The spectrophotometer is the most common instrument in any laboratory. It measures how much light a sample absorbs at a given wavelength, which is proportional to the concentration of the absorbing species.

The Beer–Lambert Law

The fundamental relationship is:

A = ε × b × c

where A is absorbance (no units), ε is the molar absorptivity (L·mol⁻¹·cm⁻¹), b is the path length (cm), and c is the concentration (mol/L). The law holds for dilute solutions (typically A < 1.5). At higher concentrations, deviations occur due to molecular interactions and instrumental stray light.

Instrument Components

• Light source: deuterium lamp for UV (190–350 nm), tungsten-halogen lamp for visible (350–900 nm). Xenon flash lamps are used in some modern instruments.
• Monochromator: a grating or prism that selects a narrow band of wavelengths. The bandwidth affects measurement quality — narrower bandwidth gives better linearity.
• Sample compartment: holds the cuvette. Most instruments accept 1 cm pathlength cuvettes. Cuvettes are quartz (for UV) or glass/plastic (for visible only).
• Detector: a photomultiplier tube or photodiode that converts transmitted light to an electrical signal.

Operation Steps

1. Turn on the instrument and allow the light source to warm up (15–30 minutes for deuterium lamps).
2. Select the measurement wavelength.
3. Fill a cuvette with the blank solution (the solvent without analyte).
4. Place the blank in the sample holder and set the absorbance to zero (autozero).
5. Measure the sample and record the absorbance.
6. For multiple samples, re-zero with the blank periodically.

Cuvette Handling

Handle cuvettes by the frosted or ridged sides — fingerprints on the optical windows scatter light and increase absorbance. Rinse cuvettes with the sample solution before filling. Ensure no bubbles are in the light path.

Common Applications

• Nucleic acid quantification: A₂₆₀ measurement (1 OD₂₆₀ ≈ 50 µg/mL dsDNA, 40 µg/mL RNA, 33 µg/mL ssDNA). Purity assessed by the A₂₆₀/A₂₈₀ ratio (≈1.8 for pure DNA, ≈2.0 for pure RNA).
• Protein quantification: Bradford, BCA, and Lowry assays all use visible spectrophotometry.
• Enzyme kinetics: continuous monitoring of NADH consumption at 340 nm or product formation at a specific wavelength.
• Bacterial growth curves: turbidity measured at 600 nm (OD₆₀₀).
• Colorimetric chemical assays: almost any analyte can be measured if it reacts to form a colored product.