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
pH= pKa + equation:
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.
The Thired Method: Using table- 3 .
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

Titration of week acid. Titration of acetic- 1
acid with strong
.base
Using the known Pka value through - 2
Handerson-Hasselbalch
.equation
Titration curve of week- 1
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.
Handerson-Hasselbalch- 2
: 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.