PROJECT FINAL REPORT COVER PAGE
GROUP NUMBER R6
PROJECT TITLE Universal Indicators and Absorption Spectrophotometry
to Determine the pH of Unknown Solutions
DATE SUBMITTED May 7, 2001
ROLE ASSIGNMENTS
ROLE GROUP MEMBER
FACILITATOR………………………………………..Adam Furman
TIME & TASK KEEPER…………………….……..…Arika Goel
SCRIBE………………………………………………..Heather Lee
PRESENTER………………………………….……….Howard Lopez
BIOENGINEER……………………………………….Franklin Shen
SUMMARY OF PROJECT
The main objective of the experiment was to determine and program an algorithm that would generate the pH (within a range of 2 to 12) of an unknown solution containing Yamada or Bogens universal indicator, given a survey scan of such a solution. To obtain solutions of differing pH values, various amounts of HCl and NaOH were added to the universal buffer to modify the pH as needed. Survey scans were taken at pH increments of 0.5 (and 0.1increments were taken for pH values from 4 to 6).
Three-dimensional graphs of absorbance vs. wavelength vs. pH for solutions containing either of the universal indicators showed that absorbance vs. wavelength graphs at various pH values had only one peak, with the appearance of another peak for survey scans of the middle pH values. Since graphs at most pHs had only one peak, an algorithm was constructed in Microsoft Excelâ to determine the pH by comparing the wavelength and absorbance at the max peak of an unknown sample solution’s survey scan with survey scans of known pH values. The pH value whose characteristics (max peak wavelength and absorption) differ least was declared to be the output pH.
The uncertainty for the algorithm was found to be +/- 1.2 for the Yamada indicator and +/- 1.3 for the Bogens indicator.
OBJECTIVES
· To design an algorithm by which the user can input survey scan data of a solution containing either the Yamada or Bogens Universal Indicator and output a pH.
· Compare the sensitivity of the Yamada and Bogens Universal Indicators
BACKGROUND
Universal Indicators
Indicators work as markers that indicate the endpoint of a titration by a change in color. They experience a distinct change in chemical structure (from the protonated to unprotonated form) when the pH of the solution that contains it is changed. The equivalence point (the point at which this structural change occurs) of an indicator is given by a value known as pKa—the pH value of the solution at the equivalence point.
Thus, a universal indicator can be made by combining indicators with different pKa values. It can therefore, show color changes at different pH values. There are many practical reasons for using a universal indicator. For example, if you had a sample solution that was present only in volumes too small to be measured with the electrode of a pH meter, a universal indicator, which changes the color of the solution that it is put in based on the pH, would be useful(1). Two such universal indicators, Yamada and Bogens, are tested in this experiment.
Universal Buffers
A buffer is a chemical system that resists a pH change when an acid or base is added. The buffer is able to maintain a pH by establishing equilibrium between a weak acid (HA) and its conjugate base (A-) which adjusts accordingly as acid or base is added to the system. (2)
In order to test the universal indicator in solutions of various pH values, it is important to use a buffered solution. This ensures that the pH will not change dramatically with the addition of universal indicator.
A universal buffer can be made to produce solutions with pH values ranging from 2-12. A recipe for a universal buffer is found in Buffers for pH and Metal Ion Control by Perrin and Dempsey (3). The buffer is “universal” in the sense that by adding certain amounts of HCl or NaOH, various pH values can be easily and accurately attained.
Absorption Spectrophotometry
In absorption spectrophotometry, when a beam of light passes through a given material, it absorbs some of this light resulting in a decrease of the intensity of the initial light beam. The amount of attenuation of the incident beam depends on the range of wavelengths of radiation comprising the beam and the molecular composition of the material.
The Spectronic Genesys 5 Spectrophotometer, if used in the survey scan mode, can measure the absorbance for every wavelength in a range from 200 to 800 nm. The absorption spectra, a plot of absorbance vs. wavelength, can be constructed from gathered data. Generally, there exists one optimum wavelength at which the solution absorbs the most incident light (4). This fact will be useful in the development of an algorithm that calculates the value of a solution of unknown pH.
APPARATUS AND MATERIALS
1
Fisher Scientific Accument Model 625 pH meter
Spectronic Genesys 5 Spectrophotometer
Mettler PB 303 Electronic Balance
3L of 1M commercial NaOH and HCl
Universal buffer ingredients:
1M Citric Acid• H2O
Potassium Phosphate
Sodium Tetraborate•10H2O
Tris
Potassium Chloride
Yamada and Bogens Universal Indicator
Deionized water
500 mL, 200 mL, 100 mL Beakers
100 mL, 50 mL volumetric flasks
2000 mL plastic containers
Plastic 10mL pipettes
Disposable droppers
Kimwipes
Cuvettes
Magnetic stirrer
2 in. Stirring bars
Plastic measuring trays
Computer for recording scan data
Cuvette holder
Foam pad
1
METHODS AND PROCEDURE
Creating a Universal Buffer
Using a universal buffer recipe obtained from the book, Buffers for pH and Metal Ion Control, solutions ranging from a pH 2 to 12 could be attained. The recipe is as follows (3):
To make 1L of Universal Buffer:
0.1 M citric acid (21.01g/L)
0.1 M potassium phosphate (13.61g/L)
0.1 M sodium tetraborate (19.07g/L)
0.1 M Tris (12.11g/L)
0.1 M potassium chloride (7.46g/L)
Since some of these ingredients were unavailable in lab, the recipe was slightly modified. Instead of using citric acid and sodium tetraborate, citric acid·H2O and sodium tetraborate·10H2O were used. With these substitutions, the modified recipe is as follows (1):
To make 1L of Universal Buffer:
0.1 M citric acid·H2O (22.81 g/L)
0.1 M potassium phosphate (13.61g/L)
0.1 M sodium tetraborate·10H2O (28.07g/L)
0.1 M Tris (12.11g/L)
0.1 M potassium chloride (7.46g/L)
Each ingredient was measured with the Mettler PB303 Electronic Balance on a weighing tray to minimize error (+/- 0.001 g). These ingredients were added to 900 mL of deionized water and mixed with a magnetic stirrer and a 2-inch stirring bar. Once all the solute was dissolved, the solution’s volume was modified with the addition of deionized water until a solution volume of 1 L was reached. This process was repeated twice every lab session so that 2 L of buffer solution were produced each day.
Preparing HCl, NaOH, and Universal Buffer Stock Solutions
For each universal indicator, two universal buffer solutions, one to be used for NaOH additions, and one for HCl additions, were made in 500 or 250 mL glass beakers after being measured in volumetric flasks—totaling four separate buffer solutions. This was done because to achieve the pH range of 2-12 of the universal buffer, varying amounts of acid or base had to be added to the universal buffer stock solution. Universal indicator was added to each of the beakers in respective quantities as seen in Table 1 with the use of a disposable dropper.
Table 1 - Amount of Reagent and Indicator Added
This was done to ensure equal concentrations of indicator in all the stock solutions. Otherwise while adding NaOH or HCl to the universal buffer, the concentration of indicator in the buffer solution would change when NaOH or HCl was added, possibly affecting the data since the different survey scans would vary in color intensity. By using equal volumetric ratios for each indicator, as indicated in Table 1, this problem is avoided and only the color change due to pH is observed. Also the amount of Bogens indicator added was greater than the amount of Yamada indicator because the Yamada indicator was fainter in the buffer solution and needed more drops to make the color more visible.
Alteration of pH of Stock Solutions
To obtain the desired solutions, NaOH or HCl was added to the universal buffer solution that was constantly stirred. A foam pad was placed under the solution, but on top of the magnetic stirrer, to reduce any heat transfer into the beaker. To produce a higher pH, NaOH would be mixed into one stock solution, and HCl would be mixed in the other stock solution to achieve a lower pH. HCl or NaOH was added to the universal buffer stock solution with a 10mL pipette motorized with a pipette aid.
In this experiment, pH increments of 0.5 were desired. To measure the pH of the solution, the electrode of the Fisher Scientific Accument Model 625 pH meter was placed in the solution. The HCl and NaOH, depending on the desired pH, were added incrementally to the universal buffer solution to obtain pH changes of approximately 0.5. In the pH range of 4 to 6, pH increments of 0.1 were used instead to gain finer data and to verify analytical methods performed later. At these incremental points, the actual pH values were recorded with the pH meter with a manufacturer’s error of +/- 0.02 pH. After each pH reading, samples of approximately 2 mL were taken from the buffer solution by a disposable dropper and used for a survey scan.
Survey Scan Procedure
Using the Spectronic Genesys 5 Spectrophotometer, a survey scan was performed evaluating the absorbance of the sample at every wavelength from 200 to 800 nm using a type 3 scan. The readings were logged by the computer and saved for data analysis.
RESULTS
Three-Dimensional Graphs
Three-dimensional graphs were produced by plotting absorbance vs. wavelength vs. pH for Yamada and Bogens indicators. These plots are shown in Figures 1 and 2.
Figure 1: 3-D graph of absorbance vs. wavelength vs. pH for the Yamada indicator from pH 2-12
Figure 2: 3-D graph of absorbance vs. wavelength vs. pH for the Bogens indicator from pH 2-12
For most pH values, survey scans of the indicator solutions (both Yamada and Bogens) yielded graphs with one noticeable peak, as seen in Figure 3.
Figure 3: Survey scan of absorbance vs. wavelength of Yamada and Bogens indicators at pH 3.5
However, in the pH range of approximately 6-8, there are two noticeable peaks, which correspond to the transition of peaks seen in the 3-D graphs for both Yamada and Bogens indicators (Figures 1 and 2). A sample Yamada scan showing the presence of two peaks at pH 7.3 is shown in Figure 4.
Figure 4: Survey scan of absorbance vs. wavelength of Yamada indicator at pH 7.3
By taking into account the properties of the survey scans, an analytical method to obtain a pH from such a scan was determined in the following section.
DISCUSSION AND ANALYSIS OF RESULTS
Sensitivity of the Universal Indicators
To find sensitivity, the data from the survey scans at every pH interval of 0.5 were compared. For every 0.5 interval, the absolute values of the differences between the two neighboring pH values were found. A larger difference signifies greater sensitivity to pH change. See figures 5 and 6 for graphical representations of sensitivity for both indicators.
Figure 5: Yamada Sensitivity
Figure 6: Bogens Sensitivity
Figures 5 and 6 show the Bogens universal indicator has a total greater difference in absorbance between adjacent pH readings in the range of pH 2 to 3 than the Yamada universal indicator. Thus, the Bogens universal indicator has a greater sensitivity in the pH range of 2 to 3 while the Yamada universal indicator has greater sensitivity over the Bogens universal indicator for the remaining of the pH range (pH 3-12).
Producing a pH Algorithm With Graphical Analysis
After examining the survey scan data from 200 to 800 nm, it was decided that only the data from 380 to 750nm would be used in constructing the pH algorithm. Below 380nm, there was noise which may be attributed to ultraviolet light which was most likely absorbed by the plastic cuvettes.
Using Excel, the data for all three days were averaged together. By doing this, the data is smoothed. The pH values of the experimentally gathered data increase by increments of 0.5. However to increase the ability for the algorithm to find pH, a weighted average calculated an approximate pH, increasing by 0.1. (See Figure A1 in the Appendix A for a graphical representation of this.)
Microsoft Visual Basic® macros were written to automate the processing of data. A routine was written that calculated the peak wavelength and its absorbance for each pH value. It calculated the maximum absorption for each pH using the Max function of Excel, then ran a loop through the column of absorbencies to find what the corresponding wavelength was. This was accomplished using the For-Next loop structure seen in Appendix A. This prepared the database for access when searching for the pH of an unknown sample.
A program was written in Excel to determine the pH of an unknown sample. First, two columns of data must be copied into this excel sheet, the first being wavelength, the second the corresponding absorbencies. This was done by the Autofill subroutine. Next, one of two check boxes must be chosen. When the code is started by clicking the "Calculate" button, the software checks the value of the two bullets and sets a string variable to either Yamada or Bogens-allowing the software to check against the correct database. Running a similar loop used when preparing the database, the program determines the peak wavelength and absorption of the unknown sample. These two values are subtracted from the entire list of wavelengths and absorbencies in the database. The lowest sum of the differences between the unknown and the database is the best curve match. Another for-next loop sifts through the column of sums, and finds the lowest value. It then relates this value to the corresponding pH value in an adjacent column. This is the pH reported by the program.
Presence of Multiple Peaks in Survey Scan Data
For the middle pH range of approximately 6-8, two distinguishable peaks appeared on the survey scans, as opposed to one peak which was present at the other pH values. This is most likely attributed to the fact that the universal indicator is composed of many different indicators. In general, the larger peak was easily distinguishable as it was much larger than the smaller peak. However, at pH 7.3 (see Figure 4), the two peaks had similar maximum absorbance values, which may have been similar enough to confuse the algorithm—this would increase the uncertainty of the output at this point.
Uncertainty of pH-determining algorithm
For each indicator, survey scan data from all of the different pH value solutions measured were put into the algorithm; the output pH values were then compared with the actual pH values. To provide a conservative figure for the uncertainty, the greatest discrepancy between output pH and actual pH was taken to be the uncertainty value for the algorithm (with two different values for Bogens and Yamada). Note that some discrepancy between output pH and actual pH occurred at a middle pH value of approximately 7.3, where two similar absorbance peaks were observed in the survey scan of the solution. This is because the algorithm uses the wavelength of max absorption in its calculation of pH. If two peaks occur simultaneously at these middle pH values, the algorithm may match the unknown to the wrong curve. The uncertainty of the algorithm for both indicators is listed below. This uncertainty is mainly attributed to possible variations in concentrations of indicator in pH solutions in trials from different lab sessions.