CALIGAN, JEFF TRISTAN Z.

DATE PERFORMED:
GARLAN, MATTHEW D. DATE SUBMITTED:
REGATALIO, KYLE DYMER L.

EXPERIMENT NO. 1
pH AND BUFFER SYSTEMS

I. Introduction

An aqueous solution can be classified as acidic or a basic solution and it can be quantitatively
determined by the value of the pH. Generally, the term pH is widely used in the different fields
of science and becomes a part of our daily life [1]. It is defined as the concentration of hydrogen
or hydronium ions present in the solution. Consider the ionization of water that produces

hydrogen and hydroxyl ion.

Figure 1: Ionization of water.

The result concentration of H+ in the solution determines the pH of water. For water, the pH
is 7 which is neutral due to the equal concentration of hydrogen and hydronium ion [2]. The
solution is considered basic if pH is greater than 7 and acidic if less than 7 [1]. The pH
dramatically changes upon increase in either of the acidic or the basic component of the
solution. The measurement of this value was first proposed by Danish Søren Peter Lauritz
Sørensen using the equation [3],
𝑝𝐻 = − log10 [𝐻 + ] [1]
The effect of different ratio of weak acid and conjugate base in a buffer system was
determined by addition of strong acid and strong base and by using the Henderson Hasslebalch
equation. [6]

On the other hand, there is a solution that can resist drastic changes in pH called buffers.
Upon addition of acid or base, it can neutralize these compounds, thus maintaining the pH
relatively constant. Buffers are made up of a weak acid and its conjugate base. Moreover,
buffer capacity is the extent where the buffer can resist the changes in pH. It is mainly
dependent on the concentration of the buffer and ratio of the two components of the buffer,
conjugate base and weak acid [4-5]. These buffers are essential in biological reactions that
require range of pH to occur and to maintain a physiological balance in an organism [5].

[𝐴− ]
𝑝𝐻 = 𝑝𝐾𝑎 + log [𝐻𝐴] [2]

Using the Henderson Hasslebalch equation, the solution of weak acid and conjugate base
can be determined if the solution acts as a buffer system. Basing on the equation, if the
concentration of weak acid and conjugate base is equal with a ratio of 1, the Henderson
Hasslebalch equation is transformed to,

𝑝𝐻 = 𝑝𝐾𝑎 [3]

since log (1.0) is equal to zero. However, as the ratio between the conjugate base and weak
acid starts to outlay from 1, the Henderson Hasslebalch equation is transformed to [6],

𝑝𝐻 = 𝑝𝐾𝑎 ± 1 [4]

An amino acid can also act as a buffer since it can react with added acids and bases and
keep the pH nearly constant this is because it contains both an acidic (COOH) and a basic group
(NH2). In an acidic media, the NH2 group will be protonated, and in basic media the COOH group
deprotonates.

In this experiment, the effect of concentration and the ratio of each component in a buffer
to its buffering capacity were determined. Also, pKa and corresponding structures of amino acid
were derived based on the titration curve.

II. Methodology
A. Reagents

Amino acid standard sample used in the titration of amino acid for pKa determination was
requested from stockroom. Stock solution of weak acid (acetic acid) and conjugate base
(acetate) for buffer system solution was prepared as a class while buffer system with different
concentration and ratio were prepared by group. Other reagents and solvents used were
requested at the stockroom.

B. Factors affecting buffering capacity

Analysis for the effect of buffer concentration and effect on conjugate base and weak acid
ration used Hanna pH meter for pH determination. Effect of buffer concentration was carried
out by comparison of the degree of change in pH from different concentration (0.005 M, 0.05M
and 0.10M) of buffer system after the addition of 2 mL of 0.1M NaOH. Effect of the conjugate
base and weak acid ration in a buffer system was determined by comparison of the magnitude
in the change of pH after the addition of 2 mL of 0.1N HCl and 2 mL of 0.1N NaOH to different
buffer system with pH of 3.7, 4.7 and 5.7 with respect to the ratio of conjugate base and weak
acid calculated using the Henderson-Hasselbalch equation.

C. Titration of Amino Acid

Potentiometric titration used Hanna pH meter for pKa determination of the assigned amino
acid. Ten mL of aspartic acid was adjusted to pH 1.5 using HCl and titrated against KOH with
1mL increments. The pKa of the amino acid was determined by plotting the change in pH
against the volume of KOH added to the sample amino acid.

III. Results and Discussion

A. Factors Affecting Buffering Capacity

Different factors can contribute on the buffering capacity of a buffering system. The
changes in pH, small or large, are affected by the differences in concentrations or the ratio of a
strong acid and its conjugate base.
The varying concentration of buffer solution with the same pH was prepared. The change in
pH of each concentration was measured with addition NaOH. Buffer capacity was calculated
using the equation [2] where dcb is the number of moles per liter of the strong base and dpH is
the change in pH upon addition of the strong base.
𝑑𝑐
𝐵
𝛽 = 𝑑𝑝𝐻 [5]
 Table 1.1 presents the summary of results on the effect of the concentration of the buffer to
its buffer capacity.
Table 1.1 Effect of concentration of buffer on the buffering capacity
Actual pH
Buffer
Concentration (M) Before addition of After addition of ∆pH
capacity
NaOH NaOH
0.005 4.471 11.544 7.073 0.01
0.050 4.34 4.66 0.32 0.31
0.100 4.401 4.903 0.502 0.20

The experimental result shows a somehow increasing trend in the buffering capacity in
terms of concentration. In dilute concentration of buffer (0.005 M), the buffer capacity is so
small that it is unable to resist the change pH upon the addition of a strong base. The addition
of only 2 ml of a strong base (NaOH) results to a pH change of 7.073. As the concentration of
the buffer is increased, the buffer capacity becomes apparent which results to minute pH
changes. Theoretically, the buffer capacity increases as its concentration increases which is
mainly due to the increase in the number of molecules of the buffer components available to
neutralize addition of acid or base [4]. In the experiment, the addition of NaOH accepts proton
from the buffer system which changes the pH by lowering the ratio of the acid to the conjugate
base. The change in pH is still small since a lot of acid is still present in the system. However,
experimental results show deviation in concentration between 0.05 M and 0.1 M, with the
latter having a larger change in pH than the former.

On the other hand, the effects of the differences in ratio of the weak acid and conjugate
base at varying pH upon the addition of a strong base (NaOH) and acid (HCl) are seen in Table
1.2 and 1.3, respectively.

Table 1.2: Effect of adding NaOH to buffer with different pH

Actual pH
Before After
Sample [A]/[HA] Calculated pH ΔpH
addition of addition of
NaOH NaOH
1 0.091 3.7 3.426 2.455 0.971
2 0.9021 4.7 4.457 4.584 0.271
 3 9.12 5.7 5.415 5.747 0.332

Table 1.3: Effect of adding HCl to buffer with different pH

Actual pH
Before After
Sample [A]/[HA] Calculated pH ΔpH
addition of addition of
HCl HCl
1 0.091 3.7 3.464 3.726 0.262
2 0.9021 4.7 4.431 4.130 0.301
3 9.12 5.7 5.414 5.080 0.334

Theoretically, samples 1 and 3 should have a greater change in pH since the concentration of
the weak acid and conjugate base is extensively different resulting to ratio which is far from 1.
However, based on the results shown in table 1.2 and 1.3 the change in pH for test tube 1 and 3
are small which significantly deviates theoretically. Test tube 2 shows a promising result and
proves that the ratio of the buffer system affects the change in pH since the ratio of weak acid
and conjugate base is near 1 and the change in pH after the addition of strong base and strong
acid is small.

The ration between the weak acid and conjugate base can be used as factor in determining
the buffer system which is at the maximum buffering capacity. A buffer that contains more
base than acid has more OH- present and results to a rise in pH seen in sample 3. In contrast, a
buffer that contains more acid than base has more H+ ions present which results to a lower pH
in sample 1. As the pH of the solution is equal to pKa of the buffering system, the solution is at
the maximum efficiency for a buffering system.

The change in pH after the addition of strong acid and strong base is minimized if the
concentration of the weak acid and conjugate base is equal since there is an equal distribution
of the dissociated weak acid and conjugate base compared to unequal ration of weak acid and
conjugate base having uneven distribution of the dissociated species resulting to imbalance of
ions and greater change in pH. The more the ratio is adjusted for a specific pH, the less efficient
the buffering capacity.[7]
 The deviations with the theoretical observation can be accounted to the differences on the
initial pH of each concentration. The buffer system should be prepared at a pH of 4.7 even at
varying concentrations but all experimental results are much lower than the target pH so the
concentrations can also deviate with stated value. Buffer and sample preparation can also
cause errors since different groups were tasked to prepare each buffer solution.

B. Titration of Amino Acid

Amino acid contains two functional groups that are opposite in nature, a basic amino group
and an acidic carboxyl group [8]. They exist as zwitterion, a dipolar ion, where the amino group
converts to ammonium ion (NH4+) and carboxyl group changes to carboxylate ion (COO-).
Aspartic acid, a nonessential amino acid, is an acidic polar amino acid and the presence of two
carboxyl group makes it hydrophilic. The titration of amino acid to determine its pKa is shown in
Figure 1. This titration curve was obtained by plotting the pH at each successive addition of
base (KOH). The pKa values are determined as the pH at the midpoint where pH slightly
changes.

Figure 1: Experimental Curve of Aspartic acid. Figure 2: Theoretical curve of Aspartic Acid [9].

The experimental curve also coincides with theoretical curve of aspartic acid which is shown
in Figure 2, when titrated with a strong base. The determined pKa values also coincide with
theoretical data. As observed, aspartic acid has three inflection points. This gives it four possible
charged species, three are chargeable and one containing no overall charges. The three
inflection points represent the pKa values or the buffering region of the amino acid. As shown
in Figure 1, moving from left to right increases the hydroxide concentration and decreases the
hydrogen ion concentration. At the first pKa, which is 1.87, the α-carboxyl dissociates. The side
chain R-group carboxyl dissociates in the second pKa at 3.70, and the last pKa which is 9.77, the
α-amino group dissociates.

The isoelectric point (pI) is the pH at which the amino acid has a net zero charge [10]. Usually
for a simple diprotic amino acid, the pI is found halfway between the two pKa values. For an
acidic amino acid like aspartic acid which contains an acidic side chain, the pI will be at a lower
pH because of the extra negative charge. The pI of aspartic acid is determined to be at pH= 2.78
using equation 1.

(𝑝𝐾𝑎1+𝑝𝐾𝑎3)
𝑝𝐼 = [6]
2

Figure 3: Different structures of amino acid at each pKa value [9].

Figure 3 shows the differences in structure of aspartic acid at each respective pKa and pI
values. At pH value less than pKa1, the R-group and α-carboxyl group is in COOH form and the
α-amino group is in NH3+. This gives a net charge of +1. As the pH increase upon the addition of
KOH, a point halfway between pKa1 and pKa3 is reached. At this point, proton from the α-
carboxyl group is removed while in the R-group it does not dissociate and the α-amino group
still has a net charge of +1. The net charge on aspartate is now 0 which is also the isoelectric
point. Further titration removes protons from both the α-carboxyl and R-group (COO-) while α-
amino group is still charged at +1. This gives a net charge to the amino acid equal to -1. The pH
level now shifts to basic and further titration with KOH reaches pKa2. This point is where all the
carboxyl groups completely dissociated and the α-amino group (NH2) has no net charge. At this
point, the overall net charge of the aspartic acid is at -2.
 Though the constructed titration curve is same with expected result, possible error may
include the purity of the amino acid. It is possible that the sample is not pure anymore and
contaminants are present in the system. Also, in determining the pH, mixing is not uniform
throughout the process and stability of the pH meter fluctuates. However, these errors are still
minimal thereby arriving at a value near with theoretical one.

IV. Conclusion

The result of the experiment shows how changes in concentration or ratio of buffer can
affect the changes in pH. As the concentration of the buffer increase, its buffering capacity also
increases. The trend was observed in table 1.1 with small errors that results to deviation in
trend. The trend where ratio of weak acid and conjugate base is near to 1 can resist pH changes
as compared to other values were observed on Tables 1.2 and 1.3. This holds true with
theoretical results as applied by Henderson-Hasselbalch equation. Also, the ratio of the
buffering system also dictates the pH of the buffer where ratio greater than 1 has a higher pH
compared to a ratio of less than 1. The actual concentrations of A - and HA influences the
effectiveness of the buffer while the ratio of [A-]/[HA] influences the pH of the solution.

In addition, amino acids were also proven to be a good buffer system. Based on the titration
curve of aspartic acid, pKa values were observed at different intervals and accompanying
structures were also determined. The unique property of amino acid as a buffer is that it can
protonate or deprotonate itself in order to resist pH changes a sseen in changes in structure of
aspartic acid at different pKa and pI.

It is recommended that preparation of samples are done by same group in order to avoid
personal errors and the use of same pH meter in the same analysis in order to avoid
instrumental errors.

V. References
[1] The Editors of Encyclopædia Britannica (Ed.). (2017, May 07). PH. Retrieved September
04, 2017, from https://www.britannica.com/science/pH
[2] PH and Buffers Defines. (n.d.). Retrieved September 04, 2017, from
http://chemcollective.org/activities/tutorials/buffers/buffers1
[3] Helmenstine A., Ph.D. (n.d.). Know the Definition of pH in Chemistry. Retrieved
September 04, 2017, from https://www.thoughtco.com/definition-of-ph-in-chemistry-
604605
[4] Skoog, D. A., West, D. M., Holler, F. J., & Crouch, S. R. (2014). Skoog and Wests
fundamentals of analytical chemistry (9th ed.). Hempshire: Cengage learning
[5] Nelson, D. L., Cox, M. M., & Cuchillo, C. M. (2008). Lehninger: Principles of Biochemistry.
Barcelona: Omega.
[6] Prep of Buffer at desired pH. Caroll Lab Chap 3. (n.d.). Retrieved September 04, 2017,
from https://www.xula.edu_Chemistry_documents_biolab_Caroll.com
[7] Boundless. “Relative Amounts of Acid and Base.” Boundless Chemistry Boundless, July
27, 2016. Retrived September 05, 2017 from
https://www.boundless.com/chemistry/textbooks/buoundless-chemistry-
textbook/acid-base-equilibria-16/buffer -effectiveness -118/relative-amounts-of-acids-
and-base-479-4880.com
[8] Vidyapeetham, A.V. (n.d.), Titration Curves of Amino Acids. Retrieved August 30, 2017
from http://vlab.amrita.edu/?sub=3&brch=63&sim=1336&cnt=1
[9] TutorVista. “Aspartic Acid”. Retrieved August 24, 2017 from
http://chemistry.tutorvista.com/biochemistry/aspartic-acid.html
[10] Chapter 27: Isoelectronic point. Retrieved August 30, 2017 from
http://www.mhhe.com/physsci/chemistry/carey5e/Ch27/ch27-1-4.html
[11] Titration of Aspartate with Hydroxide. Retrieved August 24, 2017 from
http://faculty.une.edu/com/courses/bionut/distbio/obl-512/Chap6-titration-
aspartate.html