Potentiometer and Spectroscopy: Nicotinamide-adenine dinucleotide (in its two re

Question

Potentiometer and Spectroscopy: Nicotinamide-adenine dinucleotide (in its two redox forms NAD+ and NADH) is an important biological molecule and is key in a number of electron transfer reactions needed for physiological functions. Consider the corresponding redox couple NAD^+/NADH NAD^+ H^+ 2c^- doubleheadarrow NADH (Couple NAD+/NADH) The formal potential of this half reaction is -0.1128 V vs. SHE What is the value of this potential versus Ag/AgCI electrode; repeat same question but versus the SCE reference electrode Under certain circumstances, this redox couple can be used as a reference to report other potential values. What is the potential of couple NAD+/NADH versus this same reference? What is the potential of SCE and Ag/AgCI vesus NAD+/NADH Potentiomctry and spectrophotometers can be combined on special optical fibers that can also work as electrodes; NAD+ absorb light at 260 nm but not at 340 whereas NADH absorbs at both wavelengths; with fairly easy manipulation an experimentalist, using the optical fiber coated with known 1/amounts of NADH, can access the concentration ratio of NAD+ to NADH. Explain/describe how this modified optical fiber can be used to potentiometrically determine the pH The optical fiber reports the following absorbance values at 260 nm and 340 nm A(@260) = 0.4 and A(@340) = 0.2 for the same path length (same fiber). The pH of the bathing solution is 7.00; Calculate the measured single-electrode potential for the NAD+/NADH. The molar absorptivity values for NAD+ and NADH are as follows:

Explanation / Answer

The potential of standard calomel electrode is +0.241V

The potential of reversible hydrogen electrode is 0V – 0.0591*pH

The potential of standard hydrogen electrode is 0V and activity of H+ is 1

Potential of silver chloride electrode is +0.197V

a)

The value of the NAD+/NADH potential with reference to the Ag/AgCl electrode is

-0.1128 + 0.197 = +0.0842

The value of the same electrode with reference to SCE is

-0.1128+0.241 = 0.1282

b)

Potential of NAD+/NADH with reference to Ag/AgCl and with reference to SCE respectively are

0.197-(-0.1128) and 0.241-(-0.1128)

0.3098 and 0.3538

c)

The Nernst equation is

Electrode potential, E = standard electrode potential, E0 - RT/F * ln ([Ox] / [Red])

The standard electrode potential of NAD+/NADH before the optical fiber is modified is used in the equation to calculate the current electrode potential of NAD+/NADH after the optical fiber is coated with NADH in the place of NAD+.

To calculate exactly the current electrode potential, the oxidation potential of NAD+ and reduction potential of NADH are required.

The NAD+/NADH electrode potential will give an idea of the ability of the molecule to give away a hydrogen ion or accept the hydrogen ion. So, pH can be determined with the known oxidation and reduction potentials (potentiometry) that is altered by the hydrogen ion concentration determined by spectrophotometry.

d)

Absorbance = e * L * C

Where e is molar absorptivity, L is the path length and C is the concentration of the solution inside

0.4 /0.2 = 18000*L*NAD+/ 14400*L*NADH

(2 *14400)/18000 = NAD+/NADH = 1.6 (two electrode potential)
pH=7 = -log [H+]

[H+] = 10^-7 = 0.0000001

2=0*L*NADH/6220*L*NADH

2 = 0/6220*NADH

NADH potential = 0/(2*6220) => which is almost equal to 0.0000001

So, 0.000 V is the single electrode potential for NAD+/NADH

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