Q. 35 Measurement of the absorbance of a solution containing NADH in a path length of 1 cm cuvette at
340 nm shows the value of 0.31. The molar extinction coefficient of NADH is 𝟔𝟐𝟎𝟎𝐌−𝟏𝐜𝐦−𝟏. The
concentration of NADH in the solution is ____ 𝝁𝐌 (correct to integer number).
NADH concentration is determined using Beer’s Law from given absorbance data. The correct concentration is 50 μM, calculated precisely for exam preparation in biochemistry.
Beer’s Law Basics
Beer’s Law, A = ε⋅c⋅l, relates absorbance (A) to concentration (c), molar extinction coefficient (ε), and path length (l). Here, A = 0.31 at 340 nm, ε = 6200 M⁻¹cm⁻¹, and l = 1 cm, where NADH uniquely absorbs due to its reduced form.
Step-by-Step Calculation
Rearrange for concentration: c = A / (ε⋅l) = 0.31 / (6200 × 1) = 5 × 10⁻⁵ M
Convert to μM: 5 × 10⁻⁵ M = 50 μM (integer value)
Final Answer: 50 μM
Why 340 nm for NADH
NADH exhibits peak absorbance at 340 nm (ε ≈ 6220 M⁻¹cm⁻¹), unlike NAD⁺, enabling selective quantification in enzymatic assays like dehydrogenase activity.
Common Exam Pitfalls
- Forgetting unit conversion from M to μM yields 0.05 μM (wrong).
- Misreading ε as 6220 L mol⁻¹ cm⁻¹ without path length adjustment gives incorrect scaling.
- Assuming 260 nm absorbance confuses with NAD⁺/total nucleotides.
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