4) 3ml of a solution containing both the oxidized and reduced form of NAD and NA

Question

4) 3ml of a solution containing both the oxidized and reduced form of NAD and NADH was placed in a 1.6cm spec cuvette. The absorbance at 340nm ( at which only the reduced form is measured) was 0.163. The absorbance at 260nm ( which measures both the oxidized and reduced forms together) was 0.872. The molar extinction coefficient of NADH 6220L/mol.cm and the molar extinction coeff. at 260nm is 18,000 L/mol.cm. Calculate the molar concentrations of oxidized and reduced forms of the nucleotide in the mixed solution?

Explanation / Answer

I think the main difficulty is with the awkward wording of the question you were given. Why don't they say "A student prepared a solution with a concentration of..."? Then it would be easier to imagine what was actually happening, the first step towards solving the problem. From the calibration plot, A = 1646.3 M From what you are told, for the relevant "unknown" (clearly not unknown at all) solution, M = 0.000266 Substitute that value of M into the first equation to find A Use A = - log(I/I0) or T = I/I0 = 10^-A to find fraction transmitted; x 100 for %

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