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ACID BASE BUFFERS

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 H + + A -

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ACID BASE BUFFERS

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  1. ACID BASE BUFFERS Dr Nithin Kumar U Assistant Professor Biochemistry, YMC

  2. ACIDS Acids are substances which are:- • proton donors (Lowry-Bronsted Theory) • electron acceptors (Lewis Concept) • dissociate to give hydrogen ions (Arrhenius concept) • HA H++ A- • HCl H++ Cl-

  3. STRENGTH OF ACIDS Strong acids: • Dissociate/ ionize completely • HCl H++ Cl- Weak acids • Ionize incompletely • H2CO3 H++ HCO3-

  4. BASE Bases are substances which are:- • proton acceptors (Lowry-Bronsted Theory) • electron donors (Lewis Concept) • dissociate to give hydroxyl ions (Arrhenius concept) • NH3+H+ NH4 • NaOH Na++ OH-

  5. 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

  6. CONJUGATE ACID- BASE PAIR

  7. DISSOCIATION CONSTANT • Dissociation is a reversible reaction • HA H++ A- • At equilibrium: (Undissociated/dissociated )is constant • Dissociation constant Ka= [H+]+ [A-] [HA]

  8. 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+]

  9. pH Decreasing [H+] Increasing pH

  10. pH of some biologically important fluids

  11. pKa • The pH at which the acid is half ionized • Indicates the strength of the acid • strong acids – low pKa • weak acids – high pKa

  12. 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

  13. 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]

  14. HENDERSON-HASSELBALCH EQUATION

  15. 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

  16. 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

  17. BUFFERS

  18. 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

  19. 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

  20. 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

  21. 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

  22. BUFFERS: EFFECTIVENESS A buffer is most effective when: • pH = pKa or [salt] = [acid] • ↓pKa ↑H+↑HCO3/H2CO3 ↑effectiveness of buffer

  23. BUFFERS: Example Buffer Acid Conjugate base/Salt pK • Phosphate Buffer H2PO4– HPO4– – 6.8 (HPO4– – + H2PO4–) • Bicarbonate Buffer Carbonic acid Bicarbonate 6.1 (HCO3–+ H2CO3) H2CO3 HCO3– • Protein Buffer H+-Protein Protein (Protein + H+-Protein)

  24. BUFFER SYSTEMS IN THE BODY

  25. MECHANISM OF BUFFERING • Buffer systems take up H+or release H +as conditions change Strong acid (H+) added Strong alkali (base) added

  26. 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

  27. 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

  28. ACID BASE BALANCE

  29. ACID BASE BALANCE & DISORDERS

  30. ACIDS GENERATED BY BODY METABOLISM Volatile acid • Carbonic acid (H2CO3) Carbonic anhydrase (in RBC) CO2+ H20 H2CO3 Non-Volatile acids • Lactic acid Sulphuric acid • Ketoacids Phosphoric acid • Role in H+ homeostasis/ acid-base balance in the body • Applied in separation, identification and quantitation of biomolecules in research and clinical laboratories

  31. 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

  32. 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.

  33. ACID BASE BALANCE Respiratory system: • Lungs eliminate CO2 by expiration. • Takes few minutes for compensation • 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

  34. 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 NaHCO3to H2CO3phosphate is 20:1 • Plasma Bicarbonate levels: 22 – 26 mmol/Lit • Plasma levels of H2CO3 is 1.2 mmol/L

  35. BICARBONATE BUFFER: MECHANISM OF ACTION IN ACIDOSIS CO2 Exhaled H2O + CO2 H2CO3 H++ HCO3- CA Acids H+ HCO3Reabsorbed H2CO3/HCO3

  36. BICARBONATE BUFFER: MECHANISM OF ACTION IN ALKALOSIS HCO3 Excreted H2O+ HCO3 Bases OH+ H2CO3 Regained OH-+ H2CO3 H2CO3/HCO3 CO2 Retains

  37. PHOSPHATE BUFFER • Intracellular buffer • Effective at a wide pH range • More than one ionizable groups H3PO4H+ + H2PO4- (pKa = 1.96) H2PO4 H++ HPO4-- (pKa = 6.8) HPO4 H + + PO4 ---(pKa = 12.4)

  38. 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

  39. PROTEIN BUFFER • Intracellularbuffer • 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

  40. 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

  41. HEMOGLOBIN BUFFER @ TISSUE CO2 TISSUES CO2 + H2O H2CO3 H++ HCO3- TISSUES CA CO2 RBC O2 Cl- O2 Cl- Hb HCO3-

  42. Hb Hb O2 O2 H H Hb Hb HEMOGLOBIN BUFFER @ LUNG PHOSPHATE BUFFER: COMPOSITION O2 O2 H++ HCO3 H++ HCO3 CO2 CO2 CO2 CO2 RBC RBC H2CO3 H2CO3 CA CA CO2 + H2O CO2 + H2O

  43. RESPIRATORY MECHANISMS • 2ndLine of defense against acid base imbalance • pH is regulated by altering respiratory rate changing the CO2levels • Normal pCO2 =35 - 45 mm Hg • Acidosis: hyperventilation • Alkalosis: hypoventilation

  44. RESPIRATORY MECHANISMS: ACIDOSIS Chemoreceptors Peripheral – Aorta, Carotid Arteries Central – Medulla Oblongata Acidosis: High H+ Stimulate the Respiratory Centre pH returns to Normal Increased Rate & Depth of Respiration ( hyperventilation) ↓ H + + HCO3 Expulsion of more CO2 ↓H2 CO3 ↓pCO2 of blood

  45. RESPIRATORY MECHANISMS: ALKALOSIS Chemoreceptors Peripheral – Aorta, Carotid Arteries Central – Medulla Oblongata Alkalosis: Low H+ Inhibits Respiratory Centre pH returns to Normal Decreased Rate & Depth of Respiration ( hyperventilation) ↑H + + HCO3 Retention of more CO2 ↑H2 CO3 pCO2 increases

  46. 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

  47. RENAL MECHANISMS • Excretion of H+ ions • Excretion of H as titrable acid • Reabsorption of Bicarbonate • Excretion of Ammonium ions

  48. 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

  49. RENAL MECHANISMS: EXCRETION OF H+

  50. 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

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