DO 2. Molar Absorbtivity. Consider the absorbance spectra for NAD (red) and NADH

Question

DO 2. Molar Absorbtivity. Consider the absorbance spectra for NAD (red) and NADH (blue). in the case of NADH, are the molar extinction coefficients at 260 and 340 nm the same or different? Rationalize your answer using Beers law. 1.0 Oxidized (NAD) g 0.8 0.6 0.4 0.2 Reduced (NADH) 220 240 260 280 300 320 340 360 380 Wavelength (nm) 3. Molar Absorbtivity. Explain how to determine the molar absorptivity of riboflavin- then determine its value with your data. Show your work. 4. Using Beer's Law. NADH has a molar absorbtivity of 3.3 x 103 mM1-cm2 at 366 nm. Calculate the concentration of a solution that has an absorbance of 0.175 at 366 nm when measured in a cuvette with a pathlength of 1.00 cm Show your work.

Explanation / Answer

2. The molar absorptivity of NAD+ and NADH at 260 nm and 340 nm are not the same. They have different absorbance values and thus according to the Beer's law the,

molar absorptivity = absorbance/concentration x path length

the values are different at different wavelength maxima.

3. molar absorptivity is defined as the maximum amount of light that a sample or a substance absorpbs at a particular wavelength.

For riboflavin, knowing the concentration of the solution and the path length (not given here) we could calculate value by using equation,

molar absorptivity = absorbance/concentration x path length

this is by taking absorbance reading from the plot.

4. with,

absorbance = 0.175

molar absorptivity of NADH at 366 nm = 3.3 x 10^3 mM-1.cm-1

path length = 1 cm

we get,

concentration of NADH in solution = 0.175/3.3 x 10^3 x 1

                                                       = 5.30 x 10^-5 mM

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