Principle and Definition
Principle
• The principle of chromatographic separation is very simple.
The process is achieved by distributing the substances to
be separated between a mobile phase and a stationary
phase.
• Those substances distributed preferentially in the moving
(mobile) phase pass through the chromatographic system
faster than those that are distributed preferentially in the
stationary phase.
Theoretical basis of chromatography
These theoretical concepts are applicable to all types of
chromatographic techniques.
I- Distribution equilibria and rate of travel
•
During chromatography a given solute finds itself either in the
stationary phase, which acts as a "retarder" or in the mobile
phase, which acts as a "carrier".
•
This distribution is based on distribution equilibria and is
expressed by the rate of travel.
1-Distribution equilibria
The distribution of solutes between the two phases is governed
by an equilibrium constant known as the distribution coefficient,
K (or partition coefficient in certain types of chromatography).
This allows quantifying the distribution of a compound between
the stationary and mobile phases:
K = Cstationary / Cmobile
C stationary = concentration of solute in the stationary
phase
Cmobile = concentration of solute in the mobile phase
2-Rate of travel
Factors limiting the rate of travel:
The rate of travel of a solute in a chromatographic system is limited
by:
•
The velocity of the mobile phase (or carrier), that is the same for
all components.
•
The ratio of volume of the stationary phase to the volume of the
mobile phase, that is also the same for all components.
•
The value of the distribution coefficient that is characteristic for
each component.
Classification of chromatographic techniques
Different systems of classification may be adopted relying on
criteria such as the separation mechanisms, the development
procedure, the method of holding the stationary phase.
i-According to the mechanism responsible for differential
migration:
The components will migrate through the system at different
rates based on competition between the stationary and mobile
phase for the solute molecules. The major types are:
1-Adsorption Chromatography
2-Partition Chromatography
3-Ion Exchange Chromatography (IEC)
4-Molecular Exclusion Chromatography ( Gel permeation or Gel
filtration)
5-Affinity Chromatography
Adsorption Column Chromatography
Stationary phase
Selection of the adsorbent
The ideal adsorbent should conform to the following
specifications:
•
It should be insoluble in the solvent used as mobile phase.
•
It should be inert to the adsorptives (solutes), unless
otherwise required.
•
It should be colorless, especially when zones which contain
colored substances are to be located visually.
•
It should have a suitable particle size with great surface area
to allow more efficient adsorption; but, not too fine to avoid
slowing of the rate of percolation.
Types of adsorbents and applications of each
type.
Mobile phase
Selection of the solvent
The choice of the suitable solvent (s) depends mainly on
the elution power of the solvent and the relative
adsorption of the class of compounds, which are to be
separated.
•
Strong eluents (more polar solvents) decrease
adsorption, while weak eluents (less polar) increase it.
•
Elution of the column can, therefore, be carried out with
solvents of increasing polarity. A typical series is as
follows:
•
hexane < cyclohexane < benzene < chloroform <
diethylether < ethyl acetate < acetone < ethanol <
methanol < pure water.
Types of solvents as mobile phases in
adsorption chromatography.
Factors affecting column efficiency and chromatographic
separation
1- Particle size of the supporting medium:
As the particle size of adsorbent decreases, separation is
improved. However, an adsorbent of very small particle size will
offer considerable resistance to flow.
2-Column dimensions:
The column efficiency is improved as the
length / width ratio of the column is increased.
3-Uniformity of packing of the column:
If the column is not packed uniformly, uneven and irregular
movement of the solvent front and less uniform zone formation
will result.
4-Column temperature:
As temperature is increased, the elution speeds up as the
adsorption is generally reduced at higher temperatures.
Packing of the column
This is carried by either the wet or dry packing techniques.
Wet packing:
•
A suspension (slurry) of the adsorbent, preferably the first
solvent to be used in the separation, is prepared and gradually
added to the column. The packing is allowed to settle between
additions.
•
The suspending liquid is allowed to flow out slowly but the
liquid level in the column is maintained above the packing at all
times.
Wet packing is most commonly used and results in more
homogenous columns.
Dry packing:
•
In this method, the solid stationary phase is added in
portions with vibrations between additions. The process is
repeated until the column is adequately filled.
•
The column is then washed carefully with the first solvent
•
•
•
•
Sample application
Development
Elution
Detection.
Applications
Adsorption Column Chromatography has been used for:
•
Separation of alkaloids of Cinchona, ergot, opium and nux
vomica, and cardiac glycosides from Digitalis and
anthraquinones from senna species.
•
Isolation and purification of vitamins and hormones.
•
Examination of vegetable oil and pharmaceutical
preparations.
•
Purification of tincture of alkaloids from pigments before
determination of their alkaloidal content.
Partition Chromatography
The separation of the components of a mixture is, as in countercurrent extraction, dependent on differences in the partition
coefficients of the components between an aqueous and an
immiscible organic liquid.
The liquid stationary phase is adsorbed on an inert support, which
may be either packed in a chromatographic tube (Column Partition
Chromatography) or layered on a glass plate (TLC) or in the form of
sheets of paper (PC).
Paper Chromatography
The technique was introduced as a method of partition
chromatography for the analysis of amino acid mixtures and has
extended to all classes of natural products. It remains the
method of choice for the fractionation of some groups of
substances, e.g. flavonoids
Principle and mode of action
• It is a type of planar chromatography in which the stationary
phase is a specified type of paper.
• The paper is formed of cellulose, which has a great affinity for
water and other polar solvents and holds them through formation
of hydrogen bonds thus forming a polar stationary phase.
Distribution of the solutes between the two phases is therefore
mainly by partition.
Criteria for ideal PC separation
Efficient separation is achieved by:
1-Using analytical grade chemicals and distilled water for
preparation of the mobile phase (solvent-system).
2-Maintaining the composition of the mobile phase
constant during development by using a sufficient
amount of solvent and carrying the process in a wellclosed chamber (chromatographic jar).
3-Maintaining a suitable and constant temperature through out
the process.
4-Selecting the suitable solvent in which the components of the
analyzed sample have a low but definite solubility. Highly
soluble substances will appear at or very close to the solvent
"front". Substances of very low solubility will remain near the
point of application or " start line".
Procedure
Steps followed to perform paper chromatography are:
1-Sample desalting
• The presence of inorganic salts generally causes streaking
(tailing, trailing), discoloration and other distortions on the
finished chromatogram.
• The removal of these salts can be achieved by ion exchange or
precipitation.
2-Sample application (Loading)
• Spots are applied with micropipettes, at a suitable distance
from each other, on the start line. They are kept as small as
possible (less than 5 mm in diameter).
• In case of repeated applications, spots should be completely
dried after each application.
• When large quantities of material are to be chromatographed,
the sample is applied as a streak (e.g. in preparative paper
chromatography (PPC) and quantitative chromatography).
3-Development of the chromatogram
Several techniques may be adopted. These include the
usual (or general) techniques such as the ascending,
descending, radial and horizontal methods. Special
techniques have also been devised in order to
improve resolution, examples are the stepwise and
multiple development, the continuous development
and the two-dimensional development.
4-Drying of the chromatogram
After development, the paper is withdrawn from the jar. The
front of the solvent is marked immediately with a pencil and
dried by hanging in open air at room temperature or by a
current of cold or hot air (using a hair dryer).
5-Spot detection (location or visualization)
Several methods are used for detection of the separated spots
corresponding to the components of the analyzed sample.
They include:
Thin Layer Chromatography (TLC)
• This is simple and fast and has now achieved a remarkable
success in the separation of mixtures of all classes of natural
products.
• Thin layer chromatograms often serve to identify drugs, plant
extracts and biochemical preparations, or to detect
contaminants or adulterants.
Analogy with Paper Chromatography
• Such as paper chromatography TLC is a type of planar
chromatography.
• Instead of paper (in PC) a thin layer of finely divided adsorbent
supported on a glass, plastic or aluminum sheet is used.
Analogy with Column Chromatography
• TLC is also closely related to column chromatography and has
often been called "Open-column chromatography".
Both techniques are based on the same mechanisms of
chromatographic separation: adsorption and partition.
• The major difference lies in the location of the stationary phase.
In TLC, it is a thin layer spread on a glass plate, and in column
chromatography, it is a column held in a glass tube.
Advantages of TLC over PC
• The method is more versatile, it allows the use of wide range of
stationary phases and solvents.
• Fractionation is performed more rapidly, with smaller quantities of
the mixture and is more reproducible.
• The separated spots are usually more compact and more clearly
demarcated from one another.
• Concentrated sulfuric acid and other drastic reagents can be used
for location of separated substance but not for the paper.
Applications
• Like with paper chromatography, TLC can be extended to,
quantitative evaluation and to work that involves radioactive
substances.
• Quantitative analysis can be performed by measuring the size of
the spots with a planimeter or measuring the intensity of the
color by photodensitometry. An alternative method is to scrap
the bands and elute the components with the suitable solvent
followed by colorimetric, spectrophotometric, fluorimetric,
gravimetric or volumetric methods.