The document describes how to prepare an acetate buffer using two methods: 1) Titration of acetic acid with sodium hydroxide and 2) Using the Henderson-Hasselbalch equation. For the titration method, acetic acid is titrated with sodium hydroxide and the pH is monitored to determine the pKa. For the Henderson-Hasselbalch method, the desired pH, pKa, and concentrations are used to calculate the ratios of acetic acid and sodium acetate needed to make a 0.1M buffer at pH 4.86. The solutions are then mixed and the final pH is checked to ensure it is as desired.

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 pKa.

9. To make a buffer
 Choose a compound whose pKa 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 pKa , 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 pKa
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. Titration.
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. a. 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.