Extinction Coefficient & Absorbance Calculator

Extinction Coefficient & Absorbance Calculator

mol/L (M)
cm

Extinction Coefficient & Beer-Lambert Law

The Molar Extinction Coefficient (ε), also known as molar absorptivity, is a measure of how strongly a chemical species absorbs light at a specific wavelength. It is an intrinsic property of the substance.

The Beer-Lambert Law relates absorbance to concentration and path length:

A = ε ⋅ c ⋅ l

Where:
• A = Absorbance (unitless)
• ε = Molar Extinction Coefficient (typically M⁻¹cm⁻¹ or L·mol⁻¹cm⁻¹)
• c = Molar Concentration (mol/L or M)
• l = Path Length of the cuvette (typically cm)

This law is fundamental in spectrophotometry for determining the concentration of a substance in solution by measuring how much light it absorbs. This calculator estimates ε at 280 nm for proteins (based on Trp, Tyr, Cys content) and ssDNA (based on base content), and then uses the Beer-Lambert law to calculate the expected absorbance.

Frequently Asked Questions

Why is 280 nm used for proteins?

Proteins absorb ultraviolet (UV) light primarily due to the aromatic side chains of the amino acids Tryptophan (W) and Tyrosine (Y). Both have significant absorption peaks near 280 nm. Phenylalanine (F) also absorbs but much weakly at this wavelength. Disulfide bonds (Cys-Cys) also contribute slightly. Measuring absorbance at 280 nm provides a quick, non-destructive way to estimate protein concentration, although its accuracy depends heavily on the protein's specific amino acid composition.

Why is absorbance for DNA usually measured at 260 nm instead of 280 nm?

The nitrogenous bases in nucleic acids (Adenine, Guanine, Cytosine, Thymine, Uracil) have strong UV absorption with a maximum peak around 260 nm. While they do absorb some light at 280 nm (which this calculator uses for a rough estimate based on ssDNA bases), the absorption at 260 nm is much stronger and more characteristic. The A₂₆₀/A₂₈₀ ratio is often used as an indicator of nucleic acid purity, as protein contamination increases the absorbance at 280 nm relative to 260 nm.

Are these calculated extinction coefficients exact?

No, they are estimations. The protein calculation uses average values for Trp, Tyr, and Cys-Cys, and the actual absorption can be influenced by the protein's local environment and folding. The DNA calculation uses very approximate values for individual bases at 280 nm. For precise work, the extinction coefficient should ideally be determined experimentally for the specific molecule under the specific buffer conditions used.