User:Andrew K Farag/Notebook/CHEM-581/2014/10/08

Biomaterials Design Lab

Main project page Previous entry      Next entry

Objective Make stock solutions of R6G. Thanks to Madeleine for making initial 2500μM solution of R6G. Take UV-Vis and Fluorescence spectra of prepared R6G samples Make a calibration curve using both fluorescence and UV-Vis data To finish film synthesis Notes The completion of the film synthesis used the procedure from Tami's Notebook R6G Fluorescence: Calibration and Measurement Completed by Michael Make stock concentrations (both groups can use the same solutions) Initial stock: 2500μM, secondary stock: 500μM 0.10μM Made with 500μM solution 0.50μM Made with 500μM solution 1.0μM Made with 500uM solution 1.5uM Made with 500μM solution 2.0μM Made with 2500μM solution 0.75μM Made with 500μM solution 1.2μM Made with 500μM solution Fluorimeter Settings 500nm excitation 515-700nm scan range 10.0nm slit width 200nm/min scan speed Calculations To calculate necessary amounts of stock solutions to add to enough water to have a total of 10 mL of calibration stock solutions: C1*V1 = C2*V2 secondary stock: 500μM (2500μM)*x = (500μM)*(10mL) → x = 2 mL 2500μM R6G 0.1μM (500μM)*x = (0.1μM)*(10mL) → x = 2 μL 500μM R6G 0.5μM (500μM)*x = (0.5μM)*(10mL) → x = 10 μL 500μM R6G 0.6μM (500μM)*x = (0.6μM)*(10mL) → x = 12 μL 500μM R6G 0.75μM (500μM)*x = (0.75μM)*(10mL) → x = 15 μL 500μM R6G 0.85μM (500μM)*x = (0.85μM)*(10mL) → x = 17 μL 500μM R6G 1.0μM (500μM)*x = (1.0μM)*(10mL) → x = 20 μL 500μM R6G 1.2μM (500μM)*x = (1.2μM)*(10mL) → x = 24 μL 500μM R6G 1.5μM (500μM)*x = (1.5μM)*(10mL) → x = 30 μL 500μM R6G 2.0μM (500μM)*x = (2.0μM)*(10mL) → x = 40 μL 500μM R6G Data Using the data collected from the UV-Vis Spectrometer and the Fluorometer, the following calibration curves were created. Figure 1: The figure above shows the UV-Vis absorbance spectra for various concentrations of R6G. The spectra have been corrected by subtracting the spectrum values of water from each. The spectra have also had their respective absorbance values at 800 nm subtracted from every point in order to shift the baseline to zero across the board. For those spectra that had peaks shifted down below this baseline after the subtraction, the absorbance value of each of the graphs at 560 nm was subtracted from the whole spectrum instead so that the rise of the peak would occur at the baseline like the others. Figure 2: It was noticed in figure 1 that the graph of 0.1 μM R6G had a peak at 0.003, which is extremely close to the detection limit of the instrument, so it was excluded from the calibration curve. Figure 3: The figure above shows the Fluoresence spectra for concentrations up to 1 μM of R6G. Concentrations above 1 μM gave values that were too high to show up on the fluorometer. Figure 4: The figure above shows the calibration curve for fluorescence. Integration values were determined using Riemann sums with a base of .5 nm.