Department of Pure and Applied Sciences

College of Arts and Sciences

Visayas State University

Name: Jonna Celina Y. Madrazo Date Perfomed: June 11, 2019

Group No.: 4 Date Submitted: June 18, 2019
Score: ___________

Experiment #2
pH and BUFFER SYSTEM

Abstract
This Experiment No.2 entitled pH and Buffer System entails the process involving pH and
buffer solutions together with the use of The Henderson-Hasselbalch Equation in which the
equilibrium constant for the dissociation of an acid, Ka, is rearranged. This experiment serve as a
determinant in doing the right calibration of the pH meter, knowing the factors affecting buffer
capacity considering the buffer system, knowing the effect of concentration of buffer using the
corresponding concentrations either phosphate buffer or acetate buffer as well as its effect of the
ratio of the conjugate base to the weak acid. Titration of an amino acid is also part of this
experiment wherein measuring of pH is recorded every after addition of NaOH and graphing of
the results as well as determining of the pKa’s is involved.

Introduction/Rationale
The Henderson-Hasselbalch Equation is highlighted in this experiment. This equation is
important in understanding the action of buffer systems and indicators. Its equation is written in
this form, pH = pKa + log ([A]/[HA]) , wherein [A] is considered as the conjugate base and [HA] is
an acid. It shows that the pH of the buffer solution is determined by the ratio of the base to the
acid in the buffer pair and its pKa. The buffer solution resist the changes in hydronium ion
concentration upon addition of small amounts of acid or alkali. It also shows the greatest
resistance to the pH change when the conjugate base and acid concentrations are the same, i.e.
the point of half neutralization of the weak acid. The measurement of pH may be carried out their
colorimetrically or electrometrically wherein the colorometric procedures involves the use of
indicators while electometric procedures involves the use of instruments namely reference
electrode, glass electrode, and an electrometer. Note that, to ensure measuring the accurate pH,
the standard buffer used for calibration should have a pH near that of the test solution.
Standardization with two buffer standards is preferable to the use of one standard only.
Methodology
A. Calibration of the pH meter
The laboratory instructions in the use of the pH meter was carefully followed with the
guidance of the laboratory instructor before using the instrument.
B. Factors Affecting Buffer Capacity

Effect of the Concentration of Buffer

Case 1
0.1 M phosphate buffer with a pH of 7.2 was used and 25 mL each of the buffer
solutions with a concentrations at pH 7.2 is prepared.
Case 2
0.1 M acetate buffer with a pH of 4.7 was used and 25mL each of the buffer
solutions with a concentration at pH 4.7 was prepared.

The pH of each of the buffer solutions was recorded. A 2mL of 0.1 M NaOH was
added to each of the 25mL buffer samples assigned in each group. The pH of each buffer
solution after the addition of alkali was also recorded. The magnitude of the change of pH
was accounted.

Effect of the Ratio of the Conjugate Base to Weak Acid

The ratio of the dihydrogen phosphate and monohydrogen phosphate was

calculated from the Henderson-Hasselbalch equation. The ratio of acetic acid and acetate
was calculated next to it. With the use of 0.1 M stock solution provided, 25mL of each of
the buffer solution was made. Then, the pH of each of the buffer solutions was measured
and recorded and 2mL of 0.1 M NaOH to each of the 25mL buffer samples was added.
The pH of each buffer solution after addtion of alkalli was recorded and the magnitude the
pH shift in each with reference to the direction of pH shift was accounted.
C. Choice and Preparation of a Buffer System

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.

Preparation of the Chosen Buffer System

The amount (in grams if solid and in mL if liquid) of weak acid and conjugate
base needed to be able to prepare the chosen buffer system in part A above was
calculated and measured.
 D. Titration of an Amino Acid

In this part, an amino acid, aspartic acid was assigned to the group, was titrated.
10mL of the assigned amino acid sample was drawn from the pipette into a 25mL
or 50 mL beaker. It was adjusted to a pH of 1.5. With the use of a burette a 0.1M NaOH
was added in approximately 0.5 mL increments until about Ph 12 is reached. The
accurate volume of each increment was recorded.
Every after addition it was stirred well and measured. Plotting of results of pH vs.
mL NaOH added was done appropriately. The pKa’s of the sample from the graph was
determined. The structures of the amino acids at each pKa value was drawn.

Results and Discussion

 Factors Affecting Buffer Capacity

EFFECT OF CONCENTRATION OF BUFFER

Case 1 Phosphate Buffer

pH pH with alkali Amount of Buffer Solution (mL)

0.005 M 7.4 8.1 1.25 mL
0.05 M 7.4 7.5 12.5 mL
0.10 M 7.4 7.4 25 mL

With the use of 0.1 M phosphate buffer, pH 7.2, 25 mL of each of the buffer solution was
prepared. The 0.005 M, 0.05 M, and 0.10 M concentrations have the same pH level at
7.4. After adding alkali its pH level has changed wherein the pH of 0.005 M became 8.1
with 1.25 mL buffer solution , in 0.05 M became 7.5 with 12.5 mL buffer solution, and
0.10 M became 7.4 with 25 mL buffer solution.

Case 2 Acetate Buffer

pH pH with alkali Amount of Buffer Solution (mL)

0.005 M 4.4 6.8 1.25 mL
0.05 M 4.4 4.6 12.5 mL
0.10 M 4.4 5.6 25 mL

With the use of 0.1 M acetate buffer, pH 4.7, 25 mL of each of the buffer solution was
prepared. The 0.005 M, 0.05 M, and 0.10 M concentrations have the same pH level at
4.4. After adding alkali its pH level has changed wherein the pH of 0.005 M became 6.8
with 1.25 mL buffer solution , in 0.05 M became 4.6 with 12.5 mL buffer solution, and
0.10 M became 5.6 with 25 mL buffer solution.
 EFFECT OF THE RATIO OF THE CONJUGATE BASE TO THE WEAK ACID

Case 1 Ratio Of Dihydrogen Phosphate and Monophosphate Components

Required to Produce Buffer Solution

pH H2PO4- amount HPO42- amount pH with NaOH

6.2 13.3 mL 1.86 g 6.8
7.2 7.4 mL 0.10 g 7.6
8.2 136.15 mL 0.21 g 8.3

Case 2 Ratio of Acetic Acid and Acetate Required to Produce Buffer Solution

pH Acetic Acid amount Sodium Acetate pH with NaOH

amount
3.7 5 mL 120 mL 3.9
4.7 2.67 mL 69.8 mL 4.9
5.7 0.5 mL 134. 5 mL 6.2

With the use of 0.1 M stock solution provided, 25 mL of each buffer was made. The pH
of each buffer solutions was measured and recorded. After adding 2 mL of 0.1 M NaOH
to each of the 25 mL buffer samples. The pH after addition of NaOH have changed as
shown in the table above. New pH was recorded.

 Choice and Preparation of a Buffer System

1. In Protein Precipitation, 2 L of 5Mm buffer solution with pH 5.2 is needed for isolation
of albumin. Which among the following buffer solutions is best fitted for said
purpose? Justify your answer.

Buffer Solutions pKa

Acetate Buffer 4.73
Tris-(hydroxymethyl) aminomethane 8.08
Phosphate buffer 7.20

Answer:
The best fitted buffer solution is the acetate buffer solution since its pH of 5.2 falls
within the range equal to the pKa ± 1 of acetate buffer which is 3.73-5.73 .

2. Preparation of the Chosen Buffer System

Calculation:

Given: pKa = 4.76 Let: CH3COOH be HA+

Acetic acid = CH3COOH (weak acid) C2H3O2Na be A-
Sodium acetate = C2H3O2Na (conjugate base)
Solution:
[𝐴− ]
pH = pKa + log [𝐻𝐴]
[𝐴− ]
3.7 = 4.76 + log
[𝐻𝐴]
[𝐴− ]
3.7 – 4.76 = log [𝐻𝐴]
[𝐴− ]
-1.06 = log [𝐻𝐴]
[𝐴− ]
antilog(-1.06) = log [𝐻𝐴]
0.087096359 [𝐴− ]
1
= [𝐻𝐴]

1.087096359

Decimal Fraction
0.087096359
C2H3O2Na : 1.087096359 = 0.080118343
1
CH3COOH : 1.087096359 = 0.91988657

M of each component
MA = 0.1 M x 0.080118343 = 0.080118343
MA = 0.1 M x 0.91988657 = 0.091988657
Moles of each component
moles A- = 0.080118343 x 1.5 L = 0.012017751 moles
moles HA = 0.091988657 x 1.5 L = 0.013798298 moles

C2H3O2Na = 0.1 M
CH3COOH = 3 M
C2H3O2Na
0.012017751
x 1000 mL = 120 mL
0.1 𝑀

CH3COOH
0.013798298
3𝑀
x 1000 mL= __5 mL

125 mL

C2H3O2Na = 120 mL
CH3COOH = 5 mL
 Titration of an Amino Acid

ASPARTIC ACID
pH mL
1.5 1
1.6 2
1.7 3
1.8 4
1.9 5
2.0 6
2.1 7
2.2 8
2.3 9
2.4 10
3.3 20
4.3 30
9.0 40
9.3 50
9.4 60
9.5 70
9.6 80
9.7 90
9.8 100
9.9 120
10.3 140
11 160
11.8 180
11.9 200
12 205

This part of experiment titration was conducted. It was observed that after adding drop
by drop of 0.1 M NaOH the pH level of the asigned amino acid which is the Aspartic acid
at pH of 2.8 has increased and it turn out that after the whole drop of the NaOH, it was
205 ml of NaOH was consumed, it had reach the pH of 12. Results was recorded
accurately. (see line graph below)
 Acid-Base Titration Curve
Aspartic Acid
14

12

10

pH
6 pH

0
0 50 100 150 200 250
mL
0.1 M NaOH
  ph of Blood

Through the table of pKa values provided, suggest some compounds that could act as
buffers at physiological pH values (i.e. around pH 7.4)

The pH of blood is tightly regulated by a complex system of buffers that are continuously
at work to maintain a range of 7.3 to 7.41, which is slightly more alkaline than pure water. The
Tris(hydroxymethyl)aminomethane, carbonic acid, and phosphoric acid are one of the
compounds that could act as a buffers for the blood it is because their pKa is close to the pH of
blood.

Conclusion
Therefore, it is concluded that during the conduction of experiment the purpose of this
experiment has been attained and that is to calibrate the pH meter, to choose and prepare
appropriate buffer systems, and to titrate an amino acid.

References
Chem31 Lab Manual
Laboratory Instruction Manual in Introductory Biochemistry, Biochemistry & Agricultural
Chemistry Division, IC, CAS, UPLB
http://www. Realfarmacy. com/5-cancer-facts-that-big-pharma-is-now-aggresively-claiming-are-
myths/