What you are given
- Wavelength (nm): 340
- Extinction coefficient: 6220
- Absorbance (A): 0.622
- Show result in: µM
Formula: A = ε · c · l
Spectrophotometry
How do you use the Beer-Lambert law to find concentration?
A = epsilon x c x l, so c = A / (epsilon x l). Keep the extinction coefficient and the path length in matching units, and stay inside the linear range of the instrument.
Worked example
Every number below is computed by the calculator, not typed in
The working
- Beer-Lambert:
c (mol/L) = A · DF / (ε · l) - Substitute:
c = 0.622 · 1 / (6220 · 1)=0.0001 M
Your sample is 100 µM at 340 nm.
What each symbol means
- A: absorbance
- ε: molar extinction coefficient (M⁻¹cm⁻¹)
- c: concentration (mol/L)
- ℓ: path length (cm)
A printable one-page version is on the formula sheet.
Where this usually goes wrong
- Reading above about A = 1.5, where the response is no longer linear. Dilute and re-read instead.
- Forgetting the path length when the cuvette is not 1 cm, which most plate readers are not.
- Not blanking against the same buffer as the sample.
Try it yourself
Two problems, with the working revealed
Problem 1 Beer-Lambert (absorbance to concentration)
Work out the concentration from the absorbance.
- Wavelength (nm): 400
- Extinction coefficient type: E0.1% (0.1% w/v = 1 g/L)
- Extinction coefficient: 0.667
- Absorbance (A): 0.667
Show the answer and the working
Your sample is 1 mg/mL at 400 nm.
- Beer-Lambert:
c (mg/mL) = A · DF / (E0.1% · l) - Substitute:
c = 0.667 · 1 / (0.667 · 1)=1 mg/mL
Open this in the calculator to change the numbers.
Problem 2 Beer-Lambert (absorbance to concentration)
Work out the concentration from the absorbance.
- Wavelength (nm): 750
- Extinction coefficient: 6220
- Absorbance (A): 0.25
- Show result in: µM
Show the answer and the working
Your sample is 40.19 µM at 750 nm.
- Beer-Lambert:
c (mol/L) = A · DF / (ε · l) - Substitute:
c = 0.25 · 1 / (6220 · 1)=0.00004019 M
Open this in the calculator to change the numbers.
Ten more problems on Beer-Lambert (absorbance to concentration)
Do it with your own numbers.
The Beer-Lambert (absorbance to concentration) calculator is free, shows every step, flags the hazards, and works offline once loaded. No account, no signup.
Open the calculator