Molecular Fluorescence Instrumental : Part 2: Actual Excitation and Emission Maxima

In this part of the experiment, you will determine the optimal instrument operation parameters. Using the approximate excitation wavelengths determined above from the UV-Vis absorbance spectrum, obtain two emission spectra. The emission maximum observed in the two emission spectra will then be used to collect an excitation spectrum to "refine" the choice of excitation wavelength. You will see that the fluorescence excitation spectrum closely follows the absorbance spectrum.

You should collect the following spectra:

1. Two emission spectra of the 50 ppb standard solution will be taken. Collect the first spectrum from the first excitation wavelength (~370 to 700 nm). Collect the second spectrum from the second excitation wavelength (~440 to 700 nm). The two approximate excitation wavelengths determined in part 1 should be used to determine the wavelength ranges.
2. Two excitation spectra. One spectrum of the 50 ppb standard and one spectrum of the milk sample you prepared. (280 to ~500nm) Select the scan maximum by taking the larger of the two emission wavelength and subtracting 30 nm as described in step 5 of the "Using the Varian Eclipse Fluorimeter."
3. Print all of the spectra from this section.