Spectrophotometers report absorbance, but you almost always want concentration. This tool converts between them in two modes: directly through the Beer-Lambert law when you know the molar absorptivity, or by fitting a linear calibration curve when you have a set of standards and an unknown reading.
The Beer-Lambert law: single-point mode
When the molar absorptivity (extinction coefficient) of your analyte is known, one measurement is enough:
A = ε × c × l
c = A / (ε × l)where:
- A is the measured absorbance (dimensionless)
- ε (epsilon) is the molar absorptivity in M⁻¹ cm⁻¹
- c is the concentration in mol/L (molar, M)
- l is the path length in cm (usually 1 cm in a standard cuvette)
Worked example — NADH at 340 nm: NADH has a well-established molar absorptivity of 6,220 M⁻¹ cm⁻¹ at 340 nm. If your spectrophotometer reads an absorbance of 0.62 in a 1 cm cuvette:
c = 0.62 / (6220 × 1) = 0.0000997 mol/L ≈ 99.7 µMThe tool automatically converts the molar result to µM and mM for convenience.
Standard curve (calibration curve) mode
When you do not know ε — for example, because your assay involves a colour-development reaction like Bradford protein assay or BCA — use a standard curve. Prepare known concentrations, measure their absorbances, and the tool fits a straight line by least squares:
A = m × c + b → c = (A − b) / mThe fitted slope m corresponds to ε × l for your specific assay conditions, and the intercept b accounts for blank background. Enter the absorbance of your unknown and the tool interpolates its concentration using the fitted line.
Worked example: Five protein standards with concentrations 0.1, 0.25, 0.5, 0.75, 1.0 mg/mL give absorbances of 0.08, 0.19, 0.38, 0.57, 0.75. The tool fits a line with slope ≈ 0.75 and intercept ≈ 0.004. An unknown reading of 0.45 gives approximately (0.45 − 0.004) / 0.75 ≈ 0.59 mg/mL.
Interpreting R-squared on your calibration
The R² value measures how well the straight line fits your standards. A good calibration typically exceeds 0.99. A lower R² points to a stray standard, a pipetting error, or readings at the extremes of the linear range. Inspect the curve visually — one outlying point often explains most of the variance.
Best practices for accurate readings
Stay within the linear range (roughly 0.1 to 1.0 absorbance). Below about 0.1 the signal is close to detector noise. Above roughly 1.5 (depending on the instrument) the relationship between concentration and absorbance is no longer linear because most of the light has already been absorbed — the detector cannot distinguish between “a lot absorbed” and “even more absorbed.” Dilute samples that read above 1.5 and re-measure.
Blank correctly. Measure a blank containing everything except the analyte (buffer, reagents, solvent) and use it to zero the instrument. This removes background absorbance from reagents and the cuvette walls. In standard-curve mode, including a zero-concentration standard achieves the same effect by anchoring the intercept.
Measure at the right wavelength. Each compound has a characteristic absorption maximum (λmax). Measuring there gives maximum sensitivity and the flattest part of the absorption curve, where small errors in wavelength setting matter least.
Temperature. Molar absorptivity is temperature-dependent for some compounds. Run standards and unknowns at the same temperature.
All calculations run in your browser. Nothing you enter is uploaded or stored.