Extinction Coefficient Calculators

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The extinction coefficient (also called the molar absorptivity or molar attenuation coefficient) is a measure of how strongly a chemical substance absorbs light at a given wavelength. It is a fundamental parameter in spectrophotometry — combining with concentration and path length in the Beer-Lambert law to relate absorbance to concentration. Extinction coefficients are essential for calculating the concentration of proteins, nucleic acids, enzymes, and chromophores from UV-Vis absorbance measurements, a daily task in biochemistry and molecular biology laboratories.

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What Is the Extinction Coefficient?

The molar extinction coefficient (ε, epsilon) describes how strongly one mole per liter of a substance absorbs light at a specific wavelength, in a 1 cm path length cell. It appears in the Beer-Lambert Law:

A = ε × c × l

Where:

  • A — absorbance (unitless; = log₁₀(I₀/I))
  • ε — molar extinction coefficient (M⁻¹cm⁻¹ or L·mol⁻¹·cm⁻¹)
  • c — concentration (mol/L)
  • l — path length (cm)

Rearranging: c = A / (ε × l)

Extinction Coefficients of Common Biochemical Molecules

  • Proteins at 280 nm: Depends on Trp, Tyr, and Cys content. Typical range: 10,000–100,000 M⁻¹cm⁻¹. Calculated from sequence using the Pace formula or ProtParam tool.
  • NADH at 340 nm: ε = 6,220 M⁻¹cm⁻¹. Used in enzyme assays monitoring NADH consumption or production.
  • dsDNA at 260 nm: Approximated as 50 μg/mL per A260 unit (not molar ε).
  • Heme (oxyhemoglobin at 415 nm): ~125,000 M⁻¹cm⁻¹ (Soret band)

Calculating Protein Concentration from A280

For a purified protein with known sequence:

c (mg/mL) = A280 / (ε_mg × l)

Where ε_mg = ε (M⁻¹cm⁻¹) / MW (g/mol) × 1000 (in mL·mg⁻¹·cm⁻¹).

The ProtParam tool (ExPASy) calculates ε for any protein sequence from its Trp, Tyr, and Cys residue content.

Limitations of Beer-Lambert Law

The linear relationship between absorbance and concentration (Beer-Lambert law) holds only at:

  • Absorbance values typically between 0.1 and 1.0 (A > 1.5 becomes inaccurate — too little light transmitted)
  • Dilute solutions (< ~0.01 M for most molecules)
  • Monochromatic light
  • No scattering (clear solutions)

Glossary

Molar Extinction Coefficient (ε)
The absorbance of a 1 M solution of a substance in a 1 cm path length cell at a specific wavelength. Units: M⁻¹cm⁻¹. Used in Beer-Lambert law (A = εcl) to calculate concentration from absorbance.
Beer-Lambert Law
A = ε × c × l. Absorbance equals the product of molar extinction coefficient, concentration (mol/L), and path length (cm). Linear relationship between absorbance and concentration, valid for A < ~1.5.
A280 (Absorbance at 280 nm)
UV absorbance at 280 nm used to quantify proteins, primarily from Trp and Tyr residues. Combined with the protein's known molar extinction coefficient, A280 is used to calculate protein concentration.

Frequently Asked Questions

The molar extinction coefficient (ε, or molar absorptivity) quantifies how strongly a substance absorbs light at a specific wavelength, expressed in M⁻¹cm⁻¹. In the Beer-Lambert law (A = ε × c × l), a higher ε means greater absorbance at a given concentration. It is an intrinsic property of the molecule and wavelength — independent of concentration or path length.

Use Beer-Lambert law: c = A280 / (ε × l). You need the protein's molar extinction coefficient at 280 nm (available from ProtParam/ExPASy for known sequences, or experimentally determined). Convert molar concentration to mg/mL by multiplying by the molecular weight. For crude protein estimates without the specific ε, A280 = 1.0 roughly corresponds to ~1 mg/mL for most globular proteins, but this is only approximate.

NADH absorbs at 340 nm with ε = 6,220 M⁻¹cm⁻¹; NAD⁺ does not absorb at 340 nm. This difference is exploited in enzyme assays: reactions that produce or consume NADH are monitored by the change in A340 over time. The rate of A340 change (ΔA/min) divided by ε gives the rate of NADH production or consumption, and thus enzyme activity in units of μmol/min.

Above A ≈ 1.0, very little light (< 10%) reaches the detector, making the measurement increasingly noisy. At A = 2.0, only 1% of incident light is transmitted; small variations in transmission produce large errors in calculated absorbance. Most spectrophotometers have adequate accuracy between A = 0.1 and 1.0. For highly concentrated samples, dilute to bring A into this range before measuring.