Calculator
Example data table
| Sample | Absorbance (AU) | Blank (AU) | ε (L·mol⁻¹·cm⁻¹) | Path (cm) | Dilution | Concentration (mol/L) |
|---|---|---|---|---|---|---|
| S1 | 0.742 | 0.012 | 12,500 | 1.00 | 10 | 0.000584 |
| S2 | 0.318 | 0.010 | 9,800 | 1.00 | 1 | 0.0000314 |
| S3 | 1.085 | 0.020 | 15,000 | 0.50 | 5 | 0.000710 |
Formula used
The Beer–Lambert relationship links absorbance to concentration:
A = ε · l · c
- A is absorbance (unitless, AU).
- ε is molar absorptivity (L·mol⁻¹·cm⁻¹).
- l is path length in centimeters (cm).
- c is concentration (mol/L).
With blank correction and dilution, the calculator uses: Acorr = Asample − Ablank and cstock = (Acorr / (ε·l)) · dilution. For calibration mode: A = slope·c + intercept.
How to use this calculator
- Select a method: Beer–Lambert law or a calibration curve.
- Enter sample absorbance and blank absorbance from your spectrometer.
- For Beer–Lambert, provide ε and the cuvette path length.
- If you diluted the sample, enter the dilution factor used.
- Choose a display unit; provide molar mass if using g/L.
- Press Calculate to show results above the form.
- Use the download buttons to export CSV or PDF reports.
Professional notes and background
1) Why absorbance maps to concentration
In many UV-Vis and visible assays, absorbance rises linearly with analyte concentration because the sample attenuates transmitted light exponentially while the instrument reports the logarithmic absorbance scale. Under stable optics and homogeneous solutions, the Beer–Lambert model provides a practical working line.
2) Typical working ranges in routine spectroscopy
Many benches target absorbance between 0.10 and 1.00 AU for best signal-to-noise and linearity. Below about 0.05 AU, baseline drift and stray light can dominate. Above about 1.2 AU, the detector may approach limits, and small measurement errors inflate concentration uncertainty.
3) Units and what ε actually means
Molar absorptivity ε (L·mol⁻¹·cm⁻¹) is wavelength dependent and can vary with pH, solvent, ionic strength, and temperature. A value tabulated at 260 nm for nucleic acids will not match a dye assay at 595 nm. Always confirm ε for your chosen wavelength and matrix.
4) Path length effects and microvolume cuvettes
Standard cuvettes use 1.00 cm path length, but microvolume devices and half-height cuvettes may use 0.2–0.5 cm. Because absorbance is proportional to path length, a 0.50 cm path yields half the absorbance for the same concentration, improving performance for strongly absorbing samples.
5) Blank correction and baseline control
Blank subtraction removes solvent and reagent background, improving accuracy for low concentrations. Use the same cuvette, buffer, and handling as the sample. A stable blank close to 0.000–0.050 AU typically indicates clean optics and consistent cuvette positioning.
6) Dilution strategy and uncertainty
When absorbance is high, dilute the sample so the final reading is within the linear region. For example, a 1:10 dilution reduces absorbance roughly tenfold. Record the exact dilution factor; a 2% dilution error directly adds about 2% error to the final reported stock concentration.
7) When to prefer a calibration curve
Calibration curves are preferred for complex matrices and colorimetric assays where ε is not constant or where the signal depends on reaction yield. Fit standards using A = slope·c + intercept, verify residuals, and bracket unknowns within the standards range. Rebuild curves when reagents or lamp intensity changes.
8) Data reporting and audit-friendly exports
Good records include wavelength, cuvette path length, blank composition, dilution steps, and the method used. This calculator exports a compact CSV for spreadsheets and a PDF summary for lab notebooks. Store both alongside raw instrument files for traceable, reproducible concentration reporting.
FAQs
1) What is the difference between absorbance and transmittance?
Transmittance is the fraction of light passing through a sample. Absorbance is the logarithmic measure of light loss, so it scales linearly with concentration over a useful range.
2) Why does the calculator subtract the blank?
The blank captures background from solvent, cuvette, and reagents. Subtracting it isolates the analyte contribution, reducing bias—especially when your sample absorbance is low.
3) What should I do if absorbance is above 1.2 AU?
Dilute the sample and re-measure so the reading falls near 0.1–1.0 AU. Enter the dilution factor to report the original concentration accurately.
4) Can I use this for proteins or DNA measurements?
Yes, if you use the correct wavelength and ε or a validated calibration curve. For biomolecules, ε depends on sequence/composition and buffer conditions, so choose method parameters carefully.
5) When is a calibration curve better than Beer–Lambert?
Use calibration when chemistry or matrix effects change response, such as dye binding assays, turbid samples, or reactive color development. Curves anchor unknowns to measured standards.
6) Why does ε change with wavelength?
Absorption is spectral. Molecules absorb differently across wavelengths, so ε is specific to the chosen wavelength and conditions. Always match ε to your measurement settings.
7) How do I display concentration in g/L?
Select g/L and enter the molar mass in g/mol. The calculator converts from mol/L to g/L by multiplying by molar mass, preserving the applied blank correction and dilution factor.