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BCH 202 General Biochemistry [Practical]

Lab (1) Preparation of Buffer Solutions

Emtenan Mohammed Alkhudair

Office: Building 5, 3rd floor, Office No. 269

Email: [email protected]
Website: http://fac.ksu.edu.sa/ealkhudair
Office hours: Sun, Tue 9-11
 Hydrogen number (pH):
Ø The acidity of certain solutions can be described by using hydrogen number (pH).

Ø pH defined as: The negative logarithm of the hydrogen ion concentration.

pH= - Log[H+]

Ø When the pH increase the concentration of hydrogen ion decrease and vice versa.
Ø The pH range goes from 0 to 14.

Acidic à pH< 7

Neutral à pH=7

Basic à pH>7
Acidic solution : has higher concentration of hydrogen
H+ > OH-
ions [H+] than hydroxyl ions [OH-]

Neutral solution: has equal concentration of hydrogen

ions [H+] and hydroxyl ions [OH-] H+ = OH-

Acidic solution : has lower concentration of hydrogen

ions [H+] than hydroxyl ions [OH-]
H+ < OH-
Measuring of hydrogen number:
Ø To measure the hydrogen number in certain solution in very accurate way, we use a special
instrument called pH meter. It’s consist of two electrodes:
1. Reference electrode: contains silver-silver chloride wire immersed in saturated KCl solution.
2. Glass electrode: which contains a very thin bulb, that is sensitive to pH.
This device measures the difference between the electrodes, and converts it into a pH from 0 to 14.

Ø Supporting materials:
1. How pH meter works: shorturl.at/fkpL3
2. How to use the pH meter: shorturl.at/lKXZ4
Measuring of hydrogen number cont.:

Test strip (inaccurate):

A pH test strip is a strip of litmus paper with which you can measure the pH value
of a liquid which show a different color at different acidities.
pH and biological system:
Ø All biochemical reactions occur under strict conditions of the concentration of
hydrogen ion.

Ø Biological life can not withstand large changes in hydrogen ion concentrations
which we measure as the pH.

Ø So how to resist changes in pH ? Buffers.

Buffers:
Ø So, buffers defines as: the solutions that have the ability to resist changes in pH
upon the addition of limited amounts of acid or base.

Ø A buffer is made up of:

a weak acid and its conjugate base.

Or
a weak base and its conjugate acid .

‫ ﻗﺎﻋدﺗﮫ اﻟﻣﻘﺗرﻧﺔ‬+ ‫اﻟﺣﻣض اﻟﺿﻌﯾف‬

HA ⇌ H+ + A-
‫ ﺣﻣﺿﮭﺎ اﻟﻣﻘﺗرن‬+ ‫اﻟﻘﺎﻋدة اﻟﺿﻌﯾﻔﺔ‬
BOH ⇌ B+ + OH-
 Two types of buffers
A buffer is made up of a weak acid and its conjugate base.
Or
A weak base and its conjugate acid.

Acidic Buffer Basic Buffer

Are made from weak acid and its Are made from weak base and its
conjugated base[ its salt]. conjugated 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-) èNH4Cl (conjugated acid –its salt-)
2. NaH2PO4 / Na2HPO4 (Pka)
 Mechanism of action:
Ø 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 react If OH- (base) is added to this buffer system à OH- will react with
with conjugated baseà to give conjugate acid. conjugated acid èto give conjugate base and H2O.

H+
OH-

A- ⇌ HA HA ⇌ A- + H2O
Mechanism of action cont.:
Ø Example:
Buffer system: CH3COOH / CH3COO- , (CH3COOH :acid - CH3COO-: conjugated base )

-When acid [H+] added: è CH3COO- + H+ è CH3COOH

-When base [OH] added: è CH3COOH + OH - è CH3COO- + H2O

Add H+

Ø NOTE: It resists pH changes when it’s two components are present in specific proportions.
Ø 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 èAs soon as you run out of one of the forms you no longer have a buffer .
Henderson-Hasselbalch equation:
Ø The Henderson-Hasselbalch equation is an equation that is often used to:
1. To prepare Buffer.
2. To calculate the pH of the Buffer.

Ø 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.
Choosing the proper buffer:
Ø 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].

è 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:

Ø Quantitative measure of buffer resistance to pH changes is called buffer capacity.

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

The number of moles of H+/OH- ions that must be added to one litre of the buffer in order to decrease /increase

the pH by one unit respectively.

Ø Buffer capacity is directly proportional to the buffer concentration.

A buffer has concentration of 0.5 M

A buffer has concentration of 0.9 M Which buffer has the highest capacity?
Practical Part
Objectives:
Ø To learn how to prepare buffers.
Ø To understand the behaviour of buffers solutions.
A. Preparation of phosphate buffer:

Prepare 50 ml from phosphate buffer with concentration 0.25M and pH=7.4, if you know that
(pKa=7.2).
You are provided with buffer solution content: Monosodium dihydrogen phosphate
NaH2PO4 and Disodium hydrogen phosphate Na2HPO4

Solution:
■ Provided:
pKa = 7.2 pH=7.4
Final volume of buffer =50 ml
Concentration of buffer = 0.25 M è [HA] + [A-]
■ Required:
50 ml
Weight (g) of NaH2PO4 (as HA) and Na2HPO4 (as A).
 Calculations:
-To prepare a buffer Henderson-Hasselbalch equation is used:
pH = pKa+log [A-]\[HA]

1. First calculate the concentration of the weak acid and its conjugated base that make up the buffer with
0.25 M:
è Assume [A-] = y and [HA] = 0.25 – y
So:
y y
7.4 = 7.2 +log 0.25-y
è 0.2 = log 0.25-y

By taking the “Anti log for both sides” :

y
1.6 = 0.25-y è y= 1.6 x (0.25 -y) è y= 0.4 – 1.6 y è y + 1.6 y = 0.4 è 2.6 y = 0.4

y= 0.15 M [which is the concentration of [A-] in the buffer ]

0.15+ 0.1 = 0.25M
So, [HA] = 0.25 – 0.15 = 0.1 M [which is the concentration of [HA] in the buffer ]
 Molecular weight
(A) Na2HPO4 = 142 g/mol
Calculations cont.: (HA) NaH2PO4 = 120 g/mol

2. Calculate the weight in (g) needed from [A-] to prepare the buffer, so number of mole of [A- ] should be calculated
first :
è Calculate moles of A- in buffer: [1]
No. of mole (of A-) = molarity (of A- calculated in the buffer) X volume L (volume of the buffer)
= 0.15 x 0.05 = 0.0075 mole
è Calculate weight of A- needed: [2]
Weight in (g) of [A-] = No. of moles x MW
= 0.0075 x 142 = 1.065 g

3.Calculate the weight in (g) needed from [HA] to prepare the buffer, so number of mole of [HA] should be calculated
first :
è Calculate moles of HA in buffer: [1]
No. of mole (of HA) = molarity (of HA calculated in the buffer) X volume L (volume of the buffer)
= 0.1 x 0.05 = 0.005 mole
è Calculate weight of HA needed: [2]
Weight in (g) of [HA] = No. of moles x MW No. of moles of solute
[1] Molarity = Volume (L)
= 0.005 x 120 = 0.6 g
Wt(g)
[2] No. of moles = Mwt
 Homework:
Ø You are provided with 0.15 M acetic acid and sodium acetate.
Prepare 100 ml of a 0.2M acetate buffer, pH =5.2 if you know that pKa =4.76.

Hint: you will calculate ml of acetic acid and g of sodium acetate.