ABSTRACT

A buffer is a solution that can resist ph change upon the addition of an acidic or basic
component. It is able to neutralized small amounts of added acid or base, maintaining the ph
relatively stable. Worth mentioning that each buffer has each own capacity and ph range, which
dictate how much acid or base can be neutralized before ph change, and the quantity by which may
change.

In this experiment there are two parts of experiment which are phosphate buffer and acetate buffer.
In part A, the experimenters are going to prepare the buffers in phosphate as well as acetate buffers.
This part is introduced to determine the properties of weak acids and bases using the two samples
that mentioned above. From the result, it shown that phosphate buffer is a very good buffer for
maintaining the Ph of blood resulting in 7.0 ph level, buffers like acetate buffer is good for
particular solution resulting in 4.0 ph level.

For experiment part B which is the buffering effect of four given mixtures namely: Phosphate
Buffer, Acetate Buffer, Distilled Water, Distilled Water with HCL, Phosphate Buffer with HCL
and Acetate Buffer with HCL. By using ph meter and ph universal paper, reading of ph of each
solution are able to determine. The theoretical values of each solution are Phosphate Buffer is 7.0,
Acetate Buffer is 4.0, Distilled Water is 7.0, Distilled Water with HCL is 4.0, Phosphate Buffer
with HCL is 7.0, and last sample is Acetate Buffer with HCL is 4.0.
 I. INTRODUCTION

The term pH was introduced by S.P.L. Sorensen, a Danish biochemist, to describe the degree
of acidity or basicity. He defined ph as a negative logarithm of the molar concentration of hydrogen
ions. Ph stands for potential hydrogen with the “p” meaning potential and “H” standing for
hydrogen.

The background in this experiment is to introduce review in concept of ph and buffers of weak
acids and bases. There are to specific samples to illustrate the properties of weak acids and weak
bases by using Henderson- Hasselbach Theory. The first sample that we use is Phosphate Buffer,
the second sample is Acetate Buffer.

A buffer solution is one which resists changes in ph when small quantities of an acid or an alkali
which are strong acids and bases in the solution are added to it is called buffered. To be able to
add a strong acid or base to a solution without causing a large change in the ph, it is needed to
create a buffer solution. A buffer solution contains both a weak acid (HA) and its conjugate base
(A¯ ). This reaction occurs because of the buffers solution can react both acids and bases. A basic
solution will have a ph above 7.0, while an acidic solution will have ph below 7.0, wherein buffers
can maintain the overall steady of the ph solution because buffers contain a weak acid and its
conjugate base, as such, they can absorb excess H+ ions or OH ¯ ions.

According to Bronsted-Lowry Definition, acids are proton donor and bases are proton acceptor.
The focus of this definition is on donating and accepting protons, and it is not limited to aqueous
solution. Many biochemicals are weak acids. It is then important to know how to calculate the ph
of acidic and basic solutions.
 II. METHODOLOGY

To perform the experiment of ph and buffers these are the materials needed:

A. Equipments

(4) 50 ml Beaker (2) 200 ml Beaker

(2) 10 ml Volumetric Pipette ph Meter

(1) 10 ml Graduated Cylinder

B. Reagents
1M KH2PO4 1M K2HPO4
1M CH3COOH 1M CH3COO¯Na+
1M HCL Distilled Water
A. PREPARATION OF BUFFERS

Prepare 100 ml of 1 M Take 5 ml of KH2PO4 and

solutions of KH2PO4 (monobasic), K2HPO4, mix in 50 ml
K2HPO4 (dibasic potassium phosphate), beaker. Repeat procedure in
CH3COOH (acetic acid) acetic acid.
and CH3COO¯Na+ (sodium acetate).

Record your observation Measure ph using ph

meter and universal ph
meter.
 B. BUFFERING EFFECT

Prepare 4 pcs. of 50 ml In the first beaker, place In second beaker,

beaker and labeled in 1-4. 10 ml distilled water. place 10 ml
distilled water & 1
drop HCL.

In third beaker,
Determine the ph of all In fourth beaker, place place 10 ml of
solution using ph meter 10 ml of 1M Acetate 1M Phosphate
& universal ph paper. buffer & 1 drop HCL buffer & 1 drop
HCL.

Record your observations

 III. DATA AND RESULTS

Table 1: The results of each mixtures using Ph meter and Universal Ph Paper to determine the
ph level of each solution
MIXTURE Ph Meter Ph Paper
KH2PO4/K2HPO4 7.0 7.0
CH3COOH/CH3COO¯Na+ 4.0 4.5
Distilled Water 7.0 6.0
Distilled Water + HCL 4.0 4.5
Phosphate Buffer + HCL 7.0 7.0
Acetate Buffer + HCL 4.0 4.5

IV. DISCUSSION

The overall trend in this experiment was that the results in conducting the ph scale of each
buffer solution is closer in two devices that been used for determining the ph. Given the table
above, it can be said that the objectives prior to this experiment was supported by the obtained
results wherein the greatest buffering capacity would be obtained if the buffer solution contains
equimolar concentrations of weak acids and its conjugate base.

V. CONCLUSION

Buffer can be said to be effective if it can maintain the ph of a certain solution from its ph range
such that the Phosphate Buffer is very good buffer for maintaining the ph of blood which is 7.4 since
the maximum ph of the phosphate buffer is 8.4 based from its range thus, buffers like acetate buffer
is good for particular solution which has the maximum range of 5.74 and minimum range of 3.74.
Moreover, knowing the principle of buffers and the Henderson- Hasselbach equation is very crucial
for buffer preparation and for better understanding.
Overall, this experiment provides a knowledge on how buffer work in a particular solution. It can be
used upon in the future to be able to know the properties of buffer and to fully understand the sense
of buffer.
VI. ANSWER TO QUESTIONS

1. Show the equilibrium equations for the ionization of acetic acid and KH2PO4.

CH3COOH (aq) + H2O (l)  H3O + (aq) + C2H302¯ (aq)

KH2PO4  K+ + H2PO4¯

2. Derive Henderson- Hasselbach Equation

Ka= [H=][A-]
[HA]

-log Ka = -log = [H=][A=]

[HA]

-log Ka= -log [H] ˗log [A-] / [HA]

pKa= ph -log [A] / [HA]
ph= pka + log [A-] / [HA]

3. An acetate buffer was prepared by mixing 10 ml of 0.1 M acetic acid and 100 ml of
0.1 M sodium acetate. What is the ph of the buffer solution?

Dilution Formula: (M1 V1) = (M2 V2)

(0.1 M acetic acid) (0.01 ml) = (?) (0.11 ml)
? = 0.001 / 0.11
M= 0.009 CH3COOH
(0.1 M sodium acetate) (0.1 ml) = (?) (0.11 ml)
? = 0.01 / 0.11
M= 0.09 CH3COOHNa+
Ph= pka + log [salt] / [acid]
= 4.74 + log [0.09] / [0.009]
Ph= 5.74
 4. Can a buffer solution be prepared from a mixture of NaNO3 and HNO3? Explain.

Technically from the meaning of buffer it must a combination of weak base and acid and its salt,
the combination of NaNO, and HNO3 is a strong acid and salt mixture thus it will not form buffer
solution.
WRITTEN REPORT IN BIOCHEMISTRY
LABORATORY
Ph and Buffers
Experiment No. 1

Submitted by:
Group (Afternoon Session)
Sadiwa, Claudine
Salgado, Ma. Ysabelle Xaxa
Subingsubing, Queen Jyil
Tenerife, Rizza Maica

Submitted to:
Mr. Nelson Villarante