CHE3704 Study Guide 0 B

CHE3143/1/2009-2011
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Prof. M.M. NINDI

Pretoria, 2008.
ii CHE3143/1/2009-2011
iii CHE3143/1/2009-2011
CONTENTS PAGE
AN OVERVIEW OF THE CHE314-3 MODULE ………… 1
STUDY TOPIC 1 ELECTRCHEMICAL METHODS ………… 7
Study Unit 1 Voltammetry ………… 8

1.1 Polarography ............... 9
1.2 Diffusion current ............... 10
1.3 Qualitative analysis ............... 11
1.4 Quantitative analysis ............... 11
1.4 Other Voltammetric methods ............... 11
STUDY TOIPC 2 SPECTROSCOPIC TECHNIQUES ……….. 12
Study Unit 2 Introduction to spectrometry ………… 12

2.1 Wave theory approach ............... 13
2.2 Particle theory approach ............... 14
2.3 Beer’s law ............... 15
2.4 Analytical spectrometry ............... 17
2.4.1 Application of Beer’s law ............... 18
2.5 Absorption spectra ............... 18
2.5.1 Limitation of Beer’s law ............... 19
2.6 Emission electromagnetic radiation ............... 19
2.7 Instrument ............... 20
2.8 Radiation source ............... 20
2.9 Wavelength selector ............... 21
2.10 Monochromator ............... 21
2.10.1 Holographic grating ............... 22
2.11 Analyzer ............... 22
2.11.1 Detector ............... 23
2.11.1 Semiconductor ............... 23
2.11.2 Diode Array detector ............... 23
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Study Unit 3 Ultraviolet/visible spectroscopic ………… 24
Study Unit 4 Infrared Spectrophotometry Techniques ………… 26

4.1 Instrumentation ............... 26
4.1.1 Radiation source ............... 26
4.1.1 Wavelength selector ............... 27
4.2 Detector ............... 27
4.3 Fourier transform instrument ............... 27
4.3 Applications of Infrared spectrometry ............... 28
4.3.1 Sample preparation ............... 28
4.3.1.1 Solid sample ............... 28
4.3.1.2 Liquid sample ............... 29
4.3.1.3 Gas sample ............... 29
4.4.1 Deviation from Beer’s law ............... 29
4.4 .2 Mid IR Reflection Spectrometry ............... 30
Study Unit 5 Flame emission spectrometry ………… 31

5.1 FES ............... 31
5.2 Atomisation process ............... 32
5.1.1 Instrumentation ............... 32
5.2 Radiation source ............... 32
5.3.1 Flame source ............... 32
5.3.2 Plasma source ............... 34
5.3.3 Arc source ............... 34
5.3.4 Arc spark source ............... 35
5.5 Qualitative analysis ............... 35
Study Unit 6 Atomic Absorption Spectrophotometry ………… 36

6.1 Instrumentation ............... 36
6.1.1 Primary radiant source ............... 37
6.1.2 Atomisation sources ............... 37
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6.1.2.1 Flame sources ............... 38

6.1.2.2 Electrothermal sources ............... 38
6.1.3 Dispersive system ............... 40
6.1.4 Detectors ............... 40
6.2 Interferences ............... 40
6.3. Background Absorption ............... 41
STUDY TOPIC 3 SEPARATION TECHNIQUES …………. 42

Study Unit 7 Introduction to Separation Techniques ............... 42
7.1 Principle of separation ............... 43
7.2 Definitions ............... 43
7.3 Chromatography ............... 44
7.3.1 Classification of Chromatography ............... 45
7.3.1.1 Adsorption ............... 45
7.3.1.2 Partition ............... 46
7.3.1.3 Ion exchange ............... 46
7.3.1.4 Gel permeation ............... 46
Study Unit 8 Solvent Extraction ............... 47

8.1 Principle of solvent extraction ............... 47
8.2 Distribution relations ............... 47
8.3.1 Partition coefficient ............... 48
8.3.2 Distribution ratio ............... 48
8.3 Completeness of extraction ............... 49
8.4 Extraction of covalent & neutral ............... 51
8.4.1 The effect of pH on extraction ............... 51
Study Unit 9 Theory of Chromatography ............... 53

9.1 Definitions of important parameters ............... 54
9.1.1 Retention time ............... 55
9.1.2 Dead volume ............... 55
9.1.3 Retention factor ............... 57
9.1.4 Separation factor ............... 58
9.2 Column efficiency ............... 60
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9.3 Resolution ............... 62

9.4 Band broadening process ............... 63
9.5 Sorption isotherms ............... 65
Study Unit 10 Thin layer chromatography ............... 68

10.1 Classification of TLC ............... 68
10.2 Classification based on mobile phase ............... 69
10.3 Criteria of chromatographic performance ............... 70
10.3.1 TLC theoretical plates ............... 70
10.4 TLC sorbents ............... 70
10.5 Solvent system ............... 71
10.6 Elutropic series ............... 71
10.7 Modes of development ............... 72
10.8 Detection systems ............... 72
Study Unit 11 Gas Chromatography ............... 73

11.1 GC major components ............... 73
11.1.1 GC mobile phase ............... 74
11.1.2 Sample introduction system ............... 75
11.1.3 Injection port in GC ............... 75
11.1.4 Column ............... 76
11.1.4.1 Process of separation ............... 77
11.1.4.2 Solid support ............... 77
11.1.4.3 Stationary phase ............... 77
11.1.5 Detector ............... 77
11.2 Analysis of sample by GC ............... 79
11.2.1 Qualitative analysis ............... 79
11.2.1 Quantitative analysis ............... 80
11.1.3 GC combination with other techniques ............... 80
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Study Unit 12 High pressure liquid Chromatography ............... 81

12.1 HPLC major components ............... 82
12.1.1 Solvent delivery system ............... 83
12.1.2 Pump ............... 84
12.1.3 Sample injection system ............... 85
12.2 Column ............... 85
12.2.1 Stationary phase ............... 86
12.2.2 Scavenger column ............... 87
12.2.3 Guard column ............... 87
12.2.4 Separation columns ............... 88
12.3 Mobile phase ............... 88
12.4 Detectors ............... 88
2.5 Analysis of samples by HPLC ............... 89
12.5.1 Qualitative analysis ............... 89
12.5.2 Quantitative analysis ............... 89
12.6 Other forms of HPLC ............... 90
STUDY TOPIC 4 SAMPLE PRETREATMENT ………… 91
Study Unit 13 Introduction Sample Preparation …........... 91

13.1 Analysis of Real Sample ............... 92
13.1.1 Sample ............... 92
13.1.1.1 Analysis of Sample ............... 92
13.2 Sample pre-treatment ............... 93
13.2.1 Isolation method ............... 95
13.2.2 Isolation & concentration ............... 95
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Study Unit 14 Preparing Samples for Analysis ………… 98

14.1 Dissolution of sample ………… 98
14.1 Derivatization ………… 100
14.1 Microwave assisted ………… 102
Study Unit 15 Samples Clean-up ………… 104

15.1 Solvent extraction ………… 104
15.2 Supercritical fluid extraction ………… 105
Study Unit 16 Samples Pre-concentration ………… 107

16.1 SLM ............... 107
16.2 SPE ............... 107
16.2.1 RP-SPE ............... 108
16.2.2 Normal-SPE ............... 109
16.2.3 Ion exchange ............... 110
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INTRODUCTION
Dear student,
Welcome to the study guide for Analytical Chemistry module, CHE314-3. In

this course we will cover the following topics;
i) Electrochemical techniques
ii) Spectroscopic techniques
iii) Separation techniques
iv) Analytical sample preparation or sample pre-treatment
This schematic diagram gives an outline of the content of each topic in the
study guide. It also gives the study units as chapters in Skoog et al (SWHC
8th, 2004) where the contents of the study unit(s) are discussed in details.
Electrochemical Spectro- Separation Methods Sample preparation

Methods scopic/metric or pre-treatment
Methods
CHAPTERS CHAPTERS CHAPTERS CHAPTERS

23 24 – 28 30 – 33 35 – 36
SWHC, 2004 SWHC, 2004 SWHC, 2004 SWHC, 2004
Figure 1: Overview of the CHE314-3 MODULE. Resource: Skoog, West,

Holler & Crouch (SWHC), 2004
About this Course
At level two you were introduced to analytical chemistry as well as sampling

techniques. At level 3 we would like to extend our knowledge of sampling to
sample preparation or pre-treatment in analytical chemistry. In addition we
will introduce you to the other three topics which should be very interesting
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and would allow us to build on to quantitative analysis which was introduced

at level 2. At this level we will demonstrate how instruments are used for
qualitative or quantitative analysis. The Study Guide is therefore designed to
probe you to see the connection between; i) electrochemical techniques, ii)
spectroscopic techniques, iii) Separation techniques and iv) sample
preparation or sample pre-treatment,
This should allow you to be able to answer the fundamental questions that we
as analytical chemist we are always asked; How much (quantitative)? or What
is it (qualitative)?, and what does it look like ( structural)?
Purpose of the module
The module is intended to introduce students to the sample pre-treatment and

instrumental analysis and how they could be applied in solving real life
problems. Students accredited with this module should be able to prepare
real samples for analysis, select suitable instrument to analysis sample to
generate quality data to address the fundamental question of how much and
what is it.
To answer the three questions a student would need to develop silks needed
for the task of acquiring qualitative (degree of excellence) and quantitative
(amount or numbers) information about the composition and structure of
matter or substance being analysed. You recall; that analytical methods are
often classified as classical or instrumental. Classical methods are also known
as wet chemical methods. Examples of classical methods include titration,
gravimetric methods, etc.
Instrumental methods can either be spectroscopic/spectrometric or

electrochemical. The interactions between the analyte (element of interest in
the sample) and energy give rise to analytical signals, which gives more
information about the analyte or species of interest. Your understanding of the
physical properties of molecules, atoms and ions you have gained in previous
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modules or courses would help you in making informed choices about the
analytical techniques to use.
Electrochemical properties can also be taken advantage of to provide
information to answer the two questions (how much and what is it).
Learning outcomes
By the end of the course the student should be able to understand and
demonstrate the following;
i) Real and ideal sample
ii) Sample preparation processes necessary for preparing real
processes involved in sample preparation and/or sample pre-
treatment
iii) Voltammetric methods of analysis of real samples
iv) Spectroscopic methods of analysis
v) Different forms of spectroscopic techniques, i.e. molecular (such
as uv/vis, ir, nmr & ms) and atomic spectroscopic (atomic
emission and absorption spectrophotometry)
vi) Separation techniques in solving analytical problems
vii) Theory of chromatography
viii) How all these instrumental methods can be use to solve real life
problems.
Self-study:
Self-study is required in open distance learning (ODL) environment as you

may by know. In this module I am introducing you to the use instrumental
techniques which link laboratory practicals with previous theoretical chemistry
knowledge. Therefore, you will probably need to refer back to your first and
second year modules while you are grappling with these applications. For
example, in understanding spectroscopic methods that deals with the
interaction between matter and radiation, you need to manipulate the
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knowledge of elements such as sodium, Na, and its electron configurations. In

level one, in order to further predict behaviour of electrons within the atom of
this element when radiated with electromagnetic radiations. We can then
deduce the matter’s qualities and quantities under specified conditions.
Therefore, you need to be exact, you need to control conditions and be able
to predict or solve analytical problems – This is indeed an art!
Analytical chemistry is a field that requires you to engage in regular periods of

self-study during which you attempt a number of problems to ensure that you
understand the principles and skills you need to develop.
In this Study Guide I will expand certain topics that I as the lecturer feel the
prescribed textbook has not covered sufficiently.
The Study Guide is organized under “STUDY TOPICS” which are further
subdivided into “Study Units”, that you will find in the “CONTENTS” page.
Also remember to make notes of the bibliographic detail of the ideas or
arguments you refer to, as much as you will note the details of the sampling
sites.
Resources for learning
This module uses the following resources:

• Textbooks (prescribed and recommended)
• Tutorial letters
• Other such as trustworthy internet resources we provide or you may
find.
• Articles that we provide or you may find.
• MyUnisa (this is a very important tool that will benefit you). I would
encourage you to register so that you can communicate with the other
students in your class as well as with me).
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The prescribed textbook is: Abbreviated as SWHC throughout the Study

Guide
• SKOOG, D.A., WEST, D.M., Holler, F.J. and Crouch, S. R. 2004.
Fundamentals of Analytical Chemistry. 8th Edition. Brooks/Cole-
Thomson Learning, International Student Edition: Belmont CA.
The recommended textbooks:

• The 7th edition (1998) of the prescribed textbook may also be used.
• Rouessac, F. & Rouessac, A. 2000. Chemical Analysis: Modern
Instrumentation Methods and Techniques. English Edition. John Wiley
& Son: NY.
How to use this study guide
This Study Guide should be used as a foundation to topics which are covered
in details in the textbook. As you work through the Study Guide, you should
go through the activities and responses. Each topic contains background
information and questions/learning outcomes which are meant not only to give
you practice and enhance your understanding but also to provoke your
thinking. It is recommended that you read through the chapter(s) in the
resource materials corresponding to each topic in the study guide, and then
work through the study guide.
NOTE:
This Study Guide supplements the textbook; it does not replace the textbook.
There are sections you will be expected to study in details which are not
extensively covered in this study guide. You are also encouraged to make use
of any other relevant resource materials, including articles, other textbooks,
and online resources such as Google Scholar (www.scholar.google.com). In
the event that you have serious problems understanding a particular topic,
please do not hesitate to contact me.
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Student evaluation
To pass this module, you are expected to complete a number of experiments,

which will contribute as part of your year mark. The assignments are listed in
the tutorial letter 101. The first assignment is compulsory. All the assignment
marks contribute 30 % towards your final mark. The examination mark
contributes 70 % of your final mark.
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TOPIC I Voltammetry
Outcome of this Topic
In Topic I of the study guide the students are expected to understand the
concepts of voltammetric electroanalytical method. By end of this topic the
students would be expected to able to apply voltammetric methods of analysis
to solve analytical problems.
1. Introduction
In the voltammetric process current is determined solely by rate diffusion of

the electroactive species in which a potential difference is applied in an
electrolysis cell. It comprises of a microelectrolysis in which the working
electrode potential is forced by means of external instrumentation to a known
potential-time function. The resultant current-potential relations and current-
time curves are used to obtain information on the solution composition. The
current obtained is directly proportional to the concentration of the
electroactive species.
Voltammetric techniques can be subdivided based on the shape of potential–

time curves; i) linear potential sweep (DC) voltammetry, ii) potential step
methods, iii) hydrodynamic methods, and iv) stripping voltammetry. These
techniques are particularly useful in the determination of many organic and
inorganic species (metals ions) which undergo reduction or oxidation reaction
even at trace levels.
In solution containing various metals, the will electrolyze only when; i) the
applied negative potential exceeds the reduction potentials, ii) the potential is
more negative than reduction potential. When the working electrode is a
microelectrode, the sample solution volume is relatively large and the analysis
time is very short. Hence, the bulk concentration of the analyte is not
changed significantly during the analysis time. This means we can carry out
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several analyses in the same solution. It is important that the solution should
be unstirred and temperature of the cell thermostatically controlled. In
addition a very high concentration of inert background or supporting
electrolyte is added to suppress migration of electroactive towards electrode
by electrostatic attraction.
One of the most common voltammetric techniques is Polarography.

Polarography is the study of the relationship between the current flowing
through the solution and the applied voltage to a working electrode, i.e.
dropping mercury electrode (DME). In addition to the DME as a polarisable
electrode, a Pt tip electrode can also be used. It is important to note that the
term Polarography is restricted to DME. There are two modes polarography;
i) normal pulse polarography and ii) differential pulse polarography.
1.1 Linear sweep voltammetry with DME (polarography)
A typical modern system consists of three-electrodes for aqueous work; i)

DME as the working electrode, ii) Pt wire as auxiliary electrode, and iii)
saturated calomel electrode (SCE) for reference (page 667-672, SWHC 2004,
2004). A polarogram is a resultant plot of current vs potential and it can be
used for quantitative and qualitative analysis. The current is measured during
continuously during a life of a growing Hg drop, producing a polarogram (see
Figure 5.1). A classical polarogram has three current regions, i) residual
current region, where the polarogram deviates from Ohm’s law due to
polarization of the cathode, ii) a charged transfer controlled region, in between
where qualitative (half wave potential, E1/2 or id/2) and quantitative (diffusion
current, id ) information are contained, iii) the residual current (ir)
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Figure 1.1: Typical polarogram for a reduction of hypothetical species A to

give a product P. The limiting current il is proportional to the
analyte concentration and is used for quantitative analysis.
Activity 1.1
What are the advantages of mercury electrodes for electrochemical

measurements?
Feedback 1.1
Read sections 23B-2 – 23B-4 of your assigned textbook.
1.2 Diffusion current
It is the limiting current observed in polarography when the current is limited

only by the rate of diffusion to the DME surface. The resulting current flow is
proportional to the rate of diffusion which dependant on the concentration
gradient;
i = k{C – Co} 1.1

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When the concentration gradient reaches maximum, the rate of diffusion and
current flowing in the cell reaches a limiting value;
Id = kC 1.2
What it means is means that any further increase in the potential will not
increase the current and the cell is said to be completely polarized and hence
id is directly proportional to the bulk concentration of the electroactive species
(see pages 672-678, SWHC, 2004).
1.3 Qualitative Analysis
Polarography can be used for qualitative analysis by utilising half wave

potentials (E1/2), which are characteristics to the species.
1.4 Quantitative Analysis
We have stated several times that id is proportional to the electroactive

species. It is important to first dissolve the sample in a suitable supporting
electrolyte that been adequately degassed. DME is used in determining
species in solution using Ilkovic equation;
Id = AnD1/2m2/3 t1/6C 5.3
Where;
Id = maxima current of drop (uA)
n = donates the number of faradays
D = diffusion coefficient for electroacive species
m = rate of flow of mercury (mg/sec)
t = drop time (sec)
C = concentration in the bulk solution (mM)
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A mathematical description of the diffusion current, in which il is measured at

the top of each oscillation just before the drop dislodges is given by the
following equation;
Id = 708nD1/2m2/3 t1/6C 5.4
m2/3t1/6 is constant for particular capillary and pressure

head of mercury
Activity 1.2
i) In polarography indicate how many regions of currents are present

and what analytical information is extracted from the polarogram?
ii) Under what conditions is a mixture analysis possible by classical

DC-polarography
Feedback 1.2
Read sections 23B-5 of your assigned textbook. Draw a typical a well labelled
polarogram.
1.5 Other Voltammetric Methods
Besides polarography using DME other voltammetric methods include (see

pages 689-703);
i) Differential Pulse polarography
ii) Square-wave polarography
iii) Cyclic voltammetry
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TOPIC II SPECTROMETRIC METHODS

In Topic II of the study guide the students are expected to understand the
concepts of spectroscopic methods of analysis. By end of this topic the
students would be expected to able to apply spectrometric methods of
analysis to solve analytical problems.
UNIT 2 Introduction to Spectrometric Techniques
2. Introduction
Spectrometric techniques forms the largest and the most important group of
techniques used in Analytical Chemistry and provides the following
information; i) Qualitative and ii) Quantitative.
All spectroscopic techniques depend upon absorption or emission of

electromagnetic radiation (EMR) characteristic of certain energy (E) changes
within the atomic or molecular system. Energy changes are associated with
complex series of discrete quantized energy levels in which atoms or
molecules are considered to exist. Therefore studies of such transitions
between energy levels can yield analytical information.
An understanding of the property of EMR and nature of atomic and molecular

E is essential. Spectroscopic/spectrometric methods are subdivided into two
types;
i) Molecular spectrometry, examples include infrared (IR),
ultraviolet/visible (UV/Vis), fluorescence, phosphorescence, nuclear
magnetic resonance (NMR) & mass spectrometry (MS)
ii) Atomic, examples include atomic absorption (AAS), flame emission

(FES) and X-ray (XRS)
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Electromagnetic radiation, as its name implies contains both the magnetic and
electric components and has its origins in atomic and molecular processes.
EMR is known for its dual properties, i.e. wave and particle properties.
Wave-like properties of EMR are reflection, refraction, diffraction &

interferences phenomena. Whereas its emission, absorption are best
explained by particle theory.
2.1 Wave-Theory of Radiation Approach
Radiant energy is viewed as periodic or oscillating, force field having electric

and magnetic components that maintain each other and interact with matter
(see Figure 24.1, SWHC pg 711). We will review the mathematical treatment
of radiation in this study Unit. Important parameters associated with the wave
are;
• Frequency (υ)
• Length (λ)
• Amplitude (Å)
Where:
υ = # of complete cycles on oscillation/sec
λ = linear distance between any two equivalent points in successive
cycles
Å = max values reached by vectors in a cycle
Å & υ are dependent on intensity of radiation and E associated with it

respectively. Hence
I α Å
E α υ
Where:
I = intensity
E = Energy
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Wave motion may be described in terms of λ or υ, which is the number of

waves passing a fixed point per unit time.
λυ = c (1)
υ = c/λ
Where:
c = velocity of light in vacuo
= 2.998 x 108 m s-1
Note:
E α υ (2)
E α 1/λ
2.2 Particle theory
A beam of radiant consists of a steam of discrete particles called photons,

each processing no Rest mass, but are viewed as particle of radiant energy
(see Figure 24.1, pg 711, SWHC, 2004)
The E of radiant is directly proportional to frequency
E = hυ = hc/λ
Where:
E = Energy of photon in Joule (J)
υ = frequency in herts (Hz), cycles/sec
h = Planck’s constant (6.62 x 10-34 J. sec)
ν = wavenumber
Combine Equation 1 & 2

E = hc/λ
= (6.62 x 10-34 J.s)(2.998 x 108 m s-1) (1 nm)
λ nm (10-9 m)
= 1.985 x 10-16 J
15 CHE3143/1/2009-2011
Also given in eV, Use conversion factor - !!!!!!!
2.3 BEER’S LAW
• The amount of light absorbed by a species in solution will depend on;

– # of absorbing species (ions or molecules)
– the pathway (light pathway) of the photon beam
More light will be absorbed as the concentration is increased. Similarly the

longer the path followed by photon beam through the solution, the more
photon absorbed. The third factor that governs the amount of light absorbed
is probability of photon being absorbed & causing an electronic transmission
in a chemical species.
• These facts are the basis of fundamental law of spectrophotometry
(Lambert-Beer Law or Beer’s Law)
• This law states that the amount of light; visible or UV or IR radiant

energy or transmitted by solution is an exponentially function of
concentration of absorbing substance present and the sample path
length.
• For discussion See Fig 24.7 (page 718)
• If you divide solution into small, equal sections, the ǻ in radiant power
(ǻP) will depend on a number (#) of absorbing species in the section
(ǻN)
• Amount of light absorbed by each section will depends on a number of

absorbing species in that section & proportional to radiant power of
light entering that section
ǻP = - kP (ǻN) (1)
- ǻP/ ǻN = kP (2)
Where;
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k = proportionality const
If the sections are infinitely small, Equation 1 may be written in differential

form (see SWHC pg 721)
The number of absorbing particles will depend on;

i) Concentration of absorbing species in sol
ii) Thickness of absorbing solution traversed by light beam
• Convert Equation 3 to base 10 and combine with Equation 4
log P/Po = - εbC (3)
log Po/P = εbC (4)
Where;
ε = proportionality constant (Molar absorptivity)
b = pathlength, in cm
C = concentration of analyte
NOTE: Beer’s law is only applicable to monochromatic radiant E
A = εbC
Absorptivity & Molar Absorptivity
ε = depends on the units used for expressing the
concentration.
C = Concentration Units such as ppm, mg/L, g/100 ml, M are
used
ε (episilon) is commonly used instead of a, when concentration is
expressed as molarity (M)
A = εbC (units of ε is L mol-1 cm-1)

17 CHE3143/1/2009-2011
Transmittance & % T
Given by T = P/Po = - log T = εbc
2.4 Analytical Spectrometry
The set of E levels is associated with a particular substance is a unique

characteristic of that substance, and determines the frequencies (υ) at
which EMR can be absorbed or emitted.
Activity 2.1
i) What information do you expect to extract from spectroscopic

techniques?
ii) A solution contains 1.25 mg of KMnO4 per litre. When
measured in a 1 cm cell at 525 nm, the transmittance was 0.42.
When measured under similar conditions 500 nm, the
transmittance was 0.49. (a) Calculate the molar absorbance A
at each wavelength, (b) Calculate the molar absorptivity at each
wavelength, (c) what would T be if the cell length were increased
to c cm?
Feedback 2.1
Qualitative and quantitative information can be extracted from spectrometric

methods. How is this information obtained would direct you to the (i).
Question (ii) should be simple once you have finished reading the section on
quantitative analysis
Note:
• Sample maybe itself fulfil the function of a
• Also positioned between a & b
• Or between b & c
18 CHE3143/1/2009-2011
2.4.1 Application of Beer’s Law
The Beer’s law (A = εbC) is very useful in quantitative analysis. It is therefore
important that you understand it well. We can calculate molar absortivity of a

species provided by measuring the absorbance of known concentration using
a known cell path. The molar absorptivity (ε) is a parameter that is condition
dependent and therefore in most cases we do not know it. Conditions of
published ε date are specified and therefore their use should under the same
conditions. In real life it is preferable to measure standard solution using the
same conditions (solvent, temperature, etc) that are used for the analyte, and
then the molar absorptivity can be calculated. The calculated value can then
be used for the calculation involving the sample. However, this approach is
also not popular. It is preferable to prepare serial standard solutions and
measure their absorbance. A calibration curve is constructed which is a plot
of absorbance of standard solution against concentration. As each solution
would obey the Beer’s law we would expect the plot to give a linear line. If the
analyte in the unknown solution is measured under the same conditions,
making sure that its absorbance is not higher than the most concentrated
standard. The unknown concentration can be determined by extrapolating
from the calibration curve. This is a very important application of
spectrometric methods. The urine sample you submit at the clinic or hospital
laboratory for determination of lead, or sugar for example uses this very
principle - !!!!!!!
Most importantly Beer’s law can be used to determine components present in

a mixture. The total absorbance for multi-components that do not react with
each other is the sum of the individual absorbance;
ATotal = ε1b1c1 + ε2b2c2 + ε2b2c2 + …………. + εnbncn
2.5 Absorption Spectra
The spectrum is the result of a plot of absorbance against wavelength. When

molecules are involved, a molecular absorption spectrum is produced. Figure
19 CHE3143/1/2009-2011
24.12 shows electronic transition showing infrared (IR), visible (Vis) and
ultraviolet (UV).
2.5.1 Limitation of Beer’s Law
Beer’s law is applicable only when the solutions are dilute. In high
concentrated solutions Beer’s law fails, i.e. a non-linear curve of absorbance
plotted against concentration is obtained. It is important to know why and how
this occurrence can be minimized or eliminated. The observed deviations are
classified into three types;
i) Real deviation
ii) Instrument
iii) chemical deviation
For further reading (see SWHC pg 729-734)
Activity 2.2
Briefly explain how you would carry out quantitative analysis?
Feedback 2.2
The approach used is important because all the methods used for quantitative
analysis could use the same or similar approach for quantitative analysis.
2.6 Emission of electromagenetic radiation
In absorption spectrum, is the product of atoms or molecules absorbing EMR

resulting that induces electronic transition, that show the relationship between
of the energy absorbed against number of excited electrons. An absorption
spectrum is observed. However, electrons in the excited state are not stable
and have to lose the energy and drop back to ground state, the extra energy
is therefore given up form of an emission spectrum.
20 CHE3143/1/2009-2011
2.7 Instrumentation
A typical instrument would consist of source, monochromator, cell, detector

and recorder. The instrument used for absorption and emission have more or
similar major components. The arrangement of components of the instrument
will dependent on the technique; i.e. Absorption spectrometer is as shown
below (Figure 2.1).
2.8 Radiation Source
A suitable radiation source must be a beam that has sufficiently powerful

energy for the molecules to undergo the necessary process to produce
characteristic interaction, i.e. electronic transition. For UV/Vis region the most
common sources are D2 and tungsten/halogen lamps (see pg 747-750
SWHC, 2004). It is important that the energy of the source induce the
expected interaction to the sample molecules or atoms. Radiation sources
can be classified as; i) line sources, which give atomic spectral line and ii)
continuum source, which emit radiation whose intensity varies smoothly over
an extended range of wavelengths (λ). Radiation sources have two basic
requirements; i) to provide sufficient radiation energy over the wavelength
region where absorption is measured, ii) to maintain a constant intensity over
the time interval during which measurement is made. There are two types of
lamps available;
- Hydrogen and Deuterium lamp for the ultraviolet region
- Incandescent filament or tungsten lamps
Radiation Monochromator Sample cell Detector Recorder

Source
Figure 2.1 Show the schematic arrangement for a typical UV/Vis

Absorption spectrometer. Note that the radiation passes
through the monochromator before passing through the
sample cell.
21 CHE3143/1/2009-2011
Activity 2.3
What is the significance of the arrangement of the radiation source in the

UV/Visible spectrometer?
Feedback 2.3
The arrangement is particularly important for one of the principal analysis

approach.
2.9 Wavelength selector
The source used for the UV/Visible spectrometry must be of known energy not
only for quantitative analysis but also for qualitative analysis. The function for
the wavelength selector is to isolate narrow desired wavelength band of
radiation source. There are basically two approaches used to achieve that;
i) Filter
ii) Monochromator using deflecting prism or diffraction grating
The main purpose of wavelength selector is for the selection of UV, Vis, IR
regions of the electromagnetic spectrum.
i) Filters
• Used in simple instruments and these are rather poor with respect to
selectivity
• A filter may be employed which selectively absorb all except the
required range of frequencies (Filter Photometry)
2.10 Monochromators
Monochromators generally consists of the following; i) entrance slit, ii)

collimating lens, iii) prism or reflection grating, iv) focusing lens and v) exit slit.
{It is extremely important that you draw a diagram of monochromator in order
22 CHE3143/1/2009-2011
to ensure you understand how it functions, see {pg 751, Figure 25.6 SWHC
2004}.
Diffraction grating is made of a surface ruled with a large number of parallel

grooves that are approximately one-light λ wide. For example grating for
UV/Vis region contains somewhere in the order of 300 – 2000 grooves/mm.
The gratings are replica of a master grating which requires a lot of time and
skills to fabricate.
• Light striking the grating is directed so that different λ come off at
diffraction angles
• Rotating the grating allows radiation of desired λ to be selected
• Standard diffraction grating maybe of either the;
• Transmission
• Reflection
• Both allow significant amount of stray light to reach detector at high
absorbing values
2.10.1 Holographic grating
• Newer holographic grating with reduced degree of stray light so well as

transmit more radiant energy reaches the detectors
2.11 ANALYSER
• Function of the component is to present so called monochromatic

radiation to the detector
• It separate or disperse the radiation so that selected frequencies
corresponding to a particular energy transition within the sample maybe
individually examined
23 CHE3143/1/2009-2011
11.4.1 Detectors
• Simple type of detector for UV/Vis spectral region is the photodiode

(see Figure 25.12, pg 763-765, SWHC 2004)
• Two classes:
– Vacuum photodiode (phototube)
– Semiconductor phototube
• The vacuum photodiode or phototube contains a cathode that acts as
photosensitive element
• The blue-sensitive phototube is standard equip on routine
spectrophotometers
• Photomultiplier is an important detector & contains a photosensitive
cathode & multiplying chain of dynodes & anode (Fig 25.12)
2.11.2 Semiconductor detector
• Semiconductor phototube contains a solid p-n junction as the

photosensitive element
• A photon striking p-n junction generates electron (e-) holes pairs,
producing current.
• This type of detector is used in Spectronic 21 series
2.11.3 Diode Array detector (DAD)
• Consist of silicon integrated circuit (IC) chips incorporating up to one or

two hundred pairs of Photodiode (PD) and capacitors
• Each PD measures about 0.05 x 0.05 mm and is sensitive to radiation
over a wide range (200 – 950 nm)
• An array of several 100s PDs on one or two pieces chips, positioned to
receive dispersed radiation from monochromator, allow a wide range of
λs to be monitored simultaneously without mechanical scanning.
• Current (I) generated by each PD proportional to Intensity of radiation
it receives
24 CHE3143/1/2009-2011
UNIT 3: Ultraviolet/Visible Spectroscopic Techniques
3. Introduction
The UV/Vis region spectrum is found roughly around 190 - 950 nm. The
instrument used for observing this region has been described in the previous
section (Unit 2). This region is very important analytical especially for
quantitative analysis. Limited qualitative information can also be obtained
from this region. Compounds that have ability to absorb UV/Vis radiations are
said to contain a chromophore. Chromophores are part of the molecule
responsible for light absorption. However, our eyes see the colour that the
substance has not observed from white light (see Table 3.1).
Table 3.1 Colours of visible light
wavelength Colour absorbed Colour observed

380-420 Violet Green-yellow
420-440 Violet-blue Yellow
440-470 Blue Orange
470-500 Blue-green Red
500-520 Green Purple
520-550 Yellow-green Violet
550-580 Yellow Violet-blue
580-620 Orange Blue
620-680 Red Blue-green
680-780 Purple Green
For example bromophenol blue has visible absorption max at 591 and is seen
as blue. In quantitative analysis the sample in cuvette (cell) is measured at a
specific wavelength and also the blank sample, which consist of the solvent
without the sample is measured at the same wavelength. The difference
between the absorbance of sample and that of the blank is taken as the actual
measure of the sample. The process is usually carried out in two ways; i) use
25 CHE3143/1/2009-2011
of a single beam instrument and ii) double beam instrument. Using the single
beam is more time consuming whereas the double beam instrument is more
suitable. The figure below shows a typical beam instrument. In a double
beam Instrument, the radiant absorbed or emitted by sample is automatically
compared with that associated with blank or standard solution. This process
is responsible for the correction of matrix effects or Instrument noise or drift.
Figure 3.1 Double beam instrument showing source, monochromator,

silvered mirror to slit the beam into two and second lens to
reflect the second beam through the Ref cell.
In text book three examples of instrumental arrangements are shown (see pg

Figures 25.19 - 25.21, 772-774, SWHC 2004). Please study them well and
practice to draw them. You will need to understand them well to explain each
instrumental function.
Activity 3.1
What do we understand by the following terms; i) Spectroscopy, ii)

Spectrophotometry and iii) Spectrophotometer
Feedback 3.1
Please read section 25B (pg 771, SWHC, 2004)

26 CHE3143/1/2009-2011
Unit 4 Infrared Spectrophotometry Techniques
4. Introduction
The infrared (IR) region spectrum is found roughly around 250 – 4000 cm-1
(2.5 – 40 µm). The most commonly used is 670 – 4000 cm-1. The principle
of infrared spectrometry is based on the ability of molecules to absorb
electromagnetic (EM) radiation in the infrared region of the EM spectrum.
This absorption results in changes of their vibrational, bending, rotational and
twisting motions of atoms in molecules. This spectroscopic technique is
useful for qualitative analysis of organic molecules. We will need to know
characteristic functional absorption groups in order to use the technique for
qualitative measurement. Tables of infrared spectral correlation charts that
can be used for structural identification of organic molecules have been
published. Polyatomic molecules absorb energy due to the presence of a net
dipole moment during a particular vibration. The resultant absorption spectra
are often very complex. However, IR has limitation with respect to
quantitative analysis.
4.1 Instrumentation
A typical infrared instrument would consist of source, cell, monochromator,

detector and recorder. The IR absorption instrument has more or less similar
major components as we have seen in other absorption and emission
spectrometric instruments. There are three types of IR absorption
instruments;
- Dispersive spectrophotometers
- Fourier Transform spectrometers
- Non dispersive photometers
The arrangement of components of the instrument will dependent on the

technique. Infrared absorption spectrometer is shown below;
4.1.1 Source
Infrared spectrometers use a Nernst glower as a radiation source. This

source consists of zirconium and yttrium oxides that when heated electrically
27 CHE3143/1/2009-2011
would generate IR radiation. Other sources include, i) the globar, ii)

incandescent wire source, iii) the mercury arc and tungsten filament and iv)
carbon dioxide laser source.
Radiation Sample cell Monochromator Detector Recorder

Source
Figure 14.1 Schematic diagram of a typical infrared spectrometer
4.1.2 Wavelength Selector
In a dispersive instrument, a source provides a beam of infrared radiation

which is focused through a sample where selective absorption takes place.
The beam that comes out of the sample cell follows the optical path into the
monochromatic where radiation is dispersive by a series of different granting
for a variety of frequency ranges.
4.1.3 Detector
The infrared traducers are divided into three general types; i) pyro-electric
transducers, ii) photo-conducting transducer iii) thermal transducer.
Dispersive instruments use the deuterated triglycine sulphate (DTGS)
detector. Fourier Transform-infrared (FT-IR) instrument uses mercury
cadmium telluride (MCT), a high sensitive and fast response detector. These
types of detectors are based on temperature-dependent voltage that is implied
and measured.
4.2 Fourier Transform Instruments
This is a very interesting instrument that has replaced the use of dispersive IR
spectrophotometers in particular for the mid- and far IR measurements.
However, in this course we will not discuss the instrumentation of FTIR.
28 CHE3143/1/2009-2011
4.3 Application of infrared spectrometry
The most commonly used region is the mid-IR (670- 4000 cm-1 0.75 – 0.25
µm). Sample preparation is important for IR analysis.
4.3.1 Sample preparation
In the previous discussed techniques, i.e. UV/Vis we have seen that dilute
solution are used for analysis. However, in IR due to the limitation of the
solvents over the region of interest sample preparation is a major part of the
analysis. It is the most time consuming and difficult step. Infrared is one of
the few techniques that can analyse samples in three different forms; solid,
liquid and gas. An important requirement for IR sample holder is that the
material used must be transparent to IR radiation. The lack of rugged window
material for cuvette that is transparent and also inert over the IR region poses
challenges in IR sample preparation. Therefore the selection is limited to
certain salts such as NaCl or KBr and on the wavelength range to be
examined.
4.3.1.1 Solid samples
Three methods are available for preparing solid for IR analysis. The methods
are as follows;
- The sample is ground into powder. The powder is then made into a
thick slurry, or mull by grinding it with a greasy, viscous liquid such as
Nujol (paraffin oil) or chlorofluorocarbon greases.
- In this method powered KBr is mixed with a finely ground solid sample.
The mixture is then compressed under pressure (at least 25, 000 psig)
to construct a small disk of about 1 cm in diameter. The sample is
measured directly since the formed disk is transparent to IR radiation.
- In the third method the sample in solution is deposited on the surface of

KBr or NaCl cell. The deposited solution is evaporated to dryness.
29 CHE3143/1/2009-2011
The sample is analysed by irradiating IR source through the thin

deposit.
4.3.1.2 Liquid samples
The sample in solution is analysed by placing a cell made of NaCl or KBr. In

a double beam instrument a blank cell will contain solvent used to dissolve the
sample.
N.B. It is important to ensure that cell does not come into contact with water
since it is made out of material that is water soluble. In addition organic
solvents used to dissolve the sample should be dried before placing the
sample into the IR cells.
4.3.1.3 Gas sample
Low boiling point or gas samples are analysed in special gas cells. These
cells can be a few centimetres to 10 metres long.
4.4 Quantitative analysis
Technically IR can also be used for quantitative analysis. However, due to the
complexity of spectra and the narrowness of the absorption band there are
major challenges and limitations of the IR instrumentation. This observation is
true especially with the dispersive infrared instrumentation.
4.4.1 Deviation from Beer’s Law
Deviations are more visible in IR than in UV/Vis due to the narrowness of the
relative IR absorption bands. In addition the dispersive instruments have
lower intensity of the radiant source and less sensitive traducers.
30 CHE3143/1/2009-2011
4.4.2 Mid IR Reflection Spectrometry
This approach has found use in solid samples that are usually difficult to
handle such as polymers films, agricultural products, etc. There are four
approaches to reflection radiation;
- Specular reflection
- Diffused reflection
- Internal reflection
- Attenuated total reflection (ATR)
Detail discussion of this topic would be covered in level 4.

31 CHE3143/1/2009-2011
Unit 5 Atomic Spectrophotometry Techniques
5. Introduction
Atomic spectroscopic techniques involve interaction of gaseous atoms with

EMR with the UV/visible region. There are three atomic methods observed in
UV/Visible region;
- Absorption
- Emission
- Fluorescence
In this Unit we will only consider two methods, i.e. atomic emission and
absorption.
5.1 Flame Emission Spectrometry
The technique involves the emission of electromagnetic radiation in the

ultraviolet and visible regions of the spectrum by atoms after electronic
excitation in a flame. The emission produced when a solution of metal is
introduced into a flame is measured. The wavelength (λ) indicates the
presence of an element and the intensity the amount of the element present.
The method is particularly useful for the group I and II elements of the periodic
Table.
Activity 5.1
Why is atomic flame emission spectroscopy useful for group I and II

elements?
Feedback 5.1
Review periodicity that you covered at level I

32 CHE3143/1/2009-2011
5.2 Process of atomisation
Neutral atoms are a requirement for the atomic spectroscopic techniques. It is

therefore essential to transform sample in solution to gaseous atoms and ions.
Figure 5.1 shows steps followed to convert sample in solution to ground sate
atoms (see pgs 843-854, SWHC 2004).
Nebulizer, where Flame along with Air

Sample in solution solution is converted or O2 & fuel gas
into a mist or aerosol
Atoms
Relaxation of excited atoms,

Atoms Thermal excitation by
Ground State Atoms hence emission of radiation flame of atoms to higher
characteristic atom level
Figure 5.1: The process of atomisation of elements in solution
5.3 Instrumentation
The flame emission spectrometric instruments consist of a nebulizer or

burner, wavelength selector (filter or grating), and detector.
5.3.1 Radiation source
Radiation sources in atomic emission spectroscopy can be classified into two

categories; i) sources used in solution analysis i.e. flames and plasma, ii)
sources used for solids samples, e.g. arc, spark arc
5.3.1.1 Flame source

33 CHE3143/1/2009-2011
The source of atoms is produce by a burner, which has two functions; i)

vaporise the sample, ii) introduce the sample to the flame for atomization. A
flame is an exoenergetically-controlled chemical reaction between fuel and
oxidant. Examples of fuel are acetylene and/or propane and of oxidants are
air, oxygen and/or nitrous oxide. The flame of the burner also should excite
the atoms and cause them to emit radiant energy. The stability of the
emission is important for analytical purposes and it should be maintained for a
period of about 1 – 2 min. There are two commonly used burners; i) total
consumption burner, ii) Lundegardh burner
In a typical consumption burner, the sample must be in solution form and it is

aspirated completely into the flame. Air or oxygen aspirates the sample into
the base of the flame. In the Lundegardh burner the sample in liquid is first
aspirated into a spray. Larger droplets coalesce on the side of the spray
chamber and drain away. The small portion (~ 5 %) of very small droplets of
the sample reach the flame and are easily decomposed. The process of
atomisation of this type of burner is very efficient. The temperature of about
1700 – 2400 oC occurs with various fuels in combination with air as the
oxidants. At these temperatures only easily decomposed samples such as
the alkali and alkaline earth metals can be atomised. Table 5.1 shows the
properties of Flame.
Table 5.1 Characteristics of some typical gas mixtures
Fuel Oxidants Temperature, oC Max Burning Velocity

Natural gas Air 1700 - 1900 39 - 43 cm sec-1
Natural gas Oxygen 2700 – 2800 370 - 390 cm sec-1
Hydrogen Air 2000 – 2100 300 - 440 cm sec-1
Hydrogen Oxygen 2550 – 2700 900 - 1400 cm sec-1
Acetylene Air 2100 – 2400 158 - 266 cm sec-1
Acetylene Oxygen 3050 – 3150 1100 - 2480 cm sec-1
Acetylene Nitrous oxide 2600 - 2800 285 cm sec-1
34 CHE3143/1/2009-2011
5.3.1.2 Plasmas
Plasma is described as ionised gas that is macroscopically neutral, i.e. have

equal number of negative (e-) and positive ions. In plasma an external energy
in the form of electrical energy is supplied in order to ionise the gas and to
sustain the plasma. Part of this energy is will be transmitted to the sample to
atomise and excite it. Temperatures of about 8 000 – 10 000 K are produced
by within the plasma. Three examples of plasma are given;
i) direct current plasma (DCP)
ii) inductively coupled plasma (ICP)
iii) microwave induced plasma (MIP)
Inductively coupled plasma is the most commonly used of the three plasmas.
A typical ICP source is a torch (see Figure 10.1, SHC 6th 2007, pg 255). A
high frequency is used to produce a high frequency field through an induction
coil. Argon gas at 10 – 15 L min-1 is used to generate the plasma. It is
important to draw the schematic diagram of the ICP torch and to briefly, but
informatively describe it.
5.3.1.2 Arc
The use of electric arc as a source for volatilisation and dissociation of liquid
or solids and finally excitation of atomic species goes back to decades of
years. An arc is a stable electrical discharge that forms from a high current
and low voltage between two or more electrodes. In modern analytical
chemistry these sources are no longer popular due to instability of electrical
discharges and matrix effects that limits precision of the technique. There are
three types of electrical sources; DC arc, the AC arc, and AC spark. The most
commonly used material for electrodes is graphite due to its interesting
characteristics, such as minimum contamination, excellent conductivity and
refractive properties and low cost.
35 CHE3143/1/2009-2011
5.3.1.4 Arc spark
A spark discharge on the other hand is formed by applying a high voltage and
low current between two electrodes. It is customary to have one electrode
consisting of the sample to be analysed whereas the other is usually
constructed of tungsten. The life time of the spark is in the order of few µs.
Temperatures of 4 000 – 10 000 K are achievable for both electrical arch and
spark sources.
Other sources that have been used include;

i) glow discharge lamps (GDL)
ii) low pressure discharge
iii) laser produced plasma
5.4 Quantitative Analysis
The intensity of the characteristic wavelength is proportional to the

concentration of the emitting element in the sample. The concentration is
determined by one of two methods; i) calibration curve or ii) standard addition.
5.5 Qualitative Analysis
The position of the emitted wavelength is used for qualitative analysis,

because elements emit characteristic wavelengths.
36 CHE3143/1/2009-2011
Unit 6 Atomic Absorption Spectrophotometry Techniques
6. Introduction
In contract to the atomic emission spectrometry (AES), atomic absorption

spectrometry (AAS) is a relatively new analytical technique. It was developed
by Welsh in 1953, and it is known for its easy of use and relatively minimum
interferences. Atomic absorption was until recently the most widely used
technique for trace metal analysis. In many laboratories the inductively
coupled plasma atomic emission spectrometry and inductively coupled
plasma mass spectrometry have superseded the use of flame atomic
absorption spectrometry for many applications. The sample, usually in
solution, is sprayed into the flame following the generation of an aerosol by
means of a nebulizer. The atomic absorption spectrometry (AAS) basic
instrumentation required is described in a previous Unit. AAS is very sensitive
method and can detect different metals in concentrations as low as 1 ppm.
However, a major disadvantage of AAS is that only one metal can be
analyses per time due to the need to change light source and analytical
wavelength every time another metals is required to be determined (see pgs
858-867, SWHC 2004).
In this Unit we will briefly review the nature of the flames employed in AAS,
the specific requirements of the instrumentation for use with flame AAS, and
the atomization processes that take place within the flame. An overview is
given of possible interferences and various modifications that may provide
some practical advantage over conventional flame cells. Finally, a number of
application notes for common matrices are given.
6.1 Instrumentation
A typical atomic absorption spectrometry instrument would consist of radiant

source, atomizer, monochromator, detector and recorder. The AAS absorption
instrument has more or less similar major components as we have seen in
other absorption and emission spectrometric instruments.
37 CHE3143/1/2009-2011
HCL Source Flame or Monochromator Detector Recorder

Flameless
Figure 6.1 Schematic diagram of ASS
6.1.1 Primary radiant source
The most commonly used resonance sources in AAS is the hollow cathode
lamp (HCL) and the electrodeless discharge lamp (EDL). The hallow cathode
lamp emits intense, narrow line (about 0.002 nm) that is characteristic of the
element of interest. It is important that we draw a diagram of HCL so that we
could describe the function of the lamp (see pg 860 and Figure 28.17, SWHC
2004). The cathode is constructed with metal of interest, i.e. if we are
interested in determining iron (Fe), the cathode would be made of Fe.
Activity 6.1
Draw a well labelled diagram of HCL and briefly describe how it works. How
would you define sputtering?
Response 6.1
With the aid of the diagram you can explain the functions of the lamps. A
picture tells the story is truer in instrumentation than anywhere else.
6.1.2 Atomisation sources
The function of the atomizer is to efficiently convert the sample into ground
state free atoms. In AAS, there two methods of producing ground state
atoms;
i) Flame
ii) Electrothermal
38 CHE3143/1/2009-2011
6.1.2.1 Flames
Two types of flame are employed to achieve this;

- the premixed combustion flame consisting of a fuel and oxidant gas
- the diffusion flame where the fuel is also the carrier gas that burns on
contact with air.
Premixed flames commonly employ either air or dinitrogen oxide as the

oxidant, and either acetylene, propane, or hydrogen as the fuel gas. It is
important to note that the temperature of diffusion flames is lower than that of
premixed flames. As many as over 30 elements may be determined using an
air–acetylene flame, although the flame conditions may have to be adjusted to
create a suitable environment for some elements. However, the alkaline earth
metals require a fuel rich (reducing flame), whilst the noble metals are
determined using a lean (oxidizing) flame.
In addition ionization will occur in an air–acetylene flame for a number of

easily ionized elements such as the alkali metals. In such cases, an ionization
suppressor or buffer consisting of a large excess of an easily ionized element
(e.g. cesium or potassium) may be added. This results in suppression of the
ionization (e.g. sodium) by a simple mass action effect resulting from the
excess cesium in the flame. The alkali metals may be determined using an
air–propane flame, although such flames are not commonly used these days.
Atomic emission spectrometry is the preferred technique for the determination
of such elements.
6.1.2.2 Electrothermal atomiser
Electrothermal (ET) atomization was first used for analytical work by L’vov in
the late 1950s. It typically consist of a tube of electrically conducting material,
usually manufactured from graphite (hence the name graphite furnace),
mounted in the light path of a spectrometer. Metals such as tungsten and
tantalum have also been used to construct ET atomizers, in particular for the
determination of carbide-forming elements. After the sample to be analyzed
39 CHE3143/1/2009-2011
has been introduced, via a small aperture in the upper tube wall, the atomizer
is heated resistively through a series of controlled temperature stages. In
modern instruments computer-controlled programs regulate progressive
heating stages, the starting and final temperature, the rate and timing of the
graphite tube heating, and even the gas (Ar or N2) flush of the tube. The
following are steps followed in ET atomisation;
- The sample is dried and thermally pretreated (pyrolyzed), facilitating

the selective volatilization of matrix components, which are purged from
the atomizer by a flow of argon gas.
- The second stage is ashing (pyrolysis, charring) of solid residue

remaining after drying
- The third stage is the atomisation. The tube is rapidly heated (1000 -
2000 oC s-1) to a sufficiently high temperature (1000 - 2700 oC) to
atomize the element of interest, during which time the transient
absorbance signal is recorded.
- The fourth stage is the cleaning. To clean the residual analyte and
matrix from the furnace tube the tube is heated to 2900 oC for about 5
sec.
Material that is used to form the tube surface is practically impermeable to gas
(pyrolytic graphite), therefore the atomic vapour is contained in the atomizer
for a relatively long time normally for a few tenths of a second. This long
residence time, together with the high degree of analyte atomization caused
by the reducing environment, leads to a factor of ~ 103 increase in sensitivity
compared with flame atomic absorption spectrometry (FAAS). In contrast to
the latter, solutions, slurries, and solids can be conveniently analyzed and the
possibility for in situ removal of matrix components significantly reduces the
risk from interferences. The high optical transparency of the argon
atmosphere in the ET atomizer favours light transmission, which is important
for measurements in the wavelength region 190 – 230 nm.
40 CHE3143/1/2009-2011
6.1.3 Dispersive System
Most commercial AAS system is based on single-element determination. A

monochromator system is used for the selection of one wavelength by rotating
plane grating. For multi-element determination, an echelle-grating based
polychromator is used. Both single beam and double beam instruments are
available.
6.1.4 Detectors
A photomultiplier tube is used for monochromators, whereas the echelle-

grating based polychromators use a new solid-state detector.
6.2 Interferences in AAS
Both flame and flameless AAS suffer from matrix interferences, which affect
the population of free atoms. For example the interference of phosphate on
calcium which results in the formation of Ca phosphate. In an air-acetylene
flame, the absorbance of Ca decreases in the presence of phosphates. A
buffer such as La which forms stable salts with phosphate so that the Ca is
freed is used. The problems can also be solved by using a hotter flame such
as N2O-acetylene.
Another form of chemical interference is ionisation interference which is

solved by the use of ionisation buffer such as alkali metals which easily
ionises and thereby supplying a large population of electrons. In addition to
chemical interferences, atomic absorption also suffers from spectral and
physical interferences. However, spectral interferences are not common due
to the simplicity of absorption spectrum and the sharpness of the absorption
lines.
41 CHE3143/1/2009-2011
6.3 Background Absorption and its Correction
The presence of molecular absorption although not common with flame

atomizers is very common with flameless atomizers. To minimise absorption
several approaches have been developed to correct for background. These
include; i) use of “blank” sample that is similar to the sample of interest, ii) the
use of continuum source, such as deuterium or hydrogen lamp, iii) Zeeman
effect background correction method, iv) Smith-Hieftje background correction
and v) two line method.
42 CHE3143/1/2009-2011
TOPIC-III: SEPARATION METHODS
In Topic III of the study guide the students are expected to understand the
concepts of separation methods. By end of this topic the students would be
expected to able to apply separation, and designed a separation protocol to
solve analytical problems.
UNIT 7: Introduction to Separation Techniques
7 Introduction
Separation techniques are some of the most important analytical techniques.

In this topic we will review techniques that could be used for achieving
separation as well as the most commonly used separation methods. We will
highlight techniques that have been extended for use as sample pre-treatment
technique.
Many techniques are available for separation of components from complex

matrices and these include the following;
• Solvent extraction
• chromatography
• Iso-electric focusing
• Precipitation
• Centrifugation, Filtration,
• Distillation, etc
In this topic we will discuss only solvent extraction and chromatographic

techniques. The principles of both techniques are reviewed here.
43 CHE3143/1/2009-2011
7.1 Principle of Separation
Separation depends primarily upon some physical characteristics of the

compound of interest. Table 6.1 shows examples of properties used to
achieve separations and corresponding techniques. In this section we will
also define some of the terms commonly used in separation.
Table 7.1 Physical property utilised and examples of separation

techniques used
Physical Molecular Separation Technique

Property characteristic
Polarity Volatility Gas-Liquid Chromatography

Solubility (GLC)
Adsorptivity Liquid-Liquid Chromatography
(LLC) Liquid-Solid
Chromatography (LSC)
Ionic Charge Ion-exchange Chromatography
(IEC)
Electrophoresis
Size (Mass) Diffusion Gel permeation Chromatography
(GPC)
Sedimentation Dialysis
Ultracentrifugation
Shape Ligand Affinity Chromatography
binding
7.2 Definitions
What is solvent extraction?
Solvent extraction is a selective transfer of material between two immiscible

liquid phases.
What is Chromatography?
Definition of chromatography is based on the distribution of solute between

two immiscible phases. (Note the differences between SE and
chromatography)
IUPAC Definition of chromatography

44 CHE3143/1/2009-2011
Chromatography is a method used primarily for separation of components of a

sample, in which the components are distributed between two phases, one of
which is stationary while the other is mobile.
7.3 Chromatography
The IUPAC defines is very important because it captures what

chromatography is! In chromatography one phase is stationary and is known
as the stationary phase and the other is mobile and is known as mobile
phase. We may add that chromatography is a physical technique used for
carry out separations. It does not involve processes that rely on chemical
changes, such as precipitation, complexation or redox-oxidation.
The two phases involved in chromatography

Mobile phase gaseous or liquid
Stationary phase maybe solid, liquid supported
on a solid, or a gel
The stationary phase maybe:
- packed in a column (e.g. column chromatography)

- spread as thin layer on a supporting surface distributed as a thin film on
an inert support surface consisting of porous packing material of large
surface areas (e.g. TLC)
- immobilized on the inner walls of a tube or capillary (capillary column)
Examples chromatographic systems include:
i) Thin layer chromatography (TLC) or Paper chromatography (PC)

ii) High pressure liquid chromatography (HPLC)
iii) Gas liquid Chromatography (GLC)
iv) Counter current chromatography (CCC), not common
45 CHE3143/1/2009-2011
7.3.1 Classification of chromatography
There are several ways of classifying chromatography. In this course we will

only use the following modes (i.e. based on);
i) Adsorption
ii) Partition
iii) Ion exchange
iv) Gel permeation or size exclusion chromatography
7.3.1.1 Adsorption
In adsorption chromatography separation is based on polarities. In adsorption

mode, methods based on polarities are; i) a system where gas mobile phase
and - liquid phase ii) Liquid (m. p) - liquid (s.p) and iii) Liquid (m. p) - solid (s.p)
are used.
The question is, in such systems what are the major processes that control
separation and/or retention of the analyte?
Retention of an analyte under adsorption phase conditions is primarily due to

interactions between polar functional groups of the analyte and polar groups
on the sorbent stationary phase. These include hydrogen bonding, pi-pi
interactions, dipole-dipole interactions, and dipole-induced dipole interactions,
among others. A compound adsorbed by these mechanisms is eluted by
passing a solvent that disrupts the binding mechanism — usually a solvent
that is more polar than the sample’s original matrix.
In system TLC adsorption is the major separation systems whereas in HPLC

and GLC the separation is controlled by solubility. Recall “like dissolve like”.
Hence polar liquid mobile phase will dissolve polar solutes.
46 CHE3143/1/2009-2011
6.3.1.2 Partition:
Partition chromatography involves two immiscible liquid phases (more like

Solvent Extraction Method). The difference is that one liquid phased is
immobilised and the other is mobile. Separation in partition chromatography
is based on the solubility of solute between the liquid stationary phase and
liquid mobile phase.
6.3.1.3 Ion exchange
Ion exchange chromatography involves the separation of ionic or charged

compounds when in solution on charged stationary resin. The primary
retention mechanism of the compound is based mainly on the electrostatic
attraction of the charged functional group on the analyte to the charged group
stationary phase surface. To ensure retention of the analyte by ion exchange
resin from an aqueous solution, the pH of the sample matrix must be one at
which both the analyte of interest and the functional group on the stationary
resin are charged.
6.3.1.4 Gel permeation (GP)
Gel permeation chromatography or size exclusion chromatography involves

the separation of analyte according to its size. In this technique the gel
contains pores of varying diameters up to a maximum. Penetration of solute
depends upon size of the gel’s pores. Analyte of diameters smaller than pore
sizes of the gel would penetrate through the pores, whereas those with
diameters larger than the gel’s pores would travel through between the walls
of the column and the gel. The distance thus travel by the “solute” this way
would be shorter than tortuous route taken by the “solute” through pores due
to the fact that the solute would be dogging the gel particles as it travels.
47 CHE3143/1/2009-2011
Unit 8: Solvent Extraction (Liquid –Liquid Extraction)
8. Introduction
Solvent extraction is a classical separation method that uses two immiscible

liquid phases to achieve separation. Several properties of analyte or “solute”
are exploited to achieve separation. Recall in Unit 5, we noted that
techniques achieve separation by exploiting differences in physico – chemical
properties between various components of a mixture, some of which are listed
below;
¾ Volatility ¥
¾ Solubility
¾ Charge
¾ Molecular size
¾ Shape
¾ Polarity ¥
In solvent extraction two properties, i.e. solubility and polarities play essential
roles in achieving separation.
8.1 Principle
The principle of SE is based on selective transfer of “solute” between two

immiscible liquid phases. Separations due to solubility differences and
selectivity can be achieved by pH control and complexation. It should be
noted that the distribution of solute between TWO IMMISCIBLE SOLVENTS is
important, i.e. Organic/Aqueous. A solute which is soluble in both phases will
distribute between TWO phases in a definite proportion.
8.2 Distribution Relations
Partition of a solute molecule is a dynamic process that involves a constant

interchange of the solute molecules across the region of contact of the TWO
48 CHE3143/1/2009-2011
immiscible phases. For multi-component samples a more complex

separation procedure is required!
The extraction of solute in solvent extraction is governed by the Nernst

partition or Distribution law. It states that at equilibrium a given solute will
distribute between two essentially immiscible phases in the same proportions,
up to the point where one or both phases becomes saturated with a given
solute.
There are two important terms that are used in solvent extraction and
distribution of solute between two immiscible phases which are;
i) Partition coefficient (KD)
ii) Distribution ratio (D)
8.2.1 Partition coefficient (KD)
Partition coefficient or equilibrium distribution is independent of the total

“solute” concentration and is given by;
KD = [A]org/[A]aq (8.1)
Where A is the solute and it must exit in one same form in both phases, [ ] is
the concentration or activities. The equilibrium is usually achieved within a
few minutes after vigorously shaking the mixture. The KD values obtained are
a reflection of the relative KD solubilities of solute in both phases.
7.2.2 Distribution ratio (D)
In practice solute A may dissociate, polymerise or form complexes with other

components or even interact with solvents. Therefore the values of KD do not
reflect the overall distribution of solute between the two phases; it reflects only
the distribution species. The distribution ratio is given by;
D = (AA)org/(AA)aq (8.2)
Where
49 CHE3143/1/2009-2011
(AA) = Total concentration of all forms, however D § KD

where solute has one form in both phases
8.3 Completeness of Extraction
The efficiency of an extraction of solute from one solvent system to another

depends on;
i) the magnitude of D
ii) relative volume of the liquid phases
iii) number of extractions performed (n)
Variation of the above parameters will affect the efficiency of extraction. The
percentage efficiency of extraction is given by;
E = 100D/{D + Vaq/Vorg} (8.3)
However when Vaq = Vorg, i.e. the volume of aqueous and organic phases are
equal the above equal would be;
E = 100D/{D + 1} (8.4)
When the distribution ratio (D) is large for example > 102 only single transfers
would be required to achieve quantitative (> 99%) transfer of solute.
However, when < 102 more transfers would be required to quantitatively
remove solute from one solvent to another.
Activity 8.1
What is quantitative transfer of material?
Response 8.1
Obviously we would like a system that can remove 100 % of material from one
solvent to another. However, in the real world 99 % is good enough!
50 CHE3143/1/2009-2011
It is important to assess how many transfers are required to achieve

quantitative transfers. Two equations are introduced that are used here; i) to
calculate the amount of material remaining after a single transfer and ii)
number of transfers required to achieve quantitative transfers.
i) The amount of material remaining in aqueous phase after a number of

transfer (extraction) with equal volume of organic phase is;
(Caq)n = (Caq) {Vaq/DVorg + Vaq}n (8.5)

Where;
(Caq) = amount of solute remaining in the aqueous phase
Vaq = volume of aqueous phase
Vorg = volume of organic phase
n = transfers with volumes of organic phase
Caq = amount of solute originally in the aqueous phase
ii) Using equation 8.5 the numbers of transfers required to quantitatively

remove solute from one solvent system to another can be calculated.
It is important that equal volumes of organic solvents are used. For
example if the total volume of organic solvent is 50 ml, and you need
3 transfer. Then you should use 50/3 ml equal volume of organic
solvent.
Activity 8.2
What is the equation required to calculate the amount of solute remaining in

the aqueous phase after a single transfer (one equilibration)?
Response 8.2
From equation 8.5, what happens when n = 1?

51 CHE3143/1/2009-2011
Example 8.1
For a complete removal of 0.127 g of iodine from 57 cm3 of an aqueous

solution of I2 and NaCl is required. Assuming the value of D for CCl4/H2O is
93. What would be the % E given that 27 cm3 of organic phase was used for?
i) Single extraction
ii) 3 extractions
Solution:
Will be given in a tutorial letter
8.4 Extraction of covalent and neutral molecules
In the absence of competing reaction in either phases and under controlled

conditions, the extraction of a simple molecule can be predicted using
Equations 6.3 – 6.5. The value of the distribution ratio, D may be; i) pH
dependent or ii) it may alter in the presence of complexing agent and iii) the
effect of association.
To demonstrate the effect of the three parameters, we will use effect pH as an

example.
8.4.1 The effect of pH on extraction
KD = [R COOH]org/[R COOH]aq (8.6)
In the presence of water, dissociation of the acid occurs

R COOHaq ⇔ R COO- aq + H+aq (8.7)
The dissociation constant of acid given as;
Ka = [R COO-]aq[ H+]aq/[R COOH]aq (8.8)

52 CHE3143/1/2009-2011
The distribution ratio, D is given by
D = [RCOOH]org/{[R COOH]aq + [R COO-]aq (8.9)
Recall that D involves the total concentration of solute in each phase in all
forms.
Substituting for [RCOO-]aq in Equation 8.9
[RCOO-]aq = Ka[RCOOH]aq/ [H+]aq
D = [RCOOH]org/{[RCOOH]aq + Ka[RCOOH]aq/[H+]aq}
D = [RCOOH]org/[RCOOH]aq {1 + Ka /[H+]aq}
If you divide both numerator and denominator by /[RCOOH]aq. Also we know

that;
KD = [R COOH]org/[R COOH]aq
Therefore
D = KD/{1 + Ka}/[H+]aq
From the equation, you can see that D is dependent on pH. At low pH, the
acid is undissociated, D § KD and the acid is extracted with greatest efficiency.
At high pH, where dissociation of the acid is virtually complete D approaches
zero and extraction of the acid is negligible. Similarly, the effect of complex
formation and dissociation would be treated in the same manner as the effect
of pH.
53 CHE3143/1/2009-2011
Unit 9 Theory of Chromatography
9. Introduction
In Unit 6 we defined chromatography. The word chromatography was first

used by Tswett, a Russian scientist in 1903. He used the technique to
separate plant pigment using petroleum ether as the mobile phase through a
glass column packed with calcium carbonate as the stationary phase. The
process resulted in coloured zones as the extract migrated through the
packed column due to mobile phase. The driving force or process that
caused the separation of the mixture in Tswett’s experiment, is the same
basic principle, i.e. different components of the mixture will migrate through
the stationary phase under the influence of the mobile phase.
In this Unit we will review various mathematical relations of the separation

processes that will allow us to optimise a given system. A number of factors
are involved in establishing and optimizing chromatographic systems. We will
review the following important parameters in chromatography;
i) Resolution
ii) Time taken for analysis
iii) Efficiency of separation
iv) Means of detecting the analyte
Resolution and time of analysis are interrelated and can be improved

significantly by understanding of the separation processes. There are
important differences between planar and column chromatography, which
require separate treatment.
Activity 9.1
Define the following chromatography terms: i) Elution, ii) Development, iii)

Band and iv) Peak
54 CHE3143/1/2009-2011
Response 9.1
Review other textbooks in chromatography. Which term is associated with a

particular technique?
9.1 Definition of important chromatographic parameters
Figure 8.1 shows a chromatogram (results of separation) of a separation of a

mixture. We will use the chromatogram to define a number of separation
parameters. A chromatogram is a plot of concentration of solute in mobile
phase emerging from the column (the elution) vs volume of eluate or time of
elution.
Figure 9.1 Shows a typical chromatogram
tR = ret. time from injection

to = column void volume
wb = width of peak at base
wh = width of peak at half height
t R′ = adjusted retention time
≡ (tR – to)
t R′ = adjusted retention time, which represent the actual time
during which true retention occurs
55 CHE3143/1/2009-2011
∴ tR′ directly related to the interaction of analyte with the stationary phase
The relation between retention volume VR and partition ratio k′ is:-
VR = (Vm + k′ Vm) (9.1)

= Vm (1 + k′) (9.2)
k′ = (VR - VM)/ VM (9.3)
9.1.1 Retention time (tR):
This can be obtained by monitoring the amount of solute appearing in mobile

phase (MP) as it is eluted from the column.
tR = time required for the max concentration of solute to

appear in MP as it is eluted from the column
Retention time is a very important chromatographic parameter that is used as

a qualitative measure. In a sample mixture, components with the same tR
under the same separation conditions strongly imply that the components are
the same with the exception of co-eluting components.
Similarly,
VR = volume of mobile phase required to elute sample

component to its max concentration
If solute is neither dissolved nor adsorbed, i.e. solute is un-retained, by

stationary phase, i.e. k′ = 0 and fraction in MP = 1. In this case, the solute
travels along column at the same rate as the mobile phase. The term is
commonly referred to as dead volume or void time.
9.1.2 DEAD VOLUME
Retention volume = volume of MP present in column at any given time

56 CHE3143/1/2009-2011
i. DEAD (SPACE) VOLUME, VOID VOLUME, OR

HOLD UP volume { VM or (to) }
Retention time is also given as;
tR = tM( 1 + k′ ) (9.3)
Substitute k′ from the following Eq.
K = k′ *VM/VS (9.4)
tR = tM( 1 + K VS/VM) (9.5)
This equation relates the experimental variables tR and tM to the

thermodynamic distribution coefficient, K. If the average linear mobile phase
velocity is denoted by u, then for a column of length, L
u = L/ tM (9.4)
By substituting tM from the following Equation;
tR = tM( 1 + K VS/VM) (9.5)
tR = L/u( 1 + K VS/VM) (9.6)
Activity 9.2
Will be provided in the Tutorial letter
Feedback 9.2
Therefore this equation (9.6) allows for the prediction of the effect;
i) of changes in column length
57 CHE3143/1/2009-2011
ii) linear velocity of mobile phase

iii) phase ratio and
iv) distribution coefficient on retention time
If a mixture of two substances is placed at the inlet of a column and mobile

phase is passed through it, the components will move through column
independently at rates controlled by their k′ values and the linear velocity of
mobile phase. The separation occurs if k′ values are sufficiently different and
the components of the mixture can then be eluted one after another from the
column. The extent of separation relates directly to the extent of the
differences in retention factors, k′.
Ideally, the extent to which 2 components move apart from one another
increases in proportion to the distance traversed, whereas the extent of
spreading increases only in proportion to the square root of the distance.
Thus components separate more rapidly than they spread.
9.1.3 Retention factor, k′
k′ = (tR - tM)/ tM or (9.7)

= (VR - VM)/ VM (9.8)
k′ = (mass of analyte)S/(mass of analyte)M

= mS/mM
The relationship between k′ and the distribution coefficient, K
K = CS/CM (9.9)
But
= {mS/VS}/{mM/VM} (9.10)
Need to look at this Equation carefully!
= {mS/mM}*{VM/VS} (9.11)
58 CHE3143/1/2009-2011
= k′ * VM/VS (9.12)
Where, VM and VS are volumes of mobile and stationary phases respectively.

The ratio VM/VS, is known as β, the phase ratio
The k′ compares the adjusted retention time of the analyte to the retention
time of the mobile phase. This is effectively the ratio of the time the analyte
molecules spend in the stationary phase (not moving) to their time in the
mobile phase (where they are moving down the column). This ratio should be
independent of the flow rate of the mobile phase or the physical dimensions of
the column.
∴ if the tR is known, and the dead space is known, we can

calculate k′ for a solute under particular column conditions.
The value of retention time (volume) varies according to the k′ of solute.

However, VM is independent of solute and depends only on column design.
8.1.4 Separation factor, Į
For 2 solutes, the relative retention time or volume, α
α = {t2 – tm}/{t1 – tm} (9.13)

α = {v2 – vm}/{v1 – vm} (9.14)
α = v2′/ v1′ (9.15)
= k2′/k1′ (9.16)
Where:
v2 & v1 = retention volume of the two solutes
α = separation ratio factors i.e. relative capacity factors or

relative adjusted retention times is used for comparing retention of columns on
different dimensions or with different pumps or instruments.
59 CHE3143/1/2009-2011
Activity 9.3
Will be provided in Tutorial letter
Feedback 9.3
The retention of an analyte is dependent only;

i) on the distribution constant
ii) the separation ratio (i.e. α),
and should be independent of small amounts of other components of the
sample. This conclusion has the important consequence that the retention of
an analyte will be the same, whether it is injected as a pure compound or as a
component in a mixture. Chromatographic retention times can therefore be
used for quantity identification.
For two compounds to be separated on a particular chromatographic system,

they must have different distribution constants. The condition of stationary
phase and mobile phase in HPLC or temperature in GLC, are usually adjusted
such that an analyte of interest has a k′ value between 1 and 10. If wish to
improve separation of 2 compounds with very similar retentions, either mobile
phase stationary phase or for GLC temperature of system must be altered.
Some guidance can be obtained from the structure of the analytes and
knowledge of the proportion of different stationary phases.
Usually best method is to “suck and see”. The extent of spreading of a

component can be observed either from the point of view of distribution within
a column, as in TLC, or elution from a column, as in GC and HPLC.
Distribution of solute within a column is commonly referred to as a band, or
zone of solute.
60 CHE3143/1/2009-2011
Its locations position is described in terms of band position and band width.
Thus a band may be defined as a record of the concentration of a substance
as a function of distance on a column. More commonly, elution from a column
is considered, and here the observed distribution is ≡ peak ≡ record of
concentration of a substance in the eluate as a function of its time (volume).
8.2 Column efficiency
The sharpness of the peak is a property of chromatographic column and is

described as column efficiency. The efficiency of the column can be
expressed as; the # of theoretically plates or plate # (N) or height equivalent to
theoretically plate height (HETP or simply H). It is given by;
i) N = # of theoretically plates or plate #
Three important equations for calculating efficiency from a separation

chromatogram is as follows;
N = (tR/σt)2 (8.17)
N = 5.54(tR/W0.5)2 (8.18)
N = 16(tR/ Wb)2 (8.19)
It should be noted that N is dimensionless and also σt is usually not known or

not easily determined.
Where:
σt = peak variance in time unit (0.882h)
W0.5 = width at ½ (0.50) height
Wb = width at base of the peak
tR = retention time
61 CHE3143/1/2009-2011
Activity 9.3
Which measure of N do you think would be the best to use and why?
Feedback 9.3
Since N ∝ L & increase in length will always lead to better separation because
of increase N #
N = L/H (9.20)
N is independent of column length and also is an over estimation of efficiency

since it uses uncorrected tR. A more accurate and better measure of column
efficiency is;
Neff = k′/(1 + k′)2* N (9.21)
The effective plate # (Neff) is given by;
Neff = 5.54 {(tR – tm)/w1/2}2 (9.22)

Heff = L/ Neff (9.23)
Neff and N are also related by equation;
Neff = k′/(1 + k′)2* N (9.24)
As the tR increases the value of Neff leads to N
Activity 8.4
Feedback 9.4
62 CHE3143/1/2009-2011
9.3 Resolution (Rs)
Selectivity factor or retention factor describe the separation of zone centres,

but it takes no account of peak widths.
A better measure of separation is provided by resolution, Rs, which takes into

accounts both retention difference and column efficiency; and for symmetric
peaks of Gaussian shape is given by;
Rs = 2{tr2 - tr1}/{wb1 + wb2} (9.25)
Note:
This equation can be written in terms of volumes by replacing tr1 and tr2 with
vr1 and vr2 respectively and using volume units for the baseline widths such
that Rs has the same dimensionless value.
Rs = 1.5 (achieved resolution of a Gaussian peak). For quantitative

work, a 2 % overlap is acceptable. The ability to recognize two peaks
becomes progressively difficult as Rs decreases, especially when the peak
intensities are different.
The above equation does not provide any useful information about the kinetic
or thermodynamic properties of column, or as to how resolution could be
improved.
However, by combining the equations for efficiency (Equation 9.26 & 9.27)
and resolution (Equation 9.28) with equations (9.29 & 9.30) and (9.31-9.33)
for two peaks with similar retentions, a more useful form of the resolution
equation for this purpose is obtained (9.34 & 9.35)
N = 16(tR/ Wb)2 (9.26)

N = 5.54(tR/W0.5)2 (9.27)
Rs = 2{tr2 - tr1}/{wb1 + wb2} (9.28)
k′ = (tR - tM)/ tM (9.29)
63 CHE3143/1/2009-2011
= (VR - VM)/ VM (9.30)

α = {V2 – Vm}/{V1 – Vm} (9.31)
= V2′/ V1′ (9.32)
= k2′/k1′ (9.33)
Rs = ½ * (α - 1)/( α + 1) * k2′/(1 + k1′)*√N (9.34)
Rs = ½ * (α - 1)/( α + 1) * k2′/(1 + k1′)*√(L/h) (9.35)
The resolution Equation parameters (Equations 9.34 & 9.35)

* The selectivity term dependent on α,
* Rate of migration dependent on k′
* Efficiency term dependent on L & h
The first two terms are essentially thermodynamic, whereas the L/h is mainly
associated with kinetics features of the separation process.
Activity 9.5
What is the ultimate goal of a chromatographer?
Response 9.5
Need to review the measure of efficiency of separation of mixtures. What kind
of chromatogram is suitable in a separation?
tr1 = L/u(1 + k1′) (9.36)

tr2 = L/u(1 + k2′) (9.37)
9.4 Band broadening process
Recall, the extent of spreading of a component can be observed either from

the point of view of distribution within a column, as in TLC, or elution from a
column, as in GC and HPLC.
In GC all the three processes in the stationary phase are important whilst in
LC molecular diffusion is often ignored. But mass transfer in both the
64 CHE3143/1/2009-2011
stationary and mobile phase is of important. The variances for each of the
band broadening process are additive to give overall variances for the system,
and thus is a measure of column efficiency.
Expressed in terms of H rather than σ2 & related by the column length, L. The
overall value at H is a function of the average linear velocity of the mobile
phase, ǌ.
H = {1/A + 1/Cmǌ}-1 + {B/ǌ} + Cs ǌ + Cm ǌ (9.38)
The simple version of the equation is;

H = A + B/ǌ + Cǌ (9.39)
The A term:
Defined as Multiple path effect, the flow of solute through a bed of
granular material is very tortuous
A = 2 λ dp (8.40)
Where: λ = packing constant ∼ 0.50 for a well packed column
dp = the particle diameter
The B term:
Defined as longitudinal “Molecular Diffusion” as the solute band moves
through the column diffusion in the direction of flow (longitudinal or
axial) will occur Not only in the fluid phase i.e. gas or liquid but to a
much lesser extent at interfaces between surfaces e.g. on the surface
of a solid adsorbent
The plate height contribution of longitudinal diffusion is given by;

B = 2 γ Dm (9.41)
Where;
γ = obstruction factor which accounts for the
fact that diffusion is hindered by the column
packing.
65 CHE3143/1/2009-2011
In packed column 0.60 - 0.80

Capillary column 1.0
Dm = coefficient of diffusion of solute in the mobile phase

In liquid ≈ 10-5 cm2 s-1
In gas ≈ 10-1 cm2 s-1
∴ ignore in liquids
The C term:
Defined as mass transfer relates to the rate at which solute species
are adsorbed and desorbed and diffuse within each phase. This rate
is controlled by two mechanisms;
i) Sorption-desorption kinetics
ii) Diffusion controlled kinetics in stationary phase is simple mobile
phase is complex
9.5 Sorption isotherms
When a separation behaves an ideal manner, the solute moves through the
stationary phase giving an over Gaussian profile. However, some separation
would show non Gaussian profile giving fronting or tailing. Both effects are
undesirable and they lead to poor separations. {Read more on these effects}.
Activity 9.6
Use a well labeled diagram to describe the causes of fronting and tailing.
Indicate how the two effects would affect quantification in chromatography.
66 CHE3143/1/2009-2011
Feedback 9.6
Use other textbook besides your assigned textbook. This effect is very
common in chromatography.
67 CHE3143/1/2009-2011
8.5 Summary of Important Equations in Chromatography

68 CHE3143/1/2009-2011
Unit 10 Thin Layer Chromatography
10. Introduction
Planner chromatography includes paper (PC) and thin layer chromatography

(TLC). In TLC the stationary phase is supported on glass, plastic and/or metal
support. TLC can be regarded as modified form of liquid chromatography
(LC). Planner chromatography uses the same stationary and mobile phases
as LC. Recall in Unit 5, we noted that techniques achieve separation by
exploiting differences in physico–chemical properties between various
components of a mixture, a list of properties exploited included the following;
¾ Volatility
¾ Solubility ¥
¾ Charge ¥
¾ Molecular size ¥
¾ Shape ¥
¾ Polarity ¥
In TLC FIVE properties (ticked) play essential roles in achieving separation.

Most importantly, the methods that are developed in TLC can be exported to
liquid chromatography, i.e. HPLC.
9.1 Classification of TLC
The classification of TLC depends on its nature. The layer separates

molecules by;
i) Physical sorption of solutes
ii) Dissolution of solutes into SP
iii) Attraction of ions to active sites
iv) Retention or rejection of solutes on the basis of molecular size
and/or shape.
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Activity 9.1
What is characteristic with TLC classification above? Do recognize anything

familiar with earlier classification?
Feedback 9.1
Need to go back to the introduction of chromatography (Unit 7).
In Unit 7 we reviewed and classified chromatography. TLC as a

chromatographic method can also be classified into four main modes. It can
also be classified according to phases, i.e. normal and reverse (RP). In
normal phase Chromatography for example, polar stationary phase and a non
polar mobile phase is used, whereas reverse phase chromatography was
non-polar stationary phase and polar mobile phase.
In reverse phase partition TLC the stationary liquid is impregnated into the
layer is less polar than the mobile liquid, therefore non-polar solutes are
strongly retained Adsorption TLC is very sensitive to differences in
configuration that affect the free energy of adsorption.
Adsorption is therefore suited for separation of components differing in polarity

& structural isomers partition suitable in components slightly differing in
solubility.
10.2 Classification based on mobile phase;

¾ capillary flow TLC
¾ High performance thin layer chromatography (HPTLC, forced-flow
TLC)
¾ Electric flied TLC
¾ Centrifugal force TLC
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10.3 Criteria of chromatographic performance & their relationship
The main aim is to resolve components of a mixture. In linear

chromatography retention is measured by Rf and it is defined as;
Rf = distance moved by solute/distance moved by solvent front
The resolution in TLC is given by;
R = distance between zone centres divided by average of the widths

of zones
10.3.1 TLC Theoretical Plates
The conventional TLC is known to have efficiency of about 600 theoretical

plates, whereas HPTLC of about 5000 theoretical plates for a 10 cm
development and up to 50, 000 plates per meter. However, the normal
development in TLC is about 3 – 6 cm.
10.4 Examples of sorbents, layer and pre-coated plates
There are several types of stationary phases that could be used for TLC.
Examples include;
• Silica gel
• Alumina
• Cellulose
• Polyamide
• Size-exclusion
• Ion exchange
• Kieselguhr
• Miscellaneous inorganic sorbents
• Miscellaneous organic sorbents
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9.5 Solvent Systems
The mobile is usually chosen by trial and error based on the analyst’s
experience and search of the literature. Usually the mobile phase is chosen
to match the nature of the analytes and sorbent layer used. Mobile phase
competes with separating substances for sorbent sites, polar substance will
require a polar solvent to cause migration on a silica gels or alumina.
10.6 The Elutropic Series
Solvents must be of the purest form to be used as mobile phase in a

chromatographic system. Solvents are grouped together in the order of their
elution strength, i.e. elutropic series (εo).
¾ Elutropic series (εo)

¾ Use one solvent
¾ Combination of two or more solvents
Table 9.6: Solvent Strength Data on Alumina Adsorbent
Solvent εo Solvent εo
n-pentane 0.00 Acetone 0.56
1-pentane 0.08 Ethylacetate 0.58
CCl4 0.18 Acetonitrile 0.65
CH3Cl 0.40 Ethanol 0.88
Methyl chloride 0.42 Methanol 0.95
Methy ethyl ketone 0.65 Ethylene 1.1
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10.7 Modes of Development
The process of separation of the sample mixture by migration of mobile phase

through the layer is known as development. The modes of development are
classified as;
¾ Ascending which is the most frequent mode of linear development in
TLC
¾ Descending
¾ Two-dimensional (2D)
9.8 Detection
Separated zones are detected in by various means that include;
i) Coloured substances which are observed at a particular λ

ii) By means of chromatographic reagent (producing coloured
zones)
iii) Fluorogenic reagents (producing fluorescent)
iv) Biological methods
v) Radioactivity (Geiger Müller)
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Unit 11 Gas Chromatography (GC)
11. Introduction:
The primary objective of the Unit is to select basic aspect of Gas

chromatography (GC) technique and present them in a simplified manner for
you understand. GC is a separation technique that separates components
due to the differential migration rate of components in a mixture between two
phases. One of these phases is a stationary phase (solid or liquid) and the
other is a mobile phase which is a “gas” that percolates through the stationary
phase. There are two types of gas chromatographic techniques, i.e. gas solid
chromatography (GSC) and gas liquid chromatography (GLC). In GSC
separation is based on adorptive properties of the solid stationary phase,
whereas in GLC separation is based on partitioning of the sample between
the two liquid phases. A quick review and/or summary of physico-chemical
properties that are exploited by are indicated below;
¾ Volatility ¥
¾ Solubility ¥
¾ Charge
¾ Molecular size
¾ Shape
¾ Polarity ¥
In GC TWO (volatility & polarity) of the three properties ticked play essential
roles in achieving separation in GSC whereas solubility plays a role in GLC. A
major requirement for the sample to be separated by GC is that it should be
volatile and thermally stable under separation conditions.
11.1 Major components of a Gas Chromatography
The GC instrumentation consists of the following components;

• Regulated carrier gas supply (mobile phase)
• Injector port
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• Heated metal oven

• Column
• Detector
• Recorder and/or computer for the modern instrument*
Injection Port
Column
Detector
Data Processor
Qualitative Analysis Quantitative Analysis
Figure 10.1 Show the block diagram of major components of GC. The
dotted box indicates heated components.
11.1.1 Mobile phase for GC
The carrier gas in GC instrumentation comes in high pressure gas cylinders

and should be of high purity. The most common carrier gases include
hydrogen (99.99 %), ii) helium (99.995 %) and nitrogen (99.999 %) are used.
The carrier gas used as a mobile phase is expected to be; i) inert, ii) minimize
gaseous diffusion, iii) pure, iv) inexpensive and v) detector compatible.
The higher purity of the carrier gas will prolong the column life and improve
the detector sensitivity. Therefore, impurities traps or filters should be
installed at the gas lines. The recommended flow rate of carrier gas for
packed column is 40 ~ 60 ml/min and 0.5 ~ 20 ml/min for capillary column.
75 CHE3143/1/2009-2011
11.1.2 Sample introduction system
In GC the sample should be injected into the carrier gas stream a narrow
band to ensure the best possible efficiency and resolution. This is achieved
by introducing the sample instantaneously into the GC column. Solids
samples are first dissolved in suitable solvents before being introduced into
GC system by means a hypodermic syringe needle through a self-sealing
septum. In addition, special direct injection devices are also used to introduce
solid samples to a GC system. A special designed gas apparatus or gas-tight
syringe is used for the introduction of gaseous samples into a GC system.
Volumes introduced into the GC are dependent on the type of column being
used, i.e. 0.1-50 µl for gases and 0.1-1 µl for liquid in packed column and 0.1-
1 µl and 0.004-2.0 µl for gases and liquids respectively in capillary columns.
11.1.3 Injection port in GC
Injection chamber accommodates the injection port and plays an important

function in the GC system. The injection chamber or injection port should be
hot enough to vaporise the sample rapidly to minimize band broadening.
Injection methods used in GC are dependant of the type of column used, i.e.
packed or capillary. There are several examples of injectors used for capillary
columns. These include;
- Split injector
- Splitless injector
- PTV(Programming Temperature Vaporiser)
- Cool on-column injector
- Wide-bore capillary column attachment
- Solventless sample injector or moving needle injector
- Wide precolumn injector
Capillary columns have very small capacity and hence require the use of split
injector to avoid over loading the column.
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11.1.4 Column
The column tubing can be made from metal such as stainless steel, copper,
nickel, aluminium and from glass-Pyrex and fused silica. Copper is not
suitable for separation of amines, acetylenes, terpenes and steroids due to
adsorption and reaction with the compounds of interest. Packed column
typically vary from few centimetres to about twenty metres long (~ 0.5-20 m)
with inner diameters of 2 – 4 millimetres, capillary columns on the other hand
are typically from about 10 to 100 metres long and have I.D. of 0.1~0.53 mm.
Capillary columns are known for their very high resolution in comparison with
packed columns. However, their major limitation is their very small sample
capacity and hence they need to use small sample volume to avoid over
loading the column. Table 11.1 shows examples of capillary columns and
their possible applications.
Table 11.1: Examples of capillary columns and their applications1
Bore ID/mm Length/m Film Thickness/µm Applications

High speed separation, fast
0.1 10-15 0.1 analysis, very small column
loads
High speed separation,
0.2 25-100 0.25-0.5 generally used in split type
analysis
Used in splitless, on column
0.3 25-50 0.5-1 injection methods, column
load > 0.2 µl
Separation ability equals that
0.5 10-12 1-5 of packed column. Large
column loads
Nowadays the most widely used columns are capillary columns. For example
capillary column separation is more suitable for the sample extracted from
77 CHE3143/1/2009-2011
textile clothes because it has a lot of components and the concentration of

some of these components is usually low.
11.1.4.1 Process of separation in the column
To ensure reproducible separation the conditions in which separation takes

place must be carefully controlled. The column is therefore enclosed in a
thermostatically controlled oven whose temperature can be controlled within ±
0.1 oC. The separation is carried out from ambient to about 400 oC and can
remain constant during the entire process (isothermal analysis) or is varied
during the entire separation process (temperature programming analysis).
11.1.4.2 Solid support
The function of solid support is to provide a large uniform, inert surface area
for distributing the liquid phase. Solid support commonly used consists of
calcined diatomaceous earth and firebrick, both mainly silica. This material is
marketed as Celite, Chromasorb and Stermachol.
10.1.4.3 Stationary phase
Stationary phase plays a major role in the success of a separation. The

stationary phases are classified as either non-polar or polar according to their
structure and separation abilities. The choice of stationary phase is an
extremely important task and it is usually dictated by the nature of sample. In
general the stationary phase that is similar to the sample is most suitable for
separation, i.e. “like dissolve like” e.g. polar compounds are separated on
polar stationary phase. The chemistry of stationary phases can be
manipulated to achieve selectivity.
10.1.5 Detectors
The function of a detector is to monitor or indicate the presence and measure

the amount of components in the carrier gas as it emerges from the column.
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The ideal characteristics of a detector should include; i) high sensitivity, ii)

rapid response, iii) wide linearity response, iv) rugged, v) response to all types
of compounds, vi) insensitive to flow and temperature changes, vii)
inexpensive, viii) low noise level, and ix) stability of operation. Gas
chromatography detectors respond on changes in some physical property of
the carrier gas such as; i) thermal conductivity, ii) density, iii) flame ionisation,
iv) electrolytic conductivity, iv) β–ray ionisation. The response of GC
detectors is of two types; concentration or mass flow. There are several types
of detectors used for GC and the most common ones are given in Table 9.2.
Table 11.2 GC detectors characteristics1
Detector Minimum Linear range Temp limit Comments

o
detectable ( C)
-1
quantity (gs
Thermal Sample not destroyed,
-9 4
Conductivity 10 10 450 sensitive to temp &
Detector (TCD) flow
Flame Ionization Sample destroyed,
-12 7
Detector (FID) 10 10 400 excellent stability
Electron Capture Sample not destroyed,
-13
Detector (ECD) 10 102 – 103 350 prone to
contamination
sensitive to temp
Phosphorus
-14
10 105 400 Similar to FID
Nitrogen
-13
10 105 400 Similar to FID
Flame Photometric
Detector (FPD) Signal approximately
-12 4
P compound 10 10 250 proportional to square
-10
S compound 10 of S concentration
-13
Flame Thermionic 10 for N
-14
Detector (FTD) 10 for P 104 to 105
cont.....
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Table 11.2 cont.....
Detector Carrier gas Fuel gas Support gas Make-up gas
TCD He, N2, Ar - - He (99.995 %)
FID N2, He H2 (99.95 %) Air N2 (99.95 %)
ECD N2 - - N2 (99.999 %)
FTD He (99.995 %)
N2, He H2 (99.95 %) - N2 (99.95 %)
FPD
N2, He H2 (99.95 %) N2 (99.95 %)
11.2 Analysis of samples by GC
We are now familiar with gas chromatography as a separation technique. In

addition to its fantastic ability to separate components, GC like any other
analytical technique can also be used for qualitative and quantitative analysis.
We will briefly discuss the two analysis approaches.
10.2.1 Qualitative analysis
One of the challenges in analytical chemistry is to be able to correctly identify

unknown components in a mixture. Identifying numerous peaks emerging
from a GC column, especially capillary column is a major problem.
Components are identified chromatographically by means of retention data,
i.e. retention volume or time. Retention time (tR) or volume (vR) is
characteristic of the sample and stationary phase and can therefore be used
to identify the sample at constant conditions. The identification is based on
comparison of the retention data of the unknown with that of a known
standard separated under identical conditions.
Homologous series can also be identified by plotting of the log of the retention
times against the number of the carbon atoms. The log of the retention times
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is known to be proportional to some increasing property of the homologous

series. The use of Kovats index is another way of identifying (see FK).
11.2.2 Quantitative analysis
The great majority of analysis carried out on GC is on quantitative analysis.

GC as a technique is capable of providing very accurate results with minimal
errors associated with the measurement. The use of absolute calibration
curve method, a relationship between the concentration of an analyte and its
peak area (or height), and the concentration of the unknown can be calculated
from this relationship. External and internal standards can be used for the
11.3 Gas chromatography combined with other techniques
Gas chromatography has been coupled to other techniques to enhance

identification of the analyte of interest. Examples of hyphenation include; i)
gas chromatography-mass spectrometry (GC-MS), ii) gas chromatography-
infrared spectrometry (GC-IR), iii) gas chromatography-gas chromatography
(GC-GC). These techniques will be covered in details in level IV (honours).
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Unit 12 High Pressure Liquid Chromatography (HPLC)
12. Introduction
High pressure liquid chromatography HPLC is comparative to GC for best

efficiency & resolution and it is more versatile. Unlike GC it is not limited by
volatility and thermal stability of the sample for separation to be achieved.
The ‘inventors’ of modern chromatography, Martin and Synge were aware as
far back as 1941 that, in theory, the stationary phase requires very small
particles and hence a high pressure is essential for forcing the mobile phase
through the column. As a result, HPLC is sometimes referred to as high-
pressure liquid chromatography and in some textbooks it is referred to as high
performance liquid chromatography.
Activity 12.1
Important Question: Why choose liquid chromatography? What are the

advantages of HPLC over GC?
Feedback 12.1
You need to consider the main chromatographic separation mechanisms for

the HPLC and GC? You should also look at the requirement for sample to be
separated by GC and HPLC
There are four common types of stationary phases used in HPLC which
include;
¾ solid adsorbents
¾ chemical modified adsorbents
¾ ion exchange
¾ size exclusion or gel permeation
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It should be noted that all four sorption mechanism (adsorption, partition, ion
exchange & size exclusion) can be exploited in HPLC. In comparison with GC
only two mechanisms (partition and adsorption) of the chromatographic
separation processes are exploited. Recall the summary of physico-chemical
properties earlier discussed. In HPLC all but volatility are exploited in
separating various components of a mixture as indicated below;
¾ Volatility
¾ Solubility ¥
¾ Charge ¥
¾ Molecular size ¥
¾ Shape ¥
¾ Polarity ¥
12.1 HPLC Apparatus & Instrumentation
HPLC instrumentation consists of the following major components; i) Injection

port, ii) solvent delivery system, iii) stainless steel or polyetherether-ketone
(PEEK) column, iv) flow through cell, and v) detector. Figure 10.1 shows a
general schematic of a typical HPLC system. The presence or absence of
scavenger and guard columns will depend on the user. The use of either of
the columns will be discussed further under the subsection of columns.
The HPLC system that uses a liquid mobile phase at pressure as high as
3000 psi and flow rate as high as 1 – 5 ml/min depending on the size of
particles (3 µm, 5 µm and 10 µm) packed in 10 – 25 cm length column.
Recently, the particle size has been reduced to below 2 µm. Naturally you
would expect the pressure demands to be much higher than 3 000 psi. The
smaller the particle size (< 2 µm) packings the higher the back pressure
experienced within the column.
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12.1.1 Solvent Delivery system
Solvent delivery system consists of; i) solvent reservoirs, ii) solvent filters, iii)
pump and v) mixing chamber (dotted box in Fig 10.1). The solvent reservoirs
can be 1 – 4. The very old systems would have one reservoir. However,
modern systems have as many as four reservoirs. Most common systems are
quaternary (4 solvents) system. Two and three solvents systems are known
as binary and ternary respectively.
Mixing
Chamber Pump Injector
SC
G
C
A
C
F
C
Figure 12.1 Schematic diagram of an HPLC system. System consist of

solvent reservoirs (SR) (1 up to 4), pump (P), mixing chamber
(MC), degasser, scavenger column (SC), Injection port, guard
column (GC), analytical column (AC), flow through cell (C) and
detector (D).
The solvents are drawn through the filters attached at the end solvent tubing
from the solvent reservoir. The function of these filters is to prevent small
particle that could clog the system from entering the separation process. The
mixing chamber allows solvents from different reservoirs to mix according to
84 CHE3143/1/2009-2011
the required proportions depending on the sophistication of the instrument.

For example, in a solvent system of methanol/water (40/60), the pump will mix
40 % methanol to 60 % methanol. The pump is a very important component
of the solvent system. The quality of the pump determines the cost of the
instrument. Some pumps allow high pressure mixing of the solvents,
therefore achieving rapid mixing of the solvent composition. This function is
important in gradient separation (to be discussed later).
HPLC as a technique has wider choices of more mobile phases than GC.
This results in a very considerable variation in the selectivity of the separation
process. Separation processes in HPLC can be subdivided into two types of
Mobile phases; i) isocratic elution and gradient elution. In isocratic mode, a
single solvent composition system is maintained during the entire separation
process, for example methanol/H2O (50:50) or acetonitrile/H2O (60:40).
Whereas, in gradient mode the composition of the solvent changes during the
separation process
12.1.2 Pump
The function of the pump is to deliver mobile phase through the column to
achieve separation. The particle sizes of the packing materials and the inner
diameter of column determine the type of pump required for the solvent
delivery. As already mentioned the common packing (3 µm , 5 µm & 10 µm)
would required a pump with high pressure capabilities of 3 000 psi. The
recent development of smaller packings (< 2 µm) has resulted in the
development of pumps that are capable of very high pressure (> 8 000 psi).
These pumps are capable of high speed separation as well as higher
efficiency in separation. However, there are very expensive and are not yet
common in laboratories.
Several types of pumps are available for use in HPLC. The pumps are
expected to deliver; i) constant, ii) reproducible, and iii) pulse free supply of
mobile phase. As stated earlier the pump delivers the mobile phase at flow
rate of 0.10 - 5 ml/min for standard analytical column, at pressured of 3 000
psi (200 bar). It is important that the pump is of material that is inert. For
85 CHE3143/1/2009-2011
gradient separation it is also important for the pump to have a small hold up
volume. Pumps can be classified as; i) constant pressure pump (+ ve flow)
and ii) constant flow pump (+ ve flow). The constant pressure pumps are
known for their simplicity, freedom from pulsation, smooth baseline,
affordability and ease to operate. However, their disadvantages are; i) that
flow must be monitored carefully and ii) should be constant for qualitative &
quantitative work. Changes in flow will affect both qualitative & quantitative
analysis.
12.1.3 Sample injection system
The method of sample introduction is more critical than in GC. The samples
are introduced by means of syringe and high pressure valve injector (~ 7 000
psi). Unlike the GC syringe that is sharp, the HPLC is flat to allow it to flush
straight into the injection port.
12.2 Column
HPLC columns are the heart of chromatographic separation and require

careful handling and storage than GC to avoid disturbance of the packing
material. It consists of hardware that is made of straight lengths of precision
bore stainless steel and polyetherether-ketone (PEEK) of 10 – 25 cm long & 4
or 5 mm i.d. packed with stationary phase. The typical life of an HPLC column
is about 6 months or more depending on their handling. Life can be
prolonged by use of; i) guard column and ii) scavenger column. HPLC
columns are readily available commercially. Previously these columns were
made in laboratory by slurry method. There are different blend of column
available from a number of vendors.
It was earlier noted that the four commonly known modes of separations are
taken advantage of in HPLC. What this means is that we expect to have
packing materials with; i) adsorption, ii) partition, iii) ion exchange and iv) size
exclusion properties. This is fundamentally very important because your
decision in selecting an appropriate column will depend on your knowledge of
the nature of analyte and chemistry of your packing material.
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In chromatography there are three types of column that we will to consider

here; i) separation column, ii) scavenger column and iii) guard column. In
HPLC separation columns are classified according to their inner diameters.
Recently, great strides have been made in the development of HPLC column
with respect to reduction of particle sizes of packing, inner diameter (i.d.) of
the column and chemistry of packing. This has resulted in reclassification of
separation columns in the last decade. Table 12.1 shows the classification of
the HPLC column. It should be noted, that this classification would possibly
change again in the near future as we continue to develop smaller and smaller
inner diameters.
12.2.1 Stationary Phases
The most common stationary phase is silane and it is characterized by its

chemistry, i.e. length of ligand (mono, di or tri) functional groups. Surface
coverage is expressed in µMmole/m2 of ligand. Silica stationary phase is
used for normal phase chromatography. However, chemical modification a
silica by attaching a long alkyl group (non polar) resulted in the development
of non-polar stationary phase used in reverse phase chromatography,
commonly known as bonded phase. In addition functionalizing bonded phase
resulted in formation of stationary phase with some degree of polarities.
Limitations with silica based columns includes; i) sensitive to pH, meaning that
separations at very acidic and basic condition are avoided, ii) higher
temperatures, separation at 60 oC and above avoided
Recent development in chemistry of packing has resulted in columns that can

be used in wide range of pH (1-13) and at higher temperature. Examples
hybrid columns with wider pH range and higher temperature include X-Terra
X-Bridge, Hypercarb and PBD-Zirconia/Titania. The desire to develop faster
separations led to the developments of monolithic stationary phase. These
columns allowed much higher flow rates than silica phase columns of about 1
ml/min.
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12.2.2 Scavenger column
A scavenger column is a short length of tube packed with large particle silica
and positioned between the pump and the injection valve. The function of a
scavenger column is to saturate aqueous mobile phase with silica to decrease
attack on the packings of the analytical column by high pH (> 8) buffer.
Table 12.1: Classification of HPLC columns
Typical Column i.d. Flow rate Separation Technique
> 10 mm > 20 ml/min Preparative HPLC
4.6 mm 1 ml/min Conventional HPLC
2.1 mm 200 µl/min Narrowbore HPLC or macro
1.0 mm 50 µl/min Micro LC
300 µm 4 µl/min Capillary LC
75 µm 300 nl/min Nano LC
< 10 µm < 10 nl/min Open Tubular LC
12.2.3 Guard column
Guard column consist of a very short length of column placed between

injection port & analytical column. The function of guard column is to protect
the analytical column by trapping strongly retained species or particulates
matter originating from the mobile phase, sample or wear and tear of injection
valve. The guard column is usually packed with large particles (ca 30 µm) of
the same or similar stationary phase with that used in an analytical column.
The guard column is renewed on regular basis.
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12.2.4 Separation columns
As stated earlier the separation column is the heart of the HPLC separation.
A column with i.d. 4.6 mm, here stated as conventional, has also been
referred to as analytical or standard column. Most analytical work at 1.0
ml/min. has for several years been performed with the standard column. It is
very important to view separation columns as consumables as such we
expect to dispose of them after some time depending on their use and
treatment.
12.3 Mobile Phase
In HPLC the mobile phase is in liquid form. Mobile phase plays a very
important function in the separation of components of interest. In HPLC there
are two modes phase that can be used; i) isoctratic and ii) gradient. In
achieving separation optimising chromatographic performance is essential.
To optimise the separation process the following are important; i) overall
polarity, ii) polarity of stationary phase and iii) nature of sample components.
In normal phase separation, the polar stationary/non-polar mobile phase

elution power increases with increase in solvent polarity. Whereas, in reverse
phase separation non polar stationary/polar mobile phase elution power
increases with decrease in solvent polarity.
12.4 Detectors
A detector is one of the major components of the HPLC system. It is the eyes
that see what has been separated. The characteristic of an HPLC detector
are very similar to those of a GC system. An HPLC detector should have
some of the following characteristics; i) have rapid response, ii) reproducible
response tosolutes, iii) wide range of linear response, iv) high sensitivity, v)
have stability of operation. Examples of HPLC detectors are highlighted,
however, you are expected to read further and make you own notes.
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A flow through cell is required for HPLC detectors. It should be noted that No
true universal detector has yet been developed yet. The following are
examples of HPLC detectors that could be used; i) Uv/visible radiation, ii)
Refractive index, iii) Fluorescence, iv) IR absorption, v) Electrical conductivity,
vi) Diffusion current (Amperes), vii) Radioactivity, viii) mass spectrometry, ix)
nuclear magnetic resonance. The principles and functions of the most
common detectors such as UV/Vis, Fluorescence, Refractive index,
electrochemical and mass spectrometry is found under their respective
spectrometric topics.
12.5 Analysis of samples by HPLC
High pressure liquid chromatography is a separation technique that separates

a variety of compounds including non volatile that GC is unable to separate.
HPLC like any other analytical technique can also be used for qualitative and
12.5.1 Qualitative analysis
One of the challenges in analytical chemistry is to be able to correctly identify

unknown components in a mixture. Identifying peaks emerging from an HPLC
column can be very challenging. Components are identified
chromatographically by means of retention data, i.e. retention volume or time.
Retention time (tR) or volume (vR) is characteristic of the sample and
stationary phase and can therefore be used to identify the sample at constant
conditions. The identification is based on comparison of the retention data of
the unknown with that of a known standard separated under identical
conditions.
12.5.2 Quantitative analysis
HPLC as a technique has grown in its capability to provide accurate results

especially when coupled to a sophisticated detector as such mass
spectrometer. The use of absolute calibration curve method, a relationship
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between the concentration of an analyte and its peak area (or height), and the
concentration of the unknown can be calculated from this relationship.
External and internal standards can also be used for the quantitative analysis.
12.5 Other forms of HPLC
Examples of others forms of liquid chromatography include;
i) Ion Pair Chromatography
ii) Supercritical fluid Chromatography
These topics would be covered in level 4 course.

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Topic IV: SAMPLE PREPARATION or PRE-TREATMENT in

ANALYTICAL CHEMISTRY
In this Topic students are expected to understand the concepts of what

constitute a real and ideal sample, as well as the processes involved in
sample preparation, isolation and pre-concentration. By the of the topic the
students would be expected to able to competently select appropriate sample
preparation and/or pre-treatment method for a particular sample.
Study Unit 13: Introduction to Sample Preparation
13 Introduction
Analytical chemistry is one of the most important disciplines of chemistry. It is

applied throughout industry, medicine, agriculture and all the sciences. For
example, quantitative determination of steel during its production permits
adjustment in the concentrations of elements such as carbon, nickel and
chromium to achieve a desired strength or quality of steel (Skoog, et. al.,
2004: 3). Quantitative measurements also play a role in research.
Measurements can be expressed as a mass or volume of the sample being
analyzed. Therefore, a typical analysis involves a series of steps, from method
selection to the results. These steps were discussed in level 2 of this course.
One such definition by Working Party on Analytical Chemistry of Federation of
European Chemical Societies of Analytical Chemistry is the following;
“Analytical chemistry is a scientific discipline that develops & applies

methods, instruments, and strategies to obtain information on composition
and nature of matter in space and time.”
It is very clear from the definition that acquiring information (quantitative or

qualitative) is fundamental to analytical chemistry.
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1.1 ANALYSIS OF REAL SAMPLES
Generally, analyzing samples is complex and all inclusive process. It involves

a number of steps that include identifying the study area, deciding on the type
and amount of samples to be collected, deciding on the sequence and period
of sampling, storage conditions, transportation of samples to the laboratory,
identifying element/s of interest, choosing the sample pre-treatment methods
and analytical technique to use. In most cases, analytical samples or
materials are not ideal in terms of their solubility, volatility, stability and
homogeneity. All these physical and chemical characteristics must be
considered when deciding about a sample analysis.
In order to appreciate the nature of a real sample and the challenges

associated with its analysis we need to define what we mean by sample?
13.1.1 Sample
A sample consists of two distinct components, mainly the analyte of interest

and the matrix. In your textbook the term “real sample” is used to describe the
sample that we are most likely to encounter in the real world. A sample that is
not artificially prepared in the laboratory.
13.1.1.1 Analysis of sample
The instrumental techniques used to analyse for the analyte usual do not
function well in the presence of the matrix. It is therefore, a requirement that
as much matrix as possible be removed and/or isolated from the analyte.
Some form of pre-treatment is used to allow analytical instrumental
measurement to be performed without any interference from the matrix. For
example, an apple orchard is irrigated with water from a mining source such
as Gauteng Gold mine. The apples are suspected to contain high content of
lead and cadmium. Analysing for lead and cadmium residues in apples would
definitely fall under real samples. We expect the sample to have a lot of other
93 CHE3143/1/2009-2011
components, here termed interferences that would affect the analysis of lead
or cadmium (analytes of interest).
13.2 Sample pre-treatment
Sample preparation or sample pre-treatment plays an essential role in

analytical methodology. In this Study Guide we will use the term sample pre-
treatment. The role of sample pre-treatment role is to isolate the analytes of
interest from the matrix and hence improve the selectivity, detectability,
reliability, accuracy, and reproducibility of analysis.
It is commonly known that the error associated with analysis is mainly due to
the sample pre-treatment step. Sample pre-treatment is therefore, a
prerequisite step prior to the determination step. However, sample pre-
treatment process is dependent on the nature of the sample. It is therefore,
important to review the nature of the sample first and then classify the sample
as either organic or inorganic. The sample is further subdivided according to
its form; solids, liquids or gases. Therefore, the method that would be used
would depend on the type of sample.
Sample pre-treatment can be further subdivided into three categories (see

Figure 13.1);
i) sample preparation
ii) isolation only
iii) isolation and pre-concentration (see Table 13.1 below).
Activity 13.1
What do you understand by the terms selectivity and detectability?
Feedback 13.1
How do you make one component visible in the presence of others?

Selectivity is the ability to detect one component in the presence of others.
94 CHE3143/1/2009-2011
Our ability to see something that is very small or smallest object that we can
see is how can relate to what detectability is.
We will briefly review some of the methods that can be used for preparing the
sample for analysis. Figure 13.1 shows a variety of methods used for sample
pre-treatment and their classification.
SAMPLE PRE-TREATMENT
Sample Preparation Sample isolation only Sample isolation &

Concentration
Analyte of interest that is detectable

by instrument
Figure 1.1: Flow Chart for the of Sample Pre-treatment Methods
Examples of methods of sample pre-treatment given in Table 1.1 are not

exhaustive, but they are most commonly used techniques. The type of
sample and classification is also provided in the table to assist you in selecting
the most suitable method for a particular sample. For example, acid digestion
is used for preparing a solid or biological sample.
Activity 13.2
What is the function of the following sample preparation methods; i) soxhlet, ii)
freeze drying, iii) supercritical fluid extraction and iv) solid phase extraction
95 CHE3143/1/2009-2011
Feedback 13.2
You will have to read through the topic for you to be able to answer the
question.
13.2.1 Isolation methods
They function is to selectively isolate the analyte of interest from matrix. A

classical example, is distillation, it allows for selectively separation of
components by differences in boiling points. An analyte of interest can be
separated from the matrix provided they have distinctly different boiling points
that could be taken advantage of.
13.2.2 Isolation and concentration methods
Methods that fall into this category have dual functions. They allow for not only
isolation but also concentration and/or clean-up of the analyte of interest.
Detailed discussion of these methods is presented in Units 13 – 16.
Activity 13.3
Supposed you are provided with a sample that contains cadmium (Cd) metal
of 0.5 ng/mg concentration level in waste water. The laboratory is equipped
with a Perkin Elmer atomic absorption spectrometer instrument available for
you. The detection limit of the instrument is given as 2 ppb. Does your
sample need to undergo sample pre-treatment process? Explain or justify
your answer. If yes then! What would be the appropriate sample pre-
treatment what you would use?
Feedback 13.3
Do you remember what is meant by parts per billion (ppb)? These units are
very important and are commonly used in our practicals. If you have forgotten
I suggest you revise the section on this topic in your textbook. Now check if
96 CHE3143/1/2009-2011
the level of metal is within the detectable level of the instrument. If not what is
the procedure that should be followed to arrive to an answer. The sample
concentration (0.5 ppb) is lower than the detection limit (2 ppb) of the
instrument of analysis. A sample pre-treatment method which includes pre-
concentration would be appropriate in this case.
97 CHE3143/1/2009-2011
Table 1.1 Example of some Common Methods used for Sample Pre-
treatment
Method Sample type Classification

Crushing Solid Sample preparation
Grinding Solid Sample preparation
Digestion Solid & biological Sample preparation
Blending Solid Sample preparation
Sonication Liquid, solid Sample preparation
Freeze drying Solid & biological Sample preparation
Derivatization Liquid, solid Sample preparation
Microwave assisted extraction Liquid, solid Sample preparation
Distillation Liquid Sample clean-up

Filtration Liquid Sample clean-up
Soxhlet extraction Solid Sample clean-up
Supercritical fluid extraction Solid Sample clean-up
Column chromatography Liquid (extracts) Sample clean-up
Precipitation Biological Sample clean-up
Membrane extractions Liquid, gaseous Sample clean-up &/or

pre-concentration
Solid phase extraction Liquid, gaseous Sample clean-up &/or
pre-concentration
98 CHE3143/1/2009-2011
Study Unit 14 Sample Preparation
14. Introduction
Real samples may be homogenous or heterogeneous, gaseous, liquid or even

in solid form. For solids samples, the particle sizes of laboratory samples need
to be reduced prior to analysis by crushing and grinding operations. Common
methods used to reduce sample size include grinders, pulverisers, mixers,
pestle and mortars, etc.
Drying processes are also important and are undertaken to remove moistures
from samples. Absorbed moisture for example, is normally removed from solid
samples by drying at 110 oC and at atmospheric pressure. In general, sample
contamination must be avoided as much as possible during these processes.
Activities 14.1
1. Write brief notes on “preparing laboratory samples

2. Why is it wise to decrease the particle size of the gross samples in the
laboratory?
3. Explain the differences between different “forms of water in solids”
4. Differentiate between terms such as “adsorbed”, “sorbed” and “occluded”
waters that are contained in chemical compounds
Feedback 14.1
FURTHER READINGS
SWHC, 2004: pg 1034 –1040
14.1 Decomposing and Dissolving the Sample
Most analytical measurements are performed on solutions of the analyte

(SWHC, 2004: pg 1041-1050). Some samples are readily soluble in water or
aqueous solutions of acids or bases. Other samples require powerful
99 CHE3143/1/2009-2011
chemical reagents to release the analyte from the sample matrix. Air, water
and soil samples have different matrices and require different processes.
Choosing appropriate reagents and chemical techniques is critical to the
success of an analysis. Common methods of releasing an analyte from the
sample matrix include acid decomposition, microwave digestion, fusion and
combustion methods. These methods differ in their functional processes. The
reagents used also differ in the temperature and their strengths. Table 2.1
shows acids commonly used for dissolution of a variety of inorganic samples.
Table 14.1 Most common acids used to dissolve inorganic materials
Acid Typical Composition Examples of inorganic materials

(wt % & density) dissolved
HCl 37 %, 1.19 g/ml Metal; oxides, sulfides, carbonates,

phosphates
HBr 48-65 % Metal; oxides, sulfides, carbonates,
phosphates
H2SO4 95-98 %, 1.84 g/ml Will attack metals, dehydrates & oxidizes
organic compounds
H3PO4 85 %, 1.70 g/ml When hot will dissolve refractory oxides, that
are insoluble in other acids
HF 50 %, 1.16 g/ml Used mainly to dissolve silicates. Very
Harmful use with extreme care!!!
HClO4 60-72 %, 1.54 -1.67 g/ml Hot concentrated acid is extremely powerful
oxidant. Cold & dilute acid are not. Before
using as much organic is destroyed with
hot HNO3 & evaporated to dryness.
100 CHE3143/1/2009-2011
ACTIVITY 14.2
1. List the type of errors that are encountered in the sample decomposition
step.
2. Differentiate between “wet ashing” and “dry ashing”.
3. Define the term “flux”.
4. What fluxes are suitable for determining alkali metals in silicates?
5. Name major advantages of microwave decompositions compared with
the conventional methods.
Feedback 14.2
Common substances such as silicates, some mineral oxides and a few iron
alloys are attacked slowly during the decomposition process. To allow or
speed up the decomposition of these types of substances, a flux is added. A
flux is therefore an alkali metal salt that is mixed or fused with the sample to
form water-soluble product called the melt.
Fluxes succeed in decomposing most substances by virtue of high

temperature required for their use and high concentrations of reagents
brought into contact with the sample.
REFERENCES
1. SWHC, 2004: 1041–1050

2. http://chemistry.brookscole.com/skoogfac/
http://infotrac.thomsonlearning.com.
14.2 Derivatization
Derivatization is a sample pre-treatment method that involves a chemical

process (acylation, alkylation or silylation) to convert the analyte that originally
might not be detectable prior to the process. The interaction of the analyte
with the derivatizing reagent results in a product that is detectable. Normally
101 CHE3143/1/2009-2011
derivatization is used in the following instrumental methods of analysis; gas

chromatography (GC), high pressure liquid chromatography (HPLC) and mass
spectrometry (MS). In GC it is used to enhance volatility and therefore allow
non-volatile components to be separated and monitored.
In the GC analysis of polar target analytes such as alcohols (sterols, phenols

including chlorophenols) and fatty acids, require preferably hydrophobic
derivatives which can be handled much more easily. Dicarboxylic acids
require inevitably a derivatization step for GC detection. Reaction of target
functional groups of silylation include OH group (phenols, fatty and
dicarboxylic acids, carbohydrates), as well as the amino group with
derivatization reagents such as silylation agents result in the product
molecules that is volatile and hence separated and detectable by GC
instrumentation. Other examples include 2-hydrazinobenzothiazole and
pentafluorophenylhydrazine (PFPH) as derivatization agents for the GC
analysis of carbonyl compounds present in lipid matrices.
In the HPLC two detection systems are used for monitoring the separation, i.e.
UV/Vis and fluorescence. In HPLC with UV/Vis detection method, the sample
detectability (visibility) is enhanced by introduction chromophore nature in the
analyte of interest and hence making the components UV/Vis active.
Similarly, in fluorescence detection system derivatization is known to enhance
the fluorescence nature of the analyte therefore allowing the analyte to be
detected.
Compounds that lack chromophores will not absorb within the UV/vis region
and hence would be UV/Vis inactive. Such compounds when separated by
HPLC would need to be derivatized to increase detection sensitivity and
improve selectivity resulting in the detecting of the analyte indirectly. Analysis
of amino acid compounds by HPLC will require derivatisation step, since
these compounds are very poor UV/Vis or fluorescence. Derivatization
agents are utilised to enhance spectrophotometric detection capabilities for
the amino acids. The most common derivatization agents being phenyl
isothiocyanate (PITC), benzoyl chloride, 4-nitrobenzoyl chloride and 3,5-
102 CHE3143/1/2009-2011
dinitrobenzoyl chloride. In addition, a number of different types of fluorescent

tagging reagents, such as o-phthalaldehyde (OPA), 5-
dimethylaminonaphthalene-1-sulphonyl-chloride (dansyl-Cl),
phthalimidylbenzoyl chloride, 3,4-dihydro-6,7-dimethoxy-4-methyl-3-
oxoquinoxaline-2-carbonyl chloride and 3-chloro-7-nitrobenzofurazan have
also been used for the determination of amino compounds. Table 2.2 shows
examples of derivatization agents and application methods.
In mass spectrometry derivatization agents induce and/or enhance

characteristic fragmentation that would be more informative. Sometimes
when the molecular ion is non-existent a derivatization agent could product a
product that provides a molecular ion.
Table 14.2 Shows examples of derivatization agents and their

applications
Derivative Analyte Technique used

2-hydrazinobenzothiazole carbonyl GC
pentafluorophenylhydrazine (PFPH) carbonyl GC
Dichlorodimethylsilane (DCMS) hydroxyl GC
o-phthalaldehyde (OPA) Amino group Fluorescence/HPLC
benzoyl chloride, 4-nitrobenzoyl chloride Amino group HPLC
phenyl isothiocyanate (PITC) Amino group HPLC
3,5-dinitrobenzoyl chloride Amino group HPLC
tetramethyl ammonium hydroxide triglycerides MS
(TMAH)
3,17-bistrimethylsilyl ether (TMS) steroids MS
14.3 Microwave assisted extraction (MAE)
To appreciate microwaves as sample pre-treatment tool we need to review

their heating processes and how this is achieved. In conversional heating,
heating is from outside to inside of the body, whereas in microwave heating is
from the core to the outside of the body. This is the key process in terms of its
effectiveness in sample pre-treatment. The selection of solvent plays a key
role to the success of extraction of analyte. Selection should be based on
103 CHE3143/1/2009-2011
solvents that have microwave absorbing properties (high dipole moments, e.g.
acetone 2.69 Debye), minimum interaction with matrix and higher dissolution
power with analyte of interest.
Assisted microwave extractions are performed using a microwave sample

preparation unit. Portions of solid samples such as soil (e.g. 1-2 g) are
accurately weighed into a PTFE liner. To each vessel a certain amount of
solvent (e.g. acetone, acetone-hexane, dichloromethane-acetone) is added.
The vessels are then placed symmetrically on the microwave turntable
together with the control containing the temperature and pressure sensory
equipment. The magnetron power set to a certain level such as 30 %, with a
constant temperature (e.g. 120 °C) for a certain extraction time. After the
extraction is completed, the vessels are allowed to cool. The contents of each
vessel are then quantitatively transferred through glass microbore filter. The
extracts can used as is or concentrated by reducing the volume to a smaller
about 2 - 5 ml using a rotary evaporator before the addition of internal
standards. There are other forms of microwave sample preparation methods
that have been developed for sample pre-treatment, such as atmospheric
microwave-assisted extraction and pressurised microwave-assisted
extraction.
Activity 14.3
How do microwaves achieve the process of sample pre-treatment? What are

the attractive parameters in microwave?
Feedback 14.3
Review page 5, SWHC 8th, 2004 and pages 1044-1047. The popularity of
microwaves assisted extraction as a suitable sample preparation method.
104 CHE3143/1/2009-2011
Study Unit 15 Sample clean-up
15. Introduction
Analytical samples as described earlier in section 14.1, consists of the analyte

of interest and the matrix. In real samples, the matrix more often than not
interferes with the analysis of the analyte of interest. The separation and/or
isolation of the analyte of interest from the matrix because very important. To
achieve sample pre-treatment a process that selectively isolate the analyte
from the matrix is required. There are several methods that have been used
for the isolation of the analyte from the matrix (see Table 14.1). These
include classical methods such as distillation, precipitation, solvent extraction
(SE) and more modern methods such as supercritical fluid extraction (SFE)
and chromatography such as solid phase chromatography (SPE). Each of
these methods takes advantage of different properties of the analyte from the
matrix. However, the most commonly used methods are solvent extraction,
supercritical fluid extraction and chromatographic (SPE) methods. We will
briefly discuss some of these methods in the following section in this Unit.
15.1 Solvent extraction (SE):
Solvent extraction or liquid-liquid extraction (LLE) is an interesting technique

to analytical chemist that electively transfers material between two
immiscible liquid phases. Separations are based on solubility differences
and selectivity can be achieved by pH control and complexation. This
technique is commonly used in isolation of trace and minor levels of inorganic
metals and organic residue.
In solvent extraction components can be selectively extracted from aqueous

solvent into organic solvents or re-extracted from organic phase to aqueous
one. In monitoring of pesticides in lake or river water, a sample of pesticides
in water is mixed with an organic solvent. The pesticide would transfer from
water to the organic solvent due to affinity (non polar nature) to organic
solvent, whereas the polar components would remain in water. By
105 CHE3143/1/2009-2011
manipulating the conditions of the solvent system we can selectively isolate

the component of interest.
The most attractive nature of SE is its simplicity. It does not require much in
terms of equipment. All that is required is a glass separating flask and the two
immiscible solvents. The main disadvantage is its use of high volume of
organic solvent some of which are environmental unfriendly.
Activity 15.1
Will be provided through tutorial letter
Feedback 15.1
Will be provided through tutorial letter
15.2 Supercritical fluid extraction (SFE)
Supercritical fluids extraction (SFE) is finding wide acceptance as a sample

pre-treatment method for a variety of analytical problems due its ability to
release the analyte from all sorts of matrices. In SFE the extractant
(supercritical fluid) is in its supercritical state, which means that both pressure
and temperature are above their critical values. At these conditions
supercritical fluids possess unique properties that are intermediate between
those of gas and liquids. Above critical conditions the SF viscosity is lower
than that of liquids, and diffusion coefficient is higher than that of liquids
enabling more efficient extractions.
A typical SFE instrumentation consists of a high pressure pump that delivers

the fluid through the sample in an extraction cell, an oven for the cell,
restrictor, collector and liquid carbon dioxide cylinder. The system should
have the ability to vary both temperature and pressure thus enabling it ability
to selectively extract components. Most of the extractions are carried out
using carbon dioxide. Sometimes organic modifiers are added to the fluid to
106 CHE3143/1/2009-2011
enhance its solvating properties. A second pump is required for use of organic
modifiers.
107 CHE3143/1/2009-2011
Study Unit 16: Sample clean-up &/or pre-concentration
16. Introduction
When a sample pre-treatment process leads to a sample of interest with

concentration level within the detection limit of the method of analysis
(detection), the process requires only a clean-up method. However, when
the analyte of interest exist at levels lower and/or much lower than the
detection limits, then the sample pre-treatment would require in addition to the
clean-up process a concentration step. The function of such a process would
be to enhance the concentration level of the analyte to detectable levels. The
process of enhancement of concentration is also known as enrichment or pre-
concentration {simply put it is the opposite of dilution}. Two major techniques
are commonly used for enrichment purposes of trace levels (< 0.01 ppb) of
samples in analytical chemistry. These are solid phase extraction (SPE) and
supported liquid membrane (SLM) techniques. To a small extent solvent
extraction could also be used as pre-concentration process. This is achieved
by reducing the organic solvent volume for example by evaporation to a very
small volume.
16.1 Supported liquid Membrane (SLM)
The use of semi-permeable membrane, such as supported liquid membrane

(SLM) extraction has gained popularity since its introduction by Audunsson.
Non-polar or uncharged substances diffuse across a liquid membrane from
the donor side to the acceptor side. The analyte usually diffuse across the
membrane due to the difference in water/organic liquid partition coefficient of
the analyte in the ionic and non-ionic forms. To enhance such differences, the
pH of analyte in the donor is adjusted such that at least 99 % of the analyte is
non ionic. Similarly the pH of the acceptor phase is adjusted to promote
ionization of the extracted analyte. Parameters that promote maximum
extraction of the analyte are of major interest in most applications.
108 CHE3143/1/2009-2011
16.2 Solid phase extraction (SPE)
Solid phase extraction (SPE), a technique well known for its large enrichment
capacity, has been used for pre-concentration and/or cleanup of organic
residues such as pesticides in aqueous samples. This is a technique that
uses the same principle as chromatography to achieve its separation. To
appreciate the principles involved you will need to read through Topic III of
this Study Guide. SPE is found in four common phases as in
chromatography;
i) Reversed phase packings
ii) Normal phase packings
iii) Ion exchange packings
iv) Size exclusion packings
16.2.1 Reverse phase SPE:
This type of SPE involves the use of non polar packing and polar mobile
phase (usually aqueous) and moderately to nonpolar sample matrix. Several
SPE materials, such as the alkyl- or aryl-bonded silicas (such as Superlco LC-
18, ENVI-18, HypersilSEP C-18, C-8, LiChrolu® RP-18 etc.) are examples of
reversed phase packings.
Attractiveness (retention) of organic analytes from polar solutions (e.g. water)

onto these SPE materials is due primarily to the attractive forces between the
carbon-hydrogen bonds in the analyte and the functional groups on the silica
surface. These “nonpolar-nonpolar” attractive forces are commonly called van
der Waals forces, or dispersion forces. A non-polar solvent is used to elute
any adsorbed compounds from a reversed phase SPE tube or disk. It does
so by disrupting the forces that bind the analyte to the packing. It should be
noted that all silica based bonded phases have some percentage of residual
unreacted silanols that act as secondary interaction sites. These secondary
interactions may be useful in the extraction or retention of highly polar
analytes or contaminants, but may also irreversibly bind compounds of
interest.
109 CHE3143/1/2009-2011
SPE does not have the limitation associated with solvent extraction, such as
incomplete phase separations, less-than-quantitative recoveries, use of
expensive and disposal of large quantities of organic solvents. It is however,
more efficient than solvent extraction, yields quantitative extractions that are
easy to perform, is rapid, and can be automated. Solvent use and laboratory
time are significantly reduced. SPE products are excellent for sample
extraction, concentration and cleanup. They are available in a wide variety of
chemistries, adsorbents, and sizes. Selecting the most suitable product for
each application and sample is important.
16.2.2 Normal phase SPE
This type of SPE consists of polar packings and nonpolar mobile phase.
Polar-functionalized bonded silicas (such Supelco LC-CN, LC-NH2, and LC-
Diol), and polar adsorption media (LC-Si, LC-Florisil, ENVI-Florisil, and LC-
Alumina) are typically examples of normal phase. In normal phase SPE
retention of the analyte is primarily due to interactions between polar
functional groups of the analyte and polar groups on the sorbent surface. The
forces involved include hydrogen bonding, pi-pi (ʌ-ʌ) interactions, dipole-
dipole interactions, and dipole-induced dipole interactions, among others.
Elution of the adsorbed compound is achieved by solvent that is more polar
than the sample’s original matrix. Such a solvent would disrupt the binding
mechanism between the sorbent and the sample.
The bonded silicas (such as LC-CN, LC-NH2, and LC-Diol) have short alkyl
chains with polar functional groups bonded to the surface. These packings
due to their polar functional groups are much more hydrophilic relative to the
bonded reversed phase silicas. As with typical normal phase silicas, these
packings can be used to adsorb polar compounds from nonpolar matrices.
Such SPE tubes/disks have been used to adsorb and selectively elute
compounds of very similar structure (e.g. isomers), or complex mixtures or
classes of compounds such as drugs and lipids.
110 CHE3143/1/2009-2011
16.2.3 Ion exchange SPE
Ion exchange SPE consists of charged resins packings that can be used for
isolation of analytes that are charged when in a solution. There are two types
of ion exchange resins depending on the charge carried, i.e. anionic
(negatively charged) and cationic (positively charged) resins. These resins
are normally classified according to their strength, i.e. strong cation or anion
exchange resin, commonly abbreviated as SAX or SCX respectively, and
weak cation or anion exchange resin would be WAX or WCX respectively.
Compounds can be isolated on either SAX or SCX.
The primary attractiveness mechanism of the compound is based mainly on

the electrostatic attraction of the charged functional groups on the analyte to
the charged group that is bonded to the silica resin surface. The pH of the
sample matrix must be one at which both the analyte of interest and the
functional group on the bonded silica resins are charged. The analyte is
simply eluted by having a pH that neutralizes either the analyte’s functional
group or the functional group on the sorbent resin surface. When one of these
functional groups is neutralized, the electrostatic force that binds the two
together is disrupted and the compound is eluted. Alternatively, a solution
that has a high ionic strength, or that contains an ionic species that displaces
the adsorbed compound, is also used to elute the compound.
FURTHER REFERENCES:
1. Skoog et al., 2004: 1024 –1033
2. http://chemistry.brookscole.com/skoogfac/
http://infotrac.thomsonlearning.com

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CHE3143/1/2009-2011

Prof. M.M. NINDI

AN OVERVIEW OF THE CHE314-3 MODULE ………… 1

STUDY TOPIC 1 ELECTRCHEMICAL METHODS ………… 7

Study Unit 1 Voltammetry ………… 8

STUDY TOIPC 2 SPECTROSCOPIC TECHNIQUES ……….. 12

Study Unit 2 Introduction to spectrometry ………… 12

Study Unit 3 Ultraviolet/visible spectroscopic ………… 24

Study Unit 4 Infrared Spectrophotometry Techniques ………… 26

Study Unit 5 Flame emission spectrometry ………… 31

Study Unit 6 Atomic Absorption Spectrophotometry ………… 36

6.1.2.1 Flame sources ............... 38

STUDY TOPIC 3 SEPARATION TECHNIQUES …………. 42

Study Unit 8 Solvent Extraction ............... 47

Study Unit 9 Theory of Chromatography ............... 53

9.3 Resolution ............... 62

Study Unit 10 Thin layer chromatography ............... 68

Study Unit 11 Gas Chromatography ............... 73

Study Unit 12 High pressure liquid Chromatography ............... 81

STUDY TOPIC 4 SAMPLE PRETREATMENT ………… 91

Study Unit 13 Introduction Sample Preparation …........... 91

Study Unit 14 Preparing Samples for Analysis ………… 98

Study Unit 15 Samples Clean-up ………… 104

Study Unit 16 Samples Pre-concentration ………… 107

Welcome to the study guide for Analytical Chemistry module, CHE314-3. In

Electrochemical Spectro- Separation Methods Sample preparation

CHAPTERS CHAPTERS CHAPTERS CHAPTERS

Figure 1: Overview of the CHE314-3 MODULE. Resource: Skoog, West,

About this Course

At level two you were introduced to analytical chemistry as well as sampling

and would allow us to build on to quantitative analysis which was introduced

Purpose of the module

The module is intended to introduce students to the sample pre-treatment and

Instrumental methods can either be spectroscopic/spectrometric or

Self-study is required in open distance learning (ODL) environment as you

knowledge of elements such as sodium, Na, and its electron configurations. In

Analytical chemistry is a field that requires you to engage in regular periods of

Resources for learning

This module uses the following resources:

The prescribed textbook is: Abbreviated as SWHC throughout the Study

The recommended textbooks:

How to use this study guide

To pass this module, you are expected to complete a number of experiments,

Outcome of this Topic

In the voltammetric process current is determined solely by rate diffusion of

Voltammetric techniques can be subdivided based on the shape of potential–

One of the most common voltammetric techniques is Polarography.

1.1 Linear sweep voltammetry with DME (polarography)

A typical modern system consists of three-electrodes for aqueous work; i)

Figure 1.1: Typical polarogram for a reduction of hypothetical species A to

What are the advantages of mercury electrodes for electrochemical

Read sections 23B-2 – 23B-4 of your assigned textbook.

1.2 Diffusion current

It is the limiting current observed in polarography when the current is limited

i = k{C – Co} 1.1

1.3 Qualitative Analysis

Polarography can be used for qualitative analysis by utilising half wave

1.4 Quantitative Analysis

We have stated several times that id is proportional to the electroactive