User:Matthew R Skorski/Notebook/471 - Exp BioChem/2016/03/15

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Objective

  • Because our most recent attempt at the creation of a fluorescence calibration curve relating the integrated fluorescence response to the concentration of Rhodamine B in solution yielded very scattered results (some lower concentrations would display higher integrated fluorescence responses than higher concentration samples) it was hypothesized that dimerization may occur at lower concentrations of Rhodamine B. In order to determine if dimerization is occuring, it is necessary to perform UV-Vis analysis and determine if the peak location is shifting. Thus today, was used to analyze standard solutions using UV-Vis.

Protocol

  • Take Spectra of Rhodamine B standards from 200 nm to 800 nm. All standards were prepared by Nicole (see her notebook for details on sample preparation).
  • Determine wavelength of peak absorbance and determine if it changes with concentration.

Results

Figure 1: UV-Vis spectra for 0.1-1 μM Rhodamine B standards. All standards peaked at 555 nm except for samples of concentrations 0.2, 0.3, and 0.5 μM which peaked at 553 nm. The differences in absorbances between these two wavelength's were 1/1000th of an absorbance unit (which may simply be the result of instrument noise or the way the instrument rounds absorbance values to the thousandths place.

Figure 2: UV-Vis calibration curve for Rhodamine B. From the figure above, it was evident that the linear range for UV-Vis of Rhodamine B may end at around 0.6 μM. Below that concentration, the UV-Vis response is far less predictable. As a result, higher concentration samples were analyzed.

Figure 3: UV-Vis spectra for 2-10 μM Rhodamine B standards. Higher concentrations of Rhodamine B were used to determine if these concentrations could be used practically and analyzed using UV-Vis.

Figure 4: UV-Vis calibration curve for Rhodamine B at higher concentrations. The figure above shows a calibration curve that extends the concentrations of Rhodamine B used. Note that the calibration curve is linear and under 1 absorbance unit up to 10 μM Rhodamine B. This indicates that UV-Vis analysis could be a better option to determine the amount of Rhodamine B incorporated by AuNP fibers since higher and more manageable concentrations can be used with the UV-Vis.



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