Separation, Electroanalytical, and Spectrochemical Techniques

MATERIALS

Resume

This module encompasses three interconnected subjects: Separation Techniques and Chromatographic Techniques, Electroanalytical Techniques, and Spectroscopic Methods It will be delivered through six learning units that highlight shared concepts and methodologies.

The unit on Separation Techniques and Chromatographic Techniques will explore fundamental separation methods commonly taught in schools, leading into a comprehensive discussion on various chromatography techniques, which will be introduced through an overview of general chromatographic principles.

Electro Analytical Techniques encompass the foundational principles of potentiometry, highlighting its widespread applications The discussion will then transition to voltammetry, covering a range of methods from polarographic techniques to cyclic and anodic stripping voltammetry.

This unit on Spectroscopy and Atomic Spectroscopic Techniques will explore the interaction between energy and matter, delve into the energy levels of atoms and molecules, and conclude with an overview of various atomic spectroscopic techniques.

Molecular Spectroscopy 1 explores the theory behind UV-Visible spectroscopy, detailing its origins and applications in both qualitative and quantitative analysis The course covers the instrumentation of modern UV-Visible spectrophotometers, culminating in a discussion on infrared spectroscopy This section focuses on the generation of infrared spectra, the significance of specific peaks associated with various functional groups, and the practical application of IR in identifying functional groups and compounds.

Molecular Spectroscopy 2 explores the phenomenon of nuclear magnetic resonance (NMR), focusing on proton NMR and its correlation with chemical shifts in various molecular environments This section highlights the application of proton NMR in identifying functional groups within compounds The unit concludes by examining carbon NMR and its complementary role alongside proton NMR in the comprehensive analysis of chemical structures.

The final learning unit focuses on Mass Spectrometry, exploring the generation of mass spectra and their application in identifying organic compounds, culminating with an overview of the instrumentation used in mass spectrometry.

Outline

UNITI I SEPARATION AND CHROMATOGRAPHIC TECHNIQUES- 25 hours

• Separation Techniques o Solvent Extraction o Distillation

• 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

UNIT III SPECTROSCOPY AND ATOMIC SPECTROSCOPIC TECHNIQUES- 20 hours

• Spectroscopy: o Electromagnetic Radiation o The Atom and Atomic Spectroscopy o Beers law

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

SEPARATION, ELECTROANALYTICAL, AND SPECTROCHEMICAL TECHNIQUES

INTRODUCTION TO SPECTROSCOPY AND ATOMIC SPECTROSCOPY

SPECTROSCOPY 1 o Proton NMR o Chemical Shift o Correlation of HNMR With Structure

UNIT IX MASS SPECTROMETRY- 15 hours

• Mass Spectrometry o Fragmentation Patterns o Finger Print Spectrum

General Objective

This module aims to achieve three key objectives: to clarify the fundamental concepts of modern analytical techniques, to equip learners with essential skills for applying these concepts to simulated real-world scenarios, and to enhance students' understanding of the chemical principles that underpin these techniques.

Specific Objectives

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

At the End of this unit the student willbe able to:

• Recall the theory on which potentiometry is based

Unit Learning objective(s) titration stations

• Recall the theory of Voltammetry

• Explain the concept of on which

• Interpret polarographic data to identify and quantify chemical Species

At the end of the unit learners be able to:

• Name the parts of the electromagnetic spectrum

• Recall effects of radiation on atoms and molecules

• Recall Plank’s law and apply it to spectroscopic problems

• Electronic energy levels in atoms and molecules

• Recall Beers law and apply to quantitative problems

• Explain electronic energy levels in atoms and transitions caused by absorption of radiation.

• Explain the concepts on which AAS, AES, AFS is based

• Recall AES, AFS and AAS Instrumentation

• 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.

• Explain how the phenomenon of NMR arises

• Recall nuclei that exhibit NMR

• 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

Unit Learning objective(s) their functions.

6 Mass Spectrometry At the end of the unit learners be able to:

• 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.

1) A beryllium atom has 4 protons, 5 neutrons, and 4 electrons What is the mass number of this atom?

2) The lowest principal quantum number for an electron is

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

7) Which of the following conversions requires an oxidizing agent? a) Mn 3+ > Mn 2+ b) C2H4 > C2H6 c) (2CrO4)2- > (Cr2O7) 2- d) SO2 > SO3

8 The hydrogen halides are all polar molecules which form acidic solutions Which of the following is the weakest acid? a) HI

9 Calculate the [H+] in a solution that has a pH of 8.38.

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 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

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?

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)?

20) Which element has the highest first ionization energy? a) Aluminium b) Sodium c) calcium d) phosphorus

Answer Key

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

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

A Gaussian-shaped peak is characterized by its symmetry, and the peak asymmetry factor is defined as the ratio of the distance from the peak apex to the back side of the chromatographic curve compared to the distance from the peak apex to the front side, measured at 10 percent of the peak height When this ratio exceeds 1, it indicates a tailing peak, whereas a ratio less than 1 signifies a fronting peak.

Baseline: The baseline is the line drawn by the data system when the only signal from the detector is from the mobile phase.

Chromatography: A chemical separation technique based on the differential distribution of the constituents of a mixture between two phases, one of which moves relative to the other. phase.

Dead volume (V d ): The volume outside of the column packing itself The interstitial volume

Dead volume is the total of intraparticle and interparticle volume, along with the extracolumn volume contributed by components such as the injector, detector, connecting tubing, and end fittings This volume can be quantified by injecting an inert compound that does not interact with the column packing It is commonly abbreviated as Vo or Vm.

UV/Visible light absorbance, differential refractive index, electrochemical, conductivity, and fluorescence.

In chromatographic processes, a sample is introduced at the top of a column and is subsequently displaced by a more strongly sorbed compound, leading to the sharpening of eluting sample solute zones This technique involves the displacement of sample molecules by one another and by the stronger sorbent Displacement methods are primarily utilized in preparative EPLC applications, enhancing the efficiency of separation.

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, commonly known as water-loving, refers to both water-compatible stationary phases and water-soluble molecules Most columns designed for protein separation are inherently hydrophilic, ensuring they do not adsorb or denature proteins in aqueous environments.

An injector is a crucial mechanism designed to accurately introduce a specific volume of sample into the mobile phase stream It can range from a basic manual device to an advanced auto sampler, which allows for automated injections of various samples, enabling unattended operation.

Partition coefficient (K): The amount of solute in the stationary phase relative to the amount of solute in the mobile phase It can also be the distribution coefficient, KD.

Retention time (t R ’): The time between injection and the appearance of the peak maximum.

The adjusted retention time tR’ adjusts for the column void volume

Retention volume (V R) refers to the amount of mobile phase needed to elute a substance from a chromatography column It is calculated using the void volume (Vm), the distribution coefficient (KD), and the stationary phase volume (Vs).

Stationary phase: The immobile phase involved in the chromatographic process The

In electroanalytical chemistry, several key concepts play a crucial role in understanding the behavior of electrochemical systems The background current, which is not due to the chemical reaction of the analyte, consists of two components: the capacitive current, resulting from the electrode acting as a capacitor and becoming charged, and the charging current, also known as the capacitive current The movement of ions in a solution is driven by two main factors: diffusion, which occurs due to a concentration gradient, and migration, which is caused by a potential gradient The half-cell, a fundamental component of an electrochemical cell, consists of an electrode, an electrically conductive solvent system, analyte, and a salt bridge connection The half-wave potential, a measure of the formal potential, is the potential at half of the peak or limiting current.

Absorption spectroscopy is a technique that measures the amount of radiation absorbed by a sample at specific wavelengths The absorption of radiation varies according to the molecular structure of the sample, as different electronic transitions lead to absorption at distinct wavelengths.

BEER-LAMBERT LAW An equation relating absorbance, path length, molar absorption coefficient and concentration of the sample: A = cl

CHROMOPHORE A chromophore is a functional group that is responsible for absorption e.g. an alkene absorbs at max7nm.

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

A double bond occurs when one atom, such as carbon, hybridizes its s orbital and two of its p orbitals to create three sp² orbitals, which form sigma bonds with other atoms like hydrogen or carbon The remaining p orbital then forms a pi bond with another carbon that is also sp² hybridized.

ELECTROMAGNETIC SPECTRUM The range of electromagnetic energy with wavelengths ranging from 10 5 m (radio waves) to 10 -14 m (gamma radiation) In between these extremes is the infrared, visible, ultraviolet and X-ray regions.

Fluorescence is a process where an electron transitions from its ground state to an excited state As the electron loses energy, it initially dissipates heat through vibrational relaxation, and subsequently emits light, known as fluorescence, when it returns to its 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.

Molecular orbitals are formed through the interaction of atomic orbitals during bond formation These orbitals can be categorized into bonding orbitals, which have lower energy than the original atomic orbitals; anti-bonding orbitals, which possess higher energy; and non-bonding orbitals, which maintain an equal energy level.

SPECTROSCOPY The method of producing and analyzing data to allow determination of the structure of molecules.

Transmittance refers to the proportion of radiation that successfully passes through a sample without being absorbed It is calculated by dividing the amount of incoming radiation by the amount of radiation that transmits through the sample When this ratio is multiplied by 100, it yields the percentage of transmission.

ULTRAVIOLET REGION The region of the electromagnetic spectrum which has wavelengths between 400-4nm The UV region is split into three more regions: near UV (400-300nm), far

UV radiation is categorized into two types: UV (300-200nm) and extreme or vacuum UV (less than 200nm) The vacuum UV region is characterized by its absorption by oxygen, necessitating the evacuation of the apparatus when utilizing vacuum UV radiation.

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 -14 m (0.00000000000001m).

Mass spectrometry is a powerful analytical technique that generates ions from atoms or molecules and separates them based on their charge-to-mass ratios (m/z) using an analyzer, followed by detection.

Double focusing utilizes a combination of electrostatic (E) and magnetic (B) fields to correct energy variations in ions produced by the source, thereby enhancing the resolution of the analyzer This technique is essential for achieving precise measurements and is relevant in both forward and reverse geometries.

Reference 1

Title: Spectrometric Methods of identification of organic Compounds,

Authors: Robert M Silverstein; Francis X Webster; David J Kiemle

This book serves as a comprehensive guide to the three primary types of spectrometry utilized in spectrometric identification: mass spectrometry, infrared spectrometry, and nuclear magnetic resonance spectrometry Employing a problem-solving methodology, it includes a wealth of reference charts and tables Additionally, the text presents a diverse array of real-data problems designed to challenge practicing chemists.

This reference guide is designed to help students develop practical skills in interpreting spectra Learners are encouraged to utilize this book for practice questions to master various techniques By working through two to three problems for each technique, students can achieve complete mastery of spectroscopy This book serves as the essential reference for mastering these skills.

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.

Reference 3

Title: The Essential Guide to Analytical Chemistry

This book serves as a concise reference guide for the module, presenting all covered material in an easily accessible pocket-sized format Its unique layout features vibrant diagrams alongside succinct text, facilitating quick access to relevant information Additionally, clear schematic diagrams highlight essential procedures and instrumentation, complemented by straightforward spectra that showcase real-world applications.

This book serves as a supplementary resource to enhance learners' comprehension of the module, but it is not intended for initial learning of the topics due to its less comprehensive depth compared to references 2 and 3.

Address: 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

Learners should focus on chapter thirteen, which simplifies the module's content and makes it more accessible This chapter serves as an alternative study resource, offering theoretical concepts, tutorials, and practical examples to enhance understanding.

Justification: The worked examples in this reference provide practice in solving problems associated with the module and reinforcing understanding concepts

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

This website offers concise summaries of key concepts from the module, aiding learners in retaining important facts and understanding essential topics By providing these streamlined notes, the site enhances the educational experience and supports effective study practices.

Summary: This site contains a collection of practice problems

Justification: These problems are useful for mastering molecular spectroscopy and mass spectrometry.

Address: http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/spectro.htm#contnt

Summary: This site contains a comprehensive organic Chemistry Course

Justification: The approach of this course can provide deeper understanding of material covered in this module. 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. 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

The treatment provided exceeds the module's requirements, so learners are encouraged to utilize it solely as a reference.

Summary: This is a student report on AAS

Justification: This page presents atomic spectroscopy in a simplified form http://www.chemguide.co.uk/index.html#top

Originally designed for UK A level chemistry students, the site has expanded to include resources for various UK syllabuses such as A level, IB, Scottish Advanced Higher, and Cambridge International It is now utilized by learners in equivalent courses globally, including those starting university-level studies.

Justification: This site contains very simplified explanations of the material covered in this module Justification

4 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

Justification: this site provides many multimedia tools for reinforcing the learning of this unit 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

UNIT I SEPARATION AND CHROMATOGRAPHIC TECHNIQUES

4.1 Summary of the Learning Activity

• Name common column and plane chromatographic techniques.

Required Readings

is a crucial method that transforms a mixture or solution into distinct product mixtures, aiming for purity by exploiting differences in physical or chemical properties This process is essential for the industrial economy, as raw materials often require separation to be useful Separation techniques can be analytical, focusing on identification without harvesting, or preparative, preparing fractions for further processes Examples of separation methods include distillation, centrifugation, and chromatography, each tailored to achieve specific purification goals Both complete and incomplete separations may involve multiple operations to yield desired products, highlighting the importance of separation science in various applications.

Chromatography http://en.wikipedia.org/wiki/HPLC http://en.wikipedia.org/wiki/Chromatography

4.2.1 List of Relevant Useful Links http://www.chromatography-online.org/Principles/Introduction/rs1.html http://antoine.frostburg.edu/chem/senese/101/matter/chromatography.shtml http://www.chemguide.co.uk/analysis/chromatogrmenu.html#top http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html#top http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm http://www.forumsci.co.il/HPLC/modes/modes3.htm http://www.chem.ubc.ca/courseware/121/tutorials/exp3A/columnchrom/ http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm2.htm

Separation Techniques

Liquid-liquid extraction, also known as solvent extraction or partitioning, is a technique used to separate compounds based on their solubility in two immiscible liquids, typically water and an organic solvent This method involves transferring a substance from one liquid phase to another, where an aqueous solution containing solutes is mixed with an organic solvent The two liquids are vigorously shaken to enhance contact, and then allowed to stand, enabling the phases to separate effectively.

The partition or distribution coefficient (KD) quantifies the concentration ratio of a compound in two immiscible solvents at equilibrium, serving as an indicator of the compound's differential solubility between these solvents.

Water is commonly selected as one of the solvents, with a hydrophobic solvent like octanol as the second choice Both the partition and distribution coefficients serve as indicators of a chemical substance's hydrophilicity (water-loving) or hydrophobicity (water-hating).

The solubility of a solute is influenced by the polarity of both the solute and the solvent Generally, polar solutes dissolve more readily in polar solvents, while non-polar solutes exhibit greater solubility in organic solvents However, non-polar solutes that contain a sufficient number of hydrophilic functional groups, such as hydroxyl and sulfonic groups, can enhance their solubility in polar solvents.

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.

Distillation

Laboratory scale distillations primarily utilize batch distillation methods The distillation apparatus, commonly known as a still, includes essential components: a reboiler or pot for heating the source material, a condenser for cooling the vapor back into liquid form, and a receiver for collecting the purified liquid, known as the distillate Various techniques for laboratory scale distillation are available.

In simple distillation, hot vapours are directed into a condenser, where they are cooled and condensed As a result, the distillate obtained is not pure; its composition mirrors that of the vapours at specific temperature and pressure conditions, which can be determined using Raoult's law.

Simple distillation is typically employed to separate liquids with significantly different boiling points, generally those differing by at least 25 °C This method is also effective for isolating liquids from non-volatile solids or oils, as the vapor pressures of the components are usually distinct enough to facilitate separation according to Raoult's law.

When the boiling points of components in a mixture are closely aligned, fractional distillation becomes essential for effective separation This process utilizes repeated vaporization and condensation cycles within a packed fractionating column to achieve optimal separation of the components.

Figure 1: Fractional distillation apparatus: http://en.wikipedia.org/wiki/Fractional_distillation

A spinning band distillation system enhances separation efficiency by utilizing a spinning band made of Teflon or metal, which promotes intimate contact between rising vapors and descending condensate This interaction effectively increases the number of theoretical plates, leading to improved distillation outcomes.

As the solution is heated, its vapours ascend the fractionating column, cooling and condensing on the condenser walls and packing material The condensate is then reheated by the rising vapours, leading to further vaporization According to Raoult's law, the composition of the newly formed vapours is determined at each stage Each vaporization-condensation cycle, known as a theoretical plate, results in a progressively purer solution of the more volatile component However, it's important to note that these cycles do not occur at the same position within the column, making the theoretical plate a conceptual rather than a precise representation.

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

Steam distillation is an effective technique for extracting heat-sensitive compounds by passing steam through a heated mixture of raw materials According to Raoult's law, the target compound vaporizes based on its partial pressure The resulting vapor mixture is then cooled and condensed, typically producing distinct layers of oil and water.

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.

Certain compounds exhibit high boiling points, making it more effective to reduce the pressure rather than increase the temperature for boiling By lowering the pressure to match the compound's vapor pressure at a specific temperature, boiling and subsequent distillation can begin This method, known as vacuum distillation, is frequently utilized in laboratories, often using a rotary evaporator.

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

In chromatography, components that strongly interact with the stationary phase are retained longer than those that prefer the mobile phase This differential retention leads to the elution of solutes at varying times, based on their distribution coefficients relative to the stationary phase, ultimately achieving effective separation.

Samples can be gaseous, liquid, or solid, varying from simple enantiomer blends to complex mixtures with diverse chemical species Analysis can be performed using either advanced, costly instruments or basic, affordable thin layer plates Chromatography underpins many analytical techniques, and this unit focuses on the most prevalent chromatographic methods and their applications.

Development is the term used to describe how components are separated during the chromatographic process.

There are three basic methods of chromatographic development frontal development, displacement development and elution development Most of the analytical chromatographic development is done by elution development

Elution development is best described as a series of absorption-extraction processes which

When a solute is introduced into a chromatographic system, its concentration in the mobile phase exceeds the equilibrium concentration for distribution within the stationary phase This prompts an immediate transfer of the solute into the stationary phase As additional mobile phase flows in, the concentration of the solute in the stationary phase rises until it reaches equilibrium, at which point desorption into the mobile phase occurs again, allowing the solute to be transported to a new location on the stationary phase.

The mobile phase continuously shifts the solute concentration profile forward, leading to an imbalance with the stationary phase This results in a higher solute concentration at the front of the peak in the mobile phase, prompting a net influx of solute into the stationary phase to restore equilibrium Conversely, at the rear of the peak, the stationary phase experiences an excess concentration of solute as the profile advances, further disrupting the equilibrium.

To re-establish equilibrium in a chromatographic system, a net amount of solute must exit the stationary phase and enter the mobile phase This movement occurs as the solute transitions through the system, entering the mobile phase at the rear of the peak while simultaneously transferring back to the stationary phase in the front half of the peak The net transfer of solute is balanced by the solute migrating from the stationary phase to the mobile phase at the rear half of the peak.

The distribution of analytes between phases can often be described quite simply An analyte is in equilibrium between the two phases;

The equilibrium constant, known as the partition coefficient (K), is defined as the ratio of the molar concentration of an analyte in the stationary phase to its molar concentration in the mobile phase.

Retention time (tR) refers to the duration from sample injection until an analyte peak reaches the detector at the end of the column, with each analyte exhibiting a unique retention time Additionally, the time required for the mobile phase to traverse the column is known as tM.

Figure 5: The Concept of Retention Time

The retention factor, commonly referred to as k' or the capacity factor, quantifies the migration rate of an analyte on a column For analyte A, the retention factor is specifically defined to measure its movement within the chromatographic process.

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

4.5.4 Band Broadening and Column Efficiency

Achieving optimal separations in chromatography requires the production of sharp and symmetrical peaks, which indicates that band broadening should be minimized Additionally, evaluating the efficiency of the column is essential for enhancing separation quality.

4.5.4.1 The Theoretical Plate Model of Chromatography

The plate model in chromatography posits that the chromatographic column consists of numerous distinct layers known as theoretical plates Each plate facilitates the separate equilibrations of the sample between the stationary and mobile phases As the analyte moves through the column, it transfers from one equilibrated mobile phase to the next across these plates.

Plates are a key theoretical concept used to assess column efficiency in chromatography, represented by the number of theoretical plates (N), where a higher number indicates better performance Additionally, the Height Equivalent to a Theoretical Plate (HETP) is another important metric, with a smaller value signifying enhanced efficiency.

If the length of the column is L, then the HETP is

The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution;

Where w1/2 is the peak width at half-height.

As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture.

4.5.4.2 The Rate Theory of Chromatography

A realistic understanding of chromatographic processes recognizes the time required for solute equilibration between the stationary and mobile phases, contrasting with the plate model's assumption of instantaneous equilibration This equilibration time influences the shape of chromatographic peaks, which is further impacted by the diverse pathways available to solute molecules as they navigate the stationary phase particles By examining the mechanisms that lead to band broadening, we derive the Van Deemter equation, which quantifies plate height in chromatography.

Where, u is the average velocity of the mobile phase A, B, and C are factors which contribute to band broadening.

The mobile phase flows through a column filled with a stationary phase, leading to solute molecules taking various random paths This variability in pathways results in the broadening of the solute band, as the differing lengths of these paths contribute to the overall dispersion.

The equilibration time of the analyte between the stationary and mobile phases is crucial in chromatography When the mobile phase moves quickly and the analyte strongly adheres to the stationary phase, the analyte in the mobile phase advances more rapidly, resulting in band broadening Consequently, increased mobile phase velocity exacerbates this broadening effect.

A plot of plate height vs average linear velocity, of mobile phase.

Such plots are of considerable use in determining the optimum mobile phase flow rate.

The selectivity factor α indicates the separation of band centers but does not consider peak widths A more comprehensive assessment of species separation is achieved through resolution measurement The resolution between two species, A and B, is defined as follows.

Baseline resolution is achieved when R = 1.5

Types of Chromatographic Techniques

Paper chromatography is a method used to separate and identify compounds in a sample by applying a small dot of the solution onto chromatography paper The paper is then placed in a sealed jar with a shallow solvent layer, allowing the solvent to rise and carry the sample mixture up the paper As the solvent moves, different compounds travel varying distances based on their interactions with the paper, enabling the calculation of an Rf value This value can be compared to standard compounds for the identification of unknown substances.

Thin Layer Chromatography

Thin layer chromatography (TLC) is a technique akin to paper chromatography, but it utilizes a thin layer of adsorbent materials like silica gel or alumina on a flat substrate, such as glass or plastic, instead of paper TLC offers advantages over paper chromatography, including quicker analysis times and improved separation of compounds, as well as the flexibility to choose various adsorbents In this method, different compounds in a sample mixture move distinct distances based on their interactions with the adsorbent, enabling the calculation of Rf values that can be compared to standard compounds for identifying unknown substances.

Figure 8: Development of a TLC separation: source http://www.waters.com/watersdivision/ContentD.asp?

Thin layer chromatography (TLC) commonly utilizes silica gel as the primary coating material, although cellulose is also frequently employed To create thin layers of cellulose, an aqueous slurry is prepared by blending approximately 15 grams of cellulose powder with 90 cm³ of distilled water for about one minute This cellulose powder is specifically designed for inorganic TLC and has a unique microcrystalline structure For convenience, ready-to-use precoated glass plates and plastic foils with popular adsorbents are available, including plastic sheets precoated with cellulose that may contain fluorescent materials, making them ideal for customizable inorganic TLC applications.

For optimal results in chromatographic analysis, the sample solution should contain 0.1 to 10 mg of the cation per cm³ and can be either neutral or a dilute acid Approximately 1 µL of this solution is applied with a micro syringe or micropipette near one end of the chromatoplate, positioned about 1.5 to 2.0 cm from the edge After application, the chromatoplate should be air-dried, and there is no need for equilibration before initiating the development process.

The development of chromatographic plates typically employs the ascending technique, where the plate is submerged in a redistilled or chromatographic grade solvent to a depth of 0.5 cm To enhance solvent saturation, the chamber is ideally lined with filter paper that dips into the solvent at the bottom Development continues until the solvent front reaches a distance of 10-15 cm, after which the plate is taken out, and the solvent front is promptly marked with a pencil line.

In Thin Layer Chromatography (TLC) and paper chromatography, analytes can be identified by observing them under ultraviolet light To ensure safety during this process, it is essential to wear protective goggles or spectacles The detected spots can then be carefully marked using a needle for further analysis.

Column chromatography is a separation technique in which the stationary bed is placed within a tube.

Liquid Chromatography

Liquid chromatography (LC) is a column chromatography technique where the mobile phase is a liquid, and it can be performed in either a column or planar format The modern version of this method, known as high performance liquid chromatography (HPLC), employs small packing particles and operates under relatively high pressure for enhanced efficiency and separation.

High Performance Liquid Chromatography (HPLC) is a crucial analytical technique used for separating and identifying organic and inorganic compounds in various samples, including biological, pharmaceutical, food, environmental, and industrial sectors This method involves a liquid passing through a porous solid stationary phase, effectively eluting the solutes for analysis.

The stationary phase in chromatography is composed of tiny particles or liquid-coated particles that serve as a support within a column These particles can either fill the entire volume of the tube, creating a packed column, or be concentrated along the inner walls of the tube, allowing for an open tubular column that provides an unobstructed path for the mobile phase.

Figure 9: Column Chromatography Equipment 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

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 instrument

Column - a stainless steel tube packed with silicon beads that separates what I'm looking for (the caffeine) from other compounds (like sugar)

Detector - An optical sensor (usually) that detects changes in the characteristics of the solvent stream

Data System - A means of controlling the system components and storing, processing and

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]

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 HPLC is a chromatographic technique that separates analytes based on their polarity, utilizing a polar stationary phase and a nonpolar mobile phase This method is particularly effective for polar analytes, which associate with the stationary phase and are retained longer during the separation process As the polarity of the analyte increases, so does the strength of its adsorption to the stationary phase, leading to longer elution times due to enhanced interactions between the polar analyte and the polar stationary phase compared to the mobile phase.

Reversed phase HPLC (RP-HPLC) utilizes a non-polar stationary phase combined with a moderately polar aqueous mobile phase A widely used stationary phase is treated silica, often modified with RMe2SiCl, where R represents straight-chain alkyl groups like C18H37 or C8H17 This setup results in longer retention times for non-polar molecules, facilitating the quicker elution of polar compounds Additionally, incorporating polar solvents into the mobile phase increases retention time, while adding hydrophobic solvents decreases it.

Size exclusion chromatography (SEC), also referred to as gel permeation chromatography or gel purified proteins, is the leading method used to determine the average molecular weight of both natural and synthetic polymers.

For more details on this topic, see Ion exchange chromatography.

Ion-exchange chromatography relies on the interaction between solute ions and charged sites on the stationary phase, with ions of the same charge being excluded from retention Key types of ion exchangers include polystyrene resins, which enhance stability through cross-linking, thereby reducing swerving and improving selectivity and equilibration time Additionally, cellulose and dextrin ion exchangers feature larger pore sizes and lower charge densities, making them ideal for protein separation Controlled-pore glass and porous silica are also utilized in this chromatography technique.

The chromatographic process utilizes the ability of biologically active substances to create stable, specific, and reversible complexes through various molecular forces These forces include Van der Waals interactions, electrostatic interactions, dipole-dipole interactions, hydrophobic interactions, and hydrogen bonds A highly efficient, bio-specific bond is established when multiple forces act simultaneously in 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

ELECTROANALYTICAL TECHNIQUES

Summary of the Learning Activity

At the end of this unit the student will be able to:

• Explain the application of potentiometry to pH measurement, ion selective electrode and automatic titration stations

• Interpret Voltammetric data quantitatively and qualitatively

• Explain the concept of on which polarographic analysis is based

List of Required Readings

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

4.11.1List 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

Voltammetry