Gas Chromatography Gas Chromatography 4 an analytical separations

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Gas Chromatography

Gas Chromatography

Gas Chromatography 4 an analytical separations technique useful for separating volatile organic compounds 4

Gas Chromatography 4 an analytical separations technique useful for separating volatile organic compounds 4 consists of : – Flowing mobile phase (inert gas - Ar, Ne, N) – Injection port ( rubber septum - syringe injects sample) • kept at a higher temperature than the boiling point

Principles 4 Separation due to differences in partitioning behavior 4 selective retardation

Principles 4 Separation due to differences in partitioning behavior 4 selective retardation

Key Information 4 organic compounds separated due to differences in their participating behavior between

Key Information 4 organic compounds separated due to differences in their participating behavior between the mobile gas phase and the stationary phase in the column 4 in contrast to other types of chromatography, the mobile phase does not interact with molecules of the analyte; its only function is to transport the analyte through the column

Gas Chromatography – Separation column containing stationary phase • since partitioning behavior independent of

Gas Chromatography – Separation column containing stationary phase • since partitioning behavior independent of temperature - kept in thermostat - controlled oven – Detector

Schematic of a gas Chromatograph

Schematic of a gas Chromatograph

The Beginning 4 concept of GC announced in 1941 by Martin and Synge (also

The Beginning 4 concept of GC announced in 1941 by Martin and Synge (also did liquid partition chromatography) 4 10+ years later GC used experimentally 4 1955, first commercial apparatus for GC appeared on the market

Today 4 estimate : 200, 000 gas chromatographs are currently used through out the

Today 4 estimate : 200, 000 gas chromatographs are currently used through out the world. 4 30+ instrument manufactures 4 130 different models 4 cost 1, 500 to 40, 000 dollars 4 improvements: computers- automatic control open tubular columns-separate a multitude of analytes in relatively short times

Uses of Gas Chromatography 4 Determination of volatile compounds (gases & liquids) 4 Determination

Uses of Gas Chromatography 4 Determination of volatile compounds (gases & liquids) 4 Determination of partition coefficients and absorption isotherms 4 Isolating pure components from complex mixtures

Instrumentation

Instrumentation

Instrumentation 4 flowing mobile phase 4 injection port 4 separation column 4 detector

Instrumentation 4 flowing mobile phase 4 injection port 4 separation column 4 detector

GC detectors another powerpoint

GC detectors another powerpoint

Liquid Chromatography much slower diffusion in liquid as compared to gas

Liquid Chromatography much slower diffusion in liquid as compared to gas

Liquid liquid extraction repeated extraction is basis for LC

Liquid liquid extraction repeated extraction is basis for LC

Retardation of solutes in liquid onto a solid phase

Retardation of solutes in liquid onto a solid phase

Elution chromatography 4 Increasing polarity of pure solvents 4 hexane 4 ether 4 acetone

Elution chromatography 4 Increasing polarity of pure solvents 4 hexane 4 ether 4 acetone 4 methanol 4 water 4 acetic acid 4 Solvents mixed 4%hexane and % methanol 4 miscible 4 can be mixed continuously (solvent programming)

Types of Liquid Chromatography 4 Liquid-solid: adsorption on solid which is generally polar (silica

Types of Liquid Chromatography 4 Liquid-solid: adsorption on solid which is generally polar (silica gel, alumina, magnesium silicates) or reverse phase (cellulose, poly amides) 4 Ion exchange: specific interactions with ionic species (change relative strengths of acid or base)

Types of Liquid Chromatography 4 Liquid-liquid: partition between 2 bulk phases (one immobilized) is

Types of Liquid Chromatography 4 Liquid-liquid: partition between 2 bulk phases (one immobilized) is highly selective 4 Liquid exclusion: molecular sieve separates molecules on basis of ability to diffuse into immobile support

Retardation based on size of molecule as it diffuses into porous solid

Retardation based on size of molecule as it diffuses into porous solid

High Performance Liquid Chromatography Once called High Pressure Liquid Chromatography

High Performance Liquid Chromatography Once called High Pressure Liquid Chromatography

What is HPLC? 4 The most widely used analytical separations technique 4 Utilizes a

What is HPLC? 4 The most widely used analytical separations technique 4 Utilizes a liquid mobile phase to separate components of mixture 4 uses high pressure to push solvent through the column 4 Popularity: – sensitivity – ready adaptability to accurate quantitative determination – suitability for separating nonvolatile species or thermally fragile ones

HPLC is…. 4 Popularity: – widespread applicability to substances that are of prime interest

HPLC is…. 4 Popularity: – widespread applicability to substances that are of prime interest to industry, to many fields of science, and to the public 4 Ideally suited for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and a variety of other inorganic substances

History lesson 4 Early LC carried out in glass columns – diameters: 1 -5

History lesson 4 Early LC carried out in glass columns – diameters: 1 -5 cm – lengths: 50 -500 cm 4 Size of solid stationary phase – diameters: 150 -200 m 4 Flow rates still low! Separation times long! 4 Eureka! Decrease particle size of packing causes increase in column efficiency! – diameters 3 -10 m 4 This technology required sophisticated instruments – new method called HPLC

Advantages to HPLC 4 Higher resolution and speed of analysis 4 HPLC columns can

Advantages to HPLC 4 Higher resolution and speed of analysis 4 HPLC columns can be reused without repacking or regeneration 4 Greater reproducibility due to close control of the parameters affecting the efficiency of separation 4 Easy automation of instrument operation and data analysis 4 Adaptability to large-scale, preparative procedures

Advantages to HPLC 4 Advantages of HPLC are result of 2 major advances: –

Advantages to HPLC 4 Advantages of HPLC are result of 2 major advances: – stationary supports with very small particle sizes and large surface areas – appliance of high pressure to solvent flow

Schematic of liquid chromatograph

Schematic of liquid chromatograph

LC column LC injector

LC column LC injector

Types of HPLC 4 Liquid-solid (adsorption) chromatography 4 Liquid-liquid (partition) chromatography 4 Ion-exchange chromatography

Types of HPLC 4 Liquid-solid (adsorption) chromatography 4 Liquid-liquid (partition) chromatography 4 Ion-exchange chromatography 4 Size exclusion chromatography

Partition Chromatography 4 Most widely used 4 Bonded-phase Chromatography 4 Silica Stationary Phase: OH

Partition Chromatography 4 Most widely used 4 Bonded-phase Chromatography 4 Silica Stationary Phase: OH OH O Si 4 Siloxanes: Si O O OH O Si CH 3 Si R= C 8, C 18

Partition Chromatography II 4 Reverse Phase Chromatography – Nonpolar Stationary Phase – Polar Mobile

Partition Chromatography II 4 Reverse Phase Chromatography – Nonpolar Stationary Phase – Polar Mobile Phase 4 Normal Phase Chromatography – Polar Stationary Phase – Nonpolar Mobile Phase 4 Column Selection 4 Mobile-Phase Selection

Partition Chromatography III 4 Research Applications – Parathion in Insecticides: O – CH 3

Partition Chromatography III 4 Research Applications – Parathion in Insecticides: O – CH 3 CH 2 O P O CH 3 CH 2 O NO 2 – Cocaine in Fruit Flies: A Study of Neurotransmission by Prof. Jay Hirsh, UVa

Adsorption Chromatography 4 Classic 4 Solvent Selection 4 Non-polar Isomeric Mixtures 4 Advantages/ Disadvantages

Adsorption Chromatography 4 Classic 4 Solvent Selection 4 Non-polar Isomeric Mixtures 4 Advantages/ Disadvantages 4 Applications

What is Ion Chromatography? 4 Modern methods of separating and determining ions based on

What is Ion Chromatography? 4 Modern methods of separating and determining ions based on ion-exchange resins 4 Mid 1970 s 4 Anion or cation mixtures readily resolved on HPLC column 4 Applied to a variety of organic & biochemical systems including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations

The Mobile Phases are. . . 4 Aqueous solutions – containing methanol, water-miscible organic

The Mobile Phases are. . . 4 Aqueous solutions – containing methanol, water-miscible organic solvents – also contain ionic species, in the form of a buffer – solvent strength & selectivity are determined by kind and concentration of added ingredients – ions in this phase compete with analyte ions for the active site in the packing

Properties of the Mobile Phase 4 Must – dissolve the sample – have a

Properties of the Mobile Phase 4 Must – dissolve the sample – have a strong solvent strength leads to reasonable retention times – interact with solutes in such a way as to lead to selectivity

Ion-Exchange Packings 4 Types of packings – pellicular bead packing • large (30 -40

Ion-Exchange Packings 4 Types of packings – pellicular bead packing • large (30 -40 µm) nonporous, spherical, glass, polymer bead • coated with synthetic ion-exchange resin • sample capacity of these particles is less – coating porous microparticles of silica with a thin film of the exchanger • faster diffusion leads to enhanced efficiency

Ion-Exchange Equilibria 4 Exchange equilibria between ions in solution and ions on the surface

Ion-Exchange Equilibria 4 Exchange equilibria between ions in solution and ions on the surface of an insoluble, high molecular-weight solid 4 Cation exchange resins – sulfonic acid group, carboxylic acid group 4 Anion exchange resins – quaternary amine group, primary amine group CM Cellulose Cation Exchanger DEAE Cellulose Anion Exchanger

Eluent Suppressor Technique 4 Made possible the conductometric detection of eluted ions. 4 Introduction

Eluent Suppressor Technique 4 Made possible the conductometric detection of eluted ions. 4 Introduction of a eluent suppressor column immediately following the ion-exchange column. 4 Suppressor column – packed with a second ion-exchange resin 4 Cation analysis 4 Anion analysis

Size Exclusion Chromatography(SEC) 4 Gel permeation(GPC), gel filtration(GFC) chromatography 4 Technique applicable to separation

Size Exclusion Chromatography(SEC) 4 Gel permeation(GPC), gel filtration(GFC) chromatography 4 Technique applicable to separation of high-molecular weight species 4 Rapid determination of the molecular weight or molecular-weight distribution of larger polymers or natural products 4 Solute and solvent molecules can diffuse into pores -trapped and removed from the flow of the mobile phase

SEC(continued) 4 Specific pore sizes. average residence time in the pores depends on the

SEC(continued) 4 Specific pore sizes. average residence time in the pores depends on the effective size of the analyte molecules – larger molecules – smaller molecules – intermediate size molecules

SEC Column Packing 4 Small (~10 µm) silica or polymer particles containing a network

SEC Column Packing 4 Small (~10 µm) silica or polymer particles containing a network of uniform pores 4 Two types (diameters of 5 ~ 10 µm) – Polymer beads – silica-based particles

Advantages of Size Exclusion Chromatography 4 Short & well-defined separation times 4 Narrow bands-->

Advantages of Size Exclusion Chromatography 4 Short & well-defined separation times 4 Narrow bands--> good sensitivity 4 Freedom from sample loss, solutes do not interact with the stationary phase 4 Absence of column deactivation brought about by interaction of solute with the packing

Disadvantages 4 Only limited number of bands can be accommodated because the time scale

Disadvantages 4 Only limited number of bands can be accommodated because the time scale of the chromatogram is short 4 Inapplicability to samples of similar size, such as isomers. – At least 10% difference in molecular weight is required for reasonable resolution

Instrumentation 4 Instruments required: – Mobile phase reservoir – Pump – Injector – Column

Instrumentation 4 Instruments required: – Mobile phase reservoir – Pump – Injector – Column – Detector – Data system

Schematic of liquid chromatograph

Schematic of liquid chromatograph

Mobile phase reservoir 4 Glass/stainless steel reservoir 4 Removal of dissolved gases by degassers

Mobile phase reservoir 4 Glass/stainless steel reservoir 4 Removal of dissolved gases by degassers – vacuum pumping system – heating/stirring of solvents – sparging – vacuum filtration

Elution methods 4 Isocratic elution – single solvent of constant composition 4 Gradient elution

Elution methods 4 Isocratic elution – single solvent of constant composition 4 Gradient elution – 2 or more solvents of differing polarity used

Pumping System I 4 Provide a continuous constant flow of the solvent through the

Pumping System I 4 Provide a continuous constant flow of the solvent through the injector 4 Requirements – – pressure outputs up to 6000 psi pulse-free output flow rates ranging from. 1 -10 m. L/min flow control and flow reproducibility of. 5% or better – corrosion-resistant components

Pumping System II 4 Two types: – constant-pressure – constant-flow 4 Reciprocating pumps –

Pumping System II 4 Two types: – constant-pressure – constant-flow 4 Reciprocating pumps – motor-driven piston – disadvantage: pulsed flow creates noise – advantages: small internal volume (35 -400 L), high output pressures (up to 10, 000 psi), ready adaptability to gradient elution, constant flow rates

Pumping System III 4 Displacement pumps – syringe-like chambers activated by screw-driven mechanism powered

Pumping System III 4 Displacement pumps – syringe-like chambers activated by screw-driven mechanism powered by a stepper motor – advantages: output is pulse free – disadvantage: limited solvent capacity (~20 m. L) and inconvenience when solvents need to be changed 4 Flow control and programming system – computer-controlled devices – measure flow rate – increase/decrease speed of pump motor

Sample Injection Systems 4 For injecting the solvent through the column 4 Minimize possible

Sample Injection Systems 4 For injecting the solvent through the column 4 Minimize possible flow disturbances 4 Limiting factor in precision of liquid chromatographic measurement 4 Volumes must be small 4. 1 -500 L 4 Sampling loops – interchangeable loops (5 -500 L at pressures up to 7000 psi)

LC column LC injector

LC column LC injector

Liquid Chromatographic Column 4 Smooth-bore stainless steel or heavy-walled glass tubing 4 Hundreds of

Liquid Chromatographic Column 4 Smooth-bore stainless steel or heavy-walled glass tubing 4 Hundreds of packed columns differing in size and packing are available from manufacturers ($200$500) 4 Add columns together to increase length

Liquid Chromatographic Columns II 4 Column thermostats – maintaining column temperatures constant to a

Liquid Chromatographic Columns II 4 Column thermostats – maintaining column temperatures constant to a few tenths degree centigrade – column heaters control column temperatures (from ambient to 150 o. C) – columns fitted with water jackets fed from a constant temperature bath

Detector 4 Mostly optical 4 Equipped with a flow cell 4 Focus light beam

Detector 4 Mostly optical 4 Equipped with a flow cell 4 Focus light beam at the center for maximum energy transmission 4 Cell ensures that the separated bands do not widen

Some Properties of Detector 4 Adequate sensitivity 4 Stability and reproducibility 4 Wide linear

Some Properties of Detector 4 Adequate sensitivity 4 Stability and reproducibility 4 Wide linear dynamic range 4 Short response time 4 Minimum volume for reducing zone broadening

More Properties of Detector 4 High reliability and ease of use 4 Similarity in

More Properties of Detector 4 High reliability and ease of use 4 Similarity in response toward all analytes 4 Selective response toward one or more classes of analytes 4 Non-destructive

Types of Detector 4 Refractive index 4 UV/Visible 4 Fluorescence 4 Conductivity 4 Evaporative

Types of Detector 4 Refractive index 4 UV/Visible 4 Fluorescence 4 Conductivity 4 Evaporative light scattering 4 Electrochemical

Refractive Index I 4 Measure displacement of beam with respect to photosensitive surface of

Refractive Index I 4 Measure displacement of beam with respect to photosensitive surface of dectector

Refractive Index II 4 Advantages – universal respond to nearly all solutes – reliable

Refractive Index II 4 Advantages – universal respond to nearly all solutes – reliable – unaffected by flow rate – low sensitive to dirt and air bubbles in the flow cell

Refractive Index III 4 Disadvantages – expensive – highly temperature sensitive – moderate sensitivity

Refractive Index III 4 Disadvantages – expensive – highly temperature sensitive – moderate sensitivity – cannot be used with gradient elution

UV/Visible I 4 Mercury lamp 4 = 254 nm 4 = 250, 313, 334

UV/Visible I 4 Mercury lamp 4 = 254 nm 4 = 250, 313, 334 and 365 nm with filters 4 Photocell measures absorbance 4 Modern UV detector has filter wheels for rapidly switching filters; used for repetitive and quantitative analysis

UV/Visible II

UV/Visible II

UV/Visible III 4 Advantages – high sensitivity – small sample volume required – linearity

UV/Visible III 4 Advantages – high sensitivity – small sample volume required – linearity over wide concentration ranges – can be used with gradient elution

UV/Visible IV 4 Disadvantage – does not work with compounds that do not absorb

UV/Visible IV 4 Disadvantage – does not work with compounds that do not absorb light at this wavelength region

Fluorescence I 4 For compounds having natural fluorescing capability 4 Fluorescence observed by photoelectric

Fluorescence I 4 For compounds having natural fluorescing capability 4 Fluorescence observed by photoelectric detector 4 Mercury or Xenon source with grating monochromator to isolate fluorescent radiation

Fluorescence II 4 Advantages – extremely high sensitivity – high selectivity 4 Disadvantage –

Fluorescence II 4 Advantages – extremely high sensitivity – high selectivity 4 Disadvantage – may not yield linear response over wide range of concentrations

Conductivity 4 Measure conductivity of column effluent 4 Sample indicated by change in conductivity

Conductivity 4 Measure conductivity of column effluent 4 Sample indicated by change in conductivity 4 Best in ion-exchange chromatography 4 Cell instability

Evaporative Light Scattering I 4 Nebulizer converts eluent into mist 4 Evaporation of mobile

Evaporative Light Scattering I 4 Nebulizer converts eluent into mist 4 Evaporation of mobile phase leads to formation of fine analyte particles 4 Particles passed through laser beam; scattered radiation detected at right angles by silicon photodiode 4 Similar response for all nonvolatile solutes 4 Good sensitivity

Evaporative Light Scattering II

Evaporative Light Scattering II

Electrochemical I 4 Based on reduction or oxidation of the eluting compound at a

Electrochemical I 4 Based on reduction or oxidation of the eluting compound at a suitable electrode and measurement of resulting current

Electrochemical II 4 Advantages – high sensitivity – ease of use 4 Disadvantages –

Electrochemical II 4 Advantages – high sensitivity – ease of use 4 Disadvantages – mobile phase must be made conductive – mobile phase must be purified from oxygen, metal contamination, halides

Data System 4 For better accuracy and precision 4 Routine analysis – pre-programmed computing

Data System 4 For better accuracy and precision 4 Routine analysis – pre-programmed computing integrator 4 Data station/computer needed for higher control levels – add automation options – complex data becomes more feasible – software safeguard prevents misuse of data system

Electrophoresis…charged species migrate in electric field Separation based on charge or mobility

Electrophoresis…charged species migrate in electric field Separation based on charge or mobility

Capillary electrophoresis higher voltages can be used as the heat can be dissipated

Capillary electrophoresis higher voltages can be used as the heat can be dissipated

Capillary electrophoresis

Capillary electrophoresis