UV–Visible Spectrophotometry: Principles, Instrumentation, and Application

 🔴 What is a UV–Visible Spectrophotometer?

A UV–Visible spectrophotometer is an analytical instrument used to measure how much ultraviolet or visible light a substance absorbs.

▪️ It works in the wavelength range of:

UV region: 200–400 nm

Visible region: 400–800 nm

🔴 Principle 

The working principle is based on Beer–Lambert Law:

A = εbc

Where:

A = Absorbance

ε = Molar absorptivity

b = Path length of cuvette

c = Concentration of solution

🔴Meaning:

More concentration → more absorbance

🔴 Main Components of UV Spectrophotometer

This image showcases a UV–Visible Spectrophotometer, an essential analytical instrument widely used in chemical, pharmaceutical, and research laboratories. It operates by measuring the absorbance of ultraviolet and visible light to determine the concentration and purity of substances. Known for its accuracy, simplicity, and reliability, the UV–Vis spectrophotometer plays a vital role in quality control, research, and routine analysis.

1️⃣ Light Source

Deuterium lamp → UV region

Tungsten lamp → Visible region

2️⃣ Monochromator

Selects specific wavelength

Prism or diffraction grating

3️⃣ Cuvette (Sample Holder)

Quartz cuvette → UV region

Glass cuvette → Visible region

4️⃣ Detector

Photodiode or Photomultiplier tube

5️⃣ Display & Data System

Shows absorbance, % transmittance

🔴 How Does It Work? 

1. Light passes from the source

2. Monochromator selects wavelength

3. Light passes through the sample

4. Sample absorbs part of light

5. Detector measures remaining light

6. Instrument calculates absorbance

🔴Why UV Absorption Happens?

UV absorption occurs due to electronic transitions:

Compounds with double bonds, aromatic rings absorb strongly.

🔴 Applications of UV Spectrophotometer

• Quantitative analysis of drugs

• Assay in QC laboratories

• Reaction monitoring in R&D

• Detection of impurities

• Determination of λmax

• Protein & DNA estimation

• Color analysis

🔴 Advantages

✔ Fast & simple

✔ Non-destructive

✔ High accuracy

✔ Cost-effective

✔ Widely used in pharma & chemical industry

🔴 Limitations

❌ Cannot identify complex structures alone

❌ Not suitable for colorless solvents

❌ Interference from impurities