Separation, Electroanalytical and Spectrochemical Techniques Separation, Electroanalytical and Spectrochemical Techniques

Home , Analytical technique, Separation process

Prepared by Vincent MAKOKHA

African Virtual university Université Virtuelle Africaine Universidade Virtual Africana African Virtual University

Notice

This document is published under the conditions of the Creative Commons http://en.wikipedia.org/wiki/Creative_Commons Attribution http://creativecommons.org/licenses/by/2.5/ License (abbreviated “cc-by”), Version 2.5.

I have received kind permission to reproduce diagrams and text from • Dr. Scott Van Bramer for Mass Spectrometry • Dr. William Reusch of Michigan State University for Molecular Spectroscopy African Virtual University

Table of Contents

I. Separation, Electroanalytical, and Spectrochemical Techniques______3

II. Prerequisite Courses or Knowledge______3

III. Time______3

IV. Materials______3

V. Module Rationale______4

VI. Content______4 6.1 Resume ______4 6.2 Outline______5 6.3 Graphic Organizer______7

VII. General Objective______8

VIII. Specific Learning Objective(s)______8

IX. Teaching and Learning Activities______12

X. Key Concepts______18

XI. Learning Tips______23

XII. Compulsory Reading______24

XIII. Useful links______26

XIV. Multimedia Resources______34

XV. Learning Activities______36

XVI. Synthesis of the Module______106

XVII. Summative Evaluation______108

XIII. Main Author of the Module ______110

XIX. References______111

XX. File Structure ______111

African Virtual University

I. separation, Electroanalytical, and Spectrochemical Techniques by Vincent Makokha

II. 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

III. Time

• Separation and Chromatographic Techniques 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

IV. Materials

You will require the following tools and resources for completing this module

• 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 African Virtual University

V. 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.

VI. Content

6.1 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 elemen- tary 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. The unit on Spectroscopy and Atomic Spectroscopic techniques will review concepts of energy matter interaction, 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 African Virtual University

molecular chemical environment and how proton NMR is 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 Instru- mentation for mass spectrometry.

6.2 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

• 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 African Virtual University

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 • Carbon NMR Spectroscopy

Unit VI Mass Spectrometry- 15 Hours

• Mass Spectrometry o Fragmentation Patterns o Finger Print Spectrum African Virtual University

6.3 Graphic Organiser African Virtual University

VII. 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.

VIII. specific Learning Objectives

Unit Learning objective(s) 1. Separation and At the end of the unit learners will be able to: Chromatographic • Recall Separation methods that are taught in Techniques School • Explain the principles underlying solvent extraction • Solve hypothetical numerical solvent Extraction problems • Name and draw apparatus used for solvent extraction • Name common column and plane Chromatogra- phic Techniques. • Explain the theory underlying each column and plane Chromatographic Techniques • Recall equipment for plane and column chromatography African Virtual University

Unit Learning objective(s) 2. Electro analyti- At the End of this unit the student will be able to: cal Techniques • 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

3. Introduction to At the end of the unit learners be able to: Spectroscopic and • Name the parts of the electromagnetic spectrum Atomic Spectro- • Recall effects of radiation on atoms and molecu- scopic Techniques les • 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 African Virtual University 10

Unit Learning objective(s) 4. Molecular Spec- At the end of the unit learners will be able to: troscopy 1: UV • Recall the electronic transitions caused by ab- visible and Infra sorption of UV-Vis Radiation Red Spectroscopy • 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 At the end of the unit learners will be able to: Spectroscopy 2 • Explain how the phenomenon of NMR arises Nuclear Magnetic • Recall nuclei that exhibit NMR Resonance • Explain Proton NMR phenomena • Correlate Absorption of specific HNMR frequen- Spectroscopy cies to molecular functional groups (NMR) • Correlate Absorption of specific HNMR frequen- Spectroscopy cies 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 Spectrophotome- ter and their functions. African Virtual University 11

Unit Learning objective(s) 6. Mass Spectrom- At the end of the unit learners be able to: etry • 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. African Virtual University 12

ix. Teaching and Learning Activities

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

3) Which of the following statements is true concerning acids and bases? a) acids and bases don’t react with each other b) acids mixed with bases neutralize each other c) acids mixed with bases make stronger bases d) 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 African Virtual University 13

7) Which of the following conversions requires an oxidizing agent? a) Mn3+ --> Mn2+

b) C2H4 --> C2H6 2- c) (2CrO4)2- --> (Cr2O7)

d) SO2 --> SO3

8) The hydrogen halides are all polar molecules which form acidic solutions. Which of the following is the weakest acid? 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 African Virtual University 14

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 + Cu

b) 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. 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 +] African Virtual University 15

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 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 African Virtual University 16

Answer Key

1. b 2. a 3. b 4. c 5. a 6. c 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 African Virtual University 17

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 African Virtual University 18

X. 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.

Dead volume (Vd):The volume outside of the column packing itself. The interstitial volume (intraparticle and interparticle volume) plus extracolumn volume (contrib- uted 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: 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 African Virtual University 19

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, DK .

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 character- izes the LC mode. For example, silica gel is used in adsorption chromatography, an octadecylsilane bonded phase in reversed-phase chromatography, etc. African Virtual University 20

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

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=εCL African Virtual University 21

CHROMOPHORE A chromophore is a functional group that is responsible for absorption e.g. an alkene absorbs at λmax=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,σ, bonds (e.g. with hydrogen or carbon). The remaining p orbital forms a pi,π 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: ε = A / cl (Beer-Lambert Law). This value is a constant for a particular substance. 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. African Virtual University 22

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). Accelerating Voltage The voltage applied to the source to accelerate the ions formed into the analyser. Unified Atomic Mass UnitThe 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. African Virtual University 23

XI. learning Tips

The material in this module is presented in increasing order of complexity from the beginning of each unit to the end of the 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 care- fully 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. Summative evaluation examines the understanding of concepts and retention of facts of a given learning unit. African Virtual University 24

XII. Compulsory Reading

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.

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. African Virtual University 25

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. African Virtual University 26

XIII. Useful Links http://www.mhhe.com/physsci/chemistry/carey/student/olc/ch13ir,html

Summary: This link is part of a large course of chemistry, for purposes of this module however, 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 approach 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 associated with the module and reinforcing understanding concepts African Virtual University 27

http://ull.chemistry.uakron.edu/analytical/Spectrophotometry/

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. African Virtual University 28

http://www.nd.edu/~smithgrp/structure/workbook.html

Summary: This site contains a collection of practice problems Justification: These problems are useful for mastering molecular spectroscopy and mass spectrometry. African Virtual University 29

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. African Virtual University 30

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. African Virtual University 31

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. African Virtual University 32

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 African Virtual University 33

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 Interna- tional. 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 African Virtual University 34

XIV. 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 African Virtual University 35

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 African Virtual University 36

XV. learning Activities

Unit I Separation and Chromatographic Techniques

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

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

List of Relevant Useful Links

http://www.chromatography-online.org/Principles/Introduction/rs1.html http://antoine.frostburg.edu/chem/senese/101/matter/chromatography.shtml African Virtual University 37

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

Separation Techniques

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. 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. 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. 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. African Virtual University 38

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 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. 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: African Virtual University 39

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. Steam Distillation

Figure 2: Steam distillation: http://en.wikipedia.org/wiki/Steam_distillation accessed March 2008

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. African Virtual University 40

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.

Chromatography

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. African Virtual University 41

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, displacement and elution development. Most of the analytical chromatographic development is done by elution development 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.

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. African Virtual University 42

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 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.

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 void time ( tm.)

Figure 5: The Concept of Retention Time African Virtual University 43

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. 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. 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. 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 HETP = L / N African Virtual University 44

The number of theoretical plates that a real column possesses can be found by exa- mining 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.

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. B - 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 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. African Virtual University 45

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. 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 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, African Virtual University 46

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 thenα , 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)

Types of Chromatographic Techniques

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 a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). 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. Dif- ferent 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. 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 African Virtual University 47

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.

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. African Virtual University 48

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 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 ap- propriate reagent which produces coloured areas in the regions which they occupy. Some compounds fluoresce in ultraviolet light and may be located in this way. Al- ternatively 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. Column Chromatography Column chromatography is a separation technique in which the stationary bed is placed within a tube.

Figure 9: Column Chromatography Equipment 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 African Virtual University 49

for the mobile phase in the middle part of the tube (open tubular column).

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. Components of an HPLC

Figure 10: Components of an HPLC Solvent Delivery System pushes the solvent stream through the instrument at constant flow rate Sample injection system - introduces the sample into the liquid stream of the ins- African Virtual University 50

trument 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. 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] 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 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. 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. African Virtual University 51

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 quater- nary structure of purified proteins, and is the primary technique for determining the average molecular weight of natural and synthetic polymers. 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. 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 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 African Virtual University 52

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. 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.

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 African Virtual University 53

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. African Virtual University 54

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.

Support Dynamic Detector Type Selectivity Detectability gases range Flame ion- Hydrogen Mass flow Most organic cpds. 100 pg 107 ization (FID) and air Halides, nitrates, Electron Concentra- nitriles, perox- capture Make-up 50 fg 105 tion ides, anhydrides, (ECD) organometallics

Nitrogen- Hydrogen Nitrogen, phos- Mass flow 10 pg 106 phosphorus and air phorus

Sulphur, phospho- Hydrogen Flame rus, tin, boron, and air photometric Mass flow arsenic, germa- 100 pg 103 possibly (FPD) nium, selenium, oxygen chromium Aliphatics, aro- matics, ketones, esters, aldehydes, Photo-ion- Concentra- Make-up amines, hetero- 2 pg 107 ization (PID) tion cyclics, organo- sulphurs, 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. African Virtual University 55

Gas chromatography is purely analytical instruments for identifying and quantifying chemical species. 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.

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 2. For a typical chromatographic separation giving just-resolved peaks (Rs = 1.5), assume that N = 3600, k’ = 2, and α = 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 African Virtual University 56

1 4592 2984 1238

2 512 3527 5495

Unit II Electroanalytical Techniques

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

List of Required Readings http://en.wikipedia.org/wiki/Electroanalytical_methods

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

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. African Virtual University 57

The potential as reflected above measures the activity of the ions rather than the concentration. Where a=activity, γ=activity coefficient c= 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 Eo is the potential difference of the two electrodes at unit activity and standard conditions 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.

2.303xRXT aoxid E = Εo + Log ZXF are

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. 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 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. African Virtual University 58

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 V

dE/dV

Figure 12: A plot of dE/dV versus time African Virtual University 59

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 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.

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 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. African Virtual University 60

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

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 African Virtual University 61

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.

1/2 2/3 1/6 Id = 708nD m t c 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 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.

Current

E 1/2 Voltage African Virtual University 62

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 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.

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. 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. African Virtual University 63

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 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. African Virtual University 64

Differential Pulse 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

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 African Virtual University 65

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. 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. 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:

5 3/2 1/2 1/2 ip = (2.69x10 ) n A C D v 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 indica- tion of a nonreversible reaction. These parameters of cyclic voltammograms make CV most suitable for characterization and mechanistic studies of redox reactions at electrodes. 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 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). African Virtual University 66

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. African Virtual University 67

Unit III Spectroscopy And Atomic Spectroscopic Techniques

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

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

List of Relevant Useful Links

• http://ull.chemistry.uakron.edu/analytical/Atomic_spec/ • http://www.chem.vt.edu/chem-ed/spec/atomic/aa.html

List of Relevant Multimedia Resources

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 interac- African Virtual University 68

tions, 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.

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. 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). 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 ex- tensively 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. African Virtual University 69

Figure 16 : Electromagnetic Radiation http://en.wikipedia.org/wiki/Image:EM_Spec- trum3-new.jpg#file African Virtual University 70

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 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 spac- ing which match the quantum energy of the incident radiation, then the material will be transparent to that radiation, and it will pass through. 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

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 al- lowable 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 African Virtual University 71

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. 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. Howe- ver 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. African Virtual University 72

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.

Po P

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. African Virtual University 73

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

-εbc %T = 100 P/P0 = e 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:

African Virtual University 74

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. Molar Absorption Molar Absorption ε is a measure of the amount of light absorbed per unit concentration. 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 African Virtual University 75

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 sur- prised to learn that the molar absorption of β -carotene is 100,000 L mol-1 cm-1 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. 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.

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. African Virtual University 76

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. 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 allowa- ble 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.

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, African Virtual University 77

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. 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. 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. African Virtual University 78

Figure 22 : Atomic Absorption Phenomena

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? African Virtual University 79

Unit IV Molecular Spectrocopy 1: Uv-visible And Ir

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

List of Required Readings

http://en.wikipedia.org/wiki/Molecular_energy_state

List of Relevant Useful Links

http://www.scienceofspectroscopy.info/edit/index.php?title=UV_Absorption_Ta- ble http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab4.htm http://www.scienceofspectroscopy.info/edit/index.php?title=UV-Visible_Spec- troscopy http://ull.chemistry.uakron.edu/analytical/Spectrophotometry/

List of Relevant Multimedia Resources

http://www.cem.msu.edu/~parrill/AIRS/name_list.html African Virtual University 80

Ultraviolet- Visible Spectroscopy

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.

Figure 23 : electronic transitions arising from absorption of UV and visible Light African Virtual University 81

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. 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 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 generally shifted 15-45 nm to longer wavelength with respect to the simple unconjugated chromophore. 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 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. African Virtual University 82

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

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. African Virtual University 83

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 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

Infrared Spectroscopy

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. 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 African Virtual University 84

Relative Energies of IR Absorptions Bending vibrations generally require less energy and occur at longer wavelength (lower wave number) than stretching vibrations. 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. Conver- sely, 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.

Identifying Functional Groups by Infrared Spectroscopy

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. 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, African Virtual University 85

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

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.

African Virtual University 86

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 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 ethy- African Virtual University 87

lene 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). 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. African Virtual University 88

Formative Assessment i) Arrange the following bonds in order of IR absorption frequency of their stretching vibration 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. • 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. African Virtual University 89

Unit V Molecular Spectroscopy 2: Nuclear Magnetic Resonance

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.

List of Required Readings

http://www.scienceofspectroscopy.info/edit/index.php?title=NMR_Spectros- copy http://en.wikipedia.org/wiki/NMR_spectroscopy#Chemical_Shift http://www.mhhe.com/physsci/chemistry/carey/student/olc/ch13nmr.html#basi

List of Relevant Links

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab4.htm http://www.scienceofspectroscopy.info/edit/index.php?title=UV-Visible_Spec- troscopy http://www.scienceofspectroscopy.info/edit/index.php?title=UV_Absorption_Ta- ble- non open source 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 African Virtual University 90

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. 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 even old 1,2,3... 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.

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 7, d) carbon 13, e) proton. African Virtual University 91

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

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. African Virtual University 92

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.

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 un- dergoing various energy 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 and result in NMR phenomenon 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. 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. African Virtual University 93

Figure 27 : Chemical Shift Parameters of Different Hydrogen atoms

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 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 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 African Virtual University 94

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 CH2 is split into four peaks of relative area 1:3:3:1. This is explained by the methyl CH3 group 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 inde- pendent of the strength of the applied field. African Virtual University 95

Figure 29 : High Resolution NMR Spectrum of Ethanol

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.

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, polycarbonyl compounds such as croconic acid, and compounds incorporating triple bonds (structures below, orange colored carbons). African Virtual University 96

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 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. 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. African Virtual University 97

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.

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 African Virtual University 98

13c Chemical Shift Ranges* Low Field Region

Figure 33 : Chemical Shift Ranges

* For samples in CDCl3 solution. The δ scale is relative to TMS at δ=0.

Formative Assessment

The 60 MHz spectrum shown in Figure 34 is that of a compound C10H13NO2. Signi- ficant 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.

Figure 34 : C10H13NO2 African Virtual University 99

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? African Virtual University 100

Unit VI Mass Spectrometry

Summary Of The Learning Activity

At the end of the unit learners be able to: • 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.

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

List of Relevant Useful Links http://ull.chemistry.uakron.edu/analytical/Mass_Spec/index.html/

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 The ions are sorted and separated according to their mass and charge.- Mass Ana- lyzer 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. African Virtual University 101

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. 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 African Virtual University 102

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.

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. 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 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 = African Virtual University 103

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. 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 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 pre- viously 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. African Virtual University 104

Finger Print Spectrum The complexity of fragmentation patterns has led to mass spectra being used as “fingerprints” for identifying compounds. Environmental pollutants, pesticide re- sidues 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.

Figure 37 : A Finger print Mass Spectrum of Cocain African Virtual University 105

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

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 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? African Virtual University 106

XVI. synthesis of the Module

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. 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. African Virtual University 107

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. African Virtual University 108

XVII. 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 µm (in the infrared) 2 The carbon-13 NMR spectrum of one of the butyl acetate isomers (C4H9O- COCH3) 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?

3 a. For a typical chromatographic separation giving just-resolved peaks (Rs = 1.5), assume that N = 3600, k’ = 2, and α = 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? 4 a. 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 African Virtual University 109

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.

Number of distinct carbon atoms: ❑ A Number of distinct hydrogen groups: ❑

Number of distinct carbon atoms: ❑ B Number of distinct hydrogen groups: ❑

Number of distinct carbon atoms: ❑ C Number of distinct hydrogen groups: ❑

Number of distinct carbon atoms: ❑ D Number of distinct hydrogen groups: ❑

Number of distinct carbon atoms: ❑ E Number of distinct hydrogen groups: ❑

Number of distinct carbon atoms: ❑ F Number of distinct hydrogen groups: ❑

Determine the structure f the compound African Virtual University 110

XVIII. 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. African Virtual University 111

XIX. References

Crow D.R. : Principles and applications of Electrochemistry Chapman and Hall, 2nd Edition 1996 Galen Wood Ewing Instrumental methods of chemical analysis Publisher: MacgrawHill. 1986 Braun. D Robert Introduction to chemical Analysis Publisher: McGrawHill 1st Edition 1982. Heslop R.B, Wild Gillian M.. S I Units in chemistry Applied Science Publishers, 1971. Hobarth Willard, Lynne Merritt, John Dean, and Frank Settle, Instrumental Methods of Analysis Wadsworth Publishing Company; 7 Sub edition (Fe- bruary 1988)

XX. 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 Infra Red Spectroscopy Ion Selective Electrodes Mass Spectrometry Potentiometry Separation and Chromatography African Virtual University 112 African Virtual University 113 African Virtual University 114