2 2nd Term A.Y.

2024-
2025

BIOCHEMISTRY

BIOCHEML: LABORATORY ACTIVITY 2

BUFFERS IN ACTION (DRY LAB)

OBJECTIVES

At the end of the experiment, the students should be able to:

1. explain how buffers work.
2. describe how to prepare a buffer.
3. explain the effects of buffer on biomolecules.
4. explain the application of buffers in nursing arts and practices.

FOR REVIEW
 pH
 acids and bases, ionization of strong acids/bases, weak acids/bases
 chemical equilibrium

INTRODUCTION

Almost every biological process is pH-dependent and a small change in pH produces a large
change in the rate of the process. This is true not only for the many reactions in which the H + ion is
a direct participant but also for those in which there is no apparent role for H+ ions.

The H+ concentration affects the conformation of biomolecules as well as the concentrations of

molecular and charged species in the solutions. Since optimum activities of many biomolecules
occur within a limited range of pH, living organisms are equipped with remarkably efficient
mechanisms to maintain the intracellular H+ concentration. These mechanisms prevent changes
in [H+] as a result of either the introduction of small amounts of acidic or basic substances from
external sources or formation in the course of metabolic processes.

In humans, these mechanisms include (1) the buffer systems of the body, (2) the renal control, the
action of the kidneys by which acids and bases are excreted in the urine, and (3) the respiratory
mechanism by which the rate of carbon dioxide elimination in the lungs regulates the H+
concentration in bodily fluids.

A buffer is a solution that resists drastic changes in pH when small amounts of acid or base are
added. Buffers regulate the pH of fluids and tissues of living organisms within the limits consistent
with life and normal functions. Buffers are used in the laboratory to control the pH of culture media
for microorganisms and tissues, and are also widely used in many industrial processes like
fermentation, electroplating, manufacture of leather, photographic materials, and dyes. Table 1
shows a general scheme for the classification of buffers.

Table 1. Types of Buffers

BUFFER TYPE EXAMPLE
Acidic Buffer Solution (Solution of weak Carbonic Acid-Bicarbonate buffer (for maintaining
acid and its salt (or conjugate base) blood pH) CH3COOH + CH3COONa
HCN + NaCN, HF + NaF
Basic Buffer Solution of weak base and NH4OH + NH4Cl
its salt (or conjugate acid) NH4OH + NH4NO3
Glycine + Glycine hydrochloride
Double Salt Buffer Solution of a primary Phospate buffer, HPO42—H2PO4- (buffering renal
salt and a secondary salt tubular fluid)
NaH2PO4 + Na2HPO4
Solutions of ampholytes amino acids and proteins
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How Buffers Work

In order for a buffer to “resist” the effect of adding strong acid/base, it must have both an
acidic and a basic component. However, you cannot mix any two acid/base combinations together
and get a buffer. For example, if you mix HCl (a strong acid) and NaOH (a strong base), you will
simply neutralize the acid with the base and obtain a neutral salt, not a buffer.

For a buffer to work, a buffer must consist of a mixture of a weak conjugate acid-base pair that is
part of the same equilibrium system. In this manner, neutralizing one or the other component
(by adding a strong acid or base) will transform it into the other component and maintain the
buffer mixture.

Let’s consider the simple example of a hydrofluoric acid and sodium fluoride (HF-NaF) buffer.
Hydrofluoric acid is a weak acid and does not dissociate completely in water. Therefore, if we
obtain HF in an aqueous solution, we establish the following equilibrium with only slight
dissociation.

HF (aq) + H2O (l) ⇌ H3O+ (aq) + F- (aq) (1)

weak acid conjugate
base

We can then add and dissolve sodium fluoride (NaF) into the solution and mix the two until we reach
the desired volume and pH at which we want to buffer. When NaF dissolves in water, the reaction
goes to completion, thus we obtain:

NaF (aq) + H2O (l)  Na+ + F- (aq) (2)

The addition of NaF to the solution will increase the concentration of F- in the buffer solution, and,
consequently, by Le Chatelier’s Principle, lead to slightly less dissociation of the HF in the previous
equilibrium (Reaction 1), as well. The addition of Na+ will have no effect on the pH or reactivity of
the buffer because this is a conjugate of a strong base. The presence of significant amounts of both
the weak acid, HF, and the conjugate base, F-, allows the solution to function as a buffer.

If acid (H3O+/H+) were added to the HF-NaF buffer:

H3O+ (aq) + F− (aq) ⇌ H2O (l) + HF(aq) (3)

base conjugate acid
(salt)

When acid is added to the HF-NaF buffer, the conjugate base, F-, will neutralize the added acid,
H3O+. So long as there is more F- than H3O+, almost all of the H3O+ will be consumed and the
equilibrium will shift to the right. This will slightly increase the concentration of HF and slightly
decrease the concentration of F-, but will result in hardly any change in the amount of H3O+ present,
preventing the pH from dropping too much once equilibrium is re-established.

If base (OH-) were added to the HF-NaF buffer:

OH- (aq) + HF ⇌ H2O (l) + F− (aq) (4)

(aq)
acid conjugate base (salt)

When base is added to the HF-NaF buffer, the acid (HF) will neutralize the added base (OH -), and the
equilibrium will again shift to the right in Reaction 4. This will slightly increase the
concentration of F- in the solution and slightly decrease the amount of HF. Again, since most of the
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OH- is neutralized, a significant increase in the pH will be prevented.
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Reactions 3 and 4 can continue to alternate back and forth with little pH change.

The pH of a buffer solution made up of known concentrations of a weak acid and its salt (conjugate
base) can be calculated using the Henderson-Hasselbalch equation, also known as the buffer
equation. Buffer pH is obtained by the following expression:

𝑝𝐻 = 𝑝𝐾𝑎 + log
[𝑠𝑎𝑙𝑡]

[𝑎𝑐𝑖𝑑]
Henderson-Hasselbalch equation

Using the Henderson-Hasselbalch equation as a reference, the pH of a buffer solution after the
addition of acid or base may be calculated using the following formula:

𝑝𝐻 = 𝑝𝐾𝑎 + log 𝑝𝐻 = 𝑝𝐾𝑎 + log

[𝑆+𝑏]
[𝑆−𝑎] [𝐴−𝑏
]
wher [𝐴+𝑎]
e
S = number of moles of salt component in the
buffer A = number of moles of acid
component in the buffer a = number of moles
of acid added
b = number of moles of base added

Sample Problem 1

Given a buffer solution containing 12.0 mL of 0.10 M CH 3COONa and 8.0 mL of 0.10 M
CH3COOH. (Ka CH3COOH = 1.8x10-5)

a. What is the pH of the buffer solution? What is the [H+] concentration in the buffer?
b. What will be the change in pH of the buffer solution after the addition of 1.0 mL of 0.10 M
HCI?
c.What will be the change in pH of the buffer solution after the addition of 2.0mL of 0.15 M NaOH?

Solution

a. pKa = -log (1.8x10-5) = 4.74

pH of the
[salt]
buffer pH =
[acid]
pKa + log

[0.10 M x [0.0012 mole]

pH = 4.74 + = 4.74 +
0.012 L] [0.008 mole]
log log
[0.10M x
0.008 L]

pH = 4.74 + log 1.50 = 4.92

[H+] of the
buffer pH = -
log [H+] pH =
-log [4.92]
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[H+] = 10-4.92
[H+] = 1.20 x10-5 M
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b.
pH2 = pH after the addition of HCI
[S − a]
pH2 = pKa + log
[A + a]
[(0.10M)(0.012 L) − (0.10M)(0.001L)]
pH2 = 4.74 + log
[(0.10M)(0.008L) + (0.10M)(0.001L)]

[0.011mol
e] pH2 = 4.74 + log
[0.009 mole]

pH2 = 4.74 + log 1.22 = 4.83

change in pH = |pH2 – pH of buffer| = |4.83 – 4.92| = 0.09

c.
pH3 = pH after the addition of NaOH
[𝑆 + 𝑏]
𝑝𝐻 = 𝑝𝐾𝑎 +
log [𝐴 − 𝑏]

[(0.10M)(0.012 L) + (0.15M)(0.002L)]
pH3 = 4.74 + log
[(0.10M)(0.008L) − (0.15M)(0.002L)]

[0.0015mol
pH3 = 4.74 + log
e]
[0.0005 mole]

pH3 = 4.74 + log 3 = 5.22

change in pH = |pH3 – pH of buffer| = |5.22 – 4.92| = 0.30

Sample Problem 2

Prepare 200 mL of a 0.10 M CH3COOH-CH3COONa buffer with a pH of 4.50 from 0.20 M acid and
0.50 M salt stock solutions. Calculate the volume needed for each component of the buffer.

Solution

STEP 1. Calculate the total moles of buffer components.

Total moles buffer = (volume of buffer) x (conc. of buffer)

= (0.200 L) (0.10 M) = 0.0200 mol  moles acid
component + moles salt component

STEP 2. Using the Henderson-Hasselbalch equation, determine the number of ratio of the moles
of the acid and the salt to be used in preparing the buffer.

[salt]
pH = pKa +
[acid]
log [salt]
4.50 = 4.74 +
log [acid]
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[salt
lo ] = − 0.24
g
[aci
d]

[𝑠𝑎𝑙 [𝑎𝑐𝑖
𝑡] = 0.575 = 1.74
𝑑]
𝑜𝑟
[𝑎𝑐𝑖 [𝑠𝑎𝑙
𝑑] 𝑡]

There are two working equations to calculate for the moles of buffer components in Step 3.

STEP 3. Compute for the moles of salt and moles of acid respectively.

[𝑚𝑜𝑙𝑒
𝑠𝑎𝑙𝑡] = 0.575
[𝑚𝑜𝑙𝑒
𝑎𝑐𝑖𝑑]
mole salt = 0.575 (mole acid)

mole salt + moles acid = 0.0200 mol (from Step 1)

0.575 (moles acid) + mole acid = 0.0200 mole (let
mole acid = x) 0.575x + x = 0.0200 mole
1.57 x = 0.0200 mole
x = 0.0127 mol  mole acid

mole salt + moles acid =

0.0200 mol mole salt + 0.0127
mol = 0.0200 mol moles salt =
0.00730 mol

STEP 4. From the calculate amount of substance in moles and corresponding stock solutions, the
volume of acid and salt components can then be calculated.

volume 0.20 M CH3COOH = 0.0127 mol/0.20M = 0.0635L =

63.5 mL volume 0.50 M CH3COONa = 0.00730 mol/0.50M =
0.0146L = 14.6 mL

 To prepare the 200 mL of a 0.10 M CH3COOH-CH3COONa buffer mix 63.5 mL of 0.20 M

CH3COOH and
14.6 mL of 0.50 M CH3COONa.
 Dilute the solution to 200 mL with distilled water.
 Determine the pH of the solution with a pH meter.
 Adjust the pH if necessary, by the addition of either the acid or salt. If more than 1.00 mL is
needed for pH adjustment, the buffer must be prepared all over again.
MATERIALS
scientific calculator
VIDEO 1: https://www.youtube.com/watch?v=X_NewslAWJE
VIDEO 2: https://www.youtube.com/watch?v=P-R-Cqvb5yo
VIDEO 3: https://www.youtube.com/watch?v=KZ9zwzWLjMs

PROCEDURES
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1. Watch VIDEO 1 for the demonstration on how to prepare a buffer.

2. Watch VIDEO 2 and observe the change in pH between distilled water and a buffer solution.
Record the pH as you watch the video in your laboratory worksheet.
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GROUP NO. SCORE:

NAME/S: DATE:
SECTION:

BIOCHEML: LABORATORY ACTIVITY 2

BUFFERS IN ACTION

I. DATA & OBSERVATIONS

Table 1. pH of distilled water and Buffer Solution

DISTILLED WATER BUFFER SOLUTION
Initial pH faith
After 1-2 drops HCl xena
After 15-20 drops HCl eunice
After few drops NaOH kim
After 15-20 drops NaOH hanna

II. QUESTIONS:

1. Compare the change in pH between the distilled water and buffer solution. Was the buffer
able to resist pH change in the solution? eiseine
2. Watch VIDEO 3 and differentiate the amount of HCl needed to reach the pH of 2.43
between the distilled water and NH3/NH4+ buffer solution. hanna
3. Problem Solving
a. A buffer of an enzyme inhibition assay is prepared by the undergraduate student by
adding 1.34 moles of acetic acid and 2.31 moles sodium acetate enough to make 1L
solution. What is the pH of the resulting solution? (Ka CH3COOH = 1.8x10-5) eiseine
b. What is the pH of a solution containing 0.10 M chloroacetic acid (Ka = 1.4 x 10-3) and
0.15 M sodium chloroacetate? xena
c. What is the composition of 0.10 M sodium acetate buffer at pH 5.5 if 500 mL of this
buffer is to be prepared from 0.50 M HOAc and 1.0 M NaOH stock solutions? Compute for
the volume of each buffer component to be mixed. eunice
d. (i) Calculate the pH of a buffer prepared by mixing 575 mL of 0.50 M formic acid (HCOOH,
Ka = 1.7 x 10-4) and 425 mL of 0.50 M sodium formate, HCOONa. faith
(ii) What would be the pH if 5.0 mL of 0.15 M HCI is added to the buffer in d(i)? faith
4. How does understanding the role of biological buffers, such as bicarbonate and phosphate
systems, assist nurses in managing patients with acid-base imbalances?
kim
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BIOCHEMICAL CONNECTIONS – BUFFERS IN BIOLOGICAL SYSTEMS

The purpose of a buffer in a biological system is to maintain intracellular and extracellular pH within
a very narrow range and resist changes in pH in the presence of internal and external influences. A
change in pH can result in metabolic acidosis (too acidic-low pH) or alkalosis (too basis-high
pH), resulting in severe metabolic complications. The pH of blood should maintain a pH range
of 7.35-7.45.

The buffer systems in the human body are extremely efficient, and different systems work at
different rates. It takes only seconds for the chemical buffers in the blood to make adjustments to
pH.

The buffer systems functioning in blood plasma include plasma proteins, phosphate, and
bicarbonate-carbonic acid buffers. The kidneys help control acid-base balance by excreting
hydrogen ions and generating bicarbonate that helps maintain blood plasma pH within a normal
range. Protein buffer systems work predominantly inside cells.

Respiratory Regulation of Acid-Base Balance and Bicarbonate-Carbonic Acid Buffer System

The respiratory system contributes to the body’s balance of acids and bases by regulating the
blood levels of carbonic acid. CO2 in the blood readily reacts with water to form carbonic acid, and
the levels of CO2 and carbonic acid in the blood are in equilibrium (see reaction below).
CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3–

When the CO2 level in the blood rises (as it does when you hold your breath), the excess CO2
reacts with water to form additional carbonic acid, lowering blood pH. Increasing the rate and/or
depth of respiration (which you might feel the “urge” to do after holding your breath) allows you
to exhale more CO2. The loss of CO2 from the body reduces blood levels of carbonic acid and
thereby adjusts the pH upward toward normal levels. As you might have surmised, this process
also works in the opposite direction. Excessive deep and rapid breathing (as in
hyperventilation) rids the blood of CO 2 and reduces the level of carbonic acid, making the blood too
alkaline. This brief alkalosis can be remedied by rebreathing air that has been exhaled into a
paper bag. Rebreathing exhaled air will rapidly bring blood pH down toward normal.

Renal Regulation of Acid-Base Balance

The renal regulation of the body’s acid-base balance addresses the metabolic component of the
buffering system. Whereas the respiratory system (together with breathing centers in the brain)
controls the blood levels of carbonic acid by controlling the exhalation of CO2, the renal system
controls the blood levels of bicarbonate.
 The kidneys can either absorb or excrete bicarbonate levels depending on the body's pH.
 Acidosis causes more bicarbonate, HCO3− (base) to be reabsorbed from the renal
tubular fluid, thus causing the collecting ducts to secrete more hydrogen, H + into the
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urine (and generating even more bicarbonate, increasing pH to normal level).
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 Alkalosis causes the kidneys to excrete more bicarbonate because there is a reduced
secretion of hydrogen ions and more ammonia (base) is excreted (lowering pH to
normal level).

Protein Buffers in Blood Plasma and Cells

Nearly all proteins can function as buffers. Proteins are made up of amino acids, which contain
positively charged amino groups and negatively charged carboxyl groups. The charged regions
of these molecules can bind hydrogen and hydroxyl ions, and thus function as buffers.
Buffering by proteins accounts for two-thirds of the buffering power of the blood and most of
the buffering within cells.

Hemoglobin as a Buffer
Hemoglobin is the principal protein inside of red blood cells and accounts for one-third of the mass
of the cell. During the conversion of CO2 into bicarbonate, hydrogen ions liberated in the reaction
are buffered by hemoglobin, which is reduced by the dissociation of oxygen. This buffering helps
maintain normal pH. The process is reversed in the pulmonary capillaries to re-form CO2, which
then can diffuse into the air sacs to be exhaled into the atmosphere.

Phosphate Buffer
Phosphates are found in the blood in two forms: sodium dihydrogen phosphate (Na2H2PO4−),
which is a weak acid, and sodium monohydrogen phosphate (Na2HPO42-), which is a weak base.
When Na2HPO42- comes into contact with a strong acid, such as HCl, the base picks up a second
hydrogen ion to form the weak acid Na2H2PO4− and sodium chloride, NaCl. When Na2HPO42− (the
weak acid) comes into contact with a strong base, such as sodium hydroxide (NaOH), the weak
acid reverts back to the weak base and produces water. Acids and bases are still present, but
they hold onto the ions.

REFERENCES
Boyer, R., (2000). Modern Experimental Biochemistry, 3rd ed. Addison Wesley Longman Inc.
Institute of Chemistry. Biochemistry Laboratory Manual. University of the Philippines-Diliman, Quezon City.
Oregon State University. (2019). Acid-Base Balance. https://open.oregonstate.education/aandp/chapter/26-4-
acid-base- balance/