Partition Chromatography

By-
Dr. Ekta Khare
 Principle
• Like other forms of chromatography, partition chromatography is based on
differences in retention factor, k, and distribution coefficients, Kd, of the analytes
using liquid stationary and mobile phases.
• It can be subdivided into:
– liquid–liquid chromatography, in which the liquid stationary phase is attached to
a supporting matrix by purely physical means, and
– bonded-phase liquid chromatography, in which the stationary phase is
covalently attached to the matrix.
• An example of liquid–liquid chromatography is one in which a water stationary
phase is supported by a cellulose, starch or silica matrix, all of which have the ability
to physically bind as much as 50% (w/v) water and remain free-flowing powders.
• The advantages of this form of chromatography are that it is cheap, has a high
capacity and has broad selectivity.
• Its disadvantage is that the elution process may gradually remove the stationary
phase, thereby altering the chromatographic conditions.
• This problem is overcome by the use of bonded phases and this explains their more
widespread use. Most bonded phases use silica as the matrix, which is derivatised to
immobilise the stationary phase by reaction with an organochlorosilane.
 Normal-phase liquid chromatography
• In this form of partition chromatography, the stationary phase is polar and the
mobile phase relatively non-polar.
• The most popular stationary phase is an alkylamine bonded to silica.
• The mobile phase is generally an organic solvent such as hexane, heptane,
dichloromethane or ethyl acetate.
• These solvents form an elutropic series based on their polarity.
• Such a series in order of increasing polarity is as follows:

• The order of elution of analytes is such that the least polar is eluted first and
the most polar last.
• Indeed, polar analytes generally require gradient elution with a mobile phase
of increasing polarity, generally achieved by the use of methanol or dioxane.
• The main applications of normal-phase liquid chromatography are its use to
separate analytes that have low water solubility and those that are not
amenable to reversedphase liquid chromatography.
 Reversed-phase liquid chromatography
• In this form of liquid chromatography, which has many similarities with
hydrophobic interaction chromatography, the stationary phase is non-polar
and the mobile phase relatively polar, hence the name reversed-phase.
• By far the most commonly used type is the bonded-phase form, in which
alkylsilane groups are chemically attached to silica. Butyl (C4), octyl (C8) and
octadecyl (C18) silane groups are most commonly used.
• The mobile phase is commonly water or aqueous buffers, methanol,
acetonitrile or tetrahydrofuran, or mixtures of them.
• The organic solvent is referred to as an organic modifier.
• Reversed-phase liquid chromatography differs from most other forms of
chromatography in that the stationary phase is essentially inert and only
non-polar (hydrophobic) interactions are possible with analytes.
• Reversed-phase HPLC is probably the most widely used form of
chromatography mainly because of its flexibility and high resolution.
• It is widely used to analyse drugs and their metabolites, insecticide and
pesticide residues, and amino acids, peptides and proteins.
 Ion-pair reversed-phase liquid
chromatography
• Although the separation of some highly polar analytes, such as amino acids, peptides,
organic acids and the catecholamines, is not possible by reversed-phase
chromatography, it is sometimes possible to achieve such separations by one of two
approaches:
• Ion suppression: The ionisation of the analytes is suppressed by using a mobile phase
with an appropriately high or low pH thus giving the molecules greater hydrophobic
character.
• For weak acid analytes, for example, an acidified mobile phase would be used.
• Ion-pairing: A counter ion that has a charge opposite to that of the analytes to be
separated is added to the mobile phase so that the resulting ion-pair has sufficient
hydrophobic, lipophilic character to be retained by the non-polar stationary phase of a
reversed-phase system.
• Octyl- and octadecylsilane-bonded phases are used most commonly in conjunction
with a water/methanol or water/acetonitrile mobile phase.
• One of the advantages of ionpair reversed-phase chromatography is that if the sample
to be resolved contains a mixture of non-ionic and ionic analytes, the two groups can
be separated simultaneously because the ion-pair reagent does not affect the
chromatography of the non-ionic species.
 Chiral chromatography
• This form of chromatography allows mixtures of
enantiomers (mirror image forms, denoted either
as D or L or as S or R) to be resolved.
• One of these techniques is based on the fact that
diastereoisomers, which are optical isomers that
do not have an object–image relationship, have
different physical properties even though they
contain identical functional groups.
• They can therefore be separated by conventional
chromatographic techniques, most commonly
reversed-phase chromatography.