UNIVERSITI KUALA LUMPUR

MALAYSIAN INSTITUTE OF CHEMICAL & BIOENGINEERING TECHNOLOGY

COARSE CODE : WEB 20302

COURSE : PROFESSIONAL ENGLISH 2
SEMESTER/YEAR : SEPTEMBER 2015

TECHNICAL REPORT

PREPARATION OF BUFFER SOLUTION

LECTURER’S NAME: INTAN NORJAHAN BT AZMAN SECTION: L02-T1

NAME ID NUMBER

MUHAMMAD BIN MOHD ASRI 55213115266

AZWAN BIN AHMAD 55213115254

AHMAD NURUDDIN B. ABD KAHAR 55213115456

MOHD FIRDAUS B. ZUBIR 55213115070

NURUL FARIHAH BT. ABU SAIRI 55213115262

SUBMISSION DATE: 20 NOVEMBER 2017

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 Preparation Of Buffer Solution

ABTRACT

In this Preparation of Buffer Solution experiment, the buffer solution with pH of 5.0
was prepared by using nitric acid and sodium citrate solution. The objectives were also
determinig the characteristic of the buffer solution itself. According to Laura B. (2016),
buffer solution is a solution that resist or minimize the changes of pH when small amount of
base or acid is added. The effect of pH value were examine in this experiment by adding 1ml
of 1M hydrochloric acid and 1ml of 1M sodium hydroxide into the buffer solution and the
result is being discussed either it resist the changes of pH of both addition of acid or base, or
not. The effect of the pH then was compared between the buffer solution and the pure water
with the addition of 1ml of 1M hydrochloric acid and 1ml of 1M sodium hydroxide. The
result showing there are pH changes in pure water after the addition of the acid and base
solution while minimize in changes in buffer solution after the adding of the acid and base
solution (Laura B, 2016).

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 Preparation Of Buffer Solution

TABLE OF CONTENT

Page number

ABSTRACT ii

TABLE OF CONTENT iii-v

LIST OF FIGURES vi

LIST OF TABLES vi

LIST OF ABBREVIATION vii

LIST OF FORMULA viii

CHAPTER 1 : INTRODUCTION

1.1 Background of Study

1.1.1 Definition 1

1.1.2 Historical Background of Buffer Solution 2

1.1.3 Comparison between Natural and Synthetic Buffers 3

1.1.4 Advantages in Application of Buffer Solution 4

1.2 Objectives 5

1.3 Scope of Work/Study 6

1.4 Conclusion 6

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 Preparation Of Buffer Solution

CHAPTER 2 : LITERATURE REVIEW

2.1 Introduction 7

2.2 Maintenance of pH 7

2.3 Theory 8

2.4 Concept 9-10

2.5 pH of Buffer Solution 11

2.6 Optimization of Buffer Capacity 12

2.7 Preparation of Buffer Solution 13

2.8 Theoretical Framework 14

2.9 Conclusion 18

CHAPTER 3 : METHODOLOGY
3.1 Chemical and Apparatus Required
3.1.1 Standard Materials
3.1.1.1 Chemical required 19
3.1.1.2 Apparatus required
3.1.2 Non-standard Materials
3.1.2.1 Micropipette 21-22
3.1.2.2 pH meter 22-23
3.2Experimental Procedure
3.2.1 Overview 24
3.2.2 Description of procedural steps 24-26

CHAPTER 4 : RESULT & DISCUSSION

4.1 Introduction 27

4.2 Body 28-30

4.3 Conclusion 31

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 Preparation Of Buffer Solution

CHAPTER 5 : CONCLUSION
5.1 Conclusion 32

5.2 Future Recommendation 33

REFERENCES 34

APPENDIXES 35

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 Preparation Of Buffer Solution

LIST OF FIGURES

Page number

Figure 1: Concept of Pure Water System 10

Figure 2: Concept of Buffer Solution 11

Figure 3: Preparation of buffer solution using weak base and conjugate acids 15

Figure 4: Preparation of buffer solution by using weak acids and conjugate base 15

Figure 5: Diagram for 100mL beaker 17

Figure 6: Diagram for Hydrochloric Acid 18

Figure 7:Diagram for 25mL measuring cylinder 18

Figure 8: The structure of micropipette 19

Figure 9: Structure of pH Meter 20

Figure 10: Formation of a buffer 23

Figure 11: Illustration for effectiveness of the buffer solution 24

Figure 12: pH value before/after adding HCL and NaOH for buffer solution and distilled water 28

LIST OF TABLES

Page number
Table 1: pH values measured for each solutions 28

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 Preparation Of Buffer Solution

LIST OF ABBREVIATION
pH measure of the hydrogen ion (H+)
β buffer capacity
d[b] or d[a] changes in the concentration of a strong base or acid
dpH changes in pH
MgO magnesium Oxide
ml millilitres
M molar
NaOH sodium hydroxide
HCl hydrochloric acid
pKa logarithm value of acid dissociation
OH hydroxide
HCOOH formic acid
HCOO- formate ion
H+ hydrogen ions
Ka acid dissociation constant
mM millimolar
Na3C6H5O7 trisodium citrate
C6H8O7 citric acid
H3O+ hydronium
ECU Electronic Control Unit
% percent
L micro litre

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 Preparation Of Buffer Solution

LIST OF FORMULA

𝑑[𝑏] 𝑑[𝑎]
𝛽= =
𝑑𝑝𝐻 𝑑𝑝𝐻

𝐴−
pH=pKa+log[𝐻𝐴]

[𝐻+][𝐴−]
Ka = [𝐻𝐴]

pH = -log[H+]

[𝐴−]
pH = pKa + log([𝐻𝐴])

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 Preparation Of Buffer Solution

CHAPTER 1

INTRODUCTION

1.1 Background of Study

1.1.1 Definition

The purpose of this experiment was to determine the power and importance of buffers in regulating
the pH of solution by adding very small amounts of acid or base to different buffer solutions. Buffer
solution is an aqueous solution of which it can resist the changes of pH when strong acid or strong
base is added into the solution. Buffer solution is mainly the product of weak acid with it conjugate
base or the weak base with it conjugate acid.

A buffer solution has to contain things which will remove any hydrogen ions or hydroxide ions that
added to it. Thus, the pH of the solution will not change. Otherwise, the unremoved ions will change
the pH of the solution drastically. The particular relationship of buffer solution is:

𝑑[𝑏] 𝑑[𝑎]
𝛽= =
𝑑𝑝𝐻 𝑑𝑝𝐻

In this equation, β represents the buffer capacity, d[b] and d[a] represent the very small change in the
concentration of a strong base or acid respectively and dpH represents the very small change in pH
resulting from the additions of strong acid or base. Understanding buffer capacity is important
because it allows chemists to determine the properties of the given buffer are suitable for an
application such as the capacity limit of the buffer.

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 Preparation Of Buffer Solution

1.1.2 Historical Background of Buffer Solution

Previously through buffers solution history, it were functions to resist particular pH level in
solutions. There are no one invented the buffers because the buffers itself is a natural process. The use
of bicarbonate in the blood and cell fluid is one of the simple example (Aaron Lopes, 2010).

According to Aaron Lopes (2010), since buffering is a natural process that can be damaged,
people developed buffers to help people with acidosis amongst other diseases that affect acidity. The
first synthetic buffer was used and developed by the Chinese in pre 1000 B.C. They used saturated
charcoal to treat severe heart burn and poisoning. Later in history, chemist used buffers in drugs to
enhance their effect as for the example is aspirin. Aspirin was developed in 1853 by Charles Frederic
Gerhardt, which prevents the flow of prostaglandins or pain nerves in a particular pH range. Since the
human blood is not acidic enough for aspirin, aspirin had a weaker effect. For this reason, Bayer, a
pharmaceutical company, added Magnesium Oxide, MgO molecules to aspirin to increase the pH
level in the blood to enhance aspirin’s effect in 1908. In 1928, Jim Howe used Chinese ideals to
develop the buffers Tums to cure his wife’s indigestion. The use of this buffer was to help neutralize
acid in the stomach to relieve pressure, discomfort, and indigestion.

As a conclude, from the history until nowadays, the main purposes of synthetic buffers were either
to enhance the effect of drugs, or to neutralize acidity levels within fluids.

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1.1.3 Comparison between Natural and Synthetic Buffers

According to Aaron Lopes (2010), buffers are all around us and also are in us. We used to eat
them, and buffers are necessary for our survival. However, while buffers are doing in same terms of
function, but there are slightly two different types of buffers. They are natural buffers and synthetics
buffers.

As for natural buffers, they are acid-base reactions that are made through in a natural processes.
The body has two natural buffers that are carbonic acid and a bicarbonate base. Both of them are
produced by each other. This is because bicarbonate reacts with hydronium to form carbonic acid and
water. While the carbonic acid reacts with water to form bicarbonate and hydronium. The process
keeps repeating and cycling all over again until an equilibrium is reached. Since the acids balance to
the bases, it keeps the acidity of the blood near to neutral.

On the other hand, synthetic buffers are quite different from natural buffers. Synthetic buffers are
the buffers that been created in laboratories and often contain the basic salt, sodium bicarbonate.
These buffers are created as a function to be taken for the people who had suffered from acidosis.
Acidosis itself is known as disease in which the body fluids are too acidic or too basic. Moreover,
many drugs are lined with buffers to make blood more acidic or more basic to enhance a drug’s effect.

However, it is compulsory to notice the both natural and synthetic buffers have the same
properties and also notice that many of natural buffers can be made in laboratories too.

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 Preparation Of Buffer Solution

1.1.4 Advantages in Application of Buffer Solution

Given their resistance to changes in pH, buffer solutions are very useful for chemical
manufacturing and essential for many biochemical processes. The ideal buffer for a particular pH has
a pKa equal to pH desired, since a solution of this buffer would contain equal amount of acid and base
and be in the middle of the range of buffering capacity.

Buffer solutions are necessary to keep the correct pH for enzymes in many organism to work.
Many enzymes work only under very precise conditions, such as in a situation, if the pH strays too far
out of the margins, the enzymes slow or stop working and can denature, thus permanently disabling
its catalytic activity. Referring to New World Encyclopedia (2016), a buffer of carbonic acid and
bicarbonate is present in blood plasma, to maintain a pH between 7.35 and 7.45.

Industrially, buffer solutions are used in fermentation processes and in setting the appropriate
conditions for dyeing fabrics. They are also used in chemical analyses and synthese, and for the
calibration of pH meter.

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 Preparation Of Buffer Solution

1.2 Objectives

The main objectives in this report were:

 to prepare buffer solution with pH 5.0 by using nitric acid solution and sodium citrate solution
 to determine the characteristic of the buffer solution
 to examine the effect in pH value of the buffer solution by the adding of 1ml of 1M
hydrochloric acid and 1ml of 1M sodium hydroxide into the buffer solution either it is resist
to the changes of pH or not
 to compare the effect in pH values of both buffer solution and the pure water by the adding of
1ml of 1M hydrochloric acid and 1ml of 1M sodium hydroxide

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 Preparation Of Buffer Solution

1.3 Scope of Work/Study

In this experiment, the pH of the prepared buffer solution is measured. This will help to
provides a better understanding about the buffer solution. Buffer solution was defined as a solution
which one can resist the change in pH when a certain amount of acidic or alkali solution was added.
Buffer solution is a mixture of acid and conjugate base. The commonly pH for a buffer solution is
between 7 to 5(before the adding of acid or alkaline solution). The result of this experiment, there
will be a slight change in the pH value of the prepared buffer solution. The increase or decrease in the
pH value depend on the solution added whether it is acidic or alkali. If it is acidic, the pH will
decrease a bit and if it alkali the pH will increase a bit depends on how strong the solution is. This
experiment only used citric acid and sodium citrate for the prepared buffer solution and sodium
hydroxide and hydrochloric acid was used as strong base and strong acid respectively.

1.4 Conclusion

In conclusion buffer solution is solution mainly the product of weak acid with it
conjugate base or the weak base with it conjugate acid, in buffer solution it has a containing
that will remove any hydrogen ions or hydroxide ions that added to it, but the pH value does
not change. Moreover, undissolve ions will change the pH drastically.

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 Preparation Of Buffer Solution

CHAPTER 2
LITERATURE REVIEW
2.1 Introduction

Preparation of buffer solution requires verities review from different perspective

parameters involves such as maintenance of pH, pH of buffer solution and optimization of
buffer capacity. Those parameters involve will be supported by the theory, concept and
theoretical framework.

2.2 Maintenance of pH

Maintenance of pH is vital to all cells. Cellular processes such as metabolism are

dependent on the activities of enzymes. In turn, enzyme activity is markedly influenced by
pH. Consequently, changes in pH would be disruptive to metabolism. Hence the pH of a
living system is maintained with buffers. In humans, the pH of blood is maintained between
7.35 and 7.45. Interestingly, 5 liters of blood contains enough buffering capacity to absorb
150 ml of 1M acid. The principal buffers in blood are bicarbonate, proteins (including
hemoglobin and oxyhemoglobin)andphosphates (Chemistry.wustl.edu, 2017).

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 Preparation Of Buffer Solution

2.3 Theory

There are a variety ways to prepare a buffer solution of a specific pH. In the first
method, prepare a solution with an acid and its conjugate base by dissolving the acid form of
the buffer in about 60% of the volume of water required to obtain the final solution volume.
Then, measure the pH of the solution using a pH probe.

The pH can be adjusted up to the desired value using a strong base like NaOH. If the
buffer is made with a base and its conjugate acid, the pH can be adjusted using a strong acid
like HCl. Once the pH is correct, dilute the solution to the final desired volume.

Alternatively, other way round is prepare solutions of both the acid form and base
form of the solution. Both solutions must contain the same buffer concentration as the
concentration of the buffer in the final solution. To get the final buffer, add one solution to
the other while monitoring the pH.

In a third method, it can determine the exact amount of acid and conjugate base
needed to make a buffer of a certain pH, using the Henderson-Hasselbach equation:

𝐴−
pH=pKa+log[ ]
𝐻𝐴

Where pH is the concentration of [H+], pKa is the acid dissociation constant, and [{A}-] and
[{HA}] are concentrations of the conjugate base and starting acid.

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 Preparation Of Buffer Solution

2.4 Concept
Figure 1: Concept of pure water system

Figure 1: Concept of pure water system

Figure 1 shows the concept of pure water system. Firstly, the pure water will be added
with strong base solution sodium hydroxide. Inside the beaker is picture little dot represent
0.01 moles OH. The number of mol in the whole beaker with 10 little dots is 0.1 moles OH.
Result of both solutions is basic because still lot of OH in the beaker and still same 0.1 OH on
the whole solution.

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 Preparation Of Buffer Solution

Figure 2: Concept of buffer solution

Figure 2: Concept of Buffer Solution of pure water system

While figure 2 is explaining the concept of buffer solution instead of pure water
system. Buffer is full with material. The white dots represent HCOOH whilst the black dots
represent HCOO-. The total dots of black and white are 50. The pH is exactly same with pKa
after been calculated. Then the OH solution will be added. Those white, red and blacks dots
are neutralization reaction in small beaker.

The amounts of mol still the same which is 0.1 moles OH. Here, the white dots will be
convert to the black dots until that three dots of those dots will gone. After the neutralization
reaction occurs, the OH will definitely gone. The resulting solution shows that it is still the
same as buffer solution. The reading of the pH has the different value because the number of
mol has been change of both solution which are HCOOH and HCOO-.

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 Preparation Of Buffer Solution

2.5 The pH of Buffer Solution

pH is a measure of the hydrogen ion (H+) concentration in a solution (Boundless

Chemistry, 2017). The pH of a buffer solution can simply be determined by using the pH
probe. A calculation of the concentration from various components of the reaction can
determined the pH of the buffer solution. The equilibrium equation of the solution can
determine the acid-dissociation constant Ka, which measure the propensity of an acid to
dissociate (Boundless Chemistry, 2017). The equation can be simplified as:

[𝐻+][𝐴−]
Ka = (Boundless Chemistry, 2017).
[𝐻𝐴]

The amountof hydrogen ion (H+) formed is depend on the Ka which will increase as
the ratio of denominator and nominator increase. Thus, lower the pH of the solution.
Whereby pH can be simplified as:

pH = -log[H+] (Boundless Chemistry, 2017).

From Henderson-Hasselbalch equation, the pH of the buffer solution can be estimated

using the theoretical value of the logarithmic value of acid dissociation, pKa. Beside, PKa
also known in simple word as a quantitative measure of the strength of an acid in a solution.
The Henderson-Hasselbalch equation is defined as follows:

[𝐴−]
pH = pKa + log([𝐻𝐴]) (Boundless Chemistry, 2017).

from this equation, the exact amount of acid and its conjugate base can be determined to
produce a buffer with a desired pH.

The buffer solution can be prepared with alternative method which is by preparing a
solution with an acid and its conjugate base by dissolving the acid form of the buffer in about
60% of the volume of water required to obtain the final solution volume (Boundless
Chemistry, 2017).

Another method used to prepare buffer solution is by preparing solution of both the
acid form and base form of the solution where both solution must have the same buffer
concentration as the concentration of the buffer in the final solution (Boundless Chemistry,
2017). One solution is added to the other while monitoring the pH by using pH probe.

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 Preparation Of Buffer Solution

2.6 Optimization of Buffer Capacity

The maximum buffer capacity can be obtained when the pH of the buffer solution
approximates the pKa of the solution. This means the amount of acid or base needed to
change the pH of the buffer solution in slightly higher. For example, the buffer solution of
acetic acid (sodium) has pH of 4.7 where it is approximating to the pKa of acetic acid (New
World Encyclopedia, 2017).

The buffer capacity increase as the concentration increase. For example, the amount
of acid or base need to increase or decrease the pH of acetic acid (sodium) buffer solution is
higher at 100mM concentration than at mM. However, precipitation may occur at high
concentration.

An alternative way to produce buffer solution of 10mM phosphoric acid (sodium)

buffer solution at pH = 6.9 is by diluting 100mM of phosphoric acid (sodium) buffer solution
with pH = 6.8 ten times.

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 Preparation Of Buffer Solution

2.7 Preparation of Buffer Solution

The composition of buffer solution is weak acid and its conjugate base for acidic
buffer solution and weak base or alkali and conjugate acid for alkali buffer solution. There is
various preparation of buffer solution with various pH measurements for the buffer solution.
For example, preparing a 0.1M of phosphoric acid buffer solution with a pH value of 2.1
(Council of Europe, 2011). Sodium dihydrogen phosphate dihydrate is used with a
concentration of 0.005M and combine with phosphoric acid with the same concentration for
the weak acid. Then, add water until it reaches 1 liter. This two composition will produce a
pH measurement of 2.1. By diluting this buffer solution for ten times, it will produce the
same type of buffer solution but with a slight increase of pH value which is 2.6. There is also
an alternative way to produce this buffer solution to meet the pH value of 2.6. A lower
concentration of sodium dihydrogen phosphate dehydrate (0.0005M) and the same
concentration for the phosphoric acid. Then, add water until 1 liter (Council of Europe,
2011). Thus, a weaker acidic buffer solution was created. In other example, 0.1M boric acid
buffer solution with a pH value of 9.1. According to Council of Europe (2011), 0.1M of boric
acid is added with 0.05M of potassium hydroxide or sodium hydroxide. Then, add water until
1 liter. By using either potassium hydroxide or sodium hydroxide, the result of the pH still
will be the same which is 9.1.

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 Preparation Of Buffer Solution

2.8 Theoretical Framework

In preparation of buffer solution it can mix between any weak acid or base and
conjugate base or acid. A buffer is a solution that will resists changes in pH, If the pH of a
buffer is greater tham pka, it will be more effectiveagaints additon of acid. On the other
hand, if the pH of buffer solution less than pka, it is more affective againts of base. In a
titration curve, the pH of buffer point is simply the pka value. Even if it was a weak base, we
still use pka. Figure 3 show for the preparation of buffer solution by using weak base and
conjugate acid while for Figure 4 show preparation of bufer solution between weak acid and
conjugate base (Sciencescribe.co.nz., 2017).

Figure 3: Preparation of buffer solution using weak base and conjugate acids

Figure 4: Preparation of buffer solution by using weak acids and conjugate base

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 Preparation Of Buffer Solution

Ref
Author,
Title Objectives Focused
year

Newworldencyclopedia.org. (2017). Buffer solution - New

 Definition &
World Encyclopedia. [online] Available at:
Buffersolution 2010  Basic info of buffer how buffers
http://www.newworldencyclopedia.org/entry/Buffer_solutio
work
n [Accessed 26 Oct. 2017].
 Describe the
properties of buffer
solution
 Calculate the pH of a
 In thoerical
buffer made only
 In
from a weak acid Courses.lumenlearning.com. (2017). Buffer Solutions |
calculation
 Calculate pH using Boundless Chemistry. [online] Available at:
Buffersolutions  using
Henderson- https://courses.lumenlearning.com/boundless-
equation
Hasselbalch equation chemistry/chapter/buffer-solution/ [Accessed 26 Oct. 2017].
Henderon-
 Calculating final
Hasselbalch
changes in pH of a
solution after adding
strong acid or base
into solution

15
 Preparation Of Buffer Solution

 Basic info of
buffer Lopes, A. (2017). eochemistry - buffers - Aaron Lopes. [online]
Aaron  buffers in our daily
historically Eochemistry.wikispaces.com. Available at:
Buffersandthe blood Lopes, life
and related https://eochemistry.wikispaces.com/buffers+-
2010  preparation of buffer
in our life +Aaron+Lopes#cite_note-2 [Accessed 26 Oct. 2017].

 Mixture for
Council of European Pharmacopoeia 7.0. (2011). 7th ed. Council of
 Preparation of buffer the buffer
Buffersolutions Europe, Europe: European Directorate for the Quality of Medicines
solution solution
2011 and Healthcare: Council of Europe, pp.3-8.
composition
 Explicitly
model the
K.Ganeshh,  Determine the pH profile
Dynamicapproachtopre R.Soumen, steady state of Ganesh,K., Soumen,R., Ravinchandran,Y., and Jannarthanan.
dict pH profile of Y.Ravichan concentration of biologically (2017). Dynamic Approach to predict pH Profile of biologically
biologically relevant dran, multiple ionic species relevant relevant buffers.Biochemistry and Biophysics Reports, 9, 121-
buffers Jannarthan present in complex phosphate 127. DOI: 10.1016/j.bbrep.2016.11.017
an buffer buffer and
universal
buffer

16
 Preparation Of Buffer Solution

 Mixture for Shimadzu.com (2017). Preparing Buffer Solution. [online]

Shimadzu
Preparingbuffer  Preparing the buffer the buffer Available at:
Corporatio
solution solution solution http://www.shimadzu.com/an/hplc/support/lib/lctalk/38/38l
n
composition ab.html [Accessed 26 Oct. 2017]

17
 Preparation Of Buffer Solution

2.9 Conclusion

The preparation of the buffer solution with desired pH value conducted in this
experiment is referring to the Henderson-Hasselbalch equation to acquire the exact amount of
weak acid concentration and its conjugate base. The method of preparation is followed by the
high concentration of the buffer solution diluted until pH desired is achieved. This increase
the buffer capacity of the buffer solution. pH probe is inserted inside the solution along the
experiment conducted to measure the pH.

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 Preparation Of Buffer Solution

CHAPTER 3
METHODOLOGY
3.1 Chemical and Apparatus Required

This section presents the chemical and apparatus required to run the experiment

3.1.1 Standard Materials

3.1.1.1 Chemicals Required

The chemical required are:

 1.0M HCL
 1.0M NaOH
 0.1M Citric Acid
 0.1M Sodium Citrate

3.1.1.2 Apparatus Required

The lists of apparatus required are:

 4 of 100mL beakers
 250mL beaker
 50mL beaker
 2 of 25mL measuring cylinder

Figure 5: Diagram for 100mL beaker

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 Preparation Of Buffer Solution

Figure 6: Diagram for Hydrochloric Acid

Figure 7: Diagram for 25mL measuring cylinder

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 Preparation Of Buffer Solution

Figure 8: The structure of micropipette

3.1.2 Non-standard materials

3.1.2.1Micropipette

Micropipette are commonly used in laboratory for the purpose of transferring a small
quantities of liquid. It usually can transfer down to 0.1L. There are six sizes for the
micropipette, p2, p10, p20, p100, p200, and p1000. Each of this size can transfer volume of
liquid from range, 0.2 - 2L, 1-10L, 2-20L, 20-100L, 20-200L, and 100-1000L
respectively(Henry, 2011). The most common brand for the micropipette are Eppendorf
Pipettes, Rainin Pipettes, Gilson Pipettes, and Oxford Pipettes.

From the top of the micropipette, push button is to allow the suction process to occur
at the tip. It also can be used as volume adjuster for the micropipette but in a large scale. Next
is the tip ejector button, this button allows to remove the tips because the tips are one time use
only to avoid contamination when proceed to the next transfer. This is followed by the fine
volume adjustment ring or micrometre adjustment ring(Henry, 2011). Its is used to adjust the
volume of micropipette in a more finer scale than the push button scale. All of the adjustment
was showed at the volumeter display whether it is analogue or digital type of display. In
modern micropipettes, they usually have a digital display which showed to be more accurate

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 Preparation Of Buffer Solution

than the analogue. The tip ejector is to remove the disposable tips. The tip ejector will remove
it when the tip ejector button is pushed. This is to minimize the damage on the shaft when
removing the tips manually. Next is the shaft. The shaft consist of a long rod that holds the
disposable tips from falling off. Lastly, the disposable tips. This is the most important part of
the micropipette when running the experiment. A disposable one is used because to avoid
contamination on the solution. When the adjustment was made, all of the solution that will be
suck will be contained in the tips. Therefore, it is important to use disposable tips rather than a
fixed tips for the micropipette(Henry, 2011).

For this experiment, Eppendorf Pipettes was used with a size p1000. This is to give a
more accurate reading when transferring 1mL of HCL or NaOH into the beakers. The pipette
can be adjust up to 1000L which is equivalent to 1mL.

Figure 9: Structure of pH Meter

3.1.2.2 pH Meter

pH meter is an instrument that measure the hydrogen-ion activity in a water based

solution. The reading on pH meter is from zero to 14 which enables us to determined the
solution whether it is alkali or acidic solution(Tiwari, 2015).

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 Preparation Of Buffer Solution

pH value that is equal to seven is considered neutral solution but if the reading is lower
than seven, that prove the solution is acidic or the reading is higher than seven, that prove the
solution is an alkali solution(Tiwari, 2015). Most of the pH meter is commonly used was in
pharmaceutical industry. This is because to make sure the medicine pH are corresponding to
the human body needs.

A common pH meter consist three parts which are electrode, temperature probe, and
electronic control unit(ECU). The function of temperature probe is to measure the temperature
of the solution while taking the reading of the pH. Next is electrode. The electrode does not
read the pH of the solution but it measure the hydrogen-ion activity(Tiwari, 2015). The
electrode usually consist of glass electrode and reference electrode that was made into one
electrode. When the electrode is immersed into the solution, this will make a complete circuit
for the ion to move. When more hydrogen or hydronium ion consist in the solution, the pH
solution will decrease proportionally. Lastly, the ECU. ECU is consider the brain of the pH
meter. When the hydrogen-ions move through the circuit, a reading of voltage is obtained.
Thus, the amount of voltage gain from the solution is converted into pH value from zero to
14.

For this experiment, the pH meter is used to measured the pH of the solution and to
observed the pH value of buffer solution when adding 1mL of HCL or 1mL of NaOH. pH
meter is more preferable than the traditional method(using litmus paper) because the
traditional method cannot show or prove the function of buffer solution. This is because
litmus paper only indicate blue or red when immersed in the solution. That will showed us
only the acidity or alkali of the solution. However, the pH meter will show us an exact value
of the pH reading when the electrode is immersed into the solution.

WARNING: Do not store electrode in distilled or deionized water and never wipe the
electrod

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 Preparation Of Buffer Solution

3.2 Experimental Procedure

This section will present the experimental procedure when it was conducted
3.2.1 Overview

The main objectives in this report were to prepare buffer solution with pH 5.0 by using
citric acid solution and sodium citrate solution. Next, to determine the characteristic of the
buffer solution itself. Moreover, to examine the effect in pH value of the buffer solution by
the adding of 1ml of 1M hydrochloric acid and 1ml of 1M sodium hydroxide into the buffer
solution either it is resist to the changes of pH or not and lastly to compare the effect in pH
values of both buffer solution and the pure water by the adding of 1ml of 1M hydrochloric
acid and 1ml of 1M sodium hydroxide. The data were obtained from the laboratory
experiment in Biochemical Engineering subject by using some standard materials where were
a pH meter, beakers, measuring cylinder, spatula, stirring hotplate and stir bars also another
one non-standard material which was a micropipette. Generally, this report was carried out
the nature of a buffer solution.

3.2.2 Description of procedural steps

A. Preparation of the Buffer Solution

a) 0.1M of citric acid and sodium citrate was prepared.
b) By using the following formula:
5.0 = 4.76 + log [Citrate] [Citric acid]
0.24 = log [Citrate]/[Citric acid]
[Citric acid] = 1.74 [Citrate]
A total volume of 91.24mL of citric acid and 158.75mL of sodium citrate was added in a
250mL of beaker.
c) The buffer solution was then fill into 25mL of measuring cylinder to transfer to a 100mL
beaker.
d) By using the pH meter, the pH value for the buffer solution was recorded.

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 Preparation Of Buffer Solution

Citric Acid Sodium Acetate

C2H4O2 C2H3NaO2

Figure 10: Illustration for preparation of buffer solution

B. Effectiveness of the Buffer Solution

a) A total of 100mL of distilled water was divided into two 100mL beakers.
b) By using micropipette, 1mL of HCL was added into the distilled water and 1mL of NaOH
was added into another beaker.
c) Both the of the solution pH were measured by the pH meter.
d) A total of 100mL of buffer solution was divided into two new 100mL beakers.
e) By using the same micropipette with new tips, the same amount of HCL and NaOH in step
(b) was added in both buffer solutions separately.
f) The pH value for both beaker was measured by the pH meter and recorded.

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 Preparation Of Buffer Solution

Figure 11: Illustration for effectiveness of the buffer solution

Waste and Spills:

All of the chemicals used in this experiment can be discarded via drain disposal with running
tap water. Wet towels can be used to clean the spill, and then inside the glassware with
distilled water before the experiments starts to avoid chemical contamination.

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 Preparation Of Buffer Solution

CHAPTER 4
RESULT AND DISCUSSION

4.1 Introduction

This section discusses the preparation of buffer solution with pH 5.0 by using nitric
acid solution and sodium citrate solution by using the Henderson-Hasselbach equation and to
determine the characteristic of the buffer solution itself. Then, the buffer solution is put into
test to examine the effect in pH value of the buffer solution by the adding of 1ml of 1M
hydrochloric acid and 1ml of 1M sodium hydroxide into the buffer solution either it is resist
to the changes of pH or not. Lastly, the same procedure is conducted by using distilled water
to see the different between distilled water and buffer solution.

The laboratory experiment was earlier carried out. The raw findings were processed and
are discussed here.

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 Preparation Of Buffer Solution

4.2 Body

Table 1: pH values measured for each solution

Solutions pH
1st reading 2nd reading Average
Buffer 5.15 5.20 5.175
Buffer + 1 ML HCL 4.95 4.89 4.920
Buffer + 1 ML NaOH 5.18 5.19 5.185
Distilled water 6.81 7.00 6.905
Distilled water + 1 ML HCl 1.99 2.06 2.025
Distilled water + 1 ML NaOH 10.75 11.05 10.90

Table 1 show the result obtained from the experiment where the buffer solution and
distilled water is added with HCl and NaOH. The result tabulated in a table 1 are measured
using pH meter two times to get the average of the readings.

Figure 12: pH value before/after adding HCl and NaOH for buffer solution and distilled
water

Figure 12 shows the relationship between the type of solution and the pH value of the
solution. Pure solution of distilled water and buffer is labelled with red colour while the
addition of HCL is marked with blue colour and the label with green colour indicated the
addition of NaOH.

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 Preparation Of Buffer Solution

Preparation of buffer solution experiment was conducted to prepare buffer solution

with pH of 5.0 by using citric acid solution and sodium citrate solution, determining its
characteristics as well as to study the effect of adding hydrochloric acid (HCl) and sodium
hydroxide (NaOH) to pH of buffer solution and pure water.

In this experiment, a 250 ml buffer solution was prepared by adding 91.24 ml of 0.1M
citric acid solution and 158.76 ml of 0.1M sodium citrate solution by referring the method
cited in Council of Europe (2011). According to Boundless Chemistry,(2017) the pH was
measured by using pH meter and the average reading is 5.175. The pH of buffer solution
prepared is higher than the expected pH. Supposedly, its pH is 5.0 as has been calculated
earlier before the experiment began. The difference occurred due to the volume of weak acid
and conjugate base that are not being measured accurately. The amount of hydrogen ion (H+)
formed is depend on the Ka which will decrease as the ratio of denominator and nominator
decrease. Thus, increase the pH of the solution (Boundless Chemistry, 2017).The high pH
reading is most probably because of an excessive volume of sodium citrate solution. Other
than that, the pH meter might be not calibrating properly before used which had disturbed the
reading.

On the other hand, the changes in pH resulting in the addition of 1ml of 1M HCl and
1ml of 1M NaOH to buffer solution and pure water are studied. The pH measured after
adding HCl and NaOH into buffer solution are 4.92 and 5.185 respectively. Somehow, the
addition has cause only a slight change to the pH of buffer solution. This is because the added
HCl react with weak acid as follows:

3HCl + Na3C6H5O7 ⇔ C6H8O7 + 3NaCl

This reaction has converted HCl into a weak acid which introduces a much smaller disruption
in pH compare to strong acid, thus cause only a small change in pH of buffer solution. The
quite similar thing happens when NaOH was added. The acetic acid would react with NaOH
and converted it into a weak base that nearly would not raise the pH of the buffer solution.
The reaction is as follows:

NaOH + C2H3O2H ⇔ NaC2H3O2 + H2O

This clearly shows that the buffer solution prepared is effective as it resists any changes in pH
when acids or bases are added to them as they consist of weak acids and their conjugate bases.

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 Preparation Of Buffer Solution

Meanwhile, there is a drastic change observed in pH of pure water when HCl and
NaOH were added. The pH of water is measured as 2.025 after the addition of HCl whereas,
the addition of NaOH change the water pH to 10.9. Adding a strong acid like HCl to water
caused a relatively high change. This is due to the proton (H+) from the acid binds to neutral
water molecules to form H3O+ raising the concentration of H+ ("pH in the Absence of a
Buffer", 2016). In other word, the pH of water drops significantly because a high
concentration of H+ causes the solution becoming more acidic. As for the addition of NaOH,

the pH of water has increased significantly due to a large concentration of OH- that has makes

the water more basic. This shows that pure water cannot resists any changes when either a
strong acid or strong base is added because it does not have any conjugate base nor weak acid
to help it stabilise its pH. As explain in part 2.3, the buffer solution will maintain the pH while
the pH for non buffer solution will change along the addition of new solution.

30
4.3 Conclusion

As a conclusion, the buffer solution showed the small change in pH when there are
addition of acid and base. The differences between other solutions with buffered solution can
be easily distinguished from the behaviour of the pH changed. All the objectives of the
experiment were achieved although there are a few errors has happened throughout the
experiment.

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 Preparation Of Buffer Solution

CHAPTER 5
CONCLUSION AND RECOMMENDATION

5.1 Conclusion

The main purpose of the experiment are to study the buffer solution with pH 5.0 by
using nitric acid solution and sodium citrate solution, to determine characteristic of buffer
solution, to examine the effect in pH value of the buffer solution by the adding of 1ml of 1M
hydrochloric acid and 1ml of 1M sodium hydroxide into the buffer solution either it is resist
to the changes of pH or not, and lastly to compare the effect in pH values of both buffer
solution and the pure water by the adding of 1ml of 1M hydrochloric acid and 1ml of 1M
sodium hydroxide.
The preparation of the buffer solution with desired pH value conducted in this
experiment was referring to Henderson-Hasselbalch equation in order to acquire the exact
amount of weak acid concentration and its conjugate base. The method of preparation is
followed by the high concentration of the buffer solution diluted until pH desired is achieved.
This increased the buffer capacity of the buffer solution. The differences between other
solutions with buffered solution can be easily distinguished from the behaviour of the pH
changed. Buffer solution’s characteristics included a solution which can resist the change in
pH when certain amount of acidic or alkali solution was added. The pH of buffer solution
prepared was higher than the expected pH since supposedly, the pH is 5.0 as calculated earlier
before the experiment began. The higher pH reading is most probably because of an excessive
volume of sodium citrate solution. The difference occurred due to the volume of weak acid
and conjugate base that are not being measured accurately.
Based on the results obtained, all the objectives of the experiment were successfully
achieved even though not as ideal as the theoretical result. Some recommendations should be
made for further study.

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 Preparation Of Buffer Solution

5.2 Recommendation

For further study, some recommendations can be made included the calibration of
equipment before running the experiment. The main problem this experiment of not obtaining
the ideal results were caused by the pH meter that do not calibrate properly before used which
have disturbed the reading. Next, the pH must be checked regularly in order to make sure the
pH meter is in a good condition so, accurate reading of pH value can be achieve. The distilled
water that was used should be boiled first to remove the dissolved carbon dioxide and have
been protected from contamination of atmospheric carbon dioxide. Besides, the preparation of
buffers must be prepared correctly and consistently too by make sure all the details of the
procedure are clearly spelled out in order to prevent confusion. Moreover, preparing the
Buffer also need to concern about the temperature and the concentration used, changes in
concentration may cause shift dissociation. Lastly, a proper storage of buffer also needs to be
take care because basic buffers should not be stored in glass containers.

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 Preparation Of Buffer Solution

REFERENCES

1. Laura B. (2016). What is a Buffer Solution? How to make a buffer solution? .

Retrieved from My Tutor: https://www.mytutor.co.uk/answers/6814/A-
Level/Chemistry/What-is-a-Buffer-solution-how-to-make-a-buffer-solution
2. Lopes, A. (2017). Geochemistry - buffers - Aaron Lopes. [online]
Eochemistry.wikispaces.com. Available at:
https://eochemistry.wikispaces.com/buffers+-+Aaron+Lopes#cite_note-2 [Accessed
26 Oct. 2017].

3. Courses.lumenlearning.com. (2017). Boundless Chemistry | Simple Book Publishing.

[online] Available at: https://courses.lumenlearning.com/boundless-chemistry/
[Accessed 19 Nov. 2017].

4. Chemistry.wustl.edu. (2017). pH Buffers in the Blood. [online] Available at:

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Buffer/Buffer.html [Accessed
19 Nov. 2017].

5. Newworldencyclopedia.org. (2017). Buffer solution - New World Encyclopedia.

[online] Available at: http://www.newworldencyclopedia.org/entry/Buffer_solution
[Accessed 19 Nov. 2017].

6. Sciencescribe.co.nz. (2017). Home. [online] Available at:

http://www.sciencescribe.co.nz [Accessed 19 Nov. 2017].

7. Tiwari, R. (2015, December 17). pH Meter: Principle and Type. Retrieved November
8, 2017, from Slideshare: https://www.slideshare.net/Haddies/ph-meter-56234681
[Accessed 19 Nov. 2017].
8. Henry, K. (2011, April 13). How to Use a Micropipette. Retrieved November 8, 2017,
from Molecular, Cell, and Developmental Biology:
https://www.mcdb.ucla.edu/Research/Goldberg/.pdf [Accessed 19 Nov. 2017].

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 Preparation Of Buffer Solution

APPENDIXES

Calculation for buffer solution:

Given;
pH = 5.0 pKa = 4.76
[𝐴− ]
pH = pKa + 𝑙𝑜𝑔10 [𝐻𝐴]

[𝑐𝑖𝑡𝑟𝑎𝑡𝑒]
5.0 = 4.76 + 𝑙𝑜𝑔10 [𝑐𝑖𝑡𝑟𝑖𝑐 𝑎𝑐𝑖𝑑]

[𝑐𝑖𝑡𝑟𝑎𝑡𝑒]
0.24 = 𝑙𝑜𝑔10
[𝑐𝑖𝑡𝑟𝑖𝑐 𝑎𝑐𝑖𝑑]

1.74[citrate] = [citric acid]

Total of solution: 1.74 + 1
= 2.74 ML
1
Citric acid needed: × 250 ML
2.74

= 91.24 ML
1.74
Citrate needed: × 250 ML
2.74
= 158.76 ML

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Preparation Of Buffer Solution

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Preparation Of Buffer Solution

37