gas chromatography. 4 an analytical separations technique useful for separating volatile organic...
TRANSCRIPT
Gas Chromatography
an analytical separations technique useful for separating volatile organic compounds
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
Key Information organic compounds separated due to
differences in their participating behavior between the mobile gas phase and the stationary phase in the column
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
The Beginning
concept of GC announced in 1941 by Martin and Synge (also did liquid partition chromatography)
10+ years later GC used experimentally 1955, first commercial apparatus for GC
appeared on the market
Today
estimate : 200, 000 gas chromatographs are currently used through out the world.
30+ instrument manufactures 130 different models cost 1,500 to 40,000 dollars improvements: computers- automatic control
open tubular columns-separate a multitude of analytes in relatively short times
Uses of Gas Chromatography
Determination of volatile compounds (gases & liquids)
Determination of partition coefficients and absorption isotherms
Isolating pure components from complex mixtures
Elution chromatographyElution chromatography
Increasing polarity of pure solvents
hexane ether acetone methanol water acetic acid
Solvents mixed %hexane and %
methanol miscible can be mixed
continuously (solvent programming)
Types of Liquid ChromatographyTypes of Liquid Chromatography
Liquid-solid: adsorption on solid which is generally polar (silica gel, alumina, magnesium silicates) or reverse phase (cellulose, poly amides)
Ion exchange: specific interactions with ionic species (change relative strengths of acid or base)
Types of Liquid ChromatographyTypes of Liquid Chromatography
Liquid-liquid: partition between 2 bulk phases (one immobilized) is highly selective
Liquid exclusion: molecular sieve separates molecules on basis of ability to diffuse into immobile support
What is HPLC? The most widely used analytical separations technique Utilizes a liquid mobile phase to separate components
of mixture uses high pressure to push solvent through the column Popularity:
– sensitivity
– ready adaptability to accurate quantitative determination
– suitability for separating nonvolatile species or thermally fragile ones
HPLC is….
Popularity:– widespread applicability to substances that are of prime
interest to industry, to many fields of science, and to the public
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 Early LC carried out in glass columns
– diameters: 1-5 cm
– lengths: 50-500 cm Size of solid stationary phase
– diameters: 150-200 m Flow rates still low! Separation times long! Eureka! Decrease particle size of packing causes increase in
column efficiency!– diameters 3-10 m
This technology required sophisticated instruments– new method called HPLC
Advantages to HPLC Higher resolution and speed of analysis HPLC columns can be reused without repacking or
regeneration Greater reproducibility due to close control of the
parameters affecting the efficiency of separation Easy automation of instrument operation and data
analysis Adaptability to large-scale, preparative procedures
Advantages to HPLC
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
Types of HPLC
Liquid-solid (adsorption) chromatography Liquid-liquid (partition) chromatography Ion-exchange chromatography Size exclusion chromatography
Partition Chromatography
Most widely used Bonded-phase Chromatography Silica Stationary Phase: OH OH OH OH O O O Si Si Si Si Siloxanes: O CH3
Si O Si R R= C8, C18
O CH3
Partition Chromatography II
Reverse Phase Chromatography– Nonpolar Stationary Phase
– Polar Mobile Phase
Normal Phase Chromatography– Polar Stationary Phase
– Nonpolar Mobile Phase
Column Selection Mobile-Phase Selection
Partition Chromatography III
Research Applications– Parathion in Insecticides: O
– CH3CH2O P O NO2
CH3CH2O
– Cocaine in Fruit Flies: A Study of Neurotransmission by Prof. Jay Hirsh, UVa
Adsorption Chromatography
Classic Solvent Selection Non-polar Isomeric Mixtures Advantages/ Disadvantages Applications
What is Ion Chromatography?
Modern methods of separating and determining ions based on ion-exchange resins
Mid 1970s Anion or cation mixtures readily resolved on HPLC
column Applied to a variety of organic & biochemical systems
including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations
The Mobile Phases are...
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
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
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
Exchange equilibria between ions in solution and ions on the surface of an insoluble, high molecular-weight solid
Cation exchange resins– sulfonic acid group, carboxylic acid group
Anion exchange resins– quaternary amine group, primary amine group
CM CelluloseCation Exchanger
DEAE CelluloseAnion Exchanger
Eluent Suppressor Technique
Made possible the conductometric detection of eluted ions.
Introduction of a eluent suppressor column immediately following the ion-exchange column.
Suppressor column– packed with a second ion-exchange resin
Cation analysis Anion analysis
Size Exclusion Chromatography(SEC) Gel permeation(GPC), gel filtration(GFC)
chromatography Technique applicable to separation of high-molecular
weight species Rapid determination of the molecular weight or
molecular-weight distribution of larger polymers or natural products
Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase
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(continued)
SEC Column Packing
Small (~10 µm) silica or polymer particles containing a network of uniform pores
Two types (diameters of 5 ~ 10 µm)– Polymer beads
– silica-based particles
Advantages of Size Exclusion Chromatography Short & well-defined separation times Narrow bands--> good sensitivity Freedom from sample loss, solutes do not interact
with the stationary phase Absence of column deactivation brought about by
interaction of solute with the packing
Disadvantages
Only limited number of bands can be accommodated because the time scale of the chromatogram is short
Inapplicability to samples of similar size, such as isomers. – At least 10% difference in molecular weight is required
for reasonable resolution
Instrumentation
Instruments required:– Mobile phase reservoir
– Pump
– Injector
– Column
– Detector
– Data system
Mobile phase reservoir
Glass/stainless steel reservoir Removal of dissolved gases by degassers
– vacuum pumping system
– heating/stirring of solvents
– sparging
– vacuum filtration
Elution methods
Isocratic elution– single solvent of constant composition
Gradient elution– 2 or more solvents of differing polarity used
Pumping System I
Provide a continuous constant flow of the solvent through the injector
Requirements– pressure outputs up to 6000 psi
– pulse-free output
– flow rates ranging from .1-10 mL/min
– flow control and flow reproducibility of .5% or better
– corrosion-resistant components
Pumping System II
Two types:– constant-pressure
– constant-flow 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
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 mL) and inconvenience when solvents need to be changed
Flow control and programming system– computer-controlled devices
– measure flow rate
– increase/decrease speed of pump motor
Sample Injection Systems
For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small .1-500 L Sampling loops
– interchangeable loops (5-500 L at pressures up to 7000 psi)
Liquid Chromatographic Column
Smooth-bore stainless steel or heavy-walled glass tubing
Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500)
Add columns together to increase length
Liquid Chromatographic Columns II Column thermostats
– maintaining column temperatures constant to a few tenths degree centigrade
– column heaters control column temperatures (from ambient to 150oC)
– columns fitted with water jackets fed from a constant temperature bath
Detector
Mostly optical Equipped with a flow cell Focus light beam at the center for
maximum energy transmission Cell ensures that the separated
bands do not widen
Some Properties of Detector
Adequate sensitivity Stability and reproducibility Wide linear dynamic range Short response time Minimum volume for reducing zone broadening
More Properties of Detector
High reliability and ease of use Similarity in response toward all analytes Selective response toward one or more classes of
analytes Non-destructive
Types of Detector
Refractive index UV/Visible Fluorescence Conductivity Evaporative light scattering Electrochemical
Refractive Index II
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
Disadvantages– expensive
– highly temperature sensitive
– moderate sensitivity
– cannot be used with gradient elution
UV/Visible I
Mercury lamp = 254nm = 250, 313, 334 and 365nm with filters Photocell measures absorbance Modern UV detector has filter wheels for rapidly
switching filters; used for repetitive and quantitative analysis
UV/Visible III
Advantages– high sensitivity
– small sample volume required
– linearity over wide concentration ranges
– can be used with gradient elution
UV/Visible IV
Disadvantage– does not work with compounds that do not absorb light
at this wavelength region
Fluorescence I
For compounds having natural fluorescing capability
Fluorescence observed by photoelectric detector
Mercury or Xenon source with grating monochromator to isolate fluorescent radiation
Fluorescence II
Advantages– extremely high sensitivity
– high selectivity
Disadvantage– may not yield linear response over wide range of
concentrations
Conductivity
Measure conductivity of column effluent
Sample indicated by change in conductivity
Best in ion-exchange chromatography
Cell instability
Evaporative Light Scattering I
Nebulizer converts eluent into mist Evaporation of mobile phase leads to formation of
fine analyte particles Particles passed through laser beam; scattered
radiation detected at right angles by silicon photodiode
Similar response for all nonvolatile solutes Good sensitivity
Electrochemical I
Based on reduction or oxidation of the eluting compound at a suitable electrode and measurement of resulting current
Electrochemical II
Advantages– high sensitivity
– ease of use
Disadvantages– mobile phase must be made conductive
– mobile phase must be purified from oxygen, metal contamination, halides
Data System
For better accuracy and precision Routine analysis
– pre-programmed computing integrator Data station/computer needed for higher control levels
– add automation options
– complex data becomes more feasible
– software safeguard prevents misuse of data system