34. The concentration (in micromolar) of NADH in a solution with A340 = 0.50 is __________.
Given data: Path length = 1 cm; Molar extinction coefficient of NADH ε340 = 6220 M−1cm−1
Calculation Method
The Beer-Lambert law states: A = ε · c · l
Where:
- A = absorbance = 0.50
- ε = molar extinction coefficient = 6220 M⁻¹ cm⁻¹
- c = concentration in M (to be calculated)
- l = path length = 1 cm
Rearranging gives: c = A / (ε · l) = 0.50 / (6220 · 1) = 8.04 × 10⁻⁵ M
Convert to micromolar: 8.04 × 10⁻⁵ M = 80.4 μM (approximately 80 for fill-in-the-blank)
Step-by-Step Breakdown
- Measure absorbance
A340 = 0.50using a spectrophotometer with 1 cm cuvette. - Use NADH-specific
ε340 = 6220 M⁻¹ cm⁻¹, standard for enzymatic assays. - Compute
c = 0.50 / 6220 = 8.04 × 10⁻⁵ mol/L. - Multiply by 10⁶ for μM: 80.4 μM.
No options provided; direct numerical answer applies.
Why Measure NADH at 340 nm?
NADH absorbs strongly at 340 nm (ε=6220 M⁻¹cm⁻¹), unlike NAD⁺, enabling specific detection in dehydrogenase assays. Common in fermentation kinetics and enzyme activity tests.
Common Errors to Avoid
- Forgetting unit conversion: M to μM requires ×10⁶
- ε mix-up: NADPH has similar value at 6220
- Path length oversight: Always verify cuvette length
Lab Applications
- Enzymatic assays (LDH, GAPDH)
- Bioreactor monitoring for microbial biotech
- CSIR NET/GATE biotechnology practice
Keywords: NADH concentration, A340 0.50, Beer-Lambert law, ε340 6220, micromolar calculation, path length 1 cm
