Aim of the Experiment: Exercise involving isolation & detection of active principles :
Caffeine - from tea dust.
(b. Diosgenin from Dioscorea, c. Atropine from Belladonna, d. Sennosides from Senna
Requirements:
Discussion:
caffeine is a purine alkaloid, isolated from prepared tea leaves & leaf buds of Thea sinensis
Family: Theaceae
Also got from coffee beans, cola seeds etc
Coffee beans –coffea Arabica f : Rubiaceae
Cola seeds –Cola acuminate, Cola nitida F: Sterculiaceae/Malvaceae
Cocoa seeds Theobroma cacoa –Sterculiaceae
Alkaloids are natural, bitter, organic, heterocyclic, secondary metabolite, basic in nature,
having one or more nitrogen atoms, occurring in minute quantities in plants, having
very good and specific therapeutic activities when used in small quantities in animals
or human being, but toxic in higher doses.
Caffeine, inspite of having hetercyclic ring with nitrogen with in the ring,eventhough
answers common alkaloidal tests like dragendorff’s. Myer’s, Wagners test, Hagers etc.,
it is a pseudoalkaloid because it is not got from amino acid pathway
And Murexide test: is specific for purine derivatives.
Mix the caffeine with small amount of potassium chlorate and a drop of HCl, in a watch
glass, evaporate to dryness carefully in a water bath, cool, expose the residue to ammonia
vapour (or add 2 drops of dilute ammonia), produce a
pinkish-purple color due to formation of murexide (ammonium purpurate); Appear purple
in pure state of caffeine.
Though caffeine itself is basic in nature , the decoction of coffee or tea –both
are acidic because it contains naturally occurring organic acids in tea leaves, the more we
boil more acidic . Also caffeine occur as free base in tea leaves and not as it’s salts.
In general alkaloidal salts are soluble in water (not in organic solvents) and free bases are
not soluble in water ( but soluble in organic solvents). But caffeine is an exception in the
sense the free base in soluble in water. Caffeine is readily soluble in water 2 % -means 2 gms
in 100ml while in chlorform it is highly soluble upto 18% v/w In the presence of salicylate,
citric acid, benzoates its solubility in water increases.
Caffeine is White in color or white powder. It is anhydrous or on hydration only one
molecule of water will be attached. Needle shape, crystals. Melting point: 235-237°C.
During extraction to be economic and good quality product
1. The choice of crude drug (selection of plant material),
2. Drying and communition/pulverization-availabiltiy and storage
3 . Choice of solvent :
Low potential for artifact formation (natural production of non effective and
toxic artificial products)
Low toxicity
Low flammability
Low risk of explosion
Economical
Easily recycled by evaporation
Solutes solubility
3. Simplest possible apparatus
4. Easiet possible Extraction procedure- is to be selected.
So our choice for caffeine exgtraction and isolation is more economic dried leaves or tea
dust, decoction being the extraction procedure, cheapest and safest and easy available
and effective solvent-water solvent, using only simple apparatus for boiling, separating,
simplest procedure to apply the principles .
Tea leaves contain 3.5% caffeine, while coffee beans have 1.1–2.2%.-so we use tea leaves
for extraction.
Extraction: Alkaloids are extracted by two methods namely
1. Stass otto method (Extraction in alkaline media )
2. Manski’s method (extraction in Acidic media)
Caffeine is extraxcted in two ways :
Method 1 ( modified Stass Otto-as needed: In the extraction of caffeine, tea dust is first
boiled with alkaline water and filtered out . To the filtrate basic lead acetate is added to
precipitate out tannins and albuminoids. Then the excess of lead is removed from the
filtrate by adding sulphurinc acid. To the filtrate charcoal is added so as to decolorise the
solution and caffeine is extracted from the filtrate using chloroform. Caffeine obtained is
purified by recrystallization in water.
Method 2 ( as the caffeine decotion is already acidic –this is similar to manski’s methid _
The water soluble materials in the tea leaves are extracted into hot water including caffeine
which is freely soluble in hot water The hot solution is allowed to cool and caffeine is then
extracted from the water with dichloromethane (methylene chloride) which is an organic
solvent that is insoluble in water. Since caffeine is more soluble in dichloromethame
(140mg/ml) than it is in water (22mg/ml) it readily dissolves in dichloromethane. However
the tannins are also soluble in dichloromethane which can be prevented by converting them
in to their salts by reaction with sodium carbonate. The practical disadvantage of converting
the tannins to their salts is that they become anionic surfactants which may dissolve
deichloro-methane in water, hence care must be taken not to shake the content vigorously
to avoid formation of emulsion
Uses of caffeine:
Smooth muscle relaxant
CNS stimulant in the form of beverage : cerebral vasoconstrictor and hence stimulate CNS
Diuretic
A specific analgesic in migraine to potentiate the action it is used with ergotamine tartrate
Principle of caffeine extraction: In view of physical and chemical properties of
Caffeine in tea leaves the procedure adopted the following reagents for the said
purposes:
1. Caffeine being soluble in water –boiling water is used as the menstruum to prepare
the decoction
Water is made alkaline by adding sodium carbonate to release any caffeine present in salt
form. sodium carbonate acts as a base to deprotonate caffeine
and other acidic compounds in the tea leaves, making it easier to extract the caffeine in
organic solvent like chloroform while leaving behind unwanted components like tannins.
2. Also alkali converts tannins present -into sodium tannates which is also soluble in
water. Filtered to separate out the insoluble contents
3. To the filtrate added basic lead acetate which precipitates out tannins and
albuminoids
4. Filtered and to the filtrate, added sulphuric acid to remove excess lead acetate
, lead sulphate insoluble in
acidic water, so separated by filtration
5. Filtrate containing caffeine is decolorized by charcoal, which adsorbs coloring matter,
including chlorophyll-if discoloration is not done now, the chlorophyll would be
dissolved in chloroform which is used in next step
6. Anhydrous Sodium sulphate is used to remove any moisture associated in the
chloroform layer containing extracted caffeine
7. Purification: After filtration, filtrate is extracted with chloroform, on evaporating
chlroform we get caffeine which is purified & recrystallized by dissolving in hot water. Caffein
sublimes at 178 °C, so water boiling emperature of 100°C will not effect caffeine
Isolation, Estimation/Analysis:
Thin layer chromatography of Caffeine:
Dissolve 1 mg: 1ml of caffeine & chloroform or methanol.
Then spot the sample on TLC plates
Eluted in solvent system ethyl acetate: methanol: acetic acid (8:1:1).
Then visualize the spots on dried TLC plate by exposure to iodine vapour.
The Rf value is 0.41
HPTLC/ UV spectrophotometry is also used for estimation of caffeine in comparison with a
standard caffeine.
Procedure:
1. Mix 25 gms of tea powder and 2.5 gms of sodium carbonate with 150 ml of water in a
250ml beaker and boil for 15 minutes with constant stirring, filter hot through a
muslin cloth, pressing the marc
2. To the filtrate add lead acetate solution until no more precipitate is formed, boil and
filter it rapidly. Wash the residue held in filter paper with sufficient water ( twice with
50ml of water)
3. Boil the Filtrate and dilute sulphuric acid until the precipitation ceases
4. Filter, to the filtrate add a pinch of charcoal, boil and evaporate so that the volume is
reduced approximately 50ml
5. Filter hot
6. Cool the filtrate
7. Extract the filtrate with three successive 10 ml portions of chloroform by taking in a
separating funnel , chloroform layer collected in a beaker
8. To Combined chloroform layer add a little qty of anhydrous sodium sulphate to
remove any associated moisture , stir well and filter
9. Weigh an empty china dish, take the combined chloroform extract in it , evaporate no
a water bath-which crystallizes out crude caffeine.
10. Cool the chaina dish to room temperature and weigh again, difference between the
two weights is the amount of caffeine got from 25 gms of tea dust. Now calculate the
percentage yield of caffeine.(if chaina dish is weighed when hot you get less weight
resulting in less yield)
Calculation:
Weight of the empty china dish : w1 =--------gms
Weight of empty chaina dish + caffeine= w2 =------- gms
Weight of the caffeine got w2-w1 = C = gms
Weight of the tea dust taken T = gms
Percentage yield of caffeine = C/T x 100 = % w/w
Report: