Department of Pure and Applied Chemistry

Visayas State University

Visca, Baybay City, Leyte

Name: Castil, Joyce B. Date Submitted: January 15, 2021

Lab Schedule: TTh, 8:00 – 11:00 Rating:

Exercise No. 2
pH and Buffer System

Abstract

The objective of this exercise was to calibrate pH meter, choose and prepare appropriate

buffer systems and to titrate an amino acid. Standard buffer solutions with pH 4, 7 and 10 were

used to calibrate the pH meter. There were factors affecting the buffer capacity, the effect of

concentration and ratio of weak acid and conjugate base using phosphate and acetate buffer.

Through the exercise, it showed that buffers resist pH but, on a limit, depending on their buffering

capacities. Outside their buffer capacities, buffer changes pH. A preferred buffer solution has a Ka

close to the given pH and an A/B ratio of 1. The calculations prove that the acetate buffer was

ideal for the protein precipitation. The titration curved of glycine showed two pKa values which

fit the description of glycine that can act as a buffer in two pH ranges. Maintaining a constant

blood pH is critical for the proper functioning of our body, and the buffer that maintains the pH of

human blood involves carbonic acid (H2CO3) – bicarbonate ion (HCO3-) system.
Introduction

pH is a measure of acidity and alkalinity of a solution that is a number on a scale on which

a value of 7 represents neutrality and lower numbers indicate increasing acidity and higher

numbers increasing alkalinity and on which each unit of change represents a tenfold change in

acidity or alkalinity and that is the negative logarithm of the effective hydrogen-ion concentration

or hydrogen-ion activity in gram equivalents per liter of the solution (Merriam Webster, 2020).

The control of pH is important in organisms and their cells because chemical reactions and

processes are affected by the hydrogen ion concentration. Change in pH can alter the number of

positively and negatively charged groups. The net charge on the protein effects it’s three-

dimensional structure and thus the enzymatic activity (Parsons & Schapiro, 2018).

pH = -log [H+]

Buffers are chemicals or combinations of chemicals that tend to prevent changes in the

concentration of hydrogen ions. Buffers are composed of mixtures of weak acids and their

corresponding salts. Using the Bronsted – Lowry definition, an acid is a compound that can

donated a hydrogen ion. A weak acid is one that does not completely ionize, or dissociate, in

solution. The extent of dissociation is given by the equilibrium constant Ka for the reaction:

HA ↔ H+ + A-

The equilibrium constant for the ionization of this acid is then:

Ka = [H+][A-]/[HA]
This is the measure of the ease with which the acid donates its hydrogen ion. Higher Ka indicates

that the acid will dissociate more completely into ions. The equilibrium constant equation may

also be used to determine the hydrogen ion concentration:

[H+] = (Ka)[HA]/[A-]

And, since pH = -log[H+] and pKa = -logKa, then,

[𝐴− ]
𝑝𝐻 = 𝑝𝐾𝑎 + log ( )
[𝐻𝐴]

This last equation is the Henderson – Haselbalch equation. It reveals the relationship of pH and

pKa (Parsons & Schapiro, 2018).

Hydrogen ion concentration is usually measured with a pH meter. The pH meter is a

potentiometer, capable of accurately measuring small electrical potential differences. The glass

electrode consists of a thin bubble of soft glass that contain a solution of KCl and acetic acid in

which a platinum wire is immersed. An electrical potential is developed across the glass bubble,

which is proportional to hydrogen ion concentration, the reference electrode is simply used as a

standard against which the glass electrode can be compared. In practice, the pH meter and

electrodes are calibrated against a buffer solution of known pH and potential differences are read

directly in units of pH (Calzada, 2016).

Methodology

A. Calibration of the pH meter

The laboratory instruction was carefully followed in using the pH meter, the instrument

was checked by the laboratory instructor before it was used. It was being remembered that

the glass electrode is fragile and that minor bumps of the glass membrane will ruin it. There

were three buffers used to calibrate the pH meter, the pink buffer that will give a reading

of 4 pH, green buffer that will read 7 pH and the blue buffer that will give a reading of 10

pH.

B. Factors Affecting Buffer Capacity

1. Effect of concentration

Different concentration of phosphate and acetate buffer solutions were prepared. In

case 1, 0.1 M phosphate buffer with a pH of 7.2 was used to prepare 25 mL each of the

buffer solutions with the following concentrations; 0.005 M, 0.05 M and 0.10 M at pH

7.2. While in case 2, 0.1 M acetate buffer, pH 4.7 was used to prepare 25 mL each of

the buffer solutions with the following concentrations; 0.005 M, 0.05 M and 0.10 M at

pH 4.7. The pH of each buffer solutions was recorded. 2 mL of 0.1 M NaOH was added

to each of the 25 mL buffer samples. The pH of each buffer solution was recorded after

the addition of alkali. The magnitude of the change in pH was accounted.

2. Effect of the ratio of the conjugate base to weak acid

The ratio of dihydrogen phosphate (H2PO4-) and monohydrogen phosphate (HPO42-)

components required to produce buffer solution with pH 6.2, pH 7.2 and pH 8.2 were

calculated from the Henderson – Hasselbalch equation. Furthermore, from the

Henderson – Hasselbalch equation the ratio of acetic acid and acetate required to
 produce buffer solution with pH 3.7, pH 4.7 and pH 5.7 were calculated. Using the

stock solutions prepared a 25 mL of each of the solutions was made. The pH of each

of the buffer solutions was measured and recorded. 2 mL of 0.1 M NaOH was added

to each of the 25 mL buffer solutions, the pH of each buffer solution after the addition

was then measured and recorded. The magnitude of pH shift in each with the reference

to the direction of pH shift was accounted.

C. Choice and Preparation of a Buffer System

1. Choosing the proper buffer solution

In protein precipitation, two liters of 5 mM buffer solution with pH 5.2 was needed in

the isolation of albumin. One of the following buffer solutions was chosen as the best

fitted buffer solution for the said purpose.

Buffer solutions pKa

Acetate buffer 4.73

Tris- (hydroxymethyl) aminomethane 8.08

Phosphate buffer 7.20

2. Preparation of the chosen buffer system

The amounts (in grams if solid and in mL if liquid) of weak acid and conjugate base

needed was measured and calculated to be able to prepare the chosen buffer system in

part A above. The answer was expressed in useful units (that is, prepare it from practical

amounts or concentrations of starting materials).

D. Titration of amino acid

In this part of experiment an amino acid sample was titrated. A 10 mL of amino acid sample

was pipetted into a 25 mL or 50 mL beaker. The pH was adjusted to 1.5. a burette was used
 to add 0.1 M NaOH in approximately 0.5 mL increments until about pH 12 was reach. (the

accurate volume of each increment was recorded). The pH was stirred well and measured

after each addition. The results of pH vs. mL of NaOH added was plotted. The pKa’s of

the sample was determined from the graph. The structure of amino acid at each pKa value

was drawn.

E. pH of the blood

By studying the table of pKa values provided, compounds that could act as buffers at

physiological pH values were suggested (i.e. around pH 7.4).

Results and Discussion

Calibration of the pH meter

Table 1. pH meter calibration

Buffer Color pH meter theoretical reading
Pink 4
Green 7
Blue 10

A pH calibration is the process of adjusting your pH meter by measuring solutions of

known pH value. This is because the characteristics of your electrode will change over time and

this needs to be compensated for. A calibration does this by matching your pH meter to the current

characteristics of your pH sensor (Synotronics, 2021). Table 1 shows the three standard buffer

solution use in pH meter calibration.

 Figure 1. Standard buffer solutions

Factors affecting buffer capacity

Table 2. Effect of concentration of phosphate buffer

Prepared buffer Magnitude of
pH after adding 2
solution concentration Initial pH change
mL NaOH
(M)
0.1 7.2 7.34 0.14
0.05 7.2 7.49 0.27
0.005 7.2 10.13 3.11

Table 3. Effect of concentration of acetate buffer

Prepared buffer Magnitude of
pH after adding 2
solution concentration Initial pH change
mL NaOH
(M)
0.1 4.7 4.84 0.14
0.05 4.7 4.99 0.29
0.005 4.7 5.64 0.94

Bases on the results above from table 2 and 3, the pH of the buffers remains the same as

the concentration changes. The obtained pH 7.2 for phosphate and 4.7 for acetate were theoretical

data since there were no experimental data. After the addition of NaOH a strong base, the pH of

the buffer solutions increased which indicated that the pH are already outside the range of their
buffering capacities, due to the acids present in the buffer systems that are almost used up to react

with the added base, causing the pH of the buffers to rise.

Calculations

• pH of 25 ml 0.1 M phosphate buffer solution after addition with 2 ml 0.1 M NaOH

𝑛 𝐻2 𝑃𝑂4− + 𝑛 𝐻𝑃𝑂4−2 𝑚𝑜𝑙

= 0.1
0.025 𝐿 𝐿
𝑚𝑜𝑙
2𝑛 𝐻2 𝑃𝑂4− = (0.025𝐿)(0.1 )
𝐿
2 𝑛 𝐻2 𝑃𝑂4− = 0.0025 𝑚𝑜𝑙

𝑛 𝐻2 𝑃𝑂4− = 1.25 × 10−3

𝑛 𝐻𝑃𝑂4−2 = 𝑛 𝐻2 𝑃𝑂4− = 1.25 × 10−3

𝑯𝟐 𝑷𝑶−
𝟒 + OH- 𝑯𝑷𝑶−𝟐
𝟒 + 𝑯𝟐 𝑶

𝟏. 𝟐𝟓 × 𝟏𝟎−𝟑 2 × 10−4 1.25 × 10−3

− 𝟐 × 𝟏𝟎−𝟒 − 2 × 10−4 + 2 × 10−4

𝟏. 𝟎𝟓 × 𝟏𝟎−𝟑 0 1.45 × 10−3

[𝐴− ]
pH = pKa + log [𝐻𝐴]

[ 𝟏.𝟒𝟓 × 𝟏𝟎−𝟑 ]
pH = 7.2 + log [𝟏.𝟎𝟓 × 𝟏𝟎−𝟑 ]

pH = 7.34

• pH of 25 ml 0.05 M phosphate buffer solution after addition with 2 ml 0.1 M NaOH

𝑛 𝐻2 𝑃𝑂4− + 𝑛 𝐻𝑃𝑂4−2 𝑚𝑜𝑙

= 0.05
0.025 𝐿 𝐿
−
𝑚𝑜𝑙
2 𝑛 𝐻2 𝑃𝑂4 = (0.025𝐿)(0.05 )
𝐿
2 𝑛 𝐻2 𝑃𝑂4− = 1.25 × 10−3 𝑚𝑜𝑙
𝑛 𝐻2 𝑃𝑂4− = 6.25 × 10−4
𝑛 𝐻𝑃𝑂4−2 = 𝑛 𝐻2 𝑃𝑂4− = 6.25 × 10−4
 𝑯𝟐 𝑷𝑶−
𝟒 + OH- 𝑯𝑷𝑶−𝟐
𝟒 + 𝑯𝟐 𝑶

𝟔. 𝟐𝟓 × 𝟏𝟎−𝟒 2 × 10−4 6.25 × 10−4

− 𝟐 × 𝟏𝟎−𝟒 − 2 × 10−4 + 2 × 10−4

𝟒. 𝟐𝟓 × 𝟏𝟎−𝟒 0 8.25 × 10−4

[𝐴− ]
pH = pKa + log [𝐻𝐴]

[ 𝟖.𝟐𝟓 × 𝟏𝟎−𝟒 ]
pH = 7.2 + log [𝟒.𝟐𝟓 × 𝟏𝟎−𝟒 ]

pH = 7.49

• pH of 25 ml 0.005 M phosphate buffer solution after addition with 2 ml 0.1 M NaOH

𝑛 𝐻2 𝑃𝑂4− + 𝑛 𝐻𝑃𝑂4−2 𝑚𝑜𝑙

= 0.005
0.025 𝐿 𝐿
−
𝑚𝑜𝑙
2 𝑛 𝐻2 𝑃𝑂4 = (0.025𝐿)(0.005 )
𝐿
2 𝑛 𝐻2 𝑃𝑂4− = 1.25 × 10−4 𝑚𝑜𝑙
𝑛 𝐻2 𝑃𝑂4− = 6.25 × 10−5
𝑛 𝐻𝑃𝑂4−2 = 𝑛 𝐻2 𝑃𝑂4− = 6.25 × 10−5

𝑯𝟐 𝑷𝑶−
𝟒 + OH- 𝑯𝑷𝑶−𝟐
𝟒 + 𝑯𝟐 𝑶

𝟔. 𝟐𝟓 × 𝟏𝟎−𝟓 2 × 10−4 6.25 × 10−5

- 𝟔. 𝟐𝟓 × 𝟏𝟎−𝟓 − 6.25 × 10−5 + 6.25 × 10−5

0 1.375 × 10−4 1.25 × 10−4

Hydrolysis of the conjugate base:

𝑯𝑷𝑶−𝟐
𝟒 + 𝑯𝟐 𝑶 𝑯𝟐 𝑷𝑶−
𝟒 + OH-

𝟏. 𝟐𝟓 × 𝟏𝟎−𝟒 0 1.375 × 10−4

−𝒙 +𝑥 +𝑥
 𝟏. 𝟐𝟓 × 𝟏𝟎−𝟒 − 𝒙 +𝑥 1.375 × 10−4 + 𝑥

[𝐻2 𝑃𝑂4− ][𝑂𝐻 − ]

𝐾𝑏 = = 1.6 𝑥 10−7
[𝐻𝑃𝑂4−2 ]
𝑥][1.375 × 10−4 + 𝑥]
= 1.6 𝑥 10−7
[1.25 × 10−4 − 𝑥]
1.375 × 10−4 + 𝑥 + 𝑥 2 = 2.0 𝑥 10−11 − 1.6 𝑥 10−7
𝑥 2 + 1.3766 × 10−4 𝑥 − 2.0 𝑥 10−11 = 0
𝑥1 = 1.451324754 𝑥 10−7 ; 𝑥2 = −1.378051325 𝑥 10−4
Since there are no negative value of moles 𝑥 = 1.451324754 𝑥 10−7 is used.
New concentration of weak acid and its conjugate base is then calculated by the following:
[𝐻𝑃𝑂4− ] = 1.25 × 10−4 − 𝑥
[𝐻2 𝑃𝑂4− ] = 1.25 × 10−4 − 1.451324754 𝑥 10−7 = 1.248548675 𝑥 10−4 𝑚𝑜𝑙
[𝐻2 𝑃𝑂4− ] = 𝑥 = 1.451324754 𝑥 10−7
Using the Henderson-Hasselbalch equation, the pH is calculated by:
[𝐴− ]
pH = pKa + log [𝐻𝐴]

[1.248548675 𝑥 10−4 ]
pH = 7.2 + log [1.451324754 𝑥 10−7 ]

pH = 10.13

• pH of 25 ml 0.1 M acetate buffer solution after addition with 2 ml 0.1 M NaOH

𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 + 𝑛 𝐶𝐻3 𝐶𝑂𝑂− 𝑚𝑜𝑙

= 0.1
0.025 𝐿 𝐿
𝑚𝑜𝑙
2𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = (0.025𝐿)(0.1 )
𝐿
2 𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 0.0025 𝑚𝑜𝑙
𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 1.25 × 10−3
𝑛 𝐶𝐻3 𝐶𝑂𝑂− = 𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 1.25 × 10−3

𝑪𝑯𝟑 𝑪𝑶𝑶𝑯 + OH- 𝑪𝑯𝟑 𝑪𝑶𝑶− + 𝑯𝟐 𝑶

𝟏. 𝟐𝟓 × 𝟏𝟎−𝟑 2 × 10−4 1.25 × 10−3

− 𝟐 × 𝟏𝟎−𝟒 − 2 × 10−4 + 2 × 10−4

𝟏. 𝟎𝟓 × 𝟏𝟎−𝟑 0 1.45 × 10−3

[𝐴− ]
pH = pKa + log [𝐻𝐴]

[ 𝟏.𝟒𝟓 × 𝟏𝟎−𝟑 ]
pH = 4.7 + log [𝟏.𝟎𝟓 × 𝟏𝟎−𝟑 ]

pH = 4.84

• pH of 25 ml 0.05 M acetate buffer solution after addition with 2 ml 0.1 M NaOH

𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 + 𝑛 𝐶𝐻3 𝐶𝑂𝑂− 𝑚𝑜𝑙

= 0.05
0.025 𝐿 𝐿
𝑚𝑜𝑙
2𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = (0.025𝐿)(0.05 )
𝐿
−3
2 𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 1.25 × 10 𝑚𝑜𝑙
𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 6.25 × 10−4

𝑛 𝐶𝐻3 𝐶𝑂𝑂− = 𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 6.25 × 10−4

𝑪𝑯𝟑 𝑪𝑶𝑶𝑯 + OH- 𝑪𝑯𝟑 𝑪𝑶𝑶− + 𝑯𝟐 𝑶

𝟔. 𝟐𝟓 × 𝟏𝟎−𝟒 2 × 10−4 6.25 × 10−4

− 𝟐 × 𝟏𝟎−𝟒 − 2 × 10−4 + 2 × 10−4

𝟒. 𝟐𝟓 × 𝟏𝟎−𝟒 0 8.25 × 10−4

[𝐴− ]
pH = pKa + log [𝐻𝐴]

[ 𝟖.𝟐𝟓 × 𝟏𝟎−𝟒 ]
pH = 4.7 + log [𝟒.𝟐𝟓 × 𝟏𝟎−𝟒 ]

pH = 4.99

• pH of 25 ml 0.005 M Phosphate solution after addition with 2 ml 0.1 M NaOH

𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 + 𝑛 𝐶𝐻3 𝐶𝑂𝑂 − 𝑚𝑜𝑙
= 0.005
0.025 𝐿 𝐿
𝑚𝑜𝑙
2𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = (0.025𝐿)(0.005 )
𝐿
2 𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 1.25 × 10−4 𝑚𝑜𝑙
𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 6.25 × 10−5
 𝑛 𝐶𝐻3 𝐶𝑂𝑂− = 𝑛 𝐶𝐻3 𝐶𝑂𝑂𝐻 = 6.25 × 10−5

𝑪𝑯𝟑 𝑪𝑶𝑶𝑯 + OH- 𝑪𝑯𝟑 𝑪𝑶𝑶− + 𝑯𝟐 𝑶

𝟔. 𝟐𝟓 × 𝟏𝟎−𝟓 2 × 10−4 6.25 × 10−5

- 𝟔. 𝟐𝟓 × 𝟏𝟎−𝟓 − 6.25 × 10−5 + 6.25 × 10−5

0 1.375 × 10−4 1.25 × 10−4

Hydrolysis of the conjugate base:

𝑪𝑯𝟑 𝑪𝑶𝑶− + 𝑯𝟐 𝑶 𝑪𝑯𝟑 𝑪𝑶𝑶𝑯 + OH-

𝟏. 𝟐𝟓 × 𝟏𝟎−𝟒 0 1.375 × 10−4

−𝒙 +𝑥 +𝑥

𝟏. 𝟐𝟓 × 𝟏𝟎−𝟒 +𝑥 1.375 × 10−4 + 𝑥

−𝒙

[𝐶𝐻3 𝐶𝑂𝑂𝐻][𝑂𝐻− ]
𝐾𝑏 = = 1.7 𝑥 10−5
[𝐶𝐻3 𝐶𝑂𝑂− ]
[𝑥][1.375 × 10−4 + 𝑥]
= 1.7 𝑥 10−5
[1.25 × 10−4 − 𝑥]
1.375 × 10−4 𝑥 + 𝑥 2 = 2.125 𝑥 10−9 − 1.7 𝑥 10−5 𝑥
𝑥 2 + 1.545 × 10−4 𝑥 − 2.125 𝑥 10−9 = 0
𝑥1 = 1.270867107 𝑥 10−5 ; 𝑥2 = −1.672086711 𝑥 10−4
Since there are no negative value of moles 𝑥 = 1.270867107 𝑥 10−5 is used.
New concentration of weak acid and its conjugate base is then calculated by the following:
[𝐶𝐻3 𝐶𝑂𝑂− ] = 1.25 × 10−4 − 𝑥
[𝐶𝐻3 𝐶𝑂𝑂− ] = 1.25 × 10−4 − 1.270867107 𝑥 10−5 = 1.122913289 𝑥 10−4 𝑚𝑜𝑙
[𝐶𝐻3 𝐶𝑂𝑂𝐻] = 𝑥 = 1.270867107 𝑥 10−5
Using the Henderson-Hasselbalch equation, the pH is calculated by:
[𝐴− ]
pH = pKa + log
[𝐻𝐴]

[1.122913289 𝑥 10−4 ]
pH = 4.7 + log [1.270867107 𝑥 10−5 ]

pH = 5.64
Table 4. Ratio of dihydrogen phosphate and monohydrogen phosphate
Buffer type Concentration (M) Expected pH Ratio (in moles)
(Base:Weak acid)
Phosphate 0.1 6.2 0.0089:0.0911
Phosphate 0.1 7.2 0.0494:0.0506
Phosphate 0.1 8.2 0.0907:0.0093

In finding the ratio of the weak acid and conjugate base needed to prepare the buffer

solution with different pH (6.2, 7.2, and 8.2) of dihydrogen phosphate and monohydrogen

phosphate, the Henderson-Hasselbalch equation was used. Shown in Table 4, 0.0089:0911 is the

ratio of moles of conjugate base to weak acid at pH 6.2 in which the moles of weak acid needed to

prepare the buffer solution is greater than the moles of conjugate base needed. The ratio of moles

of conjugate base to weak acid is 0.0494:0.0506 at pH 7.2 where the moles of conjugate base

needed to prepare the buffer solution is almost the same as the moles of weak acid needed. At pH

8.2, the ratio of moles of conjugate base to weak acid is 0.0907:0.0093, the moles of conjugate

base needed to prepare the buffer solution is now greater compared to the moles of weak acid

needed.

Based on the obtained results from the calculations, as the number of moles required

for the conjugate base increases, the pH of the buffer also increases and as the number of moles

needed for weak acid decreases, the pH of the buffer increases. Since the buffer approaches

basicity with increasing pH, this explains why the number of moles of conjugate base needed also

increases while the number of moles of weak acid needed to prepare the buffer decreases.
 Figure 2. Working range of buffer.

The pH of the buffer depends on the ratio of the conjugate base and weak acid. Buffers

should be made using an acid and its conjugate base. From the Ka value and the Henderson-

Hasselbalch equation, the exact ratio of the conjugate base to the acid for a desired pH can be

determined. The buffer can be considered most effective and stable following the rule of thumb

that the relative amount of acid and base should not differ by more than tenfold at pH 7.2, while

at pH 6.2 and pH 8.2 the ratio of the conjugate base and weak acid differs greatly because the

buffering capacity is also affected which means that the pH is outside its buffer range and is not

stable.

Calculations:

Case 1.
A. pH 6.2
[A− ]
pH = pKa + log
[HA]
Ka of H2PO4- = 6.2 x 10-8
pKa = −log Ka
pKa = −log (6.2 x 10−8 )
pKa = 7.21
At pH 6.2, substitute into the Henderson-Hasselbalch equation and solve for
 [HPO42− ]
[H2 PO4− ]
[HPO42− ]
6.2 = 7.21 + log
[H2 PO4− ]
[HPO42− ]
log = 6.2 − 7.21
[H2 PO4− ]
[HPO42− ]
log = −1.01
[H2 PO4− ]
[HPO42− ]
= 10−1.01
[H2 PO4− ]
[HPO42− ]
= 0.0977
[H2 PO4− ]

To calculate for [HA] and [A-], we utilize what we know:

[HA] + [A− ] = 0.1 M
[HPO42− ]
= 0.0977
[H2 PO4− ]
Thus, let [HA] = x and [A-] = y
x + y = 0.1 (Equation 1)
y
x
= 0.0977 (Equation 2)
From equation 2, we get y = 0.0977x
Inserting equation 2 in equation 1, we get
x + 0.0977x = 0.01
1.0977x = 0.1
0.1
x= = 0.0911
1.0977
Since [HA] = x therefore, [HA] = 0.0911 M
From equation 1, we get y = 0.1 − x
y = 0.1 − 0.0911 = 0.0089
Since [A-] = y therefore [A-] = 0.0089 M
The ratio in moles of [𝐇𝐏𝐎𝟒𝟐− ]: [𝐇𝟐 𝐏𝐎𝟒− ]= 𝟎. 𝟎𝟎𝟖𝟗: 𝟎. 𝟎𝟗𝟏𝟏

B. pH 7.2
[A− ]
pH = pKa + log
[HA]
Ka of H2PO4- = 6.2 x 10-8
pKa = −log Ka
pKa = −log (6.2 x 10−8 )
pKa = 7.21
At pH 7.2, substitute into the Henderson-Hasselbalch equation and solve for
[HPO42− ]
[H2 PO4− ]
[HPO42− ]
7.2 = 7.21 + log
[H2 PO4− ]
[HPO42− ]
log = 7.2 − 7.21
[H2 PO4− ]
 [HPO42− ]
log = −0.01
[H2 PO4− ]
[HPO42− ]
= 10−0.01
[H2 PO4− ]
[HPO42− ]
= 0.9772
[H2 PO4− ]
To calculate for [HA] and [A-], we utilize what we know:
[HA] + [A− ] = 0.1 M
[HPO42− ]
= 0.9772
[H2 PO4− ]
Thus, let [HA] = x and [A-] = y
x + y = 0.1 (Equation 1)
y
x
= 0.9772 (Equation 2)
From equation 2, we get y = 0.9772x
Inserting equation 2 in equation 1, we get
x + 0.9772x = 0.01
1.9772x = 0.1
0.1
x= = 0.0506
1.9772
Since [HA] = x therefore, [HA] = 0.0506 M
From equation 1, we get y = 0.1 − x
y = 0.1 − 0.0506 = 0.0494
Since [A-] = y therefore [A-] = 0.0494 M
The ratio in moles of [𝐇𝐏𝐎𝟒𝟐− ]: [𝐇𝟐 𝐏𝐎𝟒− ]= 𝟎. 𝟎𝟒𝟗𝟒: 𝟎. 𝟎𝟓𝟎𝟔

C. pH 8.2
[A− ]
pH = pKa + log
[HA]
Ka of H2PO4- = 6.2 x 10-8
pKa = −log Ka
pKa = −log (6.2 x 10−8 )
pKa = 7.21
At pH 8.2, substitute into the Henderson-Hasselbalch equation and solve for
[HPO42− ]
[H2 PO4− ]
[HPO42− ]
8.2 = 7.21 + log
[H2 PO4− ]
[HPO42− ]
log = 8.2 − 7.21
[H2 PO4− ]
[HPO42− ]
log = 0.99
[H2 PO4− ]
[HPO42− ]
= 100.99
[H2 PO4− ]
[HPO42− ]
= 0.0977
[H2 PO4− ]
To calculate for [HA] and [A-], we utilize what we know:
 [HA] + [A− ] = 0.1 M
[HPO42− ]
= 9.7724
[H2 PO4− ]
Thus, let [HA] = x and [A-] = y
x + y = 0.1 (Equation 1)
y
= 9.7724 (Equation 2)
x
From equation 2, we get y = 9.7724x
Inserting equation 2 in equation 1, we get
x + 9.7724x = 0.01
10.7724x = 0.1
0.1
x= = 0.0093
10.7724
Since [HA] = x therefore, [HA] = 0.0093 M
From equation 1, we get y = 0.1 − x
y = 0.1 − 0.0093 = 0.0907
Since [A-] = y therefore [A-] = 0.0907 M
The ratio in moles of [HPO42− ]: [H2 PO4− ]= 0.0907: 0.0093

Table 5. Ratio of acetic acid and acetate

Buffer type Concentration (M) Expected pH Ratio (in moles)
(Base:Weak acid)
Acetate 0.1 3.7 0.0084:0.0916
Acetate 0.1 4.7 0.0477:0.0523
Acetate 0.1 5.7 0.0901:0.0099

The Henderson-Hasselbalch equation was used to calculate the ratio of the weak acid

and conjugate base needed to prepare the buffer solution with different pH (3.7, 4.7 and 5.7) of

acetic acid and acetate. Shown in Table 5, at pH 3.7 the ratio of moles of conjugate base to weak

acid is 0.0084:0916 in which the moles of weak acid needed to prepare the buffer solution is greater

than the moles of conjugate base needed. The ratio of moles of conjugate base to weak acid is

0.0477:0.0523 at pH 4.7, where the moles of conjugate base needed to prepare the buffer solution

is almost the same as the moles of weak acid needed. 0.0901:0.0099 is the ratio of moles of

conjugate base to weak acid at pH 5.7 where the moles of conjugate base needed to prepare the

buffer solution is now greater compared to the moles of weak acid needed.
 Based on the obtained results from the calculations, as the number of moles required

for the conjugate base increases, the pH of the buffer also increases while as the number of moles

needed for weak acid decreases, the pH of the buffer increases. Since the buffer approaches

basicity with increasing pH, this explains why the number of moles of conjugate base needed also

increases while the number of moles of weak acid needed to prepare the buffer decreases.

Figure 3. Working range of buffer

The pH of the buffer depends on the ratio of the conjugate base and weak acid. Buffers

should be made using an acid and its conjugate base. From the Ka value and the Henderson-

Hasselbalch equation the exact ratio of the conjugate base to the acid for a desired pH can be

determined. The buffer can be considered most effective and stable following the rule of thumb

that the relative amount of acid and base should not differ by more than tenfold at pH 4.7, while

at pH 3.7 and pH 5.7, the ratio of the conjugate base and weak acid differs greatly because the

buffering capacity is also affected which means that the pH is outside its buffer range and is not

stable.
Calculations:

Case 2.
A. pH 3.7
[A− ]
pH = pKa + log
[HA]
Ka of CH3 COOH = 1.8x10−5
pKa = −log Ka
pKa = −log (1.8 x 10−5 )
pKa = 4.74
At pH 3.7, substitute into the Henderson-Hasselbalch equation and solve for
[CH3 COO− ]
[CH3 COOH]
[CH3 COO− ]
3.7 = 4.74 + log
[CH3 COOH]
[CH3 COO− ]
log = 3.7 − 4.74
[CH3 COOH]
[CH3 COO− ]
log = −1.04
[CH3 COOH]
[CH3 COO− ]
= 10−1.04
[CH3 COOH]
[CH3 COO− ]
= 0.0977
[CH3 COOH]
To calculate for [HA] and [A-], we utilize what we know:
[HA] + [A− ] = 0.1 M
[CH3 COO− ]
= 0.0912
[CH3 COOH]
Thus, let [HA] = x and [A-] = y
x + y = 0.1 (Equation 1)
y
x
= 0.0912 (Equation 2)
From equation 2, we get y = 0.0912x
Inserting equation 2 in equation 1, we get
x + 0.0912x = 0.01
1.0912x = 0.1
0.1
x= = 0.0916
1.0912
Since [HA] = x therefore, [HA] = 0.0916 M
From equation 1, we get y = 0.1 − x
y = 0.1 − 0.0916 = 0.0084
Since [A-] = y therefore [A-] = 0.0084 M
The ratio in moles of [𝐂𝐇𝟑 𝐂𝐎𝐎− ]: [𝐂𝐇𝟑 𝐂𝐎𝐎𝐇]= 𝟎. 𝟎𝟎𝟖𝟒: 𝟎. 𝟎𝟗𝟏𝟔

B. pH 4.7
[A− ]
pH = pKa + log
[HA]
Ka of CH3 COOH = 1.8x10−5
 pKa = −log Ka
pKa = −log (1.8 x 10−5 )
pKa = 4.74
At pH 4.7, substitute into the Henderson-Hasselbalch equation and solve for
[CH3 COO− ]
[CH3 COOH]
[CH3 COO− ]
4.7 = 4.74 + log
[CH3 COOH]
[CH3 COO− ]
log = 4.7 − 4.74
[CH3 COOH]
[CH3 COO− ]
log = −0.04
[CH3 COOH]
[CH3 COO− ]
= 10−0.04
[CH3 COOH]
[CH3 COO− ]
= 0.0977
[CH3 COOH]
To calculate for [HA] and [A-], we utilize what we know:
[HA] + [A− ] = 0.1 M
[CH3 COO− ]
= 0.9120
[CH3 COOH]

Thus, let [HA] = x and [A-] = y

x + y = 0.1 (Equation 1)
y
x
= 0.9120 (Equation 2)
From equation 2, we get y = 0.9120x
Inserting equation 2 in equation 1, we get
x + 0.9120x = 0.01
1.9120x = 0.1
0.1
x= = 0.0523
1.9120
Since [HA] = x therefore, [HA] = 0.0523 M
From equation 1, we get y = 0.1 − x
y = 0.1 − 0.0523 = 0.0477
- -
Since [A ] = y therefore [A ] = 0.0477 M
The ratio in moles of [𝐂𝐇𝟑 𝐂𝐎𝐎− ]: [𝐂𝐇𝟑 𝐂𝐎𝐎𝐇]= 𝟎. 𝟎𝟒𝟕𝟕: 𝟎. 𝟎𝟓𝟐𝟑

C. pH 5.7
[A− ]
pH = pKa + log
[HA]
Ka of CH3 COOH = 1.8x10−5
pKa = −log Ka
pKa = −log (1.8 x 10−5 )
pKa = 4.74
At pH 5.7, substitute into the Henderson-Hasselbalch equation and solve for
[CH3 COO− ]
[CH3 COOH]
[CH3 COO− ]
5.7 = 4.74 + log
[CH3 COOH]
[CH3 COO− ]
log = 5.7 − 4.74
[CH3 COOH]
[CH3 COO− ]
log = 0.96
[CH3 COOH]
[CH3 COO− ]
= 100.96
[CH3 COOH]
[CH3 COO− ]
= 9.1201
[CH3 COOH]
To calculate for [HA] and [A-], we utilize what we know:
[HA] + [A− ] = 0.1 M
[CH3 COO− ]
= 9.1201
[CH3 COOH]
Thus, let [HA] = x and [A-] = y
x + y = 0.1 (Equation 1)
y
= 9.1201 (Equation 2)
x
From equation 2, we get y = 9.1201x
Inserting equation 2 in equation 1, we get
x + 9.1201x = 0.01
10.1201x = 0.1
0.1
x= = 0.0099
10.1201
Since [HA] = x therefore, [HA] = 0.0099 M
From equation 1, we get y = 0.1 − x
y = 0.1 − 0.0099 = 0.0901
Since [A-] = y therefore [A-] = 0.0901 M
The ratio in moles of [𝐂𝐇𝟑 𝐂𝐎𝐎− ]: [𝐂𝐇𝟑 𝐂𝐎𝐎𝐇]= 𝟎. 𝟎𝟗𝟎𝟏: 𝟎. 𝟎𝟎𝟗𝟗

Choice and preparation of a buffer system

Table 6. Buffer solutions and their pKa

Buffer solutions pKa
Acetate buffer 4.73
Tris- (hydroxymethy) aminomethane 8.08
Phosphate buffer 7.20

The pH of the buffer solution needed to isolate albumin must have an A/B ratio of 1.0

and Ka closest to pH 5.2, table 6 shows the three given choices buffer solutions with their pKa.
Since pH is equal to pKa at half-equivalence point, among the three given buffer solutions above,

acetate buffer having a pKa of 4.73 is the closest value to pH 5.2. The Henderson-Hasselbalch

equation was used to calculate and determine which buffer ratio has an A/B ratio of 1, the

calculations is shown below. Hence, based on the obtained results from the calculations, the

preferred buffer solution to be used for the isolation of albumin in protein precipitation is the

acetate buffer with a pKa of 4.73.

Calculations:

Acetate buffer
pKa = 4.73

At pH 5.2, substitute into the Henderson-Hasselbalch equation and solve for

[CH3 COO− ]
[CH3 COOH]
[CH3 COO− ]
5.2 = 4.73 + log
[CH3 COOH]
[CH3 COO− ]
log = 5.2 − 4.73
[CH3 COOH]
[CH3 COO− ]
log = 0.47
[CH3 COOH]
[CH3 COO− ]
= 100.47
[CH3 COOH]
[CH3 COO− ]
= 2.951209227
[CH3 COOH]
Tris- (hydroxymethy) aminomethane
pKa = 8.08

At pH 5.2, substitute into the Henderson-Hasselbalch equation and solve for

[TRISH + ]
[TRIS]
[TRISH + ]
5.2 = 8.08 + log
[TRIS]
[TRISH + ]
log = 5.2 − 8.08
[TRIS]
[TRISH + ]
log = −2.88
[TRIS]
[TRISH + ]
= 10−2.88
[TRIS]
 [TRISH + ]
= 1.318256739 x 10−3
[TRIS]
Phosphate buffer
pKa = 7.20

At pH 5.2, substitute into the Henderson-Hasselbalch equation and solve for

[HPO42− ]
[H2 PO4− ]
[HPO42− ]
5.2 = 7.20 + log
[H2 PO4− ]
[HPO42− ]
log = 5.2 − 7.20
[H2 PO4− ]
[HPO42− ]
log = −2
[H2 PO4− ]
[HPO42− ]
= 10−2
[H2 PO4− ]
[HPO42− ]
= 0.01
[H2 PO4− ]
In the preparation of the chosen buffer system, the amount of weak acid (acetic acid)

and conjugate base (acetate) needed to be able to prepare the 2 liters of 5 mM buffer solution to

isolate albumin in protein precipitation was calculated by using the Henderson-Hasselbalch

equation. The calculations were shown below, and base on the obtained results from the

calculations 520 mL of CH3COOH and 1480 mL of CH3COO- were needed to prepare 2 liters of

5mM buffer solution.

Acetate buffer as the ideal buffer to be used:

To calculate for [HA] and [A-], we utilize what we know:
[HA] + [A− ] = 0.005 M
[CH3 COO− ]
= 2.951209227
[CH3 COOH]
Thus, let [HA]= x and [A-]= y
x + y = 0.005 (Equation 1)
y
x
= 2.951209227 (Equation 2)
From equation 2, we get y = 2.951209227x
Inserting equation 2 in equation 1, we get
x + 2.951209227x = 0.005
3.951209227x = 0.005
0.005
x= = 1.265435393 x 10−3
3.951209227
Since [HA]=x therefore, [HA]= 1.265435393 x 10−3 M
From equation 1, we get y = 0.005 − x
 y = 0.005 − 1.265435393 x 10−3 = 3.734564607 x 10−3
Since [A ]=y therefore [A-]=3.734564607 x 10−3 M
-

The ratio in moles of [𝐂𝐇𝟑 𝐂𝐎𝐎− ]: [𝐂𝐇𝟑 𝐂𝐎𝐎𝐇]= 𝟎. 𝟎𝟎𝟑𝟕: 𝟎. 𝟎𝟎𝟏𝟑

Calculating amount of weak acid and conjugate base needed (mL):
pH 5.2
0.0013 mol 1000 𝑚𝐿
CH3COOH = 1𝑀 x 1 𝐿 = 260 mL
5 𝑚𝑀 𝑥
1000 𝑚𝑀
0.0037 mol 1000 𝑚𝐿
CH3COO- = 1𝑀 x = 740 mL
5 𝑚𝑀 𝑥 1𝐿
1000 𝑚𝑀
Thus, 260 mL of weak acid and 740 mL of weak base is needed to prepare 5mM of buffer
solution for every 1 liter. Accounting the volume in which 2 L of 5 mM buffer solution is to be
prepared, the amount of weak acid and conjugate needed would be:
0.0013 mol 1000 𝑚𝐿
CH3COOH =( 1𝑀 x ) 2 = 520 mL
5 𝑚𝑀 𝑥 1𝐿
1000 𝑚𝑀

0.0037 mol 1000 𝑚𝐿

CH3COO- =( 1𝑀 x ) 2 = 14800 mL
5 𝑚𝑀 𝑥 1𝐿
1000 𝑚𝑀

Titration of amino acid

Table 7. pH of 0.1 M glycine after the addition of HCl and NaOH

0.1 M HCl pH of 0.1 M glycine 0.1 M NaOH pH of 0.1 M glycine
0 mL 6.13 0 mL 6.61
0.3 mL 3.20 0.3 mL 9.75
0.6 mL 3.00 0.6 mL 10.00
0.9 mL 2.05 0.9 mL 10.35
1.2 mL 1.99 1.2 mL 11.73
1.5 mL 1.60 1.5 mL 12.00

Figure 4. Theoretical titration curve of glycine

 Figure 5. Experimental titration curve of glycine

All amino acids have an amine and carboxylic acid functional group on their root

structure with acid/base properties. Seven amino acids also have a third functional group with

acid/base properties on their side chains. Each of these acidic functional groups has its own pKa.

The functional group exists predominately in its acid form when the pH is below the pKa and in

the base form when the pH is above the pKa. Thus, when you titrate an amino acid (i.e. gradually

add base to neutralize the acids), the functional groups are neutralized sequentially from low to

high pKa. At low pH, all amino acids will have a net positive charge. At high pH they have a net

negative charge. The pH value where they have a net charge of zero is called the isoelectric point

of pI. To determine the pI, find the pH range where the isoelectric structure predominates. Midway

between the pKa values that bracket this range is the pI (Oracle, 2013).
 O

H2N
OH

Figure 6. Structure of glycine

Glycine is a diprotic amino acid which means that it has two dissociable protons, one

on the α amino group and the other on the carboxyl group. In the case of glycine, the R group does

not contribute a dissociable proton (Amrita, 2012).

Figure 7. Dissociation of glycine

The dissociation of proton proceeds in a certain order which depends on the acidity of

the proton: the one which is most acidic and having a lower pKa will dissociate first. So, the H +

on the α-COOH group (pKa1) will dissociate before that on the α-NH3 group (pKa2). The graph

above shows the theoretical and experimental (base on the video provided since there was no actual

experiment) titration curves of glycine. Figure 5 shows the experimental titration curve of glycine

where the ionic species predominate at key points in the titration. The regions of greatest buffering

power were at pK1 = 2.34 and pK2 = 9.60 which was indicated with shaded boxes. Theoretically,

glycine has a pK1 = 2.35 and pK2 = 9.78 which were shown in the graph of figure 4. In comparison
of the two graphs, the experimental data was accurate since the titration curve was exactly the

same as the theoretical data.

pH of blood

Maintaining a constant blood pH is critical for the proper functioning of our body. Ideal

buffer solutions have a Ka value close to the desired pH and a A/B ratio of 1. In choosing the ideal

buffer system from the different compounds and amino acids for the blood at a pH value of 7.4,

the pKa of each given is compared to each other. The buffer that maintains the pH of human blood

involves carbonic acid (H2CO3) – bicarbonate ion (HCO3-) system.

Figure 8. Carbonic acid – bicarbonate ion system

When any acidic substance enters the bloodstream, the bicarbonate ions neutralize the hydronium

ions forming carbonic acid and water. Carbonic acid is already a component of the buffering
system of blood. Thus, hydronium ions are removed, preventing the Ph of blood from becoming

acidic.

HCO3- + H3O+ → H2CO3 + H2O

On the other hand, when basic substance enters the bloodstream, carbonic reacts with the

hydroxide ions producing bicarbonate ions and water. Bicarbonate ions are already a component

of buffer. In this manner, the hydroxide ions are removed from blood, preventing the pH of blood

from becoming basic.

H2CO3 + OH- → HCO3- + H2O

In the process of neutralizing hydronium ions or hydroxide ions, the relative concentrations of

carbonic acid (H2CO3) and bicarbonate ions (HCO3-) fluctuate in the blood stream. But this slight

change in the concentrations of the two components of the buffering system doesn’t have any

adverse effect; the critical this is that this buffering mechanism prevents the blood from becoming

acidic or basic, which can be detrimental (Khan, 2016).

Conclusion

It is important to calibrate the pH meter for precise and accurate results upon measuring

the pH of different solutions. Buffer capacity is affected by the concentration of the buffer solution

and the ratio of the conjugate base and weak acid required to prepare a buffer solution. The change

in the concentration of the phosphate buffer and acetate buffer from 0.1 M to 0.05 M and 0.005 M

resulted to the lowering of the buffer capacity. Upon the addition of NaOH which is a strong base,

the pH of the buffers increased, indicating that the pH of the buffer solutions was already outside

the range of their buffering capacities. In order to have an effective buffer solution, the solution

must be made out of a weak acid and a conjugate base with pH close to or equal to the pH in order
to achieve a maximum buffering capacity. The pH of an aqueous solution of an acid to the acid

dissociation constant of the acid and can be used to find the ratio of conjugate base and weak acid

in buffer solutions relates to the Henderson-Hasselbalch equation which can also be used as a guide

in preparing a buffer solution with effective buffering capacity. In titrating amino acid, the

functional groups are neutralized sequentially from low to high pKa. At low pH, all amino acids

will have a net positive charge. At high pH they have a net negative charge. The pH value where

they have a net charge of zero is called the isoelectric point of pI. Buffers are important in

biological systems because of their ability to maintain constant pH conditions. Furthermore,

buffers are very important in maintaining the pH of blood which must be kept within the narrow

limits of life and health because higher or lower pH than the normal variation 7.4 could cause

death. Thus, understanding pH and buffer systems is essential for it helps maintain life.

Acknowledgement

The fulfilment of this exercise could not be possible without the help and participation of

many people; their contributions are greatly appreciated. The author would like to express deepest

gratitude to Almighty God, for his guidance, enlightenment and protection for the completion of

this exercise. Ms. Helen Mae N. Mejia for sharing her knowledge, suggestions, guidance and

teachings for the success of this exercise. Also, to the author’s family, friends and classmates for

the support and ideas. Thank you so much and God bless!
References

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