
Works across UV, visible, and IR ranges.
Non-destructive technique (sample is preserved).
Widely applicable in biology, chemistry, medicine, and industry.

Limitations of Spectrophotometer

Requires clear, non-turbid solutions for accurate readings.
Results can be affected by pH, temperature, and contaminants.
Needs proper calibration with blank solution.
Limited to substances that can absorb light in UV-Visible or IR range.
Cuvettes and optical surfaces must be very clean.

Precautions While Using Spectrophotometer

Always calibrate with blank before measurement.
Use clean cuvettes (free from scratches and fingerprints).
Avoid bubbles in solution (they scatter light).
Select the correct wavelength for accurate results.
Do not expose sensitive lamps (like deuterium lamps) to unnecessary power cycles.

FAQs on Spectrophotometer

Q1. What is the difference between a colorimeter and a spectrophotometer?
A colorimeter measures absorbance at a few fixed wavelengths, while a spectrophotometer measures across a wide range of wavelengths.

Q2. Why is quartz cuvette used in UV range?
Because ordinary glass absorbs UV light, while quartz is transparent to UV.

Q3. What law does spectrophotometer follow?
Beer–Lambert’s law, which relates absorbance to concentration.

Q4. Can spectrophotometer detect impurities?
Yes, it is often used in quality control and purity analysis.

Q5. What is the typical wavelength range of UV-Vis spectrophotometer?
200–800 nm.

Conclusion

The spectrophotometer is one of the most important instruments in modern laboratories. By measuring the absorption of light at specific wavelengths, it allows scientists to determine the concentration, purity, and structural properties of compounds.

It is extensively used in biological research, medical diagnostics, environmental monitoring, pharmaceuticals, and industry.

In short: A spectrophotometer is an essential tool for analyzing and quantifying molecules in solutions with high accuracy.

References

https://www.biochemden.com/spectrophotometer-instrumentation-principle/
https://www.azom.com/article.aspx?ArticleID=10245
https://web.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy.pdf
https://byjus.com/chemistry/spectrophotometer-principle/
https://www.slideshare.net/rey216/spect