Beer-Lambert Law Absorbance Calculator

Beer-Lambert Law A = ε × l × c A = absorbance (dimensionless, also written as OD — optical density) ε = molar absorptivity (L·mol⁻¹·cm⁻¹) — a property of the molecule at a given wavelength l = path length (cm) — the distance light travels through the sample c = concentration (mol/L = M) The law was developed by Pierre Bouguer (1729, France), Johann Heinrich Lambert (1760, Germany), and August Beer (1852, Germany).

Absorbance and Transmittance Transmittance T = I/I₀ (fraction of light that passes through) Absorbance A = −log₁₀(T) = log₁₀(I₀/I) At A = 0: T = 100% (all light transmitted — blank solution) At A = 1: T = 10% (90% absorbed) At A = 2: T = 1% (99% absorbed) At A = 3: T = 0.1% (99.9% absorbed)

Molar Absorptivity (ε) ε is a molecular property — how strongly the molecule absorbs at a specific wavelength. High ε (>10,000): strong absorber (chromophore, aromatic ring, conjugated system) Low ε (<100): weak absorber (most saturated organics in UV) DNA at 260 nm: ε ≈ 6,600 L·mol⁻¹·cm⁻¹ (per nucleotide) Myoglobin at 410 nm (Soret band): ε ≈ 128,000 L·mol⁻¹·cm⁻¹ p-Nitroaniline at 380 nm: ε ≈ 13,200 L·mol⁻¹·cm⁻¹

Common Values for Proteins Protein concentration (A280/ε) from absorbance at 280 nm: IgG (antibody): ε ≈ 210,000 at 280 nm → 1 A₂₈₀ = 0.71 mg/mL BSA (bovine serum albumin): ε ≈ 43,824 at 280 nm → 1 A₂₈₀ = 1.51 mg/mL Use online tools (ProtParam) for exact ε from amino acid sequence.

Limitations Beer-Lambert Law is valid only at low concentrations (usually < 0.01 M). At high concentrations, intermolecular interactions and saturation effects cause deviations. Scattering by particles also adds apparent absorbance. Fluorescence can reduce apparent absorbance.