Chromatography:
 Chromatographic techniques are now widely used for the separation,
identification, and
determination of the chemical components in complex mixtures.
It can separate gases, and volatile substances, as well as, nonvolatile and polymeric material
including biological substances.
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.
The stationary phase may be a solid, or a liquid supported on a solid or gel,
the mobile phase may be either a gas or a liquid.
Explanatory theories
Chromatographic separations could be explained based on one of the following two
theories:
The plate theory:
•
Views column as divided into a number (N) of adjacent imaginary segments called
theoretical plates.
•
In each of these plates equilibrium is achieved (distribution of solute between the
stationary and mobile phases).
•
The number of theoretical plates, n, is useful for description of the "column efficiency".
The plate theory:
The improve in column efficiency results in better resolution (separation). This could be
achieved by:
•
Increasing the length (L) of the column, which in most cases is not practical.
•
Increasing the number of theoretical plates (n) per unit length of the column. This is
effected by decreasing the space occupied by each theoretical plate that is a function of
its height (H or HETP) and is calculated as follows:
H=L/n
•
Where: H = Height equivalent to a theoretical plate,
•
L = Length of the column,
•
n = Total number of theoretical plate (could be increased by decreasing adsorbent
particle size).
•
Thus, decreasing HETP (H) results in better resolution (i.e. better column
performance).
The rate theory
 The behavior of the molecules of a component of the mixture to be separated within a
chromatographic system is not uniform. It is affected by factors, which deviate the
process from ideality by increasing the value of H and results in band-broadening (i.e.
no sharp peak).
The Rate Theory of Chromatography
•
It involves a more realistic description of the processes of work inside a column. It takes
account of the time taken for the solute to equilibrate between the stationary and
mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast).
•
The resulting band shape of a chromatographic peak is also affected by the rate of
elution.
•
It is also affected by the different paths available to solute molecules as they travel
between particles of stationary phase. If we consider the various mechanisms which
contribute to band broadening, we arrive at the Van Deemter equation for plate height
•
The Van Deemter equation relates column separation efficiency to the bandbroadening factors as a function of the linear flow velocity (v) of the mobile phase:
H = A n1/3 + B/n + C n
where:
H is HETP (remember want a minimum!)
n is mobile phase velocity
Theoretical basis of chromatography
These theoretical concepts are applicable to all types of chromatographic
techniques.
I- Distribution equilibrium 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 equilibrium and is expressed by the rate of
travel.
•
•
•
1-Distribution equilibrium
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
•
C mobile = concentration of solute in the mobile phase
•
The K value determines the relative population in the two phases i.e. large K value
means that more time is spent in the stationary phase, and vice versa if the K value is
small the solute will be eluted very fast with 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 value of the distribution coefficient that is characteristic for each component.


Determination of the rate of travel could be performed by:
 1-Measuring the distance traveled by each solute after a fixed time as in planar
chromatography.
 2-Measuring the time interval at which each component appears after a fixed distance
as detected in columnar chromatography
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.
1-Adsorption Chromatography
It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a
stationary solid phase. Solutes equilibrate between the mobile phase (liquid or gas)
and the stationary phase.
2-Partition Chromatography
This is based on a thin film formed on the surface of a solid inert support by a
liquid stationary phase. Solutes equilibrate between the mobile phase (liquid or gas)
and the stationary liquid.
3-Ion Exchange Chromatography (IEC)
In this type of chromatography, an ion-exchange resin, with covalently attached
anions or cations, is used as stationary solid phase. Solute ions of the opposite charge in
the mobile liquid phase are attracted to the resin by electrostatic forces.
4-Molecular Exclusion Chromatography ( Gel permeation or Gel filtration)
The mobile phase (liquid or gas) passes through a porous gel, which separates
the molecules according to their size. The pores are 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.
ii-According to the method of holding the stationary phase:
In this respect chromatographic techniques are classified into two groups.
1-Planar or Plane chromatography
In this type, the stationary phase is presented as a layer, which may be a specific
type of paper or a layer of adsorbent spread on glass, plastic or aluminum sheets.
2-Columnar or Column chromatography
In this type, the stationary phase is held into a column that may be made of glass or
metal.
iii-According to the purpose of use
1-Analytical chromatography
This term is applied, when the process is applied with the aim of identification
(qualitative chromatography) or determination of the amounts (quantitative
chromatography) of the sample components.
2-Preparative chromatography
This refers to the use of the chromatographic procedure with the aim of isolation of
the sample components.
Adsorption Chromatography
Adsorption is a surface phenomenon, when a sample of a mixture is applied to the
stationary phase,( here adsorbent);
an interaction occurs between the active groups of the sample components
(adsorptives) and the adsorbent.
In general, on a polar support, more polar molecules will be more strongly
adsorbed and therefore will elute more slowly from the column. The strength of
adsorption of polar groups (solutes) on polar support increases in the following order:
•
-C=C- < O-CH3 < -COOR < C=O
•
Olefins < ethers < esters < ketones < aldehydes < amines < phenols < acids.
< -CHO
< -NH2 < -OH < - COOH
Several chromatographic techniques are based on adsorption e.g. Liquid Solid
Chromatography (LSC) commonly known as Adsorption Column Chromatography, Gas
Solid Chromatography (GSC); others can operate under certain conditions by adsorption
e.g. Thin Layer Chromatography (TLC), Paper Chromatography (PC).
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
Different substances are used as adsorbing materials e.g. alumina, kaolin,
magnesium oxide, calcium carbonate, charcoal, and silica.
Alumina
•
It is a good adsorbent for most substances as it has a large capacity, is insoluble,
relatively inert, and available.
•
It is used for separation of sterols, dyestuffs, vitamins, esters, alkaloids and inorganic
compounds.
•
It is not used for phenolics or compounds containing carboxylic acid groups.
Silica, Silica gel
This is one of the most widely used adsorbents in both column and thin-layer
chromatography.
The adsorptive properties depend on the hydroxyl groups attached to silicon atoms,
which interact with polar or unsaturated molecules, by hydrogen bonding.
Structure of silica gel (top panel) and reverse phase silica gel (octadecylsilane,
lower panel) polymers.
The strength of the bonding will depend upon the nature of the components in the
sample.

As the mobile phase flows over the stationary phase, those weakly adsorbed
components on the stationary phase will migrate with the solvent front.

While strongly adsorbed components will move slowly.


Activation of silica
Heating silica between 150 °C and 250 °C removes the physically adsorbed water,
without loss of surface hydroxyls. This treatment gives an adsorbent of maximum
activity.
Silica samples, which are not heated above 150 °C, or which are exposed to water
vapor after activation, have variable amounts of physically adsorbed water, which
renders the adsorbent less active.
They thus act by a partition mechanism
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.
-In case of using silica as adsorbent
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
According to their function in the chromatographic process solvents are
classified into:
1-Solvents
These are almost inert towards the adsorbent and adsorptive.
They are used to bring the mixture to be separated on to the column.
2- Developers
These are stronger than the solvents.
They cause the movement of the zones in the chromatogram by competition with the
adsorptive for the stationary phase.
3- Eluents
These have a better eluting power than developers and help to remove the
adsorptive outside the column. They are still slightly less adsorbed than or even equally
adsorbed to the adsorptive.
4- Displacers
These are strong mobile phases. They are more strongly adsorbed than the
adsorptive, causing its displacement and expelling it from the adsorbent to the mobile
phase.
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.
5-Solvent flow rate
When the particle size is sufficiently small, a uniform and low flow rate will result in a
more satisfactory separation and more uniform zone formation than a fast flow rate.
Equipment and Procedure
Equipment
 The equipment consists of a vertical glass tube somewhat constricted at the bottom
end, a small plug of glass wool is introduced at the base of the tube and the column is
packed with an adsorbent.
 A fraction collector supplied with a series of receivers of suitable volume is usually used
to collect the fractions.
Procedure
The steps involved to perform column chromatography include:

packing of the column with the stationary phase (adsorbent),

application of the sample (loading),

development of the chromatogram and

detection of the sample components (Fig 1).
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
Application of the sample (Loading)
 Three techniques could be adopted

1- The sample could be dissolved in the smallest possible volume of the first
solvent and then introduced onto the column to form an initial zone at the top of the
column.

2- If the sample is not soluble in the first solvent, it is thoroughly mixed with
small amount of the adsorbent (or of inert support in case of partition chromatography)
in a small beaker to produce a friable powder, which is carefully introduced to the top of
the column.

3- Sometimes, the dissolved sample-mixture is repeatedly applied to a paper disc.
The excess solvent is allowed to drain down the column and the discs are placed on the
just dry surface.
Development of the chromatogram
•
On addition of the mobile phase, the components of the initial zone move along the
column and are adsorbed at different distances from the top depending on their relative
adsorption to the stationary phase.
•
Upon drying, the series of separated bands obtained is called the chromatogram, which
can be removed from the column as a single unit, the individual zones separated and
analyzed.
•
Detection of the components of the sample
•
1-On-column detection:
•
This is done by examination of the separated zones on the column in either visible
light (for colored substances) or in UV light.
•
2-Out-of-column detection:
•
This is carried by collecting the eluate in fractions from the whole flowing column,
followed by testing each of the eluted fractions for the presence of the mixture
components. This can be carried by using thin layer or paper chromatography (TLC or
PC).
Applications
Adsorption Column Chromatography has been used for:
•
Separation of alkaloids of Cinchona, and nux vomica, and cardiac glycosides from
Digitalis and anthraquinones from senna species.
•
Isolation and purification of vitamins and hormones.
•
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 counter-current 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).
•
The mobile phase may be gas, (Gas Liquid Chromatography, GLC) or liquid (Liquid Liquid
Chromatography, LLC)
•
The method is now largely replaced by the more sophisticated technique known as High
Pressure Liquid Chromatography or High Performance Liquid Chromatography (HPLC).
The support used is of smaller particle size and the process is carried under pressure,
thus increasing speed and resolution, and reducing solvent consumption
Paper Chromatography
It is the method of choice for the fractionation of some groups of substances, e.g.
flavonoids
Principle and mode of action
•
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.
•
The sample components are detected as circular or oval spots on the paper.
•
The degree of retention of each solute is expressed as the retardation factor (Rf) or rate
of flow.
•
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 well-closed 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".
Modification
The nature of the paper may be modified by impregnation with:
•
Hydrophilic substances e.g. formamide and hanged to dry before use for separation of
relatively polar compounds.
•
Hydrophobic substances, e.g. paraffin oil, vaseline ... etc, and in this case the stationary
phase is rendered lipophilic. Development is carried with a hydrophilic (polar) mobile
phase. These are known as "reversed phase papers" and are used for separation of
lipids.
Equipment
•
Micropipettes (capillary glass tubes with narrow bores) are used for sample application.
•
Hair dryers or an infra red lamps may be used to decrease the drying time after sample
application.
•
Airtight chromatographic chambers or jars are used for developing the chromatogram.
•
Glass sprayers filled with specific reagents are generally used for location of the spots on
the chromatogram.
Procedure
Steps followed to perform paper chromatography are:
1-Sample desalting
•
The presence of inorganic salts generally causes streaking (tailing, trailing), 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.
•
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 methods.
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:
Physical methods:
These mostly depend on the behavior of the sample components in visible and UV
light by comparison to known standards.
•
Colored substances are visible in ordinary light.
•
UV light absorbance for fluorescent substances.
•
•
Chemical methods:
•
These depend on the reaction of the substances with a certain reagents, giving
colored, black, or fluorescent spots.
•
The reagent is usually applied either as a spray or by immersion of the
chromatogram in the reagent.
•
After the application of the reagent, the chromatogram is often dried in the drying
cabinet.
Examples of reagents
•
Iodine solution or vapor or modified Dragendorff ’s reagent for alkaloids.
•
Ferric chloride for phenols.
•
Ninhydrin for amino acids.
•
Aluminum chloride for flavonoids.
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.
Advantages of TLC over PC
•
The method is more versatile, it allows the use of a 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.
Equipment and application of the layer
•
TLC is carried with materials available in most laboratories.
•
Small chromatoplates as microscopic slides, 2.5 × 7 cm, are used as glass support. The
adsorbent layers are applied in the form of suspension generally by dipping
•
Any small available container e.g. a well-covered beaker is used as a developing
chamber.
•
For adsorption chromatography, the adsorbent layers are air-dried for 10-20 min then
placed vertically in a well-ventilated oven and activated by heating to 110-120 °C for 1-2
hr.
•
For partition chromatography, the chromatogram is air-dried and the residual water
acts as the stationary phase.
Mobile phase (Solvent)
•
A wide variety of solvents including benzene, chloroform, ether, ethyl acetate and
methanol are used for development, single or in mixtures.
•
They must be pure i.e. of analytical grade.
Development of the chromatogram
•
The chromatoplates are usually developed once by the ascending technique at room
temperature.
•
Detection (Visualization)
•
1-Many substances are visible on thin layers of adsorbents without the use of any
special detection method.
•
2-Most of reagents employed in paper chromatography are also applicable to TLC.
•
3-Corrosive spray reagents can be used, e.g. acids or alkalis.
•
4-Methods of detection are 10-100 times more sensitive on thin layer than on paper
chromatograms.
•
5-Iodine vapor and concentrated sulfuric acid are used as general reagents for organic
compounds
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.
Gas Chromatography (GC)
Principle
 In this type of chromatography, the mobile phase is a gas.
 substance to be analyzed is vaporized and introduced into the moving gas. It is carried
through the column and distributed between the carrier gas and the stationary phase.
 Pure separated components come out at the end of the column, the appearance of each
is detected and registered as a peak on a recorder chart.
Techniques
According to the nature of the stationary phase two techniques are
distinguished:
Gas-liquid chromatography (GLC)
 Here, the stationary phase is liquid and occurs as a thin film that is adhering to an inert
finely divided solid support. Separation is due to partition of the solutes molecules
between the two phases.
Gas-solid chromatography (GSC)
The stationary phase is here an active solid adsorbent such as alumina, silica or
carbon. Migration of the components is based on their relative adsorption to the
stationary phase.
Applications
 Examples in the field of medicinal plants include the examination of volatile oils, and
other volatile constituents.
 Glossary of Chromatographic terms

Activation: The process of heating an adsorbent layer to drive off moisture and
convert it to its most attractive or retentive state.

Chromatogram: The series of zones on or in the column, layer or paper after
development.

Chromatographic system: The combination of the solvent, sorbent, and the
sample mixture.
Effluent: the liquid that passes out of the column.
Eluate: The part of the effluent that contains the eluted substances.
Eluents: Liquids that have a better eluting power than developers and help to
remove the adsorptive outside.
Elution : Removal of adsorbed solutes from stationary phase using eluent.