Anita Yadav, M.Pharm(Clinical Pharmacy)

1. Pokhara University
School of Health and Allied Sciences
Chromatography
Anita Yadav
First Semester, M Pharm(Clinical Pharmacy)
School of health and allied Sciences
Pokhara University, Dhungepatan, Lekhnath-12, Kaski, Nepal

2. Pokhara University
School of Health and Allied Sciences
Topics under discussion
• Introduction
• Background
• Discussion
August 8, 2017 2INS 591: Presentation

3. Pokhara University
School of Health and Allied Sciences
Introduction
• Chromatography is an analytical technique where in a
sample mixture is separated into different
components.
• This is both a qualitative and quantitative method.
• The sample gets separated under the influence of a
mobile phase (moving phase) over a stationary phase.
• These separated components are later identified and
also quantified.
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4. Pokhara University
School of Health and Allied Sciences
Origin of chromatography:-
• The Russian botanist Mikhail Tswett had
invented chromatography, a word he derived from
the Greek words for color (chroma) and writing
(graphe) in 1906.
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5. Pokhara University
School of Health and Allied Sciences
• A. J. P. Martin (1910-2002) and R. L. M. Synge (1914-
1994) developed the first theoretical explanations.
• chromatography did not come into wide use until
1952, when Martin, working with A. T. James,
described a way of using a gas instead of a liquid as
the mobile phase, and a highly viscous liquid coated
on solid particles as the stationary phase
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6. Pokhara University
School of Health and Allied Sciences
Principle of Chromatography:-
• All chromatographic methods require one static part
(the stationary phase) and one moving part (the
mobile phase).
• Interaction between the stationary and mobile phase
cause separation of compounds from the mixture.
• Rely on one of the following phenomena: adsorption;
partition; ion exchange; or molecular exclusion.
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7. Pokhara University
School of Health and Allied Sciences
• The preferential separation is done due to
differential affinities of compounds towards
stationary and mobile phase. After separation of the
compounds, they are identified by
suitable detection methods.
• The differences in affinities arise due to
relative adsorption or partition coefficient in
between components towards the both phases.
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8. Pokhara University
School of Health and Allied Sciences
Classifications of chromatography
(1)Based on the nature of mobile and stationary phase:
• Liquid chromatography: LLC, LSC
• Gas chromatography: GLC, GSC
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9. Pokhara University
School of Health and Allied Sciences
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• Based on the principle of separation:
• Adsorption chromatography: - It is based on the
different ability of component to adsorb on the
surface of SP.E.g. TLC, CC, GSC
• Partition chromatography: - It is based on the
different solubility of sample of component in the
stationary and mobile phase. E.g. GLC, LLC
• Ion exchange chromatography: - it is based on the
exchange of ionic sample with ionic group of
stationary phase and is governed by electrostatic
interaction.
• E.g. IC, IEC, TLC

10. Pokhara University
School of Health and Allied Sciences
• Gel chromatography: - Based on the size and shape
of their molecules as well as the size and shape of
the pores of the stationary phase.
• Affinity chromatography:- based on the molecular
recognition of only those components which are
complementary to stationary phase, are absorbed by
their affinity
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11. Pokhara University
School of Health and Allied Sciences
• Thin-layer chromatography (TLC) is
a chromatography technique used to separate
non-volatile mixtures.
• It is performed on a sheet of glass, plastic, or
aluminium foil, which is coated with a thin
layer of adsorbent material, usually silica
gel, aluminium oxide (alumina), or cellulose.
• This layer of adsorbent is known as
the stationary phase.
Thin Layer Chromatography
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12. Pokhara University
School of Health and Allied Sciences
STATIONARY PHASE
• Silica is commonly used as stationary phase
• The separation of sample mixture will be depent on the
polarity of sample.
• Some modified silica is also used in certain purposes.

13. Pokhara University
School of Health and Allied Sciences
Stationery phase Description Application
Silica gel G Silica gel with average
particle size 15µm
containing ca 13%
calcium sulfate binding
agent
Used in wide range
pharmacopoeial test
Silica gel G254 Silica gel G with
fluorescence added
Same application with
Silica gel G where
visualization is to be
carried out under UV
light.
Cellulose Cellulose powder of less
than 30µm particle size.
Identification of
tetracyclines

14. Pokhara University
School of Health and Allied Sciences
• The ability of mobile phase to move up is
depent on the polarity itself
• Volatile organic solvents is preferably used as
as mobile phase.
MOBILE PHASE

15. Pokhara University
School of Health and Allied Sciences
SOLVENT POLARITY INDEX
Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0
MOBILE PHASE

16. Pokhara University
School of Health and Allied Sciences
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The compounds in the sample on the TLC plate (the analyte)
can do two things as the solvent moves up the plate:
If a compound is attracted to the
coating it sticks and does not move
up the plate:
If a compound is not attracted to
the coating it will not stick and it
does move up the plate:

17. Pokhara University
School of Health and Allied Sciences
August 8, 2017 INS 591: Presentation 18
• Capillary action pulls the solvent (the mobile phase)
slowly up the plate like water being soaked up by a
sponge.
• Actually, most compounds will be partially attracted
to both the coating and the solvent.
• Compounds more attracted to the coating move up
the plate slowly, while those more attracted to the
solvent travel more quickly and separation is
achieved.

18. Pokhara University
School of Health and Allied Sciences
• A major factor is polarity of the bonds.
• For example if, the coating is composed of aluminum
oxide (Al2O3).
• The aluminum - oxygen bonds are very polar. The
solvent is usually a nonpolar or very moderately polar
organic solvent.
• In general, the more polar bonds a compound has then
the more attracted it is to the very polar aluminum
oxide and the more slowly it moves up the plate.
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What features cause some compounds to prefer the stationary phase to the
mobile phase?

19. Pokhara University
School of Health and Allied Sciences
Uses
• To determine the number of components in a mixture.
• To determine the identity of two substances.
• To monitor the progress of a reaction.
• To determine the effectiveness of a purification.
• To determine the appropriate conditions for a column
chromatographic separation.
• To monitor column chromatography.
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20. Pokhara University
School of Health and Allied Sciences
• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp
Materials

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School of Health and Allied Sciences
Methods
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School of Health and Allied Sciences
• It includes three methods:
• Spotting
• Developing
• Visualizing
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School of Health and Allied Sciences
Spotting
• Spotting: A drop of a mixture of three compounds, A,
B, and C was spotted on the TLC plate with a capillary
tube at the point marked spotting line or origin.
• The plate consists of a piece of plastic coated with
silica gel, a fine grade of sand.
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Fig.Spotting

24. Pokhara University
School of Health and Allied Sciences
Developing
• The spotted plate was placed in a beaker containing a
solvent.
• The solvent rose on the plate.
• The amount of solvent in the beaker was enough so
that the spotting line did not dip into the solvent.
• The solvent passed through the spotting line and
continued to move up the plate until the plate was
removed from the beaker.
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25. Pokhara University
School of Health and Allied Sciences
August 8, 2017 INS 591: Presentation 26
• The dotted line labeled solvent front in Figure 1
shows how far the solvent moved (i.e., from below
the spotting line to the upper dotted line) when it was
removed.
• As soon as the plate was removed from the beaker,
the experimenter scratched a line with a pencil and
straight edge across the plate to indicate the solvent
front.
• The solvent is still visible on a plate immediately after
the plate is removed from the beaker.

26. Pokhara University
School of Health and Allied Sciences
• However, the solvent quickly evaporates.
• Therefore, the solvent front must be marked with a
pencil before the solvent dries.
• After the solvent dried, the pencil line (solvent front)
and origin were all that was visible.
August 8, 2017 INS 591: Presentation 27
Fig. Developing

27. Pokhara University
School of Health and Allied Sciences
Visualizing
• As the solvent moved up the plate, compounds A, B,
and C also moved up the plate but at different rates.
• Of the three compounds, A moved furthest up the
plate because it traveled faster.
• How fast a compound, referred to as a spot, moves
up a plate is a measure of its mobility in that
particular coating and solvent combination.
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28. Pokhara University
School of Health and Allied Sciences
• To see the individual spots, the plate must be
visualized. The color samples are easy to be seen and
no need to use UV lamp to detect them.
August 8, 2017 INS 591: Presentation 29
Fig. visualizing

29. Pokhara University
School of Health and Allied Sciences
• For colorless spots,following procedure applied,
• a. Iodination
• b. Ninhydrin
• c.KMnO4
• d.Alkaline tetrazolium blue
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30. Pokhara University
School of Health and Allied Sciences
• The coating contains a fluorescent indicator, which is
visible under ultraviolet (UV) light.
• After the plate was visualized, the experimenter had
the chromatogram shown in Figure 1. The final step
was to analyze the plate.
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31. Pokhara University
School of Health and Allied Sciences
Analyzing
• After a fixed time, as measured by how far the
mobile phase has moved, the process is stopped, and
the dissolved substances have moved different
distances.
• The ratio of how far the substance moved to how far
the solvent moved is called the Rf.
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32. Pokhara University
School of Health and Allied Sciences
Thin-Layer Chromatography:
Determination of Rf Values
solvent front
component B
component A
origin
dS
dB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a decimal
fraction, generally only
reported to two decimal
places
More polar!
Less polar!

33. Pokhara University
School of Health and Allied Sciences
The use of Retention Faactor(Rf) as
separation parameter
• The distance taken through by the solvent to move
up will be assigned as solvent front
• The distance taken trrough by the sample to move
up will be assign as sample front
• Rf value is obtained by dividing the sample front
toward solvent front
• Rf = sample front
• solvent front
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34. Pokhara University
School of Health and Allied Sciences
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A
B
C
y = distance
solvent moved
x = distance
compound A
moved
xy
The Rf of compound A is x over y (x/y).

35. Pokhara University
School of Health and Allied Sciences
• In general, low polarity compounds have higher Rf
values than higher polarity compounds
• Typically an effective solvent is one that gives Rf in
the range of 0.3-0.7.
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36. Pokhara University
School of Health and Allied Sciences
Experimental Techniques of Thin Layer
Chromatography:
• Pouring
• Dipping
• Spraying
• Spreading
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37. Pokhara University
School of Health and Allied Sciences
Choice of solvents system in TLC:
• The choice of solvent or a mixture of solvents used in
TLC is solely guided by two important factors:
• (a) the nature of the constituent to be separated
i.e. whether it is polar or non-polar; and
• the nature of the process involved i.e. whether it is
a case of ‘ adsorption’ or ‘partition chromatography’.
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38. Pokhara University
School of Health and Allied Sciences
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39. Pokhara University
School of Health and Allied Sciences
a. The spot shape is too broad
- Diameter is supposed to be < 1-2mm
b. The movement of solvent
- should be straight up
- unproportionality in stationary phase surface will
inhibit the movement of solvent
c. streaking formation
- caused by too concentrated sample
Three Common Problems in TLC

40. Pokhara University
School of Health and Allied Sciences
Advantages of TLC
• Low cost and short analysis time
• Ease of sample preparation
• All spots can be visualized
• Sample cleanup is seldom necessary
• Adaptable to most pharmaceuticals
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41. Pokhara University
School of Health and Allied Sciences
• Uses small quantities of solvents
• Requires minimal training
• Reliable and quick
• Minimal amount of equipment is needed
• Densitometers can be used to increase accuracy of
spot comcentration
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42. Pokhara University
School of Health and Allied Sciences
Applications of TLC
• To identify the presence of undesirable specific
organic compounds present as impurities in a
number of pharmaceutical substances, namely:
morphin in apomorphin hydrochloride; hydrazine in
carbidopa; 3-aminopropanol in dexampanthenol;
etc.
• Related substances present in official drugs,
namely: related substances present in a wide
number of potent pharmaceutical substances e.g.,
aminophylline; baclofen; chloramphenicol;
carbamazepine,etc.August 8, 2017 INS 591: Presentation 43

43. Pokhara University
School of Health and Allied Sciences
• Foreign alkaloids present in alkaloidal drugs, for
example: atropine sulphate; codeine
• Foreign steroids present in steroidal drugs, for
example betamethasone valerate;
• Ninhydrin positive substances in official amino acids
e.g.glutamic acid; leucine.
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44. Pokhara University
School of Health and Allied Sciences
References
• http//orgchem.colorado.edu/hndbooksupport/TLC/T
LCprocedure.html.
• kalasz Huba, Bathori Maria, LC.GC Int., 1-8(2001)
• Kar Ashutosh(2015) Pharmaceutical Drug Analysis(3
Ed.) New Age International Publishers, pp471-492.
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45. Pokhara University
School of Health and Allied Sciences
Thank You
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