Preparation of BUFFER

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.
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]
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

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
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]
Buffers
 The buffering power is greatest when pH=pKa , i.e. when
the acid and the salt are at the same concentration
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.
To make a buffer

 Choose a compound whose pKa is close to the pH for

the solution
 Determine what the buffer concentration should be.
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)
 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).
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.
 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.
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.
 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.
- 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.
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.
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
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.
Titration.
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.
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
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.
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])
b. Substitute for [Base]and Solve for [Acid]

 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

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

[Base] = value
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.