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 Excel 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.
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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
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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
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
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)
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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
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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
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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
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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.
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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
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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 312).
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.
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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.
Uncertainty of algorithm:
+/- 1.2 for Yamada indicator.
+/- 1.3 for Bogens indicator.
Uncertainty of Measurements
Most of the uncertainty in the experiment is due to the limitations inherent in the
lab equipment used: the Fisher Scientific Accument Model 625 pH meter and the
Spectronic Genesys 5 Spectrophotometer. Other factors that add to uncertainty are the
possibility of using scratched or not properly cleaned cuvettes. If the cuvettes were not
properly dried, uncertainty could occur from dilution of the solution placed in the cuvette.
Uncertainty could be improved by using brand new cuvettes for each run of the
experiment.
The amount of indicator was measured by the number of drops placed into a
solution. These drops were possibly of varying volume, resulting in different
concentrations of indicator in the solutions.
This problem should have been
circumvented by using a micropipette. In addition, the uncertainty in concentration could
also have been improved by using better volumetric equipment to measure each reagent
instead of volumetric flasks. Uncertainty of the volumetric equipment only affects the
concentrations of the indicator in the stock solutions, and does not affect the measured
pH of the buffered solutions since this is dependent solely on the pH meter.
Practical Applications

Allows one to find the pH of solutions in volumes too small to be measured with
electrode of a pH meter.

Using this algorithm would allow a user to find the pH of a solution without
having to use a pH meter—an expensive piece of laboratory equipment.
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CONCLUSIONS
As a result of this experiment, an algorithm was created which enables users to find
the pH of an unknown solution by adding either Bogens or Yamada universal indicators
and taking a survey scan of the unknown sample. This algorithm outputs the pH of the
unknown solution with an uncertainty of +/-1.2 for Yamada universal indicator and +/1.3 for Bogens universal indicator. It is also determined that the two indicators are better
suited for different pH ranges as one is more sensitive than then other at varying pH
values. The Bogens universal indicator is more sensitive than the Yamada universal
indicator only for the range of pH 2 to 3. For the remaining pH values, 3 to 12, it would
be better to use the Yamada universal indicator, as it is more sensitive than the Bogens
universal indicator in this range.
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REFERENCES
1. BE210 , pH Dependence of Indicator Dye Spectra, University of Pennsylvania,1999.
2. BE 210, Biological Buffer Systems, University of Pennsylvania, 1999.
3. Perrin, D.D., and Boyd Dempsey, Buffers for pH and Metal Ion Control, Chapman and
Hall, London, 1974.
4. BE210, The Dependence of Methyl Red Molar Extinction Coefficient on the pH,
University of Pennsylvania, 2000.
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Appendix A
Figure A1: Graphical Representation of Linear Approximation by Weighted Average of Survey Scans at 3.0 and
3.5 pH values.
Figure A1 represents the linear approximation, which was found by weighted average
calculations. The experimental data is shown in red while the extrapolated data is show
in green, blue and purple.
Primary program sheet code
This is the code that runs when the calculate button is clicked. It calls upon functions and
subroutines from module2 (a global access module).
Lines in green preceded by an apostrophe are remarks and annotations giving explanation
of the code’s function
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‘variable declarations and dimensions
Dim indicator
Dim maxab
Dim maxwave
Dim min_diff
Dim pH
Private Sub CommandButton1_Click()
‘determine which indicator based upon the two bullets
If OptionButton1.Value = True Then assignindicator
("Yamada")
If OptionButton1.Value = True Then indicator = "Yamada"
If OptionButton2.Value = True Then assignindicator
("Bogens")
If OptionButton2.Value = True Then indicator = "Bogens"
'call the autofill sub routine, will copy cells from
program front to the active calculation page
autofill
Sheets(indicator).Activate
'find peak for unknown
maxab = returnmax()
'find peak wavelength of unknown
maxwave = findunknown(maxab)
're-calculate sheets—this is a built in re-calculate
function in excel.
Calculate
‘find minimum difference
min_diff = mindiff
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‘find the pH by looking up through the column of numbers
pH = findph(min_diff)
‘reactivate the program’s main sheet
Sheets("The pH Calculator").Activate
‘Display the final pH
Label3.Caption = pH
Module2 Code:
‘variable declarations and dimensions
Dim maxabs
Dim indicator As String
Dim rowcheck
Dim maxwaverow
Dim startwave
Dim endwave
Dim startwaverow
Dim endwaverow
Function returnmax()
Sheets(indicator).Activate
returnmax = WorksheetFunction.Max(Range(Cells(2, 2),
Cells(150, 2)))
End Function
Function findmaxwave(a)
findmaxwave = Cells(a, 1).Value
End Function
Function findunknown(maxabs)
Sheets(indicator).Activate
For rowcheck = 2 To 150
If Cells(rowcheck - 1, 2).Value <> maxabs And
Cells(rowcheck, 2).Value = maxabs Then startwave = rowcheck
If Cells(rowcheck + 1, 2).Value <> maxabs And
Cells(rowcheck, 2).Value = maxabs Then endwave = rowcheck
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Next rowcheck
maxwaverow = (startwave + endwave) / 2
findunknown = Cells(maxwaverow, 1)
Cells(4, 5).Value = Cells(maxwaverow, 1)
Cells(3, 5).Value = maxabs
End Function
Function mindiff()
Sheets(indicator).Activate
mindiff = WorksheetFunction.Min(Range("P2:P102"))
End Function
Function findph(mindif)
Sheets(indicator).Activate
For rowcheck = 4 To 102
If Cells(rowcheck - 1, 16) <> mindif And Cells(rowcheck,
16) = mindif Then startwaverow = rowcheck
If Cells(rowcheck + 1, 16) <> mindif And Cells(rowcheck,
16) = mindif Then endwaverow = rowcheck
Next rowcheck
maxwaverow = (startwaverow + endwaverow) / 2
findph = Cells(maxwaverow, 11) 'this is the pH
End Function
Sub autofill()
Sheets(indicator).Activate
Sheets(indicator).Select
Range("A2").Select
ActiveCell.FormulaR1C1 = "='The pH Calculator'!R[9]C"
Range("A2").Select
Selection.autofill Destination:=Range("A2:A300"),
Type:=xlFillDefault
Range("B2").Select
ActiveCell.FormulaR1C1 = "='The pH Calculator'!R[4]C[-4]"
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Range("B2").Select
ActiveCell.FormulaR1C1 = "=ABS('The pH Calculator'!R[9]C)"
Range("B2").Select
Selection.autofill Destination:=Range("B2:B154"),
Type:=xlFillDefault
Range("B2:B300").Select
End Sub
Function assignindicator(indicator1)
indicator = indicator1
End Function
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Screen shot of the program’s main screen in Microsoft Excel®:
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