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
07172013 Molar Absorptivity DAP MeOH 420nm GRAPH.png

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

07172013 Molar Absorptivity DAP MeOH 420nm GRAPH T2.png

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

07172013 Molar Absorptivity DAP MeOH 420nm GRAPH T3.png