User:Melvin Colorado Escobar/Notebook/581/2014/10/01

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Biomaterials Design Lab Main project page
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  1. determine fluorophore concentrations



  1. Make stock concentrations (both groups can use the same solutions)
    1. 0.10uM
    2. 0.50uM
    3. 1.0uM
    4. 1.5uM
    5. 2.0uM
  2. Take UV-Vis and Fluorescence spectra of these samples.
  3. Make a calibration curve based on UV-Vis.
    1. Compare your data to some published values
  4. Make a calibration curve based on the fluorescence.
    1. In order to do this, you'll need to measure the area under the fluorescence curve, not just the fluorescence peak height.


  1. One group will use a PVA film
  2. The other group will use a PVA/clay film
  3. Set the film between the two chambers
  4. Add liquid to each chamber
    1. One chamber gets water
    2. The other chamber gets 20ppm Malachite green
  5. Withdraw 200uL from each chamber at the 5 minute mark and place in their own eppy tube
    1. NOTE: I want the groups to coordinate so that their 5 minutes don't overlap with one another.
    2. NOTE: You'll want to use a small volume cuvette
    3. NOTE: You'll also want a 0minute measurement for each.
    4. NOTE: You'll likely have to dilute the sample from your chamber with the 20ppm. So, when you return the sample, only return the non-diluted sample.
  6. Measure the UV-Vis spectrum for both samples. Return each to the chamber it originally came from
  7. Take measurements at the following times
    1. 15 minutes
    2. 30 minutes
    3. 1 hour
    4. 2 hours
    5. right before you leave lab
    6. It would be great if someone could stop by on Thursday to take a measurement then.
    7. You'll be taking measurements on Friday too.
  8. During each of this, it would be best to start working up the data right away.



Preparation of stock solutions

0.0120g of R6G in 10mL needed to make the initial stock solution of 2500uM, this was used to prepare a secondary stock of 500uM, used to make the dilutions for 0.1, 0.5, 0.75, 1.0, 1.2 and 1.5. The initial stock of 2500uM was used to make a 2uM. But the 1.5 and 2 uM were not used for the calibration curve because the fluorescence was too high that it did not provide accurate data.

  • table of stock solution preparation
Concentration of stock solution (M) Amount of Stock used (mL)
0.1 0.001
0.5 0.005
0.75 0.0075
1 0.01
1.2 0.012
Fluorescent Parameters Value
Exitation Wavelength 500 nm
Range 515nm -700nm
Slit Width 10nm
Scan Speed 200 nm/min

  • Fluorescence spectra of standards

  • Calibration curve based on Fluorescence


  • UV-Vis spectra of standards

  • Calibration curve based on UV-Vis


Concentration (μM) Diluted by (mL)
1 0.5
10 0.5
20 0.5


Concentration (μM) Diluted by (mL)
1 0.5
10 0.25
20 0.1

Stock Solutions

Concentration (μM) Diluted by (mL)
1 0.05
10 0.1


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