BCH312 [Practical] 1

¨ 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

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 A buffer is made up of a weak acid and its conjugate base.
Or
A weak base and its conjugate acid.

Are made from weak acid and its conjugated Are made from weak base and its conjugated
base[ its salt]. acid [ its salt].
Example: Example:
1. CH3COOH / CH3COONa (Pka) 1. NH3 / NH4Cl (Pkb)
è CH3COOH (Weak acid) è NH3 (Weak base)
è CH3COONa (conjugated base –its salt-) èNH4Cl (conjugated acid –its salt-)
2. NaH2PO4 / Na2HPO4 (Pka)
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 q How buffers can resist the change in pH?

-Example using [HA/A-] buffer:

è Where: HA is Weak acid and A- is conjugated base [its salt].

HA ⇌ H+ + A-

If H+ (acid) is added to this buffer system à H+ will If OH- (base) is added to this buffer system à OH- will
react with conjugated baseà to give conjugate acid. react with conjugated acid èto give conjugate base and
H2O.
H+
OH-

A- ⇌ HA HA ⇌ A- +  H2O 4
OH HA  ⇌ H + + A -
H+

HA A -
-
HA A
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q Example: conjugated base conjugated acid
CH3COO- CH3COOH
q Buffer system: CH3COOH / CH3 COO-

q When acid [H+] added:

CH3COO- + H+ CH3COOH

q When base [OH-] added:

CH3COOH + OH- CH3COO- + H2O

q NOTE: It resists pH changes when it’s two components are present in specific proportions.

è Thus the buffer is effective as long as it does not run out of one of its components. ( There are enough
conjugated base and conjugated acid to absorb the H+ ions or OH- ions added to the system respectively). 6
¨ It is often used to perform:
1. To calculate the pH of the Buffer.

2. To preparation of Buffer.

¨ It relates the Ka [dissociation constant] of a weak acid , [HA] concentration of weak acid
component , [A-] concentration of conjugate base [salt of the weak acid] component and the
pH of the buffer.

¨ The equation is derived from the acid dissociation constant.

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¨ A buffer is [to act as a good buffer the pH of the solution must be
within one pH unit of the pKa].
è The buffer capacity is optimal when the ratio of the weak acid to its salt is 1:1; that is,
when pH = pKa

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¨ Quantitative measure of buffer resistance to pH changes is called

¨ Buffer capacity can be defined in many ways, it can be defined as:

è The number of moles of H+/OH- ions that must be added to one liter of the buffer in order to decrease /increase the
pH by one unit respectively.

¨ The buffer capacity is expressed as β and can be derived from Henderson Hasselbalch equation:

From the equation è the buffer

capacity is directly proportional to the
buffer concentration.

¨ Where :
β = the buffer capacity , [H+] = the hydrogen ion concentration of the buffer , [C] = concentration of the buffer and Ka= acid
dissociation constant
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For weak acid [not buffers] For weak base [not buffers] For buffers

pka + p[HA] pkb + p[OH]

pH = pOH =
2 2
* P[HA] = -log [HA] * P[OH] = -log [OH]

pH = pkw – pOH
[Pkw : number of dissociation constant of
H2O].

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¨ To understand the behaviour and nature of buffers solutions.

¨ To learn how to prepare buffers.

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1. You are provided with: 0.2M solution of CH3COOH , 0.2M solution of CH3COONa.
è 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 :
1. 100% [HA]
2. 75% [HA] , 25% [A-]
3. 50% [HA] , 50% [A-]
4. 25% [HA] , 75% [A-]

3. Calculate the pH for each solution [pKa of CH3COOH = 4.76].

4. Follow the table. 13
1. To Calculate the volume that you must take from CH3COOH and CH3COONa to prepare the
previous mixtures with final volume of the solution =20 ml:
Note:
(A) 100% [HA]:
HA : as CH3 COOH.
A- : as CH3 COONa.
The final volume is 20ml, So:
20 x 100% = (20 x 100)/100 = 20 ml
è Take 20ml of HA and measure the pH.

(B) 75% [HA], 25% [A- ]:

From HA= 20 x 75% = (75 x 20) /100 = 15 ml

From A- = 20 x 25% = (25 x 20)/ 100 = 5 ml
èMix 15ml HA and 5 ml A- and measure the pH (measured PH) note that the total volume is 20 ml
[15ml +5ml =20ml]

¨ The same way for other mixtures …

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2. To Calculate the pH for the previous mixtures with pKa of CH3COOH = 4.76 :

(A) 100% [HA]:

pH = (pKa + p[HA]) è p[HA]= -log 0.2 = 0.69 è pH = ( 4.76 + 0.69) = 2.72

2 2

(B) 75% [HA] , 25% [A-] :

pH = pka + log [A-] / [HA] è pH= 4.76 + log [A-]/[HA] è [HA] = C1 X V1 = C2 X V2

=0.2 X 15 = C2 X 20 = C2 = 0.15M

è [A-] = C1 X V1 = C2 X V2
= 0.2 X 5 = C2 X 20 = C2 = 0.05 M
So, pH = 4.76+log 0.05/0.15 è pH = 4.282
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(C) 50%[HA] , 50%[A] :

pH = Pka + log [A-] / [HA] è pH= 4.76 + log [A-]/[HA] è [HA] = C1 X V1 = C2 X V2

=0.2 X 10 = C2 X 20 = C2 = 0.1M

è [A-] = C1 X V1 = C2 X V2
= 0.2 X 10 = C2 X 20 = C2 = 0.1 M
So, pH = 4.76+log 0.1/0.1 è pH = 4.76 +0 = 4.76 [pH=pka]

(D) 25% [HA], 75% [A-] :

pH = pka + log [A-] / [HA] è pH= 4.76 + log [A-]/[HA] è [HA] = C1 X V1 = C2 X V2

=0.2 X 5 = C2 X 20 = C2 = 0.05M

è [A-] = C1 X V1 = C2 X V2
= 0.2 X 15 = C2 X 20 = C2 = 0.15 M
So, pH = 4.76+log 0.15/0.05 è pH = 5.24
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 Solutions HA A- Final volume Calculated Measured 2M HCl Measured The difference
(ml) pH (ml)
CH3 COOH CH3 COONa pH pH
(ml) (ml)
100%[HA] 20 ml 0.1

75%[HA],25%[A-] 20 ml 0.1

50%[HA],50%[A-] 20 ml 0.1

25%[HA],75%[A-] 20 ml 0.1

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¨ 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).

o Solid sodium acetate [as A-].

o 0.2M Acetic acid [as HA].
o Pka = 4.76 Provided

o Final volume of buffer =50ml

o pH=4.86
o Buffer concentration = 0.19 M
Required
o Buffer Concentration = [HA] + [A-]
0.19 = [HA] + [A-]
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¨ To prepare a buffer Henderson-Hasselbalch equation is used:
pH = pka +log [A-] / [HA]

¨ First calculate the concentration of the weak acid and its conjugated base that make up the buffer with 0.19M:
è Assume [A-] = y and [HA] = 0.19 – y
So:
y
4.86 = 4.76 +log
0.19-y
y y
0.1 = log è by taking the “Anti log for both sides” è 1.26 = 0.19-y
0.19-y

y =1.26 x (0.19-y) è y= 0.24 – 1.26 y è y + 1.26 y = 0.24 è 2.26 y = 0.24

y= 0.11 M [which is the concentration of [A-] in the buffer ]

So, 0.11+ 0.08 = 0.19M

[HA] = 0.19 – 0.11
= 0.08 M [which is the concentration of [HA] in the buffer ]
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q To calculate the volume needed from [HA] to prepare the buffer, No. of mole of [HA] should be calculated first:

No. of mole = Molarity x Volume of solution in L

= 0.08 X 0.05 = 0.004mole
So, M of stock = no. of mole / Volume in Liter
0.2 = 0.004 / V

è V = 0.02 L = 20 ml

¨ To calculate the weight needed from [A-] to prepare the buffer, No. of mole of [A- ] should be calculated first:

No. of mole = Molarity x volume of solution in L

= 0.11 X 0.05 = 0.0055 mole

weight in (g) of [A-] = No. of moles x MW

è =0.0055 x 82 = 0.451 g
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¨ Now take 20 ml from 0.2M acetic acid and 0.451 g from solid sodium acetate and then complete the
volume up to 50 ml by addition of water.

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¨ 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 HCl to for both solutions.
¨ Measure the pH after the addition.

Solution Measured pH Add 2M HCl Measured pH

0.19M acetate buffer 0.1 ml

o.2M KCl 0.1 ml

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¨ You are provided with 0.5M acetic acid and solid sodium acetate.
Prepare 100ml of a 0.3M acetate buffer pH =4.78 if you know that (pKa=4.76).
è’’individually’’

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