# User:Dhea Patel/Notebook/Hemoglobin Project/2013/07/17

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## Finding the Molar Absorptivity of 2,3-Diaminophenazine (DAP) in MeOH

1. 10.0mg of 2,3-Diaminophenazine (210.23 g/mol) was added to 1.0mL of DMSO to make a 47567μM solution.

2. 26.0μL of the 47567μM solution of DAP in DMSO was added to 24.974mL of water to make a 50μM in a 25mL volumetric flask.

3. The following table outlines the volumes of DAP stock used and the final concentration of DAP solutions created.

 [STOCK] (μM) Initial Volume (mL) Vol. Water (mL) Final Volume (mL) [Final] (μM) 47567 0.105 99.895 100.000 50.000 50.000 15.000 10.000 25.000 30.000 50.000 13.500 11.500 25.000 27.000 50.000 12.000 13.000 25.000 24.000 50.000 10.500 14.500 25.000 21.000 50.000 9.000 16.000 25.000 18.000 50.000 7.500 17.500 25.000 15.000 50.000 6.000 19.000 25.000 12.000 50.000 4.500 20.500 25.000 9.000 50.000 3.000 22.000 25.000 6.000 50.000 1.500 23.500 25.000 3.000 0.000 0.000 25.000 25.000 0.000

4. An absorbance spectrum was taken of each solution.

5. The absorbance at 420.1nm was plotted vs. the solution concentrations.

 Concentration (μM) Absorbance 30 0.908 27 0.624 24 0.739 21 0.664 18 0.606 15 0.516 12 0.291 9 0.266 6 0.196 3 0.077 0 0

6. The slope of the best fit line is equal to the molar absorptivity (ε) of DAP in MeOH. ε = 29,400M-1cm-1.

7. Steps 1-6 were repeated in the afternoon. The following is the data obtained in the second trial. ε=20,800M-1cm-1.

 Concentration (uM) Absorbance 30 0.647 27 0.555 24 0.517 21 0.429 18 0.37 15 0.291 12 0.23 9 0.179 6 0.14 3 0.061 0 0

8. The mixtures made during Trial 2 were run on the Shimadzu UV-2550 in the Beeghly 207. The following is the data obtained:

 Concentration (uM) Absorbance 30 0.545 27 0.51 24 0.434 21 0.386 18 0.326 15 0.271 12 0.218 9 0.165 6 0.115 3 0.055 0 0