Cebu Doctors’ University – College of Arts and Sciences

MC2 - Biochemistry

Lecture Notes on
PROPERTIES OF WATER
Prepared by: Joselito R. Tumulak Jr.
Biochemistry Professor

I. OVERVIEW
- Any study of the chemistry of life must include a study of water. Living organisms are mostly made up of water, which accounts for 60-
95% of living cells. In the human body, 55% of the water is intracellular fluids (within the cell). The other remaining 45% is divided
between:
 plasma (8%),
 interstitial and lymph (22%), and
 connective tissue, bone and cartilage (15%)
- Normal metabolic activity can only occur only when the cells are at least 65% H2O.

Function of Water as Solvent for Biochemical Reactions:

1. Water acts as transport medium across membranes, carrying substances in and out of cells.
2. Water helps maintain temperature of the body.
3. Water acts as solvent (carrying dissolved chemicals) in the digestive and waste excretion systems.
4. Nearly all biological molecules assume their shapes, and therefore their functions, in response to the physical and chemical
properties of water.

Water Balance in the Body

- Healthy humans experience intake and loss of water every day. A water balance must be maintained within the body. If the loss of
water in the body significantly exceeds the intake, the body experiences dehydration. If the loss of water in the body is significantly less
the intake, the body experiences edema (fluid retention in tissues).

II. PHYSICAL PROPERTIES OF WATER

- Water can be considered a hydride of oxygen due to its elemental composition H 2O. In comparison to other hydrides that are its
nearest neighbors in the periodic table, namely ammonia (NH3), hydrogen fluoride (HF), and hydrogen sulfide (H2S), water has
substantially higher:
 boiling point,
 melting point,
 heat of vaporization, and
 surface tension.
- Indeed all of these physical properties are anomalously high for a substance of this molecular weight that is neither metallic nor ionic.
These properties suggest that intermolecular forces of attraction between H2O molecules are high.
- Furthermore, the maximum density of water is found in the in the liquid state, not solid, unlike most matter. This is the reason why
solid water (ice) floats on top of liquid water.
- These eccentric properties of water are really fascinating, but should have an explanation. And the explanation lies in its unrivaled ability
to form hydrogen bonds.

Polarity of Water
- The 2 hydrogens of water are covalently linked to the oxygen atom giving a non-linear arrangement. This structure of water is known
as bent structure (See Figure below). In the O–H bonds of H2O, oxygen is more electronegative than hydrogen, meaning there is
a higher probability that the bonding electrons are closer to the oxygen. This gives rise to a partial negative charge in the oxygen atom
and partial positive charge in the hydrogen atom. The partial charges are usually depicted us δ+ and δ-, respectively.
- Bonds such as this are called polar bonds. Molecules with polar bonds are polar molecules, that is why water molecule is polar.
- In the case where the electronegativity difference is quite small, such as in the C–H bond in hydrocarbons, the sharing of electrons is
very nearly equal, and the bond is essentially nonpolar.

The Ability of Water Molecule to Form Hydrogen Bonds

- One of the important consequences of the polarity of water molecule is that water molecules attract one another through the formation
of hydrogen bonds.
- Hydrogen bond is a non-covalent interaction. It is a special case of a dipole-dipole interaction formed between a hydrogen donor and
a hydrogen acceptor. A hydrogen donor is a hydrogen atom covalently bonded to an electronegative atom, specifically fluorine (F),
oxygen (O), or nitrogen (N). A hydrogen acceptor is a lone pair of electrons on any of those mentioned electronegative atoms (F, O,
and N).
- Water can then be both hydrogen donors and hydrogen acceptors (See figure below). The H2O molecule has 2 hydrogen atoms
bonded to an electronegative oxygen atom which can potentially be hydrogen donors. The oxygen atom of H2O, on the other hand,
has 2 lone pairs of electrons which can potentially be hydrogen acceptors. In total, a water molecule has the potential to form 4
hydrogen bonds (2 donors and 2 acceptors).

Hydrogen acceptor Hydrogen donors

Hydrogen acceptor

- It must also be pointed out that hydrogen bonding in water is cooperative. That is, a hydrogen bonded water molecule serving as an
acceptor is a better hydrogen donor that an unbonded molecule. A hydrogen bonded water molecule serving as a donor is also a better
hydrogen acceptor. Thus, participation in hydrogen binding by H2O molecule is a phenomenon of mutual reinforcement.

Consequence of Hydrogen Bonding in Water

- This ability of water for hydrogen bonding is the source of the strong intermolecular attractions that endow this substance with its
anomalously high boiling point, melting point, heat of vaporization, and surface tension.
- Hydrogen bonding also enables water to dissolve many organic biomolecules that contain functional groups that can participate in
hydrogen bonding.
- Example:
1. The oxygen atoms of aldehydes, ketones, and amides provides lone pairs of electrons that can serve as hydrogen acceptor.
2. Alcohols, carboxylic acids, and amines can serve both as hydrogen acceptors and donors for formation of hydrogen bonds.

Solvent Properties of Water

1. Ionic and polar substances dissolve in water.
- Substances, such as ionic substances and polar substances, that can be dissolved in water, are known as hydrophilic.
- Why does ionic substances dissolve in water?
 Due to the polarity of water molecule, they can align themselves around ionic substances so that the partially negative
oxygen atoms of the water molecules are oriented towards the cations (positively charged ions) of the ionic substances
and the partially positive hydrogen atoms are oriented towards the anions (negatively charged ions). This intermolecular
interaction is known as ion-dipole interaction.
 As an example, consider what happens when a crystal of sodium chloride (NaCl) dissolves in water. The polar water
molecules are attracted to the charged ions in the crystal. The attraction results in sodium and chloride ion on the
surface of the crystal dissociating from another and the crystal begins to dissolve. Because there are many polar water
molecules surrounding each dissolved sodium and chloride ion, the interactions between the opposite electric charges
of theses ions become much weaker that the they in an intact crystal. As a result, more and more ions are dissociated
from the crystal.
 - Why does polar substances dissolve in water?
 Polar substances can also be hydrated by water in the same manner as ionic substances. The interaction between
water and another polar molecule is known as dipole-dipole interaction. Some polar substances may also participate
in hydrogen bonding, which enhances solubility.
 However, they do not dissociate into ions, rather the solution formed contains intact molecules disperse in water.
As an example, intact polar methanol molecules are dispersed in water when dissolved.

 An increase in number of polar groups in an organic molecule increases its solubility in water.

2. Non-polar substances do not dissolve in water.

- Non-polar substances are known as hydrophobic because of their inability to be dissolved in water.
- Why do non-polar substances and water mixed together separate into layers?
 Non-polar substances are incapable of forming dipole-dipole interaction or hydrogen bond with water, but can interact
with each other through hydrophobic interaction. When non-polar substances are mixed with water are mixed, water
molecules tend to interact with other water molecules rather than with non-polar molecules. Consequently, water
molecules exclude non-polar substances forcing them to associate with each other. This is known as hydrophobic
effect.
 The hydrophobic effect is critical for folding of proteins and the self-assembly of biological membranes.

3. Amphipathic molecules form micelles and bilayers.

- There are molecules, such as fatty acids (right) and phospholipids (left), that are both hydrophilic and hydrophobic. They
usually have a non-polar hydrocarbon tail and an ionic or polar end. These substances are said to be amphipathic, and are
known as amphiphiles.

- When amphiphiles are dispersed in water, the hydrophilic head (polar)

tends to be hydrated, while the hydrophobic tail (non-polar) tends to be
excluded. This results to the formation of structurally ordered aggregates.
- An example of these aggregates are micelles, which are globules of up to
several thousand amphipathic substances arranged so that hydrophilic head
at the globule surface can interact with the aqueous environment, while the
non-polar tails associate with one another in the center of the structure
minimizing contact with water.
- Alternatively, amphipathic molecules may arrange themselves to form
bilayer, which are sheets in which the polar groups face the aqueous phase.
- In both micelles and bilayers, the aggregate is stabilized by hydrophobic effect.

III. CHEMICAL PROPERTIES OF WATER

- Water is not just a passive component of the cell or its extracellular environment. By virtue of its physical properties, water defines the
solubility of other substances. Similarly, water’s chemical properties determine the behavior of other molecules in solution.

Auto-ionization of Water
- Water shows a small but finite tendency to ionize. This tendency is manifested by pure water’s ability to conduct electricity, a property
that clearly establishes the presence of charged species (ions). This happens because the larger, highly electronegative oxygen atoms
strips the electrons from one of its hydrogen atoms, leaving the hydrogen ion to dissociate. This is referred to as the auto-ionization
of water, and is expressed as:
H2O ↔ H+ + OH-
- Two ions are thus formed: (1) hydrogen ion (H+), also referred to as proton; and (2) hydroxide ion (OH-).
- In reality, there is no such thing as a free proton (H+) in solution. Rather, the H+ associate immediately with another water molecule to
form the hydronium ion, H3O+:
H+ + H2O ↔ H3O+
- For simplicity, however, we often represent these ions by just H+.

Ionization Constant of Water (Kw)

- Notice that the chemical equation for the auto-ionization of water uses a double-sided arrow. This means this reaction is reversible
and in equilibrium. For reactions in equilibrium, we can often describe it by an equilibrium expression in which the concentration of
the reactant is in the denominator and the concentration of the product is in the numerator.
 For example:
[𝐶]
If the reaction is A + B ↔ C, the its equilibrium expression is: 𝐾 = [𝐴][𝐵] , where K is known as the equilibrium
constant. Quantities inside square brackets in the equilibrium expression symbolize molar concentration (mol/L or M) of the
indicated substance.
- In the case of the auto-ionization of water (H2O ↔ H+ + OH-), we can express it as:
[𝐻+ ][𝑂𝐻− ]
𝐾𝑤 =
[𝐻2 𝑂]
- The Kw is known as the ionization constant of water. The molar concentration of undissociated H2O in 1 L water remains appreciably
constant and larger than that of H+ and OH- species during ionization, thus its concentration can be neglected transforming the equation
above into:
𝐾𝑤 = [𝐻+ ][𝑂𝐻− ]
- Experimentally, the concentrations of H and OH in pure water are equal to 1 × 10-7 M. Thus,
+ -

𝐾𝑤 = [𝐻+ ][𝑂𝐻− ] = (1 × 10−7 𝑀)(1 × 10−7 𝑀)

= 1 × 10−14 𝑀2
- This Kw equation of water has the virtue of revealing the reciprocal relationship between H+ and OH- concentrations in aqueous
solutions. That is when hydrogen ion concentration ([H+]) is greater than 1 × 10−7 𝑀, then [OH-] must correspondingly be less and
vice versa.
- Solutions with [H+] equal to 𝟏 × 𝟏𝟎−𝟕 𝑴 are said to be neutral, those with [H+] > 𝟏 × 𝟏𝟎−𝟕 𝑴 are said to be acidic, and those
with [H+] < 𝟏 × 𝟏𝟎−𝟕 𝑴 are said to be basic.

The pH Scale
- The values of hydrogen ion concentration ([H+]) for most solutions that we will encounter in biochemistry are conveniently small and
thus difficult to compare. A more practical quantity, which was devised in 1909 by Danish biochemist Søren Sørensen, can be used.
This is known as pH (power of hydrogen) which is defined as the negative logarithm of the hydrogen ion concentration or:
𝑝𝐻 = − log[𝐻 + ]
- This equation tells us that since neutral solution, like pure water, has a [H+] of 1 × 10−7 𝑀, then its pH is 7. It follows that solution
with pH below 7 are acidic and solution with pH above 7 are basic.
- Sample Calculations:

1. What is the pH of the solution comprising 1.3 × 10-4 M? Is it acidic or basic?

- Solution:
o pH = -log [H+] = -log[1.3 × 10-4] = 3.9
o The pH of the solution is below 7; therefore it is acidic.

2. What is the pH of the solution comprising [OH-] = 1.9 × 10-5 M? Is it acidic or basic?
- Solution:
o We can input the given value directly to the pH equation, as it is [OH-] not [H+]. However, remember that [OH-] and [H+]
in aqueous solutions have reciprocal relationship, i.e. the equation for ionization constant of water, K w. We can use this
to find [H+], that is:
Kw = 1 × 10−14 𝑀2 = [OH-][H+]
By manipulation and substituting the value of [OH-]:
1 × 10−14 1 × 10−14
[𝐻 + ] = = = 5.26 × 10−6
[𝑂𝐻 − ] 1.9 × 10−9
o Now, that we have the value of [H+], we can now substitute it to the pH equation:
pH = -log [5.26 × 10−6 ] =5.3
o The pH of the solution is below 7; therefore, it is acidic.

Acids and Bases

- We defined thus far what it meant to be acidic solution or basic solution based on hydrogen ion concentration ([H+]) and pH values.
Biological molecules, such as proteins and nucleic acids, have numerous functional groups that acts as acids or bases – for example,
carboxylic acids and amines, respectively. How exactly are these functional groups considered to be acidic and basic?
- There are plenty of theories that can be used to describe the nature of acids and bases. For describing functional groups in organic
compounds and biomolecules, the most important is the one formulated by Johannes Bronsted and Thomas Lowry in 1923 known as
the Bronsted-Lowry definition. This states that an acid is a substance that can donate a proton (H+), and a base is a substance that
can accept a proton (H+). Under this definition, an acid base reaction can be written as:
HA (acid) + B (base) ↔ A- + BH+
- In the previous general reaction, an acid (HA) reacts with a base (B) to form the conjugate base of the acid (A-) and the conjugate
acid of the base (BH+). A conjugate base is the one that is left when a H+ leaves an acid, and conjugate acid is the one that is formed
when a base accepts a H+.
- Specific examples:
1. CH3COOH + H2O ↔ CH3COO- + H3O+
acetic acid water acetate hydronium ion
o Acetic acid donates H+ to water; thus, it’s an acid. Conversely, water accepts H+ and is therefore a base.
o Acetate is what is left when H+ leaves acetic acid; thus it is the conjugate base. On the other hand, the hydronium ion is
formed when water accepts H+ and is therefore the conjugate acid.

2. NH3 + H2O ↔ NH4+ + OH-

ammonia water ammonium ion hydroxide ion
o Ammonia accepts H+ from water; thus, it’s a base. Conversely, water donates the H+ and is therefore an acid.
o Ammonium is formed when ammonia accepts H+; thus it is the conjugate acid. On the other hand, the hydroxide is what is
left when H+ leaves water and is therefore the conjugate base.

-Notice how water can act as acid or base in the examples above. Substances, such as water, that can act as an acid in the presence
of a base or act as a base in the presence of an acid are referred to as amphoteric substances.

Strength of Acids
- Acid strength is determined by their tendencies to transfer a proton (H+) to water.
- Strong acids can rapidly transfer all their protons to water. For example,
HCl + H2O → H3O+ + Cl-
hydrochloric acid

- Notice how the chemical equation above no longer uses the double-sided arrow. This indicates that the dissociation of the acid is not
reversible and goes into completion. This is why strong acids can also be considered as strong electrolytes as they dissociate
completely in water.
- The acids hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HClO4), nitric acid (HNO3), and
sulfuric acid (H2SO4) are the only acids considered to be strong. Any other acids not in the list are considered weak.
- On the other hand, weak acids are partially dissociated in water, and thus some of undissociated acids remain in the solution. For
example:
CH3COOH + H2O ↔ CH3COO- + H3O+
acetic acid

- The double-sided arrow in the chemical equation indicates that the ionization of acetic acid does not proceed to completion, and
some undissociated acetic acid remains in the solution. This is why weak acids are also considered weak electrolytes.

Acid Ionization Constant (Ka) and pKa

- Because ionization of a weak acid is reversible, we could also express its equilibrium expression. For simplicity, we could remove
water from the chemical equation.
- For example, in ionization of acetic acid (CH3COOH + H2O ↔ CH3COO- + H3O+), we could write the equation as:
CH3COOH ↔ CH3COO- + H+
- Hence, the equilibrium expression can be written as:
[𝐻 + ][𝐶𝐻3 𝐶𝑂𝑂− ]
𝐾𝑎 =
[𝐶𝐻3 𝐶𝑂𝑂𝐻]
- The Ka is known as the acid ionization constant and it describes the extent to which acid forms ions in water. Thus, the Ka values can
be used to compare relative strength of weak acids. The larger the Ka value, the higher is the extent of ionization and is therefore
a much stronger acid.
- Each weak acid has a characteristic Ka values which were previously determined experimentally. Listed below are some of these Ka
values.

- Just like that of hydrogen ion concentrations [H+], the values of Ka are also inconveniently small and difficult to compare. So we could
also express it in terms of its negative logarithm known as pKa, that is:
pKa = -log (Ka)
- Because pKa is the negative logarithm of Ka, pKa has an inverse relationship with acid strength, i.e. the lower the pKa the stronger
the acid. This is the reverse of the relationship between Ka and acid strength. The table above also list the corresponding pKa values of
the common weak acids.

Buffers
- Adding a droplet (~0.01 mL) of hydrochloric acid to 1 L of pure water changes the pH of the water from 7 to 5, which represent a 100-
fold increase in hydrogen ion concentration ([H+]). Such a huge change in pH would be intolerable to most biological systems, since
even small changes in pH can dramatically affect the structures and functions of biological molecules. Maintaining a relatively
constant pH is therefore of paramount importance for living systems.
- In living systems and in the laboratories, buffers are used to resist pH changes. These are solutions that can maintain its pH even after
the addition of small amount of acid or base.
- Typically, a buffer system is composed of a mixture of a weak acid and its conjugate base.
- Example:
1. Mixture of acetic acid (CH3COOH) and acetate (CH3COO-),
2. Mixture of formic acid (CHOOH) and formate (CHOO-)
3. Mixture of carbonic acid (H2CO3) and bicarbonate (HCO3-)
4. Mixture of phosphoric acid (H3PO4) and dihydrogen phosphate (H2PO4-)
- How can buffer resist pH change?
 Buffers work based on the nature of weak acids and their conjugate bases that compose the buffer. If acid (H+) is added to a
buffer solution, it reacts with the conjugate base to form the weak acid.
H+ + A- (conjugate base of the buffer) → HA
 Similarly, if a base (OH ) is added to the buffer, it reacts with the weak acid to form water and the conjugate base.
-

OH- + HA (weak acid of the buffer) → A- + H2O

 In this way, either added acid or based is “used up” by adding it to a buffer. This keeps the pH much more stable than if the
same acid or base had been added to an unbuffered system.
- How to determine the pH of a buffer?
 The pH of the buffer is calculated by the Henderson-Hasselbalch equation:
[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒]
𝑝𝐻 = 𝑝𝐾𝑎 + log
[𝑤𝑒𝑎𝑘 𝑎𝑐𝑖𝑑]
where:
pKa = pKa value of the weak acid (refer to the table in the previous page)
[conjugate base] = concentration of conjugate base in the solution
[weak acid] = concentration of weak acid in the solution

- Sample Calculation:

1. What is the pH of the buffer comprising 0.025 M acetic acid and 0.005 M sodium acetate?
- Solution:
o Identify first if the solution is a buffer. In the example it comprises acetic acid, a weak acid, and sodium acetate, a
salt of its conjugate base. Thus, it is a buffer.
o The pKa of acetic acid is 4.76 based on the table on the previous page. Input this value to the Henderson-
Hasselbalch equation, together with the concentrations of the conjugate base and weak acid.
[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒]
𝑝𝐻 = 𝑝𝐾𝑎 + log
[𝑤𝑒𝑎𝑘 𝑎𝑐𝑖𝑑]
[0.005 𝑀]
= 4.76 + log
[0.025 𝑀]
= 4.76 + (−0.70)
= 4.06
o Thus, the pH of the buffer is 4.06. It will maintain that pH even after the addition of small amount of H + and OH-.

- How to select an appropriate buffer?

 Maximum buffering is seen when the buffer pH is approximately equal or near to the pKa of its weak acid. This is the case
for buffers comprising mixture containing almost equal concentrations of weak acid and conjugate base.
 We choose a buffer primarily by knowing the pH that we wish to maintain. For example, if we are performing an experiment and
we want the solution to stay at pH 7.5, we look for a buffer that has a pKa of 7.5 because buffers are most effective when the
pH is close to the buffer pKa.

Bicarbonate Buffer System of Blood Plasma: A Case of Physiological Buffers

- As said, maintaining a fairly constant pH is very important in living systems. There’s plenty of physiological buffers that can be used for
this purpose. In this portion, we will specifically discuss the most important type of buffer present in the plasma of the blood – the
Bicarbonate Buffer System.
- The normal pH range of the blood is between 7.35-7.45. Below this range means there’s abundant hydrogen ions (H+) in the blood
and is therefore acidic. Conversely, above this pH means there’s little H+ and is therefore basic. Conditions when the blood pH is low is
known as acidosis, while condition when pH is high is known as alkalosis.
- Acidosis and alkalosis are prevented from occurring through the Bicarbonate Buffer System which is represented by the equation:
CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3-
carbon dioxide carbonic acid bicarbonate
- Important Features of the Buffer System:
 There are 2 key organs involved in this specific buffer system – the lungs and the kidneys. The leftmost side of the equation
(interaction between CO2 and H2O) occurs in the lungs, while the rightmost side of the equation (interaction between H+ and
HCO3-) occurs in the kidneys.
 The buffer system responds to changes in the H+ concentration of the blood by changing the partial pressure of CO2.
o When the pH of blood falls due to a metabolic process that produces excess H+ (acidosis), the concentration of H2CO3
increases. But this is momentarily, as the reactions in the buffer shift to the left side. H2CO3 rapidly loses water (H2O)
to form CO2 which enters the lungs and is expired as by exhalation. An increase in the partial pressure of CO2 (pCO2)
in the air expired from the lungs thus compensates for the increased hydrogen ions.
o Conversely, when the pH of the blood rises (alkalosis), the concentration of HCO3- increases. But the pH is rapidly
restored as the reactions in the buffer shift to the right side. Breathing rate changes and the CO2 in the lungs is
converted to H2CO3 in the capillaries of the lungs. Increase in H2CO3 will then cause an increase in the H+ concentration.
Again, the equilibrium of the blood buffer system is rapidly restored by changing the partial pressure of CO 2 in the
lungs.
- Types of Acidosis and Alkalosis:
 Recall:
o Acidosis is when there is a decrease in blood pH (increase in H+). The compensation of the body is to expel more
CO2.
o Alkalosis is when there is an increase in blood pH (decreases in H+). The compensation of the body is to keep more
CO2 in the lungs.
 Acidosis and alkalosis can be classified as either respiratory or metabolic. To help us determine which is which, we consider
again the Bicarbonate Buffer System:
CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3-
carbon dioxide carbonic acid bicarbonate
 The bicarbonate buffer equation can be viewed as a see-saw, wherein increase in one area can cause a decrease on the other.
 Let’s consider the leftmost side of the buffer first (interaction between CO2 and H2O). Recall, that this occurs in the lungs.
Acidosis or alkalosis caused by disturbance of this side of buffer is considered respiratory.
o Respiratory acidosis
 Increase of CO2 resulting to a shift of the buffer reaction to the right which produces more H+ and lowers
blood pH.
o Respiratory alkalosis
 Decrease of CO2 resulting to a shift of the buffer reaction to the left which lowers H+ and increases blood
pH.
 Let’s consider the rightmost side of the buffer first (interaction between H+ and HCO3-). Recall, that this occurs in the kidneys.
Acidosis or alkalosis caused by disturbance of this side of buffer is considered metabolic.
o Metabolic acidosis
 Decrease of HCO3- resulting to a shift of the buffer reaction to the right which produces more H+ and lowers
blood pH.
o Respiratory alkalosis
 Increase of HCO3- resulting to a shift of the buffer reaction to the left which lowers H+ and increases blood
pH.