Chromatography
Chapter 4-2
1Dr Gihan Gawish
1. Paper Chromatography
Dr Gihan Gawish
Paper chromatography is a technique that involves placing a small dot of sample solution
onto a strip of chromatography paper.
The compounds within the mixture travel are non-polar.
Adsorbent: More polar substances bond with the cellulose paper more quickly.
matrix = cellulose paper (polar substance)
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Dr Gihan Gawish
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1. Paper Chromatography
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Rƒ value (Retention Factor)
Dr Gihan Gawish
Rƒ =
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.
the ratio of the distance traveled by the substancethe ratio of the distance traveled by the substancethe ratio of the distance traveled by the substancethe ratio of the distance traveled by the substance
the distance traveled by the solventthe distance traveled by the solvent..the distance traveled by the solventthe distance traveled by the solvent..
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Dr Gihan Gawish
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)
2. Thin layer chromatography (TLC)
Dr Gihan Gawish
TLC is a widely used.
Stationary phase solid+ adsorbent
Adsorbent like silica gel (polar), alumina.
Traveling of solvent and elute via capillary action
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Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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2. Thin layer chromatography (TLC)- Analysis
Dr Gihan Gawish
A. Some samples are colorless, it can be visualize by:
Manganese-activated zinc silicate (fluorescent compound (is
added to the adsorbent
Iodine vapors
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2. Thin layer chromatography (TLC)- Analysis
Dr Gihan Gawish
3. Specific color reagents are sprayed onto the plate (ninhydrin)
4. In the case of lipids, the chromatogram may be transferred to a PVDF
membrane and then subjected to further analysis like mass spectrometry
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2. Thin layer chromatography (TLC)- Analysis
Dr Gihan Gawish
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
Dr Gihan Gawish
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
Dr Gihan Gawish
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
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2. Thin layer chromatography (TLC)- Applications
