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# 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

- [Methemoglobinemia](https://durgical.com/en/conditions/methemoglobinemia/)
- [Carbon monoxide poisoning](https://durgical.com/en/conditions/carbon-monoxide-poisoning/)

## Related

- [NAD⁺ (nicotinamide adenine dinucleotide)](https://durgical.com/en/biochemistry/molecules/nad/)
- [Hemoglobin and 2,3-BPG](https://durgical.com/en/biochemistry/enzymes/hemoglobin-and-2-3-bpg/)
- [Chromatography](https://durgical.com/en/biochemistry/topics/chromatography/)
- [Isoenzymes](https://durgical.com/en/biochemistry/topics/isoenzymes/)
- [Bilirubin](https://durgical.com/en/biochemistry/molecules/bilirubin/)

## 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.

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Educational information, not medical advice. For any symptom, see a health professional.

Sources: [Pulse Oximetry: MedlinePlus Medical Test](https://medlineplus.gov/lab-tests/pulse-oximetry/) (MedlinePlus, US National Library of Medicine, 2026); [Blood gases: MedlinePlus Medical Encyclopedia](https://medlineplus.gov/ency/article/003855.htm) (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).

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