Lab3 Buffer
Lab3 Buffer
Lab3 Buffer
All biochemical reactions occur under strict conditions of the concentration of hydrogen ion.
Biological life cannot withstand large changes in hydrogen ion concentrations which we measure as
the pH.
Those solutions that have the ability to resist changes in pH upon the addition of limited amounts of acid
or base are called Buffers.
Buffers
are solutions that have the ability to resist changes in pH. upon the addition of limited amounts of
acid or base.
A buffer is made up of a weak acid and its conjugate base. Or a weak base and its conjugate acid .
Types of Buffer:
Example: • Example:
1. CH3COOH / CH3COONa (Pka) NH3/NH4CL (Pkb)
CH3COOH (Weak acid) NH3 (Weak base)
CH3COONa (conjugated base –its salt) NH4CL (conjugated acid –its salt)
HA⇌H++A-
[a] If H+ is added to this buffer system H+ will [b] If OH- is added to this buffer system [HA/A-] OH will
react with conjugated base to give conjugate acid. react with conjugated acid to give conjugate base and H2O.
H+ OH-
A-⇌ HA HA⇌A-+H2O
Mechanism of Action (Buffer):
Example:
Buffer system: CH3COOH / CH3COO- , (CH3COOH :acid - CH3COO-: conjugated base )
NOTE: It resists pH changes when it’s two components are present in specific proportions.
Thus a buffer can protect against pH changes from added H+ or OH- ion as long as there is sufficient basic
and acidic forms respectively. As soon as you run out of one of the forms you no longer have a buffer .
Henderson-Hasselbalch equation
- It relates the Ka [dissociation constant] of a weak acid , [HA] concentration Of weak acid ,
[A-] concentration Of conjugate base [salt of the weak acid] components and the pH of the buffer.
A buffer is best used close to its pKa.[to act as a good buffer the pH of the solution must be
within one pH unit of the pKa].
The buffer capacity is optimal when the ratio of the weak acid to its salt is 1:1; that is,
when pH = pKa
Buffer capacity
The buffer capacity is expressed as β and can be derived from Henderson Hasselbalch equation:
Where : β = the buffer capacity , [H+] = the hydrogen ion concentration of the buffer , [C] = concentration
of the buffer and Ka= acid dissociation constant
pH Calculation
1- for weak acid [not buffers]: 2- for weak base [not buffers] : 3- for buffers :
pH = pkw–pOH
[Pkw : number of dissociation constant of H2O]. The concentration of HA and A-
in the buffer
Practical Part
Objective:
Method:
You are provided with: 0.2M solution of CH3COOH , 0.2M solution of CH3COONa.
1. Determine which is the weak acid and which is the conjugated base [or its salt].
2. Calculate the volume that you must take from CH3COOH and CH3COONa to prepare
the following mixtures with final volume of the solution =20 ml :
a. 100% [HA]
b. 75% [HA] , 25% [A-]
c. 50% [HA] , 50% [A-]
d. 25% [HA] , 75% [A-]
1- Volume calculation
To Calculate the volume that you must take from CH3COOH and CH3COONa to prepare previous mixtures in final
volume of 20 ml :
Mix 15ml HA and 5 ml A- and measure the PH (measured PH) note that the total volume is 20 ml [15ml +5ml =20ml]
To calculate the pH for the previous mixtures with pKa of CH3COOH = 4.76 :
You are provided with 0.2M acetic acid and solid sodium acetate.
Prepare 50ml of a 0.19M acetate buffer pH =4.86 if you know that pKa =4.76.
Calculations:
Provided: 0.2 M
Acetic acid Sodium acetate
• Solid sodium acetate [as A-] [HA] [A-]
• 0.2M Acetic acid. [as HA]
g?
• Pka = 4.76 ml ?
Required:
• Final volume of buffer =50ml
• pH=4.86
• Buffer concentration = 0.19 M
pH 4.86 50 ml
Buffer Concentration = [HA] + [A-] 0.19 M
0.19 =[HA] + [A-]
Buffer
Calculations
y y
4.86 (of buffer) = 4.76 +log 0.1 = log
0.19-y 0.19-y
2- To calculate the volume needed from [HA] to prepare the buffer, No. of mole of [HA] should be calculated first :
Calculate moles in buffer: No. of mole (of HA) = molarity (of HA calculated in buffer) X volume L (volume of buffer required)
= 0.08 X 0.05 = 0.004 mole
Calculate the volume of stock HA: M (of stock HA in Q) = no. of mole (of HA calculated) / Volume in Liter (volume of HA ?)
0.2 = 0.004/V
V = 0.02 L = 20 ml
3- To calculate the weight needed from [A-] to prepare the buffer, No. of mole of [A- ] should be calculated first :
Calculate moles in buffer: No. of mole (of A-) = molarity (of A- calculated in buffer) X volume L (volume of buffer required)
=0.11 X 0.05 = 0.0055 mole
Calculate the wight of stock A-: wt in (g) of [A-] = mole X Mw
= 0.0055 x 82 = 0.45 g
Method
Now take 20 ml from 0.2M acetic acid and 0.45 g from solid sodium acetate then complete the volume
up to 50 ml by addition of water.
Check the pH
(3)Testing for buffering behavior:
Method:
In one beaker add 10ml of 0.19M acetate buffer that you have prepared, and in another beaker add
10ml of 0.2M KCl.
Measure the pH.
Add 0.1ml from 2M HClto for both solutions.
Measure the pH after the addition.
H.W
You are provided with 0.5M acetic acid and 0.5M sodium acetate.
Prepare 100 ml of a 0.3M acetate buffer pH =5.2 if you know that pKa =4.76.