Once the sample had gone through the proton NMR, the materials required were gathered to complete fluorescence spectroscopy. The materials needed to do fluorescence spectroscopy include acetonitrile, 10 ml volumetric flasks, p1000 autopipette, auto pipette tips, fluorescence spectrophotometer (fluorometer), plastic pipettes. quartz cuvette, and 100 ml (100 µM concentration) solution of purified product. First, the quartz cuvette was conditioned with the acetonitrile and inserted as the blank into the fluorometer to determine the baseline for the experiment. Next, the cuvette was conditioned with the first dilution (in a 1:10 ratio) of the purified product solution. It was continued to condition the cuvette and dilute the solution until the fluorometer can no longer detect the 6TP in the purified solution while making sure to take measurements after inserting each dilution into the fluorometer. To fill the cuvette, plastic pipettes were used but to fill the 10 ml volumetric flasks, automatic pipettes were used in order to create perfect dilutions. After each dilution is placed in the fluorometer, data points were collected. Once, the data reached the blank limit, that is the limit of detection (LOD). Lastly, to find the linearity between concentration and fluorescence, insert multiple intervals to make the data more accurate. The intervals used are 100 µM, 70 µM, 50 µM, 30 µM, 10 µM, 5 µM, 1 µM, 700 nM, 500 nM, 300 nM, 100 nM, 70 nM, 50 nM, 30 nM, and 10 nM. The same process was repeated as above except with the dilutions mentioned in the previous line.
Figure 7. - Fluorometer and its Quartz Cuvette