CHROMATOGRAPHY

Chromatography is a set of techniques for separating the components of a mixture based on the
rates at which they are carried through a stationary phase by a gaseous or liquid mobile phase.
The stationary phase consist of a solid or a liquid supported on a solid. The mobile phase flows
through the stationary phase and carries the components of the mixture with it.
Adsorption
When ions or molecules in the mobile phase approach the stationary phase, for example, a solid
surface, they may interact with atoms or molecules at the surface. This interaction is called
adsorption. When chemical bonds are formed between a molecule or ion and the surface, the
term chemisorption is used. When the forces of attraction between the molecules and the surface
are weaker, for example, Van der Waals forces, the term physical adsorption is used. The
difference between adsorption and absorption is that in adsorption, a substance is attached to the
surface, whereas, in absorption, it penetrates into the bulk. Many chromatographic techniques
involve adsorption of components from the mobile phase on to the stationary phase.
Partition
When a solute is added to a pair of immiscible liquids, it may dissolve in both of them. In this
case, the solute will distribute itself between the two solvents. It may well be more soluble in one
solvent than the other. The tendency for a compound to divide its time between two immiscible
solvents is known as partition. Some chromatographic techniques involve partition of solutes
between two solvents.
Paper Chromatography
This method is easy to set up and has the virtue of being cheap. The paper itself acts as the
stationary phase. A suitable liquid solvent is used as the mobile phase.
Producing a paper chromatogram
-

A fine pencil line is drawn near the bottom of a sheet of chromatography paper, which is
a thick absorbent paper like filter paper.
The mixture to be separated is applied as a spot onto the pencil line drawn.
The paper is suspended in a container with a shallow layer of a suitable solvent or
mixture of solvents in it (it is important that the solvent level is below the line with the
spots on it).
The container is covered. This is to ensure that the atmosphere in the beaker is saturated
with solvent vapor. This prevents the solvent from evaporating as it rises up the paper. As
the solvent slowly travels up the paper by capillary action, each component of the
mixture is carried a different distance by the moving solvent and so are separated. The
separation is stopped when the solvent has travelled nearly to the top of the paper. The
height reached by the solvent is called the solvent front.
The solvent front is marked on the paper, which is then removed from the solvent and
dried.

The Rf value
Some compounds in a mixture travel almost as far as the solvent does. Some stay much closer to
the base line. The distance travel relative to the solvent is a constant for a particular compound
provided that the type of paper and the exact composition of the solvent is constant. The Rf
values compare the distance moved by each component of the solute with the distance moved by
the solvent during the experiment. The retention factor of a component of the solute is given by
Rf = distance moved by component of solute = x
distance moved by solvent
y

The Rf value may be used to identify a component by comparing it with the Rf values of known
substances. The separated components can be obtained by cutting the paper into strips and
dissolving out each component.
Explanation of what is happening in paper chromatography
What is happening in paper chromatography is based on partition. A sheet of paper consists
largely of cellulose fibers. Cellulose is a polysaccharide composed of glucose molecules which
have a large number of hydroxyl groups. Water molecules hydrogen bond to these groups. This
water acts as the stationary phase in the technique of paper chromatography. The mobile phase is
a solvent consisting of an aqueous solution or an organic solvent. The mixture to be separated is
dissolved in this mobile phase, which moves along the paper. The solutes are partition between
the solvent adsorbed on the paper and the mobile solvent.
Sometimes two spots in the final chromatogram are the same color and have the same Rf value
in a particular solvent but are not necessarily the same compound.
Two way paper chromatography
Two way paper chromatography gets around the problem of separating out substances which
have very similar Rf values. This time a chromatogram is made starting from a single spot of
mixture placed towards one end of the base line. It is stood in a solvent as before and left until

the solvent front gets close to the top of the paper. The paper is then dried and rotated through
90 and run again in a different solvent.

Chromatograms made from amino acids

In some cases, the separated substances are colorless. It is possible to make the spots visible by
reacting them with something which produces a colored product. An example of this is in
chromatograms produced from amino acids obtained from the hydrolysis of a protein. The
chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin
reacts with amino acids to give colored compounds, mainly brown or purple.
Paper chromatography is used to analyze mixtures such as the dyestuff in inks and the colored
additives in food.
Thin Layer Chromatography (TLC)
The solid adsorbent is in the form of a thin layer on the surface of a glass plate. The adsorbent,
e.g. silica, calcium sulphate or alumina, is made into a thick paste with water and spread evenly
over a glass plate. The thin layer of paste is allowed to dry and bake in an oven. Spots of the
mixture are applied and a chromatogram is developed in the same manner as for paper
chromatography and it can be run in two dimensions. The particle size of the stationary phase is
smaller in TLC than in paper chromatography. As a result, TLC has advantages in that a variety
of absorbents can be used and the separations are more efficient and faster. Also, smaller samples
can be used.

TLC is used for the separation and identification of amino acids. It can also be used to
investigate the several products formed in the nitration of phenol. Some of the nitrophenols are
colored and the variety of the products can be seen on the developed TLC.
Sometimes many spots are not visible unless the plates are developed.
- The stationary phase on a thin layer plate often has a substance added to it which will
glow when exposed to UV light, except the components of the mixture. The spots show
up as darker patches. While the UV light is still on, the positions of the spots are marked
by drawing a pencil circle around them.
- The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin.
- A solution of iodine is used to reveal the presence of some aromatic compounds with
electron donating groups, e.g. C6H5NH2.
- Radioactive solutes can be identified on a TLC plate by passing the plate under a gieger
counter. A recorder plots the count rate as the plate passes under the counter.

The position of each spot or peak is determine by the retention time of that component. The
area under each peak is proportional to the amount of that solute. It can be measured as
shown below.

How does TLC work?

Silica gel is a form of SiO2. The silicon atoms are joined via oxygen atoms in a giant covalent
structure. At the surface, the silicon atom are attached to OH groups. See figure below.

The surface of the silica gel is very polar and because of the OH groups can form hydrogen
bonds with suitable compounds around it as well as van der Waals dispersion forces and
dipole-dipole attractions. As the solvent begins to soak up the plate, it first dissolves the
components of the spot. These tend to move up the chromatography plate as the solvent
continue to move upwards. The rate of separation depends on two things:
1. How soluble the component is in the solvent. This will depend on how much attraction
there is between the molecules of the component and those of the solvent.
2. How much the component sticks to the stationary phase (silica gel). This will depend on
how much attraction there is between the molecules of the component and the silica gel.
Suppose the original spot contained two compounds - one of which can form hydrogen bonds,
and one of which can only take part in weaker van der Waals interactions.
The one which can hydrogen bond will stick to the surface of the silica gel more firmly than the
other one which will attached itself to the surface via dispersion forces. One is adsorbed more
strongly than the other. While it is adsorbed on the silica gel, it is temporarily stopped, the
solvent is moving on without it. That means that the more strongly a compound is adsorbed, the
less distance it can travel up the plate.
TLC can rely either on partition or on adsorption depending on the conditions. In terms of TLC:
Partition: Solutes move between the mobile and stationary phases based on their relative
solubility.

Adsorption: Solutes are held on the stationary phase depending on the differences in polarity.
Rf = distance moved by component of solute = x
distance moved by solvent
y
Column Chromatography
Column chromatography is a convenient technique for physically separating the components of a
mixture for further use rather than for identification. In column chromatography, the stationary
phase is an inert solid used to pack a column (silica gel, alumina, cellulose sephadex). Silica is
slightly acidic which can adsorb basic solutes readily. Alumina is slightly basic which can adsorb
acidic solutes. The solid is made into a slurry with the solvent, poured into the column and
allowed to settle. Solvents include aliphatic and aromatic hydrocarbons, alcohols, ketones,
halogenocompounds, esters and mixtures of these. Any excess of solvent is run out through the
tap. The stationary phase must be saturated with solvent to prevent air bubbles from forming. A
solution of the analyte (mixture to be analyzed) is poured on to the top of the column and the
components are adsorbed at the top of the column. The mobile phase is a second solvent called
the eluant, which carries the components of the mixture through the stationary phase and elutes
the components in turn. The eluate, which is the solution leaving the column is collected in
fractions and the solutes are identified by their color, fluorescence, radioactivity or by
spectrophotometry.

Each solute is partitioned between the adsorbent and the eluant. The least strongly adsorbed
solutes are desorbed first by the eluant and carried further down the column before being
readsorbed. When fresh eluant reaches them, they are desorbed again and carried further down
the column. The rates at which solutes travel down the column depend on how strongly they are
adsorbed. If the rates are sufficiently different, the components become spread out along the
column as separate bands, which are called a chromatogram. If the eluant is kept flowing, the
solutes are eluted (dissolved out of the column) in turn. A complete separation may be obtained
and the components in each fraction of eluate may be recovered by evaporating the solvent.

The technique described here is an example of liquid chromatography in that the mobile phase is
a liquid. It is also an example of adsorption chromatography because the separation depends on
adsorption of the solutes.
Gas-Liquid Chromatography
Gas-liquid chromatography (GLC) is used to separate and identify very small samples of
gases, liquids and volatile solids. In this technique, a vaporized sample is carried by an inert gas
(the mobile phase) over the surface of a liquid (the stationary phase). A diagram of the apparatus
is shown below.

The mobile phase, which is called the carrier gas, flows through the column of stationary phase
at a constant rate. The relatively unreactive gas nitrogen is frequently used as the carrier gas. The
stationary phase is a non-volatile liquid on a solid support, for example, a long-chain alkane of
high boiling point coated onto the surface of SiO2. GLC is partition chromatography.
The components of the mixture are partitioned between the mobile and stationary phases to
different extents, so that they move through the column at different rates depending on (i) their
volatility and (ii) their relative solubilities in the mobile and stationary phases. When the
stationary phase is non-polar, the rate of movement of each component through the column is
determined principally by its volatility, which is related to boiling point. But when the stationary
phase is polar, it will tend to retain polar components. For example, if a mixture of non-polar
octane and polar pentanol is separated using a polar stationary phase, the octane would leave the
column before the pentanol. Stationary phases, that is the non-volatile liquids, are selected for
their suitability for the separation of different substances.
The components of a mixture leave the column after definite intervals of time, characteristic
of each component and are monitored by a detector designed to record changes in the
composition of the carrier gas as the components are separated.
Once the sample is injected, it may condense on the stationary phase, it may dissolve in the
liquid on the surface of the stationary phase or it may remain in the gas phase.

Retention time
The retention time is the time between the start of the chromatographic separation and the
appearance of the peak at the detector. The time taken for each of these components to pass
through the column is found by measuring the distance on the chromatogram between the
injection of the mixture defined as 0 minutes and the center of the peak for that component.
Since each solute has its own retention time, we can identify an unknown compound by
comparing its retention times of known compounds. However, the conditions used in the
experiments with unknown compounds and with known reference samples must be the same:
- the same carrier gas
- the same flow rate
- the same stationary phase
- the same temperature.
The gas-liquid chromatogram also tells us how much of each component is present in the
mixture. The area under a component peak in the chromatogram is related to the amount of that
component in the mixture. Another way of identifying the separated solute as they emerge from
the column is by linking the GLC apparatus to a mass spectrometer.
Factors affecting retention times
The retention time varies depending on
- The boiling point of the compound. A compound which boils at a temperature higher than
the column temperature is going to spend nearly all of its time condensed as a liquid at
the beginning of the column. High boiling point means a long retention time.
- The solubility in the liquid phase. The more soluble a compound is in the liquid phase,
the less time it will spend being carried along by the carrier gas. High solubility in the
liquid phase means a high retention time.
- The temperature of the column. A higher temperature will excite molecules into the gas
phase. A high column temperature shortens retention times for everything in the column.
The lower the temperature of the column, the better the separation but would take a very long
time to get components through which are condensing at the beginning of the column. The
higher the temperature of the column, the more quickly the components pass through the column
but less well separated out. This means that there is not much space between their peaks on the
chromatogram. Therefore, it is necessary to start with the column relatively cool and then
gradually and very regularly increase the temperature. Compounds which spend most of their
time in the gas phase will pass quickly through the column and be detected. Increasing the
temperature a bit more will encourage those compounds which have a greater force of attraction
to pass through quickly. Increasing the temperature higher will force those molecules which have
a stronger force of attraction off the stationary phase and through the column.
Electrophoresis
Separation on basis of charge
Colloids are heterogeneous mixtures whose particles are larger than the molecules and ions that
form solutions and smaller than the particles which form suspensions. Colloidal particles migrate
under the influence of an electric field in a movement called electrophoresis, which can be used

to separate substances that migrate at different rates. Separation takes place on paper or on a gel
to which an electric field is applied.
Apparatus for electrophoresis

The more highly charged an ion, the faster it moves in an electric field. Also, the larger the ion,
the more slowly it moves across the supporting medium.
Preparing amino acids for electrophoresis
Hydrolysis of the peptide bonds is carried out by heating the protein with 6 mol dm-3 HCl at
100C to 120C for 10 24 hrs.

Effect of pH
Charged molecules move towards the electrode of opposite charge at speeds which are
proportional to the charge on the molecules. An amino acid has both a basic amine group (-NH2)
and an acidic carboxylic acid group. See figure below.
NH2
CH

COOH

There is an internal transfer of a hydrogen ion from the COOH group to the NH2 group to
leave an ion with both a negative charge and a positive charge. This is called a zwitterion. It is
the form amino acid exist even in the solid state.
+

NH2
R

R
OH

i)

NH3
C

C
O-

If an acid is added, the COO- group becomes protonated.

+

NH3
R

NH3

O
C

O-

ii)

O
C
OH

The zwitterion acts as a base and during electrophoresis would move toward the
cathode.
+
If an alkali is added, the hydrogen ion is removed from the NH3
+

NH3
R

C
H

NH2

O
C

+
O-

OH-

C
H

O
C

H2O

O-

The zwitterion acts as an acid (proton donor) and during electrophoresis would move
towards the anode.
A buffer solution is used as the electrolyte in order to keep the pH of the medium at a constant
value. This is because if changes in pH were to occur during the separation process, the direction
of movement of the amino acids could be reversed. If the pH is right, the amino acid no longer
has a net positive or negative charge. That means that it would not move towards either the
cathode or anode during electrophoresis. The pH at which this lack of movement during

electrophoresis happens is known as the isoelectric point of the amino acid. This pH varies from
amino acid to amino acid.
Separation on the basis of size
A modification of electrophoresis is gel electrophoresis which separates substances ex DNA
molecules on the basis of the size of their molecules. Method consists of a polysaccharide gel
with wells into which samples can be placed. This is prepared by allowing the liquid gel to
solidify with a mould of the required shape on top. An electric field is applied and DNA
molecules move through the gel. Smaller molecules pass through more easily under the influence
of an electric field than larger molecules. After electrophoresis, the slab is stained and the
components appear as bands.
Apparatus for gel electrophoresis

Applications of gel electrophoresis

i)
ii)

iii)

These include the separation of proteins on the basis of the charge on the molecules or
according to the size of the molecules.
It can be used to check the purity of substances. If several bands are observed, the
substance is impure. If one is observed, the substance may be pure or it may be a
mixture of substances with molecules of the same size. A second method of
separation must be tried. If electrophoresis on the basis of charge fails to produce a
separation, the substance must be pure.
DNA profiling is a technique which can identify a person from a sample of their
DNA, which contains genetic information. DNA is located in the chromosomes which
are present in the nuclei of cells. DNA has a double helix structure. Each strand
consists of a chain of molecules of the sugar deoxyribose joined by phosphate groups.
Each sugar molecule has a base attached to it, which may be adenine, thymine,
cytosine or guanine. The strands are joined together with hydrogen bonds between
pairs of bases (A with T; C with G). In 1984, Alec Jeffrey discovered that human
DNA contains sequences of base pairs (10-15) which do not carry genetic instruction.
The sequences are repeated several times to form a band in the DNA called a
minisatellite, which repeat in a pattern that is unique in each individual. For this
reason, the pattern of minisatellites is called a DNA fingerprint.

Steps in obtaining a DNA fingerprint

1.) A sample of tissue is obtain and DNA is extracted from the nuclei of the cells.
2.) The DNA is cut at certain points by restriction enzymes which recognizes a certain
sequence of 4 6 base pairs and cuts the DNA at this point. (Many bacteria make
restriction enzymes which they use to protect themselves from foreign DNA such as
viruses)
3.) The fragments of DNA are separated by gel electrophoresis. The electrophoretogram is
invisible at this stage. The gel is covered by a nylon membrane which exerts a capillary
action and draw the DNA up into the nylon membrane.
4.) The sequences must be made visible. The DNA is labeled with radioactive phosphorous32 which binds to particular bands of the DNA.
5.) An X-ray film is placed on the membrane and the radioactive DNA fragments cause
fogging of the film which reveal the position of the bands.
Some applications of genetic fingerprinting
1.) It can be used to identify whether a man and woman are the parents of a child. The
technique is used in cases of disputed paternity to established who is the father of a child.
2.) Forensic scientists use genetic fingerprints in cases of rape. DNA can be obtained from a
vaginal swab taken from the victim and compared with that of a suspect.
3.) In murder cases and assault cases, the attacker may leave some of his own blood at the
scene of the crime and this can be analyzed and compared with the genetic fingerprints of
suspects.
4.) Doctors now use genetic fingerprinting in the treatment of leukemia, which is a cancer of
the bone marrow. Bone marrow makes new blood cells; if it becomes diseased, it can be
removed and replaced by transplanted bone marrow from a donor. After the operation, a
genetic fingerprint of the patients blood will show the donors bands if the operation has
been successful.