Water

Presented By:
Sapana Subedi
M. Sc. Clinical Biochemistry
1st year
Moderator : Basanta Gelal
18/09/2023
 Overview
 Introduction
 Different Properties of Water
 Ionization of Water
 Hydrogen bonding and its importance
 pH and pK
 pH of different body compartments
 Henderson and Hasselbalch equation
 Different Analytical Grades of Water
 Importance of water

 More than 70% earth surface covered with water

 Solvent and substrate for many cellular reaction
 Helps to maintain constant body temperature
 Cell components and molecules (protein, polysaccharides, nucleic acid,
membranes ) assume their shape in response to water
 Distribution of Water in Body ,weighing 70 kg
Compartment Body Weight(%) Volume(L)
Total 60 42
Intracellular fluid 40 28
Extracellular fluid 20 14
Interstitial fluid 15 10.5
Plasma 5 3.5
Water Turnover and Balance
 Water
 Water is a compound that consists of two
hydrogen atoms and one oxygen atom
attached together by two sigma bonds and
with two lone pairs of electrons around the
oxygen
 Attachment of Hydrogen to Oxygen is a
covalent chemical bond forming tetrahedral
arrangement
Dipole Molecule with
asymmetric electrical
charge due to 104.5°
bond angle (i.e. bent)
 Properties of Water
 Partial charge on oxygen and hydrogen
allow water to participate in hydrogen
bonding
 Universal Solvent
 Polar due to high electronegativity of
Oxygen
 Strong dipoles and high dielectric constant
enable water to dissolve large quantities of
charged compounds such as salts
Physical Properties of Water
 Colorless, Odorless and Tasteless
 Anomalous expansion (Highest density at 4°C)
 Melting point: 0°C
 Boiling point: 100°C
 Heat of Vaporization: 540 cal/g (Maximum cooling by evaporation to maintain body
temperature)
 Heat capacity: 1000 cal/g (helps organism to gain or loss heat quickly)
 Heat of fusion: 79.7 cal/g
 Surface tension: 72.8
 Dielectric Constant: 80
 Exist in all 3 states of matter : solid, liquid, and gas
Chemical Properties
• Amphoteric: act both as acid and base
• As base: H2O + HCl → H3O+ + Cl-
• As acid: H2O + NH3 → NH4+ + OH-
• Auto-ionization of Water: Water is very weak electrolyte which ionizes only
to a small extent
• Water as hydrolytic agent: Most of the compounds and molecules are
hydrolyzed by water
• Water is a universal solvent due to its polarity
• Hydrogen bonding
Hydrogen Bond
 Electrostatic attraction between oxygen
atom of one water molecule and
hydrogen atom of another called hydrogen
bond.
 Hydrogen bond is relatively weaker in
comparison with covalent bond.
 Bond dissociation energy about 23 KJ/mol
compared with 470 KJ/mol for covalent O-H
bond in water or 348 KJ/mol for covalent C-C
bond.
 Life span of H- bond is 1-20 ps.
 Each water can contributes 4 Hydrogen bonds.
 Hydrogen bonding is one of the strongest electrostatic
interactions
 H bond is 10% covalent (due to overlap of bonding
orbitals) and 90% electrostatic
 Hydrogen bonding is responsible for water’s unique
properties such as high MP, BP, heat of vaporization and
surface tension

Self-
association
of water
H-bonding
 H bonds are relatively weak and short-lived (1 – 20 picosecond)
 Sum of all H bonds confer internal cohesion in liquid water
 Bond dissociation energy of Liquid water 23 kJ/mol
 Bond dissociation energy of covalent O – H bond: 470 kJ/mol
 Bond dissociation energy of C – C bond: 348 kJ/mol
 Bond dissociation energy of Van der Waal’s force: 4 kJ/mol
 There is a continuous formation of another H- bond with the same partner or
new ones upon breakage in 0.1 ps: “flickering clusters”
 Tetrahedral structure about O allows each water molecule to form hydrogen bond with
as many as 4 neighboring water molecules
 But at RT water molecules are disorganized and in continuous motion
 On average, each molecule in liquid water associates through hydrogen
bonds with 3.5 other molecules
 Hydrogen atoms covalently bonded to carbon atoms do not participate in hydrogen
bonding because carbon is only slightly electronegative than hydrogen and C – H bond
is only weakly polar.
 Example:
Butanol has relatively high MP (117°C) than Butane (- 0.5 °C) conferred by –OH group
Structure of Ice
 Density of ice is less than water in Liquid
state.
 Ice has negative volume of melting.
 Ice form hexagonal lattice by forming
Hydrogen bonds.
 Bond length of H-bond in ice is 0.274 nm
whereas in liquid state H-bond length
0.177 nm.
High melting and boiling point
 Breaking sufficient proportion of hydrogen bonds to destabilize the crystal
lattice of ice require thermal energy: High melting point.

 When ice melts or water evaporates, heat is taken up by the system .

Dielectric constant
 Water is effective in screening the electrostatic
interaction between dissolved ions because it has
high dielectric constant

 Strength or force (F) of ionic interactions in a

solution depends on magnitude of charges
(Q),distance between charge groups (r), dielectric
constant (ε) of solvent where interaction occurs

 For water at 25⁰c, ε is 78.5

 Entropy Increases as Crystalline substance Dissolve

 Salt such as NaCl dissolves Na+ and cl- ions leaving the crystal lattice.
 Resulting increase in entropy of the system is largely responsible for ease of
dissolving salt such as Nacl in water.
Rationale for water soluble carrier proteins Hb and Mb to carry oxygen, Carbon
dioxide transported in dissolved form
 Water and Non-Polar molecules

 Non-polar solutes do not readily form H-bond

to water.
 As a result such compounds tend to be only
sparingly soluble in water.
 Non-polar solutes must occupy space when
added in water.
 Consequently H-bonded water network
rearranges towards formation of local cage like
structure (CLATHRATE) surrounding each
solute molecules.
 Hydrophobic effect
 Tendency of nonpolar compounds to self-
associate in an aqueous environment
which disrupts the energetically
favourable interactions between the
surrounding water molecules
 Water molecules “cages” around them
being more well ordered than in free
solution
 However, when two nonpolar molecules
come together, some molecules are
released allowing them to interact freely
in bulk water
 Outcome - Increased tendency of
nonpolar molecules to associate with on
another
 Water and Amphipathic

 Compound containing both strongly polar and strongly

non-polar groups .
 E.g. salts of fatty acid (Have long non-polar
hydrocarbon tail and strongly polar carboxyl head
group )
 Hydrocarbon tail (Non-polar) of the substances joined
together in hydrophobic interactions whereas Carboxyl
group (polar) of substances joined in hydrophilic
fashion.
 Such clusters of amphipathic molecules are termed as
micelles.
Water and Enzyme action

 Enzymes acts on aqueous environment

 Substrate and enzymes are surrounded by

ordered water shell.

 Binding of substrate to enzyme release some

ordered water.
 Nucleophilic nature of water
 Chemicals that are electron rich (nucleophiles) seek electron deficient
chemicals (electrophiles ).
 Nucleophiles are negatively charged or have unshared pair of
electrons – attack electrophiles during substitution or addition reactions.
 E.g. of nucleophiles : oxygen, nitrogen, Sulphur, carbon etc…
Bounded water in Protein
 Action of Detergent

https://connect.collins.co.uk/repo1/Content/Live/Infuze/COL/GCSE_Science_Separate_SB_OCR
_Gateway/content/Page172.html
 Pure water is slightly ionized
 Have slightly tendency to undergo reversible ionization to yield hydrogen ion
(proton) and hydroxide ion ,giving the equilibrium.

 Hydrogen ion formed in water hydrated to hydronium ions.

 Ionization can be measured by electrical conductivity.

 Movement of hydronium ion towards cathode and hydroxide towards anode is very
fast (proton Hopping ).
 Ionization of Water
 Though water is a bad conductor, purest water
ionizes to small extent
 H2O + H2O → H3O+ + OH−
 Water ionizes to hydrogen and hydroxyl ions
 But free protons (H+) do not exist in solution so
instead, hydrogen ions formed in water are
immediately hydrated to form hydronium ions
(H3O+)
 “Proton hopping”: a series of hydrogen-
bonded water molecule results in rapid net
movement of proton over a long distance
 Equilibrium Constant for Ionization of Water
 Since ionization of water is a reversible reaction, it follows LeChatelier’s principle

 Equilibrium constant for the reversible ionization of water is:

 Equilibrium Constant (Keq): The tendency of any conjugate acid (HA) to

lose proton and form its conjugate base (A-) is defined as equilibrium
constant
Molarity of pure water at 25°C
• MOLARITY = no. of moles of solute / Volume expressed in litres
• At 25°C, 1.0 litre = 1000 ml of water = 1000 gm
• Molecular weight of water = 18.02 Dalton
• No. of moles = wt in gram/ Molecular weight
• = 1000/18.02
• = 55.49
• Substituting in above equation, we get;
• MOLARITY = 55.49 / 1.0 l = 55.49 mol/L
 Equilibrium Constant for Ionization of Water
 In pure water at 25°C, the concentration of water is 55.5 M. On rearranging the
previous equation, we get;

 Kw is the ion product of water at 25°C.

 Keq determined by electrical conductivity measurement of pure water is 1.8X10-16
M at 25°C.

The product [H+][OH-] in aqueous solution at 25°C is always a constant.

 Equilibrium Constant for Ionization of Water
With exactly equal concentration of H+ and OH-, the solution is said to be at
neutral pH

From the ion product of water we can calculate [H+] if we know [OH-], and
vice versa.
 .
pH
 We use pH scale to express hydrogen ion concentration because it is
more practical to use pH scale. Expressing hydrogen ion concentration
in molarity takes a lot of space and is not easy to work

• The pH scale is a convenient way of designating the concentration of H+ and OH- in

the range between 1.0M H+ and 1.0M OH-

• pH is defined as negative logarithm of the concentration of H+

pH

@Pearson Education Inc.

pH Indicator (Acid-Base indicators)
 The pH of an aqueous solution can be approximately measured with various indicator
dyes, which undergoes color changes as a proton dissociates from the dye molecule

 Accurate measurements of pH in the chemical or clinical laboratory are made with a

glass electrode that is selectively sensitive to H+ concentration but insensitive to Na+,
K+ , and other cations

 A pH indicator is a piece of paper or plastic soaked in indicator and then dried

Some Important pH indicators
Indicator pH range Color change
Thymol Blue 1.2 – 2.8 Red to Yellow
Bromophenol Blue 2.8 – 4.6 Red to Yellow
Congo Red 3.0 – 5.0 Blue to Red
Bromocresol Green 3.8 – 5.4 Yellow to Blue
Litmus 5.0 – 8.0 Red to Blue
Bromothymol Blue 6.0 – 7.6 Yellow to Blue
Neutral red 6.8 – 8.0 Yellow to Red
Phenol Red 6.8 – 8.4 Yellow to Red
Cresol Red 7.2 – 8.8 Yellow to Red
Phenolphthalein 8.3 – 10.0 Colorless to purple-red
 Henderson- Hasselbalch Equation
• Describes the derivation of pH as a measure of acidity using pKa as the acid dissociation
constant
• Lawrence Joseph Henderson wrote an equation in 1908 describing the use of
carbonic acid as a buffer solution; he derived an equation to calculate Hydrogen ion
concentration of a buffer solution

• Soren Peter Lauritz Sorensen introduced the pH terminology which allowed Karl
Albert Hasselbalch to re-express that equation in logarithmic terms resulting in
Henderson-Hasselbalch equation
• Hasselbalch used the formula to study metabolic acidosis which results from the
carbonic acid in blood.
 Henderson-Hasselbalch Equation

• Shows quantitative relation between pH of a buffered solution and pKa of a

buffer (restates the expression for dissociation constant of an acid)
• A weak acid [HA] dissociates into hydrogen ion[H+] and an anion or conjugate
base [A-] as:
• [HA] [H+]+[A-]
• To convert into equation, we need equilibrium constant/dissociation constant
(Ka);
Henderson-Hasselbalch Equation
Solving for [H+], we get;

Taking logarithm on both sides;

 Henderson-Hasselbalch Equation
Multiplying through -1;

Inversion of last sign removes minus sign;

 Types of Water based on Purification Method
Distilled Water (distillation): vaporizing and condensing water to purify or
concentrate a substance or separate volatile substance from less volatile
 Types of Water based on Purification Method
Deionized Water (Ion exchange process): passing feed water through columns
containing insoluble resins that exchange H+ and OH- ions for impurities
 Types of Water based on Purification Method
RO Water (Reverse Osmosis): water is forced through a semi-permeable membrane
that acts as a molecular filter, removes 95 – 99% bacteria, particulate matter and 90 –
97% of ionized and dissolved materials but fewer gaseous materials
 Types of Water based on Purification Method
 Ultrafiltration
 Water passed through semipermeable membrane of pore size ≤0.2 mm

 0.1 µm – to achieve an improved bacteria free and pyogens free product

 Removes
 Particulate matter
 Emulsified solids
 Most bacteria and pyogens
How is Laboratory Water purity assessed and
defined?
 The Conductivity of water
 The Resistivity of water
 Organic compound levels in water
 Biological contamination of water
 The presence of colloids in water
Pure Water Types
Boards setting standards of water purity
Clinical and Laboratory Standards Institute(CLSI)- formly called as
NCCLS
 Classified into six categories:
- Clinical Laboratory Reagent Water (CLRW)
- Special Reagent Water (SRW)
- Instrument Feed Water
- Water Supplied by method manufacturer
- Autoclave and Wash Water
- Commercially Bottled purified Water
 References
 Lehninger Principles of Biochemistry, 6th edition

 Harper’s Illustrated Biochemistry, 31st edition

 Voet_-_Fundamentals_of_Biochemistry_Life
Hydrangeas produce blue flower in acidic soil and pink in basic soil.

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