Calculator Inputs
Example Data Table
| Observed A | Blank A | ε | Path length | Dilution | MW | Purity | Volume | Result |
|---|---|---|---|---|---|---|---|---|
| 0.82 | 0.05 | 43,000 | 1.0 cm | 10x | 50,000 Da | 92% | 2.0 mL | 179.0698 µM, 8.9535 mg/mL |
| 1.16 | 0.08 | 62,500 | 1.0 cm | 5x | 68,000 Da | 95% | 1.5 mL | 86.4000 µM, 5.8752 mg/mL |
| 0.44 | 0.03 | 35,000 | 0.5 cm | 8x | 27,500 Da | 88% | 3.0 mL | 187.4286 µM, 5.1543 mg/mL |
Formula Used
Net absorbance = Observed absorbance − Blank absorbance
c = A / (ε × l)
Measured molarity = [Net absorbance / (ε × l)] × Dilution factor
Purity-corrected molarity = Measured molarity × (Purity / 100)
mg/mL = Molarity × Molecular weight
Total µmol = Molarity × Sample volume in mL × 1000
Estimated units = Purity-corrected total mass in mg × Specific activity
This method works best when the enzyme has a known extinction coefficient at the selected wavelength and the absorbance value stays inside the spectrophotometer’s reliable range.
How to Use This Calculator
- Enter the measured absorbance of the enzyme sample.
- Enter the blank absorbance to remove baseline signal.
- Provide the extinction coefficient for the enzyme or chromophore.
- Enter the cuvette path length, usually 1.0 cm.
- Set the dilution factor used before reading absorbance.
- Enter molecular weight to convert molarity into mass concentration.
- Add sample volume to estimate total mass and micromoles.
- Use purity and optional specific activity for adjusted outputs.
- Click calculate to show results above the form.
- Download the report as CSV or PDF if needed.
Frequently Asked Questions
1) What does this calculator estimate?
It estimates enzyme concentration from absorbance data using the Beer–Lambert relationship. It also converts results into µM, mg/mL, total mass, total micromoles, and optional activity units.
2) Why is blank absorbance included?
Blank absorbance removes signal contributed by solvent, buffer, cuvette, or reagent background. Subtracting it improves the accuracy of the enzyme absorbance used for concentration calculations.
3) When should I use the dilution factor?
Use the dilution factor whenever the measured sample was diluted before the spectrophotometer reading. The calculator multiplies the measured concentration back to the original sample concentration.
4) Why do I need molecular weight?
Molecular weight converts molar concentration into mass concentration. This is helpful when you need mg/mL, total milligrams, formulation values, or activity per milligram calculations.
5) What does purity-corrected concentration mean?
Purity-corrected concentration estimates the fraction attributable to the target enzyme when the preparation is not fully pure. It is useful for downstream activity and yield interpretation.
6) Can I use this for microplate readings?
Yes, but only if you know or estimate the effective path length for the microplate well. Path length strongly affects the calculated molarity.
7) What if the absorbance is lower than the blank?
The calculator sets net absorbance to zero in that situation. This avoids negative concentration values and signals that the sample may be too dilute or the baseline needs review.
8) Does this replace a full protein assay?
No. It is a fast concentration estimate based on absorbance and known optical constants. Full protein assays remain useful when extinction coefficients are uncertain or samples contain interfering compounds.