Spectrophotometry - Biochemical Techniques | Lecture Notes

Understanding the principles and applications of spectrophotometry in biochemical analysis, including absorbance measurements, Beer-Lambert Law, and determining concentration of biomolecules.

Spectrophotometry – Quick Reference Notes

1. What Is Spectrophotometry?

Definition: An analytical technique that measures how much a chemical substance absorbs light by quantifying the intensity of light before and after it passes through a sample.
Key Principle: According to Beer‑Lambert law, absorbance (A) is proportional to the concentration (c) of the absorbing species, the path length (l) of the cuvette, and the molar absorptivity (ε).

[ A = \varepsilon , l , c ]

2. Core Components of a Spectrophotometer

Component	Function
Light Source	Provides continuous radiation (e.g., deuterium lamp for UV, tungsten‑halogen for visible).
Monochromator / Filter	Selects a narrow wavelength band (λ) from the source.
Sample Holder (Cuvette)	Holds the solution; standard path length = 1 cm (square‑wave quartz or glass).
Detector	Converts transmitted light into an electrical signal (photodiode, PMT, CCD).
Readout/Display	Shows absorbance (A) or transmittance (T = I/I₀). Often linked to a computer for data storage.

3. Fundamental Concepts

3.1. Absorbance vs. Transmittance

Transmittance (T) = I / I₀ (ratio of transmitted to incident light intensity).
Absorbance (A) = –log₁₀(T).
Relationship: A = –log₁₀(I/I₀).

3.2. Beer‑Lambert Law

Valid for dilute solutions (generally ≤0.1 M) and when the absorbing species does not interact with the solvent or other solutes.
Linearity range: Plot of A vs. c should be straight; deviations occur at high absorbance (>1.0) due to stray light or chemical effects.

3.3. Molar Absorptivity (ε)

Units: L mol⁻¹ cm⁻¹.
Indicates how strongly a species absorbs at a given wavelength.
Large ε → strong absorber → low concentration needed for measurable absorbance.

4. Typical Procedures

Step	Action	Tips
1. Calibration / Blank	Run a blank (solvent only) to set 0 % absorbance.	Use the same cuvette type as for samples.
2. Wavelength Selection	Choose λₘₐₓ (maximum absorbance) from a spectrum scan.	Maximize sensitivity and minimize interference.
3. Sample Preparation	Dilute sample so that expected A is between 0.1–0.8.	Avoid bubbles; use matched cuvettes for reference and sample.
4. Measurement	Record absorbance at λₘₐₓ.	Perform duplicate/triplicate readings for precision.
5. Quantitation	Use calibration curve (A vs. known c) or directly apply Beer‑Lambert law.	Verify linearity; include standards that bracket sample concentration.

5. Applications

Field	Example Uses
Biochemistry	Protein concentration (Bradford, BCA assays), nucleic acid quantification (260 nm).
Environmental Chemistry	Determination of nitrate, phosphate, heavy‑metal complexes.

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