PH and Buffer CK 2022 PDF

Prof.Dr.
Cengiz KARAKAYA
Department of Medical Biochemistry-2022
The covalent bonds within the water molecule, like the
hydrogen bonds between the molecules, can also break
spontaneously. This is known as water dissociation.
H2O → H+ + OH-
Water Hydrogen Ion Hydroxide Ion
This doesn’t happen often, but enough. If you have a

beaker of pure water, 1/10,000,000 molecules are
hydrogen ions.
▪ Acid, Base, Neutral Range
▪ Acids are molecules that donate hydrogen ions to a
solution.
▪ In a chemical formula, acids start with “H”. Ex. HCl, HF
(hydrochloric acid, hydrofluoric acid)
▪ Bases are molecules that combine with hydrogen ions to
remove them from a solution
▪ In a chemical formula, end with “OH”. Ex. NaOH, KOH
(sodium hydroxide, potassium hydroxide)
▪ Scale is Logarithmic
▪ Key molecules, known as enzymes, function at set
pH values.
▪ All our cells function within a certain pH range.
▪ If the fluids bathing those cells is “off” these cells
won’t function at max capacity.
▪ Other than just cells, our blood needs to be at a
certain pH.
▪ The chemical reactions of life constantly
produce acids and bases within cells.
These have a tendency to throw off the
pH values. We need some sort of
mechanism to minimize how much the
pH is altered.
Buffers: substance that can take up or
release hydrogen ions into a solution so that
the pH can maintain equilibrium.
So...if pH is low, what about the [H+]?
It is HIGH
 ...if pH is high, what about the [H+]?
It is LOW
What would a buffer do if pH falls?
Remove hydrogen ions from the solution.
What would a buffer do if pH rises?
 Donate hydrogen ions to the solution.
 In living organisms a key buffer is known as the

Bicarbonate Ion Buffer System.
Acids,
Bases &
Buffers
Importance of
The pH Scale
in Biology
CHEMICAL BONDING AND ELECTRON
VALENCES
The electrons in an atom are located at different energy
levels.
Electrons in the highest energy level are called valence
electrons.
Number of valence electrons governs an atom’s bonding
behavior.
Q: What is the max number of valence electrons for a

full valence shell?
Atoms are much more stable, or less reactive, with a full

valence shell.
By moving electrons, the two atoms become linked. This is

known as chemical bonding.
This stability can be achieved one of two ways:
- Ionic bond
- Covalent bond
IONIC BONDS
Involves transfer of electrons between two atoms.
Found mainly … inorganic compounds.
Ion = an atom or group of atoms which have lost or gained one or more electrons,
making them negatively or positively charged.
Q: What are positively charged ions (+) called?

Q: What are negatively charged ions (-) called?
IONIC COMPOUNDS ARE MADE
OF OPPOSITELY CHARGED
IONS
▪ Ionic Bonds are atoms held
together by attraction
between
a (+) and a (–) ion
▪ Compound is neutral
overall, but still charged
on the inside.
▪ Makes solid crystals.

IONS: ACIDS & BASES
An acid is any ionic
compound that releases
hydrogen _____ (H+)
in solution.
A base is any ionic

compound that releases
hydroxide _____ (-OH)
in solution.
From the Virtual Cell Biology Classroom on ScienceProfOnline.com

ANOTHER IMPORTANT CHARACTERISTIC OF
WATER…
WATER CAN FORM ACIDS AND BASES
Dissociation of Water
Hydronium ion + Hydroxyl ion -
Neutral water has equal amounts of H+ and OH -
Acids: Excess of H+ in aqueous solution

Bases: Excess of OH- in aqueous solution
Acids & bases neutralize each other.
Measurements of Acidity & Alkalinity (pH)
Acidity of a solution > measured

by concentration of hydrogen
ions (H+).
pH ranges: 0 (very acidic) to 14

(very basic).
Change in just one unit of scale

= tenfold change in H+
concentration.
If concentration of H+ = OH -
… neutral.
pH scale is logarithmic
Change in just
one unit of scale
= tenfold
change in H+
concentration.
Ions & Acids
An acid is any ionic compound that
releases hydrogen ions (H+) in solution.
Weak acids have a sour taste.
Strong acids are highly corrosive (So don’t

go around taste-testing acids.)
Examples:
▪ Ascorbic acid (C6H8O6, Vitamin C)
▪ Citric acid (C6H8O7, a weak organic acid in

citrus fruits)
▪ Phosphoric acid (H3PO4, in pop…this stuff is

also used to remove rust…hmmm)
IONS & BASES
A base is an ionic compound that releases
hydroxyl ions (OH-) in solution.
Bases are also called alkaline substances.
Some general properties of bases include:
Taste: Bitter taste (opposed to sour taste of acids

and sweetness of aldehydes and ketones).
Touch: Slimy or soapy feel on fingers.
Reactivity: Strong bases are caustic on organic

matter, react violently with acidic substances.
Examples:
▪ Sodium hydroxide, NaOH, of lye or caustic soda used in oven

cleaners.
▪ Magnesium hydroxide, Mg(OH)2, also known as milk of
magnesia, a weak base used in antacids and laxatives.
ACID/BASE BALANCE IN BIOLOGY
pH balance is important to
homeostasis of organisms.
Homeostasis = tendency of the

body to maintain a balanced
internal environment, even when
faced with external changes. Such as
the body's ability to maintain an internal temperature
around 98.6 degrees F, whatever the temperature
outside.
Examples:
▪ Digestion needs acidic environment
(pH 2-3)
▪ Urine is slightly acidic
▪ Blood must stay in neutral range
near 7.35 to 7.45
IONS & SALTS
▪ Compounds that dissociate in water and
produce cations other than H+ and anions
other than OH- are called salts.
▪ The most familiar salt is sodium chloride,

the principal component of common table
salt.
▪ Other examples of salts:
Baking soda (NaHCO3)

Epsom Salts (MgSO4)
Salts: The Role of Buffers
- Certain salts, called buffers, can
combine with excess hydrogen (H+) or
hydroxide (OH-) ions.
- Produce substances less acidic or

alkaline.
- Act like a chemical sponge to soak

up excess acid or base, keep pH
constant.
- Buffers can be “used up”. Once used up, no longer help regulate pH.
- Buffers are vital to maintaining pH in organisms.

• Example:
Antacids are buffers made of the salt calcium carbonate (CaCo3).
Bicarbonate Buffer system is important
in maintaining proper blood pH
ACID–BASE BALANCE
❖ Normal pH : 7.35-7.45
❖ Acidosis
Physiological state resulting from abnormally low plasma
❖ Alkalosis
Physiological state resulting from abnormally high plasma
❖ Acidemia: plasma pH < 7.35
❖ Alkalemia: plasma pH > 7.45

27
Concept Map
MEASUREMENT OF PH
The pH can be measured by:
ph strips
Ph indicators
Ph meter
▪pH is a Hydrogen ion
concentration.
▪pH = - log [H+]
▪Different compartment of human
body has specific pH.
▪pH has role in Enzyme activity.
WHY BLOOD PH IS ALTERED?
⚫ pH meter
 The pH meter is a laboratory equipment which used to
measure
▪ acidity or alkalinity of a solution
 The pH meter measures the concentration of hydrogen

ions [H+] using an ion-sensitive electrode.
 It is the most reliable and convenient method for

measuring ph.
▪ Indicator dyes and test strips
▪ Less precise
▪ Each indicator is only good for a small pH range (1-2 pH units)
▪ But may be good for field usage, or measuring small volumes, or dealing
with noxious samples.
SOME IMPORTANT INDICATORS USED IN A CLINICAL
BIOCHEMISTRY LABORATORY ARE LISTED BELOW
sr,. INDICATOR Ph range Colour in Colour in
No. acidic ph basic ph
1 Phenophthalein 9.3-10.5 colourless pink
2 Methyl orange 3.1-4.6 red yellow
3 Bromophenol blue 3.0-4.6 yellow blue
4 Methyl red 4.4-6.2 Red yellow
5 Phenol red 6.8 – 8.4 yellow red
6 Litmus 4.5-8.3 red Blue

- Buffers are the solutions which resist changes in pH when
small amounts of acid or alkali is added to them.
- A buffer is a pair of weak acid and its salt.
- Buffers are of main importance in regulating the pH of the

body fluids and tissues.
- Many biochemical reactions including those catalyzed by

enzymes require pH control which is provided by buffers.
TYPES OF BUFFERS
▪ Two types :
❑ ACIDIC BUFFERS –
▪Solution of a mixture of a weak acid and a salt of this weak acid with a
strong base.
E.g. CH3COOH + CH3COONa
( weak acid ) ( Salt )
❑ BASIC BUFFERS –
Solution of a mixture of a weak base and a salt of this weak
base with a strong acid.
e.g. NH4OH + NH4Cl
( Weak base) ( Salt)
HOW BUFFERS WORK
❑ Equilibrium between acid and base.
❑ Example: ACETATE BUFFER

 CH3COOH  CH3COO- +H+
❑ If more H+ is added to this solution, it simply shifts the

equilibrium to the left, absorbing H+, so the [H+] remains
unchanged.
❑ If H+ is removed (e.g. by adding OH-) then the equilibrium

shifts to the right, releasing H+ to keep the pH constant
• HANDERSON HASSELBALCH
EQUATION
❑ Lawrence Joseph Henderson wrote an equation, in 1908,

describing the use of carbonic acid as a buffer solution.
❑Karl Albert Hasselbalch later re-expressed that formula

in logarithmic terms, resulting in the Henderson–
Hasselbalch equation.
The Henderson-Hasselbalch Equation derivation
[H+] [A-]
HA H+ +A- Ka =
[HA]
[A-]
take the -log on both sides -log Ka = -log [H+] -log
[HA]
[A-]
apply p(x) = -log(x) pKa = pH -log
[HA]
and finally solve for pH…
[A-] [Proton acceptor]

pH = pKa + log = pKa + log
[HA] [Proton donor]
❑ The greater the buffer capacity the less the pH
changes upon addition of H+ or OH-
❑ Choose a buffer whose pKa is closest to the desired pH.
❑ pH should be within pKa ± 1

BUFFERS
❖ First line of defence (> 50 – 100 mEq/day)

❖ Two most common chemical buffer groups
 Bicarbonate
 Non bicarbonate (Hb,protein,phosphate)
➢ Blood buffer systems act instantaneously
➢ Regulate pH by binding or releasing H⁺
CARBONIC ACID–BICARBONATE BUFFER SYSTEM
Carbon Dioxide
✓ Most body cells constantly generate carbon dioxide

✓ Most carbon dioxide is converted to carbonic acid, which dissociates into
H+ and a bicarbonate ion
Prevents changes in pH caused by organic acids and fixed acids in ECF
✓ Cannot protect ECF from changes in pH that result from elevated

or depressed levels of CO2
✓ Functions only when respiratory system and respiratory control
centers are working normally
✓ Ability to buffer acids is limited by availability of bicarbonate ions
THE CARBONIC ACID HYDROGEN CARBONATE BUFFER
SYSTEM
• The carbonic acid-hydrogen Bicarbonate ion buffer is

the most important buffer system.
• Carbonic acid, H2CO3, acts as the weak acid
• Hydrogen carbonate, HCO3-, acts as the conjugate base
• Increase in H+(aq) ions is removed by HCO -(aq)

3
• The equilibrium shifts to the left and most of the H+(aq)
ions are removed
 The small concentration of H+(aq) ions reacts with the OH-
(aq) ions
 H2CO3 dissociates, shifting the equilibrium to the right,

restoring most of the H+(aq) ions
 Any increase in OH-(aq) ions is removed by H2CO3

THE HEMOGLOBIN BUFFER SYSTEM
CO2 diffuses across RBC membrane
✓ No transport mechanism required
As carbonic acid dissociates

✓ Bicarbonate ions diffuse into plasma
In exchange for chloride ions (chloride shift)
✓
 Hydrogen ions are buffered by hemoglobin

molecules
✓ Is the only intracellular buffer system with an immediate effect
on ECF pH
✓ Helps prevent major changes in pH when plasma PCO is

2
rising or falling
PHOSPHATE BUFFER SYSTEM
-
❖ Consists of anion H2PO4 (a weak acid)(pKa-6.8)
❖ Works like the carbonic acid–bicarbonate buffer system
❖ Is important in buffering pH of ICF
Limitations of Buffer Systems

➢ Provide only temporary solution to acid–base
imbalance
➢ Do not eliminate H+ ions
➢ Supply of buffer molecules is limited

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Prof.Dr.

This doesn’t happen often, but enough. If you have a

 In living organisms a key buffer is known as the

Q: What is the max number of valence electrons for a

Atoms are much more stable, or less reactive, with a full

By moving electrons, the two atoms become linked. This is

Found mainly … inorganic compounds.

Q: What are positively charged ions (+) called?

▪ Makes solid crystals.

A base is any ionic

From the Virtual Cell Biology Classroom on ScienceProfOnline.com

Hydronium ion + Hydroxyl ion -

Neutral water has equal amounts of H+ and OH -

Acids: Excess of H+ in aqueous solution

Acidity of a solution > measured

pH ranges: 0 (very acidic) to 14

Change in just one unit of scale

Weak acids have a sour taste.

Strong acids are highly corrosive (So don’t

▪ Ascorbic acid (C6H8O6, Vitamin C)

▪ Citric acid (C6H8O7, a weak organic acid in

▪ Phosphoric acid (H3PO4, in pop…this stuff is

Bases are also called alkaline substances.

Some general properties of bases include:

Taste: Bitter taste (opposed to sour taste of acids

Touch: Slimy or soapy feel on fingers.

Reactivity: Strong bases are caustic on organic

▪ Sodium hydroxide, NaOH, of lye or caustic soda used in oven

Homeostasis = tendency of the

▪ The most familiar salt is sodium chloride,

▪ Other examples of salts:

Baking soda (NaHCO3)

- Produce substances less acidic or

- Act like a chemical sponge to soak

- Buffers are vital to maintaining pH in organisms.

❖ Acidemia: plasma pH < 7.35

❖ Alkalemia: plasma pH > 7.45

The pH can be measured by:

 The pH meter measures the concentration of hydrogen

 It is the most reliable and convenient method for

2 Methyl orange 3.1-4.6 red yellow

3 Bromophenol blue 3.0-4.6 yellow blue

4 Methyl red 4.4-6.2 Red yellow

5 Phenol red 6.8 – 8.4 yellow red

6 Litmus 4.5-8.3 red Blue

- A buffer is a pair of weak acid and its salt.

- Buffers are of main importance in regulating the pH of the

- Many biochemical reactions including those catalyzed by

❑ Equilibrium between acid and base.

❑ Example: ACETATE BUFFER

❑ If more H+ is added to this solution, it simply shifts the

❑ If H+ is removed (e.g. by adding OH-) then the equilibrium

❑ Lawrence Joseph Henderson wrote an equation, in 1908,

❑Karl Albert Hasselbalch later re-expressed that formula

and finally solve for pH…

[A-] [Proton acceptor]

❑ Choose a buffer whose pKa is closest to the desired pH.

❑ pH should be within pKa ± 1

❖ First line of defence (> 50 – 100 mEq/day)