Spectrophotometry Concentration Calculator
The Beer-Lambert law is the foundational principle of spectrophotometry, relating a solution's absorbance to its concentration, path length, and molar extinction coefficient. This calculator rearranges the law to solve for concentration (c = A / (epsilon * l)). Enter the measured absorbance (A), the molar extinction coefficient (epsilon) in L mol-1 cm-1, and the cuvette path length in centimetres. Results are given in molarity (mol/L) and micromolar (uM). Pre-set values for common analytes are provided below.
Beer-Lambert law
A = epsilon * l * c
c = A / (epsilon * l)
Where A is absorbance (dimensionless), epsilon is the molar extinction coefficient (L mol-1 cm-1), l is the optical path length (cm), and c is the molar concentration (mol/L). The law was formalised by Pierre Bouguer, Johann Heinrich Lambert, and August Beer; the modern combined form is attributed to Beer (1852) and Lambert (1760).
Common extinction coefficients
- NADH at 340 nm: 6,220 M-1 cm-1 (used in enzyme activity assays)
- Double-stranded DNA at 260 nm: approx. 50 ug/mL per A260 unit
- BSA at 280 nm: approx. 43,824 M-1 cm-1 (by sequence) or estimated 27,500 M-1 cm-1
- Haemoglobin at 415 nm (Soret band): approx. 16,900 M-1 cm-1
Spectrophotometry: frequently asked questions
What is the Beer-Lambert law?
The Beer-Lambert law states that the absorbance (A) of a solution equals the product of the molar extinction coefficient (epsilon), the path length (l), and the molar concentration (c): A = epsilon * l * c. It describes how light is absorbed as it passes through a solution.
What units are used in the Beer-Lambert law?
Absorbance (A) is dimensionless. Molar extinction coefficient (epsilon) is in L mol-1 cm-1 (also written M-1 cm-1). Path length (l) is in centimetres. Concentration (c) is in mol/L (Molarity, M).
What is the extinction coefficient for DNA at A260?
Double-stranded DNA has an approximate extinction coefficient of 0.020 (ug/mL)-1 cm-1 at 260 nm. An A260 reading of 1.0 corresponds to approximately 50 ug/mL of dsDNA. Single-stranded DNA gives approximately 33 ug/mL per A260 unit.
What A260/A280 ratio indicates pure DNA or RNA?
A pure DNA sample should have an A260/A280 ratio of approximately 1.8. Pure RNA should be approximately 2.0. Ratios significantly below 1.8 suggest protein contamination; ratios above 2.0 can indicate RNA contamination in a DNA sample.
Why must absorbance be below 1.0 for accurate Beer-Lambert calculations?
The Beer-Lambert law assumes a linear relationship between absorbance and concentration, which holds reliably below A = 1.0. Above A = 1.0 the detector receives very little light and readings become non-linear. Dilute the sample until A is between 0.1 and 0.8 for best accuracy.
Official sources
- NIST, Beer-Lambert law and spectrophotometry: NIST SP 250-100: UV-Vis Spectrophotometry.
- NIH, nucleic acid quantification at A260: NCBI PubMed Central: Nucleic Acid Quantification.
Reviewed by the CalculatorHub team, edited by James Graham, 14 June 2026. See our methodology.