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Objective
Last week, we haven't been able to reproduce data we collected in february for fluorescence spectrum; especially, the 166 ratio with dye solution gives very different results (it was the lowest curve in february but the highest last week). To try to explain this difference, we are going to measure pH of initial solutions (made in february) and compare it with the pH of second solutions (made in march). By comparing curves and pH, we are going to see whether or not it can explain the non reproducibility of our results.
Then, we also miss some data to be able to write our article. Currently, the data we collected are:
- Spectrum (fluo and UV) of dye in solution
- Spectrum (fluo and UV) of 70 ratio + dye (from initial and 2nd solutions without having leveled the pH)
- Spectrum (fluo and UV) of 166 ratio + dye (from initial and 2nd solutions without having leveled the pH)
- Spectrum (fluo and UV) of BSA/HCl + dye (from initial and 2nd solutions without having leveled the pH)
- Spectrum (UV) of 166 ratio alone
We still miss the following data:
- Spectrum (fluo) of 166 ratio alone
- Spectrum (fluo and UV) of 70 ratio alone
- Spectrum (fluo and UV) of BSA/HCl alone
Protocol
- 2/21 solutions(initial solutions).
- Measure the initial pH:
- 70 ratio with dye: initial pH = 8.12
- 166 ratio with dye: initial pH = 8.43
- BSA/HCl with dye: initial pH = 8.3
- Increase the pH by adding Tris buffer:
- 70 ratio with dye: add 10 drops, final pH = 8.30
- 3/21 solutions (2nd solutions).
- Measure the initial pH:
- 70 ratio with dye: initial pH = 8.00 (last week pH = 8.07)
- 166 ratio with dye: initial pH = 8.35 (last week pH = 8.13)
- BSA/HCl with dye: initial pH = 7.98 (last week pH = 8.09)
- Increase the pH by adding Tris buffer:
- 70 ratio with dye: add 6 drops, final pH = 8.29
- 166 ratio with dye: add 3 drops, final pH = 8.41
- BSA/HCl ratio with dye: add 7 drops, final pH = 8.28
- Fluorescence spectrum of 3/21 solutions (2nd solutions).
- Centrifugation (5 minutes, 13200rpm)
- Take spectrum: excitation = 600nm; emission = 620 to 800nm
- Fluorescence spectrum of 2/21 solutions (initial solutions).
- Centrifugation (5 minutes, 13200rpm)
- Take spectrum: excitation = 600nm; emission = 620 to 800nm
- Fluorescence spectrum of control solutions.
- 166 ratio alone: prepared on the 2/28
- 70 ratio alone: prepared on the 2/28
- BSA/HCl alone: prepared on the 3/20
- Dye in solution: prepared in february (same as last time)
- Take UV spectrum of all solutions.
Results
- Fluorescence spectrum: second solutions.
- Fluorescence spectrum: initial solutions.
- Fluorescence spectrum: control solutions.
- UV spectrum: second solutions.
- UV spectrum: initial solutions.
- UV spectrum: control solutions.
Notes
- We noticed initial solutions had similar pH except for the 70 ratio with. So, we decided to increase the pH of this solution to level pH value between the 3 different solutions.
- By comparing initial and second solutions pH value, we noticed that the pH of initial solutions was higher (around 8.35) than pH of second solutions (around 8.1). This difference could explain the fact that we haven't been able to reproduce our data. So, we increased pH of second solutions to match initial solutions pH. Then, we performed fluorescence spectrum and noticed that fluorescence curves are related to pH.
- We also noticed that pH of second solutions evolved during the week; pH value were different from one we measured last week. This difference can be explained by contaminants (water used to clean tubes, pH meter), or by the fact that a reaction is occuring into our solutions and affect pH value. The pHmeter has been calibrated before each set of measure, so the difference shouldn't come from the instrument.
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Chem-496
