Pamantasan ng Lungsod ng Maynila

College of Science
Biochemistry (Laboratory)
CHY 2202L SY 23-24

Experiment No. 2
Buffer Solution and pH
Date of Experiment: 03/02/24 Date Submitted: 03/16/24

I. Course: BS Psychology Year: 2 Block: 3 Group No.: 3

Name Accomplishment

Leader Dela Cruz, Christian Jacob, Introduction

P.

Members Buison, Ruelyn Angel Fevi, Conclusion, and

V. Recommendations

Datu, Stephanie Iris Abstract

Lachica, Rhyla Eryn, H. Objectives, Instrumentation

and Apparatus

Micu, Annuary Glaiza Materials and Chemical

Louise, D. Reagents

Reyes, Kathlyn, D. Methodology

Sola, Krystel Angela, T. Results and Discussion

II. Abstract

This experiment seeks to observe the pH levels of different samples using pH paper
and pH meter, and prepare a buffer solution using the Henderson-Hasselbach equation, and
test its buffer capacity. The pH of 6 different liquid samples—pineapple juice, distilled water,
soft drink, fresh milk, vinegar, and toothpaste solution—were tested. A buffer solution was
prepared with 28.5 mL of 0.20 M sodium acetate with 21.5 mL of 0.20 M HAc in a volumetric
flask. Then, 0.1 mL of 0.1 M NaOH was added to 4 samples: distilled water, 0.1M HAc, 0.1M
NaAc, and acetate buffer, and the change in pH was observed. 2.5 mL of 0.1 M HCl was
then added to the acetate buffer and its buffer capacity was calculated. After the procedures,
5 out of 6 samples were determined to be acidic using the pH paper (red = 1, orange = 3,
lime green = 6) with distilled water as the lone neutral sample. In the pH meter, however,
distilled water became acidic with a pH of 5.8; the other samples retained their acidity within
the pH range of 2.4 to 6.7. The acetate buffer has a pH of 4.417 by following the
Henderson-Hasselbalch equation. The pH levels of NaAc, HAc, and distilled water
significantly changed upon the addition of NaOH solution. Lastly, NaOH and HCl solution
added to the acetate buffer showed no significant changes in pH level with a calculated
buffer capacity of 1.25.
III. Introduction

Water is the primary liquid present in the earth, entailing different unique properties
and abilities as a medium of numerous biochemical transformations (Frenkel-Pinter et al.,
2021). Its polar molecular structure and properties such as the ability to form hydrogen
bonds, which in turn contributes to the water’s high boiling point, vapor pressure, and
surface tension, allows water to support and perform biological processes. However, the
nature of this medium to which various life processes are executed is affected by the
presence of acidic and basic substances that could pose a danger to human cells, if not
regulated (Sargen, 2019).

Buffer systems or solutions maintain their hydrogen ion concentration by neutralizing

acids and bases being added to the system. They are responsible for preventing harm from
single inhalation of chemical substances as they readily absorb excess H+ and OH- ions
(Bartee et al., 2017; Ahern et al., 2021). The importance lies in the bicarbonate and carbonic
acid buffer systems' ability to maintain blood pH and regulate enzyme activities in different
organisms. Consequently, the Henderson-Hasselbalch equation serves to determine the pH
of a buffer solution and elucidate the correlation between solution pH and the proportion of
acidic and basic substances present (Ahern et al., 2021).

Using the buffer equation, the effectiveness of a solution to resist pH change will be
determined and will be vital in analyzing more buffer systems as a response to the damaging
effects of high acidity and alkalinity, especially in the human body and other biological
processes. In this experiment, the pH levels of different liquid samples, including water, will
be determined. Moreover, a buffer—specifically acetate buffer—will be created using the
buffer equation and to be tested for its buffer capacity. This activity puts forth additional
insights into the significance of pH levels, its effects to biological processes in the human
body, and continuous maintenance in the chemical systems.

IV. Objectives

General Objectives

This activity aims to:

1. Determine the pH of the given sample using pH paper and pH meter.
2. Apply the Henderson-Hasselbalch equation in preparing buffer solutions.

Specific Objectives

a. Identify the pH levels of different samples using a pH meter and pH paper and
understand their differences;
b. Observe the changes in the initial pH and final pH of the different solutions (distilled
water, 0.10 M sodium acetate (NaAc), 0.10 M Acetic acid (HAc), and, acetate buffer)
after the addition of sodium hydroxide (NaOH); and
c. Determine the buffer capacity of Acetate buffer by adding 0.1 N HCl, then calculate
the buffer capacity of the solution.
V. Materials and Chemical Reagents

Table 1. Materials used in the experiment

Materials

(3) Aspirator

(6) pH paper

(1) Spatula

Table 2. Chemicals/Reagents used in the experiment

Reagents

Fresh Milk (20 mL)

0.20 M Sodium Acetate - NaAc (34 mL)
Pineapple Juice (20 mL)
0.10 M Sodium Acetate - NaAc (20 mL)
Sprite (20 mL)
0.10 M Acetic Acid - HAc (20 mL)
Vinegar (20 mL)
0.01 M Sodium Hydroxide - NaOH (0.4 mL)
Toothpaste Solution (20 mL)
0.01 M Hydrogen Chloride - HCl (2.5 mL)
Water (40 mL)
0.10 M Acetate Buffer (50 mL)
0.20 M Acetic Acid - HAc (16 mL)

VI. Instrumentation and Apparatus

Table 3. Instrumentation and Apparatus used in the experiment.

Instrumentation and Apparatus

(1) graduated cylinder (1) Test tube rack

(2) 10 mL pipette (2) 250 mL beaker

(2) 100 mL beaker (2) 500 mL beaker

(10) test tubes (2) Stirring rod

(1)Volumetric flask (1) Watch glass

VII. Methodology

7.1 Experiment A - Determination of pH

7.1.1 Set-up of “Experiment A - Determination of pH”

Figure 1. Set-up for Experiment A - Determination of pH. The pH of the 6 samples is

identified using pH paper and pH meter.

7.1.2 Procedure Outline of “Experiment A - Determination of pH”

 Figure 2. Flowchart of the step-by-step procedures in conducting Experiment A.

7.2 Experiment B - Preparation of Buffer solution

7.2.1 Set-up of “Experiment B -Preparation of Buffer Solution”

Figure 3. Set-up for Experiment B - Preparation of Buffer Solution. The 0.20 M NaAc is
mixed with the 0.20 M HAc in a volumetric flask.
 Figure 4. Flowchart of the step-by-step procedures in conducting Experiment B.

7.3 Experiment C - Buffering Capacity

7.3.1 Set-up of “Experiment C - Buffering Capacity”

Figure 5. Set-up for Experiment C - Buffering capacity. Testing the 4 samples’ initial and final
pH

7.3.2 Procedure Outline of “Experiment C - Buffering Capacity”

 Figure 6. Flowchart of the step-by-step procedures in conducting Experiment C.

VIII. Results and Discussion

Results for Experiment A - Determination of pH

Figure 7. Color changes in the pH paper after dipping into different samples (left). A color
chart was used to determine its specific pH level (right).

Figure 8. pH levels of different samples using the pH meter

 Table 4. The result of the pH level based on the pH paper and pH meter.
Samples pH using pH paper pH using pH meter

6
1. Fresh milk 6.771
(lime green)

3
2. Pineapple juice 3.617
(orange)

3
3. Softdrink 3.186
(orange)

1
4. Vinegar 2.485
(red)

6
5. Toothpaste 5.454
(lime green)

7
6. Distilled water 5.853
(green)

Different samples were tested for their pH levels with the use of both the pH paper
and pH meter. In the pH paper, results showed that pineapple juice and soft drinks changed
the color of the paper to orange, the vinegar sample showed red, fresh milk and toothpaste
with lime green, and distilled water showed a stronger green. Consultation with the color
chart showed the following pH level conversion: orange - pH 3.0, red - pH 1.0, lime green -
pH 6.0, green - 7.0. These values presented that the samples are acidic, aside from distilled
water that is neutral. Similarly, when the samples were tested on the pH meter the results
were closely identical to the results of pH paper. pH levels from the pH meter expressed a
more specific value of pH level with three decimal values, showing more detailed differences
between the acidity of the samples.

Discussion

In determining the pH level of a particular solution, the following samples must be

measured through the pH paper and pH meter. The difference is that the pH meter, also
known as a potentiometric pH meter, is more accurate and precise than the pH paper. It
automatically measures the pH level of a substance or solution, and it measures the
electrical potential difference between a pH electrode and a reference electrode. The pH
paper, a piece of paper, measures the pH of a solution by observing the changes in the color
of the following samples and identifies the pH level by checking the reference pH scale or
color chart (Hullgren, n.d.). To determine whether it is an acidic or a basic component, the
pH value 0-14 represents the acidity and basicity of a solution. If the solution shows 0-6, it
indicates that it is an acidic solution. Therefore, pH = 8-14 indicates that it is a basic solution,
while pH = 7 indicates neutral in the solution just like pure water (Barnes & Teeter, 2023).

In using the pH paper, five (5) of the given samples ranging from 3 to 6 showed that it
is an acidic solution, and distilled water showed that it is green or 7 in pH value means that it
is neutral. In the pH meter, however, the pH level of distilled water changed to slightly acidic
(5.8) due to the time it was exposed to the room atmosphere and its ability to absorb carbon
dioxide (Deziel, 2019). In measuring the samples using the pH meter, the results were
closely identical to the results from pH paper, allowing a more precise and accurate pH value
(in numerical value) of the following samples.
Results for Experiment B - Preparation of Buffer Solution

Figure 9. Preparation of 0.2 M acetate buffer, pH = 4.5

Figure 10. Detailed computation of the volume of NaAc and HAc needed to prepare a 50 mL
acetate buffer with a pH of 4.5 using the Henderson-Hasselbalch equation.

Discussion

The process of preparing the buffer solution involved employing the

Henderson-Hasselbalch equation to predict the pH of the solution and establish the
equilibrium in an acid-base reaction. Additionally, it facilitated the determination of the
appropriate ratio of acid to conjugate base required to achieve a desired pH for the buffer
solution. Generally, the equation only applies to buffer solutions with a ratio ranging from
0.1-10 (Moore et al., 2021). In this experiment, the calculated ratio is 0.569. The conjugate
base used in creating the acetate buffer and the calculated volume is 28.5 mL of 0.2 M
sodium acetate (NaAc), and the weak acid is 21.5 mL of 0.2 M acetic acid (HAc).
Results for Experiment C - Buffering Capacity

Figure 11. (above) Initial or actual pH of distilled water, 0.10M NaAc, 0.10M HAc, and
acetate buffer (below) pH of the samples after addition of NaOH solution.

Table 5. The results of the actual pH, pH with NaOH, and the final pH of the following
reagents.
Solution Actual pH pH with NaOH ^ In pH

a. distilled
5.317 8.101 2.784
water

b. 0.10 M
Sodium
3.072 5.527 2.455
Acetate
(NaAc)

c. 0.10 M
Acetic Acid 5.744 3.177 -2.567
(HAc)

d. acetate
4.417 4.435 0.018
buffer
 Figure 12. Actual pH (left) and final pH (right) of the acetate buffer after mixed with 2.5mL
HCl solution

Table 6. The actual pH, pH with HCl, final pH, and the buffer capacity of the acetate buffer.
Buffer
SOLUTION Actual pH pH with HCl ^ in pH
Capacity

Acetate Buffer 4.417 4.421 0.004 1.25

The pH levels of four (4) samples were determined before and after the addition of
NaOH solution. The changes in pH were then calculated using the actual and final values.
Among the four samples, Table 5 reported that distilled water and 0.10 M NaAc showed
drastic changes in their pH levels with around +2 change in pH, while the 0.10 M HAc
decreased by -2.5. The prepared acetate buffer’s pH level increased by only 0.018 upon the
addition of NaOH solution, presenting its capacity to resist pH change.

Table 6 shows that acetate buffer, added with 2.5 mL of 0.1 N HCl solution, resulted
in a decrease of only 0.004 in pH, further supporting the idea of its capacity to resist change.
The buffer capacity was calculated using the formula presented in Figure 6, with a calculated
value of 1.25. These figures show that the prepared buffer has a high resistance in pH
change and would require a large amount of strong acid or base to be added to elicit drastic
changes in pH levels.

Discussion

The buffer system is a solution that resists a change in pH when a certain acid or
base is added. The value of the buffer capacity determined this ability of the solution.
External factors such as water or detergents may reduce the buffer capacity because of the
elution of the buffer chemicals which lead to an increase in pH (Proksch, 2018). The
concentration of the components that were added had an effect on the buffer capacity, and a
buffer solution exhibited its maximum buffer capacity when the pH is equal to the pKa
because the buffer will have an easier ability to resist any changes when it comes to the pH
 when added to an acid or base. The more concentrated the buffer solution, the greater the
buffer capacity has an effect on the equilibrium and can change the pH of the buffer.

In Table 5, it shows the initial pH and the final pH value of a solution with NaOH, and
the changes in terms of the pH value of the following solutions (distilled water, 0.10 M NaAc,
0.10 M HAc & acetate buffer). The introduction of a salt containing the conjugate base into a
solution of a weak acid led to an elevation in the pH of the subsequent solutions: distilled
water, 0.10 M sodium acetate (NAc), and an acetate buffer, with the buffer exhibiting the
least alteration in pH. Conversely, the addition of sodium hydroxide (NaOH) to 0.10 M acetic
acid (HAc) resulted in a reduction in pH and a shift in the equilibrium favoring an increase in
acetate ion concentration.

Upon adding 2.5 mL of 0.1 M HCl to the acetate buffer, the pH increased by 0.004.
Introducing NaOH to the acetate buffer results in neutralization of the acetic acid by NaOH.
Consequently, adding HCl to the prepared acetate buffer neutralizes half of the sodium
acetate, leading to a solution with equal concentrations of acetic acid and sodium acetate
(Flowers et al., 2019). This demonstrates the buffer solution's capacity to withstand pH
changes when its composition is altered, thus establishing its effectiveness as a buffer
system.

IX. Conclusion and Recommendation

Conclusion

In this experiment, we aimed to determine the pH of various liquid samples using pH

paper and a pH meter, applying the Henderson-Hasselbalch equation to prepare buffer
solutions and assess their buffering capacities. The results indicated that the pH meter
provided more accurate and precise measurements compared to pH paper. The samples
exhibited varying acidity levels, with distilled water being neutral, while others, such as
pineapple juice and vinegar, showed acidity. The experiment showed the importance of
accurate pH measurement in understanding the acidic or basic nature of solutions.

Furthermore, the successful application of the Henderson-Hasselbalch equation

facilitated the preparation of an acetate buffer solution, aiding in the determination of the
buffer solution's pH and elucidating the correlation between pH and the proportion of
acidic and basic components. The efficacy of the buffer was evaluated by monitoring its
pH changes upon the addition of NaOH. The acetate buffer exhibited only a slight
increase in pH, underscoring its ability to withstand considerable pH fluctuations.
Moreover, the experiment explored the buffer capacity of the acetate buffer. When
subjected to HCl, the buffer displayed minimal pH reduction, highlighting its capacity to
maintain stability against acidic influences. The calculated buffer capacity further
corroborated this resilience, suggesting that significant amounts of strong acid or base
would be required to induce substantial pH changes.

Overall, the experiment provides valuable insights into pH measurement techniques,

buffer preparation, and buffer capacity determination. The findings contribute to a better
understanding of the role of pH in various solutions and the practical applications of
buffer systems in maintaining stable pH environments.
 Recommendations

1. Include a wider variety of liquid samples for testing, such as juices, beverages, and
household liquids. This might offer a full understanding of how different liquids affect
pH levels.
2. Experiment with other buffer solutions besides acetate buffer to determine their
efficacy in resisting pH changes. This can be useful in comparing and evaluating the
efficiency of different buffers in preventing pH changes.
3. Concentrate on using the pH meter more frequently because of its accuracy. Ensure
adequate calibration and training to improve pH measurement accuracy and reduce
dependence on pH paper.
4. Repeat the trials several times to check the consistency and dependability of results.
5. Maintain stable and regulated environmental conditions, such as temperature and
humidity, throughout the experiment. These factors may change, affecting pH
measurements and buffer capacity.
6. Extend the research to real-life circumstances, such as evaluating the pH of common
home items or biological fluids, which can give practical insights into the significance
of pH regulation in everyday situations.
7. To reduce variability, standardize pH measuring processes with pH paper and a pH
meter. Clearly define the processes for sample preparation, equipment calibration,
and data collecting.
8. Encourage collaboration across labs or institutions to compare results and improve
the dependability of findings.
X. References

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Proksch E. (2018). Buffering Capacity. Current problems in dermatology, 54, 11–18.

https://doi.org/10.1159/000489513

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