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Part 3 Chromatography
In 1903 M.S. Tswett described a technique for the separation of plant
pigments. He called this technique “chromatography" (derived from
the Greek word which means colour writing). Today chromatography
encompasses a diverse group of methods, which permit the separation,
isolation, and identification of the components in a mixture. These
separation methods include paper chromatography (PC), thin layer
chromatography (TLC), gas chromatography (GC) and liquid
Basically, chromatography is a physical separation technique, which
resolves the individual components of a mixture based on their
distribution between two immiscible phases:
a- Stationary phase (adsorbent). It is a solid porous media, which
consists of the rigid porous particles, usually silica based, with the
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specific surface properties (surface chemistry). It is non-moving and
may exist in a variety of forms (e.g., bed, layer, column).
B- Mobile phase (eluent): It is a liquid solvent or mixture of solvents, which
is moving through the stationary phase or chromatographic column
and carrying analytes. It may be either a gas (i.e., GC) or a liquid (i.e.
The chromatographic process occurs as the result of the repeated
sorption-desorption of the sample components as they move along the
stationary phase. When the individual component favors the stationary
phase, it is held longer and moves more slowly through the column. If
the velocities and hence the distribution coefficients of samples
components are different, the mixture could be resolved. The
distribution coefficient (Ka) is defined as:
𝐾𝑎 = Concentration of solute in stationary phase
Concentration of solute in mobile phase
The oldest form of liquid chromatography is the column
chromatography. In classical column chromatography the columns
were open tubes (e.g., burettes), which were individually packed with
coarse material. The mixture to be separated is loaded onto the top of
the column followed by more solvent. Different components in the
sample mixture pass through the column at different rates due to
differences in their partitioning behavior between the mobile phase
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and the stationary phase. Compounds are separated by collecting
aliquots of the column effluent as a function of time. Flow of the mobile
phase was achieved by gravity feeding; and components were
collected as colored fractions (Fig.1).
In the 1960's researchers began to look for ways to improve liquid
chromatography and developed high performance liquid
chromatography (HPLC). HPLC: means high performance liquid
chromatography, which refers to high speed, high-resolution
separation. Initially, pressure was selected as the principal criterion of
modern liquid chromatography and thus the name was "high pressure
liquid chromatography" or HPLC. This was, however, an unfortunate
term because it seems to indicate that the improved performance is
primarily due to the high pressure. This is not true because naturally,
pressure is needed only to permit a given flow rate of the mobile phase;
otherwise, pressure is a negative factor not contributing to the
improvement in separation.
Figure 1 Schematic of
a simple liquid
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Comparison between classical and modern LC:
Modern LC Classical LC
- Stainless steel columns
- Small diameter (2-5 mm). Reusable.
- Packing with very small (3, 5 and 10
mm) particle stationary phase.
Open tube ex: a burette
-Large diameter (1-4
- Prepared each time.
- Continuous development of new
substances to be used stationary
- Only few materials are
used as stationary phase
ex: silica and alumina.
2- Volume of sample:
- Precise sample introduction (in ul)
-Large amounts of
sample are loaded on
top of column.
4- Flow of mobile phase:
- Relatively high inlet pressures and
controlled flow of mobile phase.
-Flow of mobile phase is
achieved by specific
5- Detection of sample:
- Special continuous flow detectors
capable of handling small flow rates
and detecting very small amounts of
samples. Then a final chromatogram is
obtained with all the information data.
-Coloured samples are
washed out and
collected in fraction,
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6- Resolution of sample:
- High resolution.
- Resolution is not good.
- Analysis is very rapid (in few minutes).
- Analysis is very slow.
- Automated standardized instrument
- It is just a simple
II- Classification of chromatographic methods:
1- According to the states of the two phases:
Usually the mobile phase is named first
(a) Liquid-solid chromatography (LSC).
(b) Liquid-Liquid chromatography (LLC)
(c) Gas- solid chromatography (GSC).
(d) Gas- Liquid chromatography (GLC)
2-According to the shape of stationary bed:
(a) Plane or flat-bed chromatography: The stationary bed is
coated on a flat surface. The two common types of plane
chromatography are paper chromatography (PC) and thin
layer chromatography (TLC).
(b) Column chromatography: The stationary bed is contained in a
column. Examples are open-column chromatography (OCC),
gas chromatography (GC) and high pressure liquid
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(c) 3- According to the mechanism of chromatographic
According to the type of equilibration process involved between
the mobile and the stationary phases, chromatographic methods
are classified into:
(a) Adsorption chromatography:
This uses a solid stationary (like silica gel or any other silica based
packing) where the sample components are adsorbed. The mobile
phase may be a liquid (liquid-solid chromatography) or a gas (gas-solid
chromatography). The components distribute between the two phases
and the separation is based on repeated adsorption-desorption steps.
Equilibration between the
adsorbed state and the solution
accounts for the separation of
sample components, e.g. TLC,
GSC, and LSC. (Fig.2) Two
modes are defined depending
on the relative polarity of the
two phases (Fig.3). These are:
Fig. 2: Adsorption
1- The normal phase chromatography: in which the stationary bed
is strongly polar in nature (e.g., silica gel), and the mobile phase is
non polar (such as n-hexane or tetrahydrofuran). Polar samples
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are thus retained on the polar surface of the column packing
longer than less polar materials.
Fig.3: Graphical illustration of normal and reversed-phase
chromatography. Circles represent types of compounds present in
the sample; their relative position to direction of mobile phase flow
indicates their order of elution.
2- The reversed-phase chromatography: which is the inverse of normal
phase. The stationary bed is non polar (hydrophobic) in nature,
while the mobile phase is a polar liquid, such as mixtures of water and
methanol or acetonitrile.
Here the more non polar the material is, the longer it will be retained.
(b) Partition chromatography:
-In which the stationary phase is a liquid supported on an inert solid. The
mobile phase may be a liquid (liquid-liquid chromatography) or a gas