BCH4160/2011:Notebook/Andrews Lab Notebook
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50ml centrifuge tubes
1 pH Indicator
1. The spectra of both the acidic and basic forms of methyl red will be examined to determine the optimal volumes of methyl red stock solution and buffer to use in each pH assay. To do this, methyl red stock solution will be added to either phosphate citrate buffer. The spectra of the acidic and basic forms of methyl red (350-600nm) will also be used to determine optimal wavelengths for assessing the amounts of acidic and basic forms of methyl red in solution.
2. A pH meter will be used to determine the pH of the buffer solution after each addition of NaOH. After each pH adjustment, an aliquot of the buffer will be removed from the reservoir and combined with the pre-determined amount of methyl red solution (from step 1) to give samples for spectral analysis.
3. The spectra of solutions at each pH will be measured and recorded using a Vernier spectrophotometer (350nm - 600nm). If necessary, spectral files may be exported as ASCII files for import into Excel for further analysis. Spectra will be used to determine the absorbance of acidic and basic forms of methyl red in each sample, using the optimal wavelengths for each form (determined in step
Data analysis will involve creating a series of plots to show the effect of changing pH on the visible spectrum of methyl red. From these spectra, the relative amounts of acidic and basic forms of the indicator may be approximated to calculate a pKa (and Ka) for the dye. To help you with your analysis, I am showing some sample data that I obtained in a study of phenol red, another pH-sensitive dye.
1. Plot an overlay of selected spectra as a function of pH. Label the plot clearly so that the effect of changing pH on specific spectral features is clearly seen. Label the spectral peaks used in quantifying relative amounts of acidic and basic forms of the dye. (You may wish to show the spectra of the acidic and basic forms for clarity.)
2. Calculate the approximate fraction of acidic and basic forms of the dye at each pH using spectral data.
3. Plot the fraction of acidic and basic forms as a function of pH.
4. Plot log([A-] / [HA]) vs. pH. This plot should give a line with an x-intercept equal to the pKa of the dye.
Graphs and Figures
Protocol 1.Fill a 2.00 L volumetric flask with DI water and let equilibrate to room temperature
2.Carfully weigh the benzoic acid pellet in the metal sample cup on the analytical balance. Do not touch the pellet with your hands.
3.Take 10.0 cm of the fuse wire and assemble the pellet, cup, and wire. Tip the cup slightly to the side. The wire should go through the holes in the electrode posts, touch the sample, and be secured to each post: it should not have kinks in it away from the electrodes and must touch only the sample, not the metal cup.
4.Pipette 1.00 mL of DI water at room temperature into the bomb. This will be used to pre-saturate the gaseous phase with water to insure that all water produced by combustion will be in the liquid state.
5.Assemble the bomb. Be careful making sure you do not to disrupt the connection between the fuse and the benzoic acid. Also moisten the sealing rubber gasket with distilled water. Close the bomb knurled screw cap. All openings of the Bomb hand-tight only. Make sure the vent valve is closed securely.
6.Attach the hose from the O2 tank to the filling post. Fill the bomb to 20-25 atm with oxygen. After the bomb is filled slowly vent the bomb and repeat filling with O2 two more times (a total of three fills and flush). After the third flush fill once more to 20-25 atm but do not flush.
7.Place the oval reflecting tank in the white calorimeter jacket, fitting it into the stand-offs on the bottom. Gently place the bomb in the oval reflecting tank at the circular detent on the bottom; the removable handle tool may make this easier. There is only one way for the calorimeter system to be assembled. Plug the ignition leads into the two terminal posts at the top of the calorimeter.
8.Top off the 2.00 L volumetric flask and slowly and carefully fill the reflecting tank with the entire volume of water. Allow sufficient time for drainage of the flask. The entire bomb should be completely immersed. Examine for a flow of bubbles from the bomb making sure there are none.
9.Place the cover on the calorimeter jacket. Be careful; the thermometer extends about 6 inches below the calorimeter lid. Place the stirrer on the side of the reflecting tank where there is more space. Attach the stirrer belt and begin stirring.
10.Wait for the system to thermally equilibrates (about 2 minutes). Begin recording the temperature and time at 30 second intervals. Continue until four consecutive reading indicate constant temperature.
11.Ignite the bomb by pressing the button on the ignition unit. Note the time of ignition. The red light should illuminate briefly, and then go out as the fuse wire burns through. Record the temperature and time every 30 seconds. Wait about 5 minutes or until the temperature of the bomb stops rising and begins to fall. If the temperature does not increase at least 1 degree Celsius within the first minute the bomb most likely did not ignite.
12.After all measurements are recorded carefully disassemble the bomb. Look in the metal cup and make sure nothing is left. If unburned material is found the experiment must be restarted.
Below is the graph with of the data obtained from the Calorimetery experiment
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