Results
From the completion of this experiment, it was learned that fluorescence spectroscopy is a better method of detection than the previous method of detection called high pressure liquid chromatography (HPLC). When the first TLC plate was made, the Rf value of the point was 0.666 and that matched with our Rf value for our column chromatography TLC plates. Next, an NMR spectrum was created by the NMR instrument and compared it to the 6TP and 9A standards with the NMR Spectrum and the results show that there is no starting material left. This is supported because there were no peaks in the NMR Spectrum that indicated there was no un-reacted substance left in the sample.
With fluorescence
spectroscopy, the limit of detection (LOD) is 1 pM shown in table 1. However,
if HPLC is used, the LOD is 20 nM (Robus, 2005). When fluorescence spectroscopy
is used, the limit of linearity (LOL) is 70 nM (shown in table 1). Likewise,
with HPLC, the LOL is 5 µM (Robus, 2005).
Discussion
The data and results collected throughout this experiment support the hypothesis that fluorescence spectroscopy is a better method of detecting small concentrations of 6TP than high pressure liquid chromatography. As discussed in the results, the LOD and LOL for fluorescence spectroscopy was much lower than the LOD and LOL for HPLC. Like all experiments, we experienced errors throughout this experiment. When the reaction mixture was first created, the hot plate did not have enough of a magnetic force to spin the magnetic tablet at the bottom of the round bottom flask therefore not making the solution completely dissolved shown in figure 4. This was probably the cause of most of the problems (mentioned in the upcoming) that were experienced in the later steps.
To fix this in a
future trial, we would find better equipment and test them out before hand.
During column chromatography, the reaction mixture fell through in an odd
fashion (due to its improper reacting) which can be seen in the pictures below.
To fix this in a future trial, the reaction mixture would be made properly.
When doing fluorescence spectroscopy, the numbers were becoming less linear and made no sense as the concentrations of 6TP got smaller. To fix this, multiple trials using the fluid should be used as well as more stable outdoor conditions. Because of this, the fluorescence values that were at either end of the spectrum that were causing the R² value to go below 0.99 were removed in order to ensure a better LOL and R² value.
From this experiment, it can be supported that fluorescence spectroscopy is a more sensitive method of detection than HPLC. The limit of detection was much lower for fluorescence spectroscopy than HPLC as well as a better limit of linearity. All of the results collected suggest that fluorescence spectroscopy is not only a highly sensitive method to detect 6TP, but another method to detect small concentrations of other materials. Another benefit to knowing the concentration of 6TP left in a patient’s body is the fact that the doctor can know what to give the patient in order to recover completely especially since large concentrations of 6TP can have large impacts on patients such as death. This is how fluorescence spectroscopy could be used in future research and techniques in the clinic.
When doing fluorescence spectroscopy, the numbers were becoming less linear and made no sense as the concentrations of 6TP got smaller. To fix this, multiple trials using the fluid should be used as well as more stable outdoor conditions. Because of this, the fluorescence values that were at either end of the spectrum that were causing the R² value to go below 0.99 were removed in order to ensure a better LOL and R² value.
From this experiment, it can be supported that fluorescence spectroscopy is a more sensitive method of detection than HPLC. The limit of detection was much lower for fluorescence spectroscopy than HPLC as well as a better limit of linearity. All of the results collected suggest that fluorescence spectroscopy is not only a highly sensitive method to detect 6TP, but another method to detect small concentrations of other materials. Another benefit to knowing the concentration of 6TP left in a patient’s body is the fact that the doctor can know what to give the patient in order to recover completely especially since large concentrations of 6TP can have large impacts on patients such as death. This is how fluorescence spectroscopy could be used in future research and techniques in the clinic.