Chromatography

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Chromatography

• Chemistry 223
• Fall 2008

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Physical and Chemical Properties

• Density
• Boiling Point
• Melting Point
• Index of Refraction
• Solubility Polarity
• Viscosity
• Color, Odor, Phase

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Steps to Identify an Organic Compound

• Isolate compound
• From reaction mixture
• From natural source
• Purify compound
• Chromatography
• Physical Properties
• Identify pure compound
• Spectroscopy

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History of Chromatography

• 1903 Tswett - plant pigments separated on chalk
  columns (radial chromatography)
• 1931 Lederer Kuhn - LC of carotenoids
• 1938 TLC and ion exchange
• 1950 Reverse phase LC
• 1954 Martin Synge (Chemistry, 1952)
• 1959 Gel permeation
• 1965 Instrumental LC (Waters)

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Early Separation Techniques

• Liquid-Liquid extraction
• Two non-miscible liquids
• Like dissolves like
• Fractional recrystallization
• Fractional distillation
• Use distillation columns with high surface areas
  (glass helices, metal turnings, beads)
• Filtration methods
• Adsorption methods (activated charcoal)

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Liquid Chromatography

• The first separations were achieved by using
  gravity to drive the separations
• Gravity flow column chromatography
• Planar chromatography (TLC)
• Descending paper chromatography
• Radial paper chromatography
• Gravity separations could be done in two
  dimensions with planar methods

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Thin Layer Chromatography

• Process occurs on a thin (lt100 µm) of support
  placed on glass or aluminum sheets
• Sample spotted on silica, alumina, cellulose
  surface (1-10 µL)
• Plate placed in solvent to develop the plate,
  allowing the solvent to move by capillary action
• Plate dried and separated compounds visualized
  with specific reagents
• Coeluting samples can be resolved by using 2-d
  methods with two solvent systems

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Chromatographic Materials

• TLC silicic acid (H4SiO4), silica gel, alumina
  (Al2O3), cellulose
• Mess size important (5-400 mesh)
• Binders used to hold the silica gel on the
  substrate (aluminum, glass, plastic)
• Polymeric binders
• Calcium sulfate
• Plate thickness also important
• Thicker surfaces can be used preparatively to
  purify large amounts of an analyte

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Modern Techniques

• Gas-Liquid Chromatography
• Gas-Solid Chromatography
• Gravity Column Chromatography
• High Performance Chromatography
• Electrophoresis
• Gel, Paper, Cellulose acetate
• Capillary Electrophoresis

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Key Ingredient In

• Chromatography Based Instruments are essential
  to
• Genomics
• Capillary Electrophoresis
• Lipidomics
• GC-MS Systems
• Proteomics
• Gel, Capillary Electrophoresis
• Metabolomics

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Key Terminology

• Tswett separated plant pigments on chalk columns
  with a solvent mixture. He also used radial
  chromatography and coined the term chroma (color)
  graphy (writing)
• Column support
• Solvent eluent
• Fractions effluent fractions

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The Chromatographic Process

• Critical defining properties of the process
• Immiscible stationary and mobile phases
• Arrangement whereby a mixture is deposited at one
  end of the stationary phase
• Flow of the mobile phase toward the other end of
  the stationary phase
• Different rates or ratios of partitioning for
  each component of the mixture, and many cycles of
  this process during elution
• A means of visualizing bands of separated
  components on or adjacent to the stationary
  phase, or of detecting eluting bands as peaks in
  the mobile phase effluent

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Types of Chromatography
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Chromatographic Detectors

• Mass detectors (amount detectors) give signals
  proportional to the total amount of molecules
  exiting the column mass spectrometer total ion
  current (TIC)
• Concentration detectors measure the total number
  of molecules exiting the column in a given volume
  (µg/mL) at any given time. If the flow rate is
  faster, the peak will be narrower, if the flow
  rate slows, the peaks will be broader.
• The longer the residence time on the column the
  broader the peaks will be

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Rtx-5 Diphenyl Polysiloxane Stationary Phase
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GC Columns (1)

• GC first used metal columns (1/4 SS or copper)
  and packed columns with the liquid phase coating
  inert particles made of silica or crushed
  firebrick. Glass (race track, coiled ¼ inch
  columns) were used with packed column GC to avoid
  the active surfaces on the metal which caused
  oxygenated compounds to decompose.
• The next development was capillary columns (0.52
  inch OD) with the liquid phase (support) coating
  the inner surface (SCOT columns)

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GC Columns (2)

• Capillary columns have been created from 0.52 to
  0.22 in in diameter and lengths up to 100 meters.
  They are coated on the outside with a polyamide
  cladding to protect them from breaking. The
  inner surface has a chemically bonded liquid
  phase that prevents or reduces bleeding of the
  liquid phase at higher temperatures

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Packed Column GC Materials

• Early liquid phases were silicone stopcock grease
  (polar) and Apiezon (high vacuum grease) nonpolar
  with many silicone polymers in between.
• The support materials were substances like
  deactivated Kiesulghur (diatomaceous earth) and
  crushed firebrick. The liquid loading was from
  1-30 depending on the porosity of the materials.

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GC - Common Industrial Solvents
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Spectroscopy

• FT-Infrared Spectroscopy
• UV-Vis Spectrophotometry
• Proton NMR Spectrometry
• Carbon NMR Spectrometry
• Raman Spectrometry
• Mass Spectrometry