Nanotechnology: From Microelectronics to Health Care

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Food Analysis Lecture 20 (4/8/2005)
Basic Principles of Chromatography (3)
Qingrong Huang Department of Food Science Read
Material Chapter 27, page 437 Final Exam April
29

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Chromatography

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Physicochemical Principles of Separation

• Adsorption (solid-liquid) chromatography
  oldest, Tsvet in 1903
• The stationary phase is a finely divided solid
  (to maximize the
• surface area),
• The mobile phase can be either gas or liquid
• The stationary phase (adsorbent) is chosen to
  permit differential
• Interaction with the components of the sample to
  be resolved.
• The interaction forces include
• - Van der Waals forces
• - Electrostatic forces
• - Hydrogen bonds
• - Hydrophobic interactions
• Typical stationary phases silica (slightly
  acidic), alumina (slightly
• Basic), charcoal (nonpolar).

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Mechanism of AC

• The solute and solvent molecules are competing
  for active sites
• on the stationary phase.
• As the relative adsorption of the mobile phase
  increases, adsorption
• of the solute must decrease.
• A solvent strength (or polarity) scale is called
  an eluotropic series.

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Ion-Exchange Chromatography

• Ion-exchange Chromatography a
  separation/purification process
• occurring naturally, e.g. in soils, and is
  utilized in water softeners and
• deionization. Three types of separation
• Ionic from nonionic
• Cationic from anionic
• Mixtures of similarly charged species.
• Similar to adsorption chromatography
• nature of interactions - electrostatic
• Cationic exchangers contain covalently bound
  negatively charged
• functional groups.
• Anionic exchangers contain bound positively
  charged groups.

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Ion-Exchange Chromatography (2)

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Ion-Exchange Chromatography (3)

• The strongly acidic sulfonic acid moieties
  (RSO3-) of strong cation
• exchangers are completely ionized at pHgt2
• Strongly basic quarternary amine group (RNR3)
  on strong anion
• exchangers are ionized at pHlt10
• Since maximum negative or positive charge is
  maintained over a broad
• pH range, the exchange or binding capacity of
  these stationary phases is
• essentially constant
• Weak cation exchangers contain RCOO-, their
  exchange capacity
• varies considerably between 4-10
• Weak anion exchangers contain RNR2, which
  are deprotonated in
• moderately basic solution, thereby losing their
  positive charge and the
• ability to bind anions
• One way to elute solutes is to change the mobile
  phase pH a second way
• is to increase the ionic strength (e.g. use of
  NaCl) of the mobile phase, to
• Weaken the electrostatic interactions.

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Ion-Exchange Chromatography (4)

• Factors that govern selectivity ionic valence,
  radius, concentration,
• the nature of the exchanger, the composition and
  pH of the mobile
• Phase
• Materials used must be ionic and highly
  permeable
• - Crosslinked Polyelectrolytes like polystyrene
  crosslinked with divinyl
• benzene
• Polysaccharide-based, like cellulose, dextran, or
  agarose for separation
• and purification of large molecules, such as
  proteins and nuclei acids, with
• the advantage of being able to derivatized with
  strong or with weakly acidic
• or basic groups via OH moieties on the
  polysaccharide backbone.

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Size-Exclusion Chromatography (SEC)

• SEC, also known as Gel Permeation Chromatography
  (GPC), can
• be used for the resolution of macromolecules,
  such as proteins and
• carbohydrates, as well as for the fractionation
  and characterization
• of synthetic polymers
• Ideal SEC, separated based on size, no
  interaction occur between
• Solutes and the stationary phase
• Column void volume (V0) the volume of the
  mobile phase in the
• Column, measured by running a very large
  molecules, e.g MW2x106
• Total permeation volume (Vt)-column void volume
  V0 the volume
• of liquid inside the sorbent pores Vi measured
  by running a low MW
• (e.g.glycyltyrosine)

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Size-Exclusion Chromatography (SEC)

• The available partition coefficient Kav
• Ve elution volume of solute
• SEC packing materials hydrophobic media like
  crosslinked PS and
• Hydrophilic gels like polysaccharide-based
  packings (Fig. 27-5a).

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Chromatography

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Affinity Chromatography

• Affinity Chromatography separation is based on
  the specific, reversible
• interaction between a solute molecule and a
  ligand immobilized on the
• chromatographic stationary phase.
• The ultimate extension of adsorption
  chromatography
• Involved biological materials as the stationary
  phase, including antibodies,
• enzyme inhibitors,

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A The support presents the immobilized ligand
to the analyte To be isolated B The analyte
makes contact with the ligand and attaches
itself C The analyte is recovered by the
introduction of an eluent, which dissociates the
complex Holding the analyte to the ligand D
The support is regenerated, ready for the next
isolation.