Chromatography
Definition
• It is a physical method of separation, in
which the components of a sample
mixture (solutes, analytes or fractions) are
separated by their differential
distribution between two phases (a
stationery phase and a mobile phase).
Basic Concepts
• During the chromatographic process, the
mobile phase carries the sample through a
layer or column containing the stationery
phase.
• As the mobile phase flows past the
stationery phase, the solutes distribute
between the two phases.
• The distribution depends on the relative
tendency (affinity) of the analytes to
associate with one of the two phases.
 The analytes with lower affinity to the
stationery phase reside in the mobile
phase, travel faster, and separate from
analytes having greater affinity to the
stationery phase.
• Elution
* Strongly bound analytes are subsequently
displaced from the stationery phase by
changing the physical or chemical nature of the
mobile phase.
* This is called elution and the received analyte
is called eluent or fraction.
Forms of Chromatography
planar
Paper
column
TLC
(HPTLC)
GC
LC
(HPLC)
In column chromatography:
- The stationery phase is coated onto the inner
surface or packed in a column.
- Depending on the nature of the mobile phase
column chromatography is either:
a- Liquid chromatography (LC): if the mobile
phase is a liquid.
b- Gas chromatography (GC): if the mobile phase
is a gas.
• In LC and GC the eluent exits from the column
and passes through a detector system that
produces a series of electronic signals that are
displayed graphically as a series of peaks
referred to as chromatographic peaks =
chromatogram.
Chromatography process
(see animation 1)***
• The peaks are described in terms of :
peak width, peak height, and peak area.
-- The retention time is the time taken by the
analyte to exit the column and pass through the
detector. It is more or less specific for a certain
analyte.
• Thus the data represented by the
chromatogram are used to:
a- Identify the analyte (through the
retension time). This is by reference to a
standard.
b- Quantify the analyte (through the peak
area).
PLANAR CHROMATOGRAPHY
Mechanisms of Chromatography
• Chromatographic separations are classified
according the chemical or physical
mechanisms used to separate analytes into:
1- Ion Exchange Chromatography.
2- Partition Chromatography.
3- Size exclusion Chromatography.
4- Affinity Chromatography.
5- Adsorption Chromatography.
Ion Exchange Chromatography
• In this type of chromatography, the use of
a resin (the stationary solid phase) is used
to covalently attach anions or cations onto
it. Solute ions of the opposite charge in the
mobile liquid phase are attracted to the
resin by electrostatic forces.
• Thus solutes are separated by the
differences in the signs and magnitude of
their ionic charges.
Requirements
• Stationery phase having fixed functional
cationic or anionic groups.
• Counter-ions placed in close proximity of
the fixed ion to maintain electrochemical
neutrality.
----------------------------------Solute ions in the mobile phase exchange
with counter-ions.
• Elution of solute ions is done by variation
of the mobile phase pH, ionic strength or
both.
• According to the type of charge of the
particle to be separated, ion exchange
chromatography can be:
a- Cation Exchange Chromatography :
in which the resin contains fixed
negatively charged groups eg.carboxylate
(CCOOH) or carboxy sulfonyl(CSO3H)
groups. It is used to separate cationic
analytes.
• b- Anion Exchange Chromatography :
Stationery phase contains (+) positively
charged basic quaternary amines eg. aminoethyl (AE) or triethyl-amino-ethyl (TEAE)
groups, to separate anionic solutes.
Clinical Applications of Ion Exchange
Chromatography
• Separation of amino acids. Ion exchange
chromatography is the principle of the amino
acid analyser.
• Separation of peptides, nucleotides and
nucleic acids.
• Preparation of de-ionized water. Deionizers
contain mixed beds (columns) of cation and
anion exchange resins.
Size Exclusion Chromatography
• - Also known as molecular exclusion
chromatography, gel permeation or gel filtration.
• - The liquid or gaseous phase passes through a
porous gel which separates the molecules
according to its size (shape and hydration).
• -The pores are normally small and exclude the
larger solute molecules, but allows smaller
molecules to enter the gel, causing them to flow
through a larger volume. This causes the larger
molecules to pass through the column at a faster
rate than the smaller ones.
• Disadvantage: poor reslution.
AFFINITY CHROMATOGRAPHY
ٍ
This is the most selective type of
chromatography employed. It utilizes the
specific interaction between one kind of solute
molecule and a second molecule that is
immobilized on a stationary phase. For
example, the immobilized molecule may be an
antibody to some specific protein.
Partition Chromatography
BASIS
The differential distribution of solutes
between two immiscible liquids is the basis
for separation by partition chromatography.
• One of the two immiscible liquids is the
stationery phase. For its preparation, a thin
film of a liquid is adsorbed or chemically
bonded to the surface of support particles.
• The mobile phase is another immiscible
liquid or gas.
Types of Partition Chromatography.
According to the mobile phase, partition
chromatography is categorized into:
GLC gaseous mobile phase
LLC liquid mobile phase
LLPC
Normal phase
Stationery phase is a
polar liquid.
Mobile phase is a
relatively non-polar
liquid
Reversed Phase
Stationery phase is
non-polar
liquid
Mobile phase is a
relatively
polar liquid
Used to separate lipids
and non-polar peptides
Adsorptive Chromatography
• Separation of components by this method depends
upon differences in the degree of adsorption of the
components by the adsorbant stationery phase.
• An adsorbent is a solid which has the property of
holding molecules at its surface, through hydrogen
bonding or electrostatic interactions.
• Technical problems (eg. impurities) and these
associated with reproducibility of solute has reduced
this mode in clinical labs.
PLANAR CHROMATOGRAPHY
Thin Layer Chromatography
* The solvent front is marked and a ruler is held next to the plate to allow •
calculation of Rf values.
* Rf = distance component travels
distance solvent travels
• TLC may be either partition or adsorptive.
• The medium supporting the stationery phase is
a thin layer of silica gel cellulose powder or
alumina,or saphedex.
• Prepared plates are available commercially.
• When coupled with densitometry TLC is
successfully useful to quantify drugs, lipids and
others from serum and amniotic fluid.
• Reverse phase and normal phase TLC are
available.
• When the thin layer consists of small
diameter (few micron) particles, the
technique is called HPTLC (more efficientbetter resolution).
Advantages of TLC
• 1- Ease separation and visualization of
compounds.
• 2- Capacity to analyze muliple samples in
a single run.
• 3-Relative low cost.