Understand the relationship
The reasoning behind the result
Absorbance is logarithmic transmission
A = log₁₀(I₀/I) = εℓc
Absorbance compares incident intensity I₀ with transmitted intensity I. For the stated absorption model it is proportional to concentration c and optical path ℓ. The coefficient ε connects those quantities for a particular absorbing species and wavelength.
Here ε uses L/(mol·cm), path length uses centimetres and concentration uses mol/L. Their product is dimensionless absorbance. A mass-based coefficient cannot be entered as a molar coefficient without the appropriate conversion.
Path normalization and dilution are different operations
A shorter optical path reduces absorbance for the same measured concentration. A diluted sample reduces concentration itself. Dividing absorbance by εℓ handles the optical path; multiplying the resulting concentration by the dilution factor handles sample preparation.
Some instruments export absorbance normalized to a 1 cm path. In that case use the path attached to the exported reading, not the instrument's physical microvolume gap again.
Molecular specificity matters
An oligonucleotide's coefficient depends on its sequence and conditions. A protein coefficient likewise depends on molecular properties and wavelength. This calculator accepts a coefficient you have established; it does not invent one from a generic assay title.
Scattering, mixtures, stray light and chemical changes can invalidate the simple proportional relationship. A calculated concentration does not establish that these assumptions hold in your sample.
Follow the numbers
Absorbance to original concentration
- A = 0.6 AU, ε = 20,000 L/(mol·cm), and ℓ = 1 cm.
- Measured concentration = 0.6/(20,000 × 1) = 0.00003 mol/L = 30 µmol/L.
- At dilution factor five, original concentration = 150 µmol/L.
The optical path belongs inside the absorption equation; the dilution correction is applied after concentration is recovered.
Quick guide
How to use this calculator
- Select the unknown quantity and use molar concentration in mol/L.
- Enter the coefficient applicable to the specific molecule, wavelength and conditions.
- Enter the optical path associated with the absorbance value; do not correct a normalized 1 cm reading twice.
- Read the measured-solution and original-sample concentrations separately.
Calculation method
Calculation and interpretation
Use the absorption relationship for a defined species and wavelength, with a user-supplied molar extinction coefficient.
A = εℓc; c = A/(εℓ); coriginal = c × dilution factor
Worked example
Absorbance to original concentration
The optical path belongs inside the absorption equation; the dilution correction is applied after concentration is recovered.
A = εℓc; c = A/(εℓ); coriginal = c × dilution factor
Supported inputs
Precision and limits
Measured response, not experimental validation
Calculations do not establish assay specificity, recovery, matrix compatibility, sample identity or biological meaning. Use standards, blanks and controls appropriate to the actual method.
Units and dilution
Use one concentration unit throughout a calibration. A dilution factor is original concentration divided by measured diluted concentration; multiply by it once when reporting the original sample.
Entered coefficient
The molar coefficient must match species, wavelength, solvent and measurement conditions. This tool does not supply sequence-derived extinction coefficients or infer a validated linear range.
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