1. Preparation of buffers

2. Buffers 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

3. Buffers Mammalian tissues in the resting state have a pH of about 7.4 In order to maintain the required pH in an invitro biochemical experiment a buffer is always used The pH of a buffer is given by Handerson-Hasselbalch equation pH= pKa + log [A-] [HA]

4. Buffers pKa= -log Ka Ka is the dissociation constant of the acid [A-] is the concentration of the base [HA] is the concentration of the acid

5. Buffers A buffer consisting of a mixture of the weak acid(CH3COOH) and its salt (CH3COONa+) Will undergo the following changes on the addition of acid or base: If the base is added (OH) to the solution it will be buffered by the following reaction with acetic acid: CH3COOH + OH  CH3COO- + H2O So the pH will not change significantly

6. Buffers If acid (H+) is added, it will be buffered by another reaction, this time using the salt (CH3COO-) CH3COO- + H+  CH3COOH The pH will not alter significantly because the CH3COOH formed is a weak acid Addition of more base increases A- and decrases (HA) and this doesn’t alter the pH much until [A-]>>>>[HA]

7. Buffers The buffering power is greatest when pH=pKa i.e. when the acid and the salt are at the same concentration

8. Method You are provided with 0.1M sodium acetate and 0.1M acetic acid solution Prepare 50ml of the buffer solution by mixing the two standard solutions Use the following info to calculate the composition of the buffer: pH of the buffer 4.86 pKa of acetic acid at 25C 4.76

9. A compound can buffer the pH of a solution when: Its concentration is sufficient The pH of the solution is close (within about one pH unit) to its pK a.

10. To make a buffer Choose a compound whose pK a is close to the pH for the solution Determine what the buffer concentration should be.

11. Four practical methods to prepare a buffer: 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.

12. Advantages: Easy to understand. Useful when only one form of the buffer is available (in this case acetic acid) Disadvantages: 1.Slow. 2.May require lots of base (or acid).

13. Cont…. 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)

14. The Third 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.

15. Advantages: 1.Fast. 2.Easy to prepare. 3.Additional pH adjustment is rarely necessary, and when necessary, the adjustment is small. Disadvantages: 1.Requires the buffer pK a 2.and solving two equations.

16. The fourth method: 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.

17. Advantages: 1.Easy to do (with appropriate table). 2.Convenient for frequently prepared buffers. Disadvantages: 1.May be impossible to find table. 2.Table may be incorrect. 3.Requires both forms of buffer. 4.Component amounts from table will need to be adjusted to give the buffer concentration and volume in your solution.

18. In this lab: We will prepare acetate buffer

19. 1.Decide on the Buffer Properties Before making a buffer you must know; 1.what molarity you want it to be 2.what volume to make 3.what the desired pH is. Most buffers work best at concentrations between 0.1 M and 10 M. The pH should be within 1 pH unit of the acid/ conjugate base pKa.

20. 2. Determine the Ratio of Acid to Base

21. Use the Henderson-Hasselbalch equation (below) to determine what ratio of acid to base is required to make a buffer of the desired pH. Use the pKa value nearest your desired pH and the ratio will refer to the acid-base conjugate pair that correspond to that pKa.

22. HH Equation: pH = pKa + log ([Base]/[Acid])

23. 3.Substitute for [Acid] and Solve for [Base] The desired molarity of the buffer is the sum of [Acid] + [Base]. For a 1 M buffer, [Base] + [Acid] = 1 and [Base] = 1 - [Acid] By substituting this into the ratio equation, from step 2, you get: [Base] =

24. 4. Solve for [Acid] Using the equation: [Base] = 1 - [Acid], you can calculate that: [Acid] =

25. 5.Mix the Acid and Conjugate Base 6. Check the pH Use a pH meter to confirm that the correct pH for the buffer is reached. Adjust slightly as necessary. 7.Correct the Volume Once the desired pH is reached, bring the volume of buffer to ___L. 8.Dilute as Desired This same buffer can be diluted to create buffers of 0.5 M, 0.1 M, 0.05 M or anything in-between.