Determination of pKa of Bromocresol Green using pH Measurement and

Absorption Spectroscopy

Name: Amanda Wang

Lab Partner: Shu Yee Lau

3/8/18

Section 008
 1

Introduction

The use of pH indicators is important in the detection of the end-point of titration. The use

of indicators is important because it could be used to detect change in pH quickly by visuals from

the change in the color. One example is Litmus. Litmus paper is commonly used to determine the

acidity or basicity of a solution. This is important because there are a lot of biological processes

that could only take place in a certain pH range. Litmus paper is a quick way to determine the pH

change visually, just like any pH indicator dye. [1]

This experiment uses Bromocresol Green (BCG) also known as 3', 3", 5', 5"-tetrabromo-

m-cresolsulfonphthalein. BCG is a common indicator dye used in titration. Like many indicators,
[2]
BCG is a weak acid. At acidic conditions, it is yellow, while at basic condition it is blue. The

structure of BCG has an SO3- group, but can be disregarded as it is a sulfonic acid. BCG is

dissociated into a basic form that is represented by a resonance structures.

 

In short, the acidic form can be written as HX-, and the equation can be written as 𝐻𝑋 − + 𝐻2 𝑂 ↔

𝑋 2− + 𝐻3 𝑂+ . Where 𝐻𝑋 − is yellow in color and 𝑋 2− is blue.

One research that had been done was to compare the acidity constants of Neutral Red and

Bromocresol Green using solution scanometric method and comparing to spectrophotometry.

 2

Results of this experiment showed that scanometric method could also obtain results very similar

to spectrophotometry. Scanometric method involves a handheld scanner and a PC, and it measures

the intensity of the solution color. This method was said to be efficient because of the short

response time, and simultaneous tests. This method could also work in opaque solutions. However,

this method could also be less uniform in the membrane, and could consequently affect the result.
[3]
This experiment is relevant because the result of the experiment shows the closeness to literature

in comparison to using the conventional method of spectrophotometry.

This experiment uses different techniques such as UV-Vis spectrophotometry, calculations

using Beer’s Law, as well as Henderson-Hasselbalch equation. UV-Vis spectrophotometry is used

to measure absorbance of molecules in the visible range as they transition from different energy

levels. Beer’s Law shows the linear relationship of absorbance and concentration of the species

absorbed. Lastly, the Henderson-Hasselbalch equation shows the relationship of pH and pKa to

measure acidity of weak acids. pKa is the acid dissociation constant. The lower the pKa, the better

the ability of donating proton, which means the stronger the acid. The value of pKa is important

because there are many indicators to choose from. Different indicators have different colors to
[4]
indicate change in pH, and with different pH range. The ionic strength in the experiment was

also to be kept constant so that the value of the dissociation constant of BCG (Ka) does not change.

The purpose of this experiment is to use absorption spectrophotometry and pH

measurements to calculate the pKa of Bromocresol Green. The actual value of pKa is 4.7. This

experiment expects to obtain a pKa close to the actual value including the uncertainty. In this

experiment, acidic and basic solutions, and the buffers of BCG were made. The pH and

Absorbance were then measured to obtain the pKa using two different methods. One using the pH

and Absorbance, while the other uses pH and the logarithm of the ratio of the acidic and basic
 3

concentrations. The result obtained using the two methods were 4.74 and 4.6655  0.01051

respectively, which was closed to the literature value of 4.7.

Experimental

25.0  0.35 mL of 0.0400M sodium acetate, 25.0  0.35 mL of stock BCG solution, and 9

 0.2 mL of 1.00 M potassium chloride were obtained using graduated cylinder, and added to 100

mL volumetric flask. This solution was then diluted to the mark with deionized water. This solution

is the basic BCG solution.

25.0  0.35 mL of 0.04 M stock acetic acid solution, 25.0  0.35 mL stock BCG solution,

and 10  0.2 mL of potassium chloride were obtained using graduated cylinder, and added to

another 100 mL volumetric flask. This solution was diluted to the mark with deionized water. This

is the acidic BCG solution.

The acidic and basic BCG solution prepared were used to fill two burets. Each buret is

rinsed with each solution. Five buffer solutions were prepared by dispensing from acidic and basic

solutions with a total of 20 mL. Beaker 1 was filled with 5  0.02 mL of acidic solution and 15 

0.02 mL of basic solution; Beaker 2 was filled with 6  0.02 mL acidic solution and 14  0.02 mL

basic solution; Beaker 3 was filled with 10  0.02 mL of each acidic and basic solution; Beaker 4

was filled with 14  0.02mL of acidic solution and 6  0.02 mL of basic solution; and Beaker 5

was filled with 15  0.02 mL acidic solution and 5  0.02 mL basic solution.

The pH of the five buffer solutions were measured using a Thermo Electron Corporation

Orion 4 Star pH-ISE Benchtop. The pH probe was calibrated using a general red pH 4 buffer

solution, and a blue buffer solution of pH 10 manufactured by Fisher Chemical. The pH probe

should be immersed in distilled water to prevent the membrane from drying up. To take the first
 4

calibration of the red buffer solution, the pH probe was set to calibration mode. The electrode was

wiped using Kim wipe, without touching the tip of the membrane and was placed in the solution

of interest without touching the glass container. The pH icon flash as the reading was taken. The

reading was then recorded when the flashing stops. The pH probe was rinsed before taking the

next measurement. This process was repeated again with the blue buffer solution of pH 10. The

measurements of the five buffers were obtained by changing into measurement mode, and the

process was repeated.

The absorbance of the five buffers, and the acid and base calibration solution were

measured using Agilent Technologies Carry 8454 UV-Vis HP diode-array spectrophotometer. A

water blank was used to rinse the cuvet, and the absorbance was recorded at 453 nm and 615 nm

using the software, 8454 UV-Visible System. The absorbance of deionized water was then taken.

This measurement was taken into account in the software to take the difference of the measured

absorbance of the acidic, basic, and the five buffer solutions.

Results

Solution Volume added

and Buffer Acidic Basic pH
( 0.02 mL) ( 0.02 mL)
1 5 15 5.12
2 6 14 4.99
3 10 10 4.63
4 14 6 4.28
5 15 5 4.19
100% Acid 3.25
100% Base 6.67
 5

Table 1 Volume of each solution added and pH of buffer solutions recorded after measuring

with pH probe.

Figure 1 Absorption of acidic solution, basic solution, and five buffer solutions measured using

Agilent Technologies Carry 8545 UV-Vis

Using values from Table 1 and Figure 1, Table 2 can be tabulated as shown.

Subsequently, using the values in Table 2, Figure 2 is plotted.

Solution
Absorbance Absorbance
and Buffer pH
(453 nm) (615nm)

1 5.12 0.0759220 0.3705500

2 4.99 0.0932810 0.3360800
 6

3 4.63 0.1290700 0.2558000

4 4.28 0.1580200 0.1535600
5 4.19 0.1792400 0.1416900
Acidic 3.25 0.2205200 0.0383680
Basic 6.67 0.0292680 0.4989200
Table 2 pH and Absorbance at 453nm and 615nm

0.6000000

0.5000000

0.4000000
Absorbance

0.3000000

0.2000000

0.1000000

0.0000000
3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
pH
Abs (453nm) Abs (615nm)

Figure 2 Graph of pH vs Absorbance at 453nm and 615nm, where pH is in the x-axis, and the

Absorbance is in the y-axis.

To calculate the pH from Figure 2, the value of the inflection point must be obtained to

find the corresponding pH. 𝐴𝜆2 corresponds to the Absorbance at 615nm and 𝐴𝜆1 corresponds to

the Absorbance at 453nm.

𝐴𝜆2,𝑏𝑎𝑠𝑖𝑐 = 0.498920

𝐴𝜆1,𝑏𝑎𝑠𝑖𝑐 = 0.0292680

𝐴𝜆2,𝑏𝑎𝑠𝑖𝑐 − 𝐴𝜆1,𝑏𝑎𝑠𝑖𝑐 0.498920 − 0.0292680

= = 0.234826 (1)
2 2
 7

From this inflection point value of 0.234826, the corresponding pH observed from Figure 2 is 4.74.

This value is the same as the value of the pKa. This is because this is calculated at the point where

the concentrations of the acidic and basic species are equal, so the logarithm of the ratio of the two

concentrations will equal to zero, and pH will equal pKa using the Henderson-Hasselbalch

equation below.

[𝐼𝑛− ]
𝑝𝐻 = 𝑝𝐾𝑎 + log ( ) (2)
[𝐻𝐼𝑛]

The corrected absorbance was calculated by subtracting the background absorbance

obtained using the absorbance at 615nm of the acidic BCG solution and absorbance at 453nm of

the basic BCG solution, which was 0.038368 and 0.029268 respectively. The values were tabulated

in Table 3.

Solution
Corrected Corrected
and pH
Abs (453 nm) Abs (615 nm)
Buffer
1 5.12 0.0466540 0.3321820
2 4.99 0.0640130 0.2977120
3 4.63 0.0998020 0.2174320
4 4.28 0.1287520 0.1151920
5 4.19 0.1499720 0.1033220
Acidic 3.25 0.1912520 0.0000000
Basic 6.67 0.0000000 0.4605520
Table 3 pH and Corrected Absorbance at 453nm and 615nm

With the values of the corrected absorbance, the ratio of the concentration of acid and

base, and the logarithm of the ratio can be obtained using the equation below.

[𝐼𝑛− ] 𝐴𝜆2 /𝐴𝜆2,𝑏𝑎𝑠𝑖𝑐 𝐴𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑𝜆2 × 𝐴𝜆1,𝑎𝑐𝑖𝑑𝑖𝑐

= = (3)
[𝐻𝐼𝑛] 𝐴𝜆1 /𝐴𝜆1,𝑎𝑐𝑖𝑑𝑖𝑐 𝐴𝜆2,𝑏𝑎𝑠𝑖𝑐 × 𝐴𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝜆1
 8

Plugging in values for the first buffer solution,

[𝐼𝑛− ] 0.3321820 × 0.1912520

= = 3.1470546
[𝐻𝐼𝑛] 0.4605520 × 0.0466540

[𝐼𝑛− ]
log = log 3.1470546 = 0.4979043
[𝐻𝐼𝑛]

The values calculated using equation 3 were then recorded below in Table 5.

Solution and
pH [In-]/[HIn] Log([In-]/[HIn])
Buffer
1 5.12 3.1470546 0.4979043
2 4.99 2.0556313 0.3129452
3 4.63 0.9629446 -0.0163987
4 4.28 0.3954444 -0.4029146
5 4.19 0.3045087 -0.5164003
Table 4 pH and values of [In-]/[HIn]
[𝐼𝑛− ]
Using the values calculated in Table 5, the graph of pH vs log ([𝐻𝐼𝑛]) can be obtained as

shown below in Figure 6. A linear fit trendline was used to obtain the equation of the line y =

0.9409x + 4.6655. The y-intercept corresponds to the pKa, which is 4.6655. Using the LINEST

function on Excel, the uncertainty of the pKa is found to be 0.01051. Hence, the value of the pKa

obtained using this method is 4.66550  0.01051.

 9

5.2

5.0

y = 0.9409x + 4.6655 4.8

R² = 0.99759
pH

4.6

4.4

4.2

4.0
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
log([In-]/[HIn])

Figure 5 Graph of pH vs log([In-]/[HIn]) where pH is in the y-axis, and log([In-]/[HIn]) is in the

x- axis

Discussion

The value of pKa is important because there are many indicators to choose from.

Different indicators have different colors to indicate change in pH, and with different pH range.

In this experiment, there are two ways to obtain the values of pKa. The first method uses the

measured pH of the buffers and the acidic and basic solutions, using the pH probe, as well as the

measured absorbance. The result was plot on Figure 2. The inflection point was calculated, and

the pKa could be obtained from the result of the inflection point. The value of pH obtained from

Figure 2 and Equation 1 was found to be 4.74. This value is also equal to the pKa, as shown earlier

in Results. The known value for the pKa of BCG is 4.7. [5] The percent error of the experimental

pKa result can be calculated relative to the known value of 4.7 using the equation below.

|𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑣𝑎𝑙𝑢𝑒 − 𝑎𝑐𝑡𝑢𝑎𝑙 𝑣𝑎𝑙𝑢𝑒|

% 𝑒𝑟𝑟𝑜𝑟 = × 100 (4)
𝑎𝑐𝑡𝑢𝑎𝑙 𝑣𝑎𝑙𝑢𝑒
 10

The percent error was calculated to be 0.9% and this result shows that the percent error is small

and is acceptable.

The second method was obtained using the Henderson-Hasselbalch equation and Beer’s

Law to obtain a linear relationship of the pH and the logarithm of the ratio of the concentrations

of acid and base. In this method, the absorbance at 615nm is assumed to be the absorbance for the

basic form, while the absorbance at 453nm is assumed to be the absorbance for the acidic form.

So, the absorbance at both 453nm and 615nm had to be corrected to take into account the effects

of other species at each wavelength. Using the corrected absorbance, the ratio of the acid and base

can be calculated by deriving from the Henderson-Hasselbalch equation and Beer’s Law. The

logarithm of this relationship and the corresponding pH was plot on Figure 6. The value of the pKa

obtained from Figure 6 was found to be 4.66550  0.01051. This value is slightly under the actual

value of 4.7, which shows that there may be sources of error that causes this. The R2 value was

0.99759, which falls within the acceptable range of 0.995 to 0.999. This shows that the data was

precise. The purpose of this experiment is to use absorption spectrophotometry and pH

measurement to measure the pKa of BCG.

A water blank was used before running the UV-Vis Spectrophotometry instrument. The

water blank contains only distilled water, and was recorded at a wavelength of 508nm. This

measurement was saved in the software so that the difference of the measured absorbance of the

buffer and the water blank will be taken.

Besides UV-Vis Spectrophotometry using Agilent Technologies Carry 8454 UV-Vis, a

similar method could be used, which involves Spectronic 20, which is a single wavelength

spectrophotometer. This research was conducted at Suffolk University. This experiment gave a

pKa of 4.62  0.02 for Bromocresol Green, which is lower than the reported literature value of 4.7.
 11

[6]
This shows that the UV-Vis Spectrophotometry using Agilent Technologies Carry 8454 UV-

Vis gives a more accurate value.

One possible source of error could be because the pH probe was not calibrated accurately,

causing the slight difference in the experimental values. During the calibration, the acidic buffer

was found to have a higher pH of 4.24 than its actual value of 4.00. This could cause the pH of the

buffer to be less accurately measured, hence affecting the succeeding values that is used to plot

Figure 2 and Figure 3. Another source of error could be because the amount of solvent added were

not precise. The instruments chosen to measure the volumes had larger standard errors relative to

the use of volumetric pipet. As a result, the ionic strength of the solution may have changed.

Another source of error could be a result of stray light that may have entered the monochromator

in the spectrophotometer. This could cause the absorbance to stray, causing the data to be less

accurate.

The ionic strength of all the solutions were kept at a constant of 0.1. They are kept at a

constant so that the values of the concentrations of ions and compounds can be accurately

estimated in equilibrium. Changing the value of the ionic strength could also affect the rate of
[7]
reaction, such as the dissociation of acid. The molarity of the BCG in the solution was

approximately 1.1 x 10-5. The ionic strength should be at a constant so that the dissociation of BCG

will not be affected, which could cause the data to be less accurate. In the acidic solution, potassium

chloride solution was added more than in the basic solution because acetic acid will not contribute

ions to the solution as it is not strongly ionized.

Conclusion

The result of this experiment showed that the pKa of Bromocresol Green for the first

method is 4.74, while for the second method the pKa is 4.6655  0.01015. This shows that the
 12

values of the pKa were very close to the actual value of 4.7. The first method gives a percent

error of 0.9%, which is very small, and can be acceptable. For the second method, the value is a

little under the actual value of 4.7, which could be due to sources of errors. Some possible

sources of errors could be due to the pH calibration, amount of solvent added, or even stray light

entering the monochromator in the spectrophotometer.

This report focuses on the determination of pKa of Bromocresol Green using pH

measurements and absorption spectroscopy. This work could be further expanded by looking into

the pKa of different indicators. Future work could also include determining different methods to

obtain pKa of objects in nature, such as the use of scanometric method that was mentioned in the

beginning. With this, there will be an increase in understanding the pKa of different objects, which

could serve as a natural indicator.

Reference

1. What Is the Function of Litmus Paper? https://sciencing.com/function-litmus-paper-

5072700.html (accessed Mar 4, 2018).

2. Acid Base Indicators http://www.ch.ic.ac.uk/vchemlib/course/indi/indicator.html

(accessed Mar 4, 2018).

3. Shokrollahi, A.; Firoozbakht., F. Beni-Suef University Journal of Basic and Applied

Sciences 2016, 5 (1), 13-20.

4. Helmenstine, P. D. A. M. Understand the Relationship Between pH and pKa

https://www/thoughtco.com/the-ph-and-pka-relationship-603643 (accessed Mar 8, 2018).

 13

5. Bromocresol Green

https://www.chemicalbook.com/ChemicalProductProperty_EN_CB9447060.htm

(accessed Mar 8, 2018).

6. Patterson, G. S. Journal of Chemical Education 1999, 76(3), 395.

7. Kennedy, C. Biochemical Education 1990, 18 (1), 35-40.