Calculate concentration, absorbance, or molar absorptivity using Beer-Lambert law (A = εlc). Includes nucleic acid quantification and common chromophore database.
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The blank must match the sample matrix (solvent, buffer, cuvette type). A mismatched blank introduces systematic offset in every reading. For nucleic acids, blank with the elution buffer, not water.
Literature values are measured at a specific wavelength in a specific solvent. Using an measured in water for a sample in DMSO, or at 280 nm instead of 260 nm, will give incorrect concentrations.
Microvolume instruments like NanoDrop use 0.05–1 mm path lengths. Microplate readers have variable path lengths depending on well volume. Always verify and enter the actual path length.
While A260/A280 1.8 suggests pure DNA, contamination with co-absorbing substances at both wavelengths (e.g., some phenolic compounds) can yield a “normal” ratio. Gel electrophoresis or fluorometric quantification provides better purity assessment.
Direct algebraic solution of the Beer-Lambert equation (). Nucleic acid quantification uses consensus conversion factors (dsDNA: 50 µg/mL per A260, ssDNA: 33, RNA: 40). Extinction coefficients sourced from primary literature and standard biochemistry references.
Last validated 2026-04-05. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.
ConductScience Beer-Lambert Calculator (v1.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/beer-lambert-calculator
Beer A. Bestimmung der Absorption des rothen Lichts in farbigen Flüssigkeiten. Ann Phys. 1852;162(5):78–88.
Schmid FX. Biological Macromolecules: UV-visible Spectrophotometry. In: eLS. Wiley; 2001.
The Beer-Lambert law relates the attenuation of light to the properties of the material through which it travels:
Where: • A = Absorbance (unitless, also called optical density) • ε = Molar absorptivity () • l = Path length through the sample (cm) • c = Molar concentration (mol/L)
Absorbance is defined as A = –log₁₀(T), where T is transmittance (I/I₀). This means A = 1 corresponds to 10% transmittance, and A = 2 corresponds to 1% transmittance. The linear relationship holds for dilute, homogeneous solutions of non-interacting chromophores.
Several factors can cause deviations from Beer-Lambert linearity:
• High concentration: Molecular interactions and aggregation change the effective ε • Stray light: Instrument imperfections add a constant signal, compressing high-absorbance readings • Scattering: Turbid samples scatter light, mimicking absorbance • Chemical equilibria: pH-dependent speciation can shift with concentration (e.g., indicator dyes) • Fluorescence: Re-emitted photons reach the detector, reducing apparent absorbance • Wrong wavelength: Measuring off-peak reduces sensitivity and may introduce nonlinearity
Best practice: run a standard curve at your exact conditions and verify linearity before quantifying unknowns.
