, Spectrophotometric Techniques
Introduction to Spectrophotometric Techniques
Spectrophotometric techniques are analytical methods used to measure how
much light a substance absorbs. These techniques are widely used in:
• Food analysis
• Clinical biochemistry
• Pharmaceutical testing
• Environmental science
• Nutritional research
The basic principle is simple:
When light passes through a solution, some of it is absorbed. The
amount of absorbed light tells us the concentration of the substance.
Beer–Lambert’s Law
Definition
Beer–Lambert’s Law explains the relationship between absorbance and
concentration of a solution.
Mathematical Formula:
A=ε×l×c
Where:
• A = Absorbance
• ε = Molar absorptivity (constant)
• l = Path length (cm)
• c = Concentration
, Flow Chart – Beer–Lambert’s Law
Light Source
↓
Monochromatic Light passes through Solution
↓
Some light absorbed by molecules
↓
Detector measures transmitted light
↓
Absorbance calculated
↓
Concentration determined using formula
Key Points
• Absorbance is directly proportional to concentration.
• Valid only for dilute solutions.
• Requires monochromatic light.
• Used in glucose estimation, protein estimation, vitamin analysis.
Colorimetry
Definition
Colorimetry is a technique used to determine the concentration of colored
compounds in solution.
If the solution is not colored, a reagent is added to produce a colored
complex.
Principle
• Based on Beer–Lambert’s law.
• Measures absorbance in the visible region (400–700 nm).
Introduction to Spectrophotometric Techniques
Spectrophotometric techniques are analytical methods used to measure how
much light a substance absorbs. These techniques are widely used in:
• Food analysis
• Clinical biochemistry
• Pharmaceutical testing
• Environmental science
• Nutritional research
The basic principle is simple:
When light passes through a solution, some of it is absorbed. The
amount of absorbed light tells us the concentration of the substance.
Beer–Lambert’s Law
Definition
Beer–Lambert’s Law explains the relationship between absorbance and
concentration of a solution.
Mathematical Formula:
A=ε×l×c
Where:
• A = Absorbance
• ε = Molar absorptivity (constant)
• l = Path length (cm)
• c = Concentration
, Flow Chart – Beer–Lambert’s Law
Light Source
↓
Monochromatic Light passes through Solution
↓
Some light absorbed by molecules
↓
Detector measures transmitted light
↓
Absorbance calculated
↓
Concentration determined using formula
Key Points
• Absorbance is directly proportional to concentration.
• Valid only for dilute solutions.
• Requires monochromatic light.
• Used in glucose estimation, protein estimation, vitamin analysis.
Colorimetry
Definition
Colorimetry is a technique used to determine the concentration of colored
compounds in solution.
If the solution is not colored, a reagent is added to produce a colored
complex.
Principle
• Based on Beer–Lambert’s law.
• Measures absorbance in the visible region (400–700 nm).
