A Preparation of Acidic and Basic Buffer Solution

Carlene Glory P. Carurucan

Kristine May Delarmente

Jemboy Candar

CHM141.1 M78
AY 2024-2025

September 17, 2024

Introduction

The proper maintenance of the pH is important in several chemical, biochemical and

biological applications. For instance, the structure, and hence the function, of proteins, nucleic
acids, and many other cellular molecules depends on weak forces such as H bonds and ionic
interactions, both of which can be affected by pH (Garrett & Grisham, 2024). In consequence,
buffers are an important aspect in most living systems.

Buffers are aqueous systems that resist changes in pH when small amounts of acid or base are
added. Buffer solutions are composed of a weak acid (the proton donor) and its conjugate base
(the proton acceptor). Buffering results from two reversible reaction equilibria in a solution
wherein the concentration of proton donor and its conjugate proton acceptor are equal (Mohan,
1995).

The relationship between pH, pKa, and the buffering action of any weak acid and its conjugate
base is best explained by the Henderson-Hasselbach equation.

Given that for any weak acid,

[𝐻3 𝑂+ ][𝐴− ] [𝐴− ]

𝑝𝐾𝑎 = −𝑙𝑜𝑔 = − log[𝐻3 𝑂+ ] − 𝑙𝑜𝑔
[𝐻𝐴] [𝐻𝐴]

[𝐴− ]
𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔
[𝐻𝐴]

This equation states that the logarithm of the concentration of dissociated acid divided by the
concentration of undissociated acid is equal to the pH of the solution minus the pKa of the acid
(McMurry, 2015). Thus, if pH of a desired buffer and the pKa of the acid to be used is known,
the ratio of [A-] to [HA] can be calculated to be able to prepare a buffer.

It is noted that in a buffer system, the components are chosen such that the pKa of the weak
acid is close to the pH of interest as it is at the pKa that the buffer system shows its greatest
buffering capacity. At pH values more than 1 pH unit from the pKa, buffer systems become
ineffective because the concentration of one of the components is too low to absorb the influx
of H+ or OHˉ (Garrett & Grisham, 2024).

Objectives
The experiment aimed for the experimenters to be able to utilize the Henderson-Hasselbach
equation to prepare an acidic and a basic buffer solution of pH 4.5 and 9.0, respectively.
Methodology

Materials
Volumetric Flask, 25mL Pipette pH Meter
Volumetric Flask, 50 mL Aspirator
Beaker, 100mL Glass Rod

The necessary materials were obtained from the laboratory stockroom prior to the execution of
the experiment. Once the materials were obtained, the stock solutions were prepared, thereafter,
the dilute solutions were prepared. Lastly, the buffer solution was made, and the pH was
measured.

Preparation of Stock Solutions

1.0M Acetic Acid. A 1.42 mL aliquot of 17.5 M acetic acid solution was transferred using a
pipette into a 25 mL volumetric flask. The solution was then diluted with distilled water to its
final volume.

1.0 M Ammonium Hydroxide. A 1.95 mL aliquot of 25.7 M ammonium hydroxide was

transferred using a pipette to a 50 mL volumetric flask. The solution was then diluted to its
final volume with distilled water.

Preparation of Dilute Solutions

0.1M Acetic Acid. A 5.0mL acetic acid from 1.0M stock solution was withdrawn a pipette and
transferred to a 50mL volumetric flask, it was then diluted with distilled water up to the 50ml
mark.

0.1M Sodium Acetate. A 0.6804 grams of sodium acetate was accurately measured in analytical
balance. It was then dissolved with an adequate amount of distilled water and transferred in a
50mL volumetric flask and diluted until the mark.

0.1 M Ammonium Hydroxide. A 5.0mL ammonium hydroxide from 1.0M stock solution was
withdrawn a pipette and transferred to a 50mL volumetric flask, it was then diluted wit distilled
water up to the 50ml mark.

0.1M Ammonium Chloride. A 0.2675g grams of ammonium chloride was accurately measured
in analytical balance. It was then dissolved with an adequate amount of distilled water and
transferred in a 50mL volumetric flask and diluted until the mark.
Preparation of Buffer Solution
Buffer Solution, pH=4.5. A 32.26mL of the 0.1M acetic acid was measured using a pipette and
transferred into a 100mL beaker. After that, 17.74mL the 0.1M sodium acetate was added to
the same beaker and mixed thoroughly with a magnetic stirrer.

Buffer Solution, pH=9. An 18.00mL of the 0.1M ammonium hydroxide was measured using a
pipette and transferred into a 100mL beaker. After that, 32.00 mL the 0.1M ammonium chloride
was added to the same beaker and mixed thoroughly with a stirring bar and magnetic stirrer.

Measurement of pH
The pH meter was set-up, a utility clamp was used to suspend a pH electrode on an iron stand
then it was calibrated using standard buffer solution of pH 4, 7, and 10. Once it was calibrated,
the beaker containing the solution to be measured was placed in a magnetic stirrer and the
stirring bar was added, the pH is then measured.

Results and Discussion

The following figures present the results of the pH measurements of the buffer solutions
prepared.

Figure I. Measurement of pH for Acidic Buffer

Figure I. displays the pH of the acidic buffer that was prepared using acetic acid & sodium
acetate conjugate acid-base pair. It was measured to have a pH of 4.52. The resulting pH is then
compared to the desired pH, 4.5, which resulted in a 0.44% error.

Figure II. Measurement of pH for Basic Buffer

Figure II. displays the pH of the basic buffer that was prepared using ammonium hydroxide &
ammonium chloride conjugate acid-base pair. It was measured to have a pH of 8.97. The
resulting pH is then compared to the desired pH, 9.0, which resulted in a 0.33% error.

Conclusion
In this laboratory experiment, the Henderson-Hasselbach equation is utilized to prepare an
acidic and basic buffer solution of desired pH, 4.0 and 9.0, respectively. The prepared acidic
buffer solution was measured to have the pH of 4.52 with an error of 0.44% while the prepared
acidic buffer solution was measured to have the pH of 8.97 with an error of 0.33%.

It is essential to take into account the deviation from the desired pH of the solutions may be
attributed to instrumental and personal errors in the preparation of the buffer solutions.
Nonetheless, it is concluded that the Henderson-Hasselbach equation is a crucial tool in a
preparation of buffers.

References
Garrett, R. H., & Grisham, C. M. (2024). Biochemistry (7th ed.). Cengage Learning.

McMurry, J. (2015). Organic chemistry (9th ed.). Cengage Learning.

Mohan, C. (1995). Buffers: A Guide to the Preparation and Use of Buffers in Biological
Systems.
Calculations

Volume of conjugate acid-base pair

Acidic Buffer Basic Buffer
[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒] [𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑎𝑐𝑖𝑑]
𝑝𝐻 = 𝑝𝐾𝑎 + log 𝑝𝑂𝐻 = 𝑝𝐾𝑏 + log
[𝑎𝑐𝑖𝑑] [𝑏𝑎𝑠𝑒]
𝐿et X=Vconjugate acid ; 50-X=Vbase
𝐿et X=Vconjugate base ; 50-X=Vacid
𝑚𝑚𝑜𝑙
𝑚𝑚𝑜𝑙 0.1 ×𝑋
0.1 ×𝑋 [ 𝑚𝐿 ]
[ 𝑚𝐿 ] 14 − 9 = 4.75 + log 50𝑚𝐿
4.5 = 4.76 + log 50𝑚𝐿 𝑚𝑚𝑜𝑙
𝑚𝑚𝑜𝑙 0.1 × 50 − 𝑋
0.1 × 50 − 𝑋 [ 𝑚𝐿 ]
[ 𝑚𝐿 ] 50𝑚𝐿
50𝑚𝐿
𝑋
𝑋 5 = 4.76 + log
4.5 = 4.76 + log 50 − 𝑋
50 − 𝑋 𝑋
𝑋 10(5−4.76) =
10(4.5−4.76) = 50 − 𝑋
50 − 𝑋
X=32.00 mL of O.1M NH3Cl
X=17.74mL of O.1M CH3COONa
50-X=18.00 mL of 0.1M NH3OH
50-X=32.26mL of 0.1M CH3COOH

Volume of weak acid/weak base for stock solutions

𝑔 𝑐𝑚3 𝑚𝑜𝑙 1000𝑚𝐿 𝑔 𝑐𝑚3 𝑚𝑜𝑙 1000𝑚𝐿
𝑀 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 1.05 3 × × × 𝑀 𝑁𝐻3 𝑂𝐻 = 0.9 3 × × ×
𝑐𝑚 𝑚𝐿 60.05𝑔 𝐿 𝑐𝑚 𝑚𝐿 35.68𝑔 𝐿
= 17.5𝑀 = 25.7𝑀
𝑀2 ×𝑉2 1.0 𝑀×25𝑚𝐿 𝑀2 ×𝑉2 1.0 𝑀×50𝑚𝐿
𝑉1 = = =1.42mL 𝑉1 = = =1.95mL
𝑀1 17.48𝑀 𝑀1 25.7𝑀

Mass of salt used for dilute solutions

0.1𝑚𝑚𝑜𝑙 136.08𝑔 0.1𝑚𝑚𝑜𝑙 53.49𝑔
50.00𝑚𝐿 × 𝑚𝐿
× 1000𝑚𝑚𝑜𝑙 = 0.6804g 50.00 𝑚𝐿 × × = 0.2675𝑔 𝑁𝐻3 𝐶𝑙
𝑚𝐿 1000𝑚𝑚𝑜𝑙
𝐶𝐻3 𝐶𝑂𝑂𝑁𝑎

Volume of weak acid/weak base for dilute solutions

𝑀1 𝑉1 = 𝑀2 𝑉2
𝑀2 ×𝑉2 0.1𝑀×50𝑚𝐿
𝑉1 = 𝑀1
= 1𝑀
=5mL