Gas Chromatography

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Gas Chromatography
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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

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Principles

• Separation due to differences in partitioning
  behavior
• selective retardation

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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

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Gas Chromatography

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

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Schematic of a gas Chromatograph
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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

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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

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Uses of Gas Chromatography

• Determination of volatile compounds (gases
  liquids)
• Determination of partition coefficients and
  absorption isotherms
• Isolating pure components from complex mixtures

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Instrumentation
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Instrumentation

• flowing mobile phase
• injection port
• separation column
• detector

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GC detectorsanother powerpoint
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Liquid Chromatography much slower diffusion in
liquid as compared to gas
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Liquid liquid extraction repeated extraction is
basis for LC
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Retardation of solutes in liquid onto a solid
phase
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Elution 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)

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Types 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)

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Types 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

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Retardation based on size of molecule as it
diffuses into porous solid
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High Performance Liquid Chromatography

• Once called High Pressure Liquid Chromatography

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Outline

• What is HPLC?
• An Overview
• Types of HPLC
• Partition Chromatography
• Adsorption Chromatography
• Ion Chromatography
• Size-Exclusion Chromatography

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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

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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

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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

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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

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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

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Liquid chromatography

• Instrumentation
• Mobile Phase Reservoir
• Pumping Systems
• Sample Injection Systems
• Liquid-Chromatographic Columns
• Detectors

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Schematic of liquid chromatograph
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LC column
LC injector
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Types of HPLC

• Liquid-solid (adsorption) chromatography
• Liquid-liquid (partition) chromatography
• Ion-exchange chromatography
• Size exclusion chromatography

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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

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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

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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

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Adsorption Chromatography

• Classic
• Solvent Selection
• Non-polar Isomeric Mixtures
• Advantages/ Disadvantages
• Applications

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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

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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

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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

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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

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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 Cellulose Cation Exchanger
DEAE Cellulose Anion Exchanger
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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

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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

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SEC(continued)

• 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

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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

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Advantages of Size Exclusion Chromatography

• Short well-defined separation times
• Narrow bands--gt 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

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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

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Instrumentation

• Instruments required
• Mobile phase reservoir
• Pump
• Injector
• Column
• Detector
• Data system

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Schematic of liquid chromatograph
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Mobile phase reservoir

• Glass/stainless steel reservoir
• Removal of dissolved gases by degassers
• vacuum pumping system
• heating/stirring of solvents
• sparging
• vacuum filtration

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Elution methods

• Isocratic elution
• single solvent of constant composition
• Gradient elution
• 2 or more solvents of differing polarity used

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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

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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

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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

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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)

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LC column
LC injector
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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

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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

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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

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Some Properties of Detector

• Adequate sensitivity
• Stability and reproducibility
• Wide linear dynamic range
• Short response time
• Minimum volume for reducing zone broadening

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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

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Types of Detector

• Refractive index
• UV/Visible
• Fluorescence
• Conductivity
• Evaporative light scattering
• Electrochemical

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Refractive Index I

• Measure displacement of beam with respect to
  photosensitive surface of dectector

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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

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Refractive Index III

• Disadvantages
• expensive
• highly temperature sensitive
• moderate sensitivity
• cannot be used with gradient elution

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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

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UV/Visible II
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UV/Visible III

• Advantages
• high sensitivity
• small sample volume required
• linearity over wide concentration ranges
• can be used with gradient elution

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UV/Visible IV

• Disadvantage
• does not work with compounds that do not absorb
  light at this wavelength region

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Fluorescence I

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

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Fluorescence II

• Advantages
• extremely high sensitivity
• high selectivity
• Disadvantage
• may not yield linear response over wide range of
  concentrations

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Conductivity

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

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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

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Evaporative Light Scattering II
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Electrochemical I

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

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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

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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

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Electrophoresischarged species migrate in
electric fieldSeparation based on charge or
mobility
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Capillary electrophoresishigher voltages can be
used as the heat can be dissipated
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Capillary electrophoresis
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The End