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SEPARATION, ELECTROANALYTICAL, AND SPECTROCHEMICAL TECHNIQUES
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This document is published under the conditions of the Creative Commons
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I have received kind permission to reproduce diagrams and text from
Dr. Scott Van Bramer for Mass Spectrometry
DR. William Reusch for Molecular Spectroscopy of Michigan State University from his Organic
Chemistry Course
These are gratefully acknowledged, other sources are acknowledged where quoted
C.C Some Rights Reserved
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1.1 Table of Contents
1.1 Table of Contents 3
2 INTRODUCTION............................................................................................................10
3 PREREQUISITE KNOWLEDGE....................................................................................10
4 TIME...............................................................................................................................10
5 MATERIALS...................................................................................................................10
6 MODULE RATIONALE..................................................................................................11
7 OVERVIEW....................................................................................................................11
7.1 Resume 11
7.2 Outline 12
7.3 Graphic Organiser 14
7.4 General Objective 15
7.5 Specific Objectives 15
8 PRE ASSESSMENT......................................................................................................18
8.1 Answer Key 23
8.1.1 PEDAGOGICAL COMMENT FOR LEARNERS..................................................24
9 KEY CONCEPTS...........................................................................................................25
10 LEARNING TIPS............................................................................................................30
11 COMPULSORY READING............................................................................................31
11.1 Reference 1: 31
11.2 Reference 2: 31
11.3 Reference 3: 31
12 USEFUL LINKS..............................................................................................................32
13 MULTI MEDIA RESOURCES........................................................................................40
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14 UNIT I SEPARATION AND CHROMATOGRAPHIC TECHNIQUES.............................42
14.1 Summary of the Learning Activity 42
14.2 Required Readings 42
14.2.1 List of Relevant Useful Links...............................................................................43
14.3 Separation Techniques 43
14.3.1 Solvent Extraction...............................................................................................43
14.3.2 Partition Coefficient.............................................................................................43
14.3.3 Factors Affecting Solvent Extraction....................................................................44
14.4 Distillation 44
14.4.1 Simple Distillation................................................................................................44
14.4.2 Fractional Distillation...........................................................................................45
14.4.3 Steam Distillation.................................................................................................46
14.4.3.1 How Steam Distillation Works 46
14.4.4 Vacuum Distillation..............................................................................................47
14.5 Chromatography 48
14.5.1 Theory of Chromatography..................................................................................48
14.5.2 The Development Process..................................................................................48
14.5.2.1 Elution Development 48
14.5.3 Efficiency of Chromatographic Separations........................................................50
14.5.4 Band Broadening and Column Efficiency............................................................51
14.5.4.1 The Theoretical Plate Model of Chromatography 51
14.5.4.2 The Rate Theory of Chromatography 52
14.5.5 Resolution...........................................................................................................53
14.6 Types of Chromatographic Techniques 54
14.6.1 Plane Chromatography.......................................................................................54
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14.6.2 Paper Chromatography.......................................................................................55
14.7 Thin Layer Chromatography 55
14.7.1 Column Chromatography....................................................................................57
14.8 Liquid Chromatography 57
14.8.1 Components of an HPLC.....................................................................................58
14.8.1.1 Chromatography Scale: 59
14.8.2 Modes of HPLC Separation.................................................................................59
14.8.2.1 Normal phase chromatography 59
14.8.2.2 Reversed phase chromatography 59
14.8.2.3 Size exclusion chromatography 59
14.8.2.4 Ion exchange chromatography 60
14.8.2.5 Bio-affinity chromatography 60
14.9 Gas Chromatography 61
14.9.1 Gas Chromatograph Instrument..........................................................................61
14.9.1.1 Qualitative and Quantitative Application of GC 64
14.9.1.2 Formative Assessment 64
15 UNIT II ELECTROANALYTICAL TECHNIQUES...........................................................66
15.1 Summary of the Learning Activity 66
15.2 List of Required Readings 66
15.2.1 List of Relevant Useful Links...............................................................................66
15.3 Potentiometry 67
15.3.1 Ph Glass Electrodes............................................................................................67
15.3.2 Potentiometric Titrations......................................................................................68
15.3.2.1 Potentiometric Titration Stations 69
15.4 Voltammetry 70
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15.4.1 Polarography.......................................................................................................71
15.5 Pulse Polarography 73
15.5.1 Normal-Pulse Polarography (NPP)......................................................................73
15.5.2 Cyclic Voltammetry..............................................................................................77
15.5.3 Anodic Stripping Voltammetry.............................................................................77
15.5.4 Formative Assessment........................................................................................78
16 UNIT III SPECTROSCOPY AND ATOMIC SPECTROSCOPIC TECHNIQUES............79
16.1 Summary of the Learning Activity 79
16.2 List of Required Readings 79
16.3 List of Relevant Useful Links 79
16.4 List of Relevant Multimedia Resources 80
16.4.1 Spectroscopy.......................................................................................................80
16.5 Electromagnetic Radiation 80
16.5.1 Classification of Electromagnetic Radiation........................................................80
16.5.2 Units of Measurement of Energy of Electromagnetic Radiation..........................83
16.5.2.1 Interaction of Radiation with Matter 83
16.5.3 The Atom and Atomic Spectroscopy...................................................................83
16.5.4 Molecules and Molecular Spectroscopy..............................................................85
16.6 Beer’s Law 85
16.6.1.1 The Beer-Lambert Law 86
16.6.2 Molar Absorption.................................................................................................87
16.6.3 Formative Assessment........................................................................................88
16.7 Atomic Spectroscopic Techniques 89
16.7.1 Atomic Emission..................................................................................................89
16.8 Atomic Absorption 90
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16.8.1 Atomic Fluorescence...........................................................................................90
16.8.2 Quantitative Analysis by Atomic Absorption........................................................91
16.8.3 Formative Assessment........................................................................................92
17 UNIT IV MOLECULAR SPECTROCOPY 1: UV-VISIBLE AND IR................................93
17.1 Summary of the Learning Activity 93
17.2 List of Required Readings 93
17.3 List of Relevant Useful Links 93
17.4 List of Relevant Multimedia Resources 94
17.5 Ultraviolet- Visible Spectroscopy 94
17.5.1 Electronic Transitions..........................................................................................94
17.5.2 The Effect of the Structural Environment.............................................................95
17.5.3 Effects of Conjugation.........................................................................................95
17.5.4 The Molar Extinction Coefficient..........................................................................96
17.5.5 The Effect of Solvent...........................................................................................96
17.5.6 Identification of Functional Groups Using UV......................................................96
17.6 Instrumentation for UV Visible Spectrometry 97
17.6.1 Formative Exercise.............................................................................................97
17.7 Infrared Spectroscopy 99
17.7.1 Molecular Vibration and IR Spectroscopy...........................................................99
17.7.2 Fundamental and Non Fundamental Absorption Bands......................................99
17.7.3 Relative Energies of IR Absorptions....................................................................99
17.8 Identifying Functional Groups by Infrared Spectroscopy 100
17.8.1.1 IR Spectra of Saturated Hydrocarbons 100
17.8.1.2 IR absorption of O-H 101
17.8.1.3 IR absorption of C=O 102
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17.8.2 Formative Assessment......................................................................................104
18 UNIT V MOLECULAR SPECTROSCOPY 2: NUCLEAR MAGNETIC RESONANCE.107
18.1.1 Summary Of The Learning Activity....................................................................107
18.1.2 List of Required Readings.................................................................................107
18.1.3 List of Relevant Links........................................................................................107
18.2 Nuclear Magnetic Resonance Spectroscopy 108
18.2.1 NMR Phenomenon............................................................................................108
18.2.2 Formative Assessment......................................................................................109
18.3 Proton NMR 109
18.3.1 Non Radiation Effects........................................................................................110
18.3.2 Chemical Shift...................................................................................................111
18.3.3 Correlation of HNMR With Structure.................................................................111
18.3.4 Inductive and Electro Negativity........................................................................112
18.3.5 Spin-Spin Interactions.......................................................................................112
18.3.6 Hydrogen Bonding.............................................................................................114
18.4 Carbon Nmr Spectroscopy 114
18.4.1.1 Technical Problems associated with C-NMR : 116
18.4.2 13c Chemical Shift Ranges*................................................................................118
18.4.3 Formative Assessment......................................................................................118
FIGURE 34: C10H13NO2...........................................................................................................119
19 UNIT IX MASS SPECTROMETRY..............................................................................120
19.1.1 Summary Of The Learning Activity....................................................................120
19.1.2 List Of Required Readings................................................................................120
19.2 List Of Relevant Useful Links 120
19.3 Mass Spectrometry 120
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19.3.1 Isotopes.............................................................................................................121
19.4 Fragmentation Patterns 122
19.4.1 Hydrocarbons....................................................................................................122
19.4.2 Hetero Atoms....................................................................................................123
19.4.3 Finger Print Spectrum.......................................................................................124
19.4.4 Formative Assessment......................................................................................125
20 MODULE SYNTHESIS................................................................................................127
21 SUMMATIVE EVALUATION........................................................................................129
22 File Structure................................................................................................................133
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2 INTRODUCTION
3 PREREQUISITE KNOWLEDGE
Atomic structure and the concept of energy levels
RedOx introduction
Balancing RedOx equations
Standard reduction potentials
Nernst equation
Concepts of Sampling
Errors and statistics
Theories of bonding
Electrochemistry
4 TIME
Separation Techniques and Chromatographic 25 hours
Electrochemical Techniques 15 hours
Spectroscopy and Atomic Spectroscopic Techniques 20 hours
Molecular Spectroscopy 1(UV and IR) 30 hours
Molecular Spectroscopy 2 (NMR) 15 hours
Mass Spectrometry 15 hours
5 MATERIALS
You will require the following tools and resources for completing this module
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Computer, CD-ROMs, and e-library
To access this module, exams, and other relevant materials on a computer
Internet Connection to access the module and other suggested reference materials.
For interactive discussions/chat sessions
Recommended textbooks and reference materials to assist learning and further
understanding of the topics in the module
Macromedia flash player
6 MODULE RATIONALE
Separation, Electro analytical and Spectroscopic Techniques are the basis of instrumental
analysis widely applied in industry, chemistry, biochemistry, environment and school science.
These techniques are based on principles of chemistry taught at school level. Therefore in this
module we shall study the principles on which these techniques are based and acquire the
basic skills necessary to use the techniques. Studying this area deepens the understanding of
the underlying chemistry principles making the learner better able to teach them at school
level.
7 OVERVIEW
7.1 Resume
This module consists of three interrelated subject areas; Separation Techniques and
Chromatographic Techniques, Electro analytical Techniques and Spectroscopic Methods.
The module will be taught in six learning units reflecting common concepts and approaches.
Separation Techniques and Chromatographic Techniques unit will review elementary
separation techniques that are usually taught in the school system, followed by a discussion of
Chromatography Techniques these are covered by introducing the general chromatographic
theory, followed by its application in different techniques of plane and column
chromatographic techniques.
Electro Analytical Techniques will introduce principles on which potentiometry is based,
elaborate the common applications of potentiometry, this will be followed by voltammetry,
starting with polarographic techniques ending with cyclic and anodic stripping voltammetry.
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The unit on Spectroscopy and Atomic Spectroscopic Techniques will review concept of energy
matter interaction, concepts of energy levels in atoms and molecules, and the unit will end with
a discussion of atomic spectroscopic techniques.
Molecular Spectroscopy 1 will start with a discussion of the theory of UV-Visible spectroscopy,
how it arises and how it is used in qualitative and quantitative analysis, instrumentation of the
modern UV-visible spectrophotometer. The unit will end with a discussion of infrared
spectroscopy starting with how the spectra arises, the different peaks exhibited by specific
functional groups and how to apply IR in identification of functional groups and compounds.
Molecular Spectroscopy 2 will introduce nuclear magnetic resonance phenomenon, followed
by a discussion of proton NMR, the relationship of chemical shift with the molecular chemical
environment and how proton NMR used in identification of functional groups. The unit ends
with a discussion of the carbon NMR and how it compliments proton NMR in analysis of
compounds.
The last learning unit will be Mass Spectrometry starting with how mass spectra arises, how it
is used in identification of organic compounds ending with the Instrumentation for mass
spectrometry.
7.2 Outline
UNITI I SEPARATION AND CHROMATOGRAPHIC TECHNIQUES- 25 hours
Separation Techniques
o Solvent Extraction
o Distillation
Chromatography
o Theory of Chromatography
o The Development Process
Types of Chromatographic Techniques
o Plane Chromatography
o Plane Chromatography
o Liquid Chromatography
o Gas Chromatography
UNIT II ELECTROANALYTICAL TECHNIQUES- 15 Hours
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Potentiometry.
o Ion Selective Electrodes
o pH Glass Electrodes
o Potentiometric Titrations
Voltammetry
o Polarography
o Pulse Polarography
o Cyclic Voltammetry
o Anodic Stripping Voltammetry
UNIT III SPECTROSCOPY AND ATOMIC SPECTROSCOPIC TECHNIQUES- 20 hours
Spectroscopy:
o Electromagnetic Radiation
o The Atom and Atomic Spectroscopy
o Beers law
Atomic Spectroscopic Techniques
UNIT IV MOLECULAR SPECTROCOPY 1: UV-VISIBLE AND IR- 30 hours
Ultraviolet- Visible Spectroscopy
o Electronic transitions
o Identification of functional groups Using UV
o Instrumentation for UV Visible Spectrometry
Infrared Spectroscopy
o Molecular Vibration and IR Spectroscopy
o Relative energies of IR Absorptions
o Identifying Functional Groups by Infrared Spectroscopy
UNIT V MOLECULAR SPECTROSCOPY 2: NUCLEAR MAGNETIC RESONANCE 15 hours
Nuclear Magnetic Resonance Spectroscopy
o Proton NMR
o Chemical Shift
o Correlation of HNMR With Structure
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SEPARATION TECHNIQUES AND CHROMATOGRAPHIC TECHNIQUES
SEPARATION, ELECTROANALYTICAL, AND SPECTROCHEMICAL TECHNIQUES
ELECTROANALYTICAL TECHNIQUES
INTRODUCTION TO SPECTROSCOPY AND ATOMIC SPECTROSCOPY
MOLECULAR SPECTROSCOPY 1
MASS SPECTROMETRY
MOLECULAR SPECTROSCOPY 2
Carbon NMR Spectroscopy
UNIT IX MASS SPECTROMETRY- 15 hours
Mass Spectrometry
o Fragmentation Patterns
o Finger Print Spectrum
7.3 Graphic Organiser
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7.4 General Objective
The General objectives of this module are three: to explain the concepts underlying modern
analytical Techniques, give learners the basic skills to apply the concepts to simulated real
world problems and deepen the students understanding of chemistry principles governing
these techniques.
7.5 Specific Objectives
Unit Learning objective(s)
1. Separation and
Chromatographic Techniques
At the end of the unit learners will be able to:
Recall Separation methods that are taught in School
Explain the principles underlying solvent extraction
Solve numerical hypothetical problems regarding
solvent Extraction
Name and draw apparatus used for solvent extraction
Name common column and plane Chromatographic
Techniques.
Explain the theory underlying each column and plane
Chromatographic Techniques
Recall equipment for plane and column
chromatography
2. Electro analytical
Techniques
At the End of this unit the student willbe able to:
Recall the theory on which potentiometry is based
Explain the application of potentiometry to pH
measurement, ion selective electrode and automatic
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Unit Learning objective(s)
titration stations
Recall the theory of Voltammetry
Interpret Voltammetric data
quantitatively and qualitatively
Explain the concept of on which
polarographic analysis is based
Interpret polarographic data to identify and quantify
chemical Species
3. Introduction to
Spectroscopic and Atomic
Spectroscopic Techniques
At the end of the unit learners be able to:
Name the parts of the electromagnetic spectrum
Recall effects of radiation on atoms and molecules
Recall Plank’s law and apply it to spectroscopic
problems
Electronic energy levels in atoms and molecules
Recall Beers law and apply to quantitative problems
Explain electronic energy levels in atoms and
transitions caused by absorption of radiation.
Explain the concepts on which AAS, AES, AFS is
based
Recall AES, AFS and AAS Instrumentation
Calculate quantities based on hypothetical AAS, AFS
and AES observations
4. Molecular Spectroscopy 1:
UV visible and Infra Red
Spectroscopy
At the end of the unit learners will be able to:
Recall the electronic transitions caused by absorption
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Unit Learning objective(s)
of UV-Vis Radiation
Correlate Absorption of specific UV- Visible radiation
frequencies to molecular functional groups
Use hypothetical data to determine concentrations
using UV data
Recall parts of a modern UV Spectrophotometer and
their functions.
Recall the electronic transitions caused by absorption
of IR Radiation
Correlate Absorption of specific IR frequencies to
molecular functional groups
Correlate Absorption of specific IR frequencies to
molecular structure of Simple organic molecules.
Recall parts of a modern IR Spectrophotometer and
their functions.
5. Molecular Spectroscopy 2
Nuclear Magnetic Resonance
Spectroscopy (NMR)
Spectroscopy
At the end of the unit learners will be able to:
Explain how the phenomenon of NMR arises
Recall nuclei that exhibit NMR
Explain Proton NMR phenomena
Correlate Absorption of specific HNMR frequencies to
molecular functional groups
Correlate Absorption of specific HNMR frequencies to
molecular structure of Simple organic molecules.
Explain the special features of C-13 NMR phenomena
Recall the nature of information provided by C-13
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Unit Learning objective(s)
NMR
Recall parts of a modern NMR Spectrophotometer and
their functions.
6. Mass Spectrometry At the end of the unit learners be ableto:
Explain how the phenomenon of mass spectrum
arises
Explain rules followed by fragmentation in Mass
spectrum
Correlate mass spectrum to specific structural
elements in a molecule
Use the mass spectrum to identify the molecular
species.
Use high resolution mass spectrum and molecular
mass calculator to uniquely identify structural
elements
Recall parts of a modern mass Spectrometer and their
functions.
8 PRE ASSESSMENT
1) A beryllium atom has 4 protons, 5 neutrons, and 4 electrons. What is the mass number
of this atom?
A)4 B)9 C)8 D)7
2) The lowest principal quantum number for an electron is
A)1 b)0 C)2 D)-1
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3) Which of the following statements is true concerning acids and bases?
A) acids and bases don't react with each otherB) acids mixed with bases neutralize each otherC) acids mixed with bases make stronger basesD) acids mixed with bases make stronger acids
4) Neutral solutions have a pH of:?
a)0 b) 1 c) 7 d)10
5) Compared to the charge and mass of a proton, an electron has
a) the same charge and a smaller mass
b) the same charge and the same mass
c) an opposite charge and a smaller mass
d) an opposite charge and the same mass
6) What is the empirical formula of the compound whose molecular formula is P4O10
a) PO
b) PO2
c) P2O5
d) P8O20
7) Which of the following conversions requires an oxidizing agent?
a) Mn3+ --> Mn2+
b) C2H4 --> C2H6
c) (2CrO4)2- --> (Cr2O7)2-
d) SO2 --> SO3
8 The hydrogen halides are all polar molecules which form acidic solutions. Which of the
following is the weakest acid?
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a) HI
b) HF
c) HBr
d) HCL
9 Calculate the [H+] in a solution that has a pH of 8.38.
A) 1.21 x 10-2
b) 3.8 x 10-8
c) 2.40 x 108
d) 4.17 x 10-9
10) In all electrochemical cells, the process that takes place at the anode is
_________________ and the process that takes place at the cathode is _________________.
A) oxidation, reduction
b) reduction, reduction
c) reduction, oxidation
d) oxidation, oxidation
11) What is the oxidation state of S in H2SO3?
a) +4
b) +2
c) 0
d) +6
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12)The standard hydrogen electrode is assigned a potential of:
a) -1.00 volts
b) 0.76 volts
c) 0
d) 1.00 volts
13) The equation that represents a reaction that is not a redox reaction is:
a) Zn + CuSO4 → ZnSO4 + Cub) 2H2O2 → 2H2O + O2
c) H2O + CO2 → H2CO3
d) 2H2 + O2 → 2H2O
14) A mole of electrons has a charge of 96,485 coulombs per mole of electrons. This
quantity is known to chemists as:
a) 1 watt
b) 1 Ampere
c) 1 joule
d) 1 faraday
15) Which of the following properties of water explains its ability to dissolve acetic acid?
a) The high surface tension of water, which is due to the formation of hydrogen bonds
between adjacent water molecules
b) The ability to serve as a buffer, absorbing the protons given off by acetic acid.
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c) The ability to form hydrogen bonds with the carbonyl and the hydroxyl groups of acetic
acid.
d) None of the above
16) The pH of a solution is equal to:
A. the hydrogen ion concentration, [H +]
B. log [H +]
C. -log [H +]
D. ln [H +]
E. -ln [H +]
17) If the concentration of H + n a solution is 10 - 3 M, what will the concentration of OH - be in the same solution at 25° C?
A. 10 - 3 M
B. 10 - 11 M
C. 1011 M
D. 2 x 10 - 11 M
E. 10 - 14 M
18) How many ml of a 0.4 M HCl solution are required to bring the pH of 10 ml of a 0.4 M
NaOH solution to 7.0 (neutral pH)?
A. 4
B. 40
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C. 10
D. 20
E. 2
19) An atom of which of the following elements has the greatest ability to attract electrons?
a) silicon
b) sulphur
c) Nitrogen
d) Chlorine
20) Which element has the highest first ionization energy?
a) Aluminium
b) Sodium
c) calcium
d) phosphorus
8.1 Answer Key
1 b
2 a
3 b
4 c
5 a
6 c
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7 d
8 b
9 d
10) a
11) a
12) c
13) c
14) d
15) c
16) c
17) b
18) c
19) d
20) d
8.1.1 PEDAGOGICAL COMMENT FOR LEARNERS
Less than 30% Learner strongly advised review prerequisite knowledge
concepts before proceeding with the module
Between 30-60% learner is prepared to continue with the module but
may be required to refresh some of the areas
Above 60% Learner is well prepared with the prerequisite
knowledge
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9 KEY CONCEPTS
Separation and chromatography
Solvent is the term for the organic layer
Diluent is the term for an inert liquid used to dissolve an extractant, and to dilute the system.
Extractant is the term for a metal extraction agent
Raffinate is the term for the aqueous layer after a solute has been extracted from it
Scrubbing is the term for the back extraction of an unwanted solute from the organic phase
Stripping is the term for the back extraction from the organic phase
Asymmetry: A factor describing the shape of a chromatographic peak. Theory assumes a
Gaussian shape peak that is symmetrical. The peak asymmetry factor is the ratio (at 10
percent of the peak height) of the distance between the peak apex and the back side of the
chromatographic curve to the distance between the peak apex and the front side of the
chromatographic curve. A value >1 is a tailing peak, while a value <1 is a fronting peak.
Baseline:The baseline is the line drawn by the data system when the only signal from the
detector is from the mobile phase.
Chromatography:A chemical separation technique based on the differential distribution of the
constituents of a mixture between two phases, one of which moves relative to the other.
phase.
Dead volume (Vd):The volume outside of the column packing itself. The interstitial volume
(intraparticle and interparticle volume) plus extracolumn volume (contributed by injector,
detector, connecting tubing, and endfittings) all combine to create the dead volume. This
volume can be determined by injecting an inert compound. For example, a compound that
does not interact with the column packing. It is also abbreviated Vo or Vm.
Detector
An electronic device that quantitatively discerns the presence of the separated components as
they elute. There are different types of detectors. Some of the common detector types are:
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UV/Visible light absorbance, differential refractive index, electrochemical, conductivity, and
fluorescence.
Displacement chromatography
A chromatographic process in which the sample is placed onto the head of the column and is
then displaced by a compound that is more strongly sorbed than the compounds of the original
mixture. Sample molecules are displaced by each other and by the more strongly sorbed
compound. The result is that the eluting sample solute zones may be sharpened.
Displacement techniques have been used mainly in preparative EPLC applications.
External standards: A separate sample containing known quantities of the same compounds
of interest. External standards are used primarily for peak identification by comparing elution
times.
Hydrophilic: It is often referred to as water loving. It adverts both to water compatible
stationary phases, and to water soluble molecules. Most columns used to separate proteins
are hydrophilic in nature and should not sorb or denature protein in the aqueous environment.
Injector: A mechanism for accurately injecting a predetermined amount of sample into the
mobile phase stream. The injector can be a simple manual device, or a sophisticated auto
sampler that permits automated injections of many different samples for unattended operation
Partition coefficient (K): The amount of solute in the stationary phase relative to the amount
of solute in the mobile phase. It can also be the distribution coefficient, KD.
Retention time (tR’): The time between injection and the appearance of the peak maximum.
The adjusted retention time tR’ adjusts for the column void volume.
Retention volume (VR):The volume of mobile phase required to elute a substance from the
column. This is where Vm is the void volume, KD the distribution coefficient, and Vs the
stationary phase volume.
Stationary phase: The immobile phase involved in the chromatographic process. The
stationary phase in liquid chromatography can be a solid, a bonded or coated phase on a solid
support, or a wall coated phase. The stationary phase used often characterizes the LC mode.
For example, silica gel is used in adsorption chromatography, an octadecylsilane bonded
phase in reversed-phase chromatography, etc.
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Electro Analytical Chemistry
background current–current not due to chemical reaction of the analyte
capacitive current–background current due to the electrode acting as a capacitor and
becoming charged.
charging current–see capacitive current
diffusion–movement through solution due to a concentration gradient
half-cell–one half of an electrochemical cell containing an electrode, an electrically conductive
solvent system, analyte and a salt bridge connection
half-wave potential (E1/2)–potential at half of the peak or limiting current; a measure of formal
potential
indicator electrodes–electrodes used in potentiometry who potentiometry who potential
depends on the activity of analyte
ion selective electrode (ISE)–electrode used in potentiometry which gives a potential signal
in the presence of the ion for which it is selective
linear sweep voltammetry (LSV)–measuring current as the potential is systematically
changed(linearly increased or decreased)
migration–movement through a solution due to a potential gradient
polarography–linear sweep voltammetry at a dropping mercury electrode
potential (also electrochemical potential)–measurement of energy of an electrochemical
reaction
potential window–range of potentials for a given solvent/electrode system where analytical
measurements can be made
reference electrode– half-cell with known, constant electrochemical potential; unit normally
volts (V)
silver/silver chloride electrode–reference electrode of a sliver wire coated with silver chloride
and submerged in a saturated solution of aqueous
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Spectroscopy
ABSORPTION SPECTROSCOPY A type of spectroscopy where the amount of radiation, of a
particular wavelength, absorbed by the sample being analysed is measured. The wavelength
that is absorbed depends of the molecule as different electronic transitions and therefore
absorption occurs at different wavelengths.
BEER-LAMBERT LAW An equation relating absorbance, path length, molar absorption
coefficient and concentration of the sample: A = cl
CHROMOPHORE A chromophore is a functional group that is responsible for absorption e.g.
an alkene absorbs at max=177nm.
CONJUGATION An extended series of alternating single and double/triple bonds which
causes the p orbitals to overlap. Conjugated systems tend to show absorption in the visible
region
DOUBLE BOND A bond where (e.g. in Carbon) the s orbital and two of the p orbitals hybridize
to give 3 sp2 orbitals which form sigma, , bonds (e.g. with hydrogen or carbon). The
remaining p orbital forms a pi, bond with another sp2 hybridized carbon:
ELECTROMAGNETIC SPECTRUM The range of electromagnetic energy with wavelengths
ranging from 105m (radio waves) to 10-14m (gamma radiation). In between these extremes is
the infrared, visible, ultraviolet and X-ray regions.
FLUORESCENCE Fluorescence occurs when an electron is promoted from the ground state
to the excited state. When the energy is lost from the electron, it is first of all lost by heat
through vibrational relaxation then by giving out light (fluorescing) when it returns to the ground
state.
HOMO Highest Occupied Molecular Orbital the orbital in a molecule where the highest energy
level is occupied by an electron at absolute zero.
LUMO Lowest Unoccupied Molecular Orbital, the lowest energy orbital which is not occupied
by an electron at absolute zero.
MOLAR ABSORPTION COEFFICIENT A measure of the amount of radiation absorbed per
unit concentration of the sample as the equation can be rearranged to: = A / cl (Beer-
Lambert Law). This value is a constant for a particular substance.
28
MOLECULAR ORBITALS Orbitals that result from the interaction of atomic orbitals when
bonds are formed. Bonding orbitals are lower in energy than the original atomic orbitals, anti-
bonding orbitals are higher in energy and non-bonding orbitals are of equal energy.
SPECTROSCOPY The method of producing and analyzing data to allow determination of the
structure of molecules.
TRANSMITTANCE The amount of radiation which passes through the sample i.e. is not
absorbed. Amount of radiation going in/amount of radiation that passes through. When this
fraction is multiplied by 100, the value obtained is the percentage transmission.
ULTRAVIOLET REGION The region of the electromagnetic spectrum which has wavelengths
between 400-4nm . The UV region is split into three more regions: near UV (400-300nm), far
UV (300-200nm) and extreme/vacuum UV (less than 200nm). The latter is know as the
vacuum UV region as the radiation is absorbed by oxygen. This means that if vacuum UV
radiation is being used then the apparatus must be evacuated.
VISIBLE REGION The region of the electromagnetic spectrum (700-400nm) which the human
eye is sensitive to and sees as white light and colours.
WAVELENGTH The measurement of waves from peak to peak e.g. radio waves have a
wavelength of up to 10km where gamma radiation have wavelengths as short as 10 -14m
(0.00000000000001m).
Mass Spectrometry
Mass Spectrometry: This is the technique in which an instrument is employed to produce ions
from atoms or molecules (the source) which are then separated according to their charge-to-
mass-ratios (see m/z) (the analyser) and detected.
Double Focusing A combination of electrostatic (E) and magnetic (B) fields is used to
compensate for variations in the energies of the ions formed in the source and thence to
improve the resolution (qv) of the analyser. (see also forward and reverse geometry).
29
Accelerating Voltage The voltage applied to the source to accelerate the ions formed into the
analyser.
Unified Atomic Mass Unit The symbol for the mass of a particle based on 12C = 12u exactly.
m/z The ratio of charge to mass of the ion detected. z is often unity but can be a larger integer
especially in ESI-MS.
Molecular Ion The ion formed from the original molecule in the source.
Radical Ion An ion containing an unpaired electron.
Average Mass (Mr) The mass of a particle or molecule of given empirical formula calculated
using atomic weights for each element.
Accurate Mass Isotopes have unique precise masses, a consequence of which is that the
elemental composition of any molecule, or fragment of one, can be calculated from its mass if
this is sufficiently accurately determined.
10 LEARNING TIPS
The material in this module is presented in increasing order of complexity from the beginning
of each unit to the end learning unit. The learner is therefore advised to study each unit from
the beginning sequentially to the end. The learner is advised to stick to the proposed time
schedule allocated for each unit.
There are four major resources for this module namely, the module as given, the online
references, reference books and formative assessment.
The learner should use the material presented as the primary learning tool carefully covering a
complete unit before referring to the text books and the online resources.
Throughout the module where the student can profitably refer to online resources has been
indicated in the body of the module.
The formative assessments are included in the module to reinforce understanding of key
concepts and skills.
These should be attempted immediately after covering the section and used as practice
questions.
30
Summative evaluation examines the understanding of concepts and retention of facts of a
given learning unit.
11 COMPULSORY READING
11.1 Reference 1:
Title: Spectrometric Methods of identification of organic Compounds,
Authors: Robert M Silverstein; Francis X Webster; David J Kiemle
Publisher: Wiley; 7 edition, 2005
Abstract: This book provides a thorough introduction to the three areas of spectrometry most
widely used in spectrometric identification: mass spectrometry, infrared spectrometry, and
nuclear magnetic resonance spectrometry. The text uses a problem-solving approach with
extensive reference charts and tables. Offers an extensive set of real-data problems offers a
challenge to the practicing chemist.
Rationale: This reference is intended to give the student an opportunity to acquire the
practical skills of interpreting spectra. The learner is advised to use this book for practice
questions and to master the techniques. By attempting two to three problems on each
technique complete mastery of spectroscopy techniques can be achieved. This book is the
primary reference for UV, IR, NMR and MS spectroscopy.
11.2 Reference 2:
Title: Principles of Instrumental Analysis
Authors: Douglas A. Skoog, F. James Holler, and Stanley R. Crouch:
Publishers: Brooks Cole; 6th edition 2006
Abstract: This book places an emphasis on the theoretical basis of each type of instrument,
its optimal area of application, its sensitivity, its precision, and its limitations.
Rationale: This book covers all aspects of this module in a very simplified way but it is the
primary reference for Electro analytical Techniques and chromatographic methods.
31
11.3 Reference 3:
Title: The Essential Guide to Analytical Chemistry
Author: Georg Schewdt
Publishers: John Wiley and Sons 2nd edition 1997
Abstract: This book is written as a quick reference for this module it contains, all the material
covered in the module in a highly readable pocket-sized form. It has unique format with full
color diagrams facing concise text making it easy to dip into and find relevant information. The
clear, schematic diagrams illustrate important procedures and instrumentation as well as
presenting real examples of application by means of simple spectra.
Rationale: This book is included as a final reference to polish the understanding of the module
for the learners; it should not be used for initial learning of the topics as the depth is not at the
same as reference 2 and 3.
12 USEFUL LINKS
Address: http://www.mhhe.com/physsci/chemistry/carey/student/olc/ch13ir,html
32
Summary: This link is part of a large course of chemistry for purposes of this module. How-ever, the learner is required to read only chapter thirteen. It presents the material covered in the module in a simple and understandable way. This reference provides an alternative ap-proach to the study of the module, with theoretical concepts tutorials, and worked examples.
Justification: The worked examples in this reference provide practice in solving problems as-sociated with the module and reinforcing understanding concepts
Address: http://ull.chemistry.uakron.edu/analytical/Spectrophotometry/
33
Summary: This site contains summarized notes on the concepts covered in the module Justification: It is good to facilitate the learner remember facts and key concepts.
http://www.nd.edu/~smithgrp/structure/workbook.html
34
Summary: This site contains a collection of practice problems
Justification: These problems are useful for mastering molecular spectroscopy and mass
spectrometry.
35
Address: http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/spectro.htm#contnt
Summary: This site contains a comprehensive organic Chemistry Course
Justification: The approach of this course can provide deeper understanding of material
covered in this module.
36
http://www.chromatography-online.org/topics/gas/chromatography/detectors.html
Summary: This site is one of the most comprehensive chromatography sites.
Justification: The depth is a little above what is required for this module should be used as a
reference.
37
http://www.cis.rit.edu/htbooks/nmr/bnmr.htm
Summary: This site offers a good treatment of the 13-Carbon NMR with frequent explanations
accompanied by spectrums.
Justification: The treatment tends to go beyond the requirements of this module therefore the
learner is advised to use it only for reference.
38
http://weather.nmsu.edu/Teaching_Material/soil698/Student_Reports/Spectroscopy/report.htm
Summary: This is a student report on AAS
Justification: This page presents atomic spectroscopy in a simplified form
39
http://www.chemguide.co.uk/index.html#top
Summary: The site was originally intended to meet the needs of UK A level chemistry
students, but I have since been widening it to cover material on all the UK-based syllabuses
including A level, IB, Scottish Advanced Higher and Cambridge International. In fact it is now
being used by people on equivalent (16 to 18 year old) courses worldwide and by students at
the beginning of university level courses.
Justification: This site contains very simplified explanations of the material covered in this
module Justification
40
13 MULTI MEDIA RESOURCES
http://www.colby.edu/chemistry/NMR/H1pred.html
Summary: A nice collection of applications for interpreting NMR, IR and mass spectra. Created at
Colby College.
Justification: this site provides many multimedia tools for reinforcing the learning of this unit
41
http://www.shsu.edu/~chemistry/primers/primers.html
Summary: This site contains a large collection of multi media resources across all themes
covered in this module.
Justification: This site should be used a source of multimedia resources
42
14 UNIT I SEPARATION AND CHROMATOGRAPHIC TECHNIQUES
14.1 Summary of the Learning Activity
At the end of the unit learners will be able to:
Recall Separation methods that are taught in School
Explain the principles underlying solvent extraction
Solve numerical hypothetical problems regarding solvent Extraction
Name and draw apparatus used for solvent extraction
Name common column and plane chromatographic techniques.
Explain the theory underlying each column and plane Chromatographic Techniques
Recall equipment for plane and column chromatography
14.2 Required Readings
Separation
http://en.wikipedia.org/wiki/Separation_process
http://en.wikipedia.org/wiki/Distillation#Laboratory_scale_distillation
http://en.wikipedia.org/wiki/Liquid-liquid_extraction
http://en.wikipedia.org/wiki/Separating_funnel
http://en.wikipedia.org/wiki/Distillation
Chromatography
http://en.wikipedia.org/wiki/HPLC
http://en.wikipedia.org/wiki/Chromatography
http://hplc.chem.shu.edu/HPLC/index.html
http://www.waters.com/watersdivision/ContentD.asp?watersit=JDRS-5LTGBH
http://www.chemguide.co.uk/analysis/chromatography/hplc.html
http://www.chemguide.co.uk/analysis/chromatography/column.html#top
43
14.2.1 List of Relevant Useful Links
http://www.chromatography-online.org/Principles/Introduction/rs1.html
http://antoine.frostburg.edu/chem/senese/101/matter/chromatography.shtml
http://www.chemguide.co.uk/analysis/chromatogrmenu.html#top
http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html#top
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm
http://www.forumsci.co.il/HPLC/modes/modes3.htm
http://www.chem.ubc.ca/courseware/121/tutorials/exp3A/columnchrom/
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm2.htm
14.3 Separation Techniques
14.3.1 Solvent Extraction
Liquid- liquid extraction also known as solvent extraction and partitioning, is a method to
separate compounds based on their relative solubility in two different immiscible liquids,
usually water and an organic solvent. It is an extraction of a substance from one liquid phase
into another liquid phase. In this technique a solution ( usually aqueous) containing a solute or
solutes is brought into contact with a second solvent ( usually organic) with the aim of
transferring one or more of the solutes from the solution to the to the second solvent. The
solution is vigorously shaken to make intimate contact with the solvent. The apparatus is
allowed to stand to allow phases to separate.
14.3.2 Partition Coefficient
Partition or distribution coefficient (KD) is the ratio of concentrations of a compound in the two
phases of a mixture of two immiscible solvents at equilibrium hence these coefficients are a
measure of differential solubility of the compound between these two solvents.
44
Normally one of the solvents chosen is water while the second is hydrophobic such as octanol.
Hence both the partition and distribution coefficient are measures of how hydrophilic ("water
loving") or hydrophobic ("water hating") a chemical substance is.
14.3.3 Factors Affecting Solvent Extraction
Polarity of the solute and the polarity of the solvent: in general polar solvent will be distributed
into the more polar solvent and non polar solutes will be more soluble in the organic solvents
unless they incorporate sufficient number of hydrophilic functional groups like hydroxyl, and
sulphonic
Generally ionic compounds would not be expected to extract into organic compounds, these
can be extracted by reacting them with complexing agents to form large neutral non polar
entities.
14.4 Distillation
Laboratory scale distillations are almost exclusively run as batch distillations. The device used
in distillation, sometimes referred to as a still, consists at a minimum of a reboiler or pot in
which the source material is heated, a condenser in which the heated vapour is cooled back to
the liquid state, and a receiver in which the concentrated or purified liquid, called the distillate,
is collected. Several laboratory scale techniques for distillation exist
14.4.1 Simple Distillation
In simple distillation, all the hot vapours produced are immediately channelled into a condenser
which cools and condenses the vapours. Thus, the distillate will not be pure - its composition
will be identical to the composition of the vapours at the given temperature and pressure, and
can be computed from Raoult's law.
Simple distillation therefore usually used only to separate liquids whose boiling points differ
greatly (rule of thumb is 25 °C) or to separate liquids from non volatile solids or oils. For these
cases, the vapour pressures of the components are usually sufficiently different that Raoult's
law may be neglected due to the insignificant contribution of the less volatile component. In
this case, the distillate may be sufficiently pure for its intended purpose.
45
14.4.2 Fractional Distillation
For many cases, the boiling points of the components in the mixture will be too close. In this
case fractional distillation is used in order to separate the components well by repeated
vaporization-condensation cycles within a packed fractionating column.
Figure 1: Fractional distillation apparatus: http://en.wikipedia.org/wiki/Fractional_distillation
More theoretical plates lead to better separations. A spinning band distillation system uses a
spinning band of Teflon or metal to force the rising vapours into close contact with the
descending condensate, increasing the number of theoretical plates.
46
1.1.1.1 How fractional distillation works
As the solution to be purified is heated, its
vapours rise to the fractionating column. As it
rises, it cools, condensing on the condenser
walls and the surfaces of the packing material.
Here, the condensate continues to be heated by
the rising hot vapours; it vaporizes once more.
However, the composition of the fresh vapors is
determined once again by Raoult's law. Each
vaporization-condensation cycle (called a
theoretical plate) will yield a purer solution of the
more volatile component. In reality, each cycle at
a given temperature does not occur at exactly
the same position in the fractionating column;
theoretical plate is thus a concept rather than an
accurate description.
14.4.3 Steam Distillation
Figure 2: Steam distillation: http://en.wikipedia.org/wiki/Steam_distillation accessed March 2008
14.4.3.1 How Steam Distillation Works
Steam distillation is a method for distilling compounds which are heat-sensitive. This process
involves using bubbling steam through a heated mixture of the raw material. By Raoult's law,
some of the target compound will vaporize (in accordance with its partial pressure). The
vapour mixture is cooled and condensed, usually yielding a layer of oil and a layer of water.
47
14.4.4 Vacuum Distillation
Figure 3: vacuum distillation apparatus,
Steam distillation useful for compounds which boil beyond their decomposition temperature at
atmospheric pressure and which would therefore be decomposed by any attempt to boil them
under atmospheric pressure.
48
Some compounds have very high boiling
points. To boil such compounds, it is often
better to lower the pressure at which such
compounds are boiled instead of
increasing the temperature. Once the
pressure is lowered to the vapour
pressure of the compound (at the given
temperature), boiling and the rest of the
distillation process can commence. This
technique is referred to as vacuum
distillation and it is commonly found in the
laboratory in the form of the rotary
evaporator.
14.5 Chromatography
14.5.1 Theory of Chromatography
Chromatography is a separation process that is achieved by distributing the components of a
mixture between two phases, a stationary phase and a mobile phase.
Those components held preferentially in the stationary phase are retained longer in the system
than those that are distributed selectively in the mobile phase. As a consequence, solutes are
eluted from the system at different times in order of their increasing distribution coefficients
with respect to the stationary phase; in this way a separation is achieved
Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple
blend of two enantiomers to a multi component mixture containing widely differing chemical
species. Furthermore, the analysis can be carried out, at one extreme, on a very costly and
complex instrument, and at the other, on a simple, inexpensive thin layer plate.
Chromatography is the basis of a large number of analytical techniques. This unit presents the
most common chromatographic techniques and their applications.
14.5.2 The Development Process
Development is the term used to describe how components are separated during the
chromatographic process.
There are three basic methods of chromatographic development frontal development,
displacement development and elution development. Most of the analytical chromatographic
development is done by elution development
14.5.2.1 Elution Development
Elution development is best described as a series of absorption-extraction processes which
are continuous from the time the sample is injected into the chromatographic system until the
time the solutes exit from it. The elution process is depicted in the Figure below.
49
Figure 4: Elution Development
As the solute enters the chromatographic system in the mobile phase its concentration is
higher than the equilibrium concentration for distribution in the stationary phase. Therefore it
immediately starts to go into the stationary phase. As more mobile phase arrives the
concentration in the stationary phase continues to increase, soon reaching the equilibrium
concentration and it then starts to desorb into the mobile phase again. Where it is transported
to a new spot on the stationary phase
The mobile phase will continuously displace the concentration profile of the solute in the
mobile phase forward, relative to that in the stationary phase which, in a grossly exaggerated
form, is depicted in Figure 4. This displacement causes the concentration of solute in the
mobile phase at the front of the peak to exceed the equilibrium concentration with respect to
that in the stationary phase. As a consequence, a net quantity of solute in the front part of the
peak is continually entering the stationary phase from the mobile phase in an attempt to re-
establish equilibrium. At the rear of the peak, the reverse occurs. As the concentration profile
moves forward, the concentration of solute in the stationary phase at the rear of the peak is
now in excess of the equilibrium concentration.
A net amount of solute must now leave the stationary phase and enters the mobile phase to
re-establish equilibrium. Thus, the solute moves through the chromatographic system as a
result of solute entering the mobile phase at the rear of the peak and returning to the stationary
phase at the front of the peak. However, the solute is always transferring between the two
phases over the whole of the peak in an attempt to attain or maintain thermodynamic
50
equilibrium. Nevertheless, the solute band progresses through the system as a result of a net
transfer of solute from the mobile phase to the stationary phase in the front half of the peak.
This net transfer of solute is compensated by solute passing from the stationary phase to the
mobile phase at the rear half of the peak.
14.5.3 Efficiency of Chromatographic Separations
The distribution of analytes between phases can often be described quite simply. An analyte is
in equilibrium between the two phases;
Amobile Astationary
The equilibrium constant, K, is termed the partition coefficient; defined as the molar
concentration of analyte in the stationary phase divided by the molar concentration of the
analyte in the mobile phase.
The time between sample injection and an analyte peak reaching a detector at the end of the
column is termed the retention time (tR). Each analyte in a sample will have a different retention
time. The time taken for the mobile phase to pass through the column is called tM.
Figure 5: The Concept of Retention Time
A term called the retention factor, k', is often used to describe the migration rate of an analyte
on a column. You may also find it called the capacity factor. The retention factor for analyte A
is defined as;
k'A = t R - tM / tM
tR and tM are easily obtained from a chromatogram. When an analytes retention factor is less
than one, elution is so fast that accurate determination of the retention time is very difficult.
51
High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the
retention factor for an analyte is between one and five.
We define a quantity called the selectivity factor, α, which describes the separation of two
species (A and B) on the column;
α, = k 'B / k 'A
When calculating the selectivity factor, species A elutes faster than species B. The selectivity
factor is always greater than one.
14.5.4 Band Broadening and Column Efficiency
To obtain optimal separations, sharp, symmetrical chromatographic peaks must be obtained.
This means that band broadening must be limited. It is also beneficial to measure the
efficiency of the column.
14.5.4.1 The Theoretical Plate Model of Chromatography
The plate model supposes that the chromatographic column is contains a large number of
separate layers, called theoretical plates. Separate equilibrations of the sample between the
stationary and mobile phase occur on these "plates". The analyte through the column by
transfer of equilibrated mobile phase from one plate to the next.
Figure 6: Theorectical Plate concept
Plates are a theoretical concept for measuring column efficiency, either by stating the number
of theoretical plates in a column, N (the more plates the better), or by stating the plate height;
the Height Equivalent to a Theoretical Plate (the smaller the better).
If the length of the column is L, then the HETP is
52
HETP = L / N
The number of theoretical plates that a real column possesses can be found by examining a
chromatographic peak after elution;
Where w1/2 is the peak width at half-height.
As can be seen from this equation, columns behave as if they have different numbers of plates
for different solutes in a mixture.
14.5.4.2 The Rate Theory of Chromatography
A more realistic description of the processes at work inside a column takes account of the time
taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate
model, which assumes that equilibration is infinitely fast). The resulting band shape of a
chromatographic peak is therefore affected by the rate of elution. It is also affected by the
different paths available to solute molecules as they travel between particles of stationary
phase. If we consider the various mechanisms which contribute to band broadening, we arrive
at the Van Deemter equation for plate height;
HETP = A + B / u + C u
Where, u is the average velocity of the mobile phase. A, B, and C are factors which contribute
to band broadening.
A - Eddy diffusion
The mobile phase moves through the column which is packed with stationary phase. Solute
molecules will take different paths through the stationary phase at random. This will cause
broadening of the solute band, because different paths are of different lengths.
Longitudinal diffusion
The concentration of analyte is less at the edges of the band than at the centre. Analyte
diffuses out from the centre to the edges. This causes band broadening. If the velocity of the
53
mobile phase is high then the analyte spends less time on the column, which decreases the
effects of longitudinal diffusion.
C - Resistance to mass transfer
The analyte takes a certain amount of time to equilibrate between the stationary and mobile
phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the
stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the
stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase,
the worse the broadening becomes.
Van Deemter plots
A plot of plate height vs. average linear velocity, of mobile phase.
Figure 7: Van Deemeter Plot
Such plots are of considerable use in determining the optimum mobile phase flow rate.
14.5.5 Resolution
Although the selectivity factor α, describes the separation of band centres, it does not take into
account peak widths. Another measure of how well species have been separated is provided
by measurement of the resolution. The resolution of two species, A and B, is defined as
Baseline resolution is achieved when R = 1.5
54
It is useful to relate the resolution to the number of plates in the column, the selectivity factor
and the retention factors of the two solutes;
To obtain high resolution, the three terms must be maximised. An increase in N, the number of
theoretical plates, by lengthening the column leads to an increase in retention time and
increased band broadening - which may not be desirable. Instead, to increase the number of
plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the
stationary phase particles.
It is often found that by controlling the capacity factor, k', separations can be greatly improved.
This can be achieved by changing the temperature (in Gas Chromatography) or the
composition of the mobile phase (in Liquid Chromatography).
The selectivity factor α, can also be manipulated to improve separations. When α, is close to
unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable
time. In these cases, k' is optimised first, and then α, is increased by one of the following
procedures:
Changing mobile phase composition
Changing column temperature
Changing composition of stationary phase
Using special chemical effects (such as incorporating a species which complexes with one of
the solutes into the stationary phase)
14.6 Types of Chromatographic Techniques
14.6.1 Plane Chromatography
Planar chromatography are separation techniques in which the stationary phase is present as
or on a plane. The plane can be a paper, serving stationery phase (paper chromatography) or
55
a layer of solid particles spread on a support such as a glass plate (thin layer
chromatography).
14.6.2 Paper Chromatography
Paper chromatography is a technique that involves placing a small dot of sample solution onto
a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of
solvent and sealed. As the solvent rises through the paper it meets the sample mixture which
starts to travel up the paper with the solvent. Different compounds in the sample mixture travel
different distances according to how strongly they interact with the paper. This allows the
calculation of an Rf value and can be compared to standard compounds to aid in the
identification of an unknown substance.
14.7 Thin Layer Chromatography
Thin layer chromatography (TLC) is similar to paper chromatography. However, instead of
using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent
such as silica gel, alumina, or cellulose on a flat, inert substrate usually glass plates or plastic
material. Compared to paper, it has the advantage of faster runs, better separations, and the
choice between different adsorbents. Different compounds in the sample mixture travel
different distances according to how strongly they interact with the adsorbent. This allows the
calculation of an Rf value which can be compared to standard compounds to aid in the
identification of an unknown substance.
56
Figure 8: Development of a TLC separation: source http://www.waters.com/watersdivision/ContentD.asp?watersit=JDRS-5LTGBH
Equipment for TLC and Paper Chromatography
Preparation of the plate.
In thin layer chromatography a variety of coating materials are available, although silica gel is
used more often than other materials. Thin layers of cellulose are made by spreading an
aqueous slurry of cellulose powder using one of the commercially available applicators The
aqueous slurry of cellulose powder is prepared by mixing about 15 g powder in 90 cm3 of
distilled water and dispersing the powder for about 1 min using a blender. The cellulose
powder used for inorganic TLC is of a special micro crystal line nature. In partition
chromatography Ready-to-use thin layers, prepared with the most widely used adsorbents, are
available, e.g. as precoated glass plates and plastic foils. Plastic sheets precoated with
cellulose (which may also incorporate fluorescent material) are marketed* and are very
convenient for inorganic TLC work as they can be cut to the required size.
Sample application.
The sample solution to be applied should contain between 0.1 and 10mg of the cation per cm3
and may be neutral or dilute acid: about 1µl of solution is applied with a micro syringe or
micropipette near one end of the chromatoplate (about 1.5-2.0 cm from the edge of the plate)
and the latter air dried. Equilibration of the chromatoplates is not necessary and development
of the plate can start immediately after it is dried.
Development of plates. The chromatogram is usually developed by the ascending technique
in which the plate is immersed in the developing solvent (redistilled or chromatographic grade
57
solvent should be used) to a depth of 0.5cm, The tank or chamber used is preferably lined with
sheets of filter paper which dip into the solvent in the base of the chamber; this ensures that
the chamber is saturated with solvent vapour. Development is allowed to proceed until the
solvent front has traveled the required distance (usually 10-15cm), the plate is then removed
from the chamber and the solvent front immediately marked with a pencil line.
Identification of Analytes In TLC and Paper Chromatography
Coloured substances can be seen directly when viewed against the stationary phase while
colourless species may usually be detected by spraying the plate with an appropriate reagent
which produces coloured areas in the regions which they occupy. Some compounds fluoresce
in ultraviolet light and may be located in this way. Alternatively if fluorescing material is
incorporated in the adsorbent the solute can be observed as a dark spot on a fluorescent
background when viewed under ultraviolet light, (When locating zones by this method the eyes
should be protected by wearing special protective goggles or spectacles.) The spots located by
this method can be delineated by marking with a needle,
14.7.1 Column Chromatography
Column chromatography is a separation technique in which the stationary bed is placed within
a tube.
58
The stationary phase consists of very small
particles or particles coated with a liquid in which
case the solid acts as a support placed in a
column. The particles of the stationary phase
may be solid may fill the whole inside volume of
the tube (packed column) or be concentrated on
or along the inside tube wall leaving an open,
unrestricted path for the mobile phase in the
middle part of the tube (open tubular column). Figure 9: Column Chromatography Equipment
14.8 Liquid Chromatography
Liquid chromatography (LC) is a type of column chromatography in which the mobile phase is
a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day
liquid chromatography that generally utilizes very small packing particles and a relatively high
pressure is referred to as high performance liquid chromatography (HPLC).
High Performance Liquid Chromatography (HPLC) is an analytical technique for the separation
and determination of organic and inorganic solutes in many samples especially biological,
pharmaceutical, food, environmental, industrial, etc. In a liquid chromatographic process a
liquid permeates through a porous solid stationary phase and elutes the solutes into a flow-
through detector. The stationary phase is usually in the form of small-diameter (5-10 µm)
uniform particles, packed into a cylindrical column. The typical column is constructed from a
rigid material (such as stainless steel or plastic) and is generally 5-30 cm long and the internal
diameter is in the range of 1-9 mm.
14.8.1 Components of an HPLC
Figure 10: Components of an HPLC
Solvent Delivery System pushes the solvent stream through the instrument at constant flow
rate
59
Sample injection system - introduces the sample into the liquid stream of the instrument
Column - a stainless steel tube packed with silicon beads that separates what I'm looking for
(the caffeine) from other compounds (like sugar)
Detector - An optical sensor (usually) that detects changes in the characteristics of the solvent
stream
Data System - A means of controlling the system components and storing, processing and
displaying data
A high pressure pump is required to force the mobile phase through the column at typical flow
rates of 0.1-2 ml/min. The sample to be separated is introduced into the mobile phase by
injection device, manual or automatic, prior to the column.
14.8.1.1 Chromatography Scale:
HPLC can be operated for a number of purposes
Analytical - Just Data High Sensitivity
Semi-Preparative - Data and a small amount of purified analyte (gram)
Preparative - Larger quantities of purified analytes (Kilograms) [High Capacity]
14.8.2 Modes of HPLC Separation
Separation of analytes is based on a number of mechanisms and each of these mechanisms
result in a different mode of application of HPLC
14.8.2.1 Normal phase chromatography
Normal phase HPLC separates analytes based on polarity. This method uses a polar
stationary phase and a nonpolar mobile phase, and is used when the analyte of interest is
fairly polar in nature. The polar analyte associates with and is retained by the polar stationary
phase. Adsorption strengths increase with increase in analyte polarity, and the interaction
between the polar analyte and the polar stationary phase (relative to the mobile phase)
increases the elution time.
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14.8.2.2 Reversed phase chromatography
Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and an aqueous,
moderately polar mobile phase. One common stationary phase is a silica which has been
treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. The
retention time is therefore longer for molecules which are more non-polar in nature, allowing
polar molecules to elute more readily. Retention time is increased by the addition of polar
solvent to the mobile phase and decreased by the addition of more hydrophobic solvent.
14.8.2.3 Size exclusion chromatography
Size exclusion chromatography (SEC), also known as gel permeation chromatography or gel
filtration chromatography, separates particles on the basis of size. It is generally a low
resolution chromatography and thus it is often reserved for the final polishing step of
purification. It is also useful for determining the tertiary structure and quaternary structure of
purified proteins, and is the primary technique for determining the average molecular weight of
natural and synthetic polymers.
14.8.2.4 Ion exchange chromatography
For more details on this topic, see Ion exchange chromatography.
In Ion-exchange chromatography, retention is based on the attraction between solute ions and
charged sites bound to the stationary phase. Ions of the same charge are excluded. Some
types of Ion Exchangers include: (1) Polystyrene resins- allows cross linkage which increases
the stability of the chain. Higher cross linkage reduces swerving, which increases the
equilibration time and ultimately improves selectivity. (2) Cellulose and dextrin ion exchangers
(gels)-These possess larger pore sizes and low charge densities making them suitable for
protein separation. (3)Controlled-pore glass or porous silica.
14.8.2.5 Bio-affinity chromatography
This chromatographic process relies on the property of biologically active substances to form
stable, specific, and reversible complexes. The formation of these complexes involves the
participation of common molecular forces such as the Van der Waal's interaction, electrostatic
61
interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An
efficient, bio-specific bond is formed by a simultaneous and concerted action of several of
these forces in the complementary binding sites.
Formative LC and HPLC Exercise
i) Name major components of a HPLC
ii) Name three sub techniques of HPLC
iii) Name the scales of application of HPLC and their applications
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14.9 Gas Chromatography
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a
separation technique in which the mobile phase is a gas. Gas chromatography is always
carried out in a column, which is typically packed or capillary
Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid
stationary phase (often a liquid silicone-based material) and a mobile gas phase (most often
Helium or nitrogen). The stationary phase is adhered to the inside of a small-diameter glass
tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). The
high temperatures used in GC make it unsuitable for high molecular weight biopolymers or
proteins.
14.9.1 Gas Chromatograph Instrument
Figure 11: Components of Gas Chromatograph
A typical gas chromatographic system consists of six major components named above
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium,
argon, and carbondioxide. The choice of carrier gas is often dependant upon the type of
detector which is used. The carrier gas system contains units to purify the gas by removal of
moisture and oxygen.
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Sample injection port
For optimum column efficiency, the sample should not be too large, and should be introduced
onto the column as a "plug" of vapour - slow injection of large samples causes band
broadening and loss of resolution. The most common injection method is where a micro
syringe is used to inject sample through a rubber septum into a flash vaporizer port at the head
of the column. The temperature of the sample port is usually about 50oC higher than the boiling
point of the least volatile component of the sample. For packed columns, sample size ranges
from tenths of a microliter up to 20 micro-liters. Capillary columns on the other hand, need
much less sample, typically around 10-3 µL.
Columns
There are two general types of column, packed and capillary (also known as open tubular).
Packed columns contain a finely divided, inert, solid support material (commonly based on
diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m
in length and have an internal diameter of 2 - 4mm.
Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of
two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-
coated columns consist of a capillary tube whose walls are coated with liquid stationary phase.
In support-coated columns, the inner wall of the capillary is lined with a thin layer of support
material such as diatomaceous earth, onto which the stationary phase has been adsorbed.
SCOT columns are generally less efficient than WCOT columns. Both types of capillary
column are more efficient than packed columns.
Column Oven
To get reproducible results, column temperature must be controlled to within tenths of a
degree. The optimum column temperature is dependant upon the boiling point of the sample.
To maintain a reproducible temperature, the chromatography column is maintained in an oven
that can be set at different temperatures.
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Detectors
There are many detectors which are used in gas chromatography. Different detectors will give
different types of selectivity. A non-selective detector responds to all compounds except the
carrier gas. A selective detector responds to a range of compounds with a common physical or
chemical property and a specific detector responds to a single chemical compound. Detectors
can also be grouped into concentration dependant detectors and mass flow dependant
detectors. The signal from a concentration dependant detector is related to the concentration
of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas
will lower the detectors response. Mass flow dependant detectors usually destroy the sample,
and the signal is related to the rate at which solute molecules enter the detector. The response
of a mass flow dependant detector is unaffected by make-up gas.
Detector TypeSupport gases
Selectivity DetectabilityDynamic
range
Flame
ionization
(FID)
Mass flowHydrogen
and airMost organic cpds. 100 pg 107
Electron
capture
(ECD)
Concentration Make-up
Halides, nitrates, nitriles,
peroxides, anhydrides,
organometallics
50 fg 105
Nitrogen-
phosphorusMass flow
Hydrogen
and airNitrogen, phosphorus 10 pg 106
Flame
photometric
(FPD)
Mass flow
Hydrogen
and air
possibly
oxygen
Sulphur, phosphorus, tin,
boron, arsenic, germanium,
selenium, chromium
100 pg 103
Photo-
ionization
(PID)
Concentration Make-up Aliphatics, aromatics,
ketones, esters, aldehydes,
amines, heterocyclics,
2 pg 107
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organosulphurs, some
organometallics
Data Control System
Modern gas chromatography use programmable logic interfaces and computer systems to
control the chromatographs parameters like the gas flow, oven temperature are set and
controlled by a computer programme. The data generated is acquired and stored using
computers.
Gas chromatography is purely analytical instruments for identifying and quantifying chemical
species.
14.9.1.1 Qualitative and Quantitative Application of GC
Due to the complexities of the interactions between the analytes and the column each analyte
is eluted at a unique time called its retention time tR. If two analytes elute at the same time on a
chromatograph then probably they are the same compound. Confirmation is made by running
the two analytes on the chromatograph using different columns and conditions if the two elute
at the same time under all conditions then this is the same substance.
The signal produced by the detector is usually related to the quantity of the analyte either its
mass or its concentration. The concentration of the analyte in an unknown sample is got by
determining the detector response of a series of standard solutions which are then plotted
against the known concentrations. The concentration of the unknown is then read from the
graph.
14.9.1.2 Formative Assessment
1 i) Name major components of gas chromatographs
ii) Name two types of GC columns
iii) State the common carrier gases used in GC, Name one chemical property they must all
posses
iv) Name one type of samples that are not analysed by GC and explain why.
V) Clearly explain what a selective detector is
66
2. For a typical chromatographic separation giving just-resolved peaks (Rs = 1.5), assume
that N = 3600, k' = 2, and = 1.15. Sketch the effects of changing these parameters one at a
time to (a) N = 1600, (b) k' = 0.8, and (c) a = 1.10.
3. To decrease the plate height and yet increase the resolution, what courses of action are
available? What penalties may accrue for each approach?
4. The relative response factors for p-dichlorobenzene and p-xylene (relative to the value
for benzene, assigned unity) were found to be 0.624 ± 0.034 and 0.917 ± 0.018, respectively.
Upon integration of chromatographic peaks the results in the table were obtained. Calculate
the percent composition of each sample.
Peak Area
Sample Benzene p-xylene Toluene
1 4592 2984 1238
2 512 3527 5495
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15 UNIT II ELECTROANALYTICAL TECHNIQUES
15.1 Summary of the Learning Activity
At the end of this unit the student will be able to:
Recall the theory on which potentiometry is based
Explain the application of potentiometry to pH measurement, ion selective electrode and
automatic titration stations
Recall the theory of Voltammetry
Interpret Voltammetric data quantitatively and qualitatively
Explain the concept of on which polarographic analysis is based
Interpret polarographic data to identify and quantify chemical Species
15.2 List of Required Readings
http://en.wikipedia.org/wiki/Electroanalytical_methods
15.2.1 List of Relevant Useful Links
http://www.chem.vt.edu/chem-ed/echem/electroc.html
http://www.chem.vt.edu/chem-ed/echem/potentio.html
http://electrochem.cwru.edu/ed/encycl/art-a03-analytical.htm
http://ull.chemistry.uakron.edu/analytical/Voltammetry/
http://ull.chemistry.uakron.edu/analytical/index.html
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15.3 Potentiometry
In potentiometry, the measuring setup always consists of two electrodes: the measuring
electrode, also known as the indicator electrode, and the reference electrode. Both electrodes
are half-cells. When placed in a solution together they produce a certain potential.
The potential as reflected above measures the activity of the ions rather than the
concentration. The activity of the measured ion a, which is also used in the Nernst equation, is
linked to the normally interesting analytical concentration c via the activity coefficient*:
a=γC
Where;
For dilute solutions with concentration cM 0.001mol/L, the activity coefficient tends towards 1
and the activity of the ion corresponds to its concentration as a first approximation. γ is a
function of the total electrolyte content. The mathematical relationship between the activity aM
of a measuring ion in solution ions and the potential measured between the reference
electrode and the measuring electrode is described by the Nernst equation.
re
oxido a
aLog
ZXFxRXTE 303.2
Where is the difference between the standard potential of the electrode and the potential of
the standard electrode. The indicator electrode usually contains one of the forms of the desired
ions or enabling it to measure the other form that is in the solution.
By measuring E, it is therefore possible to measure the concentration of analyte is known.
15.3.1 Ph Glass Electrodes
The glass membrane of a pH glass electrode consists of a silicate framework containing
lithium ions. When a glass surface is immersed in an aqueous solution then a thin solvated
layer (gel layer) is formed on the glass surface in which the glass structure is softer. This
applies to both the outside and inside of the glass membrane. As the proton concentration in
the inner buffer of the electrode is constant (pH = 7), a stationary condition is established on
the inner surface of the glass membrane. In contrast, if the proton concentration in the
measuring solution changes then ion exchange will occur in the outer solvated layer and cause
69
E
an alteration in the potential at the glass membrane. Only when this ion exchange has
achieved a stable condition, will the potential of the glass electrode also be constant. This
means that the response time of a glass electrode always depends on the thickness of the
solvated layer. Continuous contact with aqueous solutions causes the thickness of the
solvated layer to increase continuously – even if only very slowly which results in longer
response times. This is why conditioning the electrode in a suitable electrolyte is absolutely
necessary to ensure an initial solvated layer condition that is as stationary as possible so that
results can be obtained that are as reproducible as possible.
15.3.2 Potentiometric Titrations
In potentiometry titrations, ion sensitive electrode is used to monitor potential changes in the
titration vessel.
A cell as described before is made composed of an indicator electrode and a reference
electrode. As the titrant is added to the reaction vessel it reacts with the analyte and consumes
it. At equivalence point there is a large change in potential indicating total consumption of the
analyte.
A plot of the volume of the titrant with potential shows a large inflexion at the end point or a plot
of E vs. Volume.
Titrant Volume
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V
V
Figure 12: A plot of dE/dV versus time
Consider titration of Fe2+ with Ce4+.
At the start, the solution has only Fe2+ and on adding Ce4+ a little Fe3+ is formed and the
potential is
As more Ce4+ is added, the potential increases until all the Fe2+ is consumed and the E
because
R is the universal gas constant, T is the absolute temperature, F is the Faraday constant
15.3.2.1 Potentiometric Titration Stations
Titrations stations are modern electronic titrators that are for titration of many ions. In titration
station, an electronically controlled motor driven syringe is used to deliver measured volumes
of the titrant V, while the electrode is used to measure the corresponding potential E which is
automatically plotted to identify the end point. The more sophisticated titration stations can
deliver the final results of a titration with options for retrieving the E and V information.
71
dE/dV
15.4 Voltammetry
Voltammetry refers to the measurement of current that result from the application of potential.
Unlike potentiometry measurements, which employ only two electrodes, voltammetric
measurements utilize a three electrode electrochemical cell. The use of the three electrodes
(working or micro, auxiliary, and reference) along with the potentiostat instrument allows
accurate application of potential functions and the measurement of the resultant current. The
different voltammetric techniques that are used are distinguished from each other primarily by
the potential function that is applied to the working electrode to drive the reaction, and by the
material used as the working electrode. Common techniques to be discussed here include:
The micro electrode is usually polarized i.e. the concentration of the ions at the surface
electrode is different from the concentration of the ions from the bulk of the solution. Therefore
the diffusion of the ions from the bulk of the solution to the micro electrode becomes an
important phenomenon. The total current I= Im+ Id
Im=Migration current
Id=diffusion current
In order to maintain a constant migration current another electrolyte is added to the solution of
the electrolyte, this second electrolyte is called a supporting electrolyte, usually KCl is used.
This provides the migration current.
The different voltammetric techniques that are used are distinguished from each other primarily
by the potential function that is applied to the working electrode to drive the reaction, and by
the material used as the working electrode. Common techniques to be discussed in this
module include;
Polarography
Normal-pulse polarography (NPP)
Differential-pulse polarography (DPP)
Cyclic voltammetry
Anodic-stripping voltammetry
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15.4.1 Polarography
In Polarography the micro electrode is a succession of mercury drops (falling slowly from a
capillary tube) and is usually the cathode. The anode (or counter Electrode) is usually a pool of
mercury. The electrolyte is the solution of the analyte which must be electroactive material to
which is added and excess of a supporting electrolyte usually KCl.
This type of micro electrode is called a dropping mercury electrode (DME)
Figure 13: Dropping Mercury Electrode
If a voltage is imposed on the DME an It current will flow that is composed of the following
It=Id +Im +Ir
Residual current Ir
A small current will flow due to the capacitive charging of the mercury drops and reducible
impurities in the supporting electrolyte.
Migration Current Im
The electroactive material reaches the DME by two mechanisms; by migration and by
diffusion. If the concentration of the supporting electrolyte is high, more than 100 times the
analyte, then all the migration current will be carried by the supporting electrolyte.
Id = 708nD1/2m2/3t1/6c
With the excess of the supporting electrolyte the electro active material will reach the DME by
diffusion. As the voltage at the DME is increased this diffusion current increases until it
reaches a limiting value Id
From theory
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D is a constant from diffusion theory, n is the number of electrons involved in the
electrochemical reaction, m is the mass of mercury drops and t is the interval between mercury
drops
It can clearly be seen that the diffusion current is proportional to the concentration of the
electroactive analyte.
On application of an increasing voltage to the DME, the current changes as shown in the
diagram initially there will be only the residual current which is small and constant. On
increasing the voltage further a point will be reached when the reduction potential of the
analyte is reached and starts to increase with the increasing voltage until the limiting current Id
is reached. E½ is called the half wave potential and it uniquely identifies the electroactive
material in the analyte.
There are a number of limitations to the polarography experiment for quantitative analytical
measurements. Because the current is continuously measured during the growth of the Hg
drop, there is a substantial contribution from capacitive current. As the Hg flows from the
capillary end, there is initially a large increase in the surface area. As a consequence, the initial
current is dominated by capacitive effects as charging of the rapidly increasing interface
occurs. Toward the end of the drop life, there is little change in the surface area which
diminishes the contribution of capacitance changes to the total current. At the same time, any
redox process which occurs will result in faradaic current that decays approximately as the
74
Id
Cur
rent
VoltageE 1/2
square root of time (due to the increasing dimensions of the Nernst diffusion layer). The
exponential decay of the capacitive current is much more rapid than the decay of the faradaic
current; hence, the faradaic current is proportionally larger at the end of the drop life.
Unfortunately, this process is complicated by the continuously changing potential that is
applied to the working electrode (the Hg drop) throughout the experiment.
As such, the typical signal to noise of a polarographic experiment allows detection limits of only
approximately 10-5 or 10-6 M. Better discrimination against the capacitive current can be
obtained using the pulse polarographic techniques.
15.5 Pulse Polarography
Pulse polarographic techniques are voltammetric measurements which are variants of the
polarographic techniques described above which try to minimize the background capacitive
contribution to the current by eliminating the continuously varying potential ramp, and replacing
it with a series of potential steps of short duration.
15.5.1 Normal-Pulse Polarography (NPP)
In Normal-pulse polarography (NPP), each potential step begins at the same value (a potential
at which no faradaic electrochemistry occurs), and the amplitude of each subsequent step
increases in small increments. When the mercury drop is dislodged from the capillary (by a
drop knocker at accurately timed intervals), the potential is returned to the initial value in
preparation for a new step.
75
Figure 14: The Applied Potential Wave Form for Normal pulse Polaraography
For this experiment, the polarogram is obtained by plotting the measured current vs. the
potential to which the step occurs. As a result, the current is not followed during Hg drop
growth, and normal pulse polarogram has the typical shape of a sigmoid. By using discrete
potential steps at the end of the drop lifetime (usually during the last 50-100 ms of the drop life
which is typically 2-4 s), the experiment has a constant potential applied to an electrode with
nearly constant surface area. After the initial potential step, the capacitive current decays
exponentially while the faradaic current decays as the square root of time. The diffusion
76
current is measured just before the drop is dislodged, allowing excellent discrimination against
the background capacitive current. The normal pulse polarography method increases the
analytical sensitivity by 1 - 3 orders of magnitude (limits of detection 10 -7 to 10-8 M, relative to
normal dc polarography.
Differential Pulse Polarography is a polarographic technique that uses a series of discrete
potential steps rather than a linear potential ramp to obtain the experimental polarogram. Many
of the experimental parameters for differential pulse polarography are the same as with normal
pulse polarography (for example accurately timed drop lifetimes, potential step duration of 50 -
100 ms at the end of the drop lifetime). Unlike Normal Pulse Polarography, however, each
potential step has the same amplitude, and the return potential after each pulse is slightly
negative of the potential prior to the step.
Differential pulse polarography
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Figure 15: The Applied Potential Wave Form for Differential Pulse Polarography
In this manner, the total waveform applied to the DME is very much like a combination of a
linear ramp with a superimposed square wave. The differential pulse polarogram is obtained
by measuring the current immediately before the potential step, and then again just before the
end of the drop lifetime. The analytical current in this case is the difference between the
current at the end of the step and the current before the step (the differential current). This
differential current is then plotted vs. the average potential (average of the potential before the
step and the step potential) to obtain the differential pulse polarogram. Because this is a
differential current, the polarogram in many respects is like the differential of the sigmoidal
normal pulse polarogram. As a result, the differential pulse polarogram is peak shaped.
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Differential pulse polarography has even better ability to discriminate against capacitive current
because it measures a difference current (helping to subtract any residual capacitive current
that remains prior to each step). Limits of detection with Differential Pulse Polarography are
10-8 - 10-9 M.
15.5.2 Cyclic Voltammetry
Cyclic voltammetry (CV) is an electrolytic method that uses microelectrodes and an unstirred
solution so that the measured current is limited by analyte diffusion at the electrode surface.
The electrode potential is ramped linearly to a more negative potential, and then ramped in
reverse back to the starting voltage. The forward scan produces a current peak for any
analytes that can be reduced through the range of the potential scan. The current will increase
as the potential reaches the reduction potential of the analyte, but then falls off as the
concentration of the analyte is depleted close to the electrode surface. As the applied potential
is reversed, it will reach a potential that will reoxidize the product formed in the first reduction
reaction, and produce a current of reverse polarity from the forward scan. This oxidation peak
will usually have a similar shape to the reduction peak. The peak current, ip, is described by the
Randles-Sevcik equation:
ip = (2.69x105) n3/2 A C D1/2 v1/2
Where n is the number of moles of electrons transferred in the reaction, A is the area of the
electrode, C is the analyte concentration (in moles/cm3), D is the diffusion coefficient, and v is
the scan rate of the applied potential.
The potential difference between the reduction and oxidation peaks is theoretically 59 mV for a
reversible reaction. In practice, the difference is typically 70-100 mV. Larger differences, or
nonsymmetric reduction and oxidation peaks are an indication of a nonreversible reaction.
These parameters of cyclic voltammograms make CV most suitable for characterization and
mechanistic studies of redox reactions at electrodes.
15.5.3 Anodic Stripping Voltammetry
Anodic stripping voltammetry is an electrolytic method in which a mercury electrode is held at a
negative potential to reduce metal ions in solution and form an amalgam with the electrode.
The solution is stirred to carry as much of the analyte metal(s) to the electrode as possible for
concentration into the amalgam. After reducing and accumulating the analyte for some period
79
of time, the potential on the electrode is increased to reoxidize the analyte and generate a
current signal. The ramped potential usually uses a step function, such as in normal-pulse
polarography (NPP) or differential-pulse polarography (DPP).
The concentration of the analyte in the Hg electrode, CHg, is given by:
il td
CHg = -------
n F VHg
where il is the limiting current during reduction of the metal, td is the duration of accumulation, n
is the number of moles of electrons transferred in the half reaction, F is the Faraday constant
(96,487 coulombs/mole of e-), and VHg is the volume of the electrode. The expression for
current produced by anodic stripping depends on the particular type of Hg electrode, but is
directly proportional to the concentration of analyte concentrated into the electrode. The main
advantage of stripping analysis is the preconcentration of the analyte into the electrode before
making the actual current measurement. Anodic stripping can achieve detection of
concentrations as low as 10-10 M.
15.5.4 Formative Assessment
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16 UNIT III SPECTROSCOPY AND ATOMIC SPECTROSCOPIC TECHNIQUES
16.1 Summary of the Learning Activity
At the end of the unit learners will be able to:
Name the parts of the electromagnetic spectrum
Recall the relative energies of different regions of the electromagnetic spectrum
Recall common measurement units used in Spectroscopy
Recall effects of radiation on atoms and molecules
Recall electronic energy levels in molecules and possible transitions
Recall Beers law and its application in quantitative analysis
Explain electronic energy levels in atoms and transitions caused by absorption of
radiation.
Explain the concepts on which AAS is based
Recall AES and AAS Instrumentation
Calculate quantities based on hypothetical AAS and AES observations
16.2 List of Required Readings
http://en.wikipedia.org/wiki/Atomic_Orbital
http://en.wikipedia.org/wiki/Energy_level
http://en.wikipedia.org/wiki/Atomic_absorption_spectroscopy
16.3 List of Relevant Useful Links
http://ull.chemistry.uakron.edu/analytical/Atomic_spec/
http://www.chem.vt.edu/chem-ed/spec/atomic/aa.html
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16.4 List of Relevant Multimedia Resources
16.4.1 Spectroscopy
Spectroscopy is the study of the interaction between wave radiation or light, as well as particle
radiation and matter. Spectrometry is the measurement of these interactions and an instrument
which performs such measurements is a spectrometer or spectrograph. A plot of the
interaction is referred to as a spectrum.
Spectroscopy is often used in physical and analytical chemistry for the identification and
quantification of substances through the spectrum emitted from or absorbed by them. This unit
introduces in a simple way the concept of radiation matter interactions, the common
terminology used in spectroscopy and Beer’s law which is widely applied in the quantitative
spectroscopy. The unit further discusses commonly used atomic spectroscopic techniques.
16.5 Electromagnetic Radiation
Light is a form of electromagnetic radiation. Other forms of electromagnetic radiation include
radio waves, microwaves, infrared radiation, ultraviolet rays, X-rays, and gamma rays. All of
these, known collectively as the electromagnetic spectrum, and are fundamentally similar in
that they move at 3X108 m per second, the speed of light. The only difference between them is
their wavelength, which is directly related to the amount of energy the waves carry. The shorter
the wavelength of the radiation the higher the energy.
16.5.1 Classification of Electromagnetic Radiation
Radio waves are used to transmit radio and television signals. Radio waves have wavelengths
that range from less than a centimetre to tens or even hundreds of meters.
Microwave wavelengths range from approximately one millimetre (the thickness of a pencil
lead) to thirty centimetres (about twelve inches).
82
Infrared is the region of the electromagnetic spectrum that extends from the visible region to
about one millimetre (in wavelength). Infrared waves include thermal radiation. For example,
burning charcoal may not give off light, but it does emit infrared radiation which is felt as heat
Visible Radiation is that part of radiation that can be perceived by the human eye
Ultraviolet radiation has a range of wavelengths from 400 nm to about 10 nm. Sunlight
contains ultraviolet waves which can burn your skin
X-rays are high energy waves which have great penetrating power and are used extensively in
medical applications and in inspecting welds. X-ray images of our Sun can yield important
clues to solar flares and other changes on the Sun that can affect space weather. The
wavelength range is from about ten billionths of a meter to about 10 trillionths of a meter.
Gamma rays have wavelengths of less than about ten trillionths of a meter. They are more
penetrating than X-rays. Gamma rays are generated by radioactive atoms and in nuclear
explosions, and are used in many medical applications. Images of our universe taken in
gamma rays have yielded important information on the life and death of stars, and other violent
processes in the universe.
83
Figure 16: Electromagnetic Radiation http://en.wikipedia.org/wiki/Image:EM_Spectrum3-new.jpg#file
84
16.5.2 Units of Measurement of Energy of Electromagnetic Radiation
Radiation is postulated to have a particle nature. A unit of radiation is called a photon. Each
photon of a particular frequency of radiation is associated with energy. The energy is given by;
E= hν. Where E is the energy, h is planks constant h= 6.624x10 -34 JS-1. Each particle of
radiation is called a photon. v is the frequency of the radiation that is measured in hertz. It is
usually more convenient to express the energy of radiation in terms of wave numbers cm -1
Electromagnetic radiation
16.5.2.1 Interaction of Radiation with Matter
The energy levels requirements for all physical processes at the atomic and molecular levels
are quantized, and if there are no available quantized energy levels with spacing which match
the quantum energy of the incident radiation, then the material will be transparent to that
radiation, and it will pass through.
16.5.3 The Atom and Atomic Spectroscopy
The atomic spectroscopy includes three techniques for analytical use: atomic emission, atomic
absorption, and atomic fluorescence. These depend on electronic transitions in isolated atoms.
Because the atoms are isolated their energy levels are not affected by neighbouring atoms. In
order to understand the relationship of these techniques to each other, it is necessary to have
an understanding of the atom itself and of the atomic process involved in each technique.
Figure 17: Atomic energy levels
85
The atom is made up of a nucleus surrounded by electrons. Every element has a specific
number of electrons which are associated with the atomic nucleus. The lowest energy, most
stable electronic configuration of an atom, known as the "ground state", is the normal orbital
configuration for an atom. The atom contains other allowable orbits which can hold electrons
but are of a higher energy. If energy of the right magnitude is applied to an atom, the energy
will be absorbed by the atom, and an outer electron will be promoted to a less stable
configuration or "excited state". As this state is unstable, the atom will immediately and
spontaneously return to its ground state configuration. The electron will return to its initial,
stable orbital position, and radiant energy equivalent to the amount of energy initially absorbed
in the excitation process will be emitted. The process is illustrated in Figure 18. Note that in
Step I of the process, the excitation is forced by supplying energy. The decay process in Step
2 involving the emission of light occurs spontaneously.
Figure 18: Adapted from Perkin Elmer Corporation: Excitation and Emission
The wavelength of the emitted radiant energy is directly related to the energy of electronic
transition which has occurred. Since every element has a unique electronic structure, the
wavelength of light emitted is a unique property of each individual element. As the orbital
configuration of a large atom may be complex, there are many electronic transitions which can
occur, each transition resulting in the emission of a characteristic wavelength of tight, as
illustrated in Figure 2, E= hν
The process of excitation and decay to the ground state is involved in the three fields of
atomic spectroscopy; either the energy absorbed in the excitation process or the energy
emitted in the decay process is measured and used for analytical purposes.
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PoP
b
16.5.4 Molecules and Molecular Spectroscopy
For a given excitation process the molecule or the atom absorbs only one discrete amount of
energy. This should correspond to one frequency being absorbed. However in practice a group
of molecules exists in a number of energy states each state differing from the other by a small
amount of energy. Thus a group of molecules in a sample gives rise to absorption over a small
range of energy giving rise to a small band or a peak.
Formative Question
Explain why absorption spectra for atomic species consist of discrete lines at specific
wavelengths rather than broad bands as for molecular species.
16.6 Beer’s Law
When radiation passes through a region containing atoms or molecules the radiation will be
absorbed. The diagram below shows a beam of monochromatic radiation of radiant power P0,
directed at a sample solution. Absorption takes place and the beam of radiation leaving the
sample has radiant power P.
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Figure 19: Demonstration of Beer's law
The amount of radiation absorbed is dependent on the nature of the sample, the concentration
of the sample and the length of the sample.
The amount of radiation absorbed may be measured in by a number of parameters:
Transmittance, T = P / P0% Transmittance, %T = 100 T
Absorbance,
A = log10 P0 / P A = log10 1 / T A = log10 100 / %TA = 2 - log10 %T
The last equation, A = 2 - log10 %T, is worth remembering because it allows you to easily
calculate absorbance from percentage transmittance data.
The relationship between absorbance and transmittance is illustrated in the following diagram:
So, if all the light passes through a solution without any absorption, then absorbance is zero,
and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is
zero, and absorption is infinite.
16.6.1.1 The Beer-Lambert Law
Beer’s law or Beer lambert law states as below
A=εbc
Where A is absorbance (no units, since A = log10 P0 / P).ε is the molar absorption with units of
L mol-1 cm-1, b is the path length of the sample - that is, the path length of the cuvette in which
the sample is contained.
c is the concentration of the compound in solution, expressed in mol L-1
A=εbc
%T = 100 P/P0 = e -εbc
Suppose we have a solution of copper sulphate (which appears blue because it has an
absorption maximum at 600 nm). We look at the way in which the intensity of the light (radiant
power) changes as it passes through the solution in a 1 cm cuvette. We will look at the
reduction every 0.2 cm as shown in the diagram below. The Law says that the fraction of the light absorbed by each layer of solution is the same. For our illustration, we will
suppose that this fraction is 0.5 for each 0.2 cm "layer" and calculate the following data:
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Figure 20: Plot of transmitance and absorbance versus pathlength
A = εbc tells us that absorbance depends on the total quantity of the absorbing compound in
the light path through the cuvette. If we plot absorbance against concentration, we get a
straight line passing through the origin (0, 0).
Note that the Law is not obeyed
at high concentrations. This
deviation from the Law is not
dealt with here.
Figure 21: Beers Law and Concentration
The linear relationship between concentration and absorbance is both simple and
straightforward, which is why we prefer to express the Beer-Lambert law using absorbance as
a measure of the absorption rather than %T.
16.6.2 Molar Absorption
Molar Absorption ε is a measure of the amount of light absorbed per unit concentration.
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Molar absorption is a constant for a particular substance, so if the concentration of the solution
is halved so is the absorbance, which is exactly what you would expect.
Let us take a compound with a very high value of molar absorption, say 100,000 L mol -1 cm-1,
which is in a solution in a 1 cm path length cuvette and gives an absorbance of 1.
ε = 1 / 1b c
Therefore, c = 1 / 100,000 = 1X10-5 mol L-1
Consider a compound with a very low value of ε, say 20 L mol-1 cm-1 which is in solution in a 1
cm path length cuvette and gives an absorbance of 1.
ε = 1 / 1 b c
Therefore, c = 1 / 20 = 0.05 mol L-1
β-carotene is an organic compound found in vegetables and is responsible for the colour of
carrots. It is found at exceedingly low concentrations. You may not be surprised to learn that
the molar absorption of β -carotene is 100,000 L mol-1 cm-1
16.6.3 Formative Assessment
1. a) One mole of photons (Avogadro's number of photons) is called an Einstein of
radiation.
b) Calculate the energy in calories, of one Einstein of radiation of wave length 3000 A.
2. A compound of formula weight 280 absorbed 65.0% of the radiation at a certain wave-
length in a 2-cm cell at a concentration of 15.0 mg/mL. Calculate its molar absorption at that
wavelength Wavelength/Frequency/Energy
3. One mole of photons (Avogadro's number of photons) is called an Einstein of radiation.
Calculate the energy, in calories, of one Einstein of radiation at 3000 A.
4. A 20-ppm solution of a DNA molecule (unknown molecular weight) isolated from Escherichia
coli was found to give an absorbance of 0.80 in a 2-cm cell. Calculate the molar absorbance of
the molecule.
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5. A compound of formula weight 280 absorbed 65.0% of the radiation at a certain wavelength
in a 2-cm cell at a concentration of 15.0 μg/mL. Calculate its molar absorbance at that
wavelength.
16.7 Atomic Spectroscopic Techniques
Electrons exist in energy levels within an atom. These levels have well defined energies and
electrons moving between absorb or emit energy equal to the difference between them.
In atomic spectroscopy, the energy absorbed to move an electron to a more energetic level
and/or the energy emitted as the electron moves to a less energetic energy level is in the form
of a photon (a particle of light). Because this energy is well-defined, an atom's identity (i.e.
what element it is) can be identified by the energy of this transition. The wavelength of light can
be related to its energy. It is usually easier to measure the wavelength of light than to directly
measure its energy.
Atomic spectroscopy can be further divided into absorption, emission, and fluorescence.
In atomic absorption spectroscopy, light is passed through a collection of atoms. If the
wavelength of the light has energy corresponding to the energy difference between two energy
levels in the atoms, a portion of the light will be absorbed. The relationship between the
concentrations of atoms, the distance the light travels through the collection of atoms, and the
portion of the light absorbed is given by the Beer-Lambert law.
The energy stored in the atoms can be released in a variety of ways. When it is released as
light, this is known as fluorescence. Atomic fluorescence spectroscopy measures this emitted
light. Fluorescence is generally measured at a 90° angle from the excitation source to minimize
collection of scattered light from the excitation source, often such a rotation is provided by a
Pellin-Broca prism on a turntable which will also separate the light into its spectrum for closer
analysis. The wavelength once again tells you the identity of the atoms. For low absorbances
(and therefore low concentrations) the intensity of the fluoresced light is directly proportional to
the concentration of atoms. Atomic fluorescence is generally more sensitive (it can detect
lower concentrations) than atomic absorption.
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16.7.1 Atomic Emission
In atomic emission, a sample is subjected to a high energy thermal environment in order to
produce excited state atoms, capable of emitting light. The energy source can be an electrical
arc, a flame, or more recently plasma. The emission spectrum of an element exposed to such
an energy source consists of a collection of the allowable emission wavelengths, commonly
called emission lines, because of the discrete nature of the emitted wavelengths- This
emission spectrum can be used as a unique characteristic for qualitative identification of the
element. Atomic emission using electrical arcs has been widely used in qualitative analysis.
Emission techniques can also be used to determine how much of an element is present in a
sample. For a "quantitative" analysis, the intensity of light emitted at the wavelength of the
element to be determined is measured. The emission intensity at this wavelength will be
greater as the number of atoms of the analyte element increases- The technique of flame
photometry is an application of atomic emission for quantitative analysis.
16.8 Atomic Absorption
The capability of an atom to absorb very specific wavelengths of light is utilized in atomic
absorption spectrophotometry.
The quantity of interest in atomic absorption measurements is the amount of light at the
resonant wavelength which is absorbed as the light passes through a cloud of atoms. As the
number of atoms in the light path increases, the amount of light absorbed increases in a
predictable way. By measuring the amount of light absorbed, a quantitative determination of
the amount of analyte element present can be made. The use of special light sources and
careful selection of wavelength allow the specific quantitative determination of individual
elements in the presence of others, making the technique very selective.
The atom cloud required for atomic absorption measurements is produced by supplying
enough thermal energy to the sample to dissociate the chemical compounds into free atoms.
This is achieved by aspirating a solution of the analyte into the flame. Under the proper flame
conditions, most of the atoms will remain in the ground state form and are capable of
absorbing light at the analytical wavelength from a source lamp.
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16.8.1 Atomic Fluorescence
In this technique ground state atoms created in a flame are excited by focusing a beam of light
into the atomic vapour. The emission resulting from the decay of the atoms excited by the
source light is measured. The intensity of this "fluorescence" increases with increasing atom
concentration, providing the basis for quantitative determination.
The source lamp for atomic fluorescence is mounted at an angle to the rest of the optical
system, so that the light detector sees only the fluorescence in the flame and not the light from
the lamp itself. It is advantageous to maximize lamp intensity with atomic fluorescence since
sensitivity is directly related to the number of excited atoms which is a function of the intensity
of the exciting radiation. The atoms do not emit radiation at the same wavelength as the
exciting radiation.
16.8.2 Quantitative Analysis by Atomic Absorption
The atomic absorption process is illustrated in Figure 5. Light at the resonance wavelength of
initial intensity, Io, is focused on the flame cell containing ground state atoms. The initial light
intensity is decreased by an amount determined by the atom concentration in the flame cell.
The light is then directed onto the detector where the reduced intensity, I, is measured.
Figure 22: Atomic Absorption Phenomena
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16.8.3 Formative Assessment
i) Why is a sharp-line source desirable for atomic absorption spectroscopy?
ii) Explain why flame emission spectrometry is often as sensitive as atomic absorption
spectrophotometry, even though only a small fraction of the atoms may be thermally excited in
the flame.
iii) Why is a high-temperature nitrous oxide—acetylene flame sometimes required in
atomic absorption spectrophotometry?
iv) Why is high concentration of a potassium salt sometimes added to standards and
samples in flame absorption or emission methods?
v) Chemical interferences are more prevalent in "cool" flames such as air-propane, but this
flame is preferred for the determination of the alkali metals. Suggest why.
vi) Calcium in a sample solution is determined by atomic absorption spectrophotometry. A
stock solution of calcium is prepared by dissolving 1.834 g CaCI; • 2H20 in water and diluting to
1 L. This is diluted 1:10. Working standards are prepared by diluting the second solution
respectively, 1:20, 1:10, and 1:5. The sample is diluted 1:25. Strontium chloride is added to all
solutions before dilution, sufficient to give 1% (wt/vol) to avoid phosphate interference. A blank
is prepared to give 1% SrCI. Absorbance signals on the computer when the solutions are
aspirated into an air-acetylene flame are as follows: blank, 1.5 cm; standards, 10.6, 20.1, and
38.5 cm; sample, 29.6 cm. What is the concentration of calcium in the sample in parts per
million?
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17 UNIT IV MOLECULAR SPECTROCOPY 1: UV-VISIBLE AND IR
17.1 Summary of the Learning Activity
At the end of the unit learners will be able to:
Explain electronic energy levels in molecules and transitions caused by absorption of
UV and visible radiation.
Explain the concepts on which UV visible spectroscopy is based
Use hypothetical UV-visible Spectra to identify specific functional groups in a molecule
Explain how molar extinction coefficient is used for quantitative analysis
Use hypothetical data to calculate concentrations of solutions
Name major elements of a UV-Visible spectrophotometers and their functions
Recall the electronic transitions caused by absorption of IR Radiation
Correlate Absorption of specific IR frequencies to molecular functional groups
Correlate Absorption of specific IR frequencies to molecular structure.
Recall parts of a modern IR Spectrophotometer and their functions
17.2 List of Required Readings
http://en.wikipedia.org/wiki/Molecular_energy_state
17.3 List of Relevant Useful Links
http://www.scienceofspectroscopy.info/edit/index.php?title=UV_Absorption_Table
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab4.htm
http://www.scienceofspectroscopy.info/edit/index.php?title=UV-Visible_Spectroscopy
http://ull.chemistry.uakron.edu/analytical/Spectrophotometry/
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17.4 List of Relevant Multimedia Resources
http://www.cem.msu.edu/~parrill/AIRS/name_list.html
17.5 Ultraviolet- Visible Spectroscopy
17.5.1 Electronic Transitions
The absorption of light energy by organic compounds in the visible and ultraviolet region
involves promotion of electrons in δ, π, and n orbitals from the ground state to higher-energy
states. These higher energy states are called anti bonding orbitals. The anti bonding orbital
associated with the a bond is called the δ* (sigma star) orbital and that associated with the π
bond is called the π* (pi star)
Many molecules contain atoms with valence electrons that are not directly involved in bonding;
these are called nonbonding or n electrons and are mainly located in atomic orbitals of oxygen.
sulphur, nitrogen, and the halogen.
The n electrons do not form bonds, therefore, there are no anti bonding orbitals associated
with them. The presence of an electron in an anti bonding orbital indicates that the molecule is
in a high-energy state. The electron density between the atomic nuclei is less than that at the
same distance from the nucleus in an isolated atom. In the excited state some, but not all, of
the electrons in a molecule occupy antibonding orbitals.
The electronic transitions (→) that are involved in the ultraviolet and visible regions are of the
following types: δ→ δ*, n —> δ*, n →• π *, and •n —> π*. The energy required for the δ —> δ*
transition is very high; consequently, compounds in which all valence shell electrons are
involved in single-bond formation, such as saturated hydrocarbons, do not show absorption in
the ordinary ultraviolet region.
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Compounds that contain nonbonding electrons on oxygen, nitrogen, sulphur, or halogen atoms
are capable of showing absorptions, owing to n —>δ* transitions. These transitions are of
lower energy than δ —> δ*transitions, consequently molecules containing nonbonding
electrons usually exhibit absorption in the ordinary ultraviolet region.
Transitions to antibonding π* orbitals are associated only with unsaturated centres (double or
triple bonds) in the molecule; these are of still lower energy requirement and occur at longer
wavelengths, usually well within the region of the ordinary ultraviolet spectrophotometer. The
diagram below shows the general relative electronic excitation energies for these transitions.
The high-energy transitions (δ →δ*) occur at shorter wavelength and the low-energy
transitions n→ π* occur at longer wavelength.
17.5.2 The Effect of the Structural Environment
Identical functional groups in different molecules will not necessarily absorb at exactly the
same wavelength. The energy change for a particular transition dictates the position of
absorption of a given group. Transitions in identical functional groups in different molecules will
not necessarily have exactly the same energy requirement because of different structural
environments. The neighbouring molecules have a small but measurable effect on the energy
state of a chromophore
17.5.3 Effects of Conjugation
If two or more chromophoric groups are present in a molecule and they are separated by two
or more single bonds, the effect on the spectrum is usually additive; there is little electronic
interaction between isolated chromophoric groups. However, if two chromophoric groups are
separated by only one single bond (a conjugated system), a large effect on the spectrum
results because, the π electron system is spread over at least four atomic centres. When two
chromophoric groups are conjugated, the high intensity (n→π* transitions) absorption band is
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Figure 23: electronic transitions arising from absorption of UV and visible Light
generally shifted 15-45 nm to longer wavelength with respect to the simple unconjugated
chromophore.
17.5.4 The Molar Extinction Coefficient
The magnitude of the molar extinction coefficient for a particular absorption is directly
proportional to the probability of the particular electronic transition; the more probable a given
transition, the larger the extinction coefficient. In general, a given type of chromophore will
always have an extinction coefficient of roughly the same magnitude in different molecules.
Therefore, when assigning the absorption peak to a given chromophore the extinction should
be considered, because the absorption is characterised by the energy and the probability of
the transition
17.5.5 The Effect of Solvent
The electronic structures of the high-energy states of molecules are either more polar or less
polar than in the ground state. Those that are more polar in the excited state have the
absorption of the peak shifted by 10-40 cm -1to the long wave length in polar solvents and vice
versa.
17.5.6 Identification of Functional Groups Using UV
An isolated functional group not in conjugation with any other group is said to be a
chromophore if it exhibits absorption of a characteristic nature in the ultraviolet or visible
region. If a series of compounds has the same functional group and no complicating factors
are present, all of the compounds will generally absorb at very nearly the same wavelength
and will have nearly the same molar extinction coefficient. Thus, it is readily seen that the
spectrum of a compound, when correlated with data from the literature for known compounds,
can be a very valuable aid in determining the functional groups present in the molecule
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17.6 Instrumentation for UV Visible Spectrometry
Figure 24: Double Beam Spectrophotometer
The light comes from the source and a thin ray is allowed in by slit 1, the diffraction grating
selects the required wave length and a thin ray is again selected by slit 2 the filter allows in
only selected wavelength mirror 2 rotates the through the half mirror where part of the beam is
reflected to mirror 3 and part is transmitted to mirror4, to form reference beam and sample
beam.
17.6.1 Formative Exercise.
This exercise reinforces the knowledge covered in the above section identification of
Chromophores using UV
i) Which of the following compounds will absorb in the UV region?
a). CO2 b). H2 c) CH3CO
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ii) Arrange the following compounds in order of the increasing frequency or wave number
at which they will absorb
a). CH3CO b). CH2=CH-CH=CH2 c). CH2=CH-CH=CH-CH=CH2
iii) Identify possible electronic transitions that will lead to UV absorption in the following
compounds
a). CH3CO b).CH2=CH-CH=CH2 c).CH3CN
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17.7 Infrared Spectroscopy
17.7.1 Molecular Vibration and IR Spectroscopy
A molecule is not a rigid assemblage of atoms. A molecule resembles a system of balls of
varying masses, corresponding to the atoms of a molecule, and springs of varying strengths,
corresponding to the chemical bonds of a molecule. These therefore can undergo various
vibrations. There are two kinds of fundamental vibrations for molecules: stretching, in which
the distance between two atoms increases or decreases, but the atoms remain in the same
bond axis, and bending (or deformation), in which the position of the atom changes relative to
the original bond axis. The various stretching and bending vibrations of a bond require a
certain amount of energy. For transitions involving vibrations the frequency corresponds to
infra red radiation. When infra-red light of that same frequency is incident on the molecule,
energy is absorbed and the amplitude of that vibration is increased. When the molecule reverts
from the excited state to the original ground state, the absorbed energy is released as heat.
17.7.2 Fundamental and Non Fundamental Absorption Bands
A nonlinear molecule that contains n atoms has 3n - 6 possible fundamental vibrations.
Additional (non fundamental) absorption bands may occur because of the presence of
overtones (or harmonics) that occur with greatly reduced intensity, at 1/2, 1/3, 1/4 Of the
wavelength (twice, three times, the wave number), combination bands (the sum of two or
more different wave numbers), and difference bands (the difference of two or more different
wave numbers). If all vibrations were to result in absorption of IR radiation the number of peaks
would be too many to be useful but for a vibration to result in IR absorption it must result in a
change of dipole moment. Therefore only bonds connecting two different molecules can result
in IR
17.7.3 Relative Energies of IR Absorptions
Bending vibrations generally require less energy and occur at longer wavelength (lower wave
number) than stretching vibrations.
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The triple bond (absorption at4.4-5.0 µm, 2300-2000 cm-1) is stronger than the double bond
(absorption at 5.3-6.7 µm, 1900-1500 cm-1), which in turn is stronger than the single bond (C
—C, C—N, and C—0 absorption at 7.7-12.5 µm, 1300-800 cm-1)
When the single bond involves the very small hydrogen atom (C—H, O—H, or N—H),
stretching vibrations occur at much higher frequency (2.7-3.8 µm. 3700-2630 cm-1). The O—H
bond absorbs near 2.8 µm (3570 cm-1) and obtained. For example, if the spectrum contains a
strong band at 5.82 µm (1718 cm-1), the compound almost certainly contains a carbonyl
group.
The spectrum by itself does not always provide further information as to the nature of the
group; the compound could be an aldehyde, a ketone, an acid, an ester, or an amide. Thus, in
order to define a functional group, the spectrum must be examined in detail for other diagnostic
absorption bands and used in conjunction with (and cannot always replace) classical chemical
reactions and solubility determinations. Conversely, the power of negative evidence cannot be
overemphasized; if the spectrum does not contain absorption typical of a certain functional
group, the molecule does not contain that functional group. If the spectrum contains no
absorption in the 5.4-6.3 µm (1850-1587 cm-1) regions, the sample does not contain a carbonyl
group.
Many of the absorption bands that organic compounds show in the infrared region cannot be
interpreted with assurance.
17.8 Identifying Functional Groups by Infrared Spectroscopy
17.8.1.1 IR Spectra of Saturated Hydrocarbons
Saturated hydrocarbons contains absorptions resulting from vibrations typical of groups that
are present in such molecules, C—H stretching (~3.39 µm and —3.54 µm —2950 and —2820
cm-1), —CH2— bending (-6.86 µm —1458 cm-1), and C—CH3 bending (6.86 and 7.28 µm —
1458 and ~1380 cm-1). Weak absorption near 13.85 µm (722 cm-1) is caused by bending
vibrations of the group — (CH2)n—, where n > 4.
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17.8.1.2 IR absorption of O-H
If a methylene group of a saturated hydrocarbon is replaced with an oxygen atom this causes
the appearance of absorption caused by strong C—O stretching vibrations near 9 μm (~1110
cm-1). The spectrum changes in a very predictable way; it now shows absorptions owing to O
—H and C—O stretching vibrations in addition to the hydrocarbon chromophoric groups
present. The spectrum of Propanol CH3—(CH2)—CH 2OH,
Figure 25 below is an example; absorption owing to an alcohol O—H stretching vibration is
present at ~2.9 µm (~3448 cm-1) as a strong broad absorption typical of the polymeric
association of hydroxyl groups. It is broadened due to hydrogen bonding
Figure 25: IR Spectrum of 1-Propanol
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17.8.1.3 IR absorption of C=O
Ketones
If a compound contains a carbonyl group, the absorption caused by C==0 stretching is
generally among the strongest present.
Carbonyl groups of ketones generally absorb in the region 5.7-6.0 µm (1754-1667 cm -1); the
position of absorption is sensitive to ring size and to the degree of conjugated unsaturation,
among other factors.
Aldehydes
The absorption owing to the carbonyl stretching vibration of Aldehydes appears in the same
general region as that of ketones. The other striking characteristic of the aldehyde functional
group absorption is the presence of two weak bands owing to C—H stretching vibrations. The
wavelength of this absorption is increased (the wave number is lowered) from the normal C—H
stretching position near 3.4 µm (-294.0 cm-1) to about 3.55 and 3.68 µm.
(-2820 and 2720 cm-1). The presence of two absorption bands in this region is due to the
symmetric and asymmetric stretching modes of the C—H bond and C=0 bonds.
Esters and lactones
The position of absorption of the carbonyl stretching vibration of esters and lactones is
dependent, as with ketones, on conjugated unsaturation and ring size. But it is in the general
area of the C=O abs
Carboxylic Acid
The absorption owing to the carbonyl stretching vibration of a saturated carboxylic acid (5.83
µm., 1715 cm-1) is shifted to longer wavelength (lower wave number) if conjugated with an
unsaturated group (benzoic acid, 5.88 µm 1701 cm-1).
Amides
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All amides show strong absorption owing to carbonyl stretching and Absorptions resulting from
N—H stretching vibrations of primary and secondary amides are in the 2.8-3.2 μm, (~3570-
3125 cm-1)
Amines
The most characteristic absorption of amines is that owing to N—H stretching vibrations in the
region 2.8-3.0 µm, (~3570-3333 cm-1). In dilute solution in an inert solvent, the spectra of
primary amines have two sharp bands in this region, owing to symmetric and asymmetric N—
H stretching vibrations; the spectra of secondary amines have only one band in this region,
and tertiary amines do not absorb in this region.
C=C ethylene bond
As a consequence of the weak intensity of the C=C stretching vibration absorption,
concentrated solutions of the olefins should be used; the absorptions occur in the 5.95-6.17
(~1680-1620 cm-1) region. The absorptions are more intense if the ethylene bond is
conjugated with some unsaturated group. Absorption owing to olefinic C=C—H stretching
vibration is observed as a small peak at 3.19 µm (3135 cm-1) near the larger alkane C—H
stretching vibration absorption
Triple bonds
Absorptions resulting from carbon carbon triple bond stretching vibrations of acetylenic
compounds occur in the region 4.4-4.8 µm (~2275-2085 cm-1). The absorption is weak,
especially if the acetylenic linkage is non terminal. The stretching vibration results only in a
linear expansion and contraction of the molecule, and hence the dipole moment is not much
affected. Absorption caused by the acetylenic C—H stretching vibration occurs
as a fairly strong, sharp band near 3.0 µm (~3333 cm-1).
The absorption caused by the stretching vibration of the triple bond of nitriles occurs in about
the same region as that of acetylenes, but the absorption is much more intense. This
absorption of benzonitrile appears at 4.44 µm (2252 cm-1).
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Aromatic compounds
There are four absorption bands in the 6-7 am. (1667-1429 cm-1) region that are diagnostic of
aromatic structure. These occur near 6.25, 6.32, 6.67, and 6.90 µm (—1600, 1580, 1500, and
1450 cm-1) and are caused by C=C skeletal in-plane vibrations. The second band is frequently
observed only as a shoulder of the first, but is intensified if the aromatic nucleus is conjugated
with some unsaturated group; the fourth band is frequently obscured by strong absorptions
resulting from —CH2—bending vibrations if aliphatic groups are present. The absence of
absorption by a compound in these regions is fair assurance that the compound is not
aromatic.
A number of absorption bands of variable intensity appear in the 10-15 µm (1000-670)
Region that is caused by C—H bending vibrations. These absorptions depend on the-number
of adjacent free hydrogen atoms that an aromatic nucleus contains. An aromatic compound
containing five adjacent hydrogen atoms absorbs strongly in both the 13.3 and 14.3 μm
regions (~750 and 700 cm'"1); if the compound contains four adjacent hydrogen atoms, as, for
example, an o-disubstituted benzene, it absorbs
strongly only near 13.3 μm. (~750 cm"1). The remaining absorptions owing to fewer adjacent
hydrogen atoms (higher degree of substitution on the aromatic nucleus) are usually weak and
not easily assigned. Of particular importance for benzene compounds is the absorption near
14.3 μm (~700 cm"1); if the compound does not absorb strongly in this region, it cannot be a
mono substituted benzene compound. The spectra of biphenyl Fig. above and other mono
substituted benzene compounds show these absorptions.
The region 5-6 μm (2000-1670 cm-1) of the spectra of benzenoid compounds contains
absorption bands of low intensity that are overtone or combination bands. The number and
relative position of these bands are remarkably dependent upon the particular substitution type
of the benzene ring.
17.8.2 Formative Assessment
i) Arrange the following bonds in order of IR absorption frequency of their stretching
vibration
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C-N, N-H, O-H
ii) Which of the following bonds will result in absorption IR
H-H, C-H, O=O, C=O,
Infrared spectra: It is important to remember that the absence of an absorption band can often
provide more information about the structure of a compound than the presence of a band. Be
careful to avoid focusing on selected absorption bands and overlooking others.
Interpretation of IR Spectra
Look for absorption bands in decreasing order of importance:
the C-H absorption(s) between 3100 and 2850 cm-1. An absorption above 3000 cm-1
indicates C=C, either alkene or aromatic. Confirm the aromatic ring by finding peaks at
1600 and 1500 cm-1 and C-H out-of-plane bending to give substitution patterns below
900 cm-1. Confirm alkenes with an absorption at 1640-1680 cm-1. C-H absorption
between 3000 and 2850 cm-1 is due to aliphatic hydrogens.
The carbonyl (C=O) absorption between 1690-1760cm-1; this strong band indicates
either an aldehyde, ketone, carboxylic acid, ester, amide, anhydride or acyl halide. The
an aldehyde may be confirmed with C-H absorption from 2840 to 2720 cm-1.
The O-H or N-H absorption between 3200 and 3600 cm-1. This indicates either an
alcohol, N-H containing amine or amide, or carboxylic acid. For -NH2 a doublet will be
observed.
The C-O absorption between 1080 and 1300 cm-1. These peaks are normally rounded like the
O-H and N-H peak in 3. and are prominent. Carboxylic acids, esters, ethers, alcohols and
anhydrides all containing this peak.
the CC and CN triple bond absorptions at 2100-2260 cm-1 are small but exposed.
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a methyl group may be identified with C-H absorption at 1380 cm-1. This band is split
into a doublet for isopropyl(gem-dimethyl) groups.
structure of aromatic compounds may also be confirmed from the pattern of the weak overtone
and combination tone bands found from 2000 to 1600 cm-1.
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18 UNIT V MOLECULAR SPECTROSCOPY 2: NUCLEAR MAGNETIC RESONANCE
18.1.1 Summary Of The Learning Activity
At the end of the unit learners be able to:
Explain how the phenomenon of NMR arises
Recall nuclei that exhibit NMR
Explain Proton NMR phenomena
Correlate Absorption of specific HNMR frequencies to molecular functional groups
Correlate Absorption of specific HNMR frequencies to molecular structure of Simple
organic molecules.
Explain the special features of C-13 NMR phenomena
Recall the nature of information provided by C-13 NMR
Recall parts of a modern NMR Spectrophotometer and their functions.
18.1.2 List of Required Readings
http://www.scienceofspectroscopy.info/edit/index.php?title=NMR_Spectroscopy
http://en.wikipedia.org/wiki/NMR_spectroscopy#Chemical_Shift
http://www.mhhe.com/physsci/chemistry/carey/student/olc/ch13nmr.html#basi
18.1.3 List of Relevant Links
ttp://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab4.htm
http://www.scienceofspectroscopy.info/edit/index.php?title=UV-Visible_Spectroscopy
http://www.scienceofspectroscopy.info/edit/index.php?title=UV_Absorption_Table- non open
source
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UV visible
http://ull.chemistry.uakron.edu/analytical/Spectrophotometry/
http://www.chem.ucla.edu/cgi-bin/webspectra.cgi?Problem=bp1&Type=C
http://www.chem.ucla.edu/~webspectra/search.html
18.2 Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance spectroscopy most commonly known as NMR spectroscopy is
the name given to the technique which exploits the magnetic properties of certain nuclei. The
most important applications for the organic chemist are proton NMR and carbon 13
spectroscopy.
Many types of information can be obtained from an NMR spectrum. Much like using infra Red
spectroscopy to identify functional groups, analysis of NMR spectrum provides information on
the number and type of chemical entities in a molecule.
NMR can be applied to a wide variety of samples, both in the solution and the solid state
In this unit proton NMR is introduced and its application identification of organic compounds is
demonstrated.
18.2.1 NMR Phenomenon
Nuclei possess a mechanical spin, or angular momentum. The total angular momentum
depends on the nuclear spin, or spin number which may have values of 0, ½ 1 3/2 depending
on the particular nucleus. The numerical value of the spin number is related to the mass
number and atomic number of nucleus.
Mass number Atomic number Spin number I
odd Even or odd ½ , 3/2 ,5/2, ….
even even 0
The spinning nucleus results into a magnet field around the nucleus. Thus the nucleus is
equivalent to a small magnet of magnetic moment μ. Each nucleus for which I > 0 will
therefore has a characteristic magnetic moment.
eve
n
odd 1,
2,
3…
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18.2.2 Formative Assessment
i) Differentiate between nuclear magnetic moment and Nuclear Spin number
ii) State which of the following Nuclei have a magnetic moment
a) O mass number 16, Atomic Number 8, b) Carbon Mass number 12, atomic
number 6,
c) Nitrogen Mass number 14 atomic number d) carbon 13, e)proton
18.3 Proton NMR
The most useful nuclei for organic NMR the proton Mass Num 1 A.M 1 and Carbon 13
because they occur in many organic compounds.
The proton has a spin number of ½ the magnetic nucleus may therefore assume any one of
(2I + 1) ranging from -½ , to ½ in steps of 1 orientations with respect to the direction of the
applied magnetic field. Thus, a proton (I = ½ ) will be able to assume only one of two possible
orientations that correspond to energy levels of ± μ.H in an applied magnetic field, where H is
the strength of the external magnetic field
Therefore these energy levels are said to be quantised.
Figure 26: Nuclear Magnetic Resonance
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A proton in a static external magnetic field may assume only two orientations corresponding to
energies of ±μH. The low-energy orientation corresponds to that state in which the nuclear
magnetic moment is aligned parallel to the external magnetic field, and the high-energy
orientation corresponds to that state in which the nuclear magnetic moment is aligned
antiparallel (opposed) to the applied magnetic field. It is possible to induce transitions between
these two orientations; the frequency v of electromagnetic radiation necessary for such a
transition is given by v =-2μHo,/h. where Ho is the strength of the external magnetic field. Note
unlike the absorption in UV and IR this absorption the frequency ν is dependent on the applied
field.
These Radiation induced Transitions obey the following Rules
1 The probability of an upward transition by absorption of energy from the magnetic field
is exactly equal to the probability of a downward transition by a process stimulated by the field.
2 Spontaneous transition from a higher-energy state to a lower-energy state is negligible.
18.3.1 Non Radiation Effects
Radiation effects alone there do not cause observable NMR. However there are two radiation
less effect that occur one of which makes NMR possible
1 Two neibhouring nucei can exchange spin one becoming anti parallel and the other
parallel- This is called Spin- Spin Relaxiation
2 Lattice effect which results from the aggregate presence of all other nuclei undergoing
various enegry transitions this results in some anti parallel nuclei losing energy and becoming
parallel. This creates a small excess of lower energy level nuclei. It is this small excess from
which some absorb energy
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18.3.2 Chemical Shift
Not all hydrogen atoms in a molecule will absorb at exactly the same frequency ν. The
magnetic effect felt by a hydrogen nucleus is given by. Heff=Ho- δHo δHo is called the chemical
shift and it measures the electronic effect of surrounding atoms neibhouring a given proton.
δ is the shift parameter defined as below
∆v=Frequency of Proton- Frequency of standard (TMS)
As only relative absorption values can be obtained, a standard is used. The chemical shift
values of the protons in a particular compound are then determined with reference to this
standard. A standard may be used in one of two ways: as an external reference (the standard
is usually placed in a small capillary contained within the sample tube) and as an internal
reference (the standard is dissolved in a solution of the sample to be measured). Modern
Proton NMR uses Tetra Methyl Silane (TMS) as the standard.
18.3.3 Correlation of HNMR With Structure
Proton resonance frequencies can be measured with an accuracy of about ±0.02 ppm relative
to an internal standard. Figure 27 below provides a general correlation of structural type with
absorption position. The functional groups listed in Figure 27 are attached to saturated carbon
atoms. All absorption values quoted herein are δ values.
Figure 27: Chemical Shift Parameters of Different Hydrogen atoms
18.3.4 Inductive and Electro Negativity
The electrons around the proton create a magnetic field that opposes the applied field.
Electronegative groups attached to the C-H system decrease the electron density around the
protons, and there is less shielding (i.e. deshielding) so the chemical shift increases. These
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effects are cumulative, so the presence of more electronegative groups produces more
deshielding and therefore, larger chemical shifts.
These inductive effects at not just felt by the immediately adjacent protons as the disruption of
electron density has an influence further down the chain. However, the effect does fade
rapidly as you move away from the electronegative group
18.3.5 Spin-Spin Interactions
Nearly the same energy for a given spin transition is involved for each proton in a molecule.
The absorption bands, as measured by the areas that they enclose are the ratio of the number
of protons in each group. The low resolution spectrum of ethanol Figure 28 shows three
absorption peaks in an area ratio of 1:2:3, corresponding to —OH, —CH2—, and —CH3,
respectively
Figure 28: Low Resolution NMR Spectrum of Ethanol
Under higher resolution the peaks of ethyl alcohol attributed to methylene and methyl protons
appear as multiplets.
The methyl CH3 absorption is split into three of relative area 1:2:1 and the methylene CH 2 is
split into four peaks of relative area 1:3:3:1. This is explained by the methyl CH 3 group
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interacts with CH2 group splitting into 4 and the methelene CH2 group interacts with the methyl
CH3 group splitting it into 3 This effect is called the spin-spin interactions
The magnitude of multiple separation resulting from spin-spin interactions is independent of
the strength of the applied field.
Figure 29: High Resolution NMR Spectrum of Ethanol
18.3.6 Hydrogen Bonding
Protons that are involved in hydrogen bonding (this usually means -OH or -NH) are typically
observed over a large range of chemical shift values. The more hydrogen bonding there is,
the more the proton is deshielded and the higher its chemical shift will be. However, since the
amount of hydrogen bonding is susceptible to factors such as solvation, acidity, concentration
and temperature, it can often be difficult to predict.
18.4 Carbon Nmr Spectroscopy
The power and usefulness of 1H NMR spectroscopy as a tool for structural analysis is much
appreciated. Unfortunately, when significant portions of a molecule lack C-H bonds, no
information is forthcoming. Examples include polychlorinated compounds such as chlordane,
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polycarbonyl compounds such as croconic acid, and compounds incorporating triple bonds
(structures below, orange colored carbons).
Figure 30: Molecules whose structures cannot be distinguished by HNMR
Even when numerous C-H groups are present, an unambiguous interpretation of a proton
NMR spectrum is not always possible. The following diagram depicts three pairs of isomers (A
& B) which display similar proton NMR spectra. Although a careful determination of chemical
shifts should permit the first pair of compounds (blue box) to be distinguished, the second and
third cases (red & green boxes) might be difficult to identify by proton NMR alone.
Figure 31: Molecules which may not be able to be distinguished by HNMR
These difficulties would be largely resolved if the carbon atoms of a molecule could be probed
by NMR in the same fashion as the hydrogen atoms. Since the major isotope of carbon (12C)
has no spin, this option seems unrealistic. Fortunately, 1.1% of elemental carbon is the 13C
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isotope, which has a spin I = 1/2, so in principle it should be possible to conduct a carbon NMR
experiment. It is worth noting here, that if much higher abundances of 13C were naturally
present in all carbon compounds, proton NMR would become much more complicated due to
large one-bond coupling of 13C and 1H.
18.4.1.1 Technical Problems associated with C-NMR :
1. The abundance of 13C in a sample is very low (1.1%), so higher sample concentrations
are needed.
2. The 13C nucleus is over fifty times less sensitive than a proton in the NMR experiment,
adding to the previous difficulty.
3. Hydrogen atoms bonded to a 13C atom split its NMR signal by 130 to 270 Hz, further
complicating the NMR spectrum.
These problems are solved using two techniques
Hetero nuclear decoupling in which the splitting by the hydrogen nuclear is removed from the
carbon spectrum and pulse technique in which a pulse is used to collect many signals from
one carbon atom these are added to give a strong signal.
When acquired in this manner, the carbon NMR spectrum of a compound displays a single
sharp signal for each structurally distinct carbon atom in a molecule (remember, the proton
couplings have been removed). The spectrum of camphor, shown on the left below, is typical.
Furthermore, a comparison with the 1H NMR spectrum on the right illustrates some of the
advantageous characteristics of carbon NMR. The dispersion of 13C chemical shifts is nearly
twenty times greater than that for protons, and this together with the lack of signal splitting
makes it more likely that every structurally distinct carbon atom will produce a separate signal.
The only clearly identifiable signals in the proton spectrum are those from the methyl groups.
The remaining protons have resonance signals between 1.0 and 2.8 ppm from TMS, and they
overlap badly thanks to spin-spin splitting.
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Figure 32: Carbon- 13 NMR of Camphor
Unlike proton NMR spectroscopy, the relative strength of carbon NMR signals is not normally
proportional to the number of atoms generating each one. Because of this, the number of
discrete signals and their chemical shifts are the most important pieces of information
delivered by a carbon spectrum. The general distribution of carbon chemical shifts associated
with different functional groups is summarized in the following chart. Bear in mind that these
ranges are approximate, and may not encompass all compounds of a given class. Note also
that the over 200 ppm range of chemical shifts shown here is much greater than that observed
for hydrogen shifts
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18.4.2 13c Chemical Shift Ranges*
Low FieldRegion
Figure 33: Chemical Shift Ranges
* For samples in CDCl3 solution. The δ scale is relative to TMS at δ=0.
18.4.3 Formative Assessment
The 60 MHz spectrum shown in Figure 34 is that of a compound C10H13NO2. Significant
features of the infrared spectrum are C=O stretch and one N-H stretch peak. Deduce the
structure of this compound from the chemical shift, integral and coupling data on the spectrum.
119
Figure 34: C10H13NO2
2-The carbon-13 NMR spectrum of one of the butyl acetate isomers (C4H9OCOCH3) showed
signals at δc22, 28, 80 and170. What is its structure? Why is the intensity of the peak at δ 28
much more intense than that at δ 22 (by factor of approximately eight)? How would the
multiplicity and signal intensity in the proton NMR spectrum of this compound confirm your
deductions?
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19 UNIT IX MASS SPECTROMETRY
19.1.1 Summary Of The Learning Activity
At the end of the unit learners be ableto:
Explain of mass spectrum phenomenon arises
Explain rules followed by fragmentation in Mass spectrum
Correlate mass spectrum to specific structural elements in a molecule
Use the mass spectrum to identify the molecular species.
Use high resolution mass spectrum and molecular mass calculator to uniquely identify
structural elements
Recall parts of a modern mass Spectrometer and their functions.
19.1.2 List Of Required Readings
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/spectro.htm#contnt
http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/cre_index.cgi?lang=eng
19.2 List Of Relevant Useful Links
http://ull.chemistry.uakron.edu/analytical/Mass_Spec/index.html/
19.3 Mass Spectrometry
Mass spectrometer identifies compounds by ionizing the compound and breaking the
compound into pieces called fragment and analyzing these fragments by passing the pieces
through analyser. The analyser sorts the fragments according to their mass charge ratios. The
results of the analyser are displayed as a mass spectrum.
A small sample of compound is ionized, usually to cations by loss of an electron-The Ion
Source
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The ions are sorted and separated according to their mass and charge.- Mass Analyzer
The separated ions are then detected and tallied, and the results are displayed on a computer.
Because ions are very labile (as they would react with ambient species) their formation and
analysis is conducted in a vacuum.
Ion Source
In the ion source molecules of the sample are bombarded by high energy electrons coming
from a heated filament accelerated across an electric field. Ions formed by the electron
bombardment are pushed away by a charged repeller plate, and accelerated toward other
electrodes, having slits through which the ions pass as a beam. Some of these ions fragment
into smaller cations and neutral fragments. Therefore the initial molecule results in a large
number of cations which is formed into an ion beam.
19.3.1 Isotopes
Since a mass spectrometer separates and detects ions of slightly different masses, it easily
distinguishes different isotopes of a given element. This is manifested most dramatically for
compounds containing bromine and chlorine, as illustrated by the following examples. Since
molecules of bromine have only two atoms, the spectrum on the left will come as a surprise if a
single atomic mass of 80 amu is assumed for Br. The five peaks in this spectrum demonstrate
clearly that natural bromine consists of a nearly 50:50 mixture of isotopes having atomic
masses of 79 and 81 amu respectively. Thus, the bromine molecule may be composed of two 79Br atoms (mass 158 amu), two 81Br atoms (mass 162 amu) or the more probable combination
of 79Br-81Br (mass 160 amu). Fragmentation of Br2 to a bromine cation then gives rise to equal
sized ion peaks at 79 and 81 amu.
bromine methylene chloride vinyl chloride
Figure 35: Mass spectra of Bromine, Vinyl Chloride, Methylene Chloride
122
The center and right hand spectra show that chlorine is also composed of two isotopes, the
more abundant having a mass of 35 amu, and the minor isotope a mass 37 amu. The precise
isotopic composition of chlorine and bromine is: Chlorine: 75.77% 35Cl and 24.23% 37Cl
Bromine: 50.50% 79Br and 49.50% 81Br
The presence of chlorine or bromine in a molecule or ion is easily detected by noticing the
intensity ratios of ions differing by 2 amu. In the case of methylene chloride, the molecular ion
consists of three peaks at m/z=84, 86 & 88 amu, and their diminishing intensities may be
calculated from the natural abundances given above. Loss of a chlorine atom gives two
isotopic fragment ions at m/z=49 & 51amu, clearly incorporating a single chlorine atom.
Fluorine and iodine, by contrast, are monoisotopic, having masses of 19 amu and 127 amu
respectively. It should be noted that the presence of halogen atoms in a molecule or fragment
ion does not change the odd-even mass rules given above.
Two other common elements having useful isotope signatures are carbon, 13C is 1.1% natural
abundance, and sulfur, 33S and 34S are 0.76% and 4.22% natural abundance respectively. For
example, the small m/z=99 amu peak in the spectrum of 4-methyl-3-pentene-2-one (above) is
due to the presence of a single 13C atom in the molecular ion. Although less important in this
respect, 15N and 18O also make small contributions to higher mass satellites of molecular ions
incorporating these elements.
19.4 Fragmentation Patterns
The nature of the fragments provides a clue to the molecular structure, but if the molecular ion
has a lifetime of less than a few microseconds it will not survive long enough to be observed.
Most organic compounds give mass spectra that include a molecular ion, and those that do not
if different ionisation conditions are used the molecular ion may be observed.
Among simple organic compounds, the most stable molecular ions are those from aromatic
rings, other conjugated pi-electron systems and cycloalkanes. Alcohols, ethers and highly
branched alkanes generally show the greatest tendency toward fragmentation. The stable
fragments will appear in the final spectrum.
19.4.1 Hydrocarbons
The mass spectrum of dodecane on the right illustrates the behavior of an unbranched alkane.
Since there are no heteroatoms in this molecule, there are no non-bonding valence shell
123
electrons. Consequently, the radical cation character of the molecular ion (m/z = 170) is
delocalized over all the covalent bonds. Fragmentation of C-C bonds occurs because they are
usually weaker than C-H bonds, and this produces a mixture of alkyl radicals and alkyl carbo-
cations. The positive charge commonly resides on the smaller fragment, so we see a
homologous series of hexyl (m/z = 85), pentyl (m/z = 71), butyl (m/z = 57), propyl (m/z = 43),
ethyl (m/z = 29) and methyl (m/z = 15) cations. These are accompanied by a set of
corresponding alkenyl carbocations (e.g. m/z = 55, 41 &27) formed by loss of 2 H. All of the
significant fragment ions in this spectrum are even-electron ions. In most alkane spectra the
propyl and butyl ions are the most abundant.
19.4.2 Hetero Atoms
The presence of a functional group, particularly one having a heteroatom Y with non-bonding
valence electrons (Y = N, O, S, X etc.), can dramatically alter the fragmentation pattern of a
compound. This influence is thought to occur because of a "localization" of the radical cation
component of the molecular ion on the heteroatom. After all, it is easier to remove (ionize) a
non-bonding electron than one that is part of a covalent bond. By localizing the reactive
moiety, certain fragmentation processes will be favored. These are summarized in the
following diagram, where the green shaded box at the top displays examples of such
"localized" molecular ions. The first two fragmentation paths lead to even-electron ions, and
the elimination (path #3) gives an odd-electron ion. Note the use of different curved arrows to
show single electron shifts compared with electron pair shifts.
Figure 36: Hetero atom cleavage adopted from Spectroscopy: http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/spectro.htm# contnt accessed Feb 2008
124
The charge distributions shown above are common, but for each cleavage process the charge
may sometimes be carried by the other (neutral) species, and both fragment ions are
observed. Of the three cleavage reactions described here, the alpha-cleavage is generally
favored for nitrogen, oxygen and sulfur compounds. Indeed, in the previously displayed
spectra of 4-methyl-3-pentene-2-one and N,N-diethylmethylamine the major fragment ions
come from alpha-cleavages. Further examples of functional group influence on fragmentation
are provided by a selection of compounds that may be examined by clicking the left button
below.
19.4.3 Finger Print Spectrum
The complexity of fragmentation patterns has led to mass spectra being used as "fingerprints"
for identifying compounds. Environmental pollutants, pesticide residues on food, and controlled
substance identification are but a few examples of this application. Extremely small samples of
an unknown substance (a microgram or less) are sufficient for such analysis. The following
mass spectrum of cocaine demonstrates how a forensic laboratory might determine the nature
of an unknown street drug. Even though extensive fragmentation has occurred, many of the
more abundant ions (identified by magenta numbers) can be rationalized by the three
mechanisms shown above.
125
Figure 37: A Finger print Mass Spectrum of Cocain
Odd-electron fragment ions are often formed by characteristic rearrangements in which stable
neutral fragments are lost. Mechanisms for some of these rearrangements have been
identified by following the course of isotopically labeled molecular ions
19.4.4 Formative Assessment
1) An organic compound (A) is composed of carbon, hydrogen and nitrogen, with carbon
constituting over 60% of the mass. It shows a molecular ion at m/z=112 amu in the mass
spectrum. Answer the following questions by entering numbers in the answer boxes.
a. Write a plausible Molecular Formula for compound A: C H N
126
b How many Rings + Double Bonds must be present in compound A?
2) Another compound, B, composed only of carbon, hydrogen and oxygen, also shows a
molecular ion at m/z=112 amu.
a. Write a plausible Molecular Formula for compound B, assuming it has three double bonds
and no rings. C H O
3) Compound C is composed only of carbon, hydrogen and oxygen, and shows a
molecular ion at m/z=180 amu. Carbon accounts for 60% of the molecular mass.
a) Write a plausible Molecular Formula for compound C. C H O
c) How many Rings + Double Bonds must be present in compound C?
127
20 MODULE SYNTHESIS
In Unit I separation methods taught in school were revisited these were solvent extraction and
distillation for each of the technique definitions were made, the conditions under which each
separation method is appropriately applied were discussed and the equipment for carrying out
the techniques were also presented. Later in the unit chromatography techniques were
introduced starting with the general theory, including different types of development. Principal
types of chromatography were introduced, including paper, thin layer, and equipment for their
implementation discussed. This was followed by column chromatography in which was
introduced, instrumentation discussed including different types of columns, and detectors. In
the last part of the module liquid chromatography was introduced and HPLC discussed in
detail this included scales of application of HPLC, Instrumentation and the major modes of
separation of HPLC.
In Unit II the major Electrochemical Techniques were introduced, the discussion of
potentiometry included theory of potentiometry and the application of potentiometry to pH
measurement using the glass electrode, ion selective electrodes or Red Ox electrodes were
discussed and their application to automatic titration stations highlighted. The second part of
unit discussed different techniques of voltammetry starting with a general discussion of the of
the theory of voltammetry, followed by polarographic techniques based on the dropping
mercury electrode. The unit ended with a discussion of two voltammetric techniques cyclic and
anodic stripping voltammetry.
Spectroscopy and Atomic Spectrometric Techniques, Spectroscopy was introduced by
recalling different components of the electromagnetic spectrum, their relative energies
common spectroscopic terms and measurement units. The interaction of radiation and matter
was discussed in depth and how this can be used for qualitative and quantitative analysis in
both molecular and atomic spectroscopy. The last part of the unit discussed atomic
spectroscopy defining the three major modes of atomic spectroscopy, the phenomena leading
to their existence, how they are used in qualitative and quantitative analysis. The unit ended by
discussing instrumentation used in atomic spectroscopy.
Molecular spectroscopy 1 discussed UV-Visible spectroscopy and Infrared spectroscopy
starting with how each of the two phenomena arises.
128
The transitions that give rise to UV-visible spectra were discussed their correlation to specific
functional groups was offered. The factors that affect absorption of functional groups were
elaborated. Examples of how UV is used in structural determination were presented. This
discussion was concluded by application of UV-visible spectroscopy in quantitative analysis
and UV-Visible Instrumentation. The last part of the unit presented IR spectroscopy, in this
explanation of IR spectroscopy arises was given. This was followed by discussion of typical
absorption peaks of major functional groups and correlation of IR spectrum with structure.
Molecular Spectroscopy 2 discussed nuclear magnetic resonance spectroscopy, this started
by description of the NMR phenomena, the requirement for a nucleus to exhibit NMR
phenomena and the influences of the structural environment. The discussion included
correlation of hydrogen NMR with specific functional elements in the molecule, spin-spin
interactions, correlation of magnitude of peaks with number of hydrogen atoms and peak
position with molecular environment and functional groups. The unit was concluded by
discussion carbon-13 NMR this discussion highlighted the C-13 NMR, technical limitation of
information provided by C-13 NMR, how it is used to complement Hydrogen NMR in structural
determination.
Mass spectrometry started with how mass spectrometry is effected, and the arising spectrum.
This was followed by rules of fragmentation, how the presence of the isotopes affects
fragmentation and correlation of structure with mass spectrum.
129
21 SUMMATIVE EVALUATION
1 Calculate the energy of the photons in radiation of wavelength:
(a) 635 nm (in the visible)
(b) 18.7 nm (in the ultraviolet)
(c) 58.6 m (in the infrared)
2 The carbon-13 NMR spectrum of one of the butyl acetate isomers (C4H9OCOCH3) showed signals at δc22, 28, 80 and170. What is its structure? Why is the intensity of the peak at δ 28 much more intense than that at δ 22 (by factor of approximately eight)? How would the multiplicity and signal intensity in the proton NMR spectrum of this compound confirm your deductions?
3a For a typical chromatographic separation giving just-resolved peaks (Rs = 1.5), assume that N = 3600, k' = 2, and = 1.15. Sketch the effects of changing these parameters one at a time to (a) N = 1600, (b) k' = 0.8, and (c) a = 1.10.
b To decrease the plate height and yet increase the resolution, what courses of action are
available? What penalties may accrue for each approach?
4a) Why are atomic spectra different from molecular spectra?
b Why are the atomic spectra of Ca° and Ca+ different?
c. What is the difference between atomic emission spectroscopy and atomic absorption spectroscopy?
5 A pleasant sweet smelling liquid BP 1010C bas the following IR spectra and MS spectra shown
130
6 For each of the compounds A through F indicate the number of structurally-distinct groups of carbon atoms, and also the number of distinct groups of equivalent hydrogens. Enter a number from 1 to 9 in each answer box.
A
Number of distinct carbon atoms: ...
Number of distinct hydrogen groups:
B Number of distinct carbon atoms: ...
Number of distinct hydrogen groups:
C Number of distinct carbon atoms: ...
Number of distinct hydrogen groups:
D Number of distinct carbon atoms: ...
Number of distinct hydrogen groups:
E Number of distinct carbon atoms: ...
131
Number of distinct hydrogen groups:
F Number of distinct carbon atoms: ...
Number of distinct hydrogen groups:
Determine the structure f the compound
132
MAIN AUTHOR OF THE MODULE
Vincent Makokha was educated at Makerere University Kampala earning his BSc. (Ind.
Chemistry 1991) and MSc. (Analytical Chemistry 2000). He subsequently worked in Industry
worked in industry and research organizations in various capacities as analytical chemistry
specialist. He later joined Kyambogo University were he teaches Chemistry Education and
Chemistry Technology courses.
Vincent is married to Florence and they have two children Collette and Vivienne
TEACHING TIPS
This module aims at presenting the most common instrumental analytical techniques to the
learners.
With three principal aims of providing
• Knowledge of principles of the analytical techniques
• Skills for interpreting analytical data generated by instrument s,
• Practice for the skills and knowledge delivered
For undergraduate level students there is the delicate task of managing the level of complexity
of information and skills delivered. For this module there are lots of resources many of them
intended for the practicing professional and advanced graduate student and therefore
unsuitable for our purposes. The material was therefore selected and presented in a simple
form to provide a working knowledge of the subject matter, leaving room for the more
enthusiastic student to pursue the matter further, without confusing the average student.
The basic teaching aid is the material presented in the module which forms the back borne of
the course. The recommended texts and online materials are required to add depth to the
understanding of the module by providing detail and extra practice for the learner.
The major task of the e learning teacher is to encourage the learner and pace him through
each learning unit. Each learning unit should be covered and mastered before advancing to
the next unit.
The material presented should be preferably covered in the order presented in the module.
133
REFERENCES
1. Crow D.R. : Principles and applications of Electrochemistry Chapman and Hall, 2nd Edi-tion 1996
Galen Wood Ewing Instrumental methods of chemical analysis
Publisher: MacgrawHill. 1986
2. Braun. D Robert Introduction to chemical Analysis Publisher: McGrawHill 1st Edition 1982.
3. Heslop R.B, Wild Gillian M.. S I Units in chemistry
Applied Science Publishers, 1971.
4 Hobarth Willard, Lynne Merritt, John Dean, and Frank Settle, Instrumental Methods of Analysis Wadsworth Publishing Company; 7 Sub edition (February 1988)
22 FILE STRUCTURE
Microsoft Word File
Separation, Electroanalytical, and Spectrochemical Techniques Final Version.doc
PDF File
Separation, Electroanalytical, and Spectrochemical Techniques Final Version.pdf
AAS
Atomic Absorption Instrument
Cyclic Voltammetry
Distillation
Electromagnetic Spectrum
Energy Levels
Gas Chromatography
HPLC
134
Infra Red Spectroscopy
Ion Selective Electrodes
Mass Spectrometry
Potentiometry
Separation and Chromatography
135