Compare prices and features of quantitative analysis software. The three programs most commonly used by university research departments are Statistical Analysis System (SAS), and ATLAS.ti.
Contact a vendor to purchase a software license and install the program.
Gather data for input by conducting a quenching experiment. Add increasing concentrations of a quencher, such as iodide ion, to an aqueous solution of a fluorescent agent, such as pyrenesulfonic acid and sodium salt. Irradiate the solution with a pulsed light source emitting pulses of one nanosecond or shorter after adding the quencher in each phase. Record the amount of quencher added during each phase of the experiment and the time elapsed between the solution achieving excited and ground stages, as well as the level of fluorescence emitted in nanometers.
Enter each data set in the quantitative software analysis environment and calculate the process of molecular return to the ground state using the equation A*+Q^kq yields A. Enter the concentration of excited state molecules as the value for A*. Q should be replaced with the amount of quenching agent added to the solution, while A is the concentration of molecules in the ground state. The variable kq represents the bi-molecular quenching constant and must be determined for each data set using the algebraic process.
Calculate the time of reaction completion using the formula t-initial / t = 1 + kq multiplied by t-initial multiplied by Q, where t-initial is the amount of time elapsed before the fluorescence intensity decays to its initial value and t is the fluorescence lifetime with quencher.
Generate a Stern-Volmer plot, including a best-fit line, for each phase of the experiment. A Stern-Volmer plot uses the data from the experiment performed to graph the quenching constant across the x-axis and the fluorescent intensities observed in the absence of the quencher divided by the fluorescent intensities observed in the presence of the quencher over the y-axis. In most cases, the plot should appear linear; however, in some rare cases of collisional quenching, a non-linear graph may appear.
Confirm the data is correct by verifying the intercept is 1 for each graph. Repeat calculations and generate a new graph if your information yielded different results.