Quest. # Q1 DCF curves DCF Control Reaction order Total
Marks 10 40 20 10 80
Kinetic methods with spectrophotometric detection for assaying drugs have been intensively developed during the last decade due to a number of obvious advantages, including inherent simplicity, cost-effectiveness, availability in most quality control laboratories, and improved selectivity. In this task you will:
• Perform kinetic determination of Diclofenac (DCF) in a medicine by following the progress of the drug oxidation reaction.
• Determine the reaction order with respect to DCF
Q1. Spectral changes in the course of DCF oxidation with KMnO4 are given in Fig. 4, (1 to 10 reflects the reaction progress). Complete the table below suggesting which wavelengths can be applied for photometric kinetic determination of DCF. In each case, indicate the direction of the absorbance changes (denote increasing with ↑ and decreasing with ↓).
Fig. 4. DCF oxidation with KMnO4
# Wavelength, nm Yes or No and direction
1 420 Yes ↑ 2 marks
2 480 No 2 marks
3 520 Yes ↓ 2 marks
4 580 No 2 marks
5 610 Yes ↑ 2 marks
Procedure
Part A. Assembling of laboratory equipment
Assemble the laboratory equipment as shown in Fig. 5. Connect the photometer (1), 525 nm (fixed wavelength) and thermostat (2) to the Netbook via USB slots. Connect the thermostat to the cable labeled “Thermo” to the power supply at your work place via the power adapter. Put the optical cuvette (3) on top of the magnetic stirrer (4), pass the cuvette through the photometer from aside (not possible from top down) and place the thermostat over the cuvette from top down (Fig. 5b).
Fig. 5. Laboratory equipment
Hints!
- Plug in your Netbook to the mains before switching on.
- Plug in all the equipment (the photometer and thermostat) before switching on the Netbook.
Switch on the mouse.
- If only one window (hereafter referred to as Pattern) instead of two appears after launching the software, quit and re-launch the program.
47 International Chemistry Olympiad. Baku, Azerbaijan, July 20-29, 2015. ABC-1
- Do not unplug ANY device from the USB slot while carrying out the measurements. If it still happens, you will see a warning on the screen. Quit and re-launch the program.
- If your Netbook falls asleep, click the «Setup» button in the Measurements window on the absorbance plot pattern when reverting to the measurements.
- In case you see chaotic temperature changes on the screen, stop and re-start the measurement.
Part B. Plotting of the calibration curve
All measurements needed to plot the calibration curve are carried out at 30 °C with constant KMnO4 and H2SO4 initial concentrations. The DCF concentration is varied by using 4 different aliquots (of 0.2, 0.4, 0.6, and 0.8 mL) of the DCF stock solution.
1) Transfer 5 mL of 1M H2SO4 solution using the graduated cylinder and 0.2 mL of DCF stock solution using the 2 mL pipette into the 100 mL volumetric flask, dilute to the mark with distilled water, stopper the flask and mix thoroughly.
2) Carry over the flask contents into the cuvette, put the medium-size stir-bar and switch on the magnetic stirrer. Adjust the stirring speed regulator to the mark shown on Fig. 5a to provide for intensive mixing.
3) Launch the «Chemistry-Practicum» software on the Netbook. The software will detect the external devices (sensors) automatically. You will see two plot patterns (that of absorbance/extinction/optical density, D vs. t, s; and that of temperature, T °C vs. t, s) on the display.
4) Set the following parameters in the Menu bars of the corresponding plot patterns (Fig. 6):
- Click the icon next to the button («Fixes X-axis maximum on screen») on the absorbance plot pattern. The entire plot will always fit to the screen;
- Click the button («Sets the Y range») on the absorbance plot pattern and set the absorbance range (the ordinate axis) from -0.1 to 1.1.
- Type “2” (instead of “1”) in the box of the measurements interval on the absorbance plot pattern.
- Choose «Precisely» in the «Precisely/Roughly» window on the temperature plot pattern, then click on the «T = X» button and set the required temperature of 30 °C in the pop-up window.
- Calibrate the photometer by clicking the «Setup» button in the Measurements window on the absorbance plot pattern.
Fig. 6. “Chemistry-Practicum” software interface
Note! Setting the parameters (step 4) is needed only prior to the first measurement.
5) Click the button («Start measure for chosen sensors») to switch on the thermostat and observe the lamp heating up the solution in the cuvette. Follow the current temperature reported in the line above the plot. Wait until the thermostat lamp switches off, reflecting the set up temperature is attained. Stop the measurements by clicking button (is activated and turns to red-orange when the measurement is on).
6) Click any part of the absorbance plot pattern to activate it. Take 2 mL of the KMnO4 solution using the 2 mL pipette. Click the button («Start measure for chosen sensors») in the Menu bar of the Measurements window and quickly blow out (press the pipette piston) the permanganate solution from the pipette into the cuvette.
Note! Make sure the temperature in the cuvette equals 30 °C before adding the KMnO4 solution!
7) Observe the progress of the kinetic curve on the screen. Continue measurement for 50 s after adding the KMnO4 solution, then terminate the measurement by clicking the «Stop measurements» button.
47 International Chemistry Olympiad. Baku, Azerbaijan, July 20-29, 2015. ABC-1
8) Save the data by clicking the button («Export all the data collected in an external file») in the Menu bar of the absorbance plot pattern, choose the Desktop and type the file name “DCF2”
(change the name to “DCF4”, or “DCF6”, or “DCF8” in the subsequent experiments).
Note!
- Use only the names of the given format!
- Always save the data on your Desktop before starting the next experiment, otherwise the current data set will be lost after the next click on the button.
- Make sure absorbance plot pattern is active when exporting the data. Otherwise, you will export invalid results. In case no pattern is chosen, you will get a warning.
9) Empty the cuvette into the Waste bottle, wash thoroughly the cuvette with distilled water. Use black magnet from the outer side of the cuvette to avoid your stir-bar being dropped into the Waste bottle while washing. Wipe carefully the external surfaces of the cuvette with the napkin.
Also, use the napkin to dab the thermostat lamp.
10) Repeat the steps 1), 2) 5)-9) with the other volumes of the DCF stock solution.
10 marks maximum for each of 4 measurements. Students data will be recalculated by Science Committee. 40 marks in total.
Part C.
1. Studying of the DCF containing medicine (“Control”)
1) Wash the volumetric flask and prepare the mixture as described above using a 0.4 mL aliquot of the medicine (“Control”) instead of the DCF stock solution.
2) Repeat the steps 1), 2), 5)-9) described in Part B. When saving the data, name the file
“DCFmed”.
3) Repeat the measurement of the “Control” as necessary.
2. Experimental data analysis
1) Open the Excel file on your memory stick in Excel. One by one open your saved data files in Notepad by double clicking on them on Desktop. Choose Edit/Select All in the Menu bar, then right click and copy the selected data into the Excel sheet with the corresponding name (the volume of DCF added or “DCFmed”) and choose Edit/Paste in the Menu bar. You will see the experimental data on the Excel sheet (time, s, in column A, and absorbance in column B).
2) Ignore the values before the maximum. Select columns A and B, and plot the data. Use the
“Insert Scatter” icon shown on Fig. 7.
Figure 7. Position of the “Insert Scatter” icon
3) Choose the initial linear section of the remaining curve (15 to 20 data points), apply linear approximation by adding the linear trend line and bring the parameters to the chart area. Make sure that the R2 value exceeds 0.98. If needed, decrease the number of the experimental data points plotted removing later data points. Still always search for the most wide range of the experimental data providing for the target R2 value. Determine the value of the initial rate of absorbance change, v0.
47 International Chemistry Olympiad. Baku, Azerbaijan, July 20-29, 2015. ABC-1
Note! You will get zero point for this part of the task if less than 12 values are included in the plotted data range.
4) Analyze similarly the experimental data obtained with the other DCF concentrations and with the medicine solution “Control” (“DCFmed” file).
5) Calculate the DCF concentrations in the reaction mixtures (in mg/L). Write down the DCF concentrations and initial rates in appropriate cells of the “Results” Excel sheet.
6) Plot the calibration graph on the “Results” sheet and use it to determine the DCF concentration in the analyzed mixture prepared from the medicine (“Control”). Fill in the appropriate cells of the “Results” Excel sheet with the coefficients of linear approximation of the calibration graph.
Calculate the DCF concentration in the medicine.
20 marks maximum (including 8 marks for data obtained and calculations). To be graded similarly to the procedure in Task 1.
The grading scheme takes into account two values re-measured by the Science Committee: R-squared value (R2) and obtained concentration of the control solution (Conc).
• If the value obtained is within region A, 100% of 12 marks
• If the value obtained is within region B, 0.1926*Conc-154.2857 (%) of 12 marks
• If the value obtained is within region C, 400*R2-372 (%) of 12 marks
• If the value obtained is within region D, -0.1926*Conc+188.5714 (%) of 12 marks Master value – 890.1 mg/L
7) Write down the accepted value in the cell F10 of the “Results” sheet.
8) On the “Results” Excel sheet, graphically determine the reaction order with respect to DCF and write down the exact obtained value in the cell I3.
10 marks for the determination of the reaction order
9) Once finished, save your file and invite your Lab assistant to demonstrate that you have got experimental data in the Excel file. Sign and get the Lab assistant’s signature.
Note! Only the data saved on the memory stick will be considered as the result of the Task.
Data present in Excel on the memory stick (to be ticked by the Lab assistant)
Yes No Student
______________
Lab assistant ______________