This document outlines the process of isolating and identifying caffeine from tea leaves. Tea leaves are extracted using boiling water and the extract is filtered. Caffeine is then isolated from the extract using dichloromethane in a separating funnel. The dichloromethane layer containing caffeine is evaporated to obtain solid caffeine. Thin layer chromatography is used to identify the isolated compound as caffeine using a caffeine reference standard. Murexide test, which produces a purple color, confirms the identification of caffeine. The document also lists learning objectives, extraction steps, TLC procedure, identification test, assessment questions and references.

1. DISCOVER . LEARN . EMPOWERIsolation identification and analysis of Caffiene
University Institute of Pharma
Sciences
Bachelor of Pharmacy
Neeraj Bainsal
Assistant Professor
Pharmacognosy

2. To understand isolation and identification
and analysis of caffeine.
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Isolation and identification
and analysis of caffeine
CO
Number
Title Level
CO1 to know the modern extraction techniques,
characterization and identification of the
herbal drugs and phytoconstituents
Remember
CO2 to understand the preparation and development of herbal
formulation
Understand
CO3
to understand the herbal drug interactions
Understand
CO4 to carryout isolation and identification of
phytoconstituents
Understand
Course Outcome

3. Objectives
On completion of this period, you would be able to know:
• Isolation and identification of caffeine from tea
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4. Tea
 Common name: Thea sinensis
 Botanical name- it contains the prepared leaves and leaf buds of Camellia sinensis, green tea.
 Family- Theaceae
• Geographical source-
• Tea was originally grown in China over 1000 years ago. Today it is cultivated in India, China, Sri Lanka, Japan, Indonesia,
Kenya, Turkey, Pakistan, Malawi and Argentina.
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5. Isolation of caffeine
• An Erlenmeyer flask containing 5 tea bags (12.66 g) was filled with 100 mg of water and placed on a wire ring, and heated
to boiling.
• The flask was at a boil for 15 minutes.
• When the tea was done boiling, the Erlenmeyer flask was set onto the counter to cool down to 55°C.
• The tea was then poured through the Buchner funnel that was attached to the filtration flask and through the vacuum.
• The tea was then cooled even further to 20°C.
• A 125 mL seperating funnel was then used to have the filtered tea in.
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6. Contd…..
• In the funnel, 20 mL of dichloromethane was added and the funnel was inverted a couple times.
• Once the layers have separated, the dichloromethane was drained into a 50 mL flask.
• In the seperatory funnel, 20 mL of dichloromethane was again added and drained into the same 50 mL flask.
• The tea was then drained from the separating funnel.
• A narrow stem funnel was used with a cotton ball and a drying agent called sodium sulfate.
• The 100 mL dichloromethane was filtered through the drying agent and more sodium sulfate was added in the flask.
• The extra sodium sulfide in the flask was carefully decanted.
• The solution was then put on a hot plate to evaporate.
• Then recrystallized by using as minimal acetone as possible
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7. TLC
• Put approximately 25 mg of caffeine in a small, labeled test tube.
• Add 4.0 mL of ethanol mix, and heat gently to dissolve the caffeine.
• 4. Obtain a TLC plate. The plate should be handled using forceps so as to avoid contamination. A light pencil line should be
drawn using a straight edge about 1 cm from one end of the plate.
• 5. The instructor will demonstrate the spotting technique. Use a capillary micropipette to make a small spot of the pure
caffeine solution (made in step 3) on the plate. Place the spot 1 cm from the left edge along the pencil line you drew. Also
spot the TLC plate with each of the solutions Make these thre spots at 1-cm intervals to the right of the caffeine spot. In
order to avoid confusion make sure to label the spots lightly in pencil bellow the line.
• 6. Develop the TLC plate by placing it in a beaker that has been filled with developing solvent (5% acetic acid in ethyl
acetate) to a level of less than 1 cm high (the spot on the TLC plate should be above the level of the solvent). Cover the
beaker with aluminum foil immediately after the TLC plate is immersed. Allow the solvent to migrate up the TLC plate
until it is about one centimeter from the top. Do not allow the solvent line to reach the top of the plate.
• 7. Remove the TLC plate and mark the level to which the solvent rose with a pencil. Allow the solvent to evaporate off of
the plate in the hood and then visualize the plate under UV light. Outline all spots with a pencil.
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8. Identification Test
• Murexide Test-The (solid) sample is first treated with conc. nitric acid,
which is slowly evaporated away; subsequent addition of ammonia
solution (NH4OH) gives a purple color if uric acid was present, due to
formation of murexide, or a yellow color that turns to red on heating
if a xanthine is present.
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9. Assessment Pattern
FAQ’s
Q 1 Write down biological source for caffeine?
Q 2. what is isolation.
Q3. what is murexide test?
Q4. give extraction procedure for caffeine from tea leaves.
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10. REFERENCES
• A handbook of Pharmacognosy by V.E, Tyler Evaluation of crude drugs, Mono or Polyherbal formulation of crude drugs
• A textbook of Pharmacognosy by CK Kokate.
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11. THANK YOU
For Queries
Neeraj.pharma@cumail.in