Name:_______________________________________ Course:____________________ Score:

Date:________________________________________Group No:__________________

ACTIVITY 5
BUFFERING ACTION OF THE BLOOD SERUM
Buffer action is the ability of the buffer solution to resist the changes in pH value on the
addition of small amount of an acid or a base is known as buffer action. Human blood contains a
buffer of carbonic acid (H2CO3) and bicarbonate anion (HCO3-) in order to maintain blood pH
between 7.35 and 7.45, as a value higher than 7.8 or lower than 6.8 can lead to death. In this
buffer, hydronium and bicarbonate anion are in equilibrium with carbonic acid. Furthermore, the
carbonic acid in the first equilibrium can decompose into CO2 gas and water, resulting in a
second equilibrium system between carbonic acid and water. Because CO2 is an important
component of the blood buffer, its regulation in the body, as well as that of O2 , is extremely
important. The effect of this can be important when the human body is subjected to strenuous
conditions.

Buffer solutions are extremely important in biology and medicine because most
biological reactions and enzymes need very specific pH ranges in order to work properly.

Objectives:
1. To observe the buffering action of blood serum
2. To determine the pH of blood serum

Materials/Reagents:
20ml distilled water 1 graduated cylinder centrifuge machine
1ml 0.1M NaOH 10 test tubes medicine dropper
1ml 0.1M HCl 1 test tube rack alcohol
1ml phenolphthalein 1 syringe 1 tourniquet
1ml methyl orange 5 cotton

Procedure
A. 1. Withdraw 5ml venous blood (make sure to follow aseptic technique) and place it into a test
tube. Centrifuge the sample.
2. Extract at least 1mL of serum from the blood and use it to procedure B.
B. 1. Prepare four test tubes as follows:
Test Tubes 1 2 3 4
Water 5mL 5mL 4.5mL 4.5mL
0.1 M NaOH 1 Drop - - -
0.1 M HCl - 1 Drop - -
phenolphthalein 1 Drop - 1 Drop -
Methyl orange - 1 Drop - 1 Drop
Serum - - 0.5mL 0.5 mL

2. To test tube 3, add 0.1M NaOH dropwise until it matches the color of test tube 1.
3. To test tube 4, add 0.1M HCl dropwise until it matches the color of test tube 2.
4. Record the total number of drops of 0.1M NaOH and 0.1M HCl in each case.

Biochemistry Laboratory| ACT 5 | 1

Name:_______________________________________ Course:____________________ Score:
Date:________________________________________Group No:__________________

ACTIVITY 5 PRE LAB OUTPUT:

Reminders: Answer & submit this on the day that is given by the instructor.

Create a Schematic Diagram from the Procedure Above:

Guide Questions:

1. What is acid-base indicator and how it is used in this activity?

Acid–base indicators are compounds that change color when they become protonated or deprotonated.
Because this color change occurs over a specific pH range, indicators can be used to approximate the
equivalence point of an acid–base titration. It is used to determine the pH of blood serum in this
activity.

2. Give at least two factors that would account for the buffering action of blood serum.

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3. Give the physiologic importance of buffers.
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4. What are the symptoms if the blood pH becomes lower or becomes higher than its
normal value?
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REFERENCES:

Biochemistry Laboratory| ACT 5 | 2

Name:_______________________________________ Course:____________________ Score:
Date:________________________________________Group No:__________________

Acid–base indicators. (n.d.). Khan Academy. Retrieved September 28, 2022, from

https://www.khanacademy.org/science/ap-chemistry-beta/x2eef969c74e0d802:acids-and-

bases/x2eef969c74e0d802:acidbase-titrations/v/acid-base-indicators#:%7E:text=Acid

%E2%80%93base%20indicators%20are%20compounds,of%20an%20acid

%E2%80%93base%20titration.

Chemistry of buffers and buffers in our blood (article). (n.d.). Khan Academy. Retrieved

September 28, 2022, from https://www.khanacademy.org/test-prep/mcat/chemical-

processes/acid-base-equilibria/a/chemistry-of-buffers-and-buffers-in-blood#:

%7E:text=Buffering%20system%20of%20blood&text=When%20any%20acidic

%20substance%20enters,of%20blood%20from%20becoming%20acidic.

https://www.med.unc.edu/pharm/sondeklab/wp- What Is Alkalosis? (2021, June 24). WebMD.

Retrieved September 28, 2022, from https://www.webmd.com/a-to-z-guides/what-is-

alkalosis

content/uploads/sites/868/2018/10/buffers_calbiochem.pdf

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ACTIVITY 5 POST LAB OUTPUT

Reminders: Answer & submit this activity after actual experiment.

(Must be printed and answer in handwritten)

Results: (Note: The answer should be based on the actual experiment)

Test Tubes Observations

1

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Name:_______________________________________ Course:____________________ Score:
Date:________________________________________Group No:__________________

Conclusion
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Biochemistry Laboratory| ACT 5 | 4