Extinction Coefficient Calculator
Calculate molar extinction coefficient using Beer-Lambert law: A = epsilon x c x l. Find epsilon from absorbance and concentration, or calculate concentration from epsilon. Essential for spectrophotometry.
📊 Biochemistry📐 A = epsilon x c x l🧬 Biology
Calculate
Absorbance (A)
Path length l (cm)
Concentration c (uM) OR Extinction epsilon (M-1cm-1)
Please enter valid values.
Formula & Reference
| Variable | Symbol | Formula | Units |
|---|---|---|---|
| Extinction Coefficient Calculator | — | A = epsilon x c x l | M-1cm-1 |
Step-by-Step Examples
Example 1
Determine Protein Epsilon
BSA at 10 uM gives A280=0.44, l=1cm.
- eps = A/(c x l) = 0.44 / (10e-6 x 1)
- eps = 44000 M-1cm-1
- Literature value for BSA: 43824 M-1cm-1
- Within 0.4% of literature — excellent!
✓ eps(BSA) = 44,000 M-1cm-1
Example 2
Calculate DNA Concentration
OligoDNA has eps(260)=250000 M-1cm-1. A260=0.50, l=1cm.
- c = A/(eps x l) = 0.50/250000
- c = 2.0 uM
- Convert: 2.0 uM = 2000 nM for typical oligo work
✓ c = 2.0 uM oligo
Example 3
Unknown Protein
A280=0.35, eps=27960 M-1cm-1, l=1cm.
- c = 0.35/27960 = 12.52 uM
- MW=30000 Da: mg/mL = 12.52e-6 x 30000 = 0.376
- ~0.38 mg/mL
✓ c = 12.5 uM = 0.38 mg/mL
Real-World Applications
Spectrophotometry
All UV-Vis concentration measurements use Beer-Lambert law with known epsilon.
Nucleic Acid Quantification
Oligo extinction coefficients calculated from sequence composition for accurate quantification.
Drug Analysis
HPLC peak areas converted to concentration using epsilon calibrated with standards.
Analytical Chemistry
Beer-Lambert is fundamental to analytical chemistry across all sample types.
Common Mistakes to Avoid
⚠️
Confusing molar and specific extinction coefficient
Molar: M-1cm-1 (per mole/L). Specific: mL/mg/cm = 10 x molar / MW. Always check units.
⚠️
Measuring outside linear range
Beer-Lambert is linear from A=0.1 to A~1.5. Above A~2, stray light causes deviation. Dilute samples for accurate measurements.
⚠️
Using wrong wavelength maximum
Always measure at lambda-max for maximum sensitivity. Off-peak measurements give lower epsilon and poorer sensitivity.
Frequently Asked Questions
What is the Beer-Lambert law? ▾
A = epsilon x c x l. Absorbance = molar extinction coefficient x concentration (M) x path length (cm). Linear relationship between concentration and absorbance.
When does Beer-Lambert fail? ▾
At high concentrations (A > 1.5), with highly colored solvents, when solute self-associates, or with fluorescent molecules. Use dilution to restore linearity.
What are typical epsilon values? ▾
Small molecules: 1,000-20,000 M-1cm-1. Proteins at 280nm: 10,000-100,000. DNA at 260nm: 5,000-50,000 per base. Chromophores: up to 200,000.
How is epsilon calculated from protein sequence? ▾
ExPASy ProtParam: epsilon(280) = 5500 x Trp + 1490 x Tyr + 125 x Cys(disulfide). This is additive based on residue content.
What is a specific extinction coefficient? ▾
A(1mg/mL, 1cm) = specific extinction. Also written E(0.1%). For BSA: 0.66 mL mg-1 cm-1. Multiply by 10 x MW to get molar epsilon.