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Chromatography can be used for liquid, solid and gaseous
compounds whereas electrophoresis is generally carried out on
liquid and solid compounds.
In electrophoresis the electrical property of the element is used
whereas in chromatography partition coefficient of the element is
used to carry out the investigations and separation
Mikhail Tswett, Russian, 1872-1919
In 1906 Tswett used to chromatography to separate plant pigments
He called the new technique chromatography because the result of the analysis
was 'written in color' Most materials in our surroundings are mixtures of two
or more components.
 Mixtures are either homogeneous or heterogeneous.
 Homogeneous mixtures are uniform in composition, but heterogeneous
mixtures are not.
 Air is a homogeneous mixture and oil in water is a heterogeneous mixture.
 Homogeneous and heterogeneous mixtures can be separated into their
components by several physical methods.
 The choice of separation techniques is based on the type of mixture and
difference in the chemical properties of the constituents of a mixture.
 Various types of separation processes but we
will concern on chromatography in this
 Chromatography is a widely used experimental
technique for the separation of a mixture of
compounds into its individual components. The word
chromatography means "separation of colors" but
today chromato-graphy is used for both colored
and colorless substances.
Chromatography is a physical method of separation
in which the components to be separated are
distributed between two phases
one of which is stationary (stationary phase) while
the other (the mobile phase) moves through it in
a definite direction.
 Works by allowing the molecules
present in the mixture to distribute
themselves between a stationary and a
mobile medium.
 Affinity – natural attraction or force
between things.
Adsorption” refers to the adhesion or
stickyness of a substance to the surface
of another substance, as opposed to the
term “absorption” which refers to a
substance penetrating into the inner
structure of another substance.
 The separation process is based on the fact that
porous solids adsorbs different substances to
different extremes depending upon their polarity.
 A mixture to be separated is first applied to an
immovable porous solid (like paper, or alumina, or fine
silica sand) called the stationary phase.
 The components of the mixture then get
“washed” along the porous solid by the flow of a
solvent called the mobile phase.
 Each component of a mixture to be separated will be
attracted differently to the porous stationary phase
depending on its polarity and the polarity of the
stationary phase chosen.
 Remember that “Like attracts Like”.
 If the stationary phase is polar then polar
components will be attracted or stick more to it but
non-polar components will move across the surface
 If the stationary phase is nonpolar then nonpolar
components will be more attracted to it and the
polar compounds will move along more quickly.
 Chromatography involves the sample being dissolved
in a particular solvent called mobile phase.
 The mobile phase may be a gas or liquid.
 The mobile phase is then passed through another
phase called stationary phase.
 The stationary phase may be a solid packed in a glass
plate or a piece of chromatography paper.
 Can separate complex mixtures with great precision. Even
very similar components, such as proteins that may only
vary by a single amino acid.
can purify basically any soluble or volatile substance if the
right adsorbent material, carrier fluid, and operating
conditions are employed.
Exact quantitative analysis is done even from trace
Small material consumption.
The quantization has a broad linearity range.
Analyses of several compound can be done during one run.
Chromatography is a fast analysis method.
Well establishes instrumentation with high level automation
is commercially available.
Affinity to Stationary
Affinity to Mobile
Insoluble in Mobile Phase
        
• Analyze
• Identify
• Purify
Types of
Based on
stationary phase
Based on mobile
1- Liquid chromatography: mobile phase is a liquid.
2- Gas chromatography : mobile phase is a gas.
 Planar chromatography: A separation technique in which the
stationary phase serves as a plane.
The plane can be either a paper (paper chromatography) or a
layer of solid particles sorbent (silica gel, cellulose, aluminum
oxide, ion exchange resin) spread on a support such as a glass- or
a plastic- plate (thin layer chromatography).
THIS allowing to carry out qualitative and quantitative
analysis of chemical components in complex mixtures.
1- Thin layer chromatography (TLC): the stationary phase is a thin
layer supported on glass, plastic or aluminium plates.
2- Paper chromatography (PC): the stationary phase is a thin film of
liquid supported on an inert support.
 Column chromatography (CC): stationary phase is packed in a
glass column.
 For qualitative analysis the different mobilities of substances
are used, the distances passed by different substances are
different. The distance between the starting line and the
center of the spot of substance characterizes the substance.
Retention factor, RF, provides better way to indentify
substances .
 Identification of Unknown Compounds For your initial analysis, spot
your unknown and the colored standard compounds on one plate.
Be sure to note which spot corresponds to which compound.
 For quantitative determination the intensity of the spot is
used: the bigger the amount of substance in the mixture, the
more intensive is the spot. Also the size of the spot can give
quantitative information – the bigger the spot, the bigger the
content of this compound in the mixture. Intensity of the
spots is evaluated by comparing with the intensities of analyte
spots with known amounts visually or using densitometer.
 Paper chromatography uses paper as the stationary
phase and a liquid solvent as the mobile phase.
 In paper chromatography, the sample is placed on a
spot on the paper and the paper is carefully dipped
into a solvent.
 The solvent rises up the paper due to capillary action
and the components of the mixture rise up at different
rates and thus are separated from one another.
Capillary Action – the movement of liquid within the spaces of a porous material due
to the forces of adhesion, cohesion, and surface tension.
Cohesion, phenomenon of intermolecular forces holding particles of a substance
Adhesion, attraction between the surfaces of two bodies.
The liquid is able to move up the filter paper because its attraction to itself is
stronger than the force of gravity.
Solubility – the degree to which a material (solute) dissolves into a solvent.
Solutes dissolve into solvents that have similar properties. (Like dissolves like)
This allows different solutes to be separated by different combinations of solvents.
Separation of components depends on both their solubility in the mobile phase and
their differential affinity to the mobile phase and the stationary phase.
 Thin layer chromatography is a technique used to
separate and identify compounds of interest.
 A TLC plate is made up of a thin layer of silica adhered
to glass or aluminum for support.
 The silica gel acts as the stationary phase and the
solvent mixture acts as the mobile phase.
 In the ideal solvent system the compounds of interest
are soluble to different degrees. Separation results
from the partition equilibrium of the components in
the mixture.
Different compounds in sample mixture
travel different distances according to
how strongly they interact with the stationary phase as compared to the mobile phase.
In principle, the components will differ in solubility and in the strength of their adsorption to the
adsorbent and some components will be carried farther up the plate than others.
This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action.
The specific Retention factor (Rf) of each
chemical can be used to aid in the
identification of an unknown substance.
Rf values can be calculated and compared with the reference values to identify the amino acids.
Highly polar molecules interact fairly strongly with the polar SiOH groups at the
surface of these adsorbents, and will tend to stick or adsorb onto the fine particles of
the adsorbent while weakly polar molecules are held less tightly.
Weakly polar molecules generally tend to move through the adsorbent more rapidly
than the polar species. Roughly, the compounds follow the elution order given above.
• Different compound in sample mixture travel
different distance according to how strongly they
interact with the stationary phase as compared to
mobile phase. ( adsorption)
The separation depends on several factors;
(a) solubility: the more soluble a compound is in a solvent,
the faster it will move up the plate.(polarity of mobile
(b) attractions between the compound and the silica, the
more the compound interacts with silica, the lesser it
moves, (. activity of stationary phase)
(c) . structure of substrate
silica gel
 Silica gel is a form of silicon dioxide (silica). The silicon
atoms are joined via oxygen atoms in a giant covalent
structure. However, at the surface of the silica gel, the
silicon atoms are attached to -OH groups.
 The surface of the silica gel is very polar and, because of the
-OH groups, can form hydrogen bonds with suitable
compounds around it.
The other commonly used stationary phase is alumina aluminium oxide. The aluminium atoms on the surface of
this also have -OH groups attached.
 TLC is a useful technique because it is relatively
quick and requires small quantities of material.
 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).
Eluent. [Mix n-butanol, acetic acid (purity 98 – 100 %) and
distilled water in volume ratio 5:1:5. Stir for 10 minutes, then
let the layers separate. Use upper layer as eluent].
Developing Solution: Dissolve 0.3 g of ninhydrin in 100 ml nbutanol. Add 3 ml of glacial acetic acid.
0.02 M solutions of different amino acids (e.g. leucine,
methionine, alanine and serine) in H2Odd.
Chromatographic paper
Elution chamber, Glass capillaries for spotting the samples.
Drying oven at ~ 60° C.
Rubber gloves must be used during this work to avoid contamination of
chromatographic paper with amino acids from skin, and for protecting skin from
solvents and ninhydrin while working with the sprayer or sprayed paper.
While the paper is being prepared for chromatographic analysis it should be
kept on a piece of filter paper.
3. Mark the starting line to the paper - 8-9 mm from the edge of the plate - with
graphite pencil (very slight line!). Also mark the locations where the samples will
be spotted. The distance between neighboring spots should be about 8 mm and
the spots should be at least 5 mm away from the paper’s edge. Usually the spot
of unknown substance is applied to the center of the starting line.
pencil (do not use a pen, as the dye may separate and run along with your sample),
Over spotting or inadequte spacing between samples can lead to their bleeding into each
Place one end of the tube into the sample. Through capillary action the sample will be
drawn into the tube. Place your finger over the other open end of the tube to prevent
the sample from falling out.
Hold the tube slightly above the location on the starting line that you had previously
marked for spotting. Carefully allow air into the opposite end of the capillary tube by
slightly raising your finger that is covering the opening. Allow only a single drop to
fall on the plate -- the smaller the better. If the sample is not leaving the tube, slight
tapping on the opposite end should coax out a droplet. Allow the spot to evaporate.
Repeat this procedure 2-3 times. Concentration of your spotting will definitely affect
your TLC run and it is a matter of experimentation to determine what will give you
the best results. Repeat this procedure for the remaining samples. The plates only
allow for 2-3 samples to be spotted to provide for adequate spacing between sample
spots. Over spotting or inadequate spacing between samples can lead to their
bleeding into each other during the run.
Before applying samples to the paper and filling the
elution chamber fit the length of chromatographic paper
with the height of elution chamber.
5. The spots of individual amino acids and sample solutions
are applied to the chromatographic paper. Use separate
clean and dry glass capillary for each solution. Dip the
capillary into solution – some solution is drawn into the
capillary. With the filled capillary touch the prepared
location on chromatographic paper. The spot on the paper
should not be bigger than 2-3 mm. (You can exercise
spotting on a sheet of filter paper.)
6. After application of samples let the spots dry. Meanwhile
measure with a graduated test-tube 5 ml of eluent into
the elution chamber. Cover the chamber with lids and let
the chamber atmosphere saturate with eluent vapors for
at least 10 min.
Elution is stopped when the solvent front has traveled up the plate
until 7-10 mm from the lid.
Remove the paper from elution chamber and place it on a sheet of
filter paper. After 2-3 minutes mark the eluent front with pencil and
dry the paper in oven.
When the plates are removed from the chamber, quickly trace the
solvent front (the highest solvent level on the plate) with a pencil.
The time that it takes for the solvent to rise up the plate is dependent upon
your choice of solvent. Generally a run will take only a few minutes, but you
must monitor the solvent front to be sure that it does not reach the end of
the plate. This could result in your sample being carried to the end of the
plate and an unusable TLC run. A good guideline to follow is to allow the
solvent front to reach a point about 1 cm from the end of the slide.
When the paper is dry, take it into the fume hood and spray it with
solution of ninhydrin until the paper is slightly damp. Chromatographic
paper and the paper supporting it should lie at 45° angle while
spraying. The chromatographic paper is again put in the drying oven
(60 C) for 15 min to speed up the reactions.
10. Remove chromatographic paper from drying in
the oven, draw the contours and centers of the
chromatographic bands. Calculate RF values by
the method described above.
11. Compare retention of standard substances and
components in sample and determine which amino
acids were present in the sample.
1. Adding fluorescence indicators to the sorbent
layer during the process of preparation of the
plates or spraying the plates with fluorescent
solutions and then observing under ultraviolet
2. I2 vapor as indicator .
3. Ninhydrin spray.(Visualizing Agents for amino acids
) Development of Ruhemann’s purple from ninhydrin and
amino acid.
This value is constant for any given
compound under a given set of
conditions and can be used to identify an
unknown compound.
Separation of compounds is based on the competition
of the solute and the mobile phase for binding places
on the stationary phase.
For instance, if normal phase silica gel is used as the
stationary phase it can be considered polar. Given
two compounds which differ in polarity, the more
polar compound has a stronger interaction with the
silica and is therefore more capable to dispel the
mobile phase from the binding
Consequently, the less polar compound moves
higher up the plate (resulting in a higher Rf value). If
the mobile phase is changed to a more polar solvent
or mixture of solvents, it is more capable of
dispelling solutes from the silica binding places and
all compounds on the TLC plate will move higher up
the plate
 Is this a pure substance or mixture?
TLC can be used to determine whether a sample is a single substance or a mixture.
A single pure substance gives a single spot. However, care should be taken since compounds
of similar properties, isomers for example, may not separate.
A sample that results in many spots after development is a confirmation of a mixture of
different compounds.
In that sense, TCL can also be used to determine the number of components in a mixture.
 Are these compounds identical?
TLC can be used to confirm whether two or more compounds are identical or not. The
compounds to be analyzed are spotted on the same plate and the plate developed. If the
compounds have the same Rf value, this can be taken as an indication that they have a
similar chemical nature.
However, it is important to keep in mind that many chemical compounds have the same Rf
and TLC cannot be used as the only analytical tool to decide on the identity of a
chemical compound.
 The definition of Electrochromatography.