1. Buffers

2. Introduction Buffers are important in biochemical processes. Whether they occur naturally in plasma or in the cytosol of cells, buffers assure biological reactions occur under conditions of optimal pH. The word “buffer” is so common in biochemistry its replaces the word “water” in experimental protocols.

3. Buffers 3

4. What are buffers? Buffers are solutions in which the pH remains relatively constant, even when small amounts of acid or base are added Example: Contain a weak acid (CH 3 COOH) and its salt(CH 3 COONa); H 2 CO 3 & NaHCO 3 Or a weak base (B) and its (BHCl); (NH 3 and NH 4 Cl)

5. Importance of Buffers Buffers  resist changes in pH from the addition of acid or base.  in the body, absorb H 3 O + or OH - from foods and cellular processes to maintain pH.  are important in the proper functioning of cells and blood.  in blood maintain a pH close to 7.4. A change in the pH of the blood affects the uptake of oxygen and cellular processes. 5

6. Acidic buffer solution An acidic buffer solution is simply one which has a pH less than 7. Acidic buffer solutions are commonly made from a weak acid and one of its salts - often a sodium salt. Example: acetic acid and sodium acetate CH 3 COOH and CH 3 COONa

7. Alkaline(Basic) buffer solutions An alkaline buffer solution has a pH greater than 7. Alkaline buffer solutions are commonly made from a weak base and one of its salts. A frequently used example is a mixture of ammonia solution and ammonium chloride solution. NH 3 and NH 4 Cl

8. How buffers work Equilibrium between acid and base. Example: Acetate buffer CH 3 COOH  CH 3 COO - + 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

9. Buffer Capacity The buffer capacity is the amount of acid or base that can be added before a significant change in pH This depends on the amounts of HA and A- present in the buffer Most efficient buffer is when [A-] [HA] = 1

10. Henderson-Hasselbach Equation Derived from the equilibrium expression of a weak acid and the pH equation. This equation allows you to determine the pH of a buffer solution pKa = -log Ka pH = pKa + log [A-] [HA]

11. Example 1: What is the pH of a mixture of 5 ml of 0.1 M sodium acetate and 4 ml of 0.1 M acetic acid (pKa = 4.76 at 25 0 C)? Concentration of CH 3 COOH=4/9 × 0.1 M Concentration of CH 3 COONa=5/9 × 0.1 M pH = pKa + log [NA] [HA] pH = 4.76+ log (5/4) pH = 4.76 + ( + 0.097) = 4.86

12. Learning Check Which combination(s) make a buffer solution? A. HCl and KCl B. H 2 CO 3 and NaHCO 3 C. H 3 PO 4 and NaCl D. CH 3 COOH and CH 3 COOK E. NaOH and NaCl F. NH 3 and NH 4 Cl 12

13. Solution B. H 2 CO 3 + NaHCO 3 A weak acid and its salt D. CH 3 COOH + CH 3 COOK A weak acid and its salt. F. NH 3 and NH 4 Cl A weak base and its salt 13

14. PREPARATION OF BUFFER SOLUTIONS

15. Three practical methods to prepare a buffer: 1- F irst Method : By the Titration, in the presence of one of the two buffer forms with strong base or acid: Prepare a buffer composed of an acid and its salt by adding a strong base(e.g. NaOH) to a weak acid (e.g. Acetic acid) until the required pH is obtained If the other form of buffer is available (in this case sodium acetate), a strong acid is added (e.g. HCl) until the required pH is obtained. CH3COONa+HCl  CH3COOH+NaCl So acetate buffer is formed(CH3COOH/CH3COONa )

16. 2- Second Method: Using the buffer pK a, calculate the amounts (in moles) of acid/salt or base/salt present in the buffer at the desired pH. If both forms (i.e., the acid and the salt) are available, convert the amount required from moles to grams,using the molecular weight of that component, and then weigh out the correct amounts of both forms. Or convert moles to volume if the stock is available in the liquid form.

17. .3- The Thired Method: Using table Find a table of the correct amounts of acid/salt or base/salt required for different pH's Dissolve the components in slightly less water than is required for the final solution volume. Check that the pH and correct if necessary. Add water to the final volume.