Paper chromatography and thin layer chromatography are techniques used to separate mixtures. In paper chromatography, a sample is placed on chromatography paper and placed in a solvent, allowing different compounds to travel up the paper at different rates based on their interactions with the paper and solvent. Thin layer chromatography uses a thin layer of adsorbent, like silica gel, coated on a plate as the stationary phase, and a solvent to separate compounds based on polarity. Both methods allow visualization and quantification of separated compounds through calculation of Rf values and use of detection reagents.

1. 1
Paper Chromatography
AND
Thin Layer
Chromatography

2. 1. Paper Chromatography
 Paper chromatography is a technique that
involves placing a small dot of sample solution
onto a strip of chromatography paper.
matrix = cellulose paper (polar substance)
 Adsorbent: More polar substances bond
with the cellulose paper more quickly.
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3. Dr Gihan Gawish
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1. Paper Chromatography

4. 1. The paper is placed in a jar containing a
solvent such as ethanol or water then
sealed.
2. A small concentrated spot of solution that
contains the sample of the solute is applied
to a strip of chromatography paper about 2
cm away from the base of the plate
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1. Paper Chromatography-
Procedure

5. 4. As the solvent rises through the paper, it
meets the sample mixture which starts to
travel up the paper with the solvent.
5. Paper chromatography takes from several
minutes to several hours.
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1. Paper Chromatography-
Procedure

6.  Different compounds in the sample
mixture travel at different rates due to
1. differences in solubility in the solvent
2. differences in their attraction to the fibers in
the paper.
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1. Paper Chromatography

7.  In this method, the solvent moves upward
against gravitational force.
 The only force that cause the motion is
capillary force. So the speed of the process is
slow.
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Paper Chromatography
1.1 Ascending

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9.  In this method, the solvent is kept in a trough
at the top of the chamber and is allowed to
flow down the paper.
 The liquid moves down by capillary action as
well as by the gravitational force.
 In this case, the flow is more rapid as
compared to the ascending method.
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Paper Chromatography
1.2 Descending

10.  Because of this rapid speed, the chromatography
is completed in a comparatively shorter time.
 The developing solvent is placed in a trough at
the top which is usually made up of an inert
material.
 The paper is then suspended in the solvent.
Substances that cannot be separated by
ascending method, can be separated by the
above descending method.
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Paper Chromatography
1.2 Descending

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12.  After development, the spots corresponding to
different compounds may be located by:
1. their color,
2. ultraviolet light,
3. ninhydrin
4. or by treatment with iodine vapors.
 The paper remaining after the experiment is
known as the Chromatogram.
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Paper Chromatography
Analysis

13. Rƒ value (Retention Factor)
 Rƒ =
the ratio of the distance traveled by the substance
the distance traveled by the solvent.
 If Rƒ value of a solution is zero, the solute
remains in the stationary phase and thus it is
immobile.
 If Rƒ value = 1 then the solute has no affinity for
the stationary phase and travels with the solvent
front.
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14. The final chromatogram can be
compared with other known
mixture chromatograms to
identify sample mixes, using the
Rf value in an experiment .
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Rƒ value (Retention Factor)

15. 2. Thin layer chromatography
(TLC)
 TLC is a widely used.
 Stationary phase solid+ adsorbent
 Adsorbent like silica gel (polar),
alumina (aluminium oxide), or cellulose.
 Traveling of solvent and elute via capillary
action
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16.  Silica is a typical adsorbent. It has silanol (Si-OH)
groups on its surface, which are slightly acidic, and can
interact with polar functional groups of the analyte or
eluent.
 In general, an eluent with a polarity comparable to that
of the most polar analyte in the mixture is chosen.
 Thus, alcohols would be selected if the analytes
contained hydroxyl groups,
 acetone or esters would be selected for analytes
containing carbonyl groups, and
 hydrocarbons such as hexane, heptane and toluene for
analytes that are predominantly non-polar.
 Mixtures of solvents are commonly used in the context
of gradient elution.
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17. Plate preparation
1. TLC plates are made by mixing the adsorbent
+ small amount of inert binder calcium sulfate
(gypsum) + water.
2. This mixture is spread as a thick slurry on an
unreactive carrier sheet, usually glass ,thick
aluminum foil, or plastic (support)
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2. Thin layer chromatography (TLC)-
Procedure

18. 3. The resultant plate is dried and activated by
heating in an oven for thirty minutes at 110 °C.
4. A small spot of sample is applied to a plate,
about 1 cm from the base.
5. The plate is then dipped in to a suitable
solvent ,such as hexane or ethyl acetate ,and
placed in a sealed container.
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2. Thin layer chromatography (TLC)-
Procedure

19. 6. The solvent (mobile phase) moves up the plate
by capillary action and meets the sample
mixture, which is dissolved and is carried up
the plate by the solvent.
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2. Thin layer chromatography (TLC)-
Procedure

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21. 21
2. Thin layer chromatography (TLC)-
Analysis
The scan of TLC plate (silica gel G) with 10 essential oils developed with mobile
phase toluene - ethyl acetate (93:7 v/v), next sprayed with vanillin in H2SO4 and
heated. From left to right oils from: bergamot, cedar, eucalyptus, syzygium,
lavandula, mint, orange, pine, spruce. Identified components: B1 and L1 - linalol,
B2 and L2 - linalyl acetate, E1 - cinneol, G1 - eugenol, G2 - carryophyllene.
Doubtfully identified components - C1 - cedrol, M3 - menthol, P1 - limonene..

22. A. Some samples are colorless, it can be
visualize by:
1. Manganese-activated zinc silicate
(fluorescent compound( is added to the
adsorbent
2. Iodine vapors
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2. Thin layer chromatography (TLC)-
Analysis

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IODINE has been used as a colour reagent for paper chromatography of amino-acids
and their derivatives1,2, imidazoles3, and other nitrogen-containing
compounds1,4. Steroids are also revealed by this technique, which is sufficiently
sensitive5. The method has not gained wide acceptance possibly only because it
cannot be relied on to yield a colour diagnostic of a class of compounds—some
nitrogen-containing compounds are not revealed—but its application is simple,
and if unsuccessful, other colour reagents can be applied to the same
chromatogram. In the most convenient procedure, the dried paper is exposed to
iodine vapour (supplied by a few iodine crystals in a closed battery jar) for 10–
30 sec.; or the paper may be sprayed with a solution of iodine in carbon
tetrachloride3, or may be dipped into a saturated solution of iodine in
petroleum ether (b.p. 60°–80°)5. Compounds are revealed usually as brown
spots, which fade rapidly.

24. 3. Specific color reagents are sprayed onto the
plate (ninhydrin)
4. In the case of lipids, the chromatogram may be
transferred to a PVDF (Polyvinylidene
fluoride) membrane and then subjected to
further analysis like mass spectrometry
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2. Thin layer chromatography (TLC)-
Analysis

25. B. Calculate the Rf value
 These values depend on
1. the solvent
2. the type of TLC plate.
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2. Thin layer chromatography (TLC)-
Analysis

26.  It is faster runs than paper,
 It has better separations,
 Wide choice between different adsorbents.
 It has better resolution and to allow for
quantitation
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2. Thin layer chromatography (TLC)-
Advantages

27. 1. identifying compounds present in a given
substance
2. determination of the pigments a plant contains
3. detection of pesticides or insecticides in food
4. analyzing the dye composition of fibers in
forensics
5. monitoring organic reactions
6. Assaying the radiochemical purity of
radiopharmaceuticals
1. identification of medicinal plants and their
constituents [
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2. Thin layer chromatography (TLC)-
Applications

28. Hydroxylapatite chromatography
Crystalline hydroxylapatite (Ca10(PO4)6(OH)2) is an
adsorbent used to separate mixtures of proteins or
nucleic acids. One of the most important applications of
hydroxylapatite chromatography is the separation of
single-stranded DNA from double-stranded DNA.
 Both forms of DNA bind at low phosphate buffer
concentrations but as the buffer concentration is
increased single-stranded DNA is selectively desorbed.
As the buffer concentration is increased further, double-stranded
DNA is released. This behaviour is exploited in
the technique of Cot analysis (Section 5.3.4). The affinity
of doublestranded DNA for hydroxylapatite is so high
that it can be selectively removed from RNA and
proteins in cell extracts by use of this type of
chromatography.
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