ChE 427 NOVEL TOPICS in SEPARATION PROCESSES Chp 3: Chromatography

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ChE 427NOVEL TOPICS in SEPARATION
PROCESSESChp 3 Chromatography
Instructor Prof. Dr. Hayrettin YücelAssistant
Ms. Hale Ay
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OUTLINE

• INTRODUCTION TO CHROMATOGRAPHY
• CHROMATOGRAPHIC MECHANISM
• Surface adsorption
• Partition
• Ion exchange
• Size exclusion
• CHROMATOGRAPHIC TECHNIQUES
• a. GAS CHROMATOGRAPHY
• b. THIN LAYER CHROMATOGRAPHY
• c AFFINITY CHROMATOGRAPHY
• 4 METHOD OF OPERATION
• Batch Elution Chromatography
• Continuous Rotating Annular Chromatograph
• Moving-bed Chromatography

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CHROMATOGRAPHY

• TWO IMPORTANT PART EXIST
• Flowing liquid or gas stream The mobile
  phase
• Fixed, non-moving substance Stationary phase
• The moving of the solute
• from mobile phase Stationary phase
• is called sorption

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Components that are weakly retained by stationary
phase will move through the system more rapidly
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• The substance which make up stationary phase is
  called sorbent
• Solid, a liquid supported on or bonded to a
  solid, or a gel.

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Types of Chromatography
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ADVANTAGES

• Capable of separating complex mixtures at low
  operating temperatureS
• Large scale batch or continuous operation
  possible
• Capable of separating materials according to size
  and/or chemical properties
• Separation can be achieved by a variety of
  methods
• Very pure products can be recovered

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DISADVANTAGES

• Irreversible adsorption of materials creates
  problems
• To achieve efficient separations, high operating
  pressures may be required.
• Over-loading of columns with feed material may
  cause incomplete separation
• Feed material is diluted by flowing mobile phase
• Non-uniform column packing can lead to a
  significant decrease in separation performance
• Pre-filtration of feed material is usually
  required
• Periodic column re-packing / regeneration
  required

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2. Chromatographic Mechanisms

• Chromatographic techniques are based on four
  different sorption mechanisms
• Surface adsorption
• Partition
• Ion exchange
• Size exclusion

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Surface Adsorption Chromatography

• Depends upon differences in polarity between the
  different feed components
• The more polar a molecule, the more strongly it
  will be adsorbed by a polar stationary phase

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Surface Adsorption Chromatography

• The choice of stationary phase is governed by the
  polarity of the feed components.
• If the feed components are adsorbed too strongly,
  they may be difficult to remove.
• Weakly polar mixtures should be separated on
  highly active absorbents, or little or no
  separation will occur.

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Surface Adsorption Chromatography

• The choice of mobile phase is equally important.
• Good separation is achieved by using fairly polar
  stationary phases and low polarity mobile phases
  such as hexane.
• Water a very polar solvent.

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Surface Adsorption Chromatography

• The 2 most common adsorbents used in
  chromatography are porous alumina and porous
  silica gel
• Preferred for the separation of components that
  are weakly or moderately polar

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Surface Adsorption Chromatography

• Silica gel is less polar than alumina and is an
  acidic adsorbent
• Thus preferentially retaining basic compounds
• Carbon is a non-polar (apolar) stationary phase
  with the highest attraction for larger non-polar
  molecules

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• Surface adsorption
• Partition
• Ion exchange
• Size exclusion

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Partition Chromatography

• The stationary liquid phase is coated onto a
  solid support such as silica gel, cellulose
  powder, or kieselguhr (hydrated silica).
• Assuming that there is no adsorption by the solid
  support, the feed components move through the
  system at rates determined by their relative
  solubilities in the stationary and mobile phases.

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Partition Chromatography

• It is not necessary for the stationary and mobile
  phases to be totally immiscible
• Hydrophilic stationary phase liquids are
  generally used in conjunction with hydrophobic
  mobile phases

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• Surface adsorption
• Partition
• Ion exchange
• Size exclusion

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Ion Exchange Chromatography (IEC)

• The stationary phase consists of an insoluble
  porous resinous material containing fixed
  charge-carrying groups.
• The degree of affinity between the stationary
  phase and feed ions dictates the rate of
  migration and hence degree of separation between
  the different solute species.

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Ion Exchange Chromatography (IEC)

• Most widely used type of stationary phase is a
  synthetic copolymer of styrene and divinyl
  benzene (DVB), produced as very small beads in
  the micrometer range
• The choice of a particular resin will very much
  be dependent upon a given application.
• Cation () or anion (-) exchange properties can
  be introduced by chemical modification of the
  resin.

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Ion Exchange Chromatography (IEC)

• This technique is used in the separation of
  transition metals, the removal of trace metals
  from industrial effluents
• In the purification of a wide range of organic
  compounds and pharmaceuticals.
• The resin matrix is usually relatively
  inexpensive when compared with other types of
  stationary phase.
• The most widely used large-scale chromatographic
  process, but is limited to ionisable, water
  soluble molecules.

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• Surface adsorption
• Partition
• Ion exchange
• Size exclusion

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Size Exclusion Chromatography (SEC)

• Also known as gel permeation chromatography
• Molecules of a feed material are separated
  according to their size or molecular weight.
• The stationary phase consists of a porous
  cross-linked polymeric gel.

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Size Exclusion Chromatography (SEC)

• Large molecules tend to be excluded by the
  smaller pores and move preferentially with the
  mobile phase
• smaller molecules are able to diffuse into and
  out of the smaller pores and will thus be
  retarded in the system.
• The very smallest molecules will permeate the gel
  pores to the greatest extent and will thus be
  most retarded by the system

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Size Exclusion Chromatography (SEC)

• The degree of cross-linking can be varied to
  produce beads with a range of pore sizes to
  fractionate samples over different molecular
  weight ranges
• SEC is used extensively in the biochemical
  industry to remove small molecules and inorganic
  salts from valuable higher molecular weight
  products such as peptides, proteins and enzymes.

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• Surface adsorption Polarity
• Partition Solubility
• Ion exchange Affinity
• Size exclusion Size/ MW

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3. CHROMATOGRAPHIC TECHNIQUES

• All sorbents can be used in columns
• In packed columns ID 1-mm
• In capillary columns ID 0.5-mm
• wall-coated open tubular (WCOT)
• support-coated open tubular (SCOT)
• porous-layer open tubular (PLOT)

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CHROMATOGRAPHIC TECHNIQUES

• typically made of stainless steel or quartz
• Sorbents can also be applied to sheets of glass,
  plastic, or aluminum for thin layer or planar
  chromatography
• sheet of cellulose material for use in paper
  chromatography

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3.a. GAS CHROMATOGRAPHY

• mobile phase is a gas
• comprises gas-solid chromatography (GSC) and
  gas-liquid chromatography (GLC)
• In GSC the sorption process is one of adsorption
• In GLC, the stationary phase is a liquid, thus
  partitioning is the predominant sorption process.
• Gas chromatography is very much a
  laboratory-scale process
• used to analyze rather than separate materials

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GAS CHROMATOGRAPHY (GC)

• A pulse of feed material entering the system is
  first rapidly heated so that it is vaporised,
  before being carried by the mobile phase gas
  (usually an inert gas such as nitrogen) into the
  column
• Separation occurs due to differences in affinity
  between the feed components and the stationary
  phase material

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3.b THIN LAYER CHROMATOGRAPHY (TLC)

• In thin layer chromatography samples are
  separated based on the interaction between a thin
  layer of adsorbent and a selected solvent.
• separation process occurs on a flat, essentially
  two-dimensional surface
• stationary phase is usually a polar solid, such
  as silica gel or alumina

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THIN LAYER CHROMATOGRAPHY (TLC)

• separation process is surface adsorption
• TLC is primarily an analytical tool

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THIN LAYER CHROMATOGRAPHY (TLC)

• The mixture to be separated is added as a spot
  close to the base of the plate which is then
  placed in a shallow through of mobile phase.
• The mobile phase rises up the plate by
  adsorption, thus contacting the spot sample and
  its feed components.

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3.c AFFINITY CHROMATOGRAPHY

• based on the surface adsorption
• primarily used to separate valuable biochemical
  materials

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AFFINITY CHROMATOGRAPHY

• Ligand and ligate
• the interaction between a ligate and its ligand
  should be reversible
• Agarose gels and cross-linked agarose gels
• proteins, enzymes, antibodies, antigens and
  nucleic acids

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4. Types of Methods of Operation

• Batch Elution Chromatography
• Continuous Rotating Annular Chromatography
• Moving-bed Chromatography

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4.a Batch Elution Chromatography

• Most common large-scale batch chromatography
• A scaled-up version of an analytical
  chromatograph

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Batch Elution Chromatographyfor separating a
binary mixture
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Batch Elution Chromatography

• Recycled solvent or carrier gas is fed
  continuously to the column
• The effluent is sent to the different separators
• Each one separate a particular feed
• The carrier gas is purified and sent to the
  column

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Types of Methods of Operation

• Batch Elution Chromatography
• Continuous Rotating Annular Chromatograph
• Moving-bed Chromatography

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4.b Continuous Rotating Annular Chromatograph

• Stationary phase is located in the annular space
• 2-cm wide and 30-cm in diameter
• Mobile phase (also known as eluant) is introduced
  uniformly to all points of the annular stationary
  phase at the top of the column
• The column is rotated about its axis at a speed
  of 1 - 2 revolutions per hours.

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Continuous Rotating Annular Chromatograph
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Continuous Rotating Annular Chromatograph

• The more strongly interacting component will pass
  to the stationary phase.
• The strongly retained component moves
  horizontally.

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Types of Methods of Operation

• Batch Elution Chromatography
• Continuous Rotating Annular Chromatograph
• Moving-bed Chromatography

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4.C Moving-bed Chromatography

• The feed is introduced into the mid-point of a
  column
• The "stationary" phase is flowing down the column
  
• The mobile phase material is flowing up the
  column

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Moving-bed Chromatography

• Component
• whichhave a
• greater affinity
• for the mobile
• phase
• More strongly
• Retained
• component

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Types of Methods of Operation

• Batch Elution Chromatography
• Continuous Rotating Annular Chromatograph
• Moving-bed Chromatography