MBARARA UNIVERSITY OF SCIENCE AND TECHNOLOGY

FACULTY OF SCIENCE
BACHELOR OF SCIENCE WITH EDUCATION (BIOLOGICAL)
DEPARTMENT OF CHEMISTRY
CHE2101: ANALYTICAL CHEMISTRY

ANALYTICAL CHEMISTRY NOTES

Compiled By;
TR. NIWAGABA ELIA
BSc. Education (Biological) MUST
Call; +256761426546
WhatsApp: +256706604296
Email: [email protected]
 INTRODUCTION
Analytical chemistry is a scientific discipline that develops and applies methods, instruments and
strategies to obtain information on the composition and nature of matter i.e what substances and
how much of them are present.
Many problems on which analytical chemists work ultimately involve either a qualitative or
quantitative measurement while others may involve characterizing a sample’s chemical or physical
properties. Therefore, many analytical chemists engage in fundamental studies of analytical
methods.
Qualitative analysis involves determining identity of the constituent species in a sample. For
example, identifying products of chemical reaction, screening an athlete’s urine for presence of
performance enhancing drug, determining spatial distribution of lead on the surface of an airborne
particulate.
Quantitative analysis involves determining how much of constituent species is present in a
sample. For example, elemental analysis of a newly synthesized compound, measuring
concentration of glucose in blood, determining the difference between the bulk and surface
concentration of chromium in steel. Much of the work in clinical, pharmaceutical, environmental
and industrial laboratories involves developing new methods for determining concentration of
targeted species in complex samples.
Characterization analysis involves evaluation of a sample’s chemical or physical properties. For
example, determination of chemical structure, equilibrium constants, particle size and surface
structure.
Fundamental analysis’s purpose is to improve an analytical method’s capabilities. For example,
extending and improving the theory on which a method is based, studying a method’s limitations
and designing new and modifying old methods.
Analyte is a constituent of interest in a sample.
Species is any chemical of interest in the analysis.
Matrix is all other constituents in a sample except for the analytes.

Analytical chemistry is vital in the following areas;

i) Quality control in the process industries (of starting materials, intermediates, products,
confirmation of purity and identification of impurities).
 ii) Environmental analysis;
- Monitoring and control of pollutants in streams that are to be released to the
environment (in gas, liquid or solid form).
- Measurement of pollutants in the environment (air, river, ground). For
example, NOx, SOx, hydrocarbons in atmosphere, organic chemicals
(polychlorinated biphenyls, detergents), toxic heavy metals.
iii) Clinical and biological studies;
- Measurement of nutrients, including trace metals.
- Measurement of naturally produced chemicals (cholesterol, sugar, urea).
- Measurement of drug levels in the body.
iv) Geological surveys;
- Measurement of metal concentrations in ores and minerals
- Measurement of oil/gas concentrations in rocks
v) Forensic studies;
Much legislation enacted by governments relating to misuse of drugs, control of
substances hazardous to health.
vi) Fundamental and applied research;
- Chemical engineering: how much conversion (or separation) do we obtain
under certain conditions.
- Organic molecule synthesis
Types of analysis
a) Gravimetric analysis – measure mass of analyte or related compound
b) Volumetric analysis – measure volume of solution containing sufficient reagent to react
completely with the analyte
c) Electroanalytical analysis – measure electrical properties such as potential, current,
resistance and quantity of electricity.
d) Spectroscopic analysis – measurement based on interaction of electromagnetic radiation
and analyte.
e) Mass spectrometry – measure of mass to charge ratio of analyte.
f) Others – e.g radioactive decay, heat of reaction, optical activity, refractive index, rate of
reaction.
ANALYTICAL PROCEDURES
These involve a sequence or series of steps but at times one or more of the steps can be omitted.

Identify and define the problem

Analytical chemistry begins with a problem, examples of which include evaluating amount of
dust and soil ingested by children as an indicator of environmental exposure to particulate based
pollutants, resolving contradictory evidence regarding the toxicity of perfluoro polymers during
combustion or developing rapid and sensitive detectors for chemical warfare agents.
The chemist finds out what information is needed. The type of information needed and the
problem’s context are essential to selecting or designing an appropriate experimental procedure.
Design the experimental procedure
It involves selecting an appropriate method of analysis based on established criteria such as
- Accuracy, precision, sensitivity and detection limit.
- The urgency with which results are needed
- Cost of a single analysis
- Number of samples to be analyzed
- Amount of sample available for analysis

Obtaining a representative sample

Chemical analysis is usually performed on only a small portion of the material to be characterized.
To produce meaningful results an analysis must be performed on a sample whose composition
reflects that of the bulk of material from which it was taken. Where the bulk is large and not
homogeneous great effort is required to produce a representative sample.
Sample preparation
- Sample storage: Samples should be stored at conditions that do not change or interrupt the
composition such as moisture, temperature.
- Grinding, extracting analyte: Often solid materials must be ground to reduce particle size and
then be thoroughly mixed to ensure homogeneity. This is particularly important with difficulty
soluble substances. A suitable solvent that removes an analyte from solution can also be used.
- Dissolving sample / dispensing analyte: If, as is frequently the case, it is necessary to bring the
sample into solution, the choice of solvent and the selection of optimum conditions for its use
become important preliminary steps to the analysis. The solvent chosen for an analysis should be
effective in dissolving the entire sample in a reasonable period of time. In addition, chemical
components of the solvent should not interfere with subsequent steps in the analysis or should be
easily removable if they do.
- Concentrating analyte: After dissolving the sample some solvent is evaporated off to obtain a
concentrated mixture.
- Eliminate interferents (substances that cause error in an analysis by enhancing or attenuating /
making smaller the quantity being measured): interferents prevent direct measurement of the
analyte and must be removed.
Analysis / conduct an experiment
As part of the validation process appropriate chemical or physical standards are used to calibrate
any equipment being used and any solutions whose concentrations must be known. The selected
samples are then analyzed and raw data recorded.
Interpretation and recording
The raw data collected during the experiment are analyzed. Frequently the data must be reduced
or transformed to a more readily analyzable form. A statistical treatment of data is used to evaluate
the accuracy and precision of the analysis and to validate the procedure. These results are compared
with the criteria established during the design of the experiment and then the design is
reconsidered, additional experimental trials are run, or a solution to the problem is proposed.
Drawing conclusions / propose a solution
When a solution is proposed the results are subject to an external evaluation that may result in a
new problem and the beginning of a new analytical cycle.

IMPORTANT CHEMICAL CONCEPTS

Concentration; is the amount of solute present in a known amount of solution.
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑋
[𝑋] =
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑟 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑌
X = solute

Y = solution / solvent

Molarity (M); is the number of moles of solute per litre of solution.

 𝑥 𝑚𝑜𝑙𝑒 𝑜𝑓 𝑋
𝑚𝑜𝑙𝑎𝑟𝑖𝑡𝑦 (𝑀) =
𝑦 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Example

Find the molarity of a 2 L solution containing 0.1 mole NaCl.

2 L of solution contain 0.1 mole NaCl

0.1
1 L of solution contains mole NaCl
2

= 0.05 M NaCl or 0.05 mol L-1 NaCl

Question: Calculate the molarity of a 2 L solution containing 1 g NaCl.

Molality (m); is the number of moles of solute per kilogram of solvent.

𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
Molality = 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

Formality(F); is the number of moles of solute per litre of solution. It is a substance’s total
concentration in solution without regard to its specific chemical form.

Note:- There is no difference between a substance’s molarity and formality if it dissolves without
dissociating into ions e.g molar concentration of glucose is the same as its formality.

- For substances that ionize in solution e.g NaCl, molarity and formality are different. E.g
dissolving 0.1 mol Na+ and 0.1 mol Cl-. The molarity of NaCl is zero since there is no
undissociated NaCl in solution. There fore the solution is 0.1 M Na+ and 0.1 M Cl-. The formality
of NaCl is 0.1F because it represents the total amount of NaCl in solution.

Normality (N); is the number of equivalents of solute per litre of solution. The number of
equivalents, n is based on a reaction unit, which is that part of a chemical species involved in a
reaction.

In precipitation reaction: A reaction unit is charge of the cation or anion involved in reaction.

Pb2+ + 2I - ↔ PbI 2 (s)

n=2 n=1
In acid-base reaction: A reaction unit is the number of H + ions denoted by an acid or accepted by
a base.

H2 SO4 (aq) + 2NH 3 (aq) → 2NH 4 +(aq) + SO 4 2-(aq)

n=2 n=1

In complexation reaction: A reaction unit is number of electron pairs that can be accepted by the
metal or donated by the ligand.

Ag+(aq) + 2NH 3 (aq) ↔ Ag(NH 3 )2+(aq)

n=2 n=1

In oxidation – reduction reaction: A reaction unit is number of electrons released by reducing agent
or accepted by oxidizing agent.

2Fe3+(aq) + Sn2+(aq) ↔ Sn4+(aq) + 2Fe2+(aq)

n=1 n=2

Normality is the number of equivalent weights (EW) per unit volume.

An equivalent weight is the ratio of chemical species formula weight (FW) to number of its
equivalents.

𝐹𝑊
𝐸𝑊 =
𝑛

Relationship between normality and molarity

𝑁=𝑛×𝑀

Example

Calculate equivalent weight and normality for a solution of 6 M H3PO4 given the following
reactions (H = 1, P = 31, O = 16).

(a) H 3 PO 4 (aq) + 3OH -(aq) ↔ PO 4 3-(aq) + 3H 2 O(l)

(b) H 3 PO 4 (aq) + 2NH 3 (aq)↔ HPO 4 2-(aq) + 2NH 4 +(aq)
(c) H 3 PO 4 (aq) + F-(aq) ↔ H 2 PO 4 -(aq) + HF(aq)
Solution

For H3PO4, number of equivalents is number of H+ donated to base i.e (a), (b), (c), number of
equivalents are 3, 2, 1 respectively.

𝐹𝑊 98
(a) 𝐸𝑊 = = = 32.665
𝑛 3

N=nxM =3 x 6 = 18 N
𝐹𝑊 98
(b) 𝐸𝑊 = = = 49
𝑛 2

N=nxM = 2x6 =12 N

𝐹𝑊 98
(c) 𝐸𝑊 = = = 98
𝑛 1

N=nxM = 1x6 = 6 N
Weight, volume and weight-to-volume ratio

Expresses concentration as units of solute per 100 units of sample.

Weight percent (%w/w)

Grams of solute per 100 g of solution.

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
× 100 %
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Volume percent (%v/v)

Volume of solute per 100 mL of solution.

𝑣𝑜𝑙𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
× 100 %
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Weight-to-volume percent (%w/v)

Grams of solute per 100 mL of solution. For example; a solution in which a solute has a
concentration of 23 % w/v contains 23 g solute per 100 mL of solution.

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
× 100 %
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Parts per million (ppm) and parts per billion (ppb)

Are mass ratios of grams of solute to one million or one billion grams of sample respectively.

𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
𝑝𝑝𝑚 = × 106
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
𝑝𝑝𝑏 = × 109
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Note: If density of aqueous solution is approximately 1.00 g mL -1 then

𝑚𝑔 𝜇𝑔
𝑝𝑝𝑚 = =
𝑙𝑖𝑡𝑟𝑒 𝑚𝐿
𝜇𝑔 𝑛𝑔
𝑝𝑝𝑏 = =
𝑙𝑖𝑡𝑟𝑒 𝑚𝐿

Example

A concentrated solution of aqueous ammonia is 28.0 %w/w NH 3 and has a density of 0.899 g mL -
1. What is the molar concentration of NH 3 in this solution?

Solution

Mass of NH 3 in 1000 mL = ρV

= 0.899 x 1000 = 899 g

28
Mass of 28 % NH 3 = × 899 = 251.72 𝑔
100

But, 17.04 g of NH 3 contain 1 mole

251 .72
251.72 g of NH3 contain mole
17.04

= 14.8 M

Question

The maximum allowed concentration of chloride in a municipal drinking water supply is 2.5 x 102
ppm Cl-. When the supply of water exceeds this limit, it often has a distinctive salty taste. What is
this concentration in moles Cl- / litre? Ans 7.04 x 10-3 M
p-Functions
When working with concentrations that span many orders of magnitude, it is often more
convenient to express the concentration as a p-function. The p-function of a number x is written
as pX and is defined as pX = -log(x)
For example;
1. The pH of a solution is 0.10 M H + is
2. pH = -log[H+]
= -log(0.10) = 1.00
3. What is pNa for a solution of 1.76 x 10-3 M Na3 PO4 ? Ans 2.277
4. What is the [H+] in a solution that has pH of 5.16? Ans 6.9 x 10-6 M
5. The amount of oxalic acid in a sample of rhubarb was determined by reacting with Fe 3+. In
a typical analysis, the oxalic acid in 10.62 g of rhubarb was extracted with a suitable
solvent. The complete oxidation of oxalic acid to carbon dioxide required 36.44 mL of
0.0130 M Fe3+. What is the weight percent of oxalic acid in the sample of rhubarb? Ans
0.201 %w/w H2 C2 O4

Preparing solutions; Preparing a solution of known concentration is the most common activity in
any analytical laboratory. The method for measuring out the solute and solvent depend on the
desired concentration units and how exact the solution’s concentration needs to be known. Pipets
and volumetric flasks are used when a solution’s concentration must be exact; graduated cylinders,
beakers and reagent bottles suffice when concentrations need only be approximate.
A. Preparing stock solutions
A stock solution is prepared by weighing out an appropriate portion of a pure solid or by measuring
out an appropriate volume of a pure liquid and diluting to a known volume. For example, to a
prepare a solution with a desired molarity you would weigh out an appropriate mass of reagent,
dissolve it in a portion of solvent and bring to the desired volume.
To prepare a solution where the solute’s concentration is given as a volume percent, you would
measure out an appropriate volume of solute and add sufficient solvent to obtain the desired total
volume.
Describe how you would prepare the following solutions
a) 500 mL of 0.20 M NaOH using solid NaOH.
1 L of solution contains 0.2 mole
0.5 L of solution contains (0.5 x 0.2) mole
RFM of NaOH = 40
1 mole of NaOH weighs 40 g
0.5 x 0.2 mole NaOH weighs 0.5 x 0.2 x 40 g
=4g
 Weigh 4 g of NaOH into a beaker. Add little water to make a solution. Transfer this to 500
mL volumetric flask and make up to the mark with water.

b) 1 L of 150.0 ppm Cu2+ using copper metal.

150 ppm = 150 mg L-1 or 0.15 g L-1
Weigh 0.15 g Cu into a small beaker add small portion of conc. HNO 3 and heat gently until
it completely dissolves. The resulting solution is poured into a 1 L volumetric flask. The
beaker is rinsed repeatedly with small portions of water which is added to volumetric flask.
This process i.e quantitative transfer ensures that Cu2+ is completely transferred to the
volumetric flask. Finally add water to makeup.

c) 2 L of 4% v/v acetic acid using concentrated acetic acid.

4
× 2000 = 80𝑚𝐿
100
Transfer 80 mL of CH 3 COOH into 2 L volumetric flask.

B. Preparing solutions by dilution

Solutions with small concentrations are often prepared by diluting a more concentrated stock
solution. A known volume of the stock solution is transferred to a new container and brought to a
new volume. Since the total amount of solute is the same before and after dilution,
Co V o = Cd V d
Co = concentration of stock solution
V o = volume of stock solution being diluted
Cd = concentration of dilute solution
V d = volume of dilute solution
Example
1. A laboratory procedure calls for 250 mL of 0.10 M solution of NH 3 . Describe how you
would prepare this solution using a stock solution of concentrated NH 3 (14.8M).
𝐶𝑜𝑉𝑜 = 𝐶𝑑𝑉𝑑
14.8𝑉𝑜 = 0.10 × 0.25𝐿
𝑉𝑜 = 1.69 × 10−3 𝐿
Measure 1.7 mL of conc. NH3 using a graduated cylinder transfer this to 250 mL
volumetric flask, add sufficient water to bring the total solution volume to 250 mL.

2. Describe the preparation of 100 mL of 6.0 M HCl from a concentrated solution that has a
specific gravity of 1.18 and is 37 % w/w HCl (36.5 g mol-1 ).
Mass of HCl in 1000 cm3 = ρV
= 1.18 x 1000 = 1180 g
37
Mass of 37 % conc HCl = 100 × 1180 = 436.6𝑔
 36.6 g HCl contain 1 mole
436.6
436.6 g HCl contain 36.5 𝑚𝑜𝑙𝑒
= 11.96 M ~ 12 M
𝐶𝑜𝑉𝑜 = 𝐶𝑑𝑉𝑑
12𝑉𝑜 = 6 × 100
𝑉𝑜 = 50𝑚𝐿
Thus dilute 50 mL of conc HCl to 100 mL.

Note: Specific gravity is ratio of density of a substance to density of reference substance.

It is ratio of mass of substance to mass of reference substance for the same given volume.

Specific gravity = density of substance

density of water
Reference substance for liquids is water.

BASIC APPROACH TO CHEMICAL EQUILIBRIUM

A system is at equilibrium when the concentration of products and reactants remain constant.
Although a system at equilibrium appears static on a macroscopic level, it is important to remember
that the forward and reverse reactions still occur. A reaction at equilibrium exists in a steady state
in which the rate at which any species forms equals the rate at which it is consumed.

Consider the general equilibrium reaction involving solutes A, B, C, D with stoichiometric

coefficients a, b, c, d
aA + bB cC + dD

[𝐶 ]𝑐[𝐷 ]𝑑
𝐾=
[𝐴]𝑎[𝐵]𝑏

K is equilibrium constant
N.B
i- The value of K is determined by the concentrations of solutes at equilibrium.
ii- The square brackets refer to equilibrium molar concentration of solute.
iii- Partial pressure (in atmospheres) for a gas can also be used.
iv- Concentration is assumed to be one if the species is pure liquid in excess, pure solid, solvent in
dilute solution or the solvent water.

Useful relationships when working with equilibrium constants

i- If we reverse a reaction’s direction, the equilibrium constant for the new reaction is
the inverse of that for the original reaction.
A + 2B AB2
[𝐴𝐵 ]
2
K1 = [𝐴][𝐵] 2

If the reaction is reversed

AB2 A + 2B

1 [𝐴][𝐵]2
K2 = =
𝐾1 [𝐴𝐵2 ]

ii- If you add together two reactions to obtain a new reaction, the equilibrium constant
for the new reaction is the product of the equilibrium constants for the original
reactions.

[𝐴𝐶 ]
A + C AC K 1 =[𝐴][𝐶 ]

[𝐴𝐶2 ]
AC + C AC2 K2 = [
𝐴𝐶 ][𝐶]

[𝐴𝐶 ]
A + 2C AC2 K 3 = 𝐾1 𝐾2 = [𝐴][𝐶2]2

Example
Calculate the equilibrium constant for the reaction
2A + B C + 3D given the following information
a) A + B D K1 = 0.40
b) A + E C + D + F K2 = 0.10
c) C + E B K3 = 2.0
d) F + C D + B K 4 = 5.0
Solution
A + B D
A + E C +D + F

2A + B + E C + 2D + F K1K2
Reverse eqn c
1
B C + E 𝐾3
1
2A + 2B 2C + 2D + F 𝐾1 𝐾2 𝐾
3
 F + C D + B
1
2A + B C + 3D 𝐾1 𝐾2 𝐾 𝐾4
3

𝐾𝑜𝑣𝑒𝑟𝑎𝑙𝑙 =0.4×0.1×1/2×5

= 0.1
Equilibrium constants for chemical reactions
Several types of reactions are commonly used in analytical procedures either in preparing samples
for analysis or during the analysis itself.
A. Precipitation reactions
A precipitation reaction occurs when two or more soluble species combine to form an insoluble
product i.e precipitate
e.g Pb2+(aq) + 2Cl-(aq) PbCl2 (s)
In equilibrium treatment of precipitation however, the reverse reaction describing dissolution of
the precipitate is more frequently encountered,
PbCl2 (s) Pb2+(aq) + 2Cl-(aq)
The equilibrium constant for this reaction is called solubility product, Ksp
Ksp = [Pb2+][Cl-]2
B. Acid base reactions
Arrhenius concept: An acid is a substance that produces H+ in aqueous solution.
A base is a substance that produces hydroxide ion in aqueous solution
Bronsted-Lowry concept: An acid is a substance that donates a proton in chemical reaction.
A base is a substance that accepts proton in chemical reaction.
Lewis concept: An acid is a substance that accepts an electron pair in a chemical reaction.
A base is a substance that donates an electron pair in a chemical reaction
Strong and weak acids
The reaction of an acid with its solvent (typically water) is called an acid dissociation reaction.
Acids are divided into 2 categories based on the ease with which they can donate protons to the
solvent.
Strong acid – completely transfer their protons to solvent molecules
HCl + H2O → H 3 O+ + Cl-
Examples of strong acids: HI, HBr, HNO 3 , HClO 4 , H2 SO 4
Weak acid – can not completely donate their acidic protons to the solvent.
 CH 3 COOH + H2O H3 O+ + CH3 COO -
The equilibrium constant for this reaction is acid dissociation constant
[𝐻3 𝑂 +][𝐶 𝐻3 𝐶𝑂𝑂 −]
Ka = [𝐶𝐻3 𝐶𝑂𝑂𝐻]

The magnitude of Ka provides information about relative strength of a weak acid i.e the smaller
the Ka, the weaker the acid.
Strong and weak bases
Strong base – completely dissociates to produce OH - ions
Weak base – partially accept protons from solvent and are characterized by a base dissociation
constant, K b
CH3 COO - + H2O OH- + CH 3 COOH
[𝐶𝐻3 𝐶𝑂𝑂𝐻] [𝑂𝐻 ] −
Kb = [𝐶𝐻3 𝐶𝑂𝑂 − ]

Amphiprotic species are species capable of acting as both acids and base
e.g HCO 3 -(aq) + H2O H3 O+(aq) + CO3 2-
HCO 3 -(aq) + H2O OH-(aq) + H2 CO 3 (aq)
Whether an amphiprotic species behaves as an acid or as a base depends on the equilibrium
constants for the two competing reactions
Dissociation of water
Water is an amphiprotic solvent in that it can serve as an acid or base
H 2 O(l) + H2 O(l) H 3 O+(aq) + OH -(aq)
Equilibrium constant for this reaction is called dissociation constant of water, Kw or ionic product
constant of water.
Kw = [H 3 O+][OH -]
Kw = 1 x 10-14 at 25o C. The value of Kw varies with temperature.
Example
What is the [OH -] if the [H 3 O+] is 6.12 x 10-5 M?
𝐾𝑤
[𝑂𝐻 − ] =
[𝐻3 𝑂 + ]

1×10 −14
= 6.12×10 −5

= 1.63 × 10−10 M
pH scale
Indicates the degree of acidity of a solution. When [H 3 O+] = [OH -], a solution is neither acidic nor
basic i.e it is neutral.
Let [H 3 O+] = [OH -]
Then Kw = [H 3 O+]2 = 1 x 10-14
[H3 O+] = (1x10-14 )1/2
= 1 x 10-7
pH = -log[H 3 O+]
= -log (1 x 10-7 ) = 7
For a solution to be acidic, the [H 3 O+] must be greater than that for OH- or [H 3 O +] > 1 x 10-7 M
and pH must be less than 7.
Useful relationship
Kw = [H 3 O+][OH -]
-log Kw = -log [H 3 O+][OH -]
pKw = pH + pOH = 14
Relationship between Ka and Kb
A useful observation about acids and bases is that the strength of a base is inversely proportional
to strength of its conjugate acid.
CH3 COOH + H2O H3O+ + CH3 COO - Ka
CH3 COO - + H2O OH - + CH3 COOH Kb
Adding the two reactions
2H2 O H3O+ + OH - Kw = KaKb
Thus for a weak acid HA and its conjugate base A -, Kw = KaKb
Example
1. Given that the Ka for chloroacetic acid is 1.356 x 10 -3 . Calculate the [H 3 O+] for a 0.02 M
solution.

ClCH 2 COOH + H 2 O ClCH 2 COO - + H3 O+

[𝐶𝑙𝐶𝐻2 𝐶𝑂𝑂 −] +
[𝐻3 𝑂 ]
𝐾𝑎 = [𝐶𝑙𝐶𝐻2 𝐶𝑂𝑂𝐻]
Assume [ClCH 2 COO -] = [H 3 O+]
[𝐻3 𝑂+ ]2
1.356 × 10−3 =
0.02
 [𝐻3 𝑂+ ] = 0.0052 𝑀

2. At 25 o C, what is the hydronium ion concentration in 0.01 M methyl amine? (Kb = 4.8 x
10-4 ).
CH3 NH2 + H2 O CH3 NH 3 + + OH -
[𝐶 𝐻3 𝑁𝐻3+][𝑂𝐻 − ]
𝐾𝑏 =
[𝐶𝐻3 𝑁𝐻2 ]
[𝑂𝐻 −]2
4.8 × 10−4 =
0.01
[𝑂𝐻 − ] = 0.00219 𝑀

But, 𝐾𝑤 = [𝐻3 𝑂+ ][𝑂𝐻− ]

−14
[𝐻3 𝑂+ ] = 1×10
0.00219
= 4.56 × 10−12 𝑀

c. Complexation reactions
The Lewis theory of acids and bases is more useful for treating complexation reactions between
metal ions and ligands.
Cd 2+(aq) + 4NH 3 (aq) Cd(NH 3 )4 2+(aq)
The product of this reaction is a metal-ligand complex. The formation of a metal-ligand complex
is described by a formation constant, K f.
[𝐶𝑑(𝑁𝐻3 ) 2+
4 ]
𝐾𝑓 = [𝐶𝑑2+][𝑁𝐻3 ]4
…………………………………. (x)

The reverse of the above reaction is a dissociation reaction and is characterized by dissociation
constant, K d .
Many complexation reactions occur in a stepwise fashion. For example, reaction between Cd 2+
and NH 3 involves four successive reactions.
Cd 2+(aq) + NH 3 (aq) Cd(NH 3 )2+(aq) K1
Cd(NH 3 )2+(aq) + NH 3 (aq) Cd(NH 3 )2 2+(aq) K2
Cd(NH 3 )2 2+(aq) + NH 3 (aq) Cd(NH 3 )3 2+(aq) K3
Cd(NH 3 )3 2+(aq) + NH 3 (aq) Cd(NH 3 )4 2+(aq) K4
This creates a problem since it is no longer clear what reaction is described by a formation
constant. To avoid ambiguity formation constants are divided into two categories
Stepwise formation constants: which are designated as K i for the ith step, describe the successive
addition of a ligand to the metal-ligand complex formed in the previous step. Thus, the
equilibrium constants for the reactions K 1 , K2 , K 3 , K4 .
Overall or cumulative formation constants: which are designated as βi describe addition of i
ligands to the free metal ion. The equilibrium constant expression given in (x) is identified as β 4
= K1 x K2 x K3 x K4
In general, βi = K1 x K2 x ………. x K i
d. Oxidation – reduction reactions
In an oxidation – reduction reaction (redox), electrons are not shared but are transferred from one
reactant to another. As a result of electron transfer some of the elements involved in the reaction
undergo a change in oxidation state.
2Fe3+(aq) + C2 O4 2-(aq) 2Fe2+(aq) + 2CO 2 (g)
The species being oxidized is a reducing agent because it provides electrons for the reduction
half equation while the species being reduced is an oxidizing agent. The products of a redox
reaction also have redox properties. For example, Fe 2+ can be oxidized to Fe3+ while CO 2 can be
reduced to H 2 C2 O 4 . Borrowing some terminology from acid-base reactions, Fe2+ would be
conjugate reducing agent of the oxidizing agent Fe3+ and CO 2 the conjugate oxidizing agent of
the reducing agent.
Unlike the other reactions (above), equilibrium position of a redox reaction is rarely expressed
by an equilibrium constant. Since redox reactions involve transfer of electrons from reducing
agent to oxidizing agent, it is convenient to consider thermodynamics of the reaction in terms of
the electron.

Le Chatelier’s Principle
State that the position of equilibrium will always shift in such a direction as to relieve any stress
applied to the system. An example of stress that can be applied to a reaction at equilibrium is
change in concentration of reactant or product.
Question: what is the effect on solubility of AgCl if NH 3 (aq) is added to the equilibrium
solution?
AgCl (s) Ag+(aq) + Cl-(aq)
Adding NH 3 (aq) decreases the concentration of Ag+ as Ag(NH 3 )2 + complex forms.
Ag+(aq) + 2NH 3 (aq) Ag(NH 3 )2 +(aq)
As a result of decreasing concentration of Ag +, solubility of AgCl increases so as to maintain
equilibrium constant.
Solving Equilibrium Problems
1. What is [Pb2+], [IO 3 -] and [Pb(IO 3 )2 ] in a saturated solution prepared by adding Pb(IO 3 )2
to distilled water? (K sp = 2.5 x 10-13 ).
Pb(IO 3 )2 (s) Pb2+ (aq) + IO 3 - (aq)
𝐾𝑠𝑝 = [𝑃𝑏2+ ][𝐼𝑂3− ]2

Let [Pb2+] = x
(𝑥)(2𝑥)2 = 2.5 × 10−13
2.5×10 −13
𝑥= ∛ 4

= 3.97 x 10-5
The equilibrium concentrations of Pb2+ and IO 3 - are
[𝑃𝑏2+ ] = 𝑥 = 3.97 × 10−5 𝑀

[𝐼𝑂3− ] = 2𝑥 = 2(3.97 × 10−5 = 7.9 × 10−5 𝑀

Since one mole Pb(IO 3 )2 contains one mole Pb2+, solubility of Pb(IO 3 )2 is the same as
concentration of Pb2+. Thus, solubility of Pb(IO 3 )2 is 3.97 × 10−5 𝑀.
2. How is the solubility of Pb(IO 3 )2 affected if Pb(IO 3 )2 is added to a solution of 0.10 M
Pb(NO 3 )2 ?

3. pH of a monoprotic weak acid

Two equilibria affect pH of this system
i. Acid dissociation reaction
HA(aq) + H 2 O(l) H3 O +(aq) + A -(aq)
[𝐻3 𝑂 +][𝐴 −]
𝐾𝑎 = [𝐻𝐴]
…………………………….. equation 1

ii. Dissociation of water

2H2 O(l) >> H 3 O+(aq) + OH-(aq)
𝐾𝑤 = [𝐻3 𝑂+ ][𝑂𝐻− ] ……………………….. equation 2
Consider a weak acid HA of concentration C HA
Mass balance equation 𝐶𝐻𝐴 = [𝐻𝐴] + [𝐴− ]…………………….. equation 3
Charge balance equation [𝐻3 𝑂+ ] = [𝐴− ] + [𝑂𝐻 − ]……………… equation 4

Since HA isa weak acid, we expect the solution to be acidic

Therefore, [𝐻3 𝑂+ ] ≫ [𝑂𝐻 − ]
Simplifying eqn 4 [𝐻3 𝑂+ ] = [𝐴− ]……………………. Equation 5
Since HA is weak acid, little dissociation occurs, so [𝐻𝐴] ≫ [𝐴− ]
Therefore, 𝐶𝐻𝐴 = [𝐻𝐴]………………………………. Eqn 6

Substitute in equation 1
[𝐻3 𝑂+ ][𝐻3 𝑂+ ]
𝐾𝑎 =
𝐶𝐻𝐴
[𝐻3 𝑂+ ] = √𝐾𝑎 𝐶𝐻𝐴

Example
Calculate pH of 1.0 M HF given that 𝐾𝑎 = 6.8 × 10−4

Solution
[𝐻3 𝑂+ ] = √𝐾𝑎 𝐶𝐻𝐴

= √(6.8 × 10−4 )(1.0)

= 2.6 × 10−2 𝑀
𝑝𝐻 = −𝑙𝑜𝑔[𝐻3 𝑂+ ]
= − log (2.6 × 10−2 ) = 1.59

How does the result of this calculation change if we require assumptions to have an error of less
than ± 1%?
Consider a weak acid HA of concentration C HA.
Assume [𝐻3 𝑂+ ] = [𝐴− ] the remaining undissociated [HA] = C - [A-] = C - [𝐻3 𝑂+ ]
[𝐻3 𝑂+ ][𝐻3 𝑂+ ]
𝐾𝑎 =
𝐶𝐻𝐴 − [𝐻3 𝑂+ ]
[𝐻3 𝑂+ ]2 = 𝐾𝑎 𝐶𝐻𝐴 − 𝐾𝑎 [𝐻3 𝑂+ ]
[𝐻3 𝑂+ ]2 + 𝐾𝑎 [𝐻3 𝑂+ ] − 𝐾𝑎 𝐶𝐻𝐴 = 0 This is a quadratic equation
which can be solved using the quadratic formula.
𝑎𝑥 2 + 𝑏𝑥 + 𝑐 = 0
−𝑏± √𝑏 2 −4𝑎𝑐
𝑥= 2𝑎
Calculate the pH of 1.0 M HF given that Ka = 6.8 × 10−4

Solution
−6.8×10 −4 ±√(6.8×10 −4 ) 2 −(4)(1)(−6.8×10 −4 )(1.0)
𝑥= 2(1)

𝑥 = 2.57 × 10−2 𝑜𝑟 𝑥 = −2.63 × 10−2 Only the positive root has chemical significance
since negative root implies that [𝐻3 𝑂+ ] is negative.
𝑝𝐻 = −log (2.57 × 10−2 )

= 1.59
NB: This same approach can be extended to find pH of a monoprotic weak base replacing Ka
with K b , CHA with weak base’s concentration and solving for [OH -] in place of [𝐻3 𝑂+ ].

Example
Calculate pH of 0.050 M NH 3 given K b of NH 3 = 1.75 × 10−5 .
Solution
[𝑂𝐻 − ] ≫ [𝐻3 𝑂+ ] and 𝐶𝑁𝐻3 = 0.050 M

[𝑂𝐻 − ] = √𝐾𝑏 𝐶𝑁𝐻3

=√(1.75 × 10−5 )(0.050)

=9.35 × 10−4 𝑀
𝐾𝑤
[𝐻3 𝑂+ ] =
[𝑂𝐻 − ]

1×10 −14
=9.35×10 −4 = 1.07 × 10−11

pH= − log(1.07 × 10−11 ) = 10.97

BUFFER SOLUTIONS
Buffer solution is a solution containing a conjugate weak acid/ weak base pair that is resistant to a
change in pH when a strong acid or strong base is added. Buffers are of great importance in
chemistry.
-Buffer solutions are necessary to keep the correct pH for enzymes in many organisms to work.
- A buffer of H 2 CO3 and HCO 3- is present in blood plasma to maintain pH between 7.35 and 7.45.
- Industrially buffer solutions are used in fermentation process and in setting the correct conditions
for dyes used in colouring fabrics.
-Used in chemical analysis and calibration of pH meters.
- Majority of biological samples that are used in research are made in buffers especially phosphate
buffered saline (PBS) at pH 7.4.
The equilibrium position of the buffer is governed by the reaction;
HA(aq) + H2 O (l) H3 O +(aq) + A-(aq)
Ka = [H3 O+][A-]
[HA]

Ka[HA] = [H 3 O+]
[A-]
Taking –log on both sides
-log Ka[HA] = -log[H 3 O+]
[A-]
-logKa – log[HA] = -log[H 3 O+]
[A-]
pKa + log[A -] = pH the Henderson – hassel balch equation
[HA]
Buffering occurs because of the logarithmic relationship between pH and ratio of weak base and
weak acid concentrations e.g if equilibrium [HA] and [A -] are equal, pH of the buffer is equal to
pKa.

Questions
1. A buffer is prepared by dissolving 10.00 g of acetic acid and 20.00 g sodium acetate in 300
mL of total solution. Ka = 1.75 x 10-5
a) What is the pH of the solution? Ans 4.92
b) If 0.200 g of solid NaOH is added to the buffer above, what will be the new pH? Ans
4.92

2. A solution was made by dissolving 7.2 g ethanoic acid and 12.0 g sodium acetate to make
one litre of solution. To the solution was added 0.8 cm3 of 1 M HCl. Calculate the pH of
solution. Given Ka of ethanoic acid = 1.75 x 10 -5 . Ans 4.84

3. a) i) Define the term buffer solution and give examples.

ii) Explain how buffer solutions are prepared.
iii) Explain the effect of (a) dilution (b) addition of small amount of acid or base to a buffer solution
of sodium ethanoate and ethanoic acid.
b) Derive the Henderson-Hassel balch equation for calculating pH of a buffer solution.
4. a) i) Define the term buffer capacity.
ii) Of what importance are buffer solutions?
b) What weight of sodium methanoate must be added to 400 mL of 1.0 M methanoic acid
to produce a buffer solution that has a pH of 3.50. (Kam ethanoic acid = 1.8 x 10 -4)
Ans 15.5 g

ELEMENTARY STATISTICS
After collecting preliminary data in an analysis, it is then characterized by providing a measure of
the spread of the individual measurements around a central value.
A. Measures of Central Tendency
One way to characterize data is to assume that the value of individual measurements are scattered
around a central value that provide the best estimate.
(i) Mean, X
Is the numerical average obtained by dividing the sum of individual measurements by the number
of measurements.
X = ∑i=1 n Xi
n
where Xi = ith measurements
n = number of independent measurements

Question: The masses of 7 united states pennies in circulation are 3.08, 3.094, 3.107, 3.056, 3.112,
3.174, 3.198. What is their mean?

(ii) Median
Is the middle result when the data are arranged by size. When the data include an odd number of
measurements, the median is the middle value. For an even number of measurements the median
is the average of the middle two values.
N.B: the mean and median provide similar estimates of central tendency when all data are similar
in magnitude.

B. Measures of Spread
Provide an estimate of the variability in the individual results. Although spread is often defined
relative to a specific measure of central tendency, its magnitude is independent of the central value.
(i) Range
Is the difference between the largest and smallest values in the data set. It provides information
about the total variability in the data set but does not provide information about the distribution of
individual measurements.
 (ii) Standard deviation
Describes the spread of individual measurements about the mean.
Σ𝑛 − 2
𝑖=1 (Χ𝑖 −Χ )
S.D = √ 𝑛−1

Xi 𝑋𝑖 − 𝑋 − (𝑋𝑖 − 𝑋 − )2
3.080 3.08 – 3.117 0.00137
3.094 3.094 – 3.117 0.00053
3.107 3.107 – 3.117 0.00010
3.056 3.056 – 3.117 0.00372
3.112 3.112 – 3.117 0.00003
3.174 3.174 – 3.117 0.00325
3.198 3.198 – 3.117 0.00656
Σ(Χ𝑖 − Χ − )2 = 0.01556

0.01556
S=√ = 0.051
7−1

Relative standard deviation is reported as

𝑆
𝑆= 𝑋−

Percent relative standard deviation

𝑆
× 100%
𝑋−

(iii) Variance
Is the square of standard deviation.

Characterizing Experimental Errors

Errors associated with central tendency reflect accuracy of the analysis but precision of the analysis
is determined by those errors associated with spread.

Accuracy
Is a measure of how closely the result of an experiment agrees with the expected result. It is
expressed in terms of error i.e absolute or relative error.
- Absolute error, E = Xi – Xt (where Xi is measured value and Xt is true value). The
sign of absolute error tells whether the value in question is low or high. If the
 measurement result is low, the sign is negative and if the measurement result is
high, the sign is positive.
- Relative error
Er = Xi – Xt x 100%
Xt
It is a more useful quantity than absolute error.
Errors affecting accuracy of analysis are called determinate and are characterized by a systematic
deviation from the true value I.e all the individual measurements are either too large or too small.
Determinate (systematic) error causes a measurement or result to be too high or too low; can be
traced to an identifiable source.
Determinate errors are divided into;
- sampling errors
- method errors
- measurement errors
- personal errors
Sampling error
It is introduced during the process of collecting a sample for analysis when a sampling
strategy fails to provide a representative sample. This is especially important when sampling
heterogeneous (not uniform in composition) materials e.g determining the environmental quality
of a lake by sampling a single location near a point source of pollution such as an outlet for
industrial effluent gives misleading results. Determinate errors associated with selecting a sample
can be minimized with a proper sampling strategy.
Method error
Is an error due to limitations in the analytical method used to analyse a sample. They are introduced
when assumptions about the relationship between the signal and the analyte are invalid. Method
errors involving sensitivity are minimized by standardizing the method, whereas method errors
due to interferents present in reagents are minimized by using a proper reagent blank.
N.B: method errors due to interferents in the sample cannot be minimized by a reagent blank
instead such interferents must be separated from the analyte or their concentrations determined
independently.
Measurement error
It is due to limitations in the equipment and instrument used. Analytical instruments and equipment
such as glass ware and balances are usually supplied by the manufacturer with a statement of the
item’s maximum measurement error.e.g 25mL volumetric flask might have a maximum error of
+- 0.03 mL meaning that the actual volume contained by the flask lies within the range of 24.97 –
25.03 mL. so the flask’s true volume is a fixed value within the stated range. Determinate
measurement errors can be minimized by calibration.
N.B: instruments should be frequently recalibrated as they drift out of calibration overtime.
Personal error
Analytical work is always subject to a variety of personal errors which can include the ability to
see a change in the colour of an indicator used to signal the end point of a titration; biases such as
consistently overestimating or underestimating the value of an instrument’s read out scale; failing
to calibrate glass ware and instrumentation and misinterpreting procedural directions. Personal
errors can be minimized with proper care.

Precision
When a sample is analysed several times, the individual results are rarely the same instead the
results are randomly scattered. Precision is a measure of this variability. The closer the agreement
between individual analyses, the more precise the results.
Definition: Precision is the agreement between numerical values of two or more measurements
that have been made in an identical fashion.
Precision is commonly divided into two categories that is repeatability and reproducibility.
- Repeatability is precision obtained when all measurements are made by the same
analyst during a single period of laboratory work using the same solutions and
equipment.
- Reproducibility is the precision obtained under any other set of conditions, including
that between analysts or between laboratory sessions for a single analyst.
Errors affecting precision are called indeterminate and are characterized by a random variation in
both magnitude and direction. Indeterminate or random errors cause some measurement or result
to be too high while others are too low. Indeterminate errors can be traced to several sources
including the collection of samples, manipulation of samples during the analysis and the making
of measurements.
•
When collecting a sample for instance, only a small portion of the available
material is taken, increasing the likelihood that small scale homogeneities in the
sample will affect repeatability of the analysis.
• During analysis numerous opportunities arise for random variations in the way
individual samples are treated.
• Any measuring device is subject to an indeterminate error in reading its scale
with the last digit always being an estimate subject to random fluctuations or
background noise.
Error and uncertainty
Error is the difference between a single measurement or result and its true value.
OR: it is a measure of bias in a result.
Error can be divided into determinate and indeterminate. Although determinate error can be
corrected, the indeterminate portion of error remains.
Uncertainty expresses the range of possible values that a measurement or result might be expected
to have.
Note that uncertainty and precision are different. The precision of an analysis, whether reported as
arrange or standard deviation, is calculated from experimental data and provides an estimation of
indeterminate error affecting measurements. Uncertainty accounts for all errors both determinate
and indeterminate that may affect a result.
TITRATIONS
Titration is a process of determining the amount of analyte by measurement of the quantity of
reagent required to react completely with the analyte. Ordinarily a titration is accomplished by the
controlled addition of a reagent of known concentration to a solution of the analyte until reaction
between the two is judged to be complete; the volume of reagent is then measured. Occasionally
it is convenient or necessary to add an excess of the reagent and then determine the excess by back
titration with a second reagent of known concentration.
Titrimetric methods are classified into 4 groups based on the type of reaction involved.
- Acid-base titrations in which an acidic or basic titrant reacts with an analyte that is a
base or acid.
- Complexometric titrations involve a metal-ligand complexation reaction.
- Redox titrations where the titrant is an oxidizing agent or reducing agent or reducing
agent.
- Precipitation titrations in which the analyte and titrant react to form a precipitate.
Titration principles
In a titration, the analyte reacts with a reagent added as asolution of known concentration. This is
referred to as a standard solution and it is generally added from a burette. The added solution is
called a titrant and the solution of unknown concentration is the titrand. The volume of titrant
required to just completely react with the titrand is measured. Since the concentration is known
and the reaction between the analyte and reagent is known, the amount of analyte can be calculated.
Requirements of a titration
I-The reaction must be stoichiometric that is bear a simple relationship.
II-The reaction should be rapid.
III- There should be no side reactions and the reaction should be specific.
IV-There should be a marked change in some property of the solution when the reaction is
complete.
V-The end point should coincide with the equivalence point or be at a reproducible interval from
it. Equivalence point is the point in a titration when the amount of added standard reagent is
exactly equivalent to amount of analyte. End point is the point in a titration when a physical
change occurs that is associated with the condition of chemical equivalence (i.e when the reaction
is observed to be complete).
VI-The reaction should be quantitative. That is, the equilibrium of the reaction should be far to the
right so that a sufficient sharp change will occur at end point to obtain a desired accuracy. If
equilibrium does not lie far to the right then there will be a gradual change in the property marking
the endpoint and this will be difficult to correct.
Primary standard
Is a substance that is analytically pure and when dissolved in a known volume of solvent, a solution
of known concentration can be prepared.
Requirements of primary standard
I-It must be of highest purity and established methods should be available for confirming its purity.
II-It should be stable. It should not be attacked by constituents of the atmosphere.
III-Should not be hygroscopic nor efflorescent otherwise drying and weighing would be difficult.
IV-Should be readily available and not expensive.
V-Have a large molar mass so that the relative error associated with weighing the standard is
minimized.
VI-Have reasonable solubility in the titration medium.
Very few compounds meet or even approach these criteria and only a limited number of primary
standard substances are available commercially.
Question: Give examples of primary standards
Standardizing reagents: In a standardization, the concentration of a volumetric solution is
determined by titrating it against a carefully measured quantity of a primary or secondary standard
or an exactly known volume of another standard solution. A secondary standard is a compound
whose purity has been established by chemical analysis and serves as the reference material for
titrimetric method of analysis.
To be suitable for titrimetric analysis, a chemical reaction should meet certain requirements;
I-All reactions involving titrant and analyte must be of known stoichiometry.
II-The reaction must occur rapidly. If titrant is added at a faster rate than the reaction’s rate, then
endpoint will exceed equivalence point by a significant amount.
III-A suitable method must be available for determining the end point with an acceptable level of
accuracy.
These are significant limitations and for this reason several titration strategies are commonly used.
a) Direct titration – titrant reacts with analyte
b) Back titration
If the titration is too slow, a suitable indicator is not available or there is no useful direct titration
reaction then an indirect analysis may be possible. This is back titration i.e titration in which a
reagent is added to a solution containing the analyte and the excess reagent remaining after its
reaction with the analyte is determined by a titration.
c) Displacement titration
In which the analyte displaces a species, usually from a complex and the amount of the displaced
species is determined by a titration.
d) When a suitable reaction involving the analyte does not exist, it may be converted to a
species that is easily titrated hence giving an indirect determination of analyte.
Volumetric calculations
1. Describe preparation of 2.0 L of 0.05 M AgNO 3 from the primary standard grade solid.
(molar mass of AgNO 3 = 169.87 g mol-1 ) Ans: 16.98g

2. A standard 0.01 M solution of Na+ is required to calibrate a flame photometric method to

determine the element. Describe how 500 mL of this solution can be prepared from primary
standard Na2 CO3 (105.99 g mol-1 ). Ans: 0.265g

3. A 50.0 mL portion of HCl solution required 29.71 mL of 0.01963 M Ba(OH) 2 to reach an

end point with bromocresol green indicator. Calculate the molarity of HCl. Ans: 0.0233M

4. Titration of 0.2121 g of pure Na2 C2 O4 (134.0 g mol-1 ) required 43.31 mL of KMnO 4 . What
is the molarity of KMnO 4 ? Ans: 0.0146 M

5. Titration of the I 2 produced from 0.1045 g of primary standard KIO 3 required 30.72 mL of
Na2 S2 O3 . Calculate the concentration of Na2 S2 O3 . Ans: 0.095 M

6. A 0.8040 g sample of an iron ore is dissolved in acid. The iron is then reduced to Fe 2+ and
titrated with 47.22 mL of 0.02242 M KMnO 4 solution. Calculate the results of this analysis
in terms of % Fe. (Fe = 55.847) Ans: 36.77%

7. A solution of HClO 4 was standardized by dissolving 0.4125 g of primary standard grade

HgO in a solution of KBr
HgO(s) + 4Br- + H2 O → HgBr4 2- + 2OH -
The liberated OH - consumed 46.51 mL of the acid. Calculate the molarity of HClO 4 . (Hg =
200.59). Ans: 8.19 x 10-2 M
NEUTRALISATION TITRATIONS
End points in neutralization titrations are ordinarily based upon the abrupt change in pH that occurs
in the vicinity of the equivalence point. The pH range over which such a change is observed
depends upon the nature and concentration for the substance titrated as well as the titrant. Proper
selection of an indicator for any given case requires knowledge of the general features of the
titration curve for the system.

Titration curve: Is a graph showing the progress of a titration as a function of the volume of titrant
added. It provides a visual picture of how a property e gph changes as we add titrant. The types of
titration curves include;

i. Sigmoid curve
Is a “Z” or “S” shaped curve where the y-axis is a p-function of the analyte (or reagent reacted
with analyte during titration) or the potential of an ion specific electrode.
- Equivalence point is observed in the middle of the middle segment of “Z” or “S”.
- A large number of measurements are made near and surrounding equivalence point.
ii. Linear segment curve
Generally, consists of two-line segments that intersect at an angle.
- Measurements are made well away from equivalence point (where the reaction is nearly
complete) and lines are extrapolated to intersection.
- Equivalence point is associated with intersection of the line segments.
Acid-base titration curves

The experimentally determined endpoint should coincide with the titration’s equivalence point.
For an acid-base titration, the equivalence point is characterized by a pH level that is a function of
the acid-base strengths and concentrations of the analyte and titrant. The pH at endpoint, however,
may or may not correspond to the pH at the equivalence point. To understand the relationship
between endpoints and equivalence points we must know how the pH changes during a titration.

Titrating strong acids and strong bases

The net reaction of strong acids with strong bases is the reaction of H + with OH - to form water.
Acid-base titration curves

The experimentally determined endpoint should coincide with the titration’s equivalence point.
For an acid-base titration, the equivalence point is characterized by a pH level that is a function of
the acid-base strengths and concentrations of the analyte and titrant. The pH at endpoint, however,
may or may not correspond to the pH at the equivalence point. To understand the relationship
between endpoints and equivalence points we must know how the pH changes during a titration.

Titrating strong acids and strong bases

The net reaction of strong acids with strong bases is the reaction of H + with OH - to form water.

H3 O + + OH - → 2H2 O

Titration of 50 mL of 0.1 M HCl with 0.2 M NaOH

a) Calculate the volume of NaOH needed to reach equivalence point

Before equivalence point, HCl is present in excess and pH is determined by concentration of excess
HCl.

b) Initially 0.1 M HCl

c) After adding 10 mL NaOH, concentration of excess HCl is calculated as

d) At equivalence point moles of HCl and moles of NaOH are equal. Since neither acid nor base
is in excess, pH is determined by dissociation of water.

e) After equivalence point, pH is determined by concentration of excess NaOH. For example, after
adding 30 mL NaOH, concentration of OH - is

Note: calculating titration curve for titration of strong base with strong acid is done in a similar
way, except that strong base is in excess before equivalence point and strong acid is in excess after
equivalence.

Calculate the titration curve for titration of

i) 50 mL of 0.05 M HCl with 0.1 M NaOH

ii) 20 mL of 0.1 M HCl with 0.1 M NaOH

Titrating a weak acid with strong base

Titration of 50 mL 0.1 M CH 3 COOH with 0.1 M NaOH (Ka = 1.75 × 10−5 )

a) Calculate volume of NaOH needed to reach equivalence point

b) Before adding NaOH, pH is that for 0.1 M CH 3 COOH and [𝐻3 𝑂+ ] is calculated from Ka

c) NaOH added but before equivalence point

1. Added NaOH reacts with CH3 COOH producing a buffer

2. Concentration of CH 3 COOH and CH 3 COO- are calculated from volumes reacted and substituted
into Ka expression or Henderson-hassel balch equation to calculate pH.

Adding 10 mL NaOH

d) At equivalence point

Moles CH 3 COOH initially present and moles NaOH added are identical. Since their reaction
effectively proceeds to completion the predominate ion in solution is CH 3 COO - which is a weak
base.

To calculate pH, first determine [𝐶𝐻3 𝐶𝑂𝑂− ]

e) Beyond equivalence point

In this solution, OH - arise from both excess NaOH and hydrolysis of CH3 COO -. The contribution
of OH - from salt hydrolysis may be insignificant compared with that from excess NaOH permitting
the former to be ignored.

Note: Calculations for titration of weak base with strong acid are handled in a similar way except
that initial pH is determined by weak base, pH at equivalence point by its conjugate weak acid and
pH after equivalence point by concentration of excess strong acid.

Question

Construct a titration curve for the titration of 25 mL of 0.125 M NH 3 with 0.0625 M HCl.
Sketching acid-base titration curve

1. Draw axes, place pH on y-axis and volume of titrant on x-axis.

2. After calculating volume of titrant needed to reach equivalence point, draw a vertical line
that intersects x-axis at this volume.
3. Determine pH for two volumes before equivalence point and for two volumes after
equivalence point.
4. Straight lines are drawn through each pair of points with each line intersecting the vertical
line representing equivalence point volume.
5. Draw smooth curve connecting the three straight line segments.
Acid – base indicators

Are weak organic acids or weak organic bases that change color as a function of ionization
state. Acid – base indicators of two types have different ionization equilibria.

i. Acid type indicator

HIn + H2O In- + H3O+
Acid color base color

ii. Base type indicator

In + H2O InH + + OH -
Base color acid color

The pH at which an acid – base indicator changes color is determined by its acid dissociation
constant.

Consider acid type indicator

[𝐼𝑛 −][𝐻3 𝑂 + ]
Ka = [𝐻𝐼𝑛 ]

𝐾𝑎 [𝐻𝐼𝑛]
[𝐻3 𝑂+ ] = [𝐼𝑛 −]

Introducing -log on both sides gives

[𝐼𝑛 −]
p𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 [𝐻𝐼𝑛 ]
 Note: The color of the indicator solution will always be a function of the ratio [HIn] and [In -].
A solution of the indicator is the color of HIn whenever its concentration is 10 times more than
that of In- and the color of In- whenever concentration of HIn is 10 times that of In -.

Substituting

1
p𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 10 = 𝑝𝐾𝑎 − 1

10
p𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 = 𝑝𝐾𝑎 + 1
1

Therefore, indicator changes color over a pH range of ±1 units on either side of its 𝑝𝐾𝑎 . Thus
indicator will be the the colour of HIn when pH is less than 𝑝𝐾𝑎 − 1 and colour of In- for pH
greater than 𝑝𝐾𝑎 + 1.

Variables that influence behavior of indicators

The pH interval over which a given indicator exhibits a color change is influenced by

- Temperature
- Ionic strength of the medium
- Presence of organic solvents and colloidal particles
Some important acid – base indicators

Indicator pH range Color change

acid base
Thymol blue 1.2 – 2.8 red yellow
Methyl yellow 2.9 – 4.0 red yellow
Methyl orange 3.1 – 4.4 red yellow
Bromocresol green 3.8 – 5.4 yellow blue
Methyl red 4.2 – 6.3 red yellow
Bromothymol blue 6.2 – 7.6 yellow blue
Phenolphthalein 8.0 – 10.0 colorless red
Alizarin yellow 10 - 12 colorless yellow
Litmus 5.0 – 8.0 red blue
Congo red 3.0 – 5.0 blue red
Choice of indicator

1. If concentrations of acid and base are 0.1 M or higher, it does not make much difference.
The large endpoint transition spans the color transition range of almost all indicators.
2. If concentrations drop significantly below 0.1 M, an indicator whose pk a is as close as
possible to pH 7.0±1 is best.
3. If concentrations of acid and base drop too low (i.e the endpoint transition spans less than
two pH units) no indicator will work very well.
It is important to choose an indicator whose pH range coincides with the pH of resultant
solution at equivalence point.

COMPLEXOMETRIC TITRATIONS

Is a technique that involves titrating metal ions with a complexing agent or chelating agent. The
titrations are particularly useful for determination of a mixture of different metal ions in solution.

Cu2+ + 4NH 3 → Cu(NH 3 )4 2+

Terminologies

Complex ion is one which is positive or negatively charged in which atoms or groups of atoms
with negative charges or lone pair of electrons coordinate with a central atom or ion which is
normally a metal.

The complex can form only when

- The central atom accepts an electron pair from one or more ligands
- The ligand possesses at least one electron pair to donate
- The bonding (coordinate/ covalent bonding) occurs
Ligand is any electron donating species which has the ability to bind to the metal ion and produce
a complex ion.

Coordination number is the number of bonds the central atom can form.
Classification of ligands

i. Unidentate ligands are those which make one bond per molecule with the central metal
ion. Examples: halide ions, CN -, NH 3
ii. Bidentate ligands are those which make two bonds per molecule with the central metal
ion. E.g ethylene diamine H 2 NCH 2 CH2 NH2
iii. Ligands having two or more atoms which are capable of coordinating to the central
metal ion are multidentate / polydentate. Example ethylene diamine tetraacetic acid
(EDTA)

Structure of EDTA
Chelate is a heterocyclic ring compound formed by a metal atom and a ligand with two or more
functional groups.

Chelating agent is a ligand having more than one electron donating groups. The most effective
complexing agent in ligands are amino and carboxylate ions such as dimethyl glyoxime.

Sequestering agent is a chelating agent that forms water soluble complexes with polyvalent metal
ions e.g EDTA.

EDTA forms chelates with nearly all metal ions and this reaction is the basis for general analytical
method for these ions by titration with a standard EDTA solution. Such titrations are called
complexometric or chilometric or EDTA titrations.

Principle of complexometric titration

A complexometric titration is one in which the reaction between analyte and titrant involves the
formation of a complex. In a typical complexometric titration a solution of a complexing agent is
added to the analyte solution. This leads to the formation of a stoichiometric complex that is
soluble and stays undissociated.
Many principles of acid – base titrations are used in complexometric titration. In complexometric
titration, the free metal ions disappear as they are changed into complex. In acid – base titrations,
endpoint is marked by sudden change in pH. Similarly, in EDTA titration if pM (-log metal ion
concentration) is plotted against volume of titrant, it is found that at endpoint pM rapidly increases.
This sudden pM raise results from removal of traces of metal ions from solution by EDTA. Any
method which can determine this disappearance of free metal ions can be used to detect endpoint
in complexometric titrations. Endpoint can be detected with an indicator or instrumentally by
potentiometric or conductometric method.

At the beginning of titration, some analyte metal ion is complexed by indicator

Mn+ + In- → MIn

As EDTA is added, the free metal ion concentration decreases but some metal ions remain
complexed by the indicator. If the correct indicator and conditions are chosen at equivalence point
EDTA has complexed all of the free metal ion and then removes the metal from the indicator
complex.

MIn + EDTA → MEDTA + In

MIn and In have different colors.

Most indicators for complexometric titrations are organic dyes capable of forming intensely
colored complexes with metal ions. These dyes are known as metallochromic indicators. To
function as an indicator for an EDTA titration;

- The color reaction must occur at the endpoint when nearly all the metal ion is
complexed with EDTA.
- Indicator must be very sensitive to metal ions (i.e to pM) so that the color change occurs
as near to equivalence point as possible.
- Color reaction should be specific or selective.
- Color contrast between free and metal – bound indicator complex should be readily
observable.
- The metal-indicator complex must possess sufficient stability else it would not display
a sharp color change. Further the metal – indicator complex must be less stable than
 the metal – EDTA complex. This is to ensure that at endpoint EDTA is able to remove
all metal ions from metal – indicator complex. It is desirable that the change in
equilibrium from metal – indicator complex to metal – EDTA complex should be sharp
and rapid.
Indicators used in complexometric titrations

Name Color change pH range Metals detected

Mordant black Red to blue 6–7 Ca, Ba, Mg, Zn, Cd,
Eriochrome black T Mn, Pb, Hg
Solochrome black T

Murexide or Violet to blue 12 Ca, Cu, Co

ammonium purpurate
Catechol Violet to red 8 - 10 Mn, Mg, Fe, Co, Pb
Methyl blue Blue to yellow 4–5 Pb, Zn, Cd, Hg
Thymol blue Blue to grey 10 – 12
Alizarin Red to yellow 4.3 Pb, Zn, co, Mg, Cu
Sodium alizarin Blue to red 4 Al, Th
sulphonate
Xylenol Lemon to yellow 1–3 Bi, Th
4–5 Pb, Zn
5–6 Cd, hg

EDTA: important chelating agent

EDTA is available in pure form but can’t be easily used as a primary standard. The solutions of
EDTA are prepared from its disodium salt, Na2 H2 Y.2H 2O. the disodium salt is generally preferred
as it is non-hygroscopic, water soluble and a very stable sequestering agent. In any case,
standardization of EDTA against a solution of the metal ion to be determined helps to eliminate
any errors in endpoint determination. Alternatively, the standardization can be accomplished by
titrating against a solution made from the primary standard CaCO 3 . Though EDTA has been
extensively exploited for quantitative determinations of metal ions, it can’t be used for the direct
analysis of anions or neutral ligands.

Equilibria involved in EDTA titrations

EDTA is a Lewis acid having 6 binding sites (4 ionized carboxylate groups and 2 lone pairs on the
amino groups) providing 6 pairs of electrons. In typical analytical determinations completely
deprotonated molecule of EDTA forms up to 6 coordination bonds with a single metal ion. It is
accomplished by donation of the lone pair of electrons to empty orbitals on the metal ion.

The resulting product of this reaction is a metal-chelate complex in which EDTA forms a cage-
like structure around the metal ion. The actual number of coordination sites depends on the size of
the metal ion; however all metal – EDTA complexes have a 1: 1 stoichiometry irrespective of the
valency of the ions as shown.

M2+ + [H 2 Y]2- [MY]2- + 2H+

M3+ + [H 2 Y]2- [MY]- + 2H+

M4+ + [H 2 Y]2- [MY] + 2H +

The generalized reaction between the metal ion and EDTA can be described as given below.

Y4- + Mn+ MYn-4

Where Y- is shorthand notation of fully dissociated molecule of EDTA.

The formation constant for the complex will be given as follows

[𝑀𝑌𝑛−4 ]
Kf = [
𝑀𝑛+][𝑌4− ]

For example, formation of metal – EDTA complex with Cd 2+

Cd 2+(aq) + Y 4-(aq) CdY 2-(aq)

The formation constant for this reaction

[𝐶𝑑𝑌2−]
Kf = [𝐶𝑑2+][𝑌4 −]
= 2.9 × 1016 ………………………………….. (1)

is quite large suggesting the complex is stable and the reaction goes far to the right.
EDTA is a weak acid

Besides its properties as a ligand, EDTA is also a weak acid. The fully protonated form of EDTA,
H6 Y 2+ is a hexaprotic weak acid with successive pKa value of

pKa1 = 0.1 pKa2 = 1.5 pKa3 = 2.0

pka4 = 2.66 pKa5 = 6.16 pKa6 = 10.24

The first 4 values are for the carboxylic acid protons and the last two values are for the ammonium
protons. The species Y 4- becomes the predominate form of EDTA when pH is more basic than
10.24.

Conditional metal – ligand formation constants

The formation constant for CdY 2- assumes that EDTA is present as Y 4-. Because EDTA has many
forms when we prepare a solution of EDTA we know its total concentration C EDTA not the
concentration of a specific form such as Y 4-.

To use equation (1), we need to rewrite it in terms of C EDTA. At any pH a mass balance on EDTA
requires that its total concentration equal the combined concentrations of each of its forms

CEDTA = [H 6 Y 2+] + [H 5 Y+] +[H 4 Y] + [H 3 Y-] + [H 2 Y2-] + [HY 3-] + [Y 4-] ………………….. (2)

To correct the formation constant for EDTA’s acid – base properties we need to calculate the
fraction αY4-, of EDTA present as Y 4-.

[𝑌4− ]
∝ 𝑌 4− = 𝐶 …………………………………………….. (3)
𝐸𝐷𝑇𝐴

Solving equation (1) for the CdY 2- formation constant

[𝐶𝑑𝑌2−]
Kf = [𝐶𝑑2+][𝑌4 −]
= 2.9 × 1016

Substituting for [Y 4-]

[𝐶𝑑𝑌2− ]
Kf = [𝐶𝑑2+ ]∝𝑌4−𝐶
𝐸𝐷𝑇𝐴

Rearranging
 [𝐶𝑑𝑌2− ]
K’f = Kf ∝ 𝑌 4− = [ ……………………………………….. (4)
𝐶𝑑2 +]𝐶𝐸𝐷𝑇𝐴

Where K’f is a pH dependent conditional formation constant i.e it becomes smaller and the
complex becomes less stable at more acidic pHs.

EDTA competes with other ligands

To maintain a constant pH during a complexation titration, we must add a buffering agent. If one
of the buffer’s components is a ligand that binds with Cd 2+, then EDTA must compete with the
ligand for Cd 2+.

For example, an NH 4 + / NH 3 buffer includes the ligand NH 3 which forms several stable Cd 2+ - NH3
complexes. EDTA forms a stronger complex with Cd 2+ than does NH 3 , it displaces NH 3 ; however,
the stability of the Cd 2+ - EDTA complex decreases.

We can account for the effect of an auxiliary complexing agent such as NH 3 in the same way we
accounted for the effect of pH. Before adding EDTA, a mass balance on Cd 2+, CCd is

CCd = [Cd 2+] + [Cd(NH 3 )2+] + [Cd(NH 3 )2 2+] + [Cd(NH 3 )3 2+] + [Cd(NH 3 )4 2+]

The fraction of uncomplexed Cd 2+, ∝ 𝐶𝑑 2+ is

[𝐶𝑑2+]
∝ 𝐶𝑑 2+ = …………………………………………………. (5)
𝐶𝐶𝑑

Solving equation

[𝐶𝑑𝑌2− ]
K’f = Kf ∝ 𝑌 4− = [𝐶𝑑2 +]𝐶
𝐸𝐷𝑇𝐴

[𝐶𝑑𝑌2−]
K’f = Kf ∝ 𝑌 4− = ∝𝐶𝑑2 +𝐶𝐶𝑑 𝐶
𝐸𝐷𝑇𝐴

Because the concentration of NH3 in a buffer essentially is constant, we can rewrite this equation

[𝐶𝑑𝑌 2− ]
𝐾𝑓′′ = 𝐾𝑓 ∝ 𝑌 4−
∝ 𝐶𝑑 2+
=
𝐶𝐶𝑑 𝐶𝐸𝐷𝑇𝐴

to give a conditional formation constant, 𝐾𝑓′′ that accounts for both pH and the auxiliary
complexing agent’s concentration.
Complexometric EDTA titration curves

Knowing some EDTA chemical properties, we are ready to evaluate its utility as a titrant for the
analysis of metal ions. As an acid – base titration curve shows the change in pH following the
addition of titrant, the titration curve with EDTA shows the change in pM where M is he metal ion
as a function of the volume of EDTA.

Calculating the titration curve

Calculate the titration curve for 50.0 mL of 5.00 × 10−3 M Cd 2+ with 0.0100 M EDTA at a pH
of 10 and in the presence of 0.0100 MNH 3 . The formation constant for Cd 2+ - EDTA is 2.9 × 1016 .

Solution

Since the titration is carried out at a pH of 10, some of the EDTA is present in forms other than
Y4-. In addition, the presence of NH 3 means that the EDTA must compete for the Cd2+. To evaluate
the titration curve, therefore, we must use the appropriate conditional formation constant. We find
that ∝ 𝑌 4− is 0.35 at pH 10 and that ∝ 𝐶𝑑 2+ is 0.0881 when the concentration of NH 3 is 0.0100
M. using these values, we calculate the conditional formation constant

𝐾𝑓′′ = 𝐾𝑓 ∝ 𝑌 4− ∝ 𝐶𝑑 2+ = (2.9 × 1016 )(0.35)(0.0881) = 8.9 × 1014

Because 𝐾𝑓′′ is so large, we treat the titration reaction as though it proceeds to completion.

Determine the volume of EDTA needed to reach equivalence point. At equivalence point we know
that moles EDTA = moles Cd 2+

𝑀𝐸𝐷𝑇𝐴 𝑉𝐸𝐷𝑇𝐴 = 𝑀𝐶𝑑 𝑉𝐶𝑑

(0.005 𝑀)(50.0 𝑚𝐿)

𝑉𝐸𝐷𝑇𝐴 = = 25.0 𝑚𝐿
0.01 𝑀

Before equivalence point, Cd 2+ is in excess and pCd is determined by concentration of free Cd 2+

remaining in solution. Not all the untitrated Cd 2+ is free (some is complexed with NH 3 ), so we
have to account for the presence of NH 3 .
For example, after adding 5.0 mL of EDTA, the total concentration of Cd 2+ is

𝑚𝑜𝑙𝑒𝑠 𝑒𝑥𝑐𝑒𝑠𝑠 𝐶𝑑 2+ 𝑀𝐶𝑑 𝑉𝐶𝑑 − 𝑀𝐸𝐷𝑇𝐴 𝑉𝐸𝐷𝑇𝐴

𝐶𝐶𝑑 = =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑉𝐶𝑑 + 𝑉𝐸𝐷𝑇𝐴

(0.005 𝑀)(50.0 𝑚𝐿 ) − (0.010 𝑀)(5.0 𝑚𝐿)

= = 3.64 × 10−3 𝑀
50.0 𝑚𝐿 + 5.0 𝑚𝐿

To calculate the concentration of free Cd 2+

2+
[𝐶𝑑 2+ ]
∝ 𝐶𝑑 =
𝐶𝐶𝑑

[𝐶𝑑 2+ ] = (0.0881)( 3.64 × 10−3 𝑀) = 3.21 × 10−4 𝑀

Thus pCd = − log[𝐶𝑑 2+ ] = −log (3.21 × 10−4 ) = 3.49

At equivalence point, all the Cd 2+ initially present is now present as CdY 2-. The concentration of
Cd 2+, therefore is determined by the dissociation of the CdY 2- complex. To find pCd we must first
calculate the concentration of the complex.

𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝐶𝑑 2+ 𝑀𝐶𝑑 𝑉𝐶𝑑

[𝐶𝑑𝑌 2− ] = =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑉𝐶𝑑 + 𝑉𝐸𝐷𝑇𝐴

(0.005 𝑀)(50.0 𝑚𝐿)

= = 3.33 × 10−3 𝑀
50.0 𝑚𝐿 + 25.0 𝑚𝐿

Let x be the concentration of Cd 2+ due to the dissociation of the CdY 2- complex

[𝐶𝑑𝑌 2− ] 3.33 × 10−3

𝐾𝑓′′ = = = 8.94 × 1014
𝐶𝐶𝑑 𝐶𝐸𝐷𝑇𝐴 (𝑥)(𝑥)

𝑥 = 𝐶𝐶𝑑 = 1.93 × 10−9 𝑀

To find [Cd2+], we must account for the presence of NH 3

[𝐶𝑑 2+ ] =∝ 𝐶𝑑 2+ × 𝐶𝐶𝑑 = (0.0881)(1.93 × 10−9 𝑀) = 1.70 × 10−10 𝑀

𝑝𝐶𝑑 = −𝑙𝑜𝑔(1.70 × 10−10 ) = 9.77

After equivalence point, EDTA is in excess and the concentration of Cd 2+ is determined by the
dissociation of CdY 2- complex. Examining the equation for the complex’s conditional formation
constant, we see that to calculate 𝐶𝐶𝑑 we must first calculate [𝐶𝑑𝑌 2− ] and 𝐶𝐸𝐷𝑇𝐴 . After adding
30.0 mL of EDTA

𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝐶𝑑 2+ 𝑀𝐶𝑑 𝑉𝐶𝑑

[𝐶𝑑𝑌 2− ] = =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑉𝐶𝑑 + 𝑉𝐸𝐷𝑇𝐴

(0.005 𝑀)(50.0 𝑚𝐿)

= = 3.13 × 10−3 𝑀
50.0 𝑚𝐿 + 30.0 𝑚𝐿

𝑚𝑜𝑙𝑒𝑠 𝑒𝑥𝑐𝑒𝑠𝑠 𝐸𝐷𝑇𝐴 𝑀𝐸𝐷𝑇𝐴 𝑉𝐸𝐷𝑇𝐴 − 𝑀𝐶𝑑 𝑉𝐶𝑑

𝐶𝐸𝐷𝑇𝐴 = =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑉𝐶𝑑 + 𝑉𝐸𝐷𝑇𝐴

(0.01 𝑀)(30.0 𝑚𝐿 ) − (0.005 𝑀)(50.0 𝑚𝐿)

= = 6.25 × 10−4 𝑀
50.0 𝑚𝐿 + 30.0 𝑚𝐿

Substituting these concentrations into equation

[𝐶𝑑𝑌 2− ] 3.13 × 10−3

𝐾𝑓′′ = = = 8.94 × 1014
𝐶𝐶𝑑 𝐶𝐸𝐷𝑇𝐴 𝐶𝐶𝑑 (6.25 × 10−4 )

𝐶𝐶𝑑 = 5.6 × 10−15 𝑀

[𝐶𝑑 2+ ] =∝ 𝐶𝑑 2+ × 𝐶𝐶𝑑 = (0.0881)( 5.6 × 10−15 ) = 4.93 × 10−16 𝑀

𝑝𝐶𝑑 = −𝑙𝑜𝑔(4.93 × 10−16 ) = 15.31

Complexometric techniques

1. Direct titration

The analyte is titrated with standard solution of EDTA using a suitable indicator. EDTA forms
stable complexes with all metal cations except those in group I of the periodic table. If a mixture
of metal ions is to be titrated, it may be possible to determine each one individually by selecting
titration conditions carefully. For example, adjusting pH or by adding a masking agent which
protects one of the metal ions from reacting with EDTA.
2. Back titration

It is used when a suitable indicator is not available or the analyte does not react with EDTA or
does not react quickly enough. A known quantity of EDTA is added to the sample so that all of
the analyte reacts with EDTA and some EDTA is left over. The amount of EDTA left over is then
determined by titration with a standard solution of another metal ion. It is important that the metal
used to back titrate EDTA does not form a more stable EDTA complex than the analyte. For
example, in determination of Mn. This metal can not be directly titrated with EDTA because of
precipitation of Mn(OH)2 . An excess of known volume of EDTA is added to an acidic solution of
Mn salt and then ammonium buffer is used to adjust pH to 10 and the excess EDTA remaining
after chelation is back titrated with standard Zn solution kept in burette using eriochrome black T
indicator.

3. Displacement titrations

The analyte is treated with an excess of a second metal bound to EDTA. The analyte ion displaces
the second metal from the EDTA complex and then the second metal is titrated with EDTA.

e.g Mn2+ + MgEDTA 2- → Mg2+ + MnEDTA 2-

By this method Ca, Pb, Hg may be determined using eriochrome black T indicator.

4. Indirect titrations

EDTA does not directly react with the analyte but reacts with some metal that can be related
stoichiometrically to the analyte. EDTA can be used for determination of anions such as SO 42-
which do not react with it. BaSO 4 is insoluble. So, one way to determine SO 4 2- is to precipitate
with Ba2+, filter and wash the precipitate, then boil in excess EDTA to complex all Ba. Back titrate
to determine how much Ba was there and this in turn tells the amount of SO 4 2-.

Titration selectivity, masking and demasking agents

EDTA is a very unselective reagent because it complexes with numerous doubly, triply and
quadruply charged cations. The following procedures will help to increase selectivity.

1. Use of masking and demasking agents

Masking agent is a complexing agent that reacts selectively with a component in a solution and in
so doing prevents that component from interfering in an analysis. Masking agents act either by
precipitation or by formation of complexes more stable than the interfering ion EDTA complex.

a) Masking by precipitation
Many heavy metals such as Co, Cu, Pb can be separated in form of insoluble sulphides using Na 2 S.
these are filtered, decomposed and titrated with EDTA. Other precipitating agents are SO 4 2- for Pb
and Ba; C2 O 4 2- for Ca and Pb; F- for Ca, Mg, Pb; ferrocyanide for Zn, Cu.
b) Masking by complex formation
Masking agents form more stable complexes with the interfering metal ions. The most important
aspect is that the masking agent must not form complexes with the metal ion under analysis.
Examples of masking agents
- Ammonium fluoride for Al, Fe, Ti
- Ascorbic acid is a convenient reducing agent for iron(III) which is then masked by
complexing as the very stable hexacyanoferrate(II) complex
- KCN masks Ag, Cu, Hg, Fe, Zn, Cd, Co, Ni
- KI masks Hg
Demasking

It is the process in which the masked substance regains its ability to enter into a particular reaction.
This enables determination of a series of metal ions in one solution. The cyanide complexes of Zn,
and Cd may be demasked with formaldehyde or chloralhydrate. Thus, a solution containing Mg,
Zn and Cu can be titrated as follows;

Direct titration of the mixture with EDTA using solochrome black indicator, gives the sum of the
three metals

Mg Mg-EDTA
Zn + EDTA → Zn-EDTA
Cu Cu-EDTA

Treat an aliquot with KCN and titrate as before. CN- masks Zn and Cu so titrating with EDTA
gives amount of Mg

Zn2+ + 4CN - → Zn(CN)4 2-

 Cu2+ + 4CN - → Cu(CN)4 2-

Add excess chloral hydrate or formaldehyde to the cyanide containing mixture inorder to demask
Zn from the complex and titrate until the indicator turns blue.

Chloral hydrate EDTA

Zn(CN)4 2- Zn2+ Zn-EDTA
Cu(CN)4 2-

The content of Cu may then be found by difference.

2. pH control

The formation of a metal chelate is dependent on pH of the reaction medium. In weakly acid
solution the chelates of many metals are completely dissociated such as alkaline earth metals,
whereas chelates of Bi, Fe3+ or Cr are readily formed at this pH. Thus, in acidic solution, Bi can
be effectively titrated with a chelating agent in presence of alkaline earth metals. This method is
based upon the differences in stability of the chelates formed between the metal ions and the
chelating agent.

3. Use of selective metal indicators

These indicators are the metal complexing agents which react with different metal ions under
various conditions. Several selective metal indicators have been used and they are specific for a
particular ion.

4. Solvent extraction

Zn can be separated from Cu and Pb by adding excess of ammonium thiocyanate solution and
extracting the resulting zinc thiocyanate with 4-methylpentan-2-one (isobutyl methyl ketone); the
extract is diluted with water and Zn content determined with EDTA solution.

5. Removal of anions

Anions such as orthophosphate which can interfere in complexometric titrations may be removed
using ion exchange resins.

6. Kinetic masking
This is a special case in which a metal ion does not effectively enter into the complexation reaction
because of its kinetic inertness. Thus, the slow reaction of Cr(III) with EDTA makes it possible to
titrate other metal ions which react rapidly without interference from Cr(III); e.g determination of
Fe(III) and Cr(III).

Applications of complexometric titrations

- Determination of various metals such as Ca, Mg, Pb, Zn, Al, Fe, Mn, Cr in different
formulations that are official in industrial processes.
- Determination of hardness of water.

PRECIPITATION TITRATIONS

A reaction in which the analyte and titrant form an insoluble precipitate can also form the basis for
a titration. This type of titration is a precipitation titration. Most precipitation titrations involve
Ag+ as either an analyte or titrant. Those titrations in which Ag + is the titrant are called
argentometric titrations. Precipitation titration is a perfect method to determine halogens and some
metal ions and halide-like ions (SCN -, CN -, CNO -).

Titration curve for precipitation titration

The titration curve for a precipitation follows change in either the analyte’s or titrant’s
concentration as a function of the volume of titrant.

For example, analysis for I - using Ag+ as a titrant, the titration curve may be a plot of pAg or pI as
a function of the titrant’s volume.

Ag+(aq) + I -(aq) → AgI(s)

Calculating the titration curve

Titration of 50 mL of 0.050 M Cl- with 0.10 M Ag+. Ksp of AgCl is 1.8 × 10−10

Ag+(aq) + Cl-(aq) → AgCl(s)

Equilibrium constant for the reaction K = (K sp )-1

= (1.8 × 10−10 )−1 = 5.6 × 109

Since K eqm is large, we may assume that Ag+ and Cl- react completely.

Determine volume of Ag+ needed to reach equivalence point

Moles Ag+ = moles Cl-

MAgVAg = MClVCl

𝑀𝐶𝑙 𝑉𝐶𝑙 0.05 × 50.0

𝑉𝐴𝑔 = = = 25.0 𝑚𝐿
𝑀𝐴𝑔 0.10

Before equivalence point, Cl- is in excess. The concentration of unreacted Cl- after adding 10.0
mL of Ag+

𝑚𝑜𝑙𝑒𝑠 𝑒𝑥𝑐𝑒𝑠𝑠 𝐶𝑙− 𝑀𝐶𝑙 𝑉𝐶𝑙 − 𝑀𝐴𝑔 𝑉𝐴𝑔

[𝐶𝑙 − ] = =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑉𝐶𝑙 + 𝑉𝐴𝑔

(0.05 × 50) − (0.1 × 10)

= = 2.5 × 10−2 𝑀
50 + 10

If the titration curve follows change in concentration for Cl, then we calculate pCl as;

𝑝𝐶𝑙 = − log [𝐶𝑙 − ]

= − log (2.5 × 10−2 ) = 1.60

Following change in concentration for Ag+ then, calculate [Ag+] first. To do so use K sp expression
for AgCl. 𝐾𝑠𝑝 = [𝐴𝑔+ ][𝐶𝑙 − ] = 1.8 × 10−10

1.8 × 10−10
[𝐴𝑔+ ] = = 7.2 × 10−9 𝑀
2.5 × 10−2

𝑝𝐴𝑔 = − log(7.2 × 10−9 ) = 8.14

At equivalence point, [𝐴𝑔+ ] = [𝐶𝑙 − ]

Using the solubility product expression

𝐾𝑠𝑝 = [𝐴𝑔+ ][𝐶𝑙 − ] = [𝐴𝑔+ ]2 = 1.8 × 10−10

[𝐴𝑔+ ] = [𝐶𝑙 − ] = 1.3 × 10−5 𝑀 pAg and pCl are both 4.89
After equivalence point, the titration mixture contains excess Ag +. The concentration of Ag+ after
adding 35 mL of titrant is,

𝑚𝑜𝑙𝑒𝑠 𝑒𝑥𝑐𝑒𝑠𝑠 𝐴𝑔+ 𝑀𝐴𝑔 𝑉𝐴𝑔 − 𝑀𝐶𝑙 𝑉𝐶𝑙

[𝐴𝑔+ ] = =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑉𝐶𝑙 + 𝑉𝐴𝑔

(0.1 × 35.0) − (0.05 × 50.0)

= = 1.18 × 10−2 𝑀
50 + 35

𝑝𝐴𝑔 = − log (1.18 × 10−2 ) = 1.93

The concentration of Cl is

1.8 × 10−10
[𝐶𝑙 − ] = = 1.5 × 10−8 𝑀
1.18 × 10−2

𝑝𝐶𝑙 = − log 1.5 × 10−8 = 7.82

Methods for finding endpoint in precipitation titration

1. Finding the end point with a visual indicator

Like acid-base indicators, indicators for argentometric titrations are selected to produce a colour
change at or near equivalence point. Normally the indicator is selected to react with the added
titrating agent, not the analyte. If A is the analyte, R is the titrating agent and In is the indicator.

A + R AR

In(colour) + R InR (new colour

- Note that to make the indicator change colour, excess R must be added. Obviously, the
smaller the excess added to cause the colour change, the smaller the endpoint error.
- This means that the indicator should give large colour changes at very low
concentrations.
There are three methods to find endpoint in precipitation titration with visual indicator.
a) Mohr’s method
The method uses chromate ion as indicator in titration of Cl- with AgNO 3 solution. After
all Cl- has been precipitated as white AgCl, the first excess of the titrant results in formation
of silver chromate precipitate (brick red) which signals endpoint. The reactions are
Ag+ + Cl- AgCl(s)
2Ag+ + CrO 4 2- Ag2 CrO 4 (s)
By knowing stoichiometry and moles consumed at end point, the amount of Cl - in an
unknown sample can be determined. The titration is performed in neutral or slightly basic
medium pH= (7 – 10) to prevent silver hydroxide formation at pH > 10 or formation of
chromic acid at pH < 7.
2CrO 4 2- + 2H + 2HCrO 4 2- Cr2 O 7 2- + H2 O
2Ag+ + 2OH - 2AgOH Ag2 O(s) + H 2 O
The presence of an excess of either NaHCO 3 or borax in solution tends to maintain H+
concentration within suitable limits.

b) Volhard method
Silver ions are titrated with a standard solution of thiocyanate ion.
Ag+ + SCN - AgSCN(s)
Iron(III) serves as the indicator. The end point for the titration reaction is formation of red
complex.
Fe3+ + SCN - FeSCN 2+
The titration must be carried out in acidic solution to prevent precipitation of iron(III) as
the hydrated oxide. The most important application of Volhard method is the indirect
determination of halide ions.

c) Fajan’s method
This uses an adsorption indicator whose colour when adsorbed to the precipitate is different
from that when it is in solution. For example, when titrating Cl- with Ag+ the anionic dye
dichloro-fluoroscein is used as the indicator. Before the end point, the precipitate of AgCl
has a negative surface charge due to the adsorption of excess Cl-. The anionic indicator is
repelled by the precipitate and remains in solution where it has a greenish yellow colour.
 After the end point, the precipitate has a positive surface charge due to the adsorption of
excess Ag+. The anionic indicator now adsorbs to the precipitate’s surface where its colour
is pink. The change in colour signals the end point.
Requirements of dye and precipitate upon which successful indicator action depends;
- The precipitate should be produced in a highly dispersed state i.e the precipitate
particles must be colloidal so as to adsorb.
- The precipitate must strongly adsorb its own ions.
- The indicator must be strongly held by the primarily adsorbed ions.
- The pH must favour formation of ions of the indicator.
2. Finding the end point potentiometrically

Questions

1. A mixture containing only KCl and NaBr is analysed by Mohr method. A 0.3172 g sample
is dissolved in 50 mL of water and titrated to the Ag 2 CrO 4 end point requiring 36.85 mL
of 0.112 M AgNO 3 . A blank titration requires 0.71 mL of titrant to reach the same end
point. Report the %w/w KCl and NaBr in the sample. Ans 82.17 % KCl and 17.65 % NaBr

2. The %w/w I - in a 0.6712 g sample was determined by a Volhard titration. After adding 50
mL of 0.05619 M AgNO 3 and allowing the precipitate to form, the remaining silver was
back titrated with 0.05322 M KSCN, requiring 35.14 mL to reach the end point. Report the
%w/w I - in the sample. Ans 17.76 %

3. A 0.5131 g sample containing KBr is dissolved in 50 mL of distilled water. Titrating with

0.04614 M AgNO 3 requires 25.13 mL to reach the Mohr end point. A blank titration
requires 0.65 mL to reach the same end point. Report the %w/w KBr in the sample. Ans
26.19 %

4. A 0.1093 g sample of impure Na2 CO3 was analysed by the Volhard method. After adding
50.0 mL of 0.06911 M AgNO 3 , the sample was back titrated with 0.05781 M KSCN
requiring 27.36 mL to reach the end point. Report the purity of the Na 2 CO 3 sample. Ans
90.9 %
NON- AQUEOUS TITRATIONS

Non aqueous titration is the titration of substances dissolved in solvents other than water. The need
for non aqueous titrations arises because water can behave as a weak base and a weak acid as well
and can hence compete in proton acceptance or proton donation with other weak acids and bases
dissolved in it.

H2O + H+ H3O+
Competes with RNH 2 + H+ RNH 3

H2O + B OH - + BH +
Competes with ROH + B RO- + BH +

This makes end point detection relatively more difficult. That’s why very weak acid / base can’t
be titrated in water. The procedure of non aqueous titration is very useful because;

- Some analytes are too weak acids or bases to be titrated in water

- Reactants or products are insoluble in water
- Reactants or products react with water
Brosted – Lowry theory

According to this theory an acid is a proton donor while a base is a proton acceptor. The reaction
that occurs during non aqueous titration can be explained by means of the concept of Bronsted –
lowry theory.

Strong acid in water

HCl + H 2 O → H 3 O+ + Cl-
Acid base conjugate acid conjugate base

Weak acid in water

HCOOH + H 2 O H3O+ + HCOO -

Acid base conjugate acid conjugate base

Weak acid in non aqueous solvent

HCOOH + CH 3 NH2 CH3 NH 3 + + HCOO -

 Acid base conjugate acid conjugate base

It follows from these definitions that an acid may be either

- An electrically neutral molecule e.g HCl

- A positively charged cation e.g C 6 H 5 NH3 +
- A negatively charged anion e.g HSO 4 -
A base may be

- An electrically neutral molecule e.g C 6 H5 NH2

- An anion e.g Cl-
Substances which are potentially acidic can function as acids only in the presence of a base to
which they donate protons. Conversely basic properties do not become apparent unless an acid is
present. The apparent strength of an acid or base is determined by the extent of its reaction with a
solvent. In aqueous solution all strong acids appear equally strong because they react with the
solvent to undergo almost complete conversion to H 3 O+ and the acid anion. In a weakly protophilic
solvent such as acetic acid, the extent of formation of acetonium ion (CH 3 COOH 2+) due to the
addition of a proton provides a more sensitive differentiation of the strength of acids and shows
that the order of decreasing strength of acids is HClO 4 , HBr, H 2 SO4 , HCl, HNO 3 . Acetic acid reacts
incompletely with water to form H 3 O + and is therefore a weak acid. In contrast it dissolves in a
base such as ethylene diamine and reacts completely with the solvent that it behaves as a strong
acid. This is so-called levelling effect.

Levelling effect

The acidity of the weak acids can be enhanced using basic solvents because the basic solvent has
higher affinity to take up protons from acid. So, acetic acid behaves as a strong acid in ammonia
solution. Also, the basicity of weak bases can be enhanced in the presence of acidic solvent. This
is called the levelling effect of the solvent.

Classification of non aqueous solvents

Nonaqueous solvents are of the following types

i. Aprotic solvents
these are neutral chemically inert substances. They have a low dielectric constant, do not react
with acids or bases and therefore, do not favour ionisation.

Examples: benzene, toluene, CCl4 , dioxane, chloroform, ethyl acetate

ii. Protogenic solvents

These are acidic in nature and they can donate the proton. They are used to dissolve basic analytes.
They have high dielectric constant and ionized. They exert a leveling effect on bases.

Examples: formic acid, propanoic acid, H 2 SO4 , acetic acid

iii. Protophilic solvents

These are basic in nature and possess a high affinity for protons. The overall reaction can be
represented as,

HB + S SH + + B-
Acid basic solvent solvent proton conjugate base of acid

Equilibrium in the above reaction would be influenced largely by nature of the acid and nature of
the solvent. Weak acids are normally used in the presence of strongly protophilic solvents as their
acidic strengths are then enhanced and become comparable to those of strong acids; this is known
as leveling effect.

Examples: amines and ketones, pyridine, ethylenediamine, DMF (dimethylformamide).

iv. Amphiprotic solvents

These have both protophilic and protogenic properties.

Examples: acetic acid, alcohols, water

The dissociation of acetic acid which is frequently used as a solvent for titration of basic substances
is shown.

CH 3 COOH H + + CH 3 COO -
Here acetic acid is functioning as an acid. If a very strong acid such as perchloric acid is dissolved
in acetic acid, the latter can function as a base and combine with protons donated by the perchloric
acid to form protonated acetic acid, an onium ion

HClO 4 H+ + ClO 4 -

CH 3 COOH + H + CH3 COOH 2 +

Since CH 3 COOH2 + ion readily donates its proton to a base, a solution of perchloric acid in glacial
acetic acid functions as a strongly acidic solution.

Major solvents used in non-aqueous titrations

To select a solvent, there is need to consider solubility of analyte, nature of analyte and reactivity
of the analyte. The major solvents include glacial acetic acid, acetonitrile, alcohols, dioxane, DMF.

Indicators for non-aqueous titrations

The ionized and unionized forms of indicators apply equally well for non-aqueous titrations but
their color changes at the end point vary from titration to titration as they depend on the nature of
titrant. The color corresponding to the correct end point may be established by carrying out a
potentiometric titration while simultaneously observing the color change of the indicator.

Indicator Color changes

Basic Neutral Acidic
Crystal violet violet Blue-green Yellowish-
green
α-naphtholbenzein Blue or blue-green Orange Dark green
Oracet blue B Blue Purple Pink
Quinaldine red Magenta Almost
colourless
Thymol blue Yellow Blue
Disadvantages of non-aqueous titrations

- Temperature, moisture and CO 2 should be controlled

- Solvents are expensive
- Volatile solvents can pollute the environment
- Indicator must be prepared in non-aqueous medium
Application of non-aqueous titrations

Non aqueous titrations have been used to;

- Quantify mixtures of primary, secondary and tertiary amines

- Studying sulphonamides, mixture of purines and for many other organic amino
compounds and salts of organic acids
- Titrate halogen acid salts of bases
ANALYTICAL CHEMISTRY II

INTRODUCTION TO ANALYTICAL SEPARATIONS

An important part of most analyses is dealing with foreign species that either attenuate the signal
from the analyte or produce a signal that is indistinguishable from that of the analyte. A substance
that effects an analyte signal is called an interference or interferent. Several methods are used for
dealing with interferences in an analysis basing on the difference in at least one of the chemical or
physical properties of the analyte and interference.

Classification of separation techniques

Basis of separation Separation technique

Size Filtration
Dialysis
Size-exclusion chromatography
Centrifugation
Mass and density Centrifugation
Complex formation Masking
Change in physical state Distillation
Sublimation
Recrystallisation
Change in chemical state Precipitation
Ion exchange
Electrodeposition
Volatilization
Partitioning between phases Extraction
Chromatography

A. Separation based on size

The separation is accomplished using a porous medium through which only the analyte or
interferent can pass.
I. Filtration

Here gravity, suction or pressure is used to pass a sample through a porous filter. Particulate
interferents can be separated from dissolved analytes by filtration using aa filter whose pore size
retains the interferent. The separation technique is important in analysis of many natural waters
for which the presence of suspended solids may interfere in the analysis. Filtration can also be
used to isolate analytes present as solid particulates from dissolved ions in the sample matrix.

II. Dialysis

A semi-permeable membrane is used to separate the analyte and interferent. Dialysis membranes
are usually constructed from cellulose, with pore sizes of 1 – 5 nm. The sample is placed inside a
bag or tube constructed from the membrane. The dialysis membrane and sample are then placed
in a container filled with a solution whose composition differs from that of the sample. If the
concentration of a particular species is not the same on the two sides of the membrane, the resulting
concentration gradient provides a driving force for its diffusion across the membrane. Dialysis os
used to purify proteins, hormones and enzymes.

III. Size-exclusion chromatography

A separation method in which a mixture passes through a bed of porous particles with smaller
particles taking longer to pass through the bed due to their ability to move into the porous structure.
In this technique a column is packed with small approximately 10 µm porous particles of cross-
linked dextrin or polyacrylamide. The pore size of the particles is controlled by the degree of
crosslinking with greater crosslinking in smaller pore sizes. The sample to be separated is placed
into a stream of solvent that is pumped through a column at a fixed flow rate. Particles too large
to enter the pores are not retained and pass through the column at the same rate as the solvent.
Those particles capable of entering into the pore structure take longer to pass through the column.
Size exclusion chromatography is widely used in analysis of polymers and in biochemistry wher
it is used for separation of proteins.

B. Separation based on mass or density

If there is a difference in the mass or density of the analyte and interferent, then a separation using
centrifugation may be possible.
The sample, as a suspension is placed in a centrifuge tube and spun at a high angular velocity.
Particles experiencing a greater centrifugal force have faster sedimentation rates and are
preferentially pulled towards the bottom of the centrifuge tube. For particles of equal density, the
separation on mass with heavier particles having greater sedimentation rates. When particles are
of equal mass, those with highest density have the greatest sedimentation rate.

C. Separations based on complexation reactions

A reagent is added to the solution of sample to immobilize or chemically bind the interferent as a
soluble complex preventing t from interfering in the analyte’s determination. This process is
known as masking. A masking agent must not affect the behavior of the analyte significantly.

D. Separations based on change of state

Since an analyte and interferent are usually in the same phase, a separation often can be effected
by inducing a change in one of their physical or chemical states. Changes in physical states that
have been exploited for the purpose of a separation include liquid to gas and solid to gas phase
transitions. Changes in chemical state involve one or more chemical reactions.

I. Change in physical state

- When the analyte and interferent are miscible liquids, a separation based on distillation may be
possible if their boiling points are significantly different.

- When the sample is a solid, a separation of analyte and interferent by sublimation may be
possible. The sample is heated at a temperature and pressure below its triple point where the solid
vaporizes without passing through the liquid state. The vapour is then condensed to recover the
purified solid.

- Another approach for purifying solids is recrystallization. The solid is dissolved in a minimum
volume of solvent for which the analyte’s solubility is significant when the solvent is hot and
minimal when the solvent is cold. The interferent must be less soluble in the hot solvent than the
analyte or present in much smaller amounts.
II. Change in chemical state

Chemical reactivity also can be used in a separation by effecting a change in the chemical state of
the analyte or interferent. Types of reactions that can be used to chemically separate an analyte
and interferent include precipitation, electrodeposition and ion exchange.

E. Separations based on partitioning between phases

The most important class of separation techniques is based on the selective partitioning of the
analyte or interferent between two immiscible phases. The partition of a solute between two
immiscible phases in an equilibrium phenomenon is governed by the distribution law. If the solute
species A is allowed to distribute itself between water and an organic phase, the resulting
equilibrium may be written as

A(aq) A(org)

The ratio of concentration for A in the two phases will be constant and independent of the total
quantity of A; that is at any given temperature,

[𝐴] 𝑜𝑟𝑔
K= [𝐴] 𝑎𝑞

The equilibrium constant K is called distribution constant or partition constant.

i) Extraction between two phases

when the sample is initially present in one of the phases, the separation is known as an extraction.
In a simple extraction the sample is extracted one or more times with portions of the second phase.
Simple extractions are particularly useful for separations in which only one component has a
favourable distribution ratio. Several important separation techniques are based on simple
extractions including liquid – liquid, liquid – solid, solid – liquid and gas – solid extractions.

Liquid – liquid extractions

They are usually accomplished with a separatory funnel. The two liquids are placed in the
separatory funnel and shaken to increase the surface area between the phases. When the extraction
is complete, the liquids are allowed to separate with the denser phase settling to the bottom of the
separatory funnel.
Liquid – liquid extractions also may be carried out in the sample container by adding the extracting
solvent when the sample is collected. For example, pesticides in water may be preserved for longer
periods by extracting into small volumes of hexane added to the sample in the field.

Solid phase extractions

The sample is passed through a cartridge containing solid particulates that serve as the adsorbent
material. For liquid samples the solid adsorbent is isolated in either a disk cartridge or a column.
The choice of adsorbent is determined by the properties of the species being retained and the matrix
in which it is found.

In gas – solid extractions the sample is passed through a container packed with a solid adsorbent.
One application of gas – solid extraction is in the analysis of organic compounds for carbon and
hydrogen. The sample is combusted in a flowing stream of oxygen and the gaseous combustion
products are passed through a series of solid phase adsorbents that remove the carbon dioxide and
water.

Continuous extractions

An extraction is still feasible even when the component of interest has an unfavourable partition
coefficient, provided that all other components in the sample have significantly smaller partition
coefficients. Because the partition coefficient is unfavourable, a simple extraction will not be
quantitative. Instead, the extraction is accomplished by continuously passing the extracting phase
through the sample until a quantitative extraction is achieved.

Many continuous extractions involving extractions solid samples are carried out with a Soxhlet
extractor. The extracting solvent is placed in the lower reservoir and heated to its boiling point.
Solvent in vapour phase moves upward through the tube on the left side of the apparatus to the
condenser where it condenses back to liquid state. The solvent then passes through the sample
which is held in a porous cellulose filter thimble, collecting in the upper reservoir. When the
volume of solvent in the upper reservoir reaches the upper bend of the return tube, the sovent and
any extracted components are siphoned back to the lower reservoir. Overtime, the concentration
of the extracted component in the lower reservoir increases.
Suppose Vaq mL of solution containing ao moles of A (analyte) is to be extracted with Vorg mL
of organic solvent, then the concentration of solute A in aqueous and organic layers can be
established. At equilibrium let 𝑎1 be mole of A remaining in aqueous layer. The concentration of
A in aqueous and organic layers is given by

𝑎1
[A]𝑎𝑞 = 𝑉𝑎𝑞

𝑎𝑜 −𝑎1
[A]org = 𝑉𝑜𝑟𝑔

The distribution coefficient between the two layers at equilibrium is given by

[𝐴] 𝑜𝑟𝑔
K= [𝐴] 𝑎𝑞

𝑎𝑜 −𝑎1 𝑎
=( )⁄𝑉 1
𝑉𝑜𝑟𝑔 𝑎𝑞

𝑉𝑎𝑞 (𝑎𝑜 −𝑎1 )

= 𝑎1 𝑉𝑜𝑟𝑔

𝑉𝑎𝑞
Hence, 𝑎1 = ( )𝑎𝑜 ………………………………………….. (1)
𝐾𝑉𝑜𝑔 +𝑉𝑎𝑞

Note: The number of moles 𝑎2 remaining after the second extraction with an identical volume of
organic solvent can also be established.

𝑎2
[A]𝑎𝑞 =
𝑉𝑎𝑞

𝑎1 −𝑎2
[A]org = 𝑉𝑜𝑟𝑔

[𝐴]𝑜𝑟𝑔 𝑎1 −𝑎2 𝑉𝑎𝑞

Then, K = [𝐴]𝑎𝑞
= ×
𝑉𝑜𝑟𝑔 𝑎2

𝑉𝑎𝑞
𝑎2 = ( )𝑎1 ……………………………………………… (2)
𝐾𝑉𝑜𝑟𝑔 +𝑉𝑎𝑞

Substitute for 𝑎1

𝑉𝑎𝑞 𝑉𝑎𝑞
𝑎2 = (𝐾𝑉 )(𝐾𝑉 )𝑎𝑜
𝑜𝑟𝑔 +𝑉𝑎𝑞 𝑜𝑔 +𝑉𝑎𝑞

𝑉𝑎𝑞
𝑎2 = (𝐾𝑉 ) 2 𝑎𝑜
𝑜𝑟𝑔 +𝑉𝑎𝑞
Generally, the concentration of A remaining after n extractions is given by

𝑉𝑎𝑞
𝑎𝑛 = ( ) 𝑛 𝑎𝑜
𝐾𝑉𝑜𝑟𝑔 +𝑉𝑎𝑞

Example,

An aqueous solution contains 0.5 g of solute in 30 mL of solution. To this solution is added 10 mL

of ether and the mixture shaken to equilibrium at 298 K. if the distribution coefficient is 4.7,

a) Calculate the amount of solute remaining in the aqueous phase. Ans 0.1948 g
b) If the extraction is carried out with two successive 5 mL of ether, calculate the amount of
solute remaining unextracted. Ans 0.157 g
Question

What is the minimum distribution coefficient that permits removal of 99 % of a solute from 50 mL
water with two 25 mL extractions with toluene? Ans K = 18

Efficiency of extraction

The efficiency of extraction depends on the value of the distribution ratio, D. for solvent extraction,
it also depends on the relative amounts of the two liquid phases and for solid phase extraction on
the surface area of the sorbent. With solvent extraction, the percentage of solute extracted, E is
given by

100𝐷
𝐸=
𝑉
[𝐷 + ( 𝑎𝑞⁄𝑉 )]
𝑜𝑟𝑔

𝑉𝑜𝑟𝑔 𝑎𝑛𝑑 𝑉𝑎𝑞 are volumes of organic and aqueous phases respectively.

100𝐷
Or 𝐸 = when the phases are of equal volume.
(𝐷+1)

For solutes with small values of D, multiple extractions will improve the overall efficiency and an
alternative expression enables this to be calculated.

𝑉𝑎𝑞
𝑎𝑛 = ( ) 𝑛 𝑎𝑜
𝐷𝑉𝑜𝑟𝑔 + 𝑉𝑎𝑞
𝑎𝑜 and 𝑎𝑛 are the amounts of solute in the aqueous phase initially and remaining after n extractions
respectively.

Question

1. A water sample contains 10 mg each of a halogenated pesticide and an ionic herbicide

which are to be separated by extraction of the pesticide into methylbenzene. Given that the
pesticide distribution ratio, D, for methylbenzene/water is 50, calculate the extraction
efficiency for
i) One extraction of 20 cm3 of water with 10 cm3 of methylbenzene. Ans 96.15 %
ii) One extraction of 20 cm3 of water with 30 cm3 of methylbenzene. Ans 98.68 %
iii) Three extractions of the same 20 cm3 of water with 10 cm3 portions of
methylbenzene. Ans 99.99 %

2. Suppose the complete removal of 0.1 mg of iodine from 50 cm3 of an aqueous solution of
iodine and NaCl is required and distribution ratio of CCl4 /water is 85. Calculate the
extraction efficiency for
i) A single extraction with 25 mL CCl4 . Ans 97.7 %
ii) Three extractions with 8.33 cm3 of CCl4 . Ans 99.97 %
Note: Extracting several times with small volumes of organic solvent is more efficient than one
extraction with a large volume. This is of particular significance when the value of D is less than
102 .

Selectivity of extraction

Often it is not possible to extract one solute quantitatively without partial extraction of another.
Selectivity in extraction procedures is the degree to which solutes in a mixture can be separated by
virtue of having different distribution ratios. The ability to separate two solutes depends on the
relative magnitudes of their distribution ratios. For solute A and B whose distribution ratios are
DA and D B, the separation or selectivity factor, β is defined as

𝐷
𝛽 = 𝐷𝐴 where D A > DB
𝐵
Selectivity factors exceeding 104 or 105 are necessary to achieve a quantitative separation of two
solutes as for most practical purposes, a separation would be considered complete if one solute
could be extracted with greater than 99 % efficiency whilst extracting less than 1 % of another. A
separation can ne made more efficient by adjustment of the proportions of organic and aqueous
phases. The optimum ratio for the best separation is given by the Bush – Densen equation.

𝑉𝑜𝑟𝑔 1 1
=( )2
𝑉𝑎𝑞 𝐷𝐴 𝐷𝐵

The extraction of many solutes can be enhanced or suppressed by adjusting solution conditions e.g
pH or addition of complexing agents.

ii) Chromatographic separations

Chromatography was originally developed by the Russian botanist, Michael Tswett in 1903 for
the separation of colored plant pigments by percolating petroleum ether extract through glass
column packed with powdered CaCO 3 . Colored zones were produced by the various pigments
migrating through the column at different rates, the components being isolated by extrusion and
sectioning of the CaCO 3 packing. Modern chromatographic techniques are more complex and are
used for a wide variety of separations frequently involving colourless substances to be separated
and quantified.

Basic principle; A chromatographic separation involves placing of a sample onto a liquid or solid
stationary phase and passing a liquid or gaseous mobile phase through or over it a process known
as elution. Sample components or solutes whose distribution ratios between the two phases differ
will migrate (be eluted) at different rates and this differential rate of migration will lead to their
separation over a period of time and distance.

Stationary phase is a phase that is fixed in place either in a column or on a planar surface.
Stationary phase is solid or liquid film coated on solid surface.

Mobile phase is a phase that moves over or through the stationary phase carrying with it the
analyte mixture. The mobile phase may be gas, liquid or supercritical fluid.
Chromatography is a technique I which components of a mixture are separated based on
differences in the rates at which they are carried through a fixed or stationary phase by a gaseous
or liquid mobile phase.

Elution is a process in which solutes are washed through a stationary phase by movement of
mobile phase.

Chromatographic mechanisms; During a chromatographic separation, solute molecules are

continually moving back and forth between the stationary and mobile phases. While they are in
the mobile phase, they are carried forward with it but remain virtually stationary during the time
they spend in the stationary phase. The rate of migration of each solute is therefore determined by
the proportion of time it spends in the mobile phase or in other words by its distribution ratio.

The process whereby a solute is transferred from a mobile to a stationary phase is called sorption
and the reverse is desorption. Chromatographic techniques are based on four different sorption
mechanisms, namely adsorption, partition, ion exchange and exclusion.

Adsorption is a surface effect involving electrostatic interactions e. g H – bonding, dipole – dipole

and dipole – induced dipole interactions. Solute species compete with the mobile phase for a
limited number of polar sites on the surface of the adsorbent of which silica gel is the most widely
used. Its surface comprises Si – O – Si and Si – OH (silanol) groups, the later being slightly acidic
as well as being polar which readily form H – bonds with slightly polar to very polar solutes.

If a liquid is coated onto the surface of an inert solid support the sorption process is one of partition
and movement of the solute is determined solely by its relative solubility in the two phases or by
its volatility if the mobile phase is a gas.

Note: Both adsorption and partition may occur simultaneously and the contribution of each is
determined by the system parameters i.e the nature of the mobile and stationary phases, solid
support and solute. For example, a stationary phase of Al2 O 3 is highly polar and normally exhibits
strong adsorptive properties. However, these may be modified by the presence of adsorbed water
which introduces a degree of partition into the overall sorption process by acting as a liquid
stationary phase. Conversely paper (cellulose) is relatively non polar and retains a large amount of
water which functions as a partition medium. Nevertheless, residual polar groups in the structure
of the paper can lead to adsorptive effects.

Ion exchange is a process whereby solute ions in the mobile phase can exchange with counter ions
carrying the same charge and associated with oppositely charged groups chemically bound to the
stationary phase. The stationary phase is a permeable polymeric solid such as an insoluble organic
resin or chemically modified silica, containing fixed charged groups and mobile counter ions;
which can exchange with the ions of a solute as the mobile phase carries them through the structure.
Both cationic and anionic ion-exchangers are available, the exchange processes being represented
as below;

Cation exchange nR-H+ + Xn+ (R-)nX n+ + nH+

Anion exchange nR+Cl- + Yn- (R+)nY n- + nCl-

R is a polymeric resin or silica

Xn+ and Y n- are solute cations and anions respectively of valency n.

Exclusion differs from other sorption mechanisms in that no specific interactions between solute
species and the stationary phase are necessary. The separating solutes remain in the mobile phase
throughout. Separations occur because of variations in the extent to which the solute molecules
can diffuse through an inert but porous stationary phase. This is normally a gel structure which has
a small pore size and into which small molecules upto a certain critical size can diffuse. Molecules
larger than the critical size are excluded from the gel and move unhindered through the column or
layer whilst smaller ones are retarded to an extent dependent on molecular size.

Note: In each chromatographic technique one of the four mechanisms predominates but it should
be emphasized that two or more may be involved simultaneously.

Characterization of solutes

As already described, the rate of movement of a solute is determined by its distribution ratio
defined as
 𝐶 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
𝐷=
𝐶 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒

The larger the value of D, the slower will be the progress of the solute through the system and the
components of a mixture will therefore reach the end of a column or the edge of a surface inorder
of increasing value of D. in column methods, a solute is characterized by the volume of mobile
phase required to move it from one end of the column to the other. This is known as retention
volume, V R i.e the volume passing through the column between putting the sample on top of the
column and the emergence of the solute peak at the bottom.

V R = Vm + KV m

Vm is volume of mobile phase in the column (the dead or void volume)

K is retention factor which is directly proportional to D but takes account of the volume of each
phase.

Sometimes K is used to characterize a solute rather than V R. If K = 0, then V R = Vm and the solute
is eluted without being retarded or retained by the stationary phase. Large values of K, which
reflect large values of D result in very large retention volumes and hence long retention times. At
a constant rate of flow of mobile phase F, V R is related to retention time t R by the equation

V R = Ft R

If the flow of mobile phase is monitored by a detector and recorded such as is used in gas and high
performance liquid chromatography then t R can be used as a measure of V R.

Retention time, t R is the time between injection of a sample and appearance of solute peak at the
detector.

In paper and thin layer chromatography, the separation process is halted at a stage which leaves
the separated components in situ on the surface in form of spots. The rate at which a solute has
moved is then determined by its retardation factor, Rf which is defined as

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑒𝑛𝑡𝑟𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑠𝑝𝑜𝑡

𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑟𝑜𝑛𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
Note: - Rf is inversely related to distribution ratio, D and can not be greater than 1. Distances are
measured from the point of application of the sample.

- As both t R and Rf are related to D they will depend on the conditions under which a
chromatogram is run.
A chromatogram is a display of separated components. In column chromatography,
chromatogram is obtained by plotting the detector signal against time to give a series of peaks eah
of which represents the distribution of an eluted component in the mobile phase. In planar
chromatography, chromatogram is a visualised display of the separated spots from a TLC plate or
paper chromatographic separation.

- To identify a component, comparison is made of their t R or Rf to those of the standards.

Classifying chromatographic methods

a) Column chromatography
The stationary phase is held in a narrow tube and mobile phase is forced through the tube
under pressure or by gravity,
b) Planar chromatography
The stationary phase is supported on a flat plate or in the pores of a paper. The mobile
phase moves through the stationary phase by capillary action or under influence of gravity.
Types of planar chromatography

i) Thin layer chromatography (TLC)

Separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate
covered with a thin layer of alumina or silica gel (stationary phase).
ii) Paper chromatography (PC)
Separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip
(stationary phase).
Thin layer chromatography

TLC may be either adsorptive or partition. In TLC any substance that can be finely divided and
formed into a uniform layer can be used as a stationary phase. Both organic and inorganic
substances can be used to form a uniform layer for RLC. Organic substances include: cellulose,
polyamide, polyethylene and inorganic: silica gel, aluminium oxide and magnesium silicate.
Stages in TLC

i. Sample application – capillary used to spot solution of each sample.

ii. Development – this is when the separation actually occurs.
iii. Visualization – viewed under UV light.
iv. Interpretation of results – comparison of retention factors.
Sample application (spotting) on TLC plates

- Before applying the sample, “draw guide lines” lightly with pencil 1 m away from the bottom
of TLC plate.

- Sample application is generally done by placing the solute mixture in a volatile solvent (weak
mobile phase).

- Use TLC capillary to transfer and spot dissolved samples.

Development of TLC plates

TLC developing chamber (just glass jar with solvent) is use.

- Place the spotted TLC plate in a developing chamber.

- Developing solution is drawn up the plate by capillary action.

- Remove TLC plate when the solvent reaches top line.

Note: During this approximately 20 minutes developing stage, compounds in the original spots
are being pulled through the silica gel.

Visualization of TLC results

This can be done by direct measurement or chemical derivatization.

• Direct measurement
- Allow the solvent to evaporate from surface of TLC plate.

- View results under UV light, look for grayish spots on the fluorescent green background.
- Mark spots with pencil while viewing under UV.

• Chemical derivatization
For components that do not fluoresce, a chemical substance is added to the separated component
spots that aid visualization under UV. This can be done either by iodine adsorption or oxidation
using sulphuric acid.

Interpretation of TLC results

- Determine retention factors (R f) for each spot detected. The solvent front is marked and a ruler
is held next to the plate to allow calculation of R f value.

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑠𝑝𝑜𝑡 ℎ𝑎𝑠 𝑚𝑜𝑣𝑒𝑑

𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 ℎ𝑎𝑠 𝑚𝑜𝑣𝑒𝑑

- Use Rf of reference spots to identify the other components. The spots are identified by comparison
of their Rf values with those of standard components.

Advantages of TLC

- Easy separation and visualization of compounds.

- Capacity to analyse multiple samples in a single run.
- Relative low cost.
Applications

- Separation of carbohydrates
- Separation of lipid into different classes
- Separation of triacylglycerols
Paper chromatography

It was first introduced by a German scientist Christian Friedrich Schonbein (1865). It is considered
to be the simplest and most widely used of the chromatographic techniques because of its
applicability to isolation, identification and quantitative determination of organic and inorganic
compounds. It is carried out mainly by the flow of solvents on specially designed paper.
PC is a variant of partition chromatography procedure in which the cellulose support is in the form
of a sheet or paper. Cellulose contain a large amount of bound water even when extensively dry.
Partitioning occurs between the bound water and the developing solvent.

In PC the mixture to be separated is spotted onto the paper and dried. Then the solvent flows along
the sheet either by gravity (descending chromatography) or capillary attraction (ascending
chromatography).

Procedure

- Place 25 mL of solvent in a 60 mL beaker. Cover the beaker and set it aside.

- Obtain a piece of chromatography paper and draw a line 1 cm from the bottom with a pencil.

- Place a small spot of each indicator (standard) on the line.

- Spot and label each of the indicators and the unknown.

- The spots should be about 2 cm apart.

-When the spots have dried, form the paper into a cylinder with the spots facing out. Staple the
edged together being careful to keep them straight and not allowing them to touch.

- Place the cylinder into 600 mL beaker and replace the cover. Be sure the cylinder is not touching
the sides of the beaker.

- Let the chromatogram develop until the solvent is 2 cm from top of paper.

- Remove chromatogram from beaker and immediately mark the solvent front with a pencil.

- Allow chromatogram to dry.

Take chromatogram to the hood and lightly mist it with water. Place it in the ammonia chamber.

- Remove the cylinder from ammonia chamber and unroll it. Immediately circle the coloured
regions with a pencil.
- Determine R f values for each colored spot in the knowns and unknowns.

- Use computed R f values to identify the components unknown.

Applications

- Separation of amino acids.

- Separation of mixtures of drugs.

- Identification of drugs.

- Analysis of metabolites of drugs in blood, urine.

- Separation of carbohydrates, vitamins, antibiotics, proteins e.t.c

Chromatographic performance

The ideal chromatographic process is one in which the components of a mixture form narrow
bands which are completely resolved from one another in as short a time as possible. The
performance of a particular chromatographic system can be assessed in the following ways.

a) Efficiency and resolution

The width of a band or peak is a measure of efficiency of the process. Resolution is the
ability to separate adjacent peaks of components with similar t R or Rf values. For column
chromatography, a plate number, N is used as a measure of efficiency. Assuming a
Gaussian peak profile, N is defined in terms of solute retention time, t R and peak width as
given by the standard deviation.
𝑡
𝑁 = ( 𝑅 )2
𝜎
In practice it is easier to measure baseline width of a peak, W b or the width at half height,
W½ and two alternative expressions derived from the above equation.
𝑡
𝑁 = 16(𝑊𝑅 )2 …………………………….. (a)
𝑏

𝑡
Or 𝑁 = 5.54(𝑊 𝑅 )2 ………………………… (b)
ℎ /2
Note: Some laboratories favour the use of equation (a) but some favour (b) on the grounds that
peak width at half height can be measured with greater accuracy than the base width. To make
valid comparisons of efficiencies, the same formula should be used always as the computed values
of N using each of the above formulae may differ considerably.

Measurement of chromatographic efficiency from a Gaussian peak

An alternative measure of efficiency which is independent of the length of a
chromatographic column is the plate height, H (or Height Equivalent of a Theoretical Plate,
HETP) and given by
𝐿
𝐻=
𝑁
Where L is the length of the column expressed in mm or cm.
Note: N is a dimensionless number so to ensure correct computation the units of 𝑡𝑅 and
𝑊𝑏 or 𝑊ℎ/2 must be the same.
Resolution, Rs is defined as difference between retention times of two adjacent solute
peaks, ∆𝑡𝑅 divided by their average base widths, (W1 + W2 )/2
2∆𝑡𝑅
𝑅𝑠 =
𝑊1 + 𝑊2
Measurement of the resolution of two adjacent Gaussian peaks
Note: Because of the Gaussian profile of the peaks, a 100 % separation is never attainable
but values of Rs approaching or exceeding 1.5 which is defined as baseline resolution are
satisfactory.
Question
1. A gas chromatographic method for the separation of a mixture of cyclohexane, t-
butanol and benzene on a 10 m capillary column gave the following data:

Parameter Cyclohexane t-butanol Benzene

𝑡𝑅 3 m 20 s 3 m 30 s 3 m 45 s

Wb 8s 9s 11 s

𝑊ℎ /2 4.6 s 5.1 s 6.2 s

Calculate
i) The plate numbers, using both plate number formulae for each solute
𝑡 𝑡
Plate numbers 𝑁 = 16(𝑊𝑅 )2 Or 𝑁 = 5.54(𝑊 𝑅 )2
𝑏 ℎ /2
 200 2 200 2
Cyclohexane 𝑁 = 16( ) = 10,000 𝑁 = 5.54( ) = 10473
8 4.6
210 2 210 2
t-butanol 𝑁 = 16( ) = 8711 𝑁 = 5.54( ) = 9393
9 5.1
225 2 225 2
benzene 𝑁 = 16( ) = 6694 𝑁 = 5.54( ) = 7296
11 6.2

ii) The plate heights

10000
Plate height 𝐻 =
𝑁

Cyclohexane H = 1.0 mm H = 0.95 mm

t-butanol H = 1.15 mm H = 1.06 mm
benzene H = 1.49 mm H = 1.37 mm
iii) Resolution between adjacent pairs of solutes
2 ∆𝑡𝑅
Resolution 𝑅𝑠 = 𝑊
1 +𝑊2

2(30−20)
Cyclohexane / t-butanol 𝑅𝑠 = 8+9
= 1.2
2(45−30)
t-butanol / benzene 𝑅𝑠 = = 1.5
9+11

Note: - The plate numbers are slightly higher when half height peak widths are used to
calculate them due to peak tailing increasing Wb hence comparison of efficiencies are valid
only if the same formula is used throughout.
- The cyclohexane and t-butanol peaks are not fully resolved (R s = 1.2) but the t-butanol
and benzene peaks have baseline resolution (R s = 1.5)

2. The following data are a liquid chromatographic column

Length of packing 24.7 cm

Flow rate 0.313 mL / min

Vm 1.37 mL

Vs 0.164 mL
A chromatogram of a mixture of species A, B, C, D provided the following data

Retention time (min) Width of peak base(Wb) (min)

Non retained 3.1

A 5.4 0.41
 B 13.3 1.07

C 14.1 1.16

D 21.6 1.72
Calculate
i) The number of pates from each peak. Ans A= 2775.49, B = 2472.04, C = 2363.97,
D = 2523.3
ii) The mean and standard deviation for N. Ans mean = 2533.7 SD = 0.2 x 103
iii) The plate height for the column. Ans 9.748 x 10-3
iv) The resolution for species B and C. Ans 0.717

b) Peak asymmetry (peak skew)

The concentration profile of a migrating solute is fundamentally symmetrical (Gaussian)
only if the solute distribution ratio, D remains constant over the concentration range of the
entire peak as shown by a linear sorption isotherm which is a plot of solute concentration
in stationary phase, C s against that in mobile phase, C m. However, curved isotherms
resulting from changes in the solute distribution ratio at higher concentration levels lead to
two types of peak asymmetry or skew described as tailing and fronting. Both tailing and
fronting are undesirable as closely eluting peaks will be less well separated and retention
data less reproducible. Where either occur, reducing the amount of solute chromatographed
will generally improve the peak shape but slow desorption may still cause some tailing.
 Sorption isotherms and the resulting peak profiles
c) Kinetic effects
The ultimate width of a peak is determined by the total amount of diffusion occurring
during movement of the solute through the system and on the rate of mass transfer between
two phases. Both diffusion and mass transfer effects are interdependent and complex being
made up of a number of contributions from different sources. Because they are kinetic
effects, their influence on efficiency is determined by the rate at which the mobile phase
travels through the system.

Effects of diffusion and mass transfer on peak width. (a) Concentration profiles of a solute
at the beginning of a separation. (b) Concentration profiles of a solute after passing some
distance through the system.
Qualitative and quantitative analysis
There are three approaches to qualitative chromatographic analysis.
- Comparison of retention data for unknown solutes with corresponding data for standards
(known substances) obtained under identical conditions. For planar chromatography (PC
and TLC) retardation factors, R f values for standards and unknowns are compared by
chromatographing them simultaneously so as to eliminate variations in laboratory materials
and conditions. For column separations, retention times, t R or volumes, V R are compared
by chromatographing standards and unknowns sequentially under stable conditions with as
little time between runs as possible.
- Spiking samples with known solutes
For column separations where samples are known to contain certain solutes, a comparison
is made between two or more chromatograms run under identical conditions. The first is
of original sample and subsequent ones are obtained after adding a spike of one of the
known solutes. Any peak in the original chromatogram that is of increased size in a
subsequent one can then be identified as the corresponding spiked solute.
- Interfacing the chromatograph with a spectrometer
For column separations, this provides spectral information for each separated solute in
addition to retention data. Spectra of unknown solutes can be compared with those in
computerized library databases or interpreted manually even when pure standards are not
available.

For quantitative chromatographic analysis, in addition to ensuring stable and reproducible

conditions for sample preparation and chromatography, further specific requirements must
be met, namely;
- the analyte (solute) must be identified and completely separated from other components
in the chromatogram.
- standards of known purity must be available.
- a recognized calibration procedure must be used.
For planar chromatography, solute spot areas or densities can be measured insitu, or the
solute spots can be removed, dissolved and measurements made by another analytical
technique such as UV spectrometry.
For column chromatographic separations, quantitation can be by peak area, peak height or
peak height x retention time. Integrated peak areas are directly proportional to the amount
of analyte chromatographed when working within the linear range of the detector and are
the most reliable. They can be measured by computing integrators by triangulation ( ½ base
x height of the triangle that approximates to the Gaussian peak profile) or by cutting out
and weighing peaks drawn by a chart recorder.
Detector responses for an analyte are established by the preparation of a calibration graph
using chromatographed standards with or without the addition of an internal standard by
standard addition or by internal normalization.

Internal standardization
A calibration procedure whereby a constant amount of a selected substance, the internal
standard is added to all samples and analyte standards alike compensates for variations in
sample size and other parameters. The ratio of the detector response for the analyte in each
standard to the corresponding response for the added internal standard is plotted on the y-
axis of a calibration graph against the mass or concentration of the analyte on x-axis.
Response ratios for the analyte and added internal standard in the samples can be used to
determine amount of analyte in the samples by interpolation of the graph. If only one or
two analyte standards are prepared the amount of analyte in a sample can be calculated by
simple proportion.
𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑟𝑎𝑡𝑖𝑜 𝑓𝑜𝑟 𝑠𝑎𝑚𝑝𝑙𝑒
=
𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑖𝑛 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑟𝑎𝑡𝑖𝑜 𝑓𝑜𝑟 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

Internal normalization
For some purposes, only the relative amounts of the analytes in a multicomponent mixture
are required. These are normalized to 100 or 1 by expressing each as a percentage or
fraction of the total. Internal normalization is of particular value in quantitative
chromatography where several components of a sample can be determined simultaneously
and absolute levels are not of interest. The relative composition is calculated from the
instrument response, peak area in the case of a chromatographic analysis, for each
component in the mixture using the formula
 𝐴𝑥
%𝑋𝑖 = × 100
𝜀𝐴𝑖
X i is one of n components
A is measured area or response
Example
The measured peak areas (using electronic integration with a computing-integrator,
computer and chromatography data processing software or geometric construction such as
triangulation, ½ base x height) and percentages by internal normalization, which must total
100 percent are given in table below.
Peak areas and percentage composition by internal normalization for a 5-component
mixture
Component Measure peak areas Relative percent
(arbitrary units)
1 167.8 35.9
2 31.63 6.8
3 108.5 23.2
4 80.63 17.3
5 78.38 16.8
Totals 466.94 100.0
167 .8
e.g for component 1, relative percent = × 100 = 35.9
466 .94

ION EXCHANGE CHROMATOGRAPHY

Ion exchange chromatography is a process by which ions held on a porous essentially insoluble
solid are exchanged for ions in a solution that is brought into contact with the solid. Mixtures of
similar charged ions are separated by using ion exchange resin. The mobile phase can be acid,
alkali or buffer. The stationary phase consists of beads made of polystyrene polymer cross linked
with divinyl benzene. It is used to covalently attach anions or cations onto it. The cross-linked
polymer (resin) has free phenyl groups attached to the chain which can easily be treated to add
ionic functional groups.
Principle
There is reversible exchange of ions between ions present in solution and ion exchange resin.
1. Injection

2. Adsorption

3. Elution
Classification of resins
a) According to chemical nature
Cation exchange resin – high molecular weight cross linked polymer containing acidic
functional groups added to the aromatic ring of the resin. These are divided into;
- strong acid cation exchangers which have sulphonic groups, RCH 2 SO 3-
- weak acid cation exchangers having carboxylate derived ions, RCH 2 COO -
Anion exchange resin – polymer containing positively charged solid support to which
negatively charged molecules are attracted.
- Strong base anion exchangers are fashioned using quaternary amine
- Weak base anion exchangers contain diethylamino ethane
The basic groups on the resin can be exchanged with other anions.
b) According to the source, they can be
- Natural eg clay, zeolite
- Synthetic i.e inorganic and organic resins
Organic resins are polymeric resin matrix composed of polystyrene (sites for exchangeable
functional groups) and divinyl benzene (cross linking agent) – offers stability.
Requirements of ion exchange resin
- Must be chemically stable.
- Should be insoluble in common solvents.
- Should have a sufficient degree of cross linking
- Swollenresinmust be denser than water.
- Must contain sufficient number of ion exchange groups.
Physical properties of ion exchange resins
- Cross linking; it affects swelling, strength and solubility
- Swelling; when resin swells polymer chain spreads apart. (polar solvents – swelling;
non polar solvents – contraction). Swelling is also affected by electrolyte concentration.
- Particle size and porosity; increased surface area and reduced particle size will increase
rate of ion exchange
Regeneration
Cation exchange resins are regenerated by treatment with acid, then washing with water.
Anion exchange resins are regenerated by treatment with NaOH, then washing with water
until neutral.
Practical requirements
- Column; glass, stainless steel or polymers of length: diameter ratio 20 : 100 to 100 : 1
- Packing the column; wet packing.
- Application of sample; after packing, the sample is added to the top of the column
using a syringe or pipette.
- Mobile phase; acids, alkali or buffer.
- Elution; components of the mixture separate and move down the column at different
rates depending upon the affinity of the ion for the ion exchanger. Eluates are collected
at different stages.
- Analysis of eluate; spectrophotometric, flame photometry, polarographic,
conductometric.
Generally, and in practical terms, a solution is passed through the resin, all H + will be
displaced by cations and the corresponding number of protons will be got in the eluate. By
calculation, the number of cations in solution can be got from number of protons in the
eluate. Passing a strong ac id through the column will displace the metal ions adsorbed on
the resin as cations. These cations will be removed at different times because they are
adsorbed with different strength.
Factors affecting ion exchange separations
- Nature and properties of ion exchange resins
If more cross linking, they are more rigid but swelling is less and separation of ions of
different sizes is difficult.
- Nature of exchanging ion
(valency of ion, size of ion, polarizability, concentration of solution, concentration and
charge of the ion). Highly charged ions bind more strongly than ions of lower charge. For
strong acid cation exchanger, the order
Al >Ba >Pb Ca >Ni >Cd >Cu >Co >Zn >Mg >Ag >K >NH 4 >Na+>H >Li+.
3+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ + + + +

Within a group of ions of similar charge those with a smaller hydrated radius or those that
are more polarizable bind more strongly. For strong base anion exchanger, the order is
SO42->I ->HSO4 ->NO3 ->Br->NO 2->Cl->HCO3 ->CH 3COO ->OH->F-. Ions of higher charge
and smaller hydrated radius bind more strongly than ions with lower charge and larger
hydrated radius. At low concentration, the extent of exchange increases with increasing
charge (Na+<Ca2+<Al3+).
Applications
- Softening of water
- Demineralization of water
- Purification of solutions free from ionic impurities
- Separation of inorganic ions
- Separation of sugars, amino acids
- Ion exchange columns can be substituted into the general HPLC instrument
Advantages
- It is a non denaturing technique. It can be used at all stages and scales of purification.
- Ion exchange can be controlled by changing pH, salt concentration and or the ion
exchange media.
- Serves as concentrating step. A large volume of dilute sample can be applied to a media
and the adsorbed ions subsequently eluted in a smaller volume.
Disadvantages
- Costly equipment and more expensive chemicals used.
- Turbidity should be below 10 ppm.

GAS CHROMATOGRAPHY (GC)

Is a chromatographic technique in which the mobile phase is a gas. In GC the sample which
may be a gas or liquid is injected into a stream of an inert gaseous mobile phase (often
called carrier gas). The sample is carried through a column where the sample’s components
separate based on their ability to distribute themselves between the mobile and stationary
phases.
Schematic diagram of a typical gas chromatograph

Mobile phase
The common mobile phases for GC are He, Ar, N 2 . The gas should be chemically inert
toward both the sample and stationary phase. The choice of carrier gas is determined by
the instrument’s detector.
Chromatographic columns
This is where the stationary phase is located. The column’s construction also influences
the amount of sample that can be handled, efficiency of separation, number of analytes that
can be easily separated and amount of time required for the separation. There are 2 types
of columns used in GC;
- Packed columns are fabricated from glass, stainless steel, copper or aluminium and are
2-6 m in length with an internal diameter of 2-4 mm. the columns are densely packed
with a uniform finely divided packing material or solid support that is coated with a
thin layer of the stationary liquid phase.
- Capillary or open tubular columns are fabricated from fused silica coated with a
protective polymer. They may be up to 100 m in length with an internal diameter of
approximately 150 - 300µm. capillary columns are of 2 types:
a) Wall coated open tubular columns (WCOT) contain a thin layer of stationary phase
coated on the capillary’s inner wall.
b) Support coated open tubular columns (SCOT), a thin layer of solid support eg
diatomaceous earth coated with a liquid stationary phase is attached to the
capillary’s inner wall.
Stationary phases
Selectivity in gas chromatography is influenced by choice of stationary phase. Elution
order in GLC is determined primarily by the solute’s boiling point and solute’s
interaction with stationary phase. Solutes with significantly different boiling points are
easily separated. Two solutes with similar boiling points can be separated only if the
stationary phase selectively interacts with one of the solutes. The main criteria for
selecting a stationary phase are that it should be chemically inert, thermally stable, of
low volatility and of appropriate polarity for the solutes being separated.
Sample introduction
The following considerations determine how samples are introduced to the gas
chromatograph.
- All constituents injected into the GC must be volatile.
- Analytes must be present at an appropriate concentration.
- Injecting the sample must not degrade the separation.
Preparing a volatile sample
To move through the column, the sample’s constituents must be volatile. Non volatile
solutes condense on the column degrading the column’s performance. Volatile analytes
can be separated from non-volatile matrix by solvent extraction.
Adjusting the analyte’s concentration
Analytes present at concentrations too small to give an adequate signal need to be
concentrated before analysis. When an analyte is too concentrated it is easy to over load
the column, there by seriously degrading the separation. In addition, the analyte may be
present at a concentration level that exceeds the detector’s linear response. Dissolving the
sample in a volatile solvent makes the analysis feasible.
Injecting the sample
Injections are made through a rubber septum using a micro litre syringe. The injector
block/port is heated to a temperature that is at least 50 o C above the sample component with
the highest boiling point. Capillary columns require use of specialized injections to avoid
over loading the column with sample. The common injection techniques include;
- Split injection, a technique for injecting samples onto a column in which only a small
sample enters the column.
- Splitless injection allows much higher percentage of solutes to enter the column.
For samples that decompose easily an on-column injection may be necessary. In this
method the sample is injected on the column without heating. The column temperature is
then increased volatilizing the sample with as low a temperature as practical.
Temperature control
Control of the column’s temperature is critical to attaining a good separation in GC. For
this reason, the column is located inside a thermostated oven. In an isothermal separation,
the column is maintained at a constant temperature, the choice of which is dictated by the
solutes. Normally the temperature is set slightly below that for the lowest boiling solute so
as to increase the solute’s interaction with the stationary phase.
One difficulty with an isothermal separation is that a temperature favouring separation of
low boiling solutes may cause unacceptably long retention times for higher boiling solutes.
Ovens capable of temperature programming provide a solution to this problem. The initial
temperature is set below that for the lowest boiling solute. As the separation progresses,
the temperature is slowly increased at either a uniform rate or in a series of steps.

Detectors for GC
The final part of a GC is the detector. The ideal detector has several desirable features;
- Must have adequate sensitivity e.g 10-8 – 10-15 g solute/sec.
- Must have good stability and reproducibility.
- Must give a linear response over a wide range of solute concentration.
- Must have a good temperature range from room temperature to at least 400 o C.
- There should be a concept of universality i.e the detector should be able to detect most
of the components in the sample.
- Must have a response time that is independent of the flow rate.
- High reliability and ease of use.
- Must have similarity in response toward all solutes, or alternatively a highly predictable
and selective response toward one or more classes of solutes.
- Non destructive of the sample.
1. Thermal conductivity detector (TCD)

Principle
-Based upon changes in thermal conductivity of the gas stream due to presence of analyte
molecules.
-The sensing element of TCD is an electrically heated element e.g platinum, tungsten
whose temperature at constant electrical power depends upon thermal conductivity of
surrounding gas.
-Resistance of wire is a measure of its temperature which depends upon the rate at which
surrounding gas conducts away energy.
Operation
- As the mobile phase exits the column, it passes over a wire filament.
- The filament’s electrical resistance depends on its temperature, which in turn depends
on thermal conductivity of mobile phase.
- When a solute elutes from the column, thermal conductivity of the mobile phase
decreases and temperature of wire filament and thus its resistance increases.
- A reference cell, through which only the mobile phase passes, corrects for any time
dependent variations in flow rate, pressure or electrical power, all of which may lead
to a change in the filament’s resistance.
Advantages
- Its simplicity.
- Its large linear dynamic range.
- Has general response to both organic and inorganic species.
- Non destructive character which permits collection of solutes after detection.
 Disadvantage
- Low sensitivity

2. Flame ionization detector (FID)

Principle
-Combustion of an organic compound in a hydrogen/air flame results in a flame rich in electrons
and ions.
-If a potential is applied across the flame, a small current (roughly 10 -9 – 10-12 A) develops.
-When amplified, this current provides a useful analytical signal.
Operation
- If there is nothing organic coming through the column, it is hydrogen burning in air.
- If one compound in the mixture being analysed starts to come through, it is mixed with hydrogen
and burnt producing ions and electrons that conduct electricity.
- Positive ions will be attracted to the cylindrical cathode and negative ions and electrons attracted
toward the jet which is the anode.
- At the cathode, positive ions will pick up electrons from the cathode and be neutralized.
- At the anode, any electrons in the flame will transfer to the positive electrode and negative ions
will give their electrons to the electrode and be neutralized.
- This loss of electrons from one electrode and gain at the other will result in a flow of electrons
in the external circuit from the anode to cathode i.e an electric current.
- The current can be amplified, creating a chromatogram.
Advantages
- High sensitivity
- Large linear response range
- Low noise
- Generally rugged (insensitive to changes in experimental conditions).
- Ease of use
- Response that is largely independent of changes in flow rate.
Disadvantage
-Destructive of the sample

3. Electron capture detector (ECD)

It has found use in environmental work because of its very high sensitivity to halogen-
containing components, which include most herbicides and pesticides.
Principle
A radioactive isotope usually 63 Ni in the detector cell emits beta particles. These collide
with carrier gas to create a pool of low energy free electrons. Two electrodes and a
polarizing voltage collect the electrons as current.
Operation
- The sample eluate from a column is passed over a radioactive β-emitter usually 63 Ni.
- The emitted electrons ionize the mobile phase often nitrogen resulting in the production
of additional electrons that give rise to an electric current between a pair of electrodes.
- In absecnce of organic species, a constant standing current between a pair of electrodes
results from the ionization process.
- Some molecules can capture low energy electrons to form negative ions. When such a
molecule elutes from the column, some of the electrons are captured and the collected
current decreases.
- This decrease in electric current serves as the signal which creates the chromatogram.
Advantages
- High sensitivity
- Non destructive of sample
Disadvantage
- Low linear response

4. Other detectors
The above three detectors do most of the GC work. They are augmented by others, mostly
element-specific or mass-selective.
Name Use
Nitrogen-phosphorus detector Nitrogen and phosphorus containing compounds
Flame photometric detector Sulphur and phosphorus containing compounds
Mass selective detector Identify components from mass spectra; when
combined with GC, the most powerful identification
tool.
Recording
After detection, the signals can be printed in terms of chromatogram. A chromatogram is
a plot of some function of solute concentration versus elution time or elution volume.

The small peak on the left is for a species that is not retained by the stationary phase. The
component traverse the column entirely in the mobile phase as expressed in the designation
of their retention time, t m( i.e dead time or void time). It is the time it takes for an unretained
species to pass through a chromatographic column.
The larger peak on the right is that of an analyte species. The time required for this zone to
reach the detector after sample injection is called retention time, t r. It is time between
injection of sample and appearance of a solute peak at the detector of a chromatographic
column. The analyte has been retained because it spends a time, t s in the stationary phase.

Applications of GC
- Environmental analysis
e.g analysis of numerous organic pollutants in air, water and waste water. The analysis of
volatile organics in drinking water for example is accomplished by separation on a capillary
column with a non-polar stationary phase.
- Petroleum industry
GC is suited for analysis of petroleum products including gasoline, diesel fuel and oil.
- Clinical analysis
Clinical, pharmaceutical and forensic laboratories make frequent use of GC for analysis of
drugs.
- Consumer goods
Many flavours, spices, foods, beverages and fragrances are readily analysed by GC.
- Quantitative calculations
In quantitative analysis, the height or area of an analyte’s chromatographic peak is used to
determine its concentration. Although peak height is easy to measure, its utility is limited
by the inverse relationship between height and width of a peak.
- GC is often teamed with infra-red or mass spectrometry to provide a powerful tool for
qualitative identification of individual solutes.

ELECTROPHORESIS
Electrophoresis is a separation technique in which analytes are separated based on their
ability to move through a conductive medium, usually an aqueous buffer in response to an
applied electric field. The technique was first developed by a Swedish Chemist Arne
Tiselius (1930s). it was applied to analytical separation of inorganic ions, amino acids,
drugs, vitamins, carbohydrates, proteins, nucleic acids, poly nucleotides etc. in the absence
of other effects, cations migrate toward the electric field’s negatively charged cathode, and
anions migrate toward the positively charged anode. More highly charged ions and ions of
smaller size which means they have a higher charge-to-size ratio, migrate at a faster rate
than larger ions or ions of lower charge. Neutral species do not experience the electric field
and remain stationary.
Forms of electrophoresis
Slab gel electrophoresis
The conducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs
are formed by pouring the gel between two plates separated by spacers
Capillary electrophoresis
The conducting buffer is retained within a capillary tube whose inner diameter is typically
25 – 75 µm. Samples are injected into one end of the capillary tube. As the sample migrates
through the capillary, its components separate and elute from the column at different times.
Theory of capillary electrophoresis
The sample is injected into a buffered solution retained within a capillary tube. When an
electric field is applied to the capillary tube, the sample’s components migrate as a result
of two types of mobility: electrophoretic mobility and electro osmotic mobility.
Electrophoretic mobility is a measure of a solute’s ability to move through a conductive
medium in response to an applied electric field. Cations move toward the cathode, anions
toward anode and neutral species which do not respond to electric field remain stationary.
The other contribution to a solute’s migration is electro osmotic flow, which is the
movement of the conductive medium in response to an applied electric field. Under normal
conditions the buffer solution moves toward the cathode, sweeping most solutes, even
anions toward the negatively charged cathode.
Electro osmotic mobility: when an electric field is applied to a capillary filled with an
aqueous buffer, buffer ions are expected to migrate in response to their electrophoretic
mobility. Electroosmosis occurs because the walls of the capillary tubing are electrically
charged. The surface of a silica capillary contains large numbers of silanol groups (Si-OH).
At pH levels greater than approximately 2 or 3, the silanol groups ionize to form negatively
charged silanate ions (Si-O -). Cations from the buffer are attracted to the silanate ions,
forming an inner or fixed layer. Other cations are more loosely bound forming an outer or
mobile layer. Together these two layers are called the double layer. Cations in the outer
layer migrate toward the cathode. Because these cations are solvated, the solution is also
pulled along, producing the electroosmotic flow.
Schematic diagram showing origin of electroosmotic flow
Instrumentation
Schematic diagram for capillary electrophoresis

The sample and source vial / reservoir are switched when making injections
Capillary tubes
Most capillary tubes are made from fused silica coated with a 20 – 35 µm layer of poly
imide to give it mechanical strength. The inner diameter is typically 25 – 75 µm, which is
smaller than that for a capillary GC column, with an outer diameter of 200 – 375 µm. The
narrow bore of the capillary column and relative thickness of the capillary’s walls are
important. When an electric field is applied to a capillary containing a conductive medium,
current flows through the capillary. This current leads to joule heating, the extent of which
is proportional to the capillary’s radius and magnitude of electric field. Joule heating is a
problem because it changes the buffer solution’s viscosity, with the solution at the centre
of the capillary being less viscous than that near the capillary walls. Since the solute’s
electrophoretic mobility depends on the buffer’s viscosity, solutes in the centre of the
capillary migrate at a faster rate than solutes near the capillary walls.
Injecting the sample
Two types of injection are commonly used; hydrodynamic injection and electrokinetic
injection. In both cases the capillary tube is filled with buffer solution. One end of the
capillary tube is placed in the destination vial and the other in the sample vial.
Hydrodynamic injection uses pressure to force a small portion of the sample into the
capillary tubing. To inject a sample hydrodynamically a difference in pressure is applied
across the capillary by either pressurizing the sample vial or by applying a vacuum to the
destination vial.
Electrokinetic injections are made by placing both the capillary and anode into the sample
vial and briefly applying an electric field.
NB: When a solute’s concentration in the sample is too small to reliably analyse, it may be
possible to inject the solute in a way that increases its concentration in the capillary tube.
This method of injection is called stacking. Stacking is accomplished by placing the sample
in a solution whose concentration strength is significantly less than that of the buffering
solution.
Applying the electric field
Migration in electrophoresis occurs in response to the applied electric field. The ability to
apply a large electric field is necessary because higher voltages lead to shorter analysis
times, more efficient separation and better resolution.
Detectors
Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the
more common detectors are those based on absorption of UV/VIS radiation, fluorescence,
conductivity, amperometry and mass spectrometry. Whenever possible, detection is done
“on-column” before the solutes elute from the capillary tube and additional band
broadening occurs.
Capillary electrophoresis methods
There are several different forms of capillary electrophoresis;
1. Capillary Zone Electrophoresis (CZE)
Is a form of capillary electrophoresis in which separations are based on differences in the
solutes’ electrophoretic mobilities. In CZE, the capillary tube is filled with a buffer solution
and after loading the sample, the ends of the capillary tube are placed in reservoirs
containing additional buffer solutions. Under normal conditions, the end of the capillary
containing the sample is the anode and solutes migrate toward the cathode at a velocity
determined by their electrophoretic mobility and electro osmotic flow. Cations elute first,
with smaller, more highly charged cations eluting before larger cations with smaller
charges. Neutral species elute as a single band. Finally, anions are the last species to elute
with smaller, more negatively charged anions being the last to elute.
CZE can also be accomplished without an electroosmotic flow by coating the capillary’s
wall with a non-ionic reagent. In the absence of electroosmotic flow only cations migrate
from the anode to the cathode. Anions elute into the source reservoir while neutral species
remain stationary.
Advantage: CZE provide effective separation of any charged species including inorganic
ions, organic acids, amines, proteins.
Disadvantage: CZE does not separate neutral species.

2. Micellar Electrokinetic Capillary Chromatography (MEKC)

A form of capillary electrophoresis in which neutral solutes are separated based on their
ability to partition into a charged micelle. In MEKC, a surfactant such as sodium dodecyl
sulphate (SDS) is added to the buffer solution. SDS has a long chain hydrophobic “tail”
and an ionic functional group, providing a negatively charged “head”. When the
concentration of SDS is sufficiently large, a micelle forms. A micelle is an agglomeration
of molecules containing ionic heads and hydrophobic tails which form into a structure with
a hydrophobic interior and hydrophilic interior. Because micelles are negatively charged,
they migrate toward the cathode with a velocity less than the electro osmotic flow velocity.
Neutral species partition themselves between micelles and the buffer solution. Because
there is partitioning between two phases, the term chromatography is used.
The elution order for neutral species in MEKC depends on the extent to which they
partition into the micelles. Hydrophilic neutrals are insoluble in the micelle’s hydrophobic
inner environment and elute as single band as they would in CZE. Neutral solutes that are
extremely hydrophobic are completely soluble in the micelle eluting with the micelles as a
single band. Those neutral species that exist in a partition equilibrium between the buffer
solution and the micelles elute between the completely hydrophilic and completely
hydrophobic neutrals. Those neutral species favouring the buffer solution elute before
those favouring the micelles.
Application: MEKC has been used to separate a wide variety of samples including mixtures
of pharmaceutical compounds, vitamins and explosives.

3. Capillary Gel Electrophoresis (CGE)

A form of capillary electrophoresis in which the capillary contains a gel enabling
separations based on size. In CGE, the capillary tubing is filled with a polymeric gel.
Because the gel is porous, solutes migrate through the gel with a velocity determined both
by their electrophoretic mobility and their size. The ability to effect a separation based on
size is useful when solutes have similar electrophoretic mobilities.
The capillary used for CGE is usually treated to eliminate electroosmotic flow, thus
preventing the gel’s extrusion from the capillary tubing. Samples are injected
electrokinetically because the gel provides too much resistance for hydrodynamic
injection.
Application: Separation of large biomolecules including DNA fragments, proteins and
oligonucleotides.
4. Capillary Electrochromatography (CEC)
Another approach to separating neutral species. In this technique, the capillary tubing is
packed with silica particles coated with a bonded, non polar stationary phase. Neutral
species separate based on their ability to partition between the stationary phase and the
buffer solution (which due to electroosmotic flow is the mobile phase).
Practical requirement of capillary electrophoresis
- Source vial, destination vial and capillary are filled with an electrolyte e.g aqueous
buffer solution.
- To introduce the sample, capillary inlet is placed into vial containing the sample and
then returned to the source vail.
- Migration of analytes is then initiated by electric field that is applied between the source
and destination vails and is supplied to electrodes by high voltage power supply.
- All ions positive and negative are pulled through capillary in the same direction by
electro osmotic flow.
- Analytes separate as they migrate due to their electrophoretic mobility and are detected
near the outlet end of the capillary.
- Out put of the detector is sent to a data out put and handling device e.g integrator or
computer.
- Data is the displayed as an electropherogram which reports detector response as a
function of time.
- Separated chemical compounds appear as peaks with different retention times in an
electropherogram.

*END*