University of Al Fashir

Faculty of Medical Laboratory Sciences

Department of Clinical Chemistry
Biochemistry (1) Lecture 3
Water, pH and Buffers
For MLS student 2021
Batches: 4
Modified by : Dr. Adam Osman Abaker
Uni. Al Fashir

01/09/2022 1
 Introduction: Water
 Water is the predominant chemical component of living
organisms.
 Chemically, water is a weak electrolyte.

 Hydrogen bonding profoundly influences the

physical properties of water and accounts for its
exceptionally high viscosity, surface tension, and
boiling point.
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 Introduction: Water cont.

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 Introduction: Water cont.
• Water has a slight but important tendency to dissociate
into hydroxide ions and protons:

• water can act both as an acid and as a base.

• Hydrogen and hydroxide ion continuously recombine
to form water molecules.
• At one instant it is an ion; an instant later it is part of a
water molecule.
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 Introduction: Water cont.
What is the probability that a hydrogen will be present
as an ion or as part of a water molecule?
•Since 1g of water contains 3.4 × 1022 molecules, the
ionization of water can be described statistically.
•For an Example:
•probability that a hydrogen exists as an ion is 0.01
means that it has 1 chance in 100 of being an ion and 99
chances out of 100 of being part of a water molecule.
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 Introduction: Water cont.
The actual probability of a
hydrogen atom in pure water
existing as a hydrogen ion is
approximately:
• 1.8 × 10-9
For water dissociation:
K is the dissociation constant
01/09/2022 6
 Introduction: Water cont.
• Since 1 mole (mol) of water weighs 18 g,
• 1 liter (L) (1000 g) of water contains:
• 1000 ÷ 18 = 55.56 mol.
• Pure water thus is 55.56 molar.
• In pure water the probability for hydrogen as an ion is:
• 1.8 × 10-9
01/09/2022 7
 Introduction: Water cont.
• The molar concentration of ion ( H+ ) in pure water is the
product of the probability.
• 1.8 × 10-9 times the molar concentration of water, 55.56 mol/L.
• The result is 1.0 × 10-7 mol/L.
• So we can calculate K for pure water:

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 Introduction: Water cont.
The relationship between Kw and K

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 Introduction: Water cont.
• Since:

–7 2
Kw = (10 )

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 pH
pH is the negative log of the hydrogen ion
concentration to the base 10.

For example, for pure water:

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 pH cont.

• This value is also known as the power (English),

puissant (French), hence the use of the term "p."
• Low pH values correspond to high concentrations of
H+ and high pH values correspond to low concentrations
of H+.
• The physiologic pH is 7.35 – 7.45
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 pH cont.

• Question:
• What is the pH of a solution whose
hydrogen ion concentration is:
4 x 10–4 mol/L?
• Is it an acidic or basic solution?
Where:
• Log 4 = (0.6).
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 pH cont.

The molar concentration of water, 55.56 mol/L, is too great to be

significantly affected by dissociation.
• K [H₂O] = [H⁺][OH⁻] = Kw (ionic product of water)
•Kw=10-7 × 10-7/1
•Kw = 10-14
• Any excess of [H⁺] is associated with decrease [OH⁻] and vise versa.
• Pure water that contains 10-7 H⁺ g/L is considered a neutral solution.

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 pH scale

01/09/2022 15
 pH indicator

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 Acids and Bases
General concepts about Acids and Bases
• Acids are proton donors and bases are proton
acceptors.
• Strong acids (eg, HCl, H2SO4) completely dissociate even in
strongly acidic solutions (low pH).
• Weak acids dissociate only partially in acidic solutions,
CH3-COOH (acetic acid) CH3-COO- + H+
• Similarly, strong bases (eg, KOH, NaOH) are completely
dissociated at high pH.
Alkalies are substances that give (OH-) in solution.
Note: all alkalies are bases (eg NaOH) but not all bases are alkalies.
01/09/2022 17
 Acids and Bases cont.
• Since weak electrolytes dissociate only slightly in
solution, we must use the dissociation constant (Ka).

• Many biochemical molecules possess functional

groups that are weak acids or bases.
• Carboxyl groups, amino groups, and phosphate esters.

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 Acids and Bases cont.
•The protonated species (eg, HA or R-
NH3+ ) the acid and the unprotonated
species (e.g. A– Or R— NH2) its
conjugate base (that part of acid which
remains after removal of proton).
• The relative strengths of weak acids
and bases is expressed in terms of
their dissociation constants (Ka)

01/09/2022 19
 Acids and Bases cont.
• Since the numeric values of Ka for weak acids are
negative exponential numbers, we express Ka as pKa.

pKa is related to Ka as pH is related to [H+].

The stronger the acid, the lower is its pKa value.
01/09/2022 20
 Acids and Bases cont.
• For any weak acid, its conjugate is a strong base.
Similarly the conjugate of a strong base is a weak
acid

The pKa is the pH at which the concentration of the acid

R—NH3+ equals that of the base R—NH2.
01/09/2022 21
 Acids and Bases cont.

• When the concentrations of un-dissociated acid

equals the concentrations of its conjugate base:

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 Acids and Bases cont.

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 Acids and Bases cont.

+
Since –log Ka is defined as pKa, and –log [H ]

defines pH, the equation may be rewritten as

01/09/2022 24
 Acids and Bases cont.

Thus:

• the pKa of an acid group is the pH at which the

protonated and unprotonated species are present
at equal concentrations.

01/09/2022 25
 The Henderson–Hasselbalch Equation Describes the
Behavior of Weak Acids & Buffers.
• A weak acid, HA, ionizes as follows:

• The equilibrium constant for this dissociation is:

• Cross-multiplication gives

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 Henderson–Hasselbalch Equation cont.
• Divide both sides by [A–]:

• Take the log of both sides:

• Multiply through by –1:

• +
Substitute pH and pKa for –log [H ] and –log Ka

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 Henderson–Hasselbalch Equation cont.

• Inversion of the last term removes the minus sign and

gives the Henderson–Hasselbalch equation:

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 Buffer Solutions
• Solutions of Weak Acids & Their Salts Buffer
Changes in pH
• Buffering, is the ability to resist a change in pH
following addition of strong acid or base.
• Maintenance of a constant pH involves buffering
by phosphate, bicarbonate, and proteins, which
accept or release protons to resist a change in pH.

01/09/2022 29
 Buffer Solutions cont.

01/09/2022 30
 Buffer Solutions cont.

• The value of pKa relative to the desired pH is the major

determinant of which buffer is selected.
• Buffering can be observed by using a pH meter.

• A solution of a weak acid and its conjugate base

buffers most effectively in the pH range pKa ± 1.0 pH
unit.
01/09/2022 31
pH meter
 This lecture by:
Muna Ahmed Eltayeb
MSc. Bio, U of K – MBBS U of G
Modified by Adam Osman Abaker
Ph. D. Biohem. Ain Shams University Egypt
Assistant Prof. Biological Sciences Dept. U. Al Fashir
 Thanks
01/09/2022 34