1. Preparation of BUFFER

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+), and 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 decreases (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. 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.

9. 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.

10. Three practical methods to prepare a buffer: 1- First 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)

11. 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).

12. 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.

13. 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.

14. .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.

15. 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.

16. - 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.

17. In this lab: We will prepare acetate buffer By two ways: 1- Titration of week acid. Titration of acetic acid with strong base. 2- Using the known Pka value through Handerson-Hasselbalch equation.

18. 1- Titration curve of week acid acetic acid. Titration curves are produced by monitoring the pH of given volume of a sample solution after successive addition of acid or alkali The curves are usually plots of pH against the volume of titrant added or more correctly against the number of equivalents added per mole of the sample

19. Titration At the starting point the acid form predominates (CH3COOH). As strong base is added (e.g. NaOH), the acid is converted to its conjugate base. At the mid point of the titration, where pH=pK, the concentrations of the acid and the conjugate base are equal. At the end point(equivalence point), the conjugate base predominates, and the total amount of OH added is equivalent to the amount of acid that was present in the starting point.

20. Titration.

21. Determination of pKa values: pKa values can be obtained from the titration data by the following methods: 1. The pH at the point of inflection is the pKa value and this may be read directly 2. By definition the pKa value is equal to the pH at which the acid is half titrated. The pKa can therefore be obtained from the knowledge of the end point of the titration.

22. 2- Handerson-Hasselbalch equation: Method: You are provided with 0.1M sodium acetate and 0.1M acetic acid solution. Prepare 50ml of the buffer solution (0.1 M) 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

23.  Determine the Ratio of Acid to Base. Use the Henderson-Hasselbalch equation (below) to determine what ratio of acid to base is required to make a buffer of the desired pH.

24. Use the pKa value nearest your desired pH and the ratio will refer to the acid-base conjugate pair that correspond to that pKa. HH Equation: pH = pKa + log ([Base]/[Acid])

25. 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: ==> [Acid]= value b. Substitute for [Base]and Solve for [Acid]

26. c. Solve for [Base] Using the equation: [Base] = 1 - [Acid], you can calculate that: [Base] = value

27. d.Mix the Acid and Conjugate Base e. Check the pH Use a pH meter to confirm that the correct pH for the buffer is reached. Adjust slightly as necessary. f.Correct the Volume Once the desired pH is reached, bring the volume of buffer to ___L. g.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.