1. What is the Beer-Lambert Law?

The Beer-Lambert law relates the absorption of light to the properties of the material through which the light is traveling. It's widely used in chemistry for measuring the concentration of chemical solutions.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

Where:

• \( Conc \) — Protein concentration (mg/mL)
• \( A \) — Measured absorbance (AU)
• \( \varepsilon \) — Extinction coefficient (L/mg·cm)
• \( l \) — Path length (cm)

Explanation: The equation shows that concentration is directly proportional to absorbance and inversely proportional to both the extinction coefficient and path length.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration determination is essential for experimental reproducibility, protein purification, enzyme kinetics studies, and many biochemical assays.

4. Using the Calculator

Tips: Enter absorbance (typically at 280nm), the protein's extinction coefficient (often provided by manufacturers), and cuvette path length (usually 1 cm). All values must be positive.

5. Frequently Asked Questions (FAQ)

Q1: What's a typical extinction coefficient for proteins?
A: For proteins with Trp and Tyr residues, ε ≈ 1.0 L/mg·cm at 280nm. For BSA, ε = 0.66 L/mg·cm.

Q2: Why measure at 280nm?
A: Tryptophan and tyrosine absorb strongly at 280nm, allowing protein concentration estimation without dyes.

Q3: What if my protein lacks Trp/Tyr?
A: Alternative methods like Bradford or BCA assays may be needed, or measure at 205nm where peptide bonds absorb.

Q4: How accurate is this method?
A: ±5-10% for pure proteins, less accurate for mixtures. Affected by buffer composition and light scattering.

Q5: What's the linear range for absorbance measurements?
A: Typically 0.1-1.0 AU. For higher concentrations, dilute the sample or use a shorter pathlength cell.