Indicator Dye Spectra

pH Dependence of Indicator Dye Spectra

GROUP W5

Walter Aughenbaugh Seema Patel Matthew Samuelowitz Adrianne Wenisch

Friday, May 7, 1999

1 Table of Contents

I. Abstract 3

II. Background 4 Universal Indicator vs. pH meter 4 Properties of Indicators 4 Additive Properties of Colors 4 Spectrophotometric Analysis 5 Buffered Solutions 5

III. Materials 6 Reagents 6 Equipment/Apparatus 6

IV. Methods 7 Preparation of the YUI 7 Production of the Universal Buffer 7 Production of Buffers of Set pH 8 Spectrophotometric Testing of the YUI 9 Preparation of 21 Buffers for Survey 9 Conducting the Survey 10

V. Data/Results 11 Study of Yamada 11 Survey Results 13

VI. Discussion 18 Reliability 18 Error 18 Complications with Survey 18 Complications with YUI 19 Conclusion 20

VII. Appendix 21 Absorption Spectra 21 Survey Results 23

VIII.Reference 26

2 Abstract

The main objective of the project was to evaluate a universal indicator that will prompt a color change at different units of pH. An already existing universal indicator, the Yamada Universal Indicator (YUI), was prepared and analyzed. The individual indicators that constitute YUI were separately prepared and placed into buffered solutions of whole number pH values from 4 to10, the range for which YUI was designed. Spectrophotometric analysis of the resulting test solutions shows that each of the indicators “add” at each pH value to induce the appropriate color change. A survey was conducted to test the reliability of the YUI in determining whole number pH values of test solutions for pH values of 6.0, 7.0 and 8.0  0.1,  0.2, and  0.3 based on the color of the test solutions. The results of the survey show that at least 95.0% of the participants correctly identified the nearest whole number pH of test solutions with pH’s of 5.9-6.1, 6.7-7.0, and 8.1. Thus, the YUI has only restricted uses in practical situations due to its limited reliability, as defined by the 95.0% confidence limit.

3 Background

Universal Indicator versus pH Meter

The pH of an unknown sample is generally measured by using a pH meter, although there are limited possibilities for such a measurement. A pH meter can only be used in solutions that are large enough so that the electrode can be placed into the sample. Certain microscopic samples are not nearly large enough to have their pH values measured using a pH meter. Thus, a universal indicator that denotes different pH values by a distinct color change of the sample would be beneficial to the experimenter. Such a universal indicator, the YUI, has been tested and evaluated in this experiment.

Properties of Indicators

It is necessary to understand the characteristics of different acid/base indicators since these are the constituents of a universal indicator. An indicator is a substance that marks the endpoint of a titration by a change in color. They are usually complex molecules that are weak acids or bases themselves. The indicator experiences a change in chemical structure when the acidity of the solution that contains it is changed. Thus, the pH of a sample can be measured by observing the color present when the indicator is placed in the sample solution. For instance, phenolphthalein appears colorless in acidic and neutral solutions because it does not absorb any visible light under these conditions. When base is added, the indicator loses a proton so that it has absorption in the range 550-555nm. As a result, it appears pink. Thus, phenolphthalein in its protonated form appears colorless and in its unprotonated form appears pink.

An important property of an indicator is its equivalence point, the point in titration where enough titrant has been added to react exactly with the substance being titrated. This equivalence point is denoted by a value known as pKa, which is the pH value of the solution at the equivalence point. Because each indicator has its own endpoint revealed by a color change, each indicator has its own unique pKa . The indicators are thus chosen on the basis that their equivalence points coincide as closely as possible to the endpoint of a certain titration. Thus if a solution has more than one endpoint, it would be expected to have multiple pKa‘s. One example of this type of an indicator would be thymol blue that has two pH ranges, that from 1.2-2.8 and 8.0-9.6.

Additive Property of Colors

The additive property of colors states that by combining two different adjacent primary colors on a color wheel, the secondary color between those two primary colors will result. The intensity of the newly formed secondary color may be altered by changing the amount of either of the two primary colors that were initially applied. For instance, mixing red and yellow will result in an orange color that can be altered by adjusting the amount of red or yellow. This property can be applied to pH indicators. A combination of two indicators whose pH ranges overlap can produce a color that is an addition of the two indicators’ colors at a particular pH. Using a combination of available indicators, one may develop a universal indicator that is effective for the entire pH scale.

4 Spectrophotometric Analysis

The intensity of the observed color is based upon the amount of indicator present. A method known as spectrophotometry uses radiation emission and absorbance to determine the quantity of a substance in a sample. Absorbance is directly proportional to the concentration of the absorbing material in the sample. Spectrophotometry is governed by the Beer Lambert Law, A =  C l, where A is the absorbance of the sample,  is the molar extinction coefficient, C is concentration, and l is the path length.

As was stated above, specific indicators will be added at certain concentrations to prepare the universal indicator. The concentration of the indicator is directly related to the intensity of the associated color. Because absorbance and concentration are directly related, absorbance is also related similarly to color intensity. Thus, the higher the absorbance of a particular sample, the greater the intensity of its color.

The spectrophotometer used during the lab to measure absorbance was the Spectronic Genesys 5 Spectrophotometer. This allows the wavelength to be set and the absorbance of a particular sample to be measured at this wavelength. Using the survey scan mode, it can measure the absorbance for every wavelength in a particular range, usually the visible range (350-700nm). The absorption spectra, a plot of absorbance vs. wavelength, can be constructed. The wavelength at which the peak absorbance occurs can be observed; this wavelength corresponds to the absorbed color.

Buffered Solutions

In order to test the universal indicator, buffered solutions of whole number pH values are prepared. Buffers generally contain a weak base and its salt or a weak acid and its salt. Buffered solutions need to be used because they resist changes in pH; a biological example is blood. These solutions should not experience extreme pH fluctuations when the universal indicator is dropped into them; this is crucial during experimentation.

5 Materials

Equipment/Apparatus Reagents pH meter Bromthymol blue pH buffer standards (4, 7, 10) Methyl red Magnetic stirrer Thymol blue Magnetic stirring bars Phenolphthalein 50 mL burettes Ethanol Dual burette stand 1 M NaOH Spectronic Genesys 5 Spectrophotometer 1 M HCl 1 cm cuvettes Citric Acid 1L, 500mL, 250mL, Potassium Phosphate 100mL, 50mL volumetric flasks Sodium Tetraborate P1000, P200, P20 micropipettes Tris Pipette tips Potassium Chloride Various sized beakers Deionized Water Test tubes (glass and plastic) Test tube racks Graduated cylinders 10mL automatic pipette Electronic scale Weighing dishes spatulas Wash bottles 1L plastic bottles Parafilm

6 Methods

Preparation of the YUI

YUI was produced following the YUI recipe scaled down by a factor of 2:

12.5 mg Thymol blue 31.0 mg Methyl red 125.0 mg Bromthymol blue 250.0 mg Phenolphthalein 250.0 mL 95% Ethanol Neutralize with 1.0 M NaOH, dilute to 500.0 mL

A procedure was not given with the recipe, so the following procedure was agreed upon:

1. Add 100mL 95% ethanol to a small wash bottle. 2. Add 150mL 95% ethanol to a 500mL beaker. 3. Weigh out 12.5mg thymol blue in a weighing boat, wash thymol blue into the 500mL beaker using less than 25mL of the 95% ethanol in the small wash bottle. 4. Repeat step 3 for methyl red, bromthymol blue and phenolphthalein. 5. Add unused 95% ethanol in the small wash bottle to the beaker. 6. Mix resulting solution well using magnetic stirrer. 7. Add 100mL deionized water to solution; mix well again using magnetic stirrer. 8. Insert probe of calibrated pH meter into solution and neutralize solution with 0.1M NaOH. 9. Add deionized water to bring total volume of solution to 500mL using 500mL volumetric flask.

Production of the Universal Buffer

The universal buffer was produced according to the following recipe:

Make a solution that is: 0.1 M in citric acid (21.01g/L) 0.1 M in potassium phosphate (13.61g/L) 0.1 M in sodium tetraborate (19.07g/L) 0.1 M in Tris (12.11g/L) 0.1 M in potassium chloride (7.46g/L)

The solution is “universal” in the sense that specific amounts of HCl or NaOH were added to the universal buffer to make a buffer with a pH that is dependent on the amount of HCl or NaOH added. The universal buffer recipe was adjusted due to the fact that citric acid and sodium tetraborate were supplied as citric acid•H2O and sodium tetraborate•10H2O. The corrected recipe is given in Table 1.

7 Mass (mg) Chemical 22.81 Citric acid • H2O 13.61 Potassium phosphate 28.07 Sodium tetraborate • 10H2O 12.11 Tris 7.46 Potassium chloride Table 1: The corrected masses of reagents used in preparation of 1.0L universal buffer

These reagents were placed into a volumetric flask of 1.0L. Enough deionized water was added to make a total of 1.0L of the universal buffer. This process was repeated so that a total of 2.0L of the universal buffer was prepared.

Production of Buffers of Set pH

In order to make a buffer of certain pH, the buffer recipe calls for the addition of HCl or NaOH until the desired pH is achieved and then a ¼ dilution of the solution with deionized water. Small volumes of HCl and NaOH were delivered through burets. The pH was monitored with a pH meter. The buffer recipe suggested to use X mL of HCl or NaOH to set the pH of the buffer according to the following chart:

pH X mL 0.4 M HCl or NaOH 4 10.0 HCl 5 0.4 NaOH 6 11.4 NaOH 7 22.4 NaOH 8 33.2 NaOH 9 46.2 NaOH 10 59.0 NaOH Table 2: Amounts of 0.4 M HCl and NaOH to be added to each buffered solution to obtain a specific pH

The information on this chart did not yield the exact desired pH values. The given amounts did provide a sufficient range of volumes to be added; however, small amounts of HCl and NaOH needed to be added until the desired pH was attained with an accuracy of 0.020. Buffers of whole number pH values were then made in accordance with the following procedure:

1. Increase volume of 250.0mL of universal buffer to 900.0mL with deionized water. 2. Add HCl or NaOH to adjust pH to desired whole number pH value. 3. Increase volume to 1.0L with deionized water. 4. This procedure was carried out 7 times: once for each pH value from 4 to 10, inclusive.

The majority of deionized water was added prior to the addition of acid or base in the actual experimental procedure, as indicated in step 1. This was done due to the fact that a four-fold dilution would significantly change the pH of the universal buffer-HCl/NaOH mixture. The pH change is less significant when a 900.0mL solution with set pH is

8 increased to a volume of 1.0L with deionized water than the pH change that occurs when a 250.0mL solution is increased to a volume of 1.0L. However, this dilution still proved to change the pH of the solutions; the pH’s of all of the buffered solutions were therefore readjusted the day of the survey.

Buffered solutions of pH 6.0  0.1,  0.2,  0.3, pH 7.0  0.1,  0.2,  0.3 and pH 8.0  0.1,  0.2,  0.3 were then made according to the same general procedure that was used to make buffers of whole number pH values. Only three pH values were considered due to time constraints. The pH values of 6, 7 and 8 were chosen to be studied due to the fact that many biological systems have pH values that lie within the 6-8 pH range. In the preparation of these buffers, 10.0mL of universal buffer was used to make each buffer. The ¼ dilution factor dictated that the final volume of each buffer be 40.0mL.

Spectrophotometric Testing of the YUI

200L of YUI was added to 5.0mL aliquots of each of the 7 buffered solutions. 2.5mL aliquots of each of the 7 resulting solutions were placed in spectrophotometer cuvettes. A survey scan was then run on each of the seven test solutions.

After these preliminary scans were made, four solutions were made according to the YUI recipe; however, only one of the four indicators used in YUI was included in each of the four solutions. A different indicator was included in each of the four solutions. 200L of each of these 4 solutions was separately added to 5.0mL aliquots of each of the 7 whole number pH buffered solutions. This resulted in a total of 28 total solutions. 2.5mL aliquots of each of the 28 resulting solutions were placed in spectrophotometer cuvettes. A survey scan was then run on each of the 28 test solutions.

Preparation of 21 Buffers for Survey

On the day of the survey, the pH’s of each of the 21 buffered solutions were readjusted using HCl and NaOH.

6.0mL aliquots of each of the 21 buffered were then placed in 10mL test tubes. 250L aliquots of YUI were then added to each of the test tubes. The resulting test tubes were then mixed well. The test tubes were labeled A-U randomly; i.e., there was no relation between the color of the test solution and its letter. The test tubes were placed in a test tube rack in alphabetical order.

9 Conducting the Survey

Participants were given a survey sheet and the YUI pH/color key seen in Table 3.

pH Color 4 Red 5 Orange 6 Yellow 7 Green 8 Blue 9 Indigo 10 Violet Table 3: The pH/color key given to participants in the study and provided with the original YUI recipe

Participants were not given a color strip; they were given simply the YUI pH/color key. The participants were asked to determine the whole number pH value of all 21 solutions based on the color of each solution and to record this whole number pH value on the survey sheet. It was made clear that the participants were to look directly down the test tube. Once it was clear that all participants understood what was asked of them, they were presented the tubes in alphabetical order. A white card was held under each of the tubes. Participants were permitted to look down each of these tubes as long as they needed to; the time required for each participant to look down each of the tubes generally did not exceed five seconds.

A total of 70 people participated in the survey. The survey was conducted in groups of 2 to 5 participants; a few participants were tested individually. The first 30 participants were surveyed in the Towne computer labs and the last 40 were surveyed in Hill House. The locations where the survey was taken were carefully selected so that artificial lighting was ample and very similar across all survey locations.

10 Data/Results

Study of Yamada

During the second week of testing, the aim was to conduct trials to study YUI in greater detail. Four single indicator YUI solutions, each of which contained one of the four indicators, were prepared. Each of these was placed into the 7 buffered solutions from pH 4 to 10 to yield a total of twenty-eight samples. Survey scans from wavelength 300nm to 700nm were run on all the samples. The pH 7 buffered solution containing the various constituent YUI indicators is analyzed below.

0.35 0.3 Bromthymol Blue 0.25 Phenolphthalein e

c Methyl Red n

a 0.2 b

r Thymol Blue o

s 0.15 b A 0.1 0.05 0 300 400 500 600 700 Wavelength (nm)

Figure 1: A survey scan of the 4 buffered solutions of pH 7 each containing bromthymol blue, phenolphthalein, methyl red, and thymol blue

The plot shown in Figure 1 shows that at pH 7, only two of the four indicators that compose YUI are active. The absorbances for phenolphthalein and thymol blue are zero from wavelength 300nm to 700nm. This may be attributed to the fact that the range for phenolphthalein is 8.2-10.0 and the ranges for thymol blue are 1.2-2.8 and 8.0-9.6. Thus, the endpoints of these two particular indicators do not include pH 7. Phenolphthalein and thymol blue do not absorb any light for this pH value.

However, there are definite spectra at pH 7 for bromthymol blue and methyl red. Bromthymol blue’s indicator range is 6.0-7.6 and methyl red’s indicator range is 4.8-6.0. Thus, these two 0.5indicators have working ranges419nm that are 617nmeffective at pH 7. The spectras of bromthymol 0.45blue and methyl red add to give the final spectra for pH 7. Shown below in Figure 2 is a0.4 spectra that sums the absorbances at every wavelength of the plot in Figure 1. 0.35 e c

n 0.3 a b

r 0.25 o

s 0.2 b

A 0.15 0.1 0.05 0 300 400 11 500 600 700 Wavelength (nm) Figure 2: A survey scan of the individual indicators from Figure 1 “added”

Figure 2 is very similar to the spectra of the pH 7 sample that contains the original YUI solution. This survey scan was conducted on the first day of testing and can be seen below in Figure 3. Both plots shown in Figure 2 and Figure 3 have two peaks at the same location, 419nm and 617nm. If superimposed on each other, the graphs would basically be identical. However, the absorbance values are different. For the plot shown in Figure 2, the absorbances are much higher than those seen in Figure 3. This can be attributed to the fact that the indicators were more concentrated in the separately prepared indicator solutions. Thus, the absorbances are greater as a result. Besides the magnitude of the absorbances, the absorption spectra of the separate indicators “added” and the absorption spectra of YUI are virtually identical.

419nm 617nm 0.18 0.16 0.14

e 0.12 c n

a 0.1 b r

o 0.08 s b 0.06 A 0.04 0.02 0 300 400 500 600 700 Wavelength (nm)

Figure 3: A survey scan of pH 7 buffered solution containing YUI

The similarity observed between the two survey scans shown in Figure 2 and Figure 3 indicates that the individual indicators that comprise YUI actually add together at each

12 pH value. The addition of the particular indicators explains the resulting color. For instance, at pH 7, methyl red and bromthymol blue are active. The end of the methyl red range is yellow while the bromthymol blue range is blue. Using the additive property of colors, it can be asserted that the yellow and blue actually combine to result in a green color at this pH value.

The spectra for each of the 7 buffered solutions can be analyzed. For each pH value, different indicators work depending on the indicator ranges. Similar survey scans were taken for all other pH values, and these were analyzed. The indicators that interact at each pH value can be seen in Table 4 below.

pH Indicators Involved 4 bromthymol blue, methyl red, thymol blue 5 bromthymol blue, methyl red 6 bromthymol blue, methyl red 7 bromthymol blue, methyl red 8 bromthymol blue, methyl red 9 bromthymol blue, methyl red, phenolphthalein, thymol blue 10 bromthymol blue, methyl red, phenolphthalein, thymol blue Table 4: Indicators that interact to give color changes for each buffered solution

The involvement of the indicators at each pH value may be explained by the indicators’ working ranges. Bromthymol blue and methyl red have a combined range of 4.0 to 7.6, so only these two indicators act between pH’s of 4 and 8. They do, however, continue to act over the entire range between pH 4 to 10. Thymol blue acts at the low end at pH 4 and at the high end at pH’s of 9 and 10 because of its two ranges, one at the low end of the scale (1.2-2.8) and one at the high end of the scale (8.0-9.6). Finally, phenolphthalein makes an appearance at pH’s 9 and 10 because its range is 8.2-10.0.

Survey Results

The histograms below represent the percentage of participants that responded correctly when asked to identify the pH of a solution to the nearest whole number. A 95.0% confidence limit was applied to the results from each pH value in order to determine the reliability in estimating the pH of a given solution to the nearest whole number. If more than 95.0% of the participants correctly estimated the pH of a given test solution to the nearest whole number, YUI was deemed reliable in estimating the whole number pH value. Ranges over which YUI is reliable to the nearest whole number pH were determined by identifying the consecutive pH values with percent correct responses greater than 95.0%.

The results for the test solutions with pH’s ranging from 5.7 – 6.3 can be seen below in Figure 4. The correct response was 6, but as the graph shows, not all of the participants were able to estimate this accurately. The indicator is reliable at values of 6.0  0.1 based on the 95.0% confidence limit. Due to the fact that 5.7, 5.8, 6.2, and 6.3 responses were

13 below the 95.0% confidence limit, the accuracy could not be considered greater than 0.1.

Yellow

g 98.6% 98.6%

n 97.1% i 100% 92.9% d 87.1% n

o 77.1%

p 80% s e y l t R

c 60% s e t r c r e o j 40% C b 30.0% u S

f 20% o

% 0% 5.7 5.8 5.9 6.0 6.1 6.2 6.3 pH Values

Figure 4: Percent of correct responses given for test solutions with pH values ranging from 5.7 – 6.3

There is a significant drop in correct responses between pH values of 6.2 and 6.3. This can be explained by observing at about what pH range the color changes occur. The transition from yellow to green began at a pH value that was clearly closer to 6.0 than 7.0 as shown by Figure 4. The low percentage of correct responses at the 6.3 pH value is a result of this early transition.

The reliability ranges of YUI of pH values from 6.7-7.3 can be seen on the histogram in Figure 5. The indicator is reliable for the 6.7 – 7.0 range based on the 95.0% confidence limit. However, the accuracy does not extend beyond 7.0, so an accuracy of 0.3 cannot be placed on this pH unit. Thus, only a range of 6.7-7.0 can be estimated.

98.6% 100.0% 98.6% 100.0% 92.9%

100% g n i 80.0%

d 77.1%

n 80% o p s y l e t 60% R c

e s r t r c o

e 40% j C b u S

f 20% o

% 0% 6.7 6.8 6.9 7.0 7.1 7.2 7.3 pH Values

14 This reliability range, which is shifted down the pH scale from 7.0, can be attributed to the early change from yellow to green.

The YUI is only reliable at 8.1 for the 8.0  0.3 pH range as seen in Figure 6. Less than 95% of the participants were able to correctly identify the color of the rest of the test solutions with pH 7.7 - 8.3. Even the pH 8.0 sample did not meet the 95.0% confidence limit in this test; its confidence limit was 91.4%.

95.7% 100% 91.4% 92.9% g 87.1% n i 80.0%

d 77.1% n 80% 70.0% o p s y l e t 60% R c

e s r t r c o

e 40% j C b u S

f 20% o

% 0% 7.7 7.8 7.9 8.0 8.1 8.2 8.3

pH Value s

The histograms in Figure 7, Figure 8, and Figure 9 below show the distribution of responses for a particular color. The responses include the responses for all seven of the pH values (a whole number pH 0.3). The largest bar seen in each graph is the number of participants that correctly estimated the whole number pH value of the seven test solutions at 6, 7, and 8. Those participants who estimated incorrectly make up the smaller bars to the left and right of the larger bar.

When presented with a test solution of pH 6.0  0.3 that was supposed to be estimated as yellow, only 83.1% of the participants were able to estimate the correct pH whole number pH value. This is shown in Figure 7 below. The fact that 12.0% of the participants that identified what should have been identified as a yellow test solution as green is further evidence for the early yellow-to-green transition.

15 Yellow 100% 83.1% s e

s 80% n o p

s 60% e R

t 40% n e c r 12.0% e 20%

P 4.7% 0.0% 0.2% 0.0% 0% 5.0 6.0 7.0 8.0 9.0 10.0 pH Values

Figure 7: Distribution of responses when presented with test solutions ranging from 5.7 – 6.3

A much greater percentage 92.4%, of the participants was able to correctly identify green. This can be attributed to the early pH range over which YUI appears green as seen in Figure 8.

100% 92.4% s

e 80% s n o

p 60% s e R

t 40% n e c r

e 20% 6.5% P 0.0% 0.2% 0.6% 0.2% 0% 5.0 6.0 7.0 8.0 9.0 10.0 pH Values

Figure 8: Distribution of responses when presented with test solutions ranging from 6.7–7.3

The blue range data was as confounded as the yellow range data; this can be observed in Figure 9. Only 84.9% of the participants were able to correctly estimate a pH value of 8 when presented the test solutions ranging from 7.7 – 8.3.

16 Blue

100% 84.9%

s 80% e s n o

p 60% s e R

t 40% n e c r

e 20% 8.4% 6.7% P 0.0% 0.0% 0.0% 0% 5.0 6.0 7.0 8.0 9.0 10.0 pH value

Figure 9: Distribution of responses when presented with test solutions ranging from 7.7 – 8.3

Shown below in Table 5 is the range of values for pH 6.0, 7.0, and 8.0 for which YUI may be considered reliable.

pH value Reliability 6.0 5.9-6.1 7.0 6.7-7.0 8.0 8.1 Table 5: Reliability results for three pH units of the survey testing for YUI

17 Discussion

Reliability

The results of the survey show that YUI is reliable over specific pH ranges (5.9 – 6.1, 6.7 – 7.0, 8.1) for assigning whole number pH values of 6, 7, and 8. If a sample has a pH of 5.9, 6.0, or 6.1, an individual may identify that solution as yellow with 95.0% confidence, and therefore, may estimate the pH of that solution to the nearest whole number as pH 6. Similarly, for a sample with a pH of 6.7, 6.8, 6.9, or 7.0 the sample will be observed as green with 95.0% confidence, so a pH of 7 can be assigned. Finally, a sample with a pH of 8.1 will be seen as blue with 95.0% confidence, so the pH of the solution is determined to the nearest whole number as 8.

Due to the limited reliability of YUI, its applications are limited. For instance, it is not practical in laboratory situations that demand a very accurate, precise pH measurement. Thus, it can only be applied when rough estimates of pH are required. The main application of the YUI is in situations where only approximate differences in pH need to be determined. In other cases, a particular sample needs to be identified as an acid or a base. Differences in color using the YUI could reveal various samples as acidic or basic. However, because YUI is not very accurate, it cannot replace the pH meter in practical lab usage.

There were many sources of error in the experimentation that was conducted during the course of testing. The experimentation called for the use of micropipettes, volumetric flasks, and the electronic scale. Each of these has an uncertainty associated with it that can be specifically quantified. However, it would be rather difficult to determine the effects of these systematic errors on the results. The results consisted of the YUI solution and the color changes that were created in the test solutions. To some degree, the errors that resulted from the use of these items caused deviations in the shade of the colors due to effects on the concentrations, etc.

Another major source of error is the pH meter. The survey that was conducted demanded a high level of accuracy, 0.1. Participants were shown solutions that were prepared to be 0.1 pH units different. Some, but not all, of the colors were distinctly different in pH increments of 0.1, and these colors were directly observed by survey participants. Thus, errors in the pH readings could have caused a great deal of error in the colors and the perceptions of the colors. It was found that the values read by the pH meter of a single sample fluctuated greatly, due to error of the instrument.

Complications of the Survey

When administering the survey, major variable conditions were kept constant. One condition that was not kept constant was the source of light available at the time of each survey. Because different rooms were used for testing, it is likely that the type of light

18 source, and the intensity of that light, affected the participants’ color perception, and therefore the results of the study. This could have been avoided had one room been used; because of lack of resources, this was not an option.

Another major complication that was face in conducting this survey was the “indigo dilemma.” This “dilemma” was that people are not familiar with the color indigo. It was explained to them that it is a bluish-purple color. However, some were still rather confused, and felt obligated to assign the pH value associated to indigo for a certain number of samples. In this way, they tried to randomize their answers. As a result of this problem, there were fewer correct responses in mainly the blue range, 7.7-8.3. A range may not be assigned due to the poor confidence limits for this pH value. The fact that the sample of pH 8.0 did not even meet 95% confidence limits may be partially attributed to the indigo dilemma and randomized responses.

Complications of the YUI

There is a significant drop in the percentage of participants that responded correctly between 6.2 and 6.3 due to the transition from yellow to green that begins to occur at 6.3. The relatively large amount of bromthymol blue, 125mg, that is used in YUI compared to the methyl red amount, 12.5mg, may account for this early transition. As bromthymol blue becomes active and turns blue, just above pH 6, it mixes with the already active methyl red, which is yellow at this point. As the pH increases to 6.3, the methyl red becomes inactive, and bromthymol blue is now dominant. Because of the additive properties of the YUI, the yellow of the now inactive methyl red adds with the active blue of bromthymol blue to form green. It is misleading to see green at a pH value so close to 6 when yellow is expected: this is one drawback to YUI.

Estimating pH to two significant figures is difficult because of the varying increments of color intensity across the pH scale. For the pH values between 6.1 and 6.3, participants tended to respond with pH value 7 at a high frequency. This confirms that there was an early transition from yellow to green. A decrease in the percentage of correct responses was not observed in the responses between 7.1-7.3 and 8.1-8.3. This implies that the increments of indicator color intensity were not uniform for the pH units of 6, 7, and 8. This fact is evidence that intensity is not correlated to 0.1 pH units universally throughout the pH scale.

To counteract this problem, survey scans of these samples could have been conducted. YUI could have been placed in buffered solutions from pH 6.0 to 7.0 in increments of 0.1 units. The peak absorbances could have been recorded on all ten absorption spectra. Absorbance is a measure of the concentration of indicator, which is a measure of the intensity of color. Using these peak absorbances, a plot of absorbance vs. pH would be constructed. The vertical difference between each of the ten data points would be proportional to the color intensity changes between 0.1 pH units. This could be conducted for every pH unit between 4.0 and 10.0 to give the intensity changes that could then be used to better estimate pH values to more than one significant figure based on color intensity.

19 It is also likely that if another solution of YUI were prepared, different color changes would have been observed. Only one 500mL sample was prepared very early on during the testing process. Errors in massing out the individual indicators at this time were probable. Thus, the resulting colors of this YUI solution may not have been the “correct” ones. In order to have successfully determined the appropriate colors, several samples of YUI should have been prepared and tested. Slight adjustments could have also been made to the recipe in terms of indicator masses to adjust the intensity of the colors. Unfortunately, time did not allow for this depth of testing.

Conclusion

The YUI is useful for making pH estimations of unknown solutions; thus, it has only limited reliability. It serves no purpose in practical laboratory situations where accurate pH values are required. The utility of universal indicators in real lab situations is questionable. When accurate measurements are needed, the use of the pH meter is recommended. An interesting future experiment would be one that tests the reliability of YUI for the other whole number pH values, i.e. 4, 5, 9, and 10. A future survey could be conducted that asks participants to determine the pH of test solutions to the nearest half- numbered pH values. This would provide a better understanding of the YUI’s reliabilty.

20 Appendix

Absorption Spectra

0.15 e c 0.1 n a b r o

s 0.05 b A

0 300 400 500 600 700 800 -0.05 Wavelength (nm)

Figure A: A survey scan of buffered solution of pH 4 containing YUI

0.15 e c 0.1 n a b r o

s 0.05 b A

0 300 400 500 600 700 800 -0.05 Wavelength (nm)

Figure B: A survey scan of buffered solution of pH 5 containing YUI

0.15 e c n

a 0.12 b r

o 0.09 s b

A 0.06

0 300 400 500 600 700 800 Wavelength (nm)

Figure C: A survey scan of buffered solution of pH 6 containing YUI

21 0.3

e 0.2 c n a b

r 0.15 o s b 0.1 A

0 300 400 500 600 700 800 Wavelength (nm)

Figure D: A survey scan of buffered solution of pH 8 containing YUI

0.45 0.4 0.35

e 0.3 c n

a 0.25 b r

o 0.2 s b 0.15 A 0.1 0.05 0 300 400 500 600 700 800 Wavelength (nm)

Figure E: A survey scan of buffered solution of pH 9 containing YUI

1 0.9 0.8 0.7 e c

n 0.6 a b

r 0.5 o

s 0.4 b

A 0.3 0.2 0.1 0 300 400 500 600 700 800 Wavelength (nm)

Figure F: A survey scan of buffered solution of pH 10 containing YUI

22 Survey Results

98.57% 100.00% 91.43%

g 100% n i d n

o 80% p s e R y

l t s 60% t c n e r a r p i o

c 40% i C t r a P

f 20% o

% 0% 6 7 8 pH Values

Figure G: Percentage of participants that responded correctly when presented test solutions of whole number pH

98.57% 98.57% 97.14% 95.71%

g 100% 92.86% n i d

n 80.00% o 80% p s e

s 60% t c n e r a r p i o

c 40% i C t r a P

f 20% o

% 0% 5.9 6.1 6.9 7.1 7.9 8.1 pH Values

Figure H: Percentage of participants that responded correctly when presented test solutions of whole number pH 0.1

23 100.0% 100% 92.9% 87.1% 87.1%

g 77.1% 77.1% n i 80% d n o p s y e

l 60% t R

c s e t r n r a o

p 40% C i c i t r a P

f 20% o

% 0% 5.8 6.2 6.8 7.2 7.8 8.2 pH Values

Figure I: Percentage of participants that responded correctly when presented test solutions of whole number pH 0.2

98.57% 100% 92.86%

g 78.57% 78.57% n

d 70.00% n o p

l 60% t R

c s e t r n r a o

p 40% C i 30.00% c i t r a P

f 20% o

% 0% 5.7 6.3 6.7 7.3 7.7 8.3 pH Values

Figure J: Percentage of participants that responded correctly when presented test solutions of whole number pH 0.3

e 100% s n o

p 80% s e R

60% t c e e r 40% o C t 20% n e c r e 0% P

pH Values

Figure K: Percent of correct responses given for test solutions with pH values ranging from 5.7 – 8.3

25 100%

0% 5.7 6.3 6.7 7.3 7.7 8.3

References http://chem.csustan.edu/chemistry/stkrm/recipes/Recipes-Yamada.htm

Litt, Mitchell, BE 210 Bioengineering Laboratory II Manual, Spring 1999

Perrin, D.D., and Boyd Dempsey, Buffers for pH and Metal Ion Control, Chapman and Hall, London, 1974.

Zumdahl, Steven, Chemical Principles, D.C. Heath and Company, Lexington, 1995.