FACULTY OF BASIC MDICAL SCIENCES

DEPARTMENT OF BIOCHEMISTRY
BCH2301: GENERAL BIOCHEMISTR (2 UNITS)
THE DEFINITION AND BRIEF HISTORY OF BIOCHEMISTRY
OVERVIEW OF CELL BIOLOGY
CELL DISRUPTION AND CENTRIFUGATION
LECTURE NOTES PREPARED BY DR NASIRU ABDULLAHI
Introduction:

Definition: Biochemistry is the study of chemical processes and substances that occur within living
organisms. It combines principles of biology, chemistry and physics to understand the molecular
mechanisms of life. Biochemistry deals with the structures and functions of cellular components such as
proteins, carbohydrates, lipids, nucleic acids and other biomolecules.

History: The history of biochemistry is a fascinating journey that spans several centuries, with significant
contributions from various scientists. The origins of biochemistry can be traced back to ancient times when
alchemists and natural philosophers studied the nature of life. Discovery of the basic biological molecules,
such as proteins, carbohydrates, and lipids, laid the foundation for understanding biochemical processes.
Important figures include Paracelsus, who introduced chemical medicine, and Johann Friedrich
Miescher, who discovered nucleic acids. In the 18th and 19th centuries, two opposing schools of thought
emerged regarding the nature of life.

• Vitalism proposed that living organisms possess a vital force or essence that cannot be explained
by physical and chemical laws alone. Vitalism theory says that held organic compounds could only
be synthesized by living organisms.
• Mechanism argued that all biological processes could ultimately be understood in terms of
chemistry and physics.

The debate between vitalism and mechanism influenced the early development of biochemistry. The
discovery of enzymes and their role in catalyzing biochemical reactions revolutionized the field. James
Sumner (1887 – 1955) was an American chemist and a graduate of Harvard University who discovered
that enzymes can be crystallized. He won a Nobel Prize in Chemistry in 1946. He was also the first to prove
that enzymes are proteins and is commonly referred to as the father of enzymes. The concept of metabolism,
the sum of all biochemical reactions in an organism, emerged during this period. In the early 20th century,
the study of carbohydrates and energy metabolism gained prominence. The work of Otto Meyerhof (1884
– 1951) and Gustav Embden on glycolysis and the Krebs cycle provided insights into how cells generate
energy. They won a Nobel Prize in Physiology and Medicine in 1922. The discovery of ATP (Adenosine
triphosphate) as the primary energy currency of cells by German chemist Karl Lohmann in 1929 was a

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crucial breakthrough. Elucidating the structure and function of proteins became a central focus of
biochemistry. Frederick Sanger's work on protein sequencing and Linus Pauling's studies on protein
structure were pioneering contributions. Identification and classification of amino acids and understanding
their role in protein synthesis were significant milestones.

In addition, the discovery of nucleic acid structure and function was a groundbreaking advancement in
biochemistry. The work of James Watson, Francis Crick, and Rosalind Franklin led to the
understanding of DNA's double helix structure in 1953. This marked a milestone in the history of
biochemistry and gave rise to modern molecular biology, which is largely concerned with understanding
how genes are associated with diseases. Biochemistry has continued to evolve rapidly, which is driven by
technological advancements and interdisciplinary collaborations. Fields such as genomics, epigenomics,
proteomics, transcriptomics, metabolomics, and structural biology have expanded the understanding of
biochemical processes. The advent of recombinant DNA technology and gene editing techniques like
CRISPR have revolutionized the field. Biochemistry continues to play a crucial role in fields like medicine,
agriculture, biotechnology, and drug development. These developments have led to various areas of
biochemistry such as medical biochemistry, plant biochemistry, environmental biochemistry, and industrial
biochemistry among others. Today, biochemistry continues to be a rapidly evolving field, with discoveries
made every day.

Other notable scientists are:

Carl Neuberg (1877-1956): He was a German biochemist, who is referred to as the "father of modern
biochemistry." He made numerous contributions to the understanding of enzymology, carbohydrate
metabolism, and the study of fermentation processes. Neuberg developed innovative methods for isolating
and characterizing enzymes, paving the way for further research in the field. He was a prominent figure in
the development of biochemistry, particularly in the early 20th century. However, it's important to note that
several other scientists also made several contributions to the field. Below are a few notable scientists who
played key roles in the development of biochemistry.

Friedrich Wöhler (1800-1882): Friedrich Wöhler, a German chemist, is recognized for his synthesis of
urea in 1828. These landmark experiments shattered the notion of vitalism. Wöhler's synthesis of urea from
inorganic materials demonstrated that organic compounds could be artificially produced, thereby laying the
foundation of organic chemistry linked to biochemistry.

Richard Willstätter (1872-1942): Richard Willstätter, a German chemist, received the Nobel Prize in
Chemistry in 1915 for his work on plant pigments, particularly chlorophyll. His research elucidated the
structure of chlorophyll and contributed to the understanding of photosynthesis, a fundamental biochemical
process in plants.

Frederick Gowland Hopkins (1861-1947): Frederick Gowland Hopkins, a British biochemist, was
awarded the Nobel Prize in Physiology or Medicine in 1929 for his discovery of essential nutrients and the
concept of "accessory food factors" (vitamins). Hopkins's work on the role of vitamins in maintaining health
and preventing diseases laid the groundwork for understanding the importance of micronutrients in human
physiology.

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Albert Szent-Györgyi (1893-1986): Albert Szent-Györgyi, a Hungarian physiologist and biochemist,
received the Nobel Prize in Physiology or Medicine in 1937 for his discovery of vitamin C and its role in
preventing scurvy. Szent-Györgyi's research on the biochemistry of cellular respiration and the role of
vitamin C in various biological processes advanced the field of biochemistry.

Eduard Buchner (1860 – 1917): Eduard Buchner (German chemist) was awarded the Nobel Prize in
Chemistry (1907) for his work on the fermentation of sugars and the role of enzymes in biochemical
processes. His research laid the foundation for the study of enzymology.

These are just a few examples of the many scientists who contributed to the development of biochemistry.
The collective efforts have paved the way for the current understanding of the complex chemical processes
occurring within living organisms. Table 1: shows a summary of significant breakthroughs in biochemistry
that have led to a profound impact on the understanding of biological systems and the development of new
treatments and technologies.

Table 1. Some Major Milestones in Biochemistry.

Year Name of Scientist Significant Breakthrough Comment

1828 Friedrich Wöhler Synthesizes urea He debunked the concept of vitalism
1897 Eduard Buchner Demonstrates the fermentation He won the Nobel Prize in
process. enzymes are responsible Chemistry in 1907.
for catalyzing biochemical
reactions
1902 Emil Fischer's Discovered the Lock-and-key Provided a foundation for
model of enzymes. understanding enzyme-substrate
interactions.
1906 Arthur Harden and Discovered the coenzyme, a non- Essential for the function of many
William Young protein organic molecule. enzymes.
1926 Johannes Andreas Discovers the link between cancer It opens up new avenues for
Grib and parasites. understanding the causes and
mechanisms of cancer.
1926 James B. Sumner Crystallizes the enzyme urease, Opens the door for the isolation and
and proved that enzymes are purification of other enzymes
proteins.
1930 Jansen and Donath Discovers and isolated vitamins. Leads to a better understanding of
metabolic processes
1944 Oswald Avery, discover DNA as the carrier of paving the way for the
Colin MacLeod, genetic information understanding of molecular genetics.
and Maclyn
McCarty
1953 James Watson and They proposed the structure of Revealed how genetic information is
Francis Crick DNA stored and replicated
1955 Severo Ochoa and Synthesizes RNA and DNA, This provides insights into the
Arthur Kornberg respectively replication and transcription
processes.
1970 Stanley Cohen and They developed DNA technology Leads to new applications in
Herbert Boyer medicine, agriculture, and
biotechnology.

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1970 Paul Berg Successfully combined DNA from leading to the development of
different organisms, recombinant DNA technology and
the field of genetic engineering.
1977 Fred Sanger Develops DNA sequencing Revolutionized the field of genetics
methods and enabled the sequencing of entire
genomes
1980s Edmond H. Fischer Discovered protein kinases and It gives better understanding of
and Edwin G. their role in signaling pathways cellular communication and the
Krebs. leads. development of new treatments for
cancer and other diseases.
1983 Kary Mullis Invents polymerase chain reaction Facilitates numerous applications in
(PCR) molecular biology and medicine
1985 Alec Jeffreys Developed DNA fingerprinting, a It has widespread applications in
technique that uses DNA forensics and paternity testing.
variations to identify individuals.
1990 The Human The Human Genome Project, an It provides a comprehensive
Genome Project international collaborative effort, reference for human genetics and
was initiated to map and sequence opening new avenues for
the entire human genome. personalized medicine. It was
completed in 2003,
2003 The Human Completion of the Human Genome It enables researchers to study the
Genome Project, an Project genetic basis of diseases and explore
international effort the intricacies of human biology.
2005 Andrew Fire and Discovers RNA interference This opened up possibilities for
Craig Mello (RNAi) in understanding gene therapeutic applications.
regulation.
2020 Jennifer Doudna They developed the CRISPR- They were awarded the Nobel Prize
and Emmanuelle Cas9 gene editing system. in Chemistry in 2020. The
Charpentier technology holds great promise for
the treatment of genetic diseases
2020 Pfizer-BioNTech Developed of COVID-19 vaccines The first ever rapid development of
and Moderna vaccines.

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BCH2301: GENERAL BIOCHEMISTRY (2 UNITS)
LECTURE NOTES ON CELL BIOLOGY

Cell Biology Overview:

Cell biology, also known as cytology, is the study of cells, their structure, function, and interactions. It is a
fundamental discipline in the field of biology and provides a basis for understanding the processes that
occur within living organisms. A cell is the basic structural and functional unit of all living organisms. It is
the smallest independently functioning unit within an organism that can carry out the essential processes
necessary for life. Cells can be thought of as microscopic building blocks that make up the tissues, organs,
and systems of living organisms. The cells and its organelles needs to be studied in order to understand the
chemical reactions that takes place.

The cell theory is a fundamental concept in biology that provides the foundation for our understanding of
cells. It states three main principles:

1. All living organisms are composed of cells:

o The cell is the basic structural and functional unit of life. Every organism, whether
unicellular or multicellular, is made up of one or more cells. This principle recognizes that
cells are the building blocks of all living things.
2. Cells are the basic units of structure and function in organisms:
o Cells carry out all the necessary functions for the survival and functioning of an organism.
Each cell performs specific tasks, and the collective activities of cells contribute to the
overall functioning of tissues, organs, and the entire organism. Cells have distinct structures
and organelles that enable them to carry out specialized functions.
3. Cells arise from pre-existing cells:
o New cells are formed through the division of pre-existing cells. This principle, known as
cell division or biogenesis, refutes the earlier concept of spontaneous generation, which
proposed that living organisms could arise from non-living matter. The principle of cell
division emphasizes that cells give rise to new cells, and this process is essential for growth,
development, and tissue repair in organisms.
Cell Structure:
Cell Membrane: The outer boundary of the cell that separates the internal environment from the
external environment. It is a selectively permeable barrier that separates the cell from its
environment and regulates the movement of materials in and out of the cell. It consists of
phospholipid bilayer (Figure 1), which is a double layer of phospholipid molecules, with
hydrophobic tails facing inward and hydrophilic heads facing outward. Membrane proteins are
embedded within the phospholipid bilayer, while peripheral membrane proteins are loosely
associated with the membrane surface.

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Figure 1: Phospholipid bilayer
The Nucleus: It is the control center of the cell and contains the cell's genetic material (DNA) in
the form of chromosomes. It has a nuclear envelope which surrounds the nucleus with a double
membrane. It has a nuclear pore that allow for the exchange of materials between the nucleus and
cytoplasm. DNA is organized into chromosomes, which consist of DNA wrapped around histone
proteins.
The Endomembrane System: The endomembrane system is a network of organelles (including
the endoplasmic reticulum, Golgi apparatus, and lysosomes) that work together to modify, package,
and transport proteins and lipids.
Endoplasmic reticulum: The ER is a network of interconnected membranes that can be rough
(with ribosomes attached) or smooth (without ribosomes) and is involved in protein synthesis and
lipid metabolism.
Golgi apparatus: The Golgi modifies, sorts, and packages proteins and lipids for transport to their
final destination.
Lysosomes: lysosomes are membrane-bound organelles that contain digestive enzymes and are
involved in the breakdown of macromolecules. Figure 2, illustrates the arrangement and structure
of the organelles in an animal cell.

Figure 2: The Cell Structure

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BCH2301: GENERAL BIOCHEMISTR (2 UNITS)
LECTURE NOTES ON CELL DISRUPTION TECHNIQUES

Cell disruption techniques are used to break open cells and release their contents, such as proteins, nucleic
acids, organelles, or other cellular components. This process is crucial for various applications in biological
and biochemical research, including protein purification, DNA extraction, enzyme assays, and studying
cellular pathways. Here are some commonly used cell disruption techniques:

1. Mechanical Disruption:
o Homogenization: Cells are mechanically disrupted by forcing them through a small orifice
or by using a mechanical rotor-stator system. This technique can be performed using a
blender, high-pressure homogenizer, or bead mill.
o Sonication: Ultrasonic waves are applied to disrupt cells, Figure 3. The high-frequency
vibrations cause the formation and collapse of small bubbles, generating intense shear
forces that break the cell membranes.
o It is suitable for a wide range of cell types, including bacteria, yeast, and mammalian cells,
and is relatively quick and easy to perform. However, it can also cause shear stress and
generate heat, which may damage or denature sensitive biomolecules.
2. Chemical Disruption:
o Detergents: Non-ionic or ionic detergents can disrupt cell membranes by solubilizing lipids
and disrupting lipid bilayers.
o Enzymes: Enzymes like lysozyme or cellulase can degrade the cell wall, enabling the
release of cellular contents.
o Organic Solvents: Organic solvents like methanol or chloroform can disrupt cell
membranes by dissolving lipids and disrupting their structure.
o This method is suitable for extracting membrane-bound proteins or lipids but can also
damage or denature some biomolecules.
3. Physical Disruption:
o Freeze-Thaw Cycles: Repeated freezing and thawing of cells cause the formation of ice
crystals, which disrupt the cell structure.
o High-Pressure Homogenization: Cells are disrupted by subjecting them to high-pressure
conditions, leading to the disruption of cell membranes.

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 o This method is suitable for breaking open cells that are difficult to disrupt using other
techniques, such as yeast or plant cells, but may require multiple cycles of freezing and
thawing.
4. Electrical Disruption:
o Electroporation: High-voltage electrical pulses are applied to cells, creating temporary
pores in the cell membranes and allowing the release of cellular contents. his technique,
known as electroporation, is particularly useful for introducing DNA or other molecules
into cells, but may damage some biomolecules.
5. Grinding and Abrasion:
o Mortar and Pestle: Cells are ground with a pestle in a mortar, breaking open the cell walls.
o Ball Mills: Cells are disrupted by high-energy ball milling, where grinding balls collide
with the cell sample.
6. Enzymatic Disruption:
o Specific enzymes, such as lysozyme, can break down bacterial cell walls, or proteases can
digest the extracellular matrix. This technique is particularly useful for disrupting specific
types of cells or tissues but may require longer incubation times.

It's important to note that the choice of cell disruption technique depends on the type of cells being
disrupted, the cellular components of interest, and the downstream applications. Some methods are more
suitable for specific types of cells or yield higher purity of target components.

Figure 3: Mechanical disruption Ultrasonicator Figure 4: Physical disruption (Freeze-thaw)

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BCH2301: GENERAL BIOCHEMISTRY (2 UNITS)
LECTURE NOTES ON CENTRIFUGATION

Centrifugation is a technique used to separate particles from a liquid medium based on their
sedimentation rate. It is widely used in biological research, as well as in industrial applications
such as food processing and wastewater treatment. Separating of a sample is based on their density
and size. There are two main types of centrifugation: differential centrifugation and density
gradient centrifugation.

Differential centrifugation involves multiple rounds of centrifugation at increasing speeds to

separate particles based on their size, density and sedimentation rate. This method is useful for
separating organelles and subcellular components from one another. The principle behind
differential centrifugation is to subject a cell or tissue homogenate to a series of centrifugation
steps at progressively higher speeds or gravitational forces. As the sample is centrifuged, the
components sediment based on their size and density, leading to the formation of distinct layers or
pellets (Figure 5). By adjusting the centrifugation conditions, different cellular components can
be separated and collected at different stages.

Figure 5: Differential centrifugation

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Density gradient centrifugation involves layering a sample onto a density gradient, such as sucrose
or cesium chloride, and centrifuging it until the particles reach their equilibrium density within the
gradient. This method is useful for separating particles with similar size but different densities,
such as viruses and subcellular particles. The principle behind density gradient centrifugation is
the formation of a density gradient, typically in a tube or a centrifuge rotor. The gradient is
established by layering solutions of different densities, often sucrose or cesium chloride, on top of
each other (Figure 6). When the sample containing the particles of interest is centrifuged, the
particles will migrate through the gradient until they reach a region where the density of the
gradient matches their own density.

Figure 6: Density gradient centrifugation

During centrifugation, the tube or rotor is spun at high speeds, generating centrifugal forces that
cause sedimentation of the particles. Heavier particles or molecules will sediment faster and settle
at the bottom of the tube, while lighter ones will remain higher up in the gradient. This differential
sedimentation enables the separation of particles based on their density. Once centrifugation is
complete, the tube is carefully fractionated into multiple fractions, with each fraction containing
particles that have settled at a particular position along the density gradient. The fractions can then
be analyzed individually or subjected to further purification steps.

Density gradient centrifugation has several applications. It can be used to separate and purify
subcellular components such as organelles, proteins, or nucleic acids. It is also utilized for the
isolation and characterization of viruses, lipoproteins, and other biological particles. Furthermore,
density gradient centrifugation can help fractionate and purify complex mixtures, separating
different components based on their densities.

Centrifugation can be performed using a variety of centrifuges, including benchtop centrifuges and
ultracentrifuges. Ultracentrifuges are capable of generating very high centrifugal forces and are

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used for the separation of subcellular components. In addition to separating particles,
centrifugation can also be used for the concentration of particles in a sample. This is achieved by
centrifuging the sample at a high enough speed to cause the particles to pellet at the bottom of the
tube, and then removing the supernatant. Centrifugation is a powerful technique for separating and
concentrating particles in a sample, and is widely used in both research and industrial settings.

Centrifugation has various applications including

o Protein purification and analysis.

o DNA and RNA isolation.
o Subcellular fractionation.
o Blood cell separation.
o Isolation of plasma and serum.
o Viral load determination.
o Biopharmaceutical production.
o Separation and purification of chemicals.
o Wastewater treatment

Reading Assignment: Read and discuss in group about other types of centrifugation.

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BCH2301: GENERAL BIOCHEMISTRY (2 UNITS)
LECTURE NOTES ON pH AND BUFFERS
pH: Definition:
• pH is a measure of the acidity or alkalinity (basicity) of a solution.
• It quantifies the concentration of hydrogen ions (H+) in a solution.
pH Scale:

• The pH scale ranges from 0 to 14.

• A pH of 7 is considered neutral, indicating equal concentrations of H+ and OH-
ions.
• pH values below 7 indicate acidity, with lower values indicating higher acidity.
• pH values above 7 indicate alkalinity (basicity), with higher values indicating
higher alkalinity.

Calculation of pH:

o pH is determined by taking the negative logarithm (base 10) of the concentration

of H+ ions in a solution.
o The pH equation is: pH = -log[H+], where [H+] represents the concentration of H+
ions.

pH and H+ Concentration:

o As the pH decreases by one unit, the H+ concentration increases by a factor of 10.

o For example, a solution with a pH of 4 has 10 times higher H+ concentration than
a solution with a pH of 5.

Acidic and Basic Solutions:

o Acidic solutions have pH values below 7, indicating a higher concentration of H+

ions.
o Basic (alkaline) solutions have pH values above 7, indicating a lower concentration
of H+ ions and a higher concentration of OH- ions.

Buffers:

Definition:

A buffer is a solution that resists changes in pH when small amounts of acid or base are added.
Buffers help maintain the stability of pH in biological systems. Buffers typically contain a weak
acid and its corresponding conjugate base, or a weak base and its corresponding conjugate acid.
When an acid is added to a buffer solution, the weak acid in the buffer reacts with the added acid
to form its conjugate base, thereby minimizing the increase in H+ ions and maintaining the pH of
the solution. Similarly, when a base is added to a buffer solution, the weak base in the buffer reacts
with the added base to form its conjugate acid, thereby minimizing the decrease in H+ ions and
maintaining the pH of the solution.

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Types of Buffers:

There are two main types of buffers: acidic buffers and basic buffers. Let's explore each type and
provide examples of each:

1. Acidic buffers: Acidic buffers consist of a weak acid and its conjugate base. When a small
amount of acid is added to an acidic buffer, the added acid is neutralized by the conjugate
base, preventing a significant change in pH. Here are a few examples of acidic buffers:

a. Acetic acid (CH3COOH) and sodium acetate (CH3COONa): This is a commonly used
acidic buffer, often referred to as an acetate buffer. It can maintain a pH around 4.75.

b. Citric acid (C6H8O7) and sodium citrate (C3H5Na3O7): Citrate buffer is another example
of an acidic buffer. It is used in various biochemical and molecular biology applications.

c. Carbonic acid (H2CO3) and sodium bicarbonate (NaHCO3): This buffer system helps
regulate the pH in the blood and maintain the carbon dioxide-bicarbonate equilibrium. This
buffer system is important in regulating the pH of blood, maintaining it around 7.4.

2. Basic buffers: Basic buffers consist of a weak base and its conjugate acid. When a small
amount of base is added to a basic buffer, the added base is neutralized by the conjugate
acid, preventing a significant change in pH. Here are a few examples of basic buffers:

a. Ammonia (NH3) and ammonium chloride (NH4Cl): This buffer system is commonly
used in laboratories and can maintain a pH around 9.25.

• b. Boric acid (H3BO3) and sodium borate (Na2B4O7): Borate buffer is a basic buffer system
used in various biological and biochemical applications, and has a pH range of around 9.2-
10.

c. Diethylamine (C4H11N) and acetic acid (CH3COOH): This buffer system is often used
in analytical chemistry and can maintain a pH around 8.5.

Remember that the pH range over which a buffer effectively resists changes depends on
the specific components and their concentrations in the solution.

IMPORTANTS OF PH and BUFFERS

pH measurement and the use of buffers are essential in various fields and applications due to
the following reasons:

1. Biological Systems: pH regulation is critical for the proper functioning of biological

systems. Many biological processes, such as enzyme activity, cellular signaling, and
protein structure, are highly dependent on maintaining specific pH levels. Buffer solutions
help maintain the desired pH range, ensuring optimal conditions for biochemical reactions
and cellular functions.

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 2. Chemical Reactions: The pH of a solution can significantly influence chemical reactions.
Certain reactions require specific pH conditions for optimal reaction rates and yields. By
accurately measuring pH and using appropriate buffers, chemists can control reaction
conditions and improve the efficiency and selectivity of chemical processes.
3. Industrial Processes: In various industries, such as pharmaceuticals, food and beverage,
agriculture, and water treatment, pH control is crucial. pH measurements help ensure
product quality, control microbial growth, and maintain optimal conditions for
manufacturing processes. Buffers are often employed to stabilize pH and prevent drastic
pH changes that could negatively impact product quality or process efficiency.
4. Environmental Monitoring: pH is an essential parameter in environmental monitoring and
assessment. pH affects the solubility and bioavailability of chemicals, the growth of aquatic
organisms, and the stability of ecosystems. pH measurements in natural waters, soils, and
wastewaters provide valuable information for assessing environmental health, pollution
levels, and ecological impacts.
5. Laboratory Research: pH measurement and buffer solutions are fundamental in various
scientific research fields. Whether in biology, chemistry, biochemistry, or material science,
pH control and accurate pH measurements are essential for conducting experiments,
optimizing reaction conditions, and interpreting results.

Properties and Applications of Buffers:

1. Buffers are crucial in maintaining the pH stability of biological fluids, such as blood and
intracellular fluids, to ensure proper functioning of enzymes and cellular processes.
2. They are widely used in laboratory settings, such as in biochemical and molecular biology
experiments, to maintain a specific pH for optimal reaction conditions.
3. Buffers are also employed in industrial processes, including fermentation, food production,
and pharmaceutical manufacturing, to control pH and prevent undesirable effects on
chemical reactions and product stability.
4. Buffers are crucial in maintaining the pH of biological fluids, such as blood, which needs
to remain within a narrow pH range for proper physiological function.
5. Enzymes, which are essential for biological reactions, often have optimal pH ranges.
Buffers help maintain these pH conditions for enzyme activity.
6. Cells and organisms have buffering systems to regulate the pH of their internal
environment, protecting against drastic changes in acidity or alkalinity.

The Henderson-Hasselbalch

The Henderson-Hasselbalch equation is a mathematical expression that relates the pH of

a buffer solution to the pKa of the weak acid and the ratio of the concentrations of the weak
acid and its conjugate base. In 1908, Lawrence Joseph Henderson derived this equation. Later,
this equation became famous as the Henderson-Hasselbalch equation. It shows how two factors
affect the pH of the buffer solution. The first factor is the pKa of the acid and base used, while the
second factor is the ratio of the concentration of salt and acid or a base.

The equation is:

pH = pKa + log ([A-]/[HA]),

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 where [A-] is the concentration of the conjugate base and [HA] is the concentration of the
weak acid.

Importance of the Henderson-Hasselbalch Equation

1. The Henderson-Hasselbalch equation provides insights into the behavior and effectiveness
of buffers.
2. The Henderson-Hasselbalch equation is a mathematical relationship that relates the pH of
a buffer solution to the pKa of the weak acid and the ratio of the concentrations of the weak
acid and its conjugate base.

Here is a derivation of the Henderson-Hasselbalch equation:

Let's consider a weak acid, HA, that dissociates in water to form its conjugate base, A-,
and hydrogen ions, H+:

HA ⇌ H+ + A- ----------------------------Equation 1

The dissociation constant of the weak acid, Ka, is defined as the ratio of the concentrations
of the products (H+ and A-) to the concentration of the weak acid (HA):
Ka = [H+][ A-] / [HA]---------------------------Equation 2
Multiply both side by [HA]

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

[HA]
Rearranging:
Ka [HA]= [H+][A-]
Divide both side by [A-]as to make [H+][ the subject formaular
Ka [HA] = [H+][A-]
-
[A ] [A-]
Rearranging:
Ka [HA] = [H+]
[A-]
Or
[H+] = Ka [HA] / [A-]
Taking the negative logarithm of both sides, we get:
-log [H+] = -log Ka - Log [HA]
[A-]

Since – log [H+] = pH

– log Ka= pKa
Therefore: pH = pKa - Log [HA]
[A-]
Where, [A–] = concentration of salt and [HA] = concentration of the weak acid
Further, on reversing the numerator and denominator the sign of the log changes and the above equation
becomes; pH = pKa + log [A-] / [HA]

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Calculations

Example 1. Let's consider an example of a buffer solution containing 0.1 M acetic acid
(CH3COOH) and 0.1 M sodium acetate (CH3COONa). The pKa of acetic acid is 4.76.

The Henderson-Hasselbalch equation can be used to determine the pH of the buffer solution:
pH = 4.76 + log([CH3COO-] / [CH3COOH])
pH = 4.76 + log(0.1 / 0.1)
pH = 4.76 + log(1)
pH = 4.76
Therefore, the pH of the buffer solution is 4.76.

Example 2. Let's consider an example of adding 0.01 M hydrochloric acid (HCl) to the buffer
solution we used in the previous example.

The Henderson-Hasselbalch equation can be used to predict the new pH of the buffer solution:
pH = pKa + log([A-] / [HA])
pH = 4.76 + log(0.1 / (0.1 + 0.01))
pH = 4.76 + log(0.909)
pH = 4.76 + (-0.04)
pH = 4.72
Therefore, the addition of 0.01 M hydrochloric acid to the buffer solution will cause the pH to
decrease from 4.76 to 4.72.

Example 3: Suppose you have a buffer composed of 0.05 M ammonia (NH3) and 0.1 M
ammonium chloride (NH4Cl). Calculate the pH of the buffer solution. Given: The pKa value for
ammonia is 9.25.

Solution: Using the Henderson-Hasselbalch equation: pH = pKa + log([A-]/[HA])

[A-] = 0.1 M (concentration of ammonium chloride)

[HA] = 0.05 M (concentration of ammonia)
pKa = 9.25

pH = pKa + log([A-]/[HA]
= -9.25 + log 0.1/0.05
= 9.25 + log 2
= 9.25 +0.301
= 9.551
These examples demonstrate how to solve buffer calculations using the Henderson-Hasselbalch
equation. Remember to use the correct values for pKa, concentrations, and pay attention to the
units of measurement in your specific problem.

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Example 4: Calculate the pH of a buffer solution containing 0.1 M acetic acid (CH3COOH) and
0.2 M sodium acetate (CH3COONa). The pKa of acetic acid is 4.76.

First, we need to determine the ratio of [A-] to [HA]: [A-]/[HA] = 0.2 M / 0.1 M = 2
Using the Henderson-Hasselbalch equation: pH = pKa + log([A-]/[HA])
pH = 4.76 + log(2)
pH ≈ 4.76 + 0.301
pH ≈ 5.06

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