What is the Beer-Lambert law?

It states that absorbance equals the extinction coefficient times concentration times path length, written A equals epsilon c l. Rearranged, concentration equals absorbance divided by the coefficient times the path length, which is exactly what this calculator computes.

Should I use a molar or a mass extinction coefficient?

Use a molar coefficient in inverse molar per centimetre when you know the protein sequence and want a molar concentration. Use a mass coefficient, the absorbance of a 1 mg per mL solution at 280 nm, when you only need a mg per mL value, which is common for routine A280 readings.

Why does path length matter?

Absorbance is proportional to the distance light travels through the sample. A standard cuvette is 1 cm, but microvolume instruments use much shorter paths, often around 1 mm or 0.1 cm. Using the wrong path length scales your answer by the same factor and is a frequent source of error.

How is the dilution factor applied?

If you diluted the sample before reading, the measured absorbance reflects the diluted concentration. Multiplying the result by the dilution factor recovers the concentration of the original undiluted stock, so always enter the factor you used.

How do I convert a molar result to mg per mL?

Multiply the molar concentration by the molecular weight in grams per mole. Because one molar of a substance with molecular weight in g per mol equals that many grams per litre, the product gives grams per litre, which is numerically the same as mg per mL.

Beer-Lambert Protein Concentration Calculator

The Beer-Lambert law is the backbone of UV-Vis quantitation. Given an absorbance reading, an extinction coefficient, and a path length, this calculator returns the concentration of your protein or compound, corrects for dilution, and optionally converts a molar result to mg/mL.

How it works

The law states A = eps x c x l. Solving for concentration gives c = A / (eps x l). With a molar coefficient (inverse molar per centimetre) the answer comes out in moles per litre; with a mass coefficient (the absorbance of a 1 mg/mL solution over 1 cm) it comes out directly in mg/mL.

If you diluted before reading, the displayed value is multiplied by your dilution factor to give the original concentration. Supplying a molecular weight converts a molar result to mg/mL, since one molar of a species equals its molecular weight in grams per litre.

Worked example

A purified protein with a molar extinction coefficient of 43,824 read in a 1 cm cuvette at A280 of 0.85 has a concentration of 0.85 / (43824 x 1) = 1.94e-5 M, or 19.4 µM. If its molecular weight is 14,300 g/mol that is about 0.28 mg/mL.

Tips

Keep absorbance readings in the accurate range, roughly 0.1 to 1.0, by diluting samples that read too high. Always confirm the path length of microvolume instruments, which is often 0.1 cm rather than 1 cm. For proteins, derive the molar coefficient from the sequence using tryptophan, tyrosine, and cystine content rather than guessing.