A biochemist following the oxidation of lactic acid measures the absorbance of o

Question

A biochemist following the oxidation of lactic acid measures the absorbance of one of the products, NADH, at 340 nm. The compound has a molar absorptivity at that wavelength of approximately 6010 M^-1cm^-1. (a) In a 1.00 cm cell an unknown solution of NADH had a Transmittance of 19.0%. However, it was found that a dirty cuvette was causing 1.00% of the incoming light in this measurement to be reflected, therefore, not actually reaching the detector. Based on this information, what would be the observed absorbance of the sample? Using that value, what is the apparent concentration of NADH in the solution? (b) If a 19.0 transmittance value was actually a measurement made assuming that all the incoming light went through the sample and had no reflection, what would be the concentration of NADH in a sample? (c) The stray light is causing a positive deviation from Beer's Law. What is the difference expressed as a percent, in the two concentrations?

Explanation / Answer

a)

Calculate the absorbance by using following formula

Transmittance = 19%

1% of light is reflected due to dirty cuvette

So, actual transmittance = 19 +1= 20%

A = 2 - log10 %T

A = 2- log (20)

A = 2 – 1.301

A = 0.699

Calculate concentration of NADH by using Beer’s law

A = elc

e = the extinction coefficient of the substance in units of M-1 cm-1 = 6010 M-1cm-1

l = the sample path length measured in cm = 1cm

c = the molar concentration of the solution

c = A/el

c = 0.699/(6010M-1 cm-1 x 1cm)

c = 0.000116 = 1.16 x 10^-4 M

Apparent concentration of NADH = 1.16 x 10^-4 M

b) Now transmittance is 19%

So,

A = 2 - log (19)

A = 2 – 1.279

A = 0.721

Calculate concentration of NADH by using Beer’s law

A = elc

c = A/ el

c = 0.721/6010 M-1cm-1 x 1cm

c = 0.00012M = 1.2 x 10^-4 M

c)

% difference in Concentration = (Actual concentration – Apparent concentration/Actual concentration) x 100

% difference in concentration = (1.2 x 10^-4 M – 1.16 x 10^-4 M/1.2 x 10^-4 M) x 100

                                                      = 3.33%

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