Gas Chromatography Gas Chromatography 4 an analytical separations
- Slides: 90
Gas Chromatography
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
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 temperature - kept in thermostat - controlled oven – Detector
Schematic of a gas Chromatograph
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 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 of partition coefficients and absorption isotherms 4 Isolating pure components from complex mixtures
Instrumentation
Instrumentation 4 flowing mobile phase 4 injection port 4 separation column 4 detector
GC detectors another powerpoint
Liquid Chromatography much slower diffusion in liquid as compared to gas
Liquid liquid extraction repeated extraction is basis for LC
Retardation of solutes in liquid onto a solid phase
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 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 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
High Performance Liquid Chromatography Once called High Pressure Liquid Chromatography
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 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 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 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: – stationary supports with very small particle sizes and large surface areas – appliance of high pressure to solvent flow
Schematic of liquid chromatograph
LC column LC injector
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 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 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 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 4 Applications
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 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 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 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 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 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 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 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 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--> 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 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 – Detector – Data system
Schematic of liquid chromatograph
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 – 2 or more solvents of differing polarity used
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 – 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 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 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
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 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 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 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 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 light scattering 4 Electrochemical
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 – 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 – cannot be used with gradient elution
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 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 light at this wavelength region
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 – may not yield linear response over wide range of concentrations
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 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
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 – 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 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
Capillary electrophoresis higher voltages can be used as the heat can be dissipated
Capillary electrophoresis
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