What is the Beer-Lambert law?

The Beer-Lambert law states that absorbance equals the molar absorptivity times concentration times path length, or A equals epsilon c l. It means absorbance is directly proportional to concentration, which is what lets a spectrophotometer measure how much of a substance is present.

When should I use a standard curve instead?

Use a standard curve when you do not know the molar absorptivity, when your assay involves a colour-development reaction, or when you want to correct for blank background. Fitting calibration standards captures the real slope and intercept of your specific assay rather than relying on a textbook epsilon.

Why keep absorbance between 0.1 and 1.0?

Below about 0.1 the signal is close to noise and above roughly 1.5 the detector stops responding linearly because almost all light is absorbed. Readings in the 0.1 to 1.0 window sit in the reliable linear range, so dilute and re-measure samples that read too high.

What does the R-squared on the curve tell me?

R-squared measures how well the straight line fits your standards. A good calibration usually exceeds 0.99. A lower value points to a bad standard, a pipetting error, or readings that have left the linear range, so inspect or repeat the offending point.

What units does the result use?

In single-point mode the concentration comes out in molar, since molar absorptivity is in inverse molar per cm, and the tool also shows micromolar and millimolar. In curve mode the units match whatever concentration units you used for your standards, since the fit is unit-agnostic.

Spectrophotometry Absorbance to Concentration Calculator

Spectrophotometers report absorbance, but you almost always want concentration. This tool converts between them two ways: directly through the Beer-Lambert law when you know the molar absorptivity, or by fitting a calibration curve when you do not.

How it works

The Beer-Lambert law relates absorbance to concentration:

A = epsilon x c x l   ->   c = A / (epsilon x l)

where epsilon is the molar absorptivity in inverse molar per centimetre and l is the path length, usually 1 cm. In standard-curve mode the tool instead fits your calibration points to a straight line by least squares and inverts it for the unknown:

A = m x c + b   ->   c = (A - b) / m

The fitted slope m corresponds to epsilon x l, and the intercept b absorbs any blank background.

Tips and notes

Keep absorbance readings between about 0.1 and 1.0; below that you are near noise, and above roughly 1.5 the detector is no longer linear, so dilute and re-read. Always blank the instrument against your buffer first, and in curve mode include a zero-concentration standard so the intercept is anchored. Watch the R-squared: anything below 0.99 usually means a stray standard or a reading that has drifted out of the linear range. The classic worked example is quantifying NADH at 340 nm, where the molar absorptivity is 6,220 inverse molar per centimetre.