Lecture 02- pH and Buffers..

pH and buffers Prof. Inoka Uluwaduge Dept. of Basic Sciences Lesson outcomes 1. Explain the term pH and calculate pH at different [H+] 2. State the role of a conjugate acid and a base 3. Explain the applications of Henderson-Hasselbalch equation 4. Define a “buffer” and explain buffering ca...

pH and buffers Prof. Inoka Uluwaduge Dept. of Basic Sciences Lesson outcomes 1. Explain the term pH and calculate pH at different [H+] 2. State the role of a conjugate acid and a base 3. Explain the applications of Henderson-Hasselbalch equation 4. Define a “buffer” and explain buffering capacity and buffering range of a buffer 5. Name the buffers found in body fluids and explain their buffering action 6. Understand briefly the clinical conditions “acidosis” and “alkalosis” 7/2/2024 2 pH- a measure of H+ concentration pH = -log10 [H+] pH = 7.40 (physiological “normal”) 7.35 7.45 Reference range for blood pH Neutral Increasing acidity Increasing basicity H+ = 1 mol/L (M) H+ = 10 -7 mol/L (M) H+ = 10 -14 mol/L (M) pH = 0 pH = 7 pH = 14  pH measurements pH is a numeric scale used to specify the acidity or alkalinity of an aqueous solution The higher the pH reading, the more alkaline and oxygen/OH- rich the fluid is The lower the pH reading, the more acidic and oxygen deprived (H+ rich) the fluid is The pH range is from 0 to 14, with 7.0 being neutral; above 7.0 is alkaline, below 7.0 is acidic 7/2/2024 4  Acids and Bases Acids = H+ donors Bases = H+ acceptors 1. Strong acids / bases completely dissociate HCl H+ + Cl- 2. Weak acids / bases partially dissociate CH3COOH H+ + CH3COO- NH4OH NH4+ + OH- Many biologically important molecules are either weak acids or bases e.g. carbonic acid H CO H+ + HCO - Conjugate bases and conjugate acids Conjugate base = Proton Conjugate acid= Proton donor acceptor of a weak acid of a weak base (deprotonated form of a (protonated form of a weak acid) weak base) CH3COOH H++ CH3COO- CH3NH2+ H + CH3NH3 proton donor proton weak base conjugate acid acceptor proton acceptor proton donor weak acid conjugate base Methylamine Methylammonium 7/2/2024 6 Dissociation constant of an acid Ka Quantitative relationship between concentration of weak acid (HA) and its conjugate base (A-) HA H + + A- [ H + ][ A -] Ka = Dissociation constant [ HA ] of acid Relative strength of acids (acidity) are expressed in terms of dissociation constant e.g. acetic acid Ka =1.8 x 10–5 , formic acid Ka =1.8 x 10–4 Larger the Ka stronger the acid ( formic acid >acetic acid) The tendency of any acid to lose its H+= Ka ! Henderson - Hasselbalch equation HA H+ + A- - Ka = [H+] [A ] [HA] [H+] [H+] = Ka [HA] - [A ] log log [H+] = log Ka + log [HA] - [A ] (-) -log [H+] = - log Ka + - log [HA] weak acid - [A ] conjugate base - pH = pKa + log [A ] [HA] 7/2/2024 8 Applications of H-H equation pH = pKa + log [A-] [HA] 1. To calculate the pH of a solution 2. To predict the acidity of an acid 3. To calculate the proportion of acid/salt 7/2/2024 9  Applications To predict the pH of a system when the acid is half neutralized HA H+ A- [A-] = [HA] - pH = pKa + log [A ] = pKa + log 1 = pKa + 0 [HA] 1 pH = pKa 7/2/2024 10 Applications of H-H eq To predict the pH of a system when the ratio of [A-]/[HA]is given [A-]/[HA]= 100:1 - pH = pKa + log [A ] = [HA] 7/2/2024 11 [A-]/[HA]= 100:1 pH - = pKa + log [A ] [HA] pKa + log 100 1 pKa+2 pH = pKa+2 7/2/2024 12  Buffers Definition: Solutions that resist change in pH following the addition of (small amounts) acid or base [Solutions of weak acid & their conjugate base (or weak base & their conjugate acid) behave as buffers] CH3COOH (WA) CH3COO- (CB) OH- H+ CH3COO-+ H2O CH3COOH donating H+ accepting H+  Buffers A buffer is a substance that has the ability to bind or release H+ in solution, thus keeping the pH of the solution relatively constant despite the addition of acids or base (small amounts) 7/2/2024 14 Buffer: is a mixture of a weak acid and its conjugate base Weak acid OH- HA H+ H A- Conjugate base H2O 7/2/2024 15 Prediction of buffering capacity pH= pKa + log [A-] [HA] pH= pKa + log [proton acceptor] [proton donor] pKa of a weak acid is equal to the pH of the solution at the mid point of titration [HA] = [A-] Maximum buffering capacity occurs pH= pKa Weak acid/ conjugate base pair can still serve effectively when the pH= pKa±1 16  Characteristics of buffers Effective Buffering Range III pH= pKa ±1 OH- CH3COOH CH3COO- + H20 [I] H+ [II]  Characteristics of buffers.. Buffer capacity: How well a solution resists changes in pH Depends on: 1. Concentration of acid and conjugate base Higher the [concentration ] greater the capacity 2. How close the pH of buffer is to the pKa of weak acid Maximum buffering capacity occurs at a pH= pKa Effective buffering capacity pH= pKa±1  What is acid base balance? In order to achieve homeostasis, there must be a balance between the intake/ production of hydrogen ions and the net removal of hydrogen ions from the body Homeostasis-keeping the internal environment constant 7/2/2024 19  6.8 - 8 7/2/2024 20  Acid-Base balance Body systems carefully control pH of the body within the range of 7.35-7.45 Homeostasis of pH is tightly controlled Acidosis (acidemia) below 7.35 Alkalosis (alkalemia) above 7.45 7/2/2024 21 pH value of certain body fluids Body fluid pH Blood 7.35-7.45 Saliva 5.4-7.5 Gastric juice 1.5-3.5 ? HCl Bile 6-8.5 Urine 4.5-8.0 7/2/2024 22 pH of body fluids- saliva Saliva has a pH 5.4 -7.5 which is the optimum value for the action of salivary amylase, Initiates the digestion of carbohydrates The action of salivary amylase is inhibited when food reaches stomach and is mixed with acid gastric juice 7/2/2024 23 pH of body fluids -gastric juice The highly acid pH of the gastric juice is maintained by HCl secreted by the parietal cells in the walls of the gastric glands The low pH value in the stomach provides the environment best suited to the functioning of the enzyme pepsin that begins the digestion of dietary protein 7/2/2024 24 pH of body fluids- blood Blood has a pH value between 7.35 and 7.45. The pH range of blood compatible with life is 6.8 to 8.0 The metabolic activity of the body cells produces certain acids and alkalis which alter the pH of the tissue fluid and blood To maintain the pH within the normal range, there are substances present in blood that act as buffers 7/2/2024 25  Buffers of the body fluids The body maintain the pH within narrow limits despite the fact that metabolic processes will constantly release or uptake protons Blood Bicarbonate H2CO3 H+ + HCO3- Hemoglobin H+Hb H+ + Hb- Protein HProt H+ + Prot- Intracellular Protein HProt H+ + Prot- fluid Phosphate H2PO4- H+ + HPO4- Interstitial Bicarbonate H2CO3 H+ + HCO3- fluid Urine Bicarbonate H2CO3 H+ + HCO3- Phosphate H2PO4- H+ + HPO4- Ammonia NH4+ H+ + NH3  Bicarbonate buffer system A major buffer system in blood catalyzed by CO2 + H2O H2CO3 H+ + HCO3- carbonic at lungs anhydrase at body fluids As with any other buffer system pH is balanced by Weak acid/ conjugate base? Excess acid/ bases introduced is neutralized *If [H+] is low, hold the breath. How ? If [H+] is high,increase the exhaling rate. Bicarbonate buffer system … Bicarbonate buffer system is important to regulate blood pH What will happen with the release of CO2 from lungs ? 7/2/2024 28  Bicarbonate buffer system … pKa of this system is low (6.1) relative to the pH of the blood (7.4) The most effective range for this system = 5.1-7.1 Still this system is very important [HCO3-] : = 20 at physiological pH of 7.4 [H2CO3] 1 Amount of CO2& HCO3- are physiologically regulated (by lungs & kidneys) 7/2/2024 29  Phosphate buffer system H2PO4- H+ + HPO42- reaction 1 OH- + H2PO4- H2O + H2PO42- reaction 2 pKa = 6.8, buffer range = 5.8 – 7.8 In the plasma, the phosphate concentration is too low for this system to be an important buffer But it is important intracellularly, and in the urine (Urine pH can vary from pH= 4.6 to 8) Phosphate buffer system Principal urinary buffer GFR ~80% Present in the HPO42- H2PO4- H+ + HPO42- reaction 1 Minimum urinary pH (4.6) all P in H2PO4- pKa= 6.8, buffer range = 5.8 – 7.8 Its ability to serve as an important buffer decreases when urinary pH reduces to as low as 5.8/6 7/2/2024 31  Buffering system in the RBC - Hb Hemoglobin (Hb) can act as a buffer due to the presence of Histidine residues 32 histidines / Hb (present in large amounts) H N N+ imidazole group N N pKa=6.2 H H H2N COOH H2N COOH Hb + H+ H+Hb  Acidosis & Alkalosis When arterial pH » falls below 7.35 acidosis » rises above 7.45 alkalosis 2 types; 1) Metabolic - blood HCO3- 2) Respiratory - arterial PCO2 pH measurements Ordinary litmus paper indicates whether a solution is acid or alkaline by colouring blue for alkaline and red for acid When accurate measurements of pH are required, sensitive pH meters are used 7/2/2024 36 References Lippincott’s Biochemistry; Chapter on Acid-Base Balance and Arterial Blood Gas Analysis 7/2/2024 37