This document discusses the determination of caffeine content in tea samples. It provides background on caffeine, including its structure, properties, health effects, and mechanism of action. Caffeine acts as a central nervous system stimulant by antagonizing adenosine receptors in the brain. The document also discusses the typical caffeine content in different types of tea and the methodology that will be used to determine the caffeine levels in tea samples.

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DETERMINATION OF CAFFEINE
IN TEA SAMPLES

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DETERMINATION OF CAFFEINE CONTENT IN TEA SAMPLES

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Dissertation Submitted In Partial Fulfilment Of The Degree Of Bachelor
Of Science In Chemistry Of Mahatma Gandhi University ,Kottayam, Kerala.
BY
Lakshmi D Pillai
University Register No; SBAC10171824
2010-2013
DEPARTMENT OF CHEMISTRY
ASSUMPTION COLLEGE
CHANGANACHERRY

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CERTIFICATE
This is to certify that the dissertation entitled DETERMINATION OF
CAFFIENE CONTENT IN TEA SAMPLES is an authentic record of the
project work done by Ms.ANU THOMAS, Assumption College,
Changanacherry for the Bachelor Degree of Science in Chemistry of Mahatma
Gandhi University, Kottayam under my supervision.
Approved by,
Dr. Jissy Mathew
Department Of Chemistry
Assumption College
Changanacherry

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DECLARATION
I hereby declare that the dissertation entitled ‘DETERMINATION
OF CAFFEINE CONTENT IN TEA SAMPLES’ submitted to Mahatma
Gandhi University in partial fulfillment of the requirements for the award of
the Degree of BACHELOR SCIENCE IN CHEMISTRY is a record of original
work done by me under the Supervision and Guidance of Prof Jissy Mathew
and no part of it has been submitted for any other degree or diploma.

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ACKNOWLEDGMENT
I take this opportunity to express my sincere gratitude to Prof. Jissy
Mathew, Department of Chemistry, Assumption College, Changanacherry for
her guidance and for arranging all the required facilities for this project. I am
also grateful to Prof. Rosili Philip, Head of department of chemistry and to
other professors of the department for their suggestions provided in the
completion of this project. Iam thankful to everyone who helped me in
conducting this work. I am grateful to my parents for their support which has
enabled me to conduct and complete this work.

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CHAPTER -1
INTRODUCTION
Tea is the most commonly and widely used soft beverage in the
household. It is an aromatic beverage commonly prepared by pouring hot or
boiling water over cured leaves of the tea plant Camellia sinensis. It acts as a
stimulant for central nervous system and skeletal muscles. It also increases the
capacity of thinking. The principal constituent of tea, which is responsible for all
these properties, is the alkaloid-caffeine. The amount of caffeine in tea leaves
varies from sample to sample.Orginally it was thought that caffeine is responsible
for the taste and flavour of tea .But pure caffeine has been found to be a tasteless
white substance.

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CONTENTS OF TEA
Tea contains catechins,a type of antioxidant.Catechins can comprise up
to 30% of the dry weight. They are highest in concentration in white and green
teas, while black tea has substantially fewer due to its oxidative preparation
Tea also contains L-theanine, and the stimulant caffeine at about 3% of
its dry weight, translating to between 30mg and 90 mg per 250ml cup depending
upon type, brand etc
Tea also contains theobromine and theophilline.Due to modern
environmental pollution fluoride and aluminum have also been found to occur
in tea, with certain types of brick tea made from old leaves and stems having
the highest levels. This occurs due to the tea plants high sensitivity to and
absorption of environmental pollutants.
Although tea contains various types of poly phenols and tannin, it
does not contain tannic acid.
CAFFEINE

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Systematic (IUPAC) name
1,3,7-trimethyl-1H-
purine-2,6(3H,7H)-
dione
3,7-dihydro-1,3,7-
trimethyl-1H-purine-
2,6-dione
Caffeine is a bitter, white crystalline xanthine alkaloid that acts as
stimulant drug. Caffeine is found in varying quantities in the seed, leaves and

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fruit of some plants, where it acts as a natural pesticide that paralyzes and kills
certain insects feeding on the plants .It is most commonly consumed by
humans in infusions extracted from seed of the coffee plant and the leaves of
the tea bush.
STRUCTURE AND CHEMICAL PROPERTIES
Caffeine is a methylxanthine and methylxanthines are known to
have anti-inflammatory properties. This is due to the similarity in molecular
structure to the nucleotide adenosine. Methylxanthines have a heterocyclic
ring structure; they are derived from amino acids, are basic in nature, and
can generally form water soluble salts. Caffeine blocks the action of
adenosine by acting as a competitive inhibitor for the A1 and A2 adenosine
receptor. With the initial absorption of caffeine occurring within 15 minutes
the peak time of caffeine absorption is estimated at 45 minutes. Although
varying in many studies the half-life of caffeine has been found to be
around 5.2 -6.8 hours for adults. However, the metabolic rate of caffeine
absorption differs between each species.
HEALTH EFFECTS OF CAFFEINE
In humans caffeine acts as a central nervous system stimulant,
temporarily warding of drowsiness and restoring alertness. It is the world’s
most widely consume psychoactive drug, but, unlike many other
psychoactive substances, it is both legal and unregulated in nearly all parts
of the world.
Caffeine is toxic at sufficiently high doses. Ordinary consumption can
have low health risks, even when carried on for years-there may be a modest

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protective effect against some diseases, including certain types of cancer.
Caffeine can have both positive and negative effects on anxiety disorders. Some
people experience sleep disruption if they consume caffeine, especially during
the evening hours, but others show little disturbance and the effect of caffeine on
sleep is highly variable.
Evidence of a risk to pregnancy is equivocal, but some authorities
have concluded that prudent advice is for pregnant women to limit
consumption to the equivalent of two cups of coffee per day or less.
Caffeine has diuretic properties when administered to people, who are not
used to it, but regular users develop a tolerance to this effect, and studies
have generally failed to support the common notion that ordinary
consumption contributes significantly to dehydration. With heavy use,
strong tolerance develops rapidly and caffeine can produce clinically
significant physical and mental dependence.
POSITIVE EFFECTS
❖ Increases levels of neurotransmitters such as norepinephrine,
acetylcholine, dopamine, serotonin, epinephrine and glutamate. Low doses
of caffeine show increased alertness and decreased fatigue.
❖ Caffeine has shown to increase metabolic rate.
❖ Reduces the risk of developing cancer.
❖ Reduces the risk of Parkinson’s disease.
❖ Lower the risk of developing type 2 diabetes.

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❖ Reduce certain kind of hepatic cancer.
NEGATIVE EFFECTS
❖ Increases blood pressure in non –habitual consumers.
❖ May reduce control of fine motor movements.
❖ Stimulate urination.
❖ Increases cortisol secreation, some tolerance is developed.
❖ Can contribute to increase insomnia and sleep tendency.
❖ Caffeine withdrawal produces headache, fatigue and decreased alertness.
❖ Caffiene is additive.
❖ High doses of caffeine can causes anxiety.
❖ Causes auditory hallucinations.

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❖ High caffeine consumptions accelerate bone loss at the spine in elderly post
menopausal women.
Average content of caffeine

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MECHANISM OF ACTION
Caffeine's primary mechanism of action is as an antagonist of
adenosine receptors in the brain.
Adenosine acts as an inhibitor neurotransmitter that suppresses
activity in the central nervous system. Consumption of caffeine antagonizes
adenosine and increases activity in neurotransmission including acetylcholine,
epinephrine, adenosine, dopamine, serotonin, nor epinephrine and glutamate.

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There has also been conclusive evidence that caffeine inhibits acetyl
cholinesterase, an enzyme that breaks down acetylcholine; therefore the
duration of acetylcholine is increased in the nicotinic and muscarinic receptors
in the central nervous system.
Because caffeine is both water-soluble and lipid-soluble, it readily
crosses the blood–brain barrier that separates the bloodstream from the interior
of the brain. Once in the brain, the principal mode of action is as a nonselective
antagonist of adenosine receptors (in other words, an agent that reduces the
effects of adenosine). The caffeine molecule is structurally similar to adenosine,
and is capable of binding to adenosine receptors on the surface of cells without
activating them, thereby acting as a competitive inhibitor.
Adenosine is found in every part of the body, because it plays a role
in the fundamental adenosine triphosphate (ATP) related energy producing
mechanism and is also needed for RNA synthesis, but it has additional functions
in the brain. The evidence indicates that brain adenosine acts to protect the brain
by suppressing neural activity and by increasing blood flow via receptors
located on vascular smooth muscle. Brain adenosine levels are increased by
various types of metabolic stress, including lack of oxygen and interruption of
blood flow. There is evidence that adenosine functions as a synoptically
released neurotransmitter in some parts of the brain; however, stress-related
adenosine increases appear to be produced mainly by extracellular metabolism
of ATP. Unlike most neurotransmitters, adenosine does not seem to be
packaged into vesicles that are released in a voltage-controlled manner, but the
possibility of such a mechanism has not been ruled out fully.
Several classes of adenosine receptors have been described, with
different anatomical distributions. A1 receptors are widely distributed, and act to
inhibit calcium uptake. A2A receptors are heavily concentrated in the basal

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ganglia, an area that plays a critical role in behavior control, but can be found in
other parts of the brain as well, in lower densities. There is evidence that A 2A
receptors interact with the dopamine system, which is involved in reward and
arousal. (A2A receptors can also be found on arterial walls and blood cell
membranes.)
Beyond its general neuroprotective effects, there are reasons to believe
that adenosine may be more specifically involved in control of the sleep-wake
cycle. Robert McCarley and his colleagues have argued that accumulation of
adenosine may be a primary cause of the sensation of sleepiness that follows
prolonged mental activity, and that the effects may be mediated both by
inhibition of wake-promoting neurons via A1 receptors, and activation of sleep-
promoting neurons via indirect effects on A2A receptors. More recent studies
have provided additional evidence for the importance of A2A, but not A1,
receptors.
Caffeine, like other xanthines, also acts as a phosphodiesterase inhibitor.
A number of potential mechanisms have been proposed for the athletic
performance-enhancing effects of caffeine. In the classic or metabolic theory,
caffeine may increase fat utilization and decrease glycogen utilization.
Caffeine mobilizes free fatty acids from fat and/or intramuscular triglycerides
by increasing circulating epinephrine levels. The increased availability of free
fatty acids increases fat oxidation and spares muscle glycogen, thereby
enhancing endurance performance. In the nervous system, caffeine may
reduce the perception of effort by lowering the neuron activation threshold,
making it easier to recruit the muscles for exercise.

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Metabolism
Caffeine is metabolized in the liver into three primary metabolites: paraxanthine
(84%), theobromine (12%), and theophylline (4%)
Caffeine from coffee or other beverages is absorbed by the small intestine
within 45 minutes of ingestion and then distributed throughout all tissues of the
body. Peak blood concentration is reached within one hour. It is eliminated by
first-order kinetics.[114]
Caffeine can also be absorbed rectally, evidenced by the
formulation of suppositories of ergotamine tartarate and caffeine (for the relief
of migraine) and chlorobutanol and caffeine (for the treatment of hyperemesis).
The biological half-life of caffeine – the time required for the body to eliminate
one-half of the total amount of caffeine – varies widely pregnancy, some
concurrent medications, and the level of enzymes in the liver needed for
caffeine metabolism. It can also be significantly among individuals according to

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such factors as age, liver function, altered by drugs or hormonal states. In
healthy adults, caffeine's half-life has been measured with a range of results.
Some measures get 4.9 hours, and others are at around 6 hours. Heavy cigarette
smokers show a decrease in half-life of 30–50%, oral contraceptives can double
it, and pregnancy can raise it even more, to as much as 15 hours during the last
trimester. In newborn infants the half-life can be 80 hours or more; however it
drops very rapidly with age, possibly to less than the adult value by the age of 6
months. The antidepressant fluvoxamine (Luvox) reduces the clearance of
caffeine by more than 90%, and prolongs its elimination half-life more than
tenfold; from 4.9 hours to 56 hours.
Caffeine is metabolized in the liver by the cytochrome P450 oxidase enzyme
system (to be specific, the 1A2 isozyme) into three metabolic
dimethylxanthines, each of which has its own effects on the body:
● Paraxanthine (84%): Increases lipolysis, leading to elevated glycerol and
free fatty acid levels in the blood plasma.
● Theobromine (12%): Dilates blood vessels and increases urine volume.
Theobromine is also the principal alkaloid in the cocoa bean, and
therefore chocolate.
● Theophylline (4%): Relaxes smooth muscles of the bronchi, and is used
to treat asthma. The therapeutic dose of theophylline, however, is many
times greater than the levels attained from caffeine metabolism.
Each of these metabolites is further metabolized and then excreted in the urine.
Caffeine can accumulate in individuals with severe liver disease, increasing its
half-life.

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Some quinolone antibiotics exert an inhibitory effect on the cytochrome P-450
enzyme CYP1A2, thereby reducing clearance of caffeine and thus increasing
blood levels.
A 2011 analysis published by PLoS Genetics reviewed five studies covering
more than 47,000 subjects of European descent. Researchers determined that
habitual caffeine intake is associated with variations in two genes that regulate
how quickly the body processes caffeine. Subjects who had a high-intake
mutation of either gene on both chromosomes consumed 40 mg more caffeine
per day (equivalent to a can of cola) than people who did not.

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CHAPTER -2
METHODS AND MATERIALS
Reagents Used
Kannan Devan Tea -50g
➢AVT Tea -50g
➢Red Label Tea[Brook Bond ] -50g
➢Lead Acetate -as per requirement

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➢Chloroform -20 ml
➢Distilled Water -150ml
PROCEDURE
♦ First of all, 50 grams of tea powder were taken as samples and
150 ml of water was added to it in a beaker.
♦ Then the beaker was heated up to extreme boiling.
♦ The solution was filtered and Lead f acetate was added to the
filtrate, leading to the formation of a curdy brown coloured precipitate
♦ Lead acetate was added till no more precipitate has been formed.
♦ Again solution is filtered .
♦ The filtrate so obtained was heated until it had become 50 ml.

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♦ Then the solution left was allowed to cool.
♦ After that, 20 ml of chloroform was added to it.
♦ Then the whole solution is transferred to a separating funnel.
♦ Soon after, two layers appeared in the separating funnel.
♦ The aqueous layer is separated.
♦ The organic layer then exposed to atmosphere in order to allow
chloroform to get evaporated.
♦ The residue left behind was caffeine.
♦ Then it is weighed and observations are recorded.

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CHAPTER -3
RESULT AND DISCUSSION
Caffeine content in three different tea samples are estimated as follows:
Sample1-Kannan Devan Tea
Weight of sample taken =50 g
Weight of china dish =61.9708 g
Weight of china dish with precipitate=62.9313g

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Amount of caffeine =0.9605 g
Sample 2- Brook Bond
Weight of sample taken =50 g
Weight of china dish = 63.9837 g
Weight of china dish with precipitate =64.6855g
Amount of caffeine =0.7018g

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Sample 3-AVT
Weight of sample taken =50g

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Weight of china dish =57.1205 g
Weight of china dish with precipitate =57.4490g
Amount of caffeine =0.3285g

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CONCLUSION
The caffeine content in various tea samples are found as follows:
Kannan devan > Brooke Bond > AVT
0.9605 > 0.7018 > 0.3285
Caffeine is a compound in tea which has both positive and negative
effects.Caffiene,upto a particular amount is good for health due to its anti oxidant
properties.At the same time its overuse can lead to serious health problems and
may even lead to addiction.So,caffeine content in our diet should be in a
controlled level.
Keeping on mind the above facts, AVT is the preffered brand among the samples
under consideration.

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REFERENCE
1. Food and bevereage service 6th
edition
-Dennis Lillicrap, Johncousins& Robert Smith
2. Food and Beverage Service
-Vijay Dhawan
3 .Food and Beverage Service and Management
-Bobby George & Sandeep P Chatterjee
4. Food science 5th
edition
-Norman N Potter
-Joseph H Hotchkiss
5. Foods facts & principles 2nd
edition
-N. Shakuntala Manay
-M. Shadaksharaswamy
6. Food science chemistry & experimental foods
-Dr.M Swaminathan
7. Food science & Nutrition
-M.N Ahmed
8. Food, Nutrition, & Diet Therapy
-L. Kathlen Mahan
-Sylvia Escott -Stump

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