Liquid Chromatography

(Ion Exchange, Size

Exclusion and Affinity)

CDB 3093: ANALYTICAL CHEMISTRY

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 Classification of Chromatography
Techniques

Completed

Completed

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 Ion- Exchange Chromatography (IEC)
 Separation is based on exchange of ions in the
stationary phase.

 Well suited for separation of inorganic ions, both

cations and anions, separation of amino acids.

 The stationary phase (packed)  resin (beads made

of a polystyrene polymer crosslinked with
divinylbenzene).

 Mobile phase aqueous with organic or inorganic

buffer.
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  Separate cation (M+)  Cation exchangers contain
covalently bound, negatively charged sites that
attract solute cations.

 Separate anion (A-)Anion exchangers, positively

charged groups on the stationary phase attract
solute anion.

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 Principles of Separation

More highly charged

molecules are more tightly
bound to the resin, and so
travel slowly and are eluted
later

Moderately charged molecules

equilibrating between the resin
and the moving buffer more
readily

Less charged molecules

bind less strongly to the resin,
equilibrate with the moving
buffer more readily, and so
travel rapidly and are eluted
sooner

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 Ion Exchanger (Stationary Phase)
 Polystyrene resin  copolymerization of styerene and
divinylbenzene.

 The benzene ring can be modified to produce:

i) cation-exchange resin, containing sulfonate group
(-SO3-) or
ii) anion-exchange resin-containing ammonium group
(-NR3+).

 Ion exchanger are classified as strongly or weakly acidic

or basic:
i. Strongly acidic cation exchanger : RSO 3-
ii. Weakly acidic cation exchanger : RCO2-
iii. Strongly basic anion exchanger : RNR’ 3+
iv. Weakly basic anion exchanger : RNR’ 2H+
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 Structures of styrene-divinlybenzene cross-linked
ion-exchange resins

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 Ion-Exchange Resin

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 Examples of IEC Chromatogram

Separation of cations with a cationic column

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 Separation of anions with an anionic column
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 Size-Exclusion Chromatography (SEC)
 Also called gel filtration or gel permeation
chromatography.

 Gel Filtration mobile phase is water.

 Gel Permeation mobile phase is an organic solvent.

 Molecules are separated according to their size.

 Small molecules penetrate the small pores in the

stationary phase, but large molecules do not.

 Widely used for determination of molecular weight

distribution of polymers.
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 Principles of Separation

Very small molecules enter

many pores in the gel,
equilibrating between the gel
and the moving buffer, and so
travel slowly and are eluted later

Medium sized molecules enter

some pores in the gel,
equilibrating between the gel
and the moving buffer

Large molecules enter few

pores in the gel, and so travel
rapidly and are eluted sooner

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 Stationary Phase - Type of Gel
 Common type of SEC gel: Sephadex, Bio-Gel,
Sephacry and Sepharose.

 The smallest pore size (highly cross-linked gels)

exclude molecules with a molecular weight >700.

 Largest pore sizes exclude molecules with molecular

weights >108.

 The finer the particle size of the gel, the greater the
resolution and the slower the flow rate of the column.

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 Types of Gels

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 Molecular Weight Determination
 Gel Filtration and Gel Permeation  mainly used to
separate molecules of significantly different molecular
weight.

 Estimation of the molecular weight of an unknown by

comparing its elution time/volume with those
standard.

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 Calibration graph for polystyrene using different
molecular weight standard

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 Chromatogram: Separation of proteins by size
exclusion chromatography

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 Affinity Chromatography (AFC)

 Based on specific binding of that one compound to the

stationary phase.

 Only one solute is bound when the sample passed

through the column.

 The adhering solute is eluted by changing a condition

such as pH or ionic strength to weaken its binding.

 Applicable in biochemistry and is based on specific

interactions between enzymes and substrates,
antibodies and antigens, or receptors and hormones.

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 Principles of Separation
The enzyme binds to the
immobilized substrate; all other
proteins that do not bind the
substrate are eluted in the void
volume of the column

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 Example: Isolation of Immunoglobin G (IgG) by
Affinity Chromatography on a Column
Containing Protein A
Stationary
Phase
(Bounded
Protein A)

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 Absorbance at 280 nm

 Protein A binds to one specific region of IgG at pH~ 7.2.

 When a crude mixture containing IgG and other proteins

was passed through the column at pH 7.2, everything
except IgG was eluted within 0.3 min.

 At 1 min, the eluent pH was lowered to 2.6 and IgG was

cleanly eluted at 1.3 min.
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 Example: Combination of Affinity Chromatography
and Size Exclusion Chromatography

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 End of Liquid Chromatography

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