Pharmacognosy and Phytochemistry II B. Pharm.
Semester V
EXPERIMENT NO: 5 DATE:
AIM: To isolate and detect caffeine from tea dust.
REQUIREMENTS:
Apparatus: Beaker, funnel, conical flask, measuring cylinder, separating funnel, evaporating dish.
Chemicals: Tea dust, 10% basic Lead acetate solution, Charcoal, Chloroform, distilled water, 10%
Sulphuric acid, anhydrous sodium sulfate.
THEORY:
CAFFEINE
Botanical source: Caffeine is obtained from the dried leaves of Thea sinensis. Family: Theaeae
• Plant is also known as Camellia sinensis.
• It is a shrub cultivated in India, Sri Lanka, East Africa, Mauritius, China and Japan.
• The leaves may be fermented or left unfermented. Fermented teas are referred to as black tea,
unfermented teas as green tea, and partially fermented teas as oolong.
• The leaves contain thease, an enzymic mixture containing an oxidase, which partly converts
the phlobatannin into phlobaphene. This oxidase may be destroyed by steaming for 30 s.
• Tea contains 1–5% of caffeine and 10–24% of tannin; also, small quantities of theobromine,
theophylline and volatile oil.
• The alkaloid content of the leaves is very much dependent on age and season.
Chemistry:
• Caffeine is a purine alkaloid. It is a xanthine derivative (1,3,7-trimethyl xanthine).
Caffeine
• Molecular formula: C5H10N4O2
• Molecular weight: 194.2 gm/mole
• IUPAC name: 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione
• Pharmacological actions: Caffeine stimulates the central nervous system and has a weak
diuretic action. It increases blood pressure and have stimulate action on heart and lungs. It
increases the potency of analgesics (aspirin and caffeine tablets IP 2007) and relieves asthma
attack.
• Uses: CNS stimulant, dose: 300-600 mg
Treatment of migraine.
Chemical tests:
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Pharmacognosy and Phytochemistry II B. Pharm. Semester V
PROCEDURE:
(A) Isolation of caffeine
METHOD I
1. Weigh 10gm of Tea dust, add 2 gm Calcium carbonate powder and boil it with 60ml of
distilled water in 250ml beaker for 15 minutes with constant stirring.
2. Strain the resulting hot extract through muslin, expressing as much of the liquid as possible
from the material.
3. Wash the remaining mass with 40ml of boiling water and express again. Combine this
washing to filtrate.
4. Cool, transfer to a separating funnel and extract with Chloroform (10+10+5 ml). shake gently
(Do not shake vigorously to avoid formation of emulsion).
5. Collect the combined Chloroform extract in a small beaker, decolorize with activated charcoal,
add a pinch of anhydrous sodium sulfate and filter into a dry porcelain dish.
6. Carefully evaporate Chloroform on waterbath or hot plate to dryness.
7. Weigh the residual caffeine. Find the % yield, take m. p., test its purity on TLC and submit the
product.
METHOD II
1. Weigh 10gm of Tea dust and boil it with 60ml of distilled water in 250ml beaker for 15
minutes with constant stirring.
2. Strain the resulting hot extract through muslin, expressing as much of the liquid as possible
from the material.
3. Wash the remaining mass with 40ml of boiling water and express again. Combine this
washing to filtrate.
4. Carefully add 10% basic lead acetate solution to the filtrate until no more ppt is formed.
5. Heat the mixture to boiling and filter rapidly. Wash the residue remaining on the filter with 5
ml of boiling water.
6. To the filtrate, add dilute Sulphuric acid solution until precipitation ceases. Add 0.5 g
decolorizing charcoal to the mixture, boil for few minutes and then filter.
7. Collect the filtrate in evaporating dish and evaporate it over the flame with frequent stirring
until the volume is reduced to 40-50 ml.
8. Filter the concentrated extract while hot, and if it is not clear, return the first collected
portion through the same filter paper until a clear filtrate is obtained.
9. Cool, transfer to a separating funnel and extract with Chloroform (10+10+5 ml). Collect the
Chloroform extract in a small beaker, add a pinch of anhydrous sodium sulfate and filter into
a dry beaker.
10. Rinse the sodium sulfate twice with about 3-5 ml of Chloroform, filter and add in the
Chloroform extract.
11. Transfer the combined Chloroform extract to evaporating dish and carefully evaporate
Chloroform on waterbath or hot plate to dryness.
12. Weigh the residual caffeine. Find the % yield, take m. p., test its purity on TLC and submit
the product.
(B) Detection of caffeine by TLC
Stationary phase: Silica gel G
Mobile phase: Ethyl acetate: Methanol: Acetic acid
8 : 1 : 1
Sample: 1 mg of isolated caffeine in 1 ml of methanol or chloroform
Spray reagent: Dragendorff’s reagent or iodine chamber
RESULT:
(1) % yield of Caffeine:
(2) Rf value of isolated Caffeine:
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Pharmacognosy and Phytochemistry II B. Pharm. Semester V
Reference:
https://www.youtube.com/watch?v=sPhJWBL17OQ
Calculation:
% yield
10 gm Tea dust contain a gm of caffeine
100 gm Tea dust contain (?) gm of caffeine
= 100 × a
10
=______ %w/w caffeine
TLC
Rf value
Rf = Distance traveled by solute
Distance traveled by solvent
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