Transcript Preparation of buffers

PREPARATION OF BUFFERS
DEFINITION
A 1M solution of sulfuric acid contains 98.06 g of sulfuric acid in
1 liter of total solution.
"mole" is an expression of amount
"molarity" is an expression of concentration.
"Millimolar", mM, millimole/L.
 A millimole is 1/1000 of a mole.
"Micromolar", µM, µmole/L.
 A µmole is 1/1,000,000 of a mole.
FORMULA
HOW MUCH SOLUTE IS NEEDED FOR A SOLUTION OF A
PARTICULAR MOLARITY AND VOLUME?
(g solute ) X (mole) X (L) = g solute needed
1 mole
L
or
FW
X molarity
x volume = g solute needed
EXAMPLE
How much solute is required to make 300 mL of 0.8 M CaCl2?
(111.0 g) (0.8 mole) (0.3 L) = 26.64 g
mole
L
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 in vitro biochemical
experiment a buffer is always used
 The pH of a buffer is given by Handerson-Hasselbalch equation
 pH= pKa + log [A-]
[HA]
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+)
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]
The buffering power is greatest when pH=pKa i.e. when
the acid and the salt are at the same concentration
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 information to calculate the composition of
the buffer:
pH of the buffer 4.86
pKa of acetic acid at 25C 4.76
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.
 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)
The Third 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.
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.