Beer’s Law Problem Set Spring 2013 1. Calculate the absorbances corresponding to the following values of the percentage of transmitted light: (Provide your final answer with three decimal places) a. 95% b. 88% c. 71% d. 50% e. 17.5% f. 1% 2. A solution of a compound (1.0mM) was placed in a spectrophotometer cuvette of light path 1.05cm. The light transmission was 18.4% at 470nm. Determine the molar extinction coefficient. Include units in your answer. 3. The molar extinction coefficient of reduced NADH (nicotinamide adenine dinucleotide phosphophate) at 340nm is 6220 L/mole·cm. 3ml of solution containing 0.2 micromoles of NADH were placed in a cuvette of 1.05cm light path. Calculate the percentage light transmission of this sample at 340nm. 4. 3ml of a solution containing both the oxidized and reduced form of nicotinamide adenine dinucleotide (NAD and NADH, respectively) was placed in a 1.0cm spectrophotometer cuvette.

The absorbance at 340nm (at which only the reduced form is measured) was 0.207. The absorbance at 260nm (which measures both the oxidized and reduced forms together) was 0.900. The molar extinction coefficient of NADH is 6220 L/mole·cm, and the molar extinction coefficient at 260nm is 18,000 L/mole·cm. Calculate the molar concentrations of oxidized and reduced forms of the nucleotide in the mixed solution. 5. A mixture of ortho, meta, and para cresoles dissolved in cyclohexane may be analyzed spectrophotometrically because each exhibits an absorption in a region where absorption due to the other cresols is negligible. The absorption maxima occur at 752nm, 776nm, and 815nm for ortho, meta, and para cresols, respectively. To test the validity of Beer’s Law for solutions of cresols, each is made up in cyclohexane at a series of concentrations and the absorbances measured. Data obtained are recorded below: Ortho Meta Para

Concentration (g/100ml) 0.25 0.50 1 2

Absorbance (at 752nm) 0.120 0.235 0.465 0.820

Concentration (g/100ml) 0.60 1.15 2.35

Absorbance (at 776nm) 0.115 0.220 0.460

Concentration (g/100ml) 0.50 1 2.1 3.15

Absorbance (at 815nm) 0.09 0.2 0.415 0.60

An unknown mixture of the three cresols in cyclohexane was analyzed and the percentage of light absorbed at 752, 776, and 815nm was 27.5, 50, and 41 respectively. Determine the concentration of each cresol and the percentage of the final mixture. (Calculate your answer using 3 decimal places) (Hint: if 20% of the light is absorbed by the sample, then 80% is transmitted. Percent absorbed does not equal Absorbance. Also, the table of data above should be used to generate a graph. How would this help you?) Answers: 1) a. 0.022 b. 0.056 c. 0.149 d. 0.301 2) ~700 L/mole·cm 3) 36.7% 4) NAD= 16.7uM , NADH= 33.3uM 5) ortho 0.259g/100ml 8.697% meta 1.549g/100ml 52.014% para 1.170g/100ml 39.288%

e. 0.757

f. 2.00

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