Chromatography

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Chromatography

• Organic Chemistry Laboratory
• By Joseph Martin Mikula III

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Chromatography Basics

• Definition- Any of various laboratory techniques
  used to separate mixtures of substances into
  their components. It may be used for analysis or
  for purification of organic compounds.

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Chromatography Basics

• Stationary Phase- The liquid or solid that stay
  doesn't move.
• Mobile Phase- The liquid or gas that moves and
  contains the components to be separated.
• The mobile phase carries the components through
  the stationary phase and the components separate
  based on the different affinities of substances
  as they travel through the stationary phase.

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Theory of Chromatography

• Stationary Phase silica, aluminium oxide,
  various synthetic resins
• Mobile Phase
• organic liquid (solvent),
• helium gas

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Organic Analysis

• Qualitative procedures determine the identity
  the compounds in the sample.
• Quantitative procedures determine how much of
  each component is in the sample.

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Types of Chromatography

• Thin Layer Chromatography
• Column Chromatography
• Gas Chromatography
• Ion Exchange Chromatography
• Size Exclusion Chromatography
• Affinity Chromatography
• Paper Chromatography

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Column Chromatography

• Separates mixtures based off of polarity
• The stationary phase is silica gel or alumina
• The mobile phase is a solvent like methylene
  chloride, ethyl acetate, or hexanes.
• Polar molecules interact with the silca gel and
  move slowly through the column, while nonpolar
  and slightly polar molecules move faster.
• These different interactions with the stationary
  phase cause the molecules to elute at different
  rates.
• Polar molecules will elute faster with a polar
  solvent such as ethyl acetate.

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Column Chromatography
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Column Chromatography

• Procedure First Part-Isolating Sample
• Put two tea bags in 50mL of distilled water and
  boil
• After two minutes at a boil turn off heat and
  allow to cool
• Remove the tea bags and squeeze them out
• Add 20mL of 5 HCl
• Extract solution with 4 10mL increments of
  methylene chloride (don't shake, but swirl)
• Add sodium sulfate and filter into a 100mL round
  bottom flask
• Add 0.2 grams silica gel and evaporate

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Column Chromatography

• Procedure Second Part-Building Column
• Obtain 10 x 150-mm column and 0.7g of silica gel
• Put glass wool in bottom of column
• Add 0.5cm of sand
• Add silica gel
• Add sample
• Add 0.5cm of sand again
• each layer should be gently tapped to get an
  even layer

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Column Chromatography

• Procedure Third Part-Running Column
• Add 3 5mL increments of methylene chlride trying
  not to disturb column
• Add 2 5mL increments of ethyl acetate
• Collect 5mL increments from bottom of column in
  test tubes
• Run thin layer chromatography to find which
  increment contains caffeine against known
  caffeine
• Take 5mL increments with caffeine and evaporate
• Record mass and find melting point

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Gas Chromatography

• Separates molecules by vaporizing them into gas
  phase, which then travel through tubing
  containing a stationary phase at different rates.
• The mobile phase is typically helium.
• The stationary phase is a high boiling point
  liquid absorbed onto a solid-solid diatomaceous
  earth and liquid usually a waxy polymer.
• How fast a particular compound travels through
  the column will depend on how much of its time is
  spent moving with the gas as opposed to being
  attached to the liquid in some way.

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Gas Chromatography

• The time it takes a molecule to come out the end
  of the tube is called the retention time.
• High solubility and high boiling points lead to
  longer retention times.
• High column temperature leads to shorter
  retention times and less separation, while low
  column temperature give longer retention times,
  but better separation.

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Gas Chromatography

• Chromatograph - The printed record of the
  separation.
• May be used for both qualitative and quantitative
  analysis.

Retention Time - The time required for a
component to emerge from the column from the time
of injection.
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Gas Chromatography
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Gas Chromatography
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Chromatograms
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GC/MS
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Mass Spectrometry

• A mass spectrometer ionizes the compound by
  knocking an electron out of compound.
• This gives the compound an overall positive
  charge and it will fragment into other charged
  positively charged ions.
• In a mass spectrum graph the most stable ion
  fragment is the most abundance and appears as the
  tallest peak- called the base ion peak
• The other ion fragments are recorded as relative
  abundance to the base ion peak.

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Mass Spectrometry

• Also, on the graph the peak with the largest mass
  value is the unfragmented compound and therefore
  equal to the molecular weight.
• It is called the parent ion peak.

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Mass Spectrometry
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Mass Spectrometry
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Thin Layer Chromatography

• Uses a silica plate glass plate with a thin
  layer of silica on it.
• Silica is the stationary phase and is very
  polar.
• A solvent is the mobile phase.
• How well the molecule dissolves in the solvent
  and affinity to the silica determines the
  distance it will travel up the plate.

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Thin Layer Chromatography

• Origin line - the location of the initial
  placement of a spot of the compound (marked in
  pencil).
• Solvent front - how far the solvent moved up the
  plate.
• Spots will be between solvent front and origin
  line. If colorless, they can be revealed with uv
  light (requires fluorescence in silica) or iodine
  vapor.
• The retardation factor (Rf) can be calculated to
  quantify the distance traveled by a compound.

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Thin Layer Chromatography
TLC is only qualitative, not quantitative.
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TLC Chromatogram

• Rf value distance component traveled
• distance solvent traveled
• Rf value for Quinine 0.4
• Rf value for Heroin 0.8

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Thin Layer Chromatography

• Procedure
• Dissolve 10mg of four compounds in a few drops
  methanol in a test tube
• Take 10mg of an unknown and dissolve it in a few
  drops methanol in a test tube
• Obtain 2-3 TLC plates and draw a line in pencil
  1cm up from an end on each (no pen why?)- This is
  the origin line
• Put a drop from an open ended capillary tube onto
  the penciled line creating lanes
• A technique to use is place a drop let it dry
  then put another drop in same spot let it dry and
  repeat several times
• Put TLC plate in chamber with solvent (make sure
  solvent level is not higher than origin line
• Pull out of chamber when solvent front is 0.5cm
  from top and immediately mark
• Allow plate to dry and put it in iodine chamber
  for 30 seconds and then expose to uv light
  circling the spots that appear
• Calculate Rf values and deduce compounds or
  compound in unknown