4. DEFINATION
The technique of separating
the components of a mixture is
achieved by the differential
movement of individual
components through a
stationary phase under the
influence of a mobile phase.
Chromatography is widely
used for separation ,
purification , identification
and characterisation of the
components of a mixture
wether coloured or colourless.
5. Principles of chromatography
Chromatography is a physical process.
Any chromatography system is composed of mainly
three things:
1) Stationary phase
2) Mobile phase
3) Mixture to be separated
6. Chromatography is an dynamic process in which
mobile phase moves in definite direction.
In chromatography process we can only control
stationary &mobile phase as mixture is the problem we
have to deal with.
7. TERMINOLOGIES
Differential: showing a difference.
Affinity: natural force of attraction between the
substances.
Adsorption :phenomenon of higher concentration of
molecular species(liquid or gas) on the surface of solid in
bulk.
Adsorbent: the substance’s surface on which adsorption
took place.
Adsorbate : substance which get adsorbed on adsorbent.
Stationary phase : part of apparatus that does not move
with the sample.
Mobile phase : gas or liquid that carries the sample with
it.
8. WORKING OF CHROMATOGRAPHY
The substance which has to be separated has many
components, some of them has affinity to mobile
phase & some has affinity to stationary phase.
The substance having affinity to mobile phase get
dissolved in it and travels large distance it.
While substance having affinity to stationary phase get
adsorbed on it and travels very short distance.
9. Uses of chromatography
Analyze: examine of mixture, it’s components & there
relation with one another.
Identify : determine the identy of mixture or
components based on known components.
Purify: separates components in order to isolate
components for further studies.
Quantify : dertimens the amount of substances
present in the sample.
15. STATIONARY PHASE
The stationary phase or adsorbent in column
chromatography is a solid.
The most common stationary phase for column
chromatography is silica gel, followed
by alumina. Cellulose powder has often been used in the
past.
The stationary phases are usually finely ground powders
or gels and/or are micro porous for an increased surface.
16. MOBILE PHASE
•The mobile phase or eluent is either a
pure solvent or a mixture of different
solvents.
• The eluent has also been chosen so that
the different compounds can be separated
effectively
17.
18. Column chromatography
Column chromatography in chemistry is a method used
to purify individual chemical compounds from mixtures of
compounds.
The main advantage of column chromatography is the
relatively low cost and disposability of the stationary
phase used in the process. The latter prevents cross-
contamination and stationary phase degradation due to
recycling
We can’t separate different amino acid’s &sugars. We
can’t also do quantitative analyses accurately.
20. 6 beakers or jars
6 covers or lids
Distilled H2O
Isopropanol
Graduated cylinder
6 strips of filter paper
Different colors of Sharpie
pens
Pencil
Ruler
Scissors
Tape
Materials List
21. STATIONARY PHASE
•The stationary phase or adsorbent in paper
chromatography is a liquid
Paper is made of cellulose fibres, and cellulose is a
polymer of the simple sugar, glucose.
The key point about cellulose is that the polymer
chains have –OH groups sticking out all around
them. To that extent, it presents the same sort of
surface as silica gel or alumina in thin layer
chromatography
cellulose fibres attract water vapour from
the atmosphere as well as any water that
was present when the paper was made.
You can therefore think of paper as being
cellulose fibres with a very thin layer of
water molecules bound to the surface.
22. MOBILE PHASE
The mobile phase used in paper chromatography is liquid
. non-polar solvent such as hexane, Acetone,etc, Polar
solvents water, alcohols,etc as the solvent
The solvent has also been chosen so that the different
compounds can be separated effectively
23. PRINCIPLE OF PAPER
CHROMATOGRAPHY
Capillary Action – the movement of liquid within the spaces of a
porous material due to the forces of adhesion, cohesion, and surface
tension. 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.
It can be used to separate different
amino acids & sugars but quantitative
analyses is not accurately.
24. THIN LAYER CHROMATOGRAPHY
Here the mobile phase is a liquid, flowing past
a thin layer of powder on a solid support by
CAPILLARY ACTION; a variation of column
chromatography.
Substances that are less attracted to
the solid or are more soluble in the
liquid move faster. And so move
further up the plate by the time that
the process has been stopped by
taking the plate out of the liqiud. -
larger Rf
26. . Detection
If the spots are not colored and can’t be seen by the
eye, use:
• UV lamp for UV-active compounds; most aromatics
are UV-active
• If compounds are not UV-active, use an iodine .
27. Rf value(retention factor)
The ratio of distance travelled by the component (from
origin) compared with the distance travelled by the
solvent front (from origin) is called the Rf value.
Rf = Migration distance of a substance
Migration distance of solvent front
Origin Line
Solvent Front Line
Distance traveled
by solvent
Distance traveled by spot
28. 28
••
The ratio of distance travelled by the component (from origin) compared
with the distance travelled by the solvent front (from origin) is called the Rf
value.
Solvent front
x
a
b
c
Rf of = a/x
Rf of = b/x
Rf of = c/x
29. Rf Value
•The Rf value is not
informative
•What affects the Rf value?
Temperature
Solvent
Thickness and amount
of spot
31. ?
GAS CHROMATOGRAPNY
1) In gas chromatography the moving
phase is a gas and the stationary phase
is either liquid or solid.
2) The technique is suitable for
separation of materials which are
volatile without decomposition
32. It can be used for qualitative as well
as quantitative analyses of
substances.
It can’t be used to separate non-
volatile substances.
33. HIGH PRESSURE LIQUID
CHROMATOGRAPHY
Different analytes have different equilibria between the
mobile phase and stationary phase
Equilibrium is dynamic; thus we can view it as a given
analyte molecule spending a fraction of time dissolved in
the mobile phase
Since different solutes gave different fractions, a separation
of the analytes occur as they are pushed through the
column by the mobile phase
34. Uses for Chromatography
Real-life examples of uses for chromatography:
• Pharmaceutical Company – determine amount of
each chemical found in new product
• Hospital – detect blood or alcohol levels in a
patient’s blood stream
• Law Enforcement – to compare a sample found at
a crime scene to samples from suspects, Like
Explosion residue, Arson cases Poisons in viscera.
• Environmental Agency – determine the level of
pollutants in the water supply
• Manufacturing Plant – to purify a chemical
needed to make a product
• Dyes –Identifying coloring dyes used in different
food products
