ACID BASE BUFFERS
Dr Nithin Kumar U
Assistant Professor
Biochemistry, YMC
ACIDS
Acids are substances which are:proton donors (Lowry-Bronsted Theory)
electron acceptors (Lewis Concept)
dissociate to give hydrogen ions (Arrhenius concept)
HA
HCl
H++ AH++ Cl-
STRENGTH OF ACIDS
Strong acids:
Dissociate/ ionize completely
HCl
H++ ClWeak acids
Ionize incompletely
H2CO3
H++ HCO3-
BASE
Bases are substances which are:proton acceptors (Lowry-Bronsted Theory)
electron donors (Lewis Concept)
dissociate to give hydroxyl ions (Arrhenius concept)
NH3+H+
NaOH
NH4
Na++ OH-
CONJUGATE ACID- BASE PAIR
HA
H++ A-
HA ACID since it donates protons
A- BASE since it accepts the protons
HA and A- are called as conjugate acid – base pair
CONJUGATE ACID- BASE PAIR
Conjugate Acids
Conjugate base
H2CO3
HCO3
NH4
NH3
NaH2PO4
Na2HPO4
CH3COOH
CH3COONa
DISSOCIATION CONSTANT
Dissociation is a reversible reaction
HA
H++ AAt equilibrium: (Undissociated/dissociated )is constant
Dissociation constant
Ka= [H+]+ [A-]
[HA]
pH
pH = negative Logarithm of hydrogen ion concentration
pH= -log[H+]
pH= log{1/[H+]}
Higher the hydrogen ions concentration or acidity lower will
be the pH value
pH = - log [H+]
pH


In pure water at 25o C,
[H+] = 10–7 mol/L
Therefore, pH = negative log of 10–7
=7
pH
Decreasing
[H+]
Increasing
pH
pH of some biologically important fluids
Fluid
pH
Blood/ plasma
Interstitial fluid, cerebrospinal fluid (CSF)
Intracellular fluid (cytosol)
Human milk
Saliva
Gastric juice
Pancreatic juice
Urine
7.35 – 7.45
7.2 - 7.4
7
7.4
6.4 - 7
1.5 - 3
7.5 - 8
4.6- 7.5
pKa
The pH at which the acid is half ionized
 Indicates the strength of the acid
strong acids – low pKa
weak acids – high pKa
IMPORTANCE OF pH
pH affects the structure, properties and functions of
biomolecules, proteins (enzymes) and nucleic acids
Small changes in pH can produce major disturbances in cell
functions
HENDERSON-HASSELBALCH EQUATION
Relates pH, pKa & concentrations of the acid , conjugate
base of a buffer system
pH = pKa + log [base]
[acid]
In solution weak acids dissociates
HA
H++ ADissociation constant: Ka= [H+]+ [A-]
[HA]
HENDERSON-HASSELBALCH EQUATION
HENDERSON-HASSELBALCH EQUATION:
Applications
To calculate any one of the four variables- pH, pK, [acid] or
[salt], if other three are known
Assessment of acid-base status
Measurement of pH
Measurement of salt concentration
To understand and predict dissociation behavior of buffers
and in preparation of buffers
ACIDS GENERATED BY BODY METABOLISM
Volatile acid
Carbonic acid (H2CO3)
Carbonic anhydrase (in RBC)
CO2 + H20
Non-Volatile acids
Lactic acid
Ketoacids
H2CO3
Sulphuric acid
Phosphoric acid
ACID BASE BALANCE
Reference range of pH = 7.35 -7.45
Acidosis = pH < 7.35
CNS depression, coma
Alkalosis = pH > 7.45
neuromuscular hyper excitability, tetany
Life is threatened when plasma pH goes beyond: 6.8 - 7.8
ACID BASE BALANCE & DISORDERS
REGULATION OF BLOOD pH
FIRST LINE DEFENCE -
Bicarbonate Buffer
Phosphate Buffer
Protein Buffer
Hb Buffer
2nd LINE DEFENCE -
RESPIRATORY MECHANISMS
3rd LINE DEFENCE -
RENAL MECHANISMS
ACID BASE BALANCE
Buffers:
First line of defense against change of pH.
Present in blood, ICF and urine/renal tubular fluid.
Act immediately when a change in pH occurs.
Effect is temporary- do not eliminate acids or bases from
the body.
ACID BASE BALANCE
Respiratory system:
Lungs eliminate CO2 by expiration.
Takes few minutes for compenastion
compensation is not complete or permanent
Renal system:
Permanent
Non-volatile acids are excreted by kidney takes hours to
few days to fully compensate any acid-base imbalance
BUFFERS
The solutions that resist changes in pH when acid or alkali
is added to them
Mixture of weak acid with its salt with a strong base
Ex: CH3COOH/CH3COONa
Mixture of weak base with its acidic salt
Ex: Na2HPO4 /NaH2PO4
BUFFERS
Buffer
solution
Weak
acid
Its
conjugate
base
BUFFERS: MECHANISM OF ACTION
When acid is added – salt component will take up the H+
ions & forms the weak acid
Ex: Acetate Buffer
CH3COOH/CH3COONa
HCl + CH3COONa
CH3COOH+NaCl
BUFFERS: MECHANISM OF ACTION
Base is added – the acid component will react with it &
forms the weak base & water.
Ex: Acetate Buffer
CH3COOH/CH3COONa
NaOH+ CH3COOH
CH3COONa+H20
MECHANISM OF BUFFERING
Buffer systems take up H+ or release H + as conditions
change
Strong acid (H+) added
Strong alkali (base) added
BUFFERING CAPACITY
The amount of strong acid or strong alkali required to be
added to buffer to bring about a unit change in pH
It depends on:
pKa value
Ratio between the salt to acid concentration
(molar concentration of the buffer )
Buffering capacity is maximum when the pH of the buffer is
equal to its pK value
BUFFERS: EFFECTIVENESS
In a buffer, when [salt] = [acid] :
pH = pK + log 1
pH = pK + 0
pH = pK
 A buffer has maximum buffering capacity and is most
efficient, when pH of the soln. = its pK value
BUFFERS: EFFECTIVENESS
A buffer is most effective when:
pH = pKa
or
[salt] = [acid]
↓pKa
↑H+
↑HCO3/H2CO3
↑effectiveness of buffer
BUFFERS: Example
Buffer
Phosphate Buffer
(HPO4– – + H2PO4–)
Acid Conjugate base/Salt pK
H2PO4–
HPO4– –
6.8
 Bicarbonate Buffer Carbonic acid
(HCO3– + H2CO3)
H2CO3
Bicarbonate 6.1
HCO3–
Protein Buffer
(Protein + H+-Protein)
Protein
H+-Protein
BUFFER SYSTEMS IN THE BODY
ECF
ICF
RBC
Bicarbonate
NaHCO3
HCO3
Phosphate
K2HPO4
KH2PO4
Hemoglobin buffer
K+ Hb
H+ Hb
Phosphate
Na2HPO4
NaH2PO4
Protein buffer
K+ Protein
H+ Protein
Phosphate
K2HPO4
KH2PO4
BUFFERS: IMPORTANCE
Role in H+ homeostasis/ acid-base balance in the body
Applied in separation, identification and quantitation of
biomolecules in research and clinical laboratories
ACID BASE BALANCE
BICARBONATE BUFFER
Counts for 65 % of buffering capacity in plasma
pKa : 6.1
Components:
Bicarbonate (NaHCO3)
Carbonic acid (H2CO3)
represents a high alkali reserve- for buffering against
acids
The ratio of NaHCO3 to H2CO3 phosphate is 20:1
Plasma Bicarbonate levels: 22 – 26 mmol/Lit
Plasma levels of H2CO3 is 1.2 mmol/L
BICARBONATE BUFFER: MECHANISM OF ACTION
IN ACIDOSIS
H2O + CO2
CO2
Exhaled
H2CO3
Acids
H+
CA
H++ HCO3H2CO3/HCO3
HCO3 Reabsorbed
BICARBONATE BUFFER: MECHANISM OF ACTION
IN ALKALOSIS
HCO3
Excreted
H2O + HCO3
Bases
OH+
OH-+ H2CO3
H2CO3/HCO3
H2CO3
Regained
CO2
Retains
PHOSPHATE BUFFER
Intracellular buffer
Effective at a wide pH range
More than one ionizable groups
H3PO4
H+ + H2PO4- (pKa = 1.96)
H2PO4
H+ + HPO4-- (pKa = 6.8)
HPO4
H + + PO4 ---(pKa = 12.4)
PHOSPHATE BUFFER: COMPOSITION
Sodium dihydrogen Phosphate (NaH2PO4) - acidic
Disodium Monohydrogen Phosphate (Na2HPO4)- basic
pKa= 6.8
pH = pKa + log [base] / [acid]
The ratio of base to acid phosphate is 4:1
PROTEIN BUFFER
Intracellular buffer
Ionizable side chains
Side chain with pKa nearer to the physiological pH – act as
a buffer.
pKa of Imidazole group of the Histidine: 6.1
HEMOGLOBIN BUFFER
Function: transport of O2 & CO2
RBC need a buffer system to combat acid base
derangements occurring during gas transports
Hb a buffer in RBC
Buffering action: imidazole group of histidine
Enzyme: Carbonic anhydrase
HEMOGLOBIN BUFFER @ TISSUE
TISSUES
TISSUES
CO2 + H2O
CA
CO2
CO2
O2
H2CO3
H+ + HCO3O2
Hb
RBC
ClCl-
HCO3-
HEMOGLOBIN
BUFFER
@ LUNG
PHOSPHATE
BUFFER:
COMPOSITION
O2 O2
H H Hb Hb
O2 O2 Hb Hb
++ HCO
H++ HHCO
3
3
CO2 CO2
CO2 CO2
H2COH32CO3
CA CA
CO2 CO
+ H22+O H2O
RBCRBC
RESPIRATORY MECHANISMS
2nd Line of defense against acid base imbalance
pH is regulated by
altering respiratory rate
changing the CO2 levels
Normal pCO2 = 35 - 45 mm Hg
Acidosis: hyperventilation
Alkalosis: hypoventilation
RESPIRATORY MECHANISMS: ACIDOSIS
Acidosis:
High H+
pH returns to Normal
↓ H + + HCO3
Chemoreceptors
Peripheral – Aorta, Carotid Arteries
Central – Medulla Oblongata
Stimulate the Respiratory Centre
Increased Rate & Depth of Respiration
( hyperventilation)
Expulsion of more CO2
↓H2 CO3
↓pCO2 of blood
RESPIRATORY MECHANISMS: ALKALOSIS
Alkalosis:
Low H+
pH returns to Normal
↑ H + + HCO3
Chemoreceptors
Peripheral – Aorta, Carotid Arteries
Central – Medulla Oblongata
Inhibits Respiratory Centre
Decreased Rate & Depth of Respiration
( hypoventilation)
Retention of more CO2
↑H2 CO3
pCO2 increases
RENAL MECHANISMS
Takes hours to days to act
Can eliminate large amounts of acid
Can also excrete base
Can conserve and produce bicarbonate ions
Most effective regulator of pH
If kidneys fail pH balance fails
RENAL MECHANISMS
Excretion of H+ ions
Excretion of H as titrable acid
Reabsorption of Bicarbonate
Excretion of Ammonium ions
RENAL MECHANISMS: EXCRETION OF H+
Site: PCT
Increase the alkali reserve
Generation of HCO3
Mediated by Na-H exchanger
Potassium competes with H+ for Na-H exchanger
RENAL MECHANISMS: EXCRETION OF H+
Blood
Proximal Convoluted
tubule
Tubular lumen
Na+
Na+
Na+
HCO3-
HCO3- + H+
H+
H2CO3
CA
CO2 + H2O
RENAL MECHANISMS:
EXCRETION OF H+ AS TITRABLE ACID
Site: DCT, Collecting duct
Secretion of H+ by H+ -ATPase
Titrable acidity: number of ml of 0.1N NaOH required to
titrate 1L of urine to pH 7.4
Major titrable acid: sodium acid phosphate
Urine NaH2PO4 : Na2HPO4 = 9:1
Normal H+ excreted: 10 – 30 mEq/day
RENAL MECHANISMS:
EXCRETION OF H+ AS TITRABLE ACID
Blood
Na+
HCO3-
Tubular cell
Na+
HCO3- + H+
Tubular lumen
Na2HPO4(pH=7.4)
Na+
H+
NaHPO4
H2CO3
CA
CO2 + H2O
NaH2PO4(pH=5.4)
RENAL MECHANISMS:
REABSORPTION OF BICARBONATE
Site: PCT
HCO3- filtered by glomerulus is reabsorbed
No excretion of H+
RENAL MECHANISMS:
REABSORPTION OF BICARBONATE
Blood
Na+
HCO3-
Tubular cell
Na+
HCO3- + H+
H2CO3
CA
CO2 + H2O
Tubular lumen
NaHCO3(pH=7.4)
Na+
H+
HCO3-
H2CO3
CO2 + H2O
RENAL MECHANISMS:
EXCRETION OF AMMONIUM IONS
Site: DCT
Source of NH3 Glutamine (glutaminase)
Oxidative deamination (Amino acid oxidase)
Glycine (glycine oxidase)
Acidosis: ↑glutaminase
Alkalosis: ↓glutaminase
H+ is trapped as NH4 and excreted
Normal H excreted: 30 – 50 mEq/day
RENAL MECHANISMS:
EXCRETION OF AMMONIUM IONS
Blood
Tubular cell
Glutamine
Tubular lumen
Glut. acid
NH3
Na+
HCO3-
NH3
Na+
Na+
HCO3- + H+
H
H2CO3
CA
CO2 + H2O
NH4+
59
ANION GAP
ANION GAP
Difference between measured cations and measured anions
Unmeasured anions –
Protein & organic anion
Sulphate
Phosphate
Useful in differential diagnosis of acid base disordersmetabolic acidosis
Calculated by: AG = (Na+ + K +) – (HCO3 - + Cl - )
Normal value – 15 ± 5 mmol/L
METABOLIC ACIDOSIS
Primary HCO3 deficit
Decreased HCO3-  Reduction in blood pH
Causes: Increased formation of organic acids
Decreased excretion of H+
Loss of bicarbonate
 ↓pH, ↓↓HCO3 and↓ pCO2
 Anion gap normal or ↑
 ↑ K+(due to redistribution of potassiu and proton)
 Rx: IV lactate solution
METABOLIC ACIDOSIS: CAUSES
High anion gap Acidosis:
 Renal failure
 Lactic acidosis
 Diabetic ketoacidosis
Normal anion gap Acidosis:
 Diarrhoea
 Hyperchloremic acidosis(RTA, Acetazolamide)
METABOLIC ALKALOSIS
Primary HCO3 excess
pH increased
Causes: H+ loss (severe vomiting, Cushing syndrome)
Alkali ingestion (NaHCO3,Milk alkali syndome)
↑↑HCO3, ↑pH, ↑pCO2
Respiratory compensation by hypoventilation
 Rx: Electrolyte replacement, IV chloride containing
solution, Treat underlying disorder
RESPIRATORY ACIDOSIS
Primary Carbonic acid excess
Plasma pH decreased d/t excess carbonic acid
Causes: Lung disorders(Pneumonia, asthma, COPD)
Depression of respiratory center(sedative)
Paralysis of respiratory muscles
↑pCO2, ↓pH, normal HCO3
Renal compensation
Rx: Restore ventilation, treat underlying dysfunction
/disease, IV lactate solution
RESPIRATORY ALKALOSIS
Primary Carbonic acid deficit
Causes: Due to hyperventilation
Stimulation of respiratory center (High altitude)
Hysteria, Septicemia
↑ pH, ↓↓pCO2, HCO3- unaltered
Respiratory Compensation does not take place
Rx: Breathe into a paper bag
IV Chloride containing solution – Cl- ions replace lost
bicarbonate ions
ACIDOSIS FEATURES
CNS Depression d/t ↓ in synaptic transmission
Generalized weakness
Severe acidosis causes - Disorientation
Coma
Death
ALKALOSIS FEATURES
CNS / PNS over
Numbness
Lightheadedness
Nervousness
Muscle spasms or tetany
Convulsions
Loss of consciousness
Death
DIAGNOSIS OF ACID-BASE IMBALANCES
STEP 1: Note the pH
 low (acidosis) or high (alkalosis)
STEP 2: Decide which among pCO2 or HCO3- lies outside
normal
If cause is a change in pCO2problem is respiratory
If the cause is HCO3-  problem is metabolic
 STEP 3: Look for value that doesn’t correspond to the
observed pH change for determining the compensation
THANK U