Chromatography

• The word chromatography means "color writing"
which is a way that a chemist can test liquid
mixtures
• Chromatography is defined as a procedure by which
solutes are separated by dynamic differential
migration process in a system consisting of two or
more phases, one of which moves continuously in a
given direction and in which the individual
substances exhibit different mobilities by reason of
differences in adsorption, partition, solubility, vapor
pressure, molecular size, or ionic charge density

• Chromatography is a method used by scientists for
separating organic and inorganic compounds so that
they can be analyzed and studied.
• By analyzing a compound, a scientist can figure out what
makes up that compound.
• Chromatography is a great physical method for
observing mixtures and solvents, some people use
chromatography to find out what is in a solid or a liquid.
• It is also used to determine what unknown substances
are.
• The Police, F.B.I., and other detectives use
chromatography when trying to solve a crime.
• It is also used to determine the presence of cocaine in
urine, alcohol in blood, polychlorinated biphenyls (PCB's)
in fish, and lead in water

 Chromatography is based on differential migration.
 The solutes in a mobile phase go through a stationary phase.
 Solutes with a greater affinity for the mobile phase will spend
more time in this phase than the solutes that prefer the stationary
phase.
 As the solutes move through the stationary phase they separate.
 In paper and thin-layer chromatography the mobile phase is the
solvent.
 The stationary phase in paper chromatography is the strip or piece
of paper that is placed in the solvent.
 In thin-layer chromatography the stationary phase is the thin-layer
cell.
 Both these kinds of chromatography use capillary action to move
the solvent through the stationary phase.
 This is called chromatographic development.
Principle

• The physical and chemical properties which can be
use to separate molecules are:
Physical Properties
1. Molecular weight
2. Boiling point (in case both are liquid)
3. Freezing point
4. Crystallization
5. Solubility
6. Density
Chemical Properties
1. Functional Group, for example, phenol has –OH
where as aniline has NH2.
2. Reactivity towards other reagent to form complex

Adsorbtion: Interaction of solute molecules (or atoms or ions)
with the surface of the stationary phase (note that it is different
from absorption where the molecules fill the pores of a solid).
Eluent: The mobile phase (usually for solvents)
Elution: Motion of the mobile phase through the stationary
phase
Elution time: The time taken for a solute to pass through the
system. A solute with a short elution time travels through the
stationary phase rapidly, i.e. it elutes fast.
Mobile phase: The part of the chromatography system that is
mobile. Commonly a solvent mixture (as in column
chromatography or thin layer chromatography) or a gas (as in
gas chromatography).

Normal phase: “Unmodified” stationary phase where POLAR
solutes interact strongly and run slowly
Reverse phase: “Modified” stationary phase where POLAR
solutes run fast i.e. reverse order
Resolution: Degree of separation of different solutes. In
principle, resolution can be improved by using a longer
stationary phase, finer stationary phase (e.g. column packing
or TLC plate coating) or slower elution.
Stationary phase: The part of the chromatography system that
is fixed in place. Most commonly a solid e.g. the packing in
column chromatography or gas chromatography or the
coating on a chromatographic plate.

 The retention factor, Rf, is a quantitative
indication of how far a particular compound
travels in a particular solvent.
 The Rf value is a good indicator of whether an
unknown compound and a known compound are
similar, if not identical.
 If the Rf value for the unknown compound is close
or the same as the Rf value for the known
compound then the two compounds are most
likely similar or identical.

• The retention factor, Rf, is
defined as
Rf = distance the solute
(D1) moves divided by the
distance
traveled by the solvent front
(D2)
Rf = D1 / D2
where
D1 = distance that color
traveled, measured from
center of the band
of color to the point where
the food color was applied
D2 = total distance that
solvent traveled

There are four main types of chromatography.
These are:
•Liquid Chromatography
•Gas Chromatography
•Thin-Layer Chromatography
•Paper Chromatography.

• Liquid Chromatography is used in the world to test
water samples to look for pollution in lakes and
rivers.
• It is used to analyze metal ions and organic
compounds in solutions.
• Liquid chromatography uses liquids which may
incorporate hydrophilic, insoluble molecules.
• Liquid chromatography, which encompasses methods
such as High Performance Liquid Chromatography
(HPLC) and Ion Chromatography, is a separation
technique. It is used to identify, quantify and purify
individual components in a mixture

 In high-performance liquid chromatography
(HPLC) the sample mixture is passed along with
a liquid solvent under high pressure through a
column filled with a solid adsorbent material.
 The pressure within the system is built-up with
the help of pumps. The working principle is
that each compound in the mixture interacts
slightly different with the adsorbent material in
the column, resulting in varying flow rates for
the different components.
 This leads to separation of the components as
they flow out of the column. The adsorbent
material used is typically granular, made up of
solid particles, such as silica, constituting the
‘stationary phase’.

• The pressurized liquid is a mixture of solvents, such
as water and organic liquids like methanol and
acetonitrile, which constitute the ‘mobile phase’.
• The detector is connected to a digital microprocessor
and user software for data acquisition and analysis.
• The separated compounds are visualized as peaks
with the number of peaks corresponding to the
number of separated components in the mixture. The
area of the peak is proportional to the concentration
of the compound present within the mixture.
• The resolution between two peaks in
chromatographic techniques is the extent to which
substances are separated during the experiment.
Higher resolution reflects good separation of the
compounds.

Gas Chromatography is used:
 In airports to detect bombs and is used is
forensics in many different ways.
 It is used to analyze fibers on a persons body
and also analyze blood found at a crime scene.
 In gas chromatography helium is used to move a
gaseous mixture through a column of absorbent
material

• A sample containing the solutes is injected onto a heated block where
it is immediately vaporized and swept as a plug of vapor by the
carrier gas stream into the column inlet.
• The solutes are adsorbed by the stationary phase and then desorbed
by fresh carrier gas.
• The process is repeated in each plate as the sample is moved toward
the outlet.
• Each solute will travel at its own rate through the column.
• Their bands will separate into distinct zones depending on the
partition coefficients, and band spreading.
• The solutes are eluted one after another in the increasing order of
their Kd (physico chemical constant), and enter into a detector
attached to the exit end of the column.
• Here they register a series of signals resulting from concentration
changes and rates of elution on the recorder as a plot of time versus
the composition of carrier gas stream.
• The appearance time, height, width and area of these peaks can be
measured to yield quantitative data.
• Kd = Concentration in Phase A
Concentration in Phase B
• When k' is # 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum

 Thin-layer Chromatography uses an absorbent
material on flat glass or plastic plates.
 This is a simple and rapid method to check
the purity of an organic compound.
 It is used to detect pesticide or insecticide
residues in food.
 Thin-layer chromatography is also used in
forensics to analyze the dye composition of
fibers.

• To apply sample spots, thin marks are made at the bottom
of the plate with the help of a pencil.
• Apply sample solutions to the marked spots.
• Pour the mobile phase into the TLC chamber and to
maintain equal humidity, place a moistened filter paper in
the mobile phase.
• Place the plate in the TLC chamber and close it with a lid.
It is kept in such a way that the sample faces the mobile
phase.
• Immerse the plate for development. Remember to keep the
sample spots well above the level of the mobile phase. Do
not immerse it in the solvent.
• Wait till the development of spots. Once the spots are
developed, take out the plates and dry them. The sample
spots can be observed under a UV light chamber.

 Paper Chromatography is one of the most
common types of chromatography.
 It uses a strip of paper as the stationary
phase.
 Capillary action is used to pull the solvents
up through the paper and separate the
solutes.

• Selecting a suitable type of development: It is decided based on the
complexity of the solvent, paper, mixture, etc. Usually ascending type or
radial paper chromatography is used as they are easy to perform. Also, it is
easy to handle, the chromatogram obtained is faster and the process is less
time-consuming.
• Selecting a suitable filter paper: Selection of filter paper is done based on the
size of the pores, and the sample quality.
• Prepare the sample: Sample preparation includes the dissolution of the
sample in a suitable solvent (inert with the sample under analysis) used in
making the mobile phase.
• Spot the sample on the paper: Samples should be spotted at a proper
position on the paper by using a capillary tube.
• Chromatogram development: Chromatogram development is spotted by
immersing the paper in the mobile phase. Due to the capillary action of
paper, the mobile phase moves over the sample on the paper.
• Paper drying and compound detection: Once the chromatogram is developed,
the paper is dried using an air drier. Also, detecting solution can be sprayed
on the chromatogram developed paper and dried to identify the sample
chromatogram spots.

Problem Possible causes
• Samples does not show up Not enough sample, different
visualisation method required
• Streaking This could be due to the nature of
the sample, due to sample
overload or a poor choice of
solvent mixture.
• Diffuse / blurred spots The origin line was below the
solvent level or the plate was
developed for too long so the
solvent front reached the top of the
plate.
• Sample “lanes” are not straight /
parallel
A problem with the way the solvent
ran due to the plate being crooked
in the solvent, touching the filter
paper in the developing jar or the
plate was chipped.

 Ion Chromatography
 Gel Filtration Chromatography
 Affinity Chromatography

Chromatography

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