Chemical Test for Caffeine
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We are interested in the alkaloid --- caffeine (C8H10N4O2) which is
very common substance in many drinks, such as coffee, tea, Coca-Cola
etc. Adults or even students will drink a cup of coffee to temporarily
restoring alertness and warding off drowsiness. However, how much
caffeine in our coffee? How can we determine the amount of caffeine
The following project will investigate the chemical test for caffeine.
At first, we extract caffeine from coffee. Then we need to prove that
the extraction contain caffeine. Finally we try to find out the amount of
caffeine in coffee.
We’ll first follow the common procedure, which is the solvent
extraction and the thin layer chromatography. We also do some
research on other chemical test for caffeine. We come up with the test
of iodometric back titration.
All the tests we investigate can be done in school laboratory, and
pure caffeine is bought for verifying and investigating these chemical
We hope that these tests can be further developed in order to
become more popular, more efficient, lower the cost and less pollute to
First, we try to extract caffeine from coffee by a suitable method.
Then, by using chemical tests to analysis the amount of caffeine in
coffee and extract.
Last, we want to validate the efficiency of the extraction method.
Solvent extraction 
It mainly based on the difference of solubility of caffeine in different
solvent. Caffeine is sparingly soluble in water at room temperature but
highly soluble in boiling water (100 °C). Thus we can extract most of
caffeine in raw coffee powder to a coffee solution.
Then, in order to extract crude caffeine from coffee solution, we can
base on the property that caffeine is more soluble in organic solvent
(here we use chloroform (CHCl3)) than that in water at room
temperature. Also chloroform is much denser than water and insoluble
in water, the caffeine in coffee solution are transferred to the chloroform
and separated out.
Then to prove that the extraction is mainly caffeine, we can carry out
two tests, which are Thin Layer Chromatography (TLC) and iodometric
Thin Layer Chromatography (TLC)
There are different types of chromatographic methods such as paper
chromatography, thin-layer chromatography, column chromatography,
gas chromatography, etc. They have the same principle :
(1) Different solutes have different solubility in a solvent /different
solutes have different degrees of tendency to be dissolved in the
(2) As the solution ( contains the solvent with the dissolved solutes )
moves along a stationary solid surface ( a solid surface ) , different
solutes adsorbed onto the solid surface in different extent as they
have different degree of adsorption characteristics ( due to the
different degrees of dissolve tendency).
Christian Alliance S C Chan Memorial College/2013-14 Chemistry Olympiad/Caffeine
(3) The “less soluble” solute will be retained first, and the “more
soluble” solutes will be retained afterwards.
( Note : No two substances have the same solubility and adsorption
(4) Different solutes will then be separated on the different positions of
the solid surface.
Iodometric Back Titration
Caffeine reacts with excess accurately known amount of iodine in acidic
environment, forming insoluble precipitate. Then the insoluble
precipitate is removed by filtration. Using titration by a standard
sodium thiosulphate solution with starch solution as indicator, we can
determine the amount of remaining iodine, and thus the amount of
caffeine can be found. Here are the chemical equations :
Experimental Methods and Procedures
(A) Extraction of caffeine from raw coffee power
Preparation of raw coffee solution
1. 4.58g of fine grinded raw coffee powder is weighted, add 100 cm3
of distilled water and 3.0g of sodium carbonate to increase
solubility of Caffeine (alkaloid) and boil it and brew it by following
the instruction on the Coffee package (one tablespoon of coffee
powder in 100 cm3
of hot water, about 4.58g /100 cm3
2. Cool down the raw coffee solution to room temperature
1. Rinse all apparatus with chloroform.
2. Transfer the raw coffee solution to separating funnel (Figure a).
3. Add 10 cm3
of chloroform and swirl vigorously, (*Don’t shake
the mixture because an emulsion will form*)(Figure b). Allow
the mixture to stand and separate out the bottom chloroform
layer to a beaker. Repeat this step for 5 times.
4. Add calcium sulphate to the separated chloroform to remove
water. Shake well and stop until fluffy, cloudy effect.
5. Weight the beaker which is going to hold the filtrate.
6. Filter out the excess calcium sulphate. (Figure c)
7. Put the beaker with filtrate into hot water bath to evaporate
chloroform (Boiling Point : 61.2 o
C) (Figure d)
8. Weigh the powder and calculate the amount of powder extracted.
Qualitative analysis of caffeine by Thin Layer Chromatography (TLC)
1. Dissolve the extract and pure caffeine into distilled water separately.
2. Prepare the TLC plate ( silica gel coating, and cut it to 3 cm x 10 cm ).
3. Use a pencil to draw a horizontal line 0.5 cm above the bottom of the
4. Spot the extracted caffeine and pure caffeine by 10µ L micro-capillary
tubes separately on the pencil line of the TLC plate.
5. Immerse the TLC plate into the solvent ( a mixture of 20:1 of ethyl
acetate : ethanoic acid ).
6. Let the plate stand and develop the chromatogram.
7. Visualize the result with a UV lamp, use a pencil to circle the spot
seen under UV light.
Quantitative analysis of caffeine by iodometric back titration
1. Transfer 15.00 cm3
of standard caffeine solution / the solution
prepared by the extract to a conical flask and add 10 cm3
2. Add 25.00 cm3
of standardized iodine solution to the mixture and
swirl gently, brown-red precipitate will form.
3. Filter the solution.
4. Take down the initial reading of burette.
5. Titrate the filtrate against a standard sodium thiosulphate solution
6. Add a few drops of starch solution when the titrand becomes pale
7. Slowly run Na2S2O3 until titrand become colorless from dark blue.
8. Take final reading of burette.
9. Repeat the above steps until two to three consistent results are
For the TLC test, the possible reason that we can’t see other
impurities maybe impurities can’t be visualize under UV light or they
dissolved in the solvent used to develop the plate. The detail
constituent compounds and elements can be known by some advance
instrument, e.g. HPLC etc.
For iodometric back titration, because Iodine solution is volatile,
some Iodine may loss during experiment process especially filtration.
Thus it contributes the error of the titration. The “standard iodine
solution” should be standardized by a standard sodium thiosulphate
solution from time to time to acquire its concentration.
After the above tests, we confirmed that the about solvent extraction
method is a practical experimental procedure for extracting caffeine in
school laboratory. Due to the nature of the partition coefficients of
solvent-solvent extraction, the amount of loss of caffeine could be
reduced by increasing the number of portion of chloroform used in the
process. ( Refer to the step 3 of the section of Solvent Extraction).
However, chloroform is a CFCs and a carcinogenic substance, it is not
recommended to use.
The TLC results proved that caffeine was successfully extracted from
the raw coffee solution. It also showed that no obviously impurities in
the chromatogram under UV light.
The Iodometric back titration is a simple and an accurate method to
determine the amount of caffeine in aqueous solution. It requires
simple apparatus and common chemicals only. Our result in the section
of Result E(1) showed that it is an accurate method. It has very high
accuracy, about 97%!
Besides it, different accurate known amounts of caffeine were spiked
into coffee solutions to find out the relationship between percentage
recovery and the concentration of caffeine in raw coffee solution. It
seemed that the recovery percentage of caffeine is related to the
concentration of caffeine in the coffee solution, i.e. higher concentration
of caffeine, lower the recovery percentage while the recovery
percentage of spike test in water is roughly constant (~62%). Thus the
matrix of coffee solution will affect the recovery percentage of caffeine.
The titration result showed that 32.56 mg of caffeine contained in
the extract from 100 cm3
of raw coffee solution. By using the result of
the recovery percentage in the section of Result D1, the recovery
percentage is about 78.3% , the amount of dissolved caffeine in a cup of
coffee (250 cm3
) is about = 32.56 x 250 / ( 0.783 x 100 ) = 103.9 mg !
The above spike test can be further developed for checking the
extraction efficiency of the above extraction method. Spiking different
concentrations of standard caffeine into the coffee solutions to
consolidate the relationship of caffeine concentration and recovery
percentage by constructing a curve that shows the relationship between
the recovery percentage and the concentration of caffeine in the coffee
solution. So the titration results can be “adjusted” by the recovery
percentage ( dividing the titration result by the corresponding recovery
percentage ) to find a more reliable result.
Also, find a less harmful solvent to substitute chloroform because
chloroform is a suspected carcinogenic substance and a CFC’s etc. In
order to protect the Earth, investigation on environmentally friendly
solvent can be done. Although supercritical carbon dioxide (CO2) is a
better solvent, it’s costly and impractical be made in school laboratory.
However, for the Iodometric Back Titration, we can try to reduce the
limitations and find out if other constituent substances in the extract will
react with iodine, contributing to error.
Decaffeinated coffee and other drinks also can be tested by the