Bolandos, Kim Leonard C.

Chem 161.1
Group 1

Exercise 1
pH and Buffer System
Postlab Report

Results and Discussion

Biochemical processes happening in cells are greatly influenced by the pH. Any small change in
pH may produce large change in the rate of the process. Cells and organisms keep biomolecules in their
optimal ionic state by maintaining a constant cytosolic pH, usually near pH 7. Constancy in pH is
achieved with the help of biological buffers. Extracted cell components must be maintained in their
normal pH to remain stable in the laboratory since the natural environment of biomolecules are in strict
pH control (Nelson & Cox, 2008).

A buffer system consists of a weak acid (the proton donor) and its conjugate base (the proton
acceptor) or a weak base and its conjugate acid. When H + is added to a buffer, the weak acid's conjugate
base or the weak base will accept a proton (H +), thereby "absorbing" the H + before the pH of the solution
lowers significantly. Similarly, when OH- is added, the weak acid or the weak base’s conjugate acid will
donate a proton (H+) to its conjugate base, thereby resisting any increase in pH before shifting to a
new equilibrium point. However, buffer systems have limit. The buffer capacity can be exceeded when
large amounts of H3O+ and OH- are added to a buffer; then pH changes since the buffer system is
overwhelmed (Stoker, 2010).
−¿
+¿+ A¿(aq)
Reaction 1.1 For Weak Acid-Conjugate Base
HA (aq )+ H 2 O (l) → H 3 O¿(aq )
+¿ ¿
−¿+ HA (aq)
Reaction 1.2 For Weak Base-Conjugate Acid
A (aq)+ H 2 O (l ) → O H ¿(aq)

Each conjugate acid-base pair has a characteristic pH range where it works as an effective buffer.
The buffering region is about 1 pH unit on either side of the pK a of the conjugate acid. The midpoint of
the buffering region is when one-half of the acid reacts to dissociation and where the concentration of the
proton donor (acid) equals that of the proton acceptor (base). In other words, the pH of the equimolar
solution of acid (e.g., when the ratio of the concentration of acid and conjugate base is 1:1) is equal to
the pKa. This represents the point in the titration that is halfway to the equivalence point. This region is
the most effective for resisting large changes in pH when either acid or base is added.
Each conjugate acid-base pair has a characteristic pH range where it works as an effective buffer. The
buffering region is about 1 pH unit on either side of the pK a of the conjugate acid. The midpoint of the
buffering region is when one-half of the acid reacts to dissociation and where the concentration of the
proton donor (acid) equals that of the proton acceptor (base). The Henderson-Hasselbach equation
relates the pH, pKa and the concentration of the buffer components. From this equation, the ratio of the
of the proton donor and acceptor, pKa or the pH of the compound can be calculated given that the other
components are determined.

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 −¿¿
A
¿
[¿ )
Equation 1.1
[ HA ]
¿
pH= pKa+ log ¿

Buffering deals with reversible equation equilibria that is happening on the solution and when the
pKa is equal to the pH, as said earlier, this signifies that the proton donor and acceptor is in equal
concentration.
Buffer solutions are often characterized by its range and capacity. Buffer range refers to the pH range
of the buffer capacity. This means that it is the pH range where a buffer can maintain a constant pH upon
addition of acid or base in small amounts. On the other hand, buffer capacity is the amount of acid or base
that can be added to known volume of buffer before pH changes significantly (Stoker, 2010). Buffer
capacity is illustrated by a titration curve. The buffer zone or the middle part of the curve is flat because
addition of base or acid does not drastically affect the pH of the solution. But, if the curve extends outside
the buffer region, pH will increase tremendously when small amount of base is added to a buffer system.
If too much acid is added to the buffer, extra protons remain free and the pH will fall sharply (Nelson &
Cox, 2008).

In biochemical experiments, buffer solutions are important especially when working with
biomolecules. Buffer acts to stabilize the ionic status during the course of experiment since most of
biomolecules vary their ionic structure whenever environmental pH changes. This variation in ionic
structure may denature or inactivate biomolecules or pathways that are important in biological processes.
Buffers in the laboratory are prepared using the Henderson-Hasselbalch equation (Eq. 1.1) to determine
the volume needed and also the theoretical values.
Most biological molecules in the living system perform chemical processes at constant pH.
Physiological buffers mitigate the any drastic changes in pH inside the body. Two especially important
biological buffers are the phosphate and bicarbonate systems (Berg et al., 2012).The bicarbonate buffer is
consists of carbonic acid as proton donor and bicarbonate as proton acceptor (Reaction 1.3). It is the main
buffer found in the blood plasma and the main extracellular buffer system that provides a way of
removing CO2 produced by metabolizing tissues. The pH of this buffer system depends on the
concentration of its components. Also, the concentration of the carbonic acid depends on the
concentration of dissolved CO2 in the blood.

H2CO3 ⇌ H+ + H2CO32- Reaction 1.3

The phosphate buffer is a suitable buffer system for biological fluids such as cytoplasmic and
extracellular fluids. It most effective at pH close to its pKa of 6.86, with abuffering range of 5.9 to 7.9.It
is consists of H2PO4- as the proton donor and HPO42- as the proton acceptor (Nelson & Cox, 2008). This
was also the buffer used in the exercise.

H2PO4-⇌ H+ + HPO42- Reaction 1.4

Upon addition of acid:

HPO42- + H+⇌ H2PO4- + H2O Reaction 1.5
Upon addition of base:
H2PO4- + OH-⇌HPO42- + H2O Reaction 1.6

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 A pH meter was used in this exercise to measure the pH of the solutions. Before measuring an
unknown solution, the pH meter was first standardized with buffer solution of known pH (pH 4, 7, and
10). This is an important step since this will ensure the calibration and the accuracy of the instrument to
be used. However, measurement of pH may have some errors such as temperature that affects the pKa of
the ions in the solution since this are temperature dependent species. There are also problems using pH
meter such as the sodium error which is significant at high pH, poor conductivity of the instrument and
the effect of concentration due to activity of ionic species which was corrected during the experiment by
adjusting the actual pH of the solutions near the calculated pH (Boyer, 2012).
Table 1.1. Observation on the effect of buffer concentration on buffering capacity.

Buffer Concentration Original pH pH after addition of Change in pH

base
Stock solution 7.2 7.3 0.1
1:10 7.1 10.9 3.8
1:50 6.8 11.3 4.5
1:100 6.7 11.0 4.3

In this exercise, the effect of buffer concentration on buffering capacity was tested using a
phosphate buffer. A 50 mL of 0.1 M pH 7.2 of phosphate buffer was prepared using 0.1 M Na 2HPO4 and
0.1 M NaH2PO4 solutions and also corresponding dilutions seen of Table 1.1 were prepared to observed
the effect of buffer concentration on buffering capacity. From the different concentration; stock solution,
1:10, 1:50 and 1:100, the original pH was determined before addition of the NaOH using a pH meter
(which was calibrated earlier prior to using it). After the addition of the said base, it was measured again
using a pH meter to determine the change in pH the buffer has went.
Based on the results on Table 1.1, it can somehow look that as the buffer decreases its
concentration or the more diluted the buffer is, the pH changes significantly. It can be stated that the pH
of the buffer solution on the diluted samples are not maintained. This can signify that as the concentration
of the weak acid and its conjugate or weak base and its conjugate acid gets smaller, the smaller also the
buffering capacity of the buffer system. This is because that the smaller the concentration of the
components mean that the smaller the interaction for the buffer system to respond to the added base or
acid, therefore a significant change in pH will result.
The effect of the ratio of conjugate base to weak acid on buffering capacity was also determined
in this exercise using the phosphate buffer. Several buffer solutions were prepared with varying
concentrations of conjugate base to weak acid. The ratio of the conjugate base to weak acid was also
calculated using the Henderson-Hasselbach equation. The pH values of each solutions were obtained
using a pH meter and Henderson-Hasselbalch equation. The pH was adjusted for beakers 2 to 4 when the
difference between calculated pH and measured pH was greater than ± 0.05 using 0.1 M HCl or 0.1 M
NaOH. For each prepared solution, 2.00 mL of NaOH was added and the pH was measured. The change
in pH was calculated. Then the mentioned procedures were repeated, but instead of adding NaOH, 2.00
mL of HCl was added to each solutions.
Table 1.2a. Preparation of the phosphate buffer system.

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 Test Tube No. Vol. of 0.1 M Vol. of 0.1 M pH H 2 P O2−¿
4
Na2HPO4 (mL) NaH2PO4 (mL) 2−¿
HPO 4
¿
¿
¿
1 0.60 19.40 5.7 0.0309
2 1.80 18.20 6.2 0.0989
3 10.00 10.00 7.2 1
4 18.20 1.80 8.2 10.111
5 19.40 0.60 8.7 32.330
6 20 mL distilled water

Table 1.2b. Observation on the effect of the ratio of conjugate base to weak acid ( upon addition of
NaOH).

Test Tube No. Measured pH pH after adding NaOH Change in pH

1 5.7 6.1 0.4
2 6.2 6.5 0.3
3 7.2 7.3 0.1
4 8.2 8.9 0.7
5 8.7 10.7 2.0
6 8.7 11.2 2.5

Table 1.2c. Observation on the effect of the ratio of conjugate base to weak acid (upon addition of HCl).

Test Tube No. Measured pH pH after adding HCl Change in pH

1 5.7 3.7 2.0
2 6.2 5.6 0.6
3 7.2 6.8 0.4
4 8.2 7.5 0.7
5 8.7 7.8 2.9
6 8.7 2.3 6.4

For table 1.2a, the preparation of the phosphate buffer system were shown with corresponding
mL of Na2HPO4 and NaH2PO4. Also the ratio was again calculated using Henderson-Hasselbach equation
(see Eq. 1.1). Based on the results of table 1.2b and 1.2c, when the ratio of the conjugate base to weak
acid is closer or equal to 1, there is a small or little change in the buffer system after an addition of a base
or an acid. Highest buffering capacity is observed for test tube no. 3 since the ratio is equal to 1. This is
because there is enough base or acid component of the buffer that can correspond on the addition of a
base or an acid resulting to a little change in the pH. A buffer is most effective when it contains equal
concentrations of the weak acid and its conjugate base. The buffering region is where pH = pKa ± 1. The
pKa represents the center of the buffering region. As seen in Eq 1.1, pH = pKa when [A-] is equal to [HA]
or equal to 1. This represents the point in the titration that is halfway to the equivalence point. This region
is the most effective for resisting large changes in pH when either acid or base is added (Boyer, 2012).
In table 1.2b and 1.2c, it can be observed that as the ratio of the conjugate base and weak acid of
the buffer system gets farther from 1, the change in pH proportionally increases with it.

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 For the last experiment for this exercise, titration of an unknown amino acid was done. In the
zwitterionic form, amino acid can act as a buffer because it can react to an added acid or base and kept the
pH constant or goes to little changes. The pH of the solution of unknown amino acid was adjusted to 1.5
using 1 M HCl and was titrated using 0.1 M KOH in 0.5 mL interval until about pH 12 was reached. The
solution was stirred constantly during titration using a magnetic bar. A titration curve was then plotted
using the data obtained.

Table 1.3. Data on the titration of an unknown amino acid.

Volume (mL) pH Voluma (mL) pH
0 1.5 5.5 2.7
0.5 1.5 6.0 3.2
1.0 1.6 6.5 8
1.5 1.6 7.0 8.7
2.0 1.8 7.5 9.1
2.5 1.9 8.0 9.5
3.0 2.0 8.5 9.8
3.5 2.0 9.0 10.9
4.0 2.1 9.5 11.4
4.5 2.3 10.0 11.8
5.0 2.5 10.5 12.1

14

12
C
10

8
pH
6 B

4 A
2

0
0 2 4 6 8 10 12
Volume of KOH added, mL

Figure 1.1. Titration Curve for the unknown amino acid.

Structures:

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 (A) (B) (C)

As the volume of KOH increases, this is expected to go to a more basic pH. Upon titration, the
figure 1.1 shows the changes in pH upon addition of KOH. Important piece of information derived from
the titration curve of an amino acid is the relationship between its net charge and the pH of the solution.
The characteristic pH at which the net electric charge is zero is called the isoelectric point or
isoelectric pH designated pI where the amino acid exist in the zwitterionic form. The isoelectric point is
simply the arithmetic mean of the two pKa values:

Using the titration curve, the pKa values were determined via interpolation. The pKa 1 is 2 and
pKa2 is 9.60 and IpH of 5.95.Based on the pKa and IpH values, the identity of the unknown amino acid
was valine. The pKa values of valine base on literature are 2.32 and 9.62 for pka1 and pka2 respectively
and an IpH of 5.97. This amino acid can act as a buffer at pH 1.36 to 3.36 and pH 8.60 to 10.60 using the
formula pH = pKa ±1 to get the buffering region.

Possible errors that might affected the results of the experiment include the inaccurate preparation of
the buffer solutions and effect of concentration due to activity of ionic species which was corrected during
the experiment by adjusting the actual pH of the solutions near the calculated pH. In order to achieve
optimum results, it is recommended to check the cleanliness of the glassware and follow the instructions
carefully.

Sample Calculations

1.) Preparation of 50 mL of 0.1 M phosphate buffer at pH 7.2. (MM NaH2PO4 = 141.958 g/mol and MM
of NaH2PO4 = 119.76 g/mol.
−¿¿
A
pH= pKa log + ¿
¿
¿

2−¿
HPO¿4
7.2 = pKa + log ¿
¿
¿

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 2−¿
HPO¿4
log ¿ = antilog (7.2-7.21) = 0.977237221
¿
¿

 Amount of 0.1 M Na2HPO4 (mL)

(0.1 moll )(50 mL X 10001 LmL )( 1+0.977237221

0.977237221
)
V0.1 M Na2HPO4 = = 24.71218958 mL
( 0.1 molL )( 10001 LmL )

 Amount of 0.1 M NaH2PO4 (mL)

(0.1 moll )(50 mL X 10001 LmL )( 1+0.977237221

1
)
V0.1 M NaH2PO4 = = 25.28781042 mL
( 0.1 molL )( 10001 LmL )

2.) For dilutions in determining the effect of buffer concentration to buffer capacity.

1: 10 dilution
Let X be the vol. of buffer needed
1 X
= → X = 5 mL buffer
10 50
Volume of diluent = 50-5 = 45 mL dH2O

3.) Calculation of the pH of the resulting mixture and ratio of the conjugate base to weak
acid.

 Test tube 1 , 0.60 mL 0.1 M Na2HPO4+ 19.40 mL 0.1 M NaH2PO4

2−¿
HPO¿4
pH = pKa + log ¿
¿
¿
0.60
pH = 7.21 + log 19.40
pH = 5.70034952

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 4.) Computation on the change of pH.

For test tube 1 addition of NaOH:

ΔpH = final pH – initial pH
ΔpH = 6.1-5.7 = 0.4

5.) Computation of IpH

( p Ka1 + p Ka2 )
IpH =
2
( 2.32+ 9.62 )
IpH = 2

IpH = 5.97

References:
 Boyer, Rodney. 2012. Biochemistry Laboratory: Modern theory and techniques. 2 nd ed. New Jersey:
Pearson Education, Inc.
 Berg J.M., Tymoczko J.L., Styler L. 1975. Biochemistry 5thed. W.H. Freeman and Company
 Nelson, David L. and Michael M. Cox. 2008. Lehninger Principles of Biochemistry. 5 th ed. New
York: W.H. Freeman and Company.
 Stoker, H.S. 2010. General, Organic, and Biological Chemistry. 5th ed. USA: Brooks/Cole Cengage
Learning.

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