User:Floriane Briere/Notebook/CHEM-496/2012/03/21

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Contents

Objective

According to yesterday's results, we haven't been able to reproduce the fluorescence results we obtained on the 2/22 whereas the UV results are consistent. In fact, on the 2/22 and on the 3/20 we obtained for the UV spectroscopy a curve where the BSA/HCl (with dye) is the highest, then it's the 70 ratio (with dye) and the lowest is the 166 ratio (with dye). However, for the fluorescence spectroscopy we obtained different results. On the 2/22, the highest were the 70 ratio (with dye) and the BSA/HCl (with dye); these two results were very closed to each other; the lowest was the 166 ratio. On the 3/20, the highest was the 166 ratio (with dye); the lowest were the 70 ratio (with dye) and BSA/HCl (with dye); these two results were very closed to each other. So, results are only partially reproductible. Today we are going to redo the measurements on yesterday's solutions to check our results then, we are going to figure out what could have happened and see if we can reproduce the 2/22 results.

Protocol

  • Spectroscopy measurements with same solutions as yesterday.
  1. Centrifugation (5 minutes at 13200rpm) for:
    1. 166 ratio with dye solution
    2. 70 ratio with dye solution
    3. BSA/HCl with dye solution
  2. Take fluorescence spectrum
  • Spectroscopy measurements after having changed the pH of solutions.
  1. Measure the pH:
    1. 166 ratio solution with dye: pH = 8.13
    2. 70 ratio solution with dye: initial pH = 7.62
    3. BSA/HCl solution with dye: initial pH = 7.75
  2. Add Tris buffer:
    1. 70 ratio solution with dye: added 3 drops; final pH = 8.07
    2. BSA/HCl solution with dye: added 3 drops; final pH = 8.09
  3. Centrifugation (3 solutions): 5 minutes at 13200rpm
  4. Take fluorescence spectrum

Results

  • Fluorescence spectroscopy results (same solutions as yesterday).

Image:21mar - fluorescence curve same sol.jpg

  • Fluorescence spectroscopy results (after having changed the pH of the solutions).

Image:21mar - fluorescence curve ph changedl.jpg

  • UV spectroscopy results (after having changed the pH of the solutions).

Image:21mar - UV curve ph changed.jpg

Image:21mar - UV curve ph changed zoom.jpg

Notes

We saw that we have been able to reproduce yesterday's results, our measurements were right. However, we haven't been able to reproduce 2/22 results, even after having changed and checked the pH of the solutions. In fact, we know that the dye is pH dependent and our protocol isn't very accurate (we added drops of Tris buffer until the solutions turned blue, we haven't added a precise amount of Tris buffer into our solutions and the pH is maybe very different between the different solutions).

Because we haven't been able to reproduce 2/22 results, we should measure the pH of the solutions we used on the 2/22 and see if it's very different from the one we used on the 3/20 and 3/21.

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