The millimolar extinction coefficient for NADH at 340 nm is 6.22 cm-1. You put 125 LL stock NADH solution in total volume of 3.0 mL and read the absorbance as 0.44 What is the concentration of the original stock solution of NADH? (A Ebc)
The correct answer and explanation is:
To determine the concentration of the original stock NADH solution, we use the Beer-Lambert Law:
A = ε × b × c
Where:
- A is the absorbance (0.44)
- ε is the extinction coefficient (6.22 mM⁻¹·cm⁻¹)
- b is the path length of the cuvette (usually 1.0 cm unless stated otherwise)
- c is the concentration in mM (millimolar)
We are given:
- 125 µL (or 0.125 mL) of NADH stock is diluted into a total volume of 3.0 mL
- The measured absorbance is 0.44
- ε = 6.22 mM⁻¹·cm⁻¹
- b = 1.0 cm
Step 1: Solve for concentration of diluted solution
Using Beer-Lambert Law:
c (diluted) = A / (ε × b) = 0.44 / (6.22 × 1.0) ≈ 0.07074 mM
This is the concentration of NADH in the diluted 3.0 mL solution.
Step 2: Use dilution formula to find original stock concentration
Using the dilution equation:
C₁ × V₁ = C₂ × V₂
Where:
- C₁ is the concentration of the stock solution (what we are solving for)
- V₁ is the volume of stock solution added = 0.125 mL
- C₂ is the concentration after dilution = 0.07074 mM
- V₂ is the total volume after dilution = 3.0 mL
Now plug in values:
C₁ = (C₂ × V₂) / V₁ = (0.07074 × 3.0) / 0.125 ≈ 1.698 mM
Final Answer:
The concentration of the original NADH stock solution is approximately 1.70 mM
Explanation:
This calculation relies on the principle that absorbance is directly proportional to concentration. By measuring the absorbance of the diluted solution and applying Beer-Lambert Law, the concentration of NADH in the diluted sample is determined. Then, using the dilution relationship, the original stock concentration is calculated. This process is fundamental in biochemistry labs when working with spectrophotometry to quantify nucleotides or proteins.