Experiment No.

1 and 2: Preparation of Buffers & Amino Acids as Ampholytes

Arlyn Jave B. Adlawon | Jemil Austin M. Lacson | Faith D. Villahermosa
Group No. 10, Chemistry 41- LB1A, Prof. Arvin Marasigan
September 2, 2019

ABSTRACT

The pH of the environment is an important consideration in understanding biochemical

reactions and replicating them in laboratory simulations. Likewise, part of pH regulation in
physiological systems are buffer solutions: solutions that are able to resist drastic shifts in pH
upon addition of other chemicals. In understanding the relationship between pH and the reagents
used, the Henderson-Hasselbalch equation is utilized. There exists many molecules that affect the
pH of physiological systems. One particular example is amino acids, which have the property of
acting both as bases and as acids depending on the environment they are placed. Molecules like
these are called ampholytes and can act as natural buffers in aqueous solutions. The goals of the
experiments performed were to prepare five buffer solutions with varying pH and to perform
titration on samples to observe the amphoteric property of amino acids, specifically glycine and
aspartic acid, when compared to an inorganic acid, phosphoric acid. It was observed in the five
buffer solutions that although the calculations using the Henderson-Hasselbalch equation were
correct, there exists deviations from the theoretical and actual pH that could be attributed to
factors including the environment in which the experiment was performed, the quality of the
reagents used, and human and instrument limitations. Moreover, the titration curves of glycine
and aspartic acid were able to show pH ranges in which they could act as natural buffers, a
distinguishing characteristic of an amino acid. It is recommended that in future experiments,
better reagents be used.

Keywords: Henderson-Hasselbalch equation, buffers, titration, amino acids, inorganic acid

Introduction pH when small amounts of a strong acid or a strong

pH regulation is critical in many
physiological systems as well as in economic
industries involved with drug and food production
among others. Hence, regulating mechanisms are in
place to prevent negative implications associated
with changes in pH levels. One of these mechanisms
include the presence of buffers.
Buffer preparation, as well as titration
curve analyses, are vital processes that are at risk
for human errors if there is no proper
understanding of related concepts involving acids
and bases, pH, and equivalence points, thus, it is
necessary to have a comprehensive discussion in
order to make accurate interpretations of
underlying mechanisms.
A buffer is a mixture of a weak acid and its
conjugate base (or a mixture of a weak base and its
conjugate acid). Buffer solutions resist a change in
base are added (Flowers, Theopold and Langley,
2016).
A buffer in acidic range is a mixture of a
weak acid and salt of its conjugate base while a
buffer in a basic region is a mixture of weak base
and salt of its conjugate acid. Generally, the pH of a
buffer depends on the acid-base ratio. The exact
ratio of the base to the acid for a desired pH can be
determined from the Ka value and the
Henderson-Hasselbalch equation. Good buffer
mixtures have about equal concentrations of both of
its components. In other words, when the log
[base]/[acid] equals 0 and the pH equals the pKa,
change is minimized (Lumen Learning, n.d).
However, buffer solutions do not have an
unlimited capacity to keep the pH relatively
constant. A buffer solution has accordingly lost its
usefulness when one component of the buffer pair is
less than about 10% of the other. The excessive

CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes 1

addition of base (or acid) to a buffer leading to the
depletion of the weak acid (or weak base) would
result in a loss of its buffering action. This property
refers to its buffer capacity.
The buffer capacity is the amount of acid or
base that can be added to a given volume of a buffer
solution before the pH changes significantly by one
unit. Buffer capacity depends on the amounts of the
weak acid (or weak base) and its conjugate base (or
acid) that are in a buffer mixture. The buffer
capacity has a range of about 2. This means that
when a buffer is created, the pH can be changed by
-1 by acid or +1 by base before the pH begins to
change substantially (Fong, 2019).
In the human body, the HCO3−/CO2 buffer
system is the most physiologically important
because of its quantitative capacity to buffer acids or
bases and its independent regulation of HCO3− and
PCO2 by the kidneys and lungs, respectively in order
to prevent acidosis or alkalosis (Hamm, Nakhoul and
Hering-Smith, 2015). On the other hand, production
of protein-based drugs requires the use of proteins
that are highly sensitive to pH and other
environmental factors and are therefore unstable
unless maintained under certain conditions. Thus, to
maintain a viable environment, different buffer
solutions are required for nearly all downstream
biopharmaceutical processing steps, including
filtration, capture, and chromatography operations
(Challener, 2015).
In biochemistry, most of the reactions that
will be studied happen in environmental conditions
that generally act as buffers and likewise have stable
pH’s. In order to better understand and simulate the
reactions that happen inside biological systems,
reactions must happen in conditions that are
relatively stable, hence the need for buffers.
Moreover, in the bodies of animals, aside
from the carbonate buffer system, amino acids (and
likewise proteins) have the tendency to act as both
an acid and a base in aqueous solutions (depending
on the environment) because of the presence of both
carboxyl (proton donor) and amine (proton acceptor)
group. Molecules like these are called amphoteric
because they have the ability to act both as a base
and as an acid. This gives them the natural ability to
act as buffers in aqueous solutions.
CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes
A titration curve visually demonstrates
buffer capacity. The middle part of the curve appears
flat because the addition of base or acid does not
affect the pH of the solution drastically. This refers
to the buffer zone. However, once the curve extends
out of the buffer region, it will increase
tremendously when a small amount of acid or base is
added to the buffer system and if excess acid, for
example, is added to the buffer, or if the
concentration is too strong, extra protons remain free
and the pH will fall sharply.
Titration curves define several
characteristics, the precise number of which depend
on the nature of the acid being titrated: 1) the
number of ionizing groups, 2) pKa of the ionizing
group/s and 3) the buffer region/s.
Generally, acids can be classified by the
number of protons per molecule that they can give
up in a reaction. Monoprotic acids contain one
ionizable hydrogen atom in each molecule and have
one equivalence point. Polyprotic acids, on the other
hand, can lose several protons per molecule and are
further categorized into diprotic acids with two
ionizable hydrogen atoms per molecule and triprotic
acids with three. The titration curves of polyprotic
acids display the multiple dissociation constants as
they undergo stepwise ionization (Flowers,
Theopold and Langley, 2016).
The experiments performed primarily aimed
to: 1) prepare buffer solutions with a desired pH and
2) perform titration of acidic samples in order to
observe the ampholytic properties of amino acids.
Specifically, they aimed to: 1) apply the
Henderson-Hasselbalch equation and utilize a pH
meter for standardized readings during buffer
preparation, and 2) compare the corresponding
titration curves of an amino acid and an inorganic
acid (specifically phosphoric acid) generated from
pH vs volume NaOH readings.
Materials and Methods
Preparation of Buffers
Five buffer solutions were tasked to be
prepared as seen in Table 1. To ensure maximum
buffering capacity, the weak acids whose pKa were
closest to the desired pH values were used.
Calculations were done in order to determine the
2
necessary amounts of salt and acid to be used for the Results and Discussion
preparation of the respective buffers. These
computations were obtained through the Preparation of Buffers
Henderson-Hasselbalch equation. Varying buffers with specific pH values and
volumes were prepared by the different groups. In
[A ]
pH = pKa  + log [HA] order to prepare these, the mass and volume of the
necessary salts and acids were first computed with
Equation 1: Henderson-Hasselbalch Equation the use of the Henderson-Hasselbalch equation (see
Appendix). Table 2 shows the list of the salts and
Table 1: Specific pH Buffers to be Prepared acids used to prepare the buffers, as well as their
respective mass and volume.
Reagent Conc. (M) Volume (mL) Desired
pH
Table 2: Salts and Acids Used in the Preparation
Phosphate 0.1 1000 7.4 of the Buffers
Phosphate 0.1 1000 6.7 Salt and Acid Mass and/ or
Buffer
Phosphate 0.1 100 2.0 Used Volume
Acetic 0.1 100 4.0 Phosphate Na2HPO4 10.150 g
Carbonate 0.1 100 10.0 (pH 7.4) NaH2PO4•2H20 4.446 g
Phosphate Na2HPO4 4.739 g
Once the appropriate ratios of acid and salt (pH 6.7) NaH2PO4•2H20 10.453 g
were acquired, they were then dissolved in water. A
Phosphate NaH2PO4•2H20 0.811 g
pH meter, which was first calibrated using the
standard pH 4, pH 7, and pH 10 solutions, was used (pH 2.0) H3PO4 0.805 mL
to measure the current pH of the buffer solution. The Acetic NaCH3COO 0.128 g
pH of the buffer solutions were then adjusted to the (pH 4.0) CH3COOH 1.41 mL
desired pH using 0.1M NaOH and 0.1M HCl. After
which, the solutions were transferred to volumetric Carbonate Na2CO3 0.302 g
flasks where they were diluted to their respective (pH 10.0) NaHCO3 0.601 g
volumes. Finally, the prepared buffers were stored in
appropriate containers at 4 ºC for future use. The mechanism for buffer solutions is based
on Le Chatelier's Principle which states that if a
Titration of Acids dynamic equilibrium is disturbed by changing the
In separate Erlenmeyer flasks, 10 mL of 0.1 conditions, the position of equilibrium shifts to
M phosphoric acid, 0.1 M aspartic acid with pH 1.0, counteract the change to reestablish an equilibrium.
and 0.1 M glycine with pH 1.0 were prepared. These Therefore, when H+ is added to a buffer, the weak
solutions were titrated with 0.1 M NaOH as the acid’s conjugate base will accept a proton (H+),
titrant, and for every addition of 0.5 or 1.0 mL thereby “absorbing” the H+ before the pH of the
NaOH, the pH of the solutions were recorded using solution lowers significantly. Similarly, when OH– is
the pH meter. Two trials were done for each acid added, the weak acid will donate a proton (H+) to its
titrated, and the titrations were done until the conjugate base, thereby resisting any increase in pH
solutions reached the pH of 12.0. Titration curves before shifting to a new equilibrium point (Lumen
were later constructed, showing the plot of the Learning, n.d.).
recorded pH values against the volume of 0.1 M
NaOH added. The experimental curves were then After preparing the said buffers, their pH
compared to each other as well as to their theoretical values were then recorded. Table 3 shows the initial
curves. observed pH, as well as the adjusted pH of the
prepared buffers. As shown on the table, out of the
five buffer solutions prepared, only the phosphate
buffer with pH 2.0 had an actual initial pH close to
that of the desired value. The other four prepared

CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes 3

buffers had pH’s that were lower than the desired
values, therefore, their pH’s had to be adjusted by
adding 0.1 M NaOH to these solutions.
Table 3: Initial and Adjusted pH of Prepared Buffers
Buffer Initial pH Adjusted pH
Phosphate 7.25 7.39
Phosphate 6.20 6.64
Phosphate 2.02 2.02
Acetic 3.47 4.00
Carbonate 9.51 9.98
Deviations in terms of the observed pH from
the expected pH in buffers can be attributed to
various factors. First, the standard pKas used in the
experiment were pKas obtained at standard
temperature and pressure (STP) of 25 °C and 1 atm.
Because of the limitations of the environment, such
as the location being generally above sea level and
the room temperature being relatively warmer, it is
possible that the actual pKa of the acid dissociations
of the salts could be different from the theoretical.
It is also worth mentioning that some of the
prepared buffers made use of prepared stock
solutions and not salts. The concentrations of these
stock solutions could have possibly changed since
the time it was prepared, and have thus affected the
pH of the buffers prepared during the experiment.
Other possible reasons for the deviations in
the pH measurement are attributed to uncontrollable
measurement and human errors. Some salts used in
the activity were hygroscopic, meaning they absorb
moisture from the air. Since the measurement device
used was in open air, it is possible that there were
uncontrollable errors in the measurement and
likewise the concentration of the salts.
The prepared buffer solutions, stored at low
temperatures, are expected to have a shelf life of
around 2-6 months depending on the frequency in
which the bottles were exposed to open air. This is
due to the fact that solutions (especially alkaline
ones with pH greater than 10) are likely to react with
carbon dioxide in the air forming carbonic acid
thereby changing the pH of the solutions.
CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes
Titration of Acids
Three different acids namely- phosphoric
acid, glycine, and aspartic acid- were titrated with 0.1
M NaOH as the titrant. Titrations were done by
adding the titrant to the analyte until the pH of the
solution reached the value of 12.0. Table A1 (see
Appendix) shows the full data on the pH of the three
acids against the volume of NaOH added, and the
experimental titration curves constructed from the
different trials are shown in Figures 1 to 3.
Titration curves are generally sigmoidal in
appearance with the number of curves and plateaus
dependent on the number of ionizable protons
present. Each change in curvature of the titration
curve are noted as a point of inflection and can
demarcate the existence of the pKa value or an
equivalence point (Chemistry LibreTexts, 2019).
Inflection points were computed by using the
first derivative of the curves and looking for peaks.
The first derivative was computed by dividing the
change in volume of NaOH added by the change in
pH. By definition, inflection points can be found on
points where the second derivative is equal to zero. It
is also known through calculus that if the first
derivative of a function goes through a maximum,
the second derivative is equal to zero. These were
then the indications used to compute for the
inflection points on the titration curve.
An inflection point on the steep areas of a
curve denote the equivalence point. This point is
where the amount of titrant is substantial to react
with all of the analyte. When the form of the analyte
is neutral (as in amino acids), this point can also be
referred to as the isoelectric point where in the net
charge of the solution is zero. The specific volumes
denoting the equivalence points and pKa in the
experiment are seen below in Table 4.
Table 4: Volume at Equivalence Point and at pKa of
Aspartic Acid, Glycine and Phosphoric Acid
Trial 1 Trial 2
Vol. at Vol. at Vol. at Vol. at
Equiv. Pt pKa Equiv. Pt pKa
(mL) (mL) (mL) (mL)
12.00 12.00
Aspartic 19.00 15.50 18.00 15.00
Acid 27.00 23.00 28.00 23.00
4
 37.00 32.00 38.00 33.00
Glycine 8.00 7.00
18.00 13.00 18.00 12.50
29.00 23.50 30.00 24.00
Phosphoric 4.00 4.00
Acid 10.00 7.00 10.00 7.00
19.00 14.50 19.00 14.50
27.00 23.00 25.00 22.00

On the other hand, the inflection points in the

middle of the flat areas of the curve denote the pKa
values. They demarcate the points in which the Figure 1: Experimental Titration Curve of
concentration of the acid and conjugate base are Phosphoric Acid
equal and likewise, using the Henderson-Hasselbach
equation, the pH is equal to the pKa. These points can
also be considered as half-equivalence points
because it is the point where half the analyte is
reacted, hence, the computation for the volume at
these points are done by finding for the midpoint
between 2 peaks at the steep areas of the curve. It
should be noted that at the half-equivalence point, the
buffering capacity of the solution is at its highest due
to the equal concentration of the acid and the
conjugate base. Values for the theoretical and actual
pKa can be seen in Table 5. As in the table, there is a
noticeable discrepancy between the standard pKa and
the measured pKa and this could be due to numerous
factors similar to those that affected the pH of the Figure 2: Experimental Titration Curve of Glycine
prepared buffer solutions.

Table 5: Standard and Experimental pKa of Aspartic

Acid, Glycine and Phosphoric acid
Standard Experimental pKa
pKa Trial 1 Trial 2
1.88 2.03 2.57
Aspartic 9.60 9.86 9.93
Acid 3.65 4.18 3.94
Glycine 2.34 2.44 2.40
9.60 9.59 9.70
Phosphoric 2.16 2.87 2.86
Acid 7.21 7.26 7.24 Figure 3: Experimental Titration Curve of
12.32 11.66 11.69 Aspartic Acid

Figures 1, 2 and 3 show the experimental In addition to the experimental titration

titration curves of phosphoric acid, glycine and curves, it is also necessary to compare them to
aspartic acid, respectively. theoretical curves, obtained from different sources.
Figures 4 to 6 show the theoretical titration curves of

CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes 5

phosphoric acid, glycine, and aspartic acid,
respectively.
Figure 4: Theoretical Titration Curve of Phosphoric
Acid (Source: Chemistry LibreTexts, 2019)
Figure 5: Theoretical Titration Curve of Glycine
(Source: King Saud University, n.d.)
Figure 6: Theoretical Titration Curve of Aspartic
Acid (Source: King Saud University, n.d.)
One of the first properties one may observe
in a titration curve is the acid strength. Acid strength
refers to the tendency for the ionization reaction to
proceed to the right. It is given quantitatively by the
CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes
size of an equilibrium constant, Ka. Taking the -log
of Ka gives pKa which can be determined through
titration as in the case of this experiment wherein
three acidic solutions were titrated with a strong
base, NaOH. Likewise, acid strength can be
compared based on the amount of NaOH needed to
reach the same pH. Based off of the results of the
experiment, the weakest acid was phosphoric acid
(30 mL) followed by glycine (average of 43 mL),
and then by aspartic acid (average of 46.5 mL).
In comparing the titration curves, the
plateaus may also signify the buffering regions of the
said acid. These plateaus are observed to have only
minimal changes in pH upon the addition of NaOH, a
characteristic of a buffer solution. As mentioned
above, it is when the pH of the solution is equal to
the pKa (at the half-equivalence points) that the
maximum buffering capacity of the solution is
reached, hence the plateau. Generally, the plateaus
are different for each amino acid and are defining
characteristics.
The first acid to be titrated was phosphoric
acid. Comparing the experimental and theoretical
titration curves of phosphoric acid (Figures 1 and 4),
it can be observed that there are three plateaus
present in both curves: the first one ranging from
around pH 2 to 4; the second one ranging from
around pH 6 to 8; and the third one ranging from
around pH 11 to 12.
When analyzing titration curves, the said
plateaus in the curve correspond to the number of
ionizable groups in the analyte, in this case,
phosphoric acid. Phosphoric acid (H3PO4) is a weak
polyprotic acid, particularly a triprotic one.
Accordingly, at any point along the titration curve of
a triprotic acid, there is some percentage of each acid
form present in the mixture such that the pH is
dependent on several forms of the acid. This means
that a polyprotic acid solution such as phosphoric
acid possesses three ionizable protons or hydrogen
ions that it can donate, as depicted below in its
dissociation equations, along with the pKa values.
This shows that phosphoric acid has three
opportunities to dissociate: first into H2PO4-, then
HPO42- and lastly PO43-. This also means that more
than one inflection point is observed in the titration
curves.
6
 H 3 P O4   H + + H 2 P O4    [pKa1 = 2.16]
H 2 P O4     H + + H P O4  2   [pKa2 = 7.21]
H P O4  2    H + + P O4  3   [pKa3 = 12.32]
Equation 2: Dissociation of Phosphoric Acid
Triprotic inorganic acids are known to be
sequentially deprotonated meaning the gaps between
their pKas are distributed relatively equally. This
entails that deprotonation of phosphoric acid happen
step-by-step. This could explain how the titration
curves for phosphoric acid shows the buffering
properties of the acid at relatively lower pH because
at higher pH’s, the reaction towards formation of
phosphoric acid will be favored upon addition of
bases and acids.
The titration curve of phosphoric acid was
generally observed to be distinct from common
amino acids as it is characterized by apparent
equivalence points. Meaning to say, because of the
largely varying acid dissociation constants and pKa
of phosphoric acid, protons are reacted at different
pH. As discussed in the succeeding titrations, the
presence of several species in a solution (due to
incomplete reactions) at the pH where an
equivalence point should be reached were known to
affect the shape of the titration curve.
The next two acids titrated in the activity
were glycine and aspartic acid, which are both
amino acids. Generally, amino acids are weak
polyprotic acids. They are present as zwitterions at
neutral pH and are amphoteric molecules that can be
titrated with both acid and base. The general
structure of amino acids (Figure 7) shows that all of
the amino acids have an acidic carboxylic acid group
(COOH) and a basic amino group (NH2) attached to
the α carbon, and they contain ionizable groups that
act as weak acids or bases, giving off or taking on
protons when the pH is altered.
Figure 7: General Structure of Amino Acids
(Source: WikiBooks, 2019)
CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes
When amino acids are dissolved in water,
they exist predominantly in the isoelectric form. The
isoelectric point, pI, is the pH of an aqueous solution
of an amino acid at which the molecules have no net
charge. In other words, the positively charged groups
are exactly balanced by the negatively charged
groups. When this dissolved amino acid is titrated
with acid, it acts as a base, and with base, it acts as
an acid which makes them an amphoteric molecule.
Glycine (C2H5NO2) is characterized as the
smallest α-amino acid with a nonpolar side chain
consisting of a single H atom. Based on its structure
(Figure 8), it is a diprotic amino acid therefore
having two dissociable protons, one on the α amino
group and the other on the carboxyl group. Its side
chain group does not contribute a dissociable proton.
Figure 8: Structure of Glycine
(Source: ChemSrc, 2019)
As a diprotic acid, glycine has two ionizable
protons to lose. Figure 9 shows its dissociation
process. At a very low pH, glycine is fully
protonated and has a charge of +1 due to the excess
of protons in the amino group. With the continuous
addition of OH-, the amino group becomes
deprotonated and at a very high pH, it has a negative
charge.
Figure 9: Dissociation of Glycine
(Source: Mills, 2009)
Comparing the experimental and theoretical
titration curves of glycine (Figures 2 and 5), it can
be observed that there are two plateaus present in
both curves: one ranging from around pH 1 to 3 and
another ranging from around pH 9 to 11. This
therefore confirms that glycine is indeed a diprotic
acid.
7
 In glycine, the pI is the average of the pKa of
the carboxyl and amino groups. Based from the pKa
shown in Table 5, theoretically the pI of glycine is
calculated to be (2.34 + 9.60)/2 = 5.97.
Experimentally, it was obtained at 6.0325 given by
the average of the experimental pKa results.
The last acid titrated in the activity was
aspartic acid (C4H7NO4). Its structure (shown in
Figure 10) consists of two carboxyl groups, one on
the α-carbon atom and the other on the side chain.
Unlike glycine which is diprotic in nature, aspartic
acid is triprotic, hence it has three ionizable protons.
Figure 10: Structure of Aspartic Acid
(Source: Sigma-Aldrich, 2019)
As shown in Figure 11, there are three
theoretical pKa values for aspartic acid: 1.88 for the
dissociation of the alpha-carboxyl group; 3.65 for the
dissociation of the side chain carboxyl group; and
9.68 for the dissociation of the alpha-amino group.
Initially at low pH, aspartic acid is fully protonated
with a net charge of +1, and as it is titrated with more
hydroxide ions, even more protons are removed up
until it reaches a net charge of -2 for very high pH
values.
Figure 11: Dissociation of Aspartic Acid
(Source: Hunt and Spinney, 2006)
Aspartic acid represents a type of amino acid
that has additional acidic functions in its side chain.
Theoretically, it displays three inflection points, thus,
three equivalence points in its titration curve as
shown in Figure 6. However, these are not as evident
in the experimental titration curve with only one
dominant sigmoidal curve. There was only one
apparent inflection point, accompanied with minute
changes in the curve sections.
CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes
For most acid-base titrations, the inflection
point very nearly coincides with the equivalence
point. In actuality, an inflection point precedes its
corresponding equivalence point by a small amount,
with the error approaching 0.1% for weak acids with
dissociation constants smaller than 10-9, or for very
dilute solutions. For acid dissociation constants
larger than 10-9, an inflection point is generally
visible but is missing when Ka is 10-11. The principal
limitation to using an inflection point to locate the
equivalence point, therefore, is that the inflection
point must be present.
In the titration curve of aspartic acid, the
inflection points were quite difficult to detect
primarily because the analyte is a polyprotic weak
acid with successive dissociation constants that are
quite near in magnitude (e.g. pKa 1.88 and 3.65 as
shown in Table 5). In order to see distinct inflection
points, the prior reaction must be essentially
complete before the succeeding reaction begins. In
general, separate inflection points can be detected
when successive acid dissociation constants differ by
a factor of at least 500 where a change in pKa is at
least 2.7 (Harvey, 2019).
Accordingly, if additional acidic or basic
groups are present as side-chain functions, the pI is
the average of the pKa of the two most similar acids.
In the case of aspartic acid, the similar acids are the
alpha-carboxyl function and the side-chain carboxyl
function, so theoretically pI=(1.88 + 3.65)/2 = 2.77.
Experimentally, it was obtained at 3.18.
This is because with acidic side chains, the
pI will be at a lower pH because the acidic side chain
introduces an extra negative charge so the neutral
form exists under more acidic conditions when the
extra negative charge has been neutralized
(Kennepohl, Farmer and Reusch, 2019).
In this laboratory experiment, derivative
methods for calculating the titration end point were
employed. Generally, derivative methods are used
when titrating a sample that contains more than one
analyte in order for a single titration to be sufficient,
as well as for an analyte with a poorly defined
normal titration curve. However, derivative methods
were evaluated to be accurate only if sufficient data
during the rapid increase in pH near the equivalence
8
point has been recorded. pH changes occur rapidly
near the equivalence point that a manual titration
predominantly is not able to provide enough data,
particularly during the more gently rising portions of
the titration curve before and after the equivalence
point, in order to produce a useful derivative titration
curve. This has possibly contributed to the major
deviations observed in the experimental and
theoretical titration curves obtained (Harvey, 2019).
Conclusion and Recommendations
Buffer solutions are important towards
having a better understanding of how biological
systems maintain constant pH in an environment. In
the study of biochemical reactions, buffers, likewise,
provide an environment similar to that of the
aforementioned systems. This similar environment
would then make simulations of biochemical
reactions more accurate. In the preparation of buffer
solutions, it is of utmost importance that correct
calculations and measurements be done in order to
make sure that the prepared solution is of substantial
quality. One should also note that many factors, such
as the environment, also play a part in the
preparation of buffers.
Moreover, as part of what influences the pH
levels of biological systems, an understanding of the
amphoteric property of amino acids is also
necessary. Titrimetric analysis is an important tool in
understanding this property in that different acids
have different titration curves that are able to show
their pKas, their equivalence points, and their
buffering properties. Specifically, the experiment
was able to show the titration curve of amino acids
glycine and aspartic acid and was able to compare it
with an inorganic acid: phosphoric acid.
It is recommended that in future preparation
of buffers, better quality of salts and solutions must
be used and reagent bottles and glassware used must
be clean and dry. For future iterations of the titration
analysis, more amino acids be titrated in order to
have a better understanding of the pattern of the
titration curves. Moreover, a firm background in
titration and general and organic chemistry is
required in order for error to be minimized.
CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes
References
Books
Flowers, P., Theopold, K., Langley, R. (2016).
Openstax, Chemistry by Rice University. Rice
University. Retrieved from: http://cnx.org/
contents/85abf193-2bd2-4908-8563-90b8a7ac8d
f6@9.311
Journals
Challener, C.A. (2015). Behind the scenes with
buffers. BioPharm International, 28(2), 27
Hamm, L.L., Nakhoul, N., & Hering-Smith, K.S.
(2015). Acid-base homeostasis. Clinical Journal
of the American Society of Nephrology, 10(12),
2232-2242.
Online Sources
Amrita Vishwa Vidyapeetham Virtual Laboratories
(2012). Titration Curves of Aminoacids.
Retrieved from https://vlab.amrita.edu/?sub=3&
brch=63&sim=1336&cnt=1
Bell, R.C. (n.d.). pKa and Molar Mass of a Weak
Acid. General Chemistry Laboratory – Eastern
Michigan University. Retrieved from
https://www.emich.edu/chemistry/genchemlab/d
ocuments/10-pka_and_molar_mass.pdf
Chemistry LibreTexts (2019, April 28). Titrations
and pH Curves. Retrieved from
https://chem.libretexts.org/Bookshelves/General
_Chemistry/Map%3A_A_Molecular_Approach_
(Tro)/17%3A_Aqueous_Ionic_Equilibrium/17.4
%3A_Titrations_and_pH_Curves
ChemSrc (2019, August 20). Glycine [Image].
Retrieved from https://www.chemsrc.com/en/
cas/56-40-6_311698.html
Fong, K. (2019, June 6). How Does a Buffer
Maintain pH? Chemistry LibreTexts. Retrieved
from https://chem.libretexts.org/Bookshelves/
Physical_and_Theoretical_Chemistry_Textbook
_Maps/Supplemental_Modules_(Physical_and_
Theoretical_Chemistry)/Acids_and_Bases/Buffe
rs/How_Does_A_Buffer_Maintain_Ph%3F
Harvey, D. (2019, June 6). Acid-Base Titrations.
Chemistry LibreTexts. Retrieved from
https://chem.libretexts.org/Bookshelves/Analytic
al_Chemistry/Book%3A_Analytical_Chemistry
_2.0_(Harvey)/09_Titrimetric_Methods/9.2%3A
_Acid%E2%80%93Base_Titrations
9
Hunt, I. and Spinney, R. (2006). Chapter 27- Amino I hereby certify that I have given substantial
Acids, Peptides, and Proteins. Organic contribution in this scientific report.
Chemistry On-Line Learning Center. University
of Calgary. Retrieved from
http://www.chem.ucalgary.ca/courses/351/Carey
5th/Ch27/ch27-1-4.html Arlyn Jave B. Adlawon
Kennepohl, D., Farmer, S., & Reusch, W. (2019, 2018-06180
June 6). Amino Acids, the Henderson-
Hasselbalch Equation, and Isoelectric Points.
Chemistry LibreTexts. Retrieved from
https://chem.libretexts.org/Bookshelves/Organic Jemil Austin M. Lacson
_Chemistry/Map%3A_Organic_Chemistry_(Mc 2018-09833
Murry)/Chapter_26%3A_Biomolecules%3A_A
mino_Acids%2C_Peptides%2C_and_Proteins/2
6.02_Amino_Acids%2C_the_Henderson-Hassel
balch_Equation%2C_and_Isoelectric_Points Faith D. Villahermosa
King Saud University (n.d.). Amino Acid Titration. 2018-06170
Retrieved from http://fac.ksu.edu.sa/sites/
default/files/amino_acid_titration.pdf
Lumen Learning (n.d.). Buffer Effectiveness.
Boundless Chemistry. Retrieved from
https://courses.lumenlearning.com/boundless-ch
emistry/chapter/buffer-effectiveness/
Mills, B. (2009, March 17). Protonation states of
glycine, C2H5NO2 [Image]. Retrieved from
https://commons.wikimedia.org/wiki/File:Glycin
e-protonation-states-2D-skeletal.png
Sigma-Aldrich (2019). L-aspartic acid [Image].
Retrieved from https://www.sigmaaldrich.com/
catalog/product/sigma/a9256?lang=en&region=
PH
WikiBooks (2019, August 29). Amino Acid
Structure [Image]. Retrieved from
https://en.wikibooks.org/wiki/Medical_Physiolo
gy/Basic_Biochemistry/Amino_Acids_and_Prot
eins

CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes 10

APPENDIX [HP O4  2 ] 0.3
[H 2 P O4  ] = 10 = 0.5012 0.501

Buffer Preparation Calculations

A. Phosphate buffer, 0.1 M, 1000 mL, pH 7.4 [HP O4  2 ] + [H 2 P O4  ] = 0.1M
[N a2 HP O4 ]
0.1   [N a2 HP O4 ] = 0.501
+
H 3 P O4   H + H 2 P O4   [pKa = 1.97]
H 2 P O4     H + + H P O4  2   [pKa = 7.0]
[N a2 HP O4 ] = 0.033M →   0.033 mol  ( 141.96 g
mol )  
H P O4  2    H + + P O4  3  [pKa = 12.5]
[N aH 2 P O4 • 2H 2 O] = 0.067M  →
H 2 P O4     H + + H P O4  2   0.067 mol  ( 156.01 g
mol ) 
pH = pKa  + log [H
[HP O4  2 ] [Na2HPO4] = 4.739 g
2 P O 4  ] [NaH2PO4] = 10.453 g
[HP O4  2 ]
7.4 = 7.0  + log [H
2 P O 4  ]
[HP O4  2 ] C. Phosphate buffer, 0.1 M, 100 mL, pH 2.0
0.4 = log [H
2 P O 4  ]
[HP O4  2 ] H 3 P O4   H + + H 2 P O4   [pKa = 1.97]
[H 2 P O4  ] = 100.4 = 2.5119 2.51
H 2 P O4     H + + H P O4  2   [pKa = 7.0]
H P O4  2    H + + P O4  3  [pKa = 12.5]
[HP O4  2 ] + [H 2 P O4  ] = 0.1M

0.1   [H 2 P O4  ]

H 3 P O4   H + + H 2 P O4   
[H 2 P O4  ]
= 2.5119 [H 2 P O4  ]
2.0 = 1.97  + log [H 3 P O4  ]
0.1 [H 2 P O4  ] = 2.5119[H 2 P O4  ]   [H 2 P O4  ]
0.1 = 3.5119[H 2 P O4  ]   [H 3 P O4  ] = 100.03 = 1.072
0.1
[H 2 P O4  ] = 3.5119 = 0.0285M
2
[HP O4  ] = 0.1 0.0285 = 0.0715M   [H 3 P O4  ] + [H 2 P O4  ] = 0.1M  
0.1   [H 3 P O4  ]
[H 3 P O4  ] = 2.5119
NaH2PO4:( 0.0285 mol
L ) (1L) ( ) = 3.419 g  
119.98g
mol
[H 3 P O4  ] = 0.048 M
Na2HPO4: ( 0.0715 mol
L ) (1L) ( 141.96g
mol ) =  10.15 g [H 2 P O4  ] =  0.052 M

Change in weight of NaH2PO4 since dihydrate

→ + 2 H2O:
NaH2PO4: ( 0.052 mol
L ) ( 156.01 g
mol ) (0.1L)  = 0.811 g

) (1 L) ( 156.01 g ) (0.1L)  = 8.046 x 10=4 L

mol ) =  4.446 g
( 0.0285 mol H3PO4: ( 0.048 mol
L
1L
) ( 6mol
1 L
or 0.805 mL
NaH2PO4•2H2O
D. Acetic buffer, 0.1 M, 100 mL, pH 4.0
B. Phosphate buffer, 0.1 M, 1000 mL, pH 6.7
[N aCH 3 COO]
H 3 P O4   H + + H 2 P O4   [pKa = 1.97] pH = pKa  + log [CH 3 COOH]
[N aCH 3 COO]
H 2 P O4     H + + H P O4  2   [pKa = 7.0] 4 = 4.74  + log [CH COOH]
3
H P O4  2    H + + P O4  3  [pKa = 12.5] [N aCH 3 COO] 0.74
[CH 3 COOH] = 10 = 0.18197  

H 2 P O4     H + + H P O4  2  
[HP O4  2 ]
[N aCH 3 COO] + [CH 3 COOH] = 0.1M
pH = pKa  + log [H
2 P O 4  ]
[HP O4  2 ] 0.1   [CH 3 COOH]
6.7 = 7.0  + log [H [CH 3 COOH] = 2.5119
2 P O 4  ]
[HP O4  2 ] [CH 3 COOH] = 0.0846M
0.3 = log [H [N aCH 3 COO] = 0.0154 M
2 P O 4  ]

CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes 11

 Computing for the required amount of reagents,
we have:
C1V1 = C2V2
(6M)(V1) = (0.0846 M)(0.1L)
V1 = 1.41 x 10-3 L
V1 = 1.41 mL CH3COOH

( 0.0154 mol
L ) ( 82.03 g
mol ) (0.1L)  = 0.128 g
NaCH3COO

E. Carbonate buffer, 0.1 M, 100 mL, pH 10.0

H 2 CO3   H + + H CO3   [pKa = 6.1]

H CO3     H + + C O3  2   [pKa = 10.4]

H CO3     H + + C O3  2  
2
[CO ]
   pH = pKa  + log [HCO3  ]
3 
2
[CO ]
10.0 = 10.4  + log [HCO3  ]
3 
2
[CO ]
0.4 = log [HCO3  ]
3 
[CO3  2 ] 0.4
[HCO3  ] = 10 = 0.3981 0.40

   [CO3  2 ] + [HCO3  ] = 0.1M  

0.1   [HCO3  ]
[HCO3  ]
= 0.3981
0.1 [HCO3  ] = 0.3981[HCO3  ]  
0.1 = 1.3981[HCO3  ]  
0.1
[HCO3  ] = 1.3981 = 0.0715M
2
[CO3  ] = 0.1 0.0715 = 0.0285M  

NaHCO3: ( 0.0715 mol
L ) (0.1L) ( 84.007g
mol ) =  0.601 g
NaHCO3
Na2CO3: ( 0.0285 mol
L ) (0.1L) ( 105.99g
mol ) =  0.302 g
Na2CO3

CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes 12

Data Table 22.00 3.94 3.72 9.34 8.92 11.51 11.69
23.00 4.18 3.94 9.52 9.20 11.66 11.79
Table A1: Titration pH of Aspartic acid, Glycine and
Phosphoric Acid Against Volume of 0.1 M NaOH 24.00 4.35 4.07 9.66 9.38 11.71 11.86
Aspartic Glycine pH Phosphoric 25.00 4.76 4.35 9.82 9.54 11.77 11.94
Vol Acid pH Acid pH 26.00 6.49 4.69 9.97 9.70 11.84 11.96
(mL)
T1 T2 T1 T2 T1 T2 27.00 8.68 5.52 10.11 9.86 11.94 12.02
0.00 1.15 1.12 1.04 1.05 1.85 1.84 28.00 9.10 8.67 10.27 10.01 12.00 12.06
0.50 1.16 1.07 29.00 9.33 9.30 10.45 10.18 12.02 12.12
1.00 1.17 1.17 1.13 1.12 1.95 1.90 30.00 9.53 9.38 10.93 10.37 12.08 12.16
1.50 1.21 1.17 31.00 9.70 9.58 11.22 10.63
2.00 1.27 1.26 1.21 1.21 2.06 2.01 32.00 9.86 9.76 11.42 10.76
2.50 1.29 1.26 33.00 10.02 9.93 11.58 10.90
3.00 1.31 1.34 1.30 1.31 2.17 2.15 34.00 10.17 10.10 11.68 11.20
3.50 1.39 1.35 35.00 10.36 10.28 11.76 11.45
4.00 1.55 1.42 1.40 1.41 2.33 2.31 36.00 10.56 10.51 11.83 11.62
4.50 1.55 1.45 37.00 10.87 10.78 11.89 11.73
5.00 1.55 1.50 1.50 1.51 2.46 2.47 38.00 11.04 11.06 11.94 11.88
5.50 1.61 1.54 39.00 11.35 11.34 11.96 11.94
6.00 1.63 1.59 1.60 1.62 2.67 2.64 40.00 11.49 11.53 12.01 11.99
6.50 1.65 1.65 41.00 11.63 11.67 11.67
7.00 1.70 1.67 1.70 1.72 2.87 2.86 42.00 11.72 11.78 11.78
7.50 1.76 43.00 11.80 11.86 11.86
8.00 1.68 1.76 1.81 1.85 3.24 3.18 44.00 11.86 11.92 11.92
8.50 1.88 45.00 11.91 11.97 11.97
9.00 1.76 1.86 1.94 1.96 4.59 4.00 46.00 11.96 12.02 12.02
10.00 1.78 1.96 2.05 2.09 6.16 6.09 47.00 11.99
11.00 1.82 2.05 2.18 2.22 6.53 6.49
12.00 2.24 2.18 2.31 2.33 6.77 6.75
13.00 2.37 2.29 2.44 2.47 6.97 6.95
14.00 2.50 2.42 2.59 2.61 7.16 7.13
15.00 2.68 2.57 2.75 2.75 7.36 7.34
16.00 2.85 2.72 3.00 7.58 7.56
17.00 3.07 2.89 3.30 7.92 7.85
17.50 3.47
18.00 2.91 3.08 3.88 3.58 8.54 8.41
19.00 3.44 3.26 8.20 4.16 9.84 11.17
20.00 3.59 3.43 8.88 7.67 11.09 11.40
21.00 3.76 3.60 9.14 8.42 11.36 11.57
 

CHEM 41 Lab | Preparation of Buffers & Amino Acids as Ampholytes 13