Partition Chromatography

• Based on a thin film of a

liquid stationary phase
coated on the surface of a
solid support .
• Mobile phase could be
either a liquid or a gas.
• Solute equilibrates
between the mobile phase
and the stationary liquid.
• Partition - based on the
relative solubility of
analyte in mobile and
stationary phases 1
 Partition Chromatography
 If both the stationary and the mobile phase are liquid, then the
partition chromatography is also known as Liquid-Liquid
Chromatography (LLC).

 If the mobile phrase is gas then it is called Gas-liquid

Chromatography (GLC).

 In LLC the two phase are liquids. The two liquids must be
immiscible.

 The stationary liquid is present as thin film on an inert solid

support

 Separation occurs due to the difference in partition coefficients

of solutes between the two liquids.
 Relative solubility of cpds in liquid stationary and mobile phases
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 Selection of the solid support
The support material should:
 adsorb & retain the liquid stationary phase.
 be mechanically stable.
 be easy to pack.
 not retard the solvent flow
 expose as large surface as possible to the mobile
phase

Examples of solid supports:

Silica gel, diatomaceous earth (as kieselguhr, celite etc.)
& cellulose.
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 Examples of supports:
The following materials supports the aqueous
stationary phase by hydrogen bonding.

1. Filter paper.
2. Cellulose powder.
3. Silica gel.

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 Selection of the mobile phase
 The liquid stationary & mobile phases should have a
considerable
 difference between their solvent strength
parameters.
 Pure water > Methanol > Ethanol > Propanol >
Acetone > Ethyl acetate> Ether > Chloroform >
Dichloromethane >Benzene > Toluene > Carbon
tetrachloride > Cyclohexane > Hexane > Pentane.

e.g. if the stationary phase is water (polar),

pentane/hexane (non-polar) would be the
eluent/mobile phase of choice.
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The mobile phase is usually
 saturated with the stationary phase to
overcome "stripping" (washing of the
stationary phase from the column by the
mobile phase).

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 Examples of Partition Chromatography
1. Reversed phase silica gel Chromatography:
 Silica gel is modified to become RP silica gel
 non-polar liquid stationary phase and
 the mobile phase is the more polar liquid in this case.

 Reversed phase silica gel is an example of the stationary

phases in this type such as
 RP18 (C18) / RP 8 (C8).

 Partition Chromatography is better for the

 separation of more polar solutes that may not easily
eluted from the adsorption type chromatography.
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 Reversed phase silica gel (RP)
RP are non-polar
A straight chain aliphatic groups are attached to the Si
of silica gel (by replacing OH group) by silylation.
RP silica gel are named according to the length of the
carbon chains (RP18 or C18; RP8 or C8)

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 2. Paper Chromatography
 It is an example of a partition chromatography
 Liquid-Liquid chromatography
 Cellulose fibers strips serve as inert solid support.

 The factor governing separation of mixtures of solutes on

filter paper is the partition between two immiscible
phases.
 One is usually water adsorbed on cellulose fibers in the
paper (stationary phase).

 The second is the organic solvent flows past the sample on

the paper (stationary phase). 9
 Paper Chromatography

 Partition occurs between the

 mobile phase and
 the stationary aqueous phase bound on the cellulose.

 The isolation depends on partition coefficient of the

solute.
 Ideal method for sugar analysis (Rhamnose, glucose etc)

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Paper Chromatography

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 Gel Permeation Chromatography (GPC)

This type is also known as:

Size Exclusion Chromatography (SEC)
Molecular Exclusion Chromatography (MEC)
Molecular Sieve Chromatography (MSC)
Gel Filtration Chromatography (GFC)
Gel Chromatography

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 Molecular Exclusion Chromatography

 Separation based
on size
 Molecular weight
 Small molecules get
trapped in pores &
take longer to get
out

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 Facts about Gel Chromatography
 The pores of the beads are of a controlled size and
this regulates which molecule can enter the beads.
 The larger molecules will be excluded from the beads
and will flow through the column faster than the
smaller molecules, which experience a much larger
volume.
 In practice, there exists a range of pore sizes, and
molecules are separated by their sizes (and shapes):
the largest are eluted first, and the smallest elute last.
Commonly Used Gel Filtration Materials
 Stationary phase
 Porous polymeric matrix: formed of spongy particles,
with pores completely filled with the liquid mobile phase
(gel).

 The gels (polymers) consist of open, three-dimensional

networks formed by cross-linking of long polymeric
chains.

 The pore size varies with the degree of cross-linking.

 The diameter of the pores is critical as separation is

based on that molecules above certain size are totally
excluded from the pores because they can not enter the
gel.

 The interior of the pores is reached, partially or wholly,

by smaller molecules.
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 Mobile phase
 Mobile phase is a liquid usually water or dilute
alcohol or chloroform (combination of these)

 Separation mechanism
 Based on difference between the solutes molecular
weights.
 The two mixture should have at least 10% molecular
weight difference

 Molecules will distribute themselves outside & inside

the pores according to their size.

 Larger are excluded, medium sized enter half-way &

smallest permeate all the way.
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The retention volume Vo
 is the total volume of the mobile phase
required to elute molecule from the column
 Of a substance is inversely proportional to
the molecular weight (M. Wt) of the solute.

Vo ~ 1 / M.wt
Vo = retention volume
M.wt = Molecular Weight

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 Applications of GPC to natural products
 Determination of M. wt. of peptides, proteins &
polysaccharides.

 Separation of mixture of mono- & polysaccharides.

 Separation of amino acids from peptides & proteins.

 Separation of proteins of different molecular weights.

 Separation of mucopolysaccharides & soluble RNA.

 Separation of myoglobin & haemoglobin.

 Separation of alkaloids & purification of enzymes.

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 Ion Exchange Chromatography
 Separation of either cations
or anions
 Separation based on relative
strength of ionic bond
 It works for ionic
compounds

 Used in LC exclusively

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 Ion Exchange Chromatography
Principle
 Process by which ions of an electrolyte solution are brought into
contact with an ion exchange resin.
 The ion exchange resin is
 an insoluble polymer consisting of a "matrix" (Lattice or framework)
that carries fixed charges (not exchangeable) and mobile active ions
"counter ions" which are loosely attached to the matrix.
 e.g. a cation exchanger in the free carboxylic acid form:
 X-COO- H+
X = frame/matrix; coo- =fixed charge ; H+ = active ion
 In water, the counter-ions move more or less freely in the framework
& can be replaced by ions of the same sign present in the surrounding
solution.
 The "matrix" (framework) of a "cation exchanger" is considered as a
crystalline non-ionized "polyanion" & the matrix of an "anion
exchanger" as a non-ionized "polycation". 23
Molecular formulas of cellulose based ion
exchangers.
Some common ion exchangers
 Types of Exchangers
 Ion Exchangers
These are either
(I) Cation exchangers or
(II) Anion exchangers of either organic or inorganic
nature.
 Inorganic ion exchangers
Common clays, soils, minerals e.g. zeolites used for
"softening water".
 Disadvantage:
 low ion-exchange capacity.
 Advantages:
 Great resistance to high temperatures.
 High volume capacity.
 Great selectivity towards simple inorganic ions.
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 Cation Exchangers

 Active ions (counter ions) are cations.

 The polar groups attached to the matrix are acidic
(sulphonic acids, carboxylic acids, phenols, phosphoric
acids)
 e.g. a cation exchanger in the free carboxylic acid form:
 X-COO- H+
 X = Frame work (matrix)
 -COO- = Fixed charge (anionic), Non-exchangeable
 H+ = Counter ion (cation), Exchangeable

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 Cation Exchangers

 They are usually (but not always) supplied in the Na+

form: X-COO-Na+

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 Anion Exchangers
 Active ions (counter ions) are anions.
 The polar groups attached to the matrix are
tertiary or quaternary ammonium groups (basic).
 e.g. Anion exchanger in the quaternary
ammonium form:

 X- NR3+OH –
 X = Framework (matrix)
 -NR3+ = Fixed charge (cationic), Non exchangeable
 -OH– = counter ion (anion), Exchangeable
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 Anion Exchangers
 They are supplied as the chloride rather than
the hydroxide as the chloride form is a more
stable. Represented as: X-NR3+Cl -

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 Regeneration of the resin
 Ion exchange process is generally reversible
e.g in the following:

 2 Na+ + Ca++ 2Cl - → Ca++ + 2 Na+ Cl –

 The cation exchanger could be exhausted after

exchanging all Na+ for Ca++, the exchanger
could be regenerated (loaded again with Na+)
by contacting it with excess Na+ ions e.g. a
solution of NaCl.
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 Organic exchangers
 They may be natural or synthetic.
 Preparation of organic synthetic ion exchangers :
 Polycondensation of phenols, sulpho- & carboxy-
derivatives with formaldehyde → cationic
exchangers.
 Polycondensation of aromatic amines with
formaldehyde → anionic exchangers.
 These techniques yield products linear in structure
& relatively soluble in water which are now
replaced by resin materials based on styrene
divinyl benzene copolymers and polyacrylate.
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Applications of Ion Exchange Chromatography
1- Water softening:
Removal of Ca2+, Mg2+ & other multivalent ions causing
hardness of water by filtration through a layer of strong
cation resin.
2-Water demineralization:
Removal of cations & anions dissolved in water. Usually
carried by the two step technique in which two columns
of strongly acid cation exchanger in [H+] form & strongly
basic anion exchanger in [OH-] form are used in
sequence.
3- Neutralization:
Cationic exchanger in [H+] can be used to neutralize
alkali hydroxide & anionic exchanger in [OH+] form to
neutralize the acidity.
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4- Separation of electrolytes from non-electrolytes.

5- Separation of carbohydrates & their derivatives:

 Uronic acids separated on anion exchanger.
 Sugars converted into ionized form by using
borate& separated on strong anion exchanger.
 Hexosamines separated on strong cation
exchanger.

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 Separating Ions by Ion Exchange
• Ion exchange resin
－ high molecular weight polymers
－ large numbers of an ionic functional group
－ cation exchange resin
－ anion exchange resin

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 Affinity Chromatography

 Very selective
 Specific binding site is used to
concentrate analyte on column
 Unique biological specificity of
the sample and the ligand
interaction is utilized for the
separation
 Used a lot in biological
applications

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37
Affinity Chromatography

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 Electrophoresis
Electrophoresis
Method for separating substances on the basis
of their tendency to migrate to a positively or
negatively charged electrode at a particular pH.
Separation of aas by electrophoresis (separation of
charged compounds in an electric field) is possible
because all aas have different isoelectric points
 pI (isoelectric point)
pI > pH (acidic solution) +ve  cathode
pI < pH (basic solution)  -ve  anode
 Isoelectric Points
• In acidic solution, the carboxylate and amine are in their
conjugate acid forms, an overall cation
• In basic solution, the groups are in their base forms, an
overall anion
• In neutral solution cation and anion forms are present
The isoelectric point (pI)
“is the pH at which the molecule has an average net
charge of zero and therefore does not migrate in an
electric field”
– Consider a mixture of alanine (pI=6.01), lysine
(pI=9.74), and aspartic acid (pI=2.77) in a buffer
solution at pH 6
 Gel Electrophoresis
Separation based on
size and charge
Smaller molecules
will migrate further,
less tangled

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