Biochemistry · Topic
Spectrophotometry and colorimetry (Beer–Lambert law) !
A coloured solution absorbs light in proportion to how much substance it contains. Measuring that absorbed light tells the concentration.
Updated 2026-11-01
Key facts
- Beer–Lambert law: absorbance A = ε × c × l (molar absorptivity × concentration × path length); at fixed path length, absorbance is directly proportional to concentration.
- Absorbance A = log (I₀/I) = −log T; transmittance (T) falls exponentially as concentration rises, so labs read absorbance, not transmittance.
- Colorimeter: uses filters for visible light and a coloured reaction product. Spectrophotometer: uses a monochromator for a precise wavelength, including ultraviolet.
- NADH absorbs at 340 nm and NAD⁺ does not: most enzyme assays (LDH, ALT, AST, CK, glucose by hexokinase) follow the rise or fall of absorbance at 340 nm.
- Unknown concentration = (absorbance of test ÷ absorbance of standard) × concentration of standard, always corrected with a blank.
In detail
- 1. Light source → wavelength selector (filter or monochromator) → cuvette with the sample → detector (photocell) → reading.
- 2. The blank (reagents without sample) sets absorbance to zero so that only the substance measured counts.
- 3. The chosen wavelength is the one the coloured product absorbs most (its complementary colour): a blue solution is read in the orange–red band.
- 4. The law holds only in a range: very concentrated or turbid samples deviate and must be diluted.
- 5. Proteins absorb at 280 nm (tryptophan and tyrosine) and nucleic acids at 260 nm; the 260/280 ratio checks DNA purity.
- 6. Related methods: flame photometry (emission of light by Na⁺ and K⁺ in a flame; now mostly replaced by ion-selective electrodes), fluorimetry, turbidimetry and nephelometry (scattered light, for immunoglobulins and CRP).
- Also called
- Beer–Lambert law, Absorbance, Flame photometry
Clinical notes
- Pulse oximetry applies the same principle: oxygenated and deoxygenated haemoglobin absorb red and infrared light differently.
- CO-oximeters measure several wavelengths to detect carboxyhaemoglobin and methaemoglobin, which standard pulse oximeters misread.
- Many routine chemistry tests (glucose, creatinine by Jaffe reaction, bilirubin, enzymes) are colorimetric or spectrophotometric.
Related conditions
Related
Test yourself
Think of the answer, then tap the question to see it.
What does the Beer–Lambert law state?
Absorbance equals molar absorptivity × concentration × path length, so at a fixed path length absorbance rises in direct proportion to concentration.
Why are many enzyme assays read at 340 nm?
NADH and NADPH absorb at 340 nm while NAD⁺ and NADP⁺ do not, so the change in absorbance follows the reaction.
What is the purpose of the blank in a colorimetric test?
It zeroes the instrument with reagents but no sample, so only the substance measured adds absorbance.
What is the main difference between a colorimeter and a spectrophotometer?
A colorimeter selects light with filters in the visible range; a spectrophotometer uses a monochromator for exact wavelengths, including ultraviolet.
Which haemoglobin forms are misread by a standard two-wavelength pulse oximeter?
Carboxyhaemoglobin and methaemoglobin; a CO-oximeter using more wavelengths is needed.
Educational information, not medical advice. For any symptom, see a health professional.
Sources: Pulse Oximetry: MedlinePlus Medical Test (MedlinePlus, US National Library of Medicine, 2026); Blood gases: MedlinePlus Medical Encyclopedia (MedlinePlus, US National Library of Medicine, 2026); Textbook of Biochemistry for Medical Students, 6th ed. (Jaypee Brothers Medical Publishers (Vasudevan DM, Sreekumari S, Vaidyanathan K), 2011).
Spotted a mistake?We are always improving for the community. Tell us what is wrong and our team will review it.Report a mistake
Representative model, for educational purposes only.