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Candidate code: klm897

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Topic: Determining the tea caffeine content using DCM-facilitated separation funnel

Research question: How does the source of caffeine (Lipton Earl Grey, Lipton Yellow Label,
Remsey Earl Grey, Milton Earl Grey Classic) affect its content (in g), determined using the
DCM-facilitated separation funnel technique?

Introduction

Tea is a globally consumed beverage that holds a significant place in human culture and daily
rituals. Notably, its stimulating component, caffeine, has attracted scientific interest due to its

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physiological effects and potential health benefits. The following study aims to explore how
the source of caffeine, specifically in various types of tea, influences its content. The

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experiment utilizes the Dichloromethane (DCM)-facilitated separation funnel technique, a

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method known for its efficacy in isolating caffeine from aqueous solutions. As such, the aim
of this investigation is to examine the caffeine content in commercially popular tea brands such
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as Lipton Earl Grey, Lipton Yellow Label, Remsey Earl Grey, and Milton Earl Grey Classic,
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and determine potential variations in caffeine levels among them.

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Background information
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Tea
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Tea is a widely used beverage, typically made from infusion of hot or boiling water from leaves
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of Camellia sinensis (L.) plant, native to East Asia. Harvest of leaves varies depending on the
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type of tea, however the preparation of black tea involves picking, withering, bruising,
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oxidating, shaping and drying of the leaves (www.amli.com, 2024). This allows the tea to gain
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its rich flavor. The caffeine produced by young Camellia leaves is a natural insecticide,
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however its neuromodulating effects have allowed tea to become one of the most popular
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stimulant beverages. And as several studies demonstrated potential health benefits of drinking
tea, including decrease in the risk of myocardial infarction, tea proves to be one of the best
sources of caffeine available, biochemically balanced with numerous polyphenols associated
with health markers (Ruxton, 2008).

Caffeine

Caffeine (C8H10N4O2) is a xanthine, classified more generally as a purine alkaloid. Isolated,

it presents as a white, crystalline solid, easily solvable in organic solvents and slightly less in
water. Structurally, caffeine is formed from a purine ring (containg two N atoms) fused to a
xanthine ring (containg both N and O atoms) (Dews, 1982). Additionally, caffeine features four
hydroxyl groups (-OH) attached to the xanthine ring and a methyl group (-CH3) linked to the
nitrogen atom on the purine ring, with a molecular weight of 194.19 g/mol. Synthesized from
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xanthine, a derivative compound of adenine, a nucleotide present in DNA and RNA, caffeine
synthesis predominantly occurs in the leaves, seeds, and fruits of various plants. It is believed
to function as a natural insecticide and a deterrent against herbivores (Zhao et al., 2023).

Caffeine is partially responsible for the bitter taste of coffee and tea. Functioning as a stimulant
on the central nervous system, caffeine exerts various physiological effects on the body. It is a
common constituent in beverages like coffee, tea, and soda, as well as in some pharmaceuticals
and dietary supplements. While typically considered safe in moderate consumption, excessive
intake of caffeine can lead to adverse effects (Spriet, 1995).

Dichloromethane (DCM)

Historically, a significant portion of caffeine production in the United States resulted from the

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methylation of theobromine, isolated from cocoa. This process employed methylation agents
such as dichloromethane. It is a chlorinated hydrocarbon, presenting as a transparent, colorless

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liquid that is volatile and nonflammable. It must be noted that dichloromethane has excellent

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solvent properties, with caffeine being much more soluble in DCM (140 mg/ml) than in water
(22 mg/ml), which allows for the extraction of this compound from aqueous solutions like
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various beverages (Chaugule et al., 2019).
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Dichloromethane facilitated separating funnel.

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Immiscible fluids are fluids that do not mix with each other when poured together. When two
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of such liquids are introduced into a separating funnel, distinct layers become observable as
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they retain their individuality. In the context of this experiment, the immiscible liquids are the
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tea mixture and dichloromethane. They are introduced into the funnel, and the flow of each
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liquid is regulated using a tap, allowing them to exit from the bottom of the funnel at controlled
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rates. They can then be collected in separate containers for further processing. Notably, the
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denser solvent, in this case, dichloromethane, settles in the lower layer. Then, using a tap it can
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be drained from the funnel and left to evaporate (being a very volatile substance), leaving
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crystallized caffeine in the vessel.

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Methodology

Variables

Independent variable: The type of tea (Lipton Earl Grey, Lipton Yellow Label, Remsey
Earl Grey, Milton Earl Grey Classic).

Five widely available brands were chosen for this experiment to test their varying caffeine
content and hence determine their suitability as an energizing drink.

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Dependent variable: The caffeine content of tea infusions (g).

The caffeine was isolated from the tea infusion using the DCM-facilitated separating funnel,
forming crystalline precipitate which was weighed using a digital scale. As each type of tea is
processed, packaged, and stored slightly differently, as well as coming from different plants
and plantations, the variability in caffeine content in different tea types could be quite
significant.

Controlled variables

Table 1. Controlled variables

Controlled variable Method of control Reason for control

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Temperature of the solution impacts
Tea solution was allowed
Temperature of the the rate of reactions that allow for the

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to cool to 35.0℃ before
tea solutions release of caffeine from the tea leaves,

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pouring it into the funnel.
hence it was kept constant at 35.0℃.

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The volume of the water impacted the
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200 ml of water was used concentration of the tea solution by
Volume of water
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for each trial. impacting the distribution of caffeine,

hence it needed to be kept constant.
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The number of holes directly impacted

32 holes were poked in the the evaporation rate and hence also the
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Number of holes in
aluminum foil covering the final mass of the caffeine precipitate,
the foil
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Petri dish as the water remnants could have

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increased the mass of the precipitate.

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The amount of tea leaves directly

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5.000 g of tea leaves were correlated with the amount of caffeine

Mass of tea leaves
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weighed for each trial that would be released from them into
the experimental solution.
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As the dichloromethane was directly

15 ml of dichloromethane responsible for the extraction of
Mass of DCM
was mixed with the tea caffeine from the solution, its volume
added
solution directly impacted how much caffeine
was obtained.

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Materials
● 4 x 250 ±5ml beaker ● Distilled water
● 4 x 250 ±5ml conical flask ● Weighing boats
● 1 x 250 ±0.1ml graduated cylinder ● Petri dish
1 x 25 ±0.5ml graduated cylinder ● Sticker labels
● 1 ±0.1ml pipette ● Stirrer
● 1 x 250ml separating funnel ● Spatula spoon
● 1 x 75mm funnel ● Ring stand
● 1 x Thermometer ±0.1℃ ● Aluminum foil
● 1 x Digital balance ±0.001g ● Filter paper
● Dichloromethane

Preliminary work

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As the literature (Chaugule et al., 2019) suggested, tea samples can contain up to 75 mg (0.075

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g) of caffeine per 200 ml. At the same time, the solubility of caffeine in water at 25 degrees

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Celsius was around 22 mg/ml, and in DCM up to 140 mg/ml. Hence, it was determined that
the volume of the solution and amount of tea leaves would not prove to be limiting to the
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capability of the method to extract all the caffeine. Therefore, the experimental volumes were
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determined by the size of the funnel available, which could store up to 250 ml of liquids. Using
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almost the maximum possible volume of the solution (200 ml plus 15 ml of DCM) allowed
also for extracting of as much caffeine as possible, therefore increasing its mass and decreasing
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percentage uncertainty of the results.

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A preliminary experiment was conducted using freshly boiled tea infusions. It was observed
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that tea started to react with dichloromethane, producing bubbles and unpleasantly smelling
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vapors. Therefore, particularly for safety reasons, in the main experiment tea was allowed to
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cool down before being introduced into the reaction chamber.

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Procedure
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Figure 1. Dichloromethane separating funnel (image made using Chemix software)

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1. 200ml of distilled water was boiled.

2. Approximately 5g of Lipton Earl Grey Tea leaves were weighed out.
3. The tea leaves were poured and stirred in the boiled water in the beaker, then left for 5
minutes to infuse.
4. The tea leaves were filtered and separated from the tea solution into a 250ml conical
flask using filter paper and a funnel.
5. 15ml of dichloromethane was measured into a 25ml measuring cylinder using a 1ml
pipette.
6. The separating funnel was prepared and placed on a ring stand, ensuring the separating
funnel was closed.
7. The tea solution was poured into the 250ml separating funnel once it reached 35oC.
8. 15ml of dichloromethane was poured into the tea solution in the separating funnel.
9. A cork was placed in the opening of the funnel, and the solution was shaken and mixed

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while releasing pressure every 10 seconds.
10. A conical flask was placed under the funnel, and the dichloromethane mixed solution

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was allowed out of the funnel slowly to avoid any tea entering and mixing with the

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solution (as seen on Figure 1).
11. Steps 8-10 were repeated three more times. 7@
12. The petri dish was labelled with the brand of the tea and weighed out.
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13. The final dichloromethane solution was poured into the labelled petri dish.
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14. The petri dish was covered with aluminum foil, and 32 holes were poked for
evaporation.
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15. The solution was left to evaporate, and caffeine crystallized.

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16. The petri dish was weighed, and the caffeine content was found by the difference in
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masses.
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17. Steps 1-16 were repeated with Lipton Earl Grey, Lipton Yellow Label, Remsey Earl
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Grey, and Milton Earl Grey Classic.

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Safety concerns
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● Protective gloves, glasses and laboratory coat were always worn to prevent exposure to
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dichloromethane, which acts as a potent skin irritant.

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● A face mask was always worn to minimize the risk of inhalation of dichloromethane,
which may cause symptoms such as headaches, light-headedness, and agitation.
● Dichloromethane was only poured after tea had already cooled down, as this induces a
reaction in which irritating vapors would be evolved.

Ethical issues

As no living organisms were involved in the experiment, no ethical issues were identified.

Environmental issues

The dichloromethane solution was disposed of per the local chemical waste disposal protocols
instead of being poured straight down the drain to prevent water pollution.
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Data

Qualitative data
● After pouring dichloromethane into tea, bubbles on the surface of the solution and a
strong smell of dichloromethane were noted
● The purified caffeine had the appearance of a light, crystalline solid

Quantitative data

Table 2. Raw data on the amount of caffeine obtained per each tea type (in mg)

Tea type
Mass measurement
Trial Lipton Milton

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(±0.001g) Lipton Remsey
Yellow Earl Grey
Earl Grey Earl Grey

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Label Classic

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gm
Petri dish without caffeine 37.121 46.286 40.494 34.451
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1 Petri dish with caffeine 37.148 46.310 40.518 34.475
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Difference (caffeine mass) 0.027 0.024 0.024 0.024

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Petri dish without caffeine 37.121 46.286 40.494 34.451

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Petri dish with caffeine 37.149 46.312 40.515 34.474

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0.028 0.026 0.021 0.023

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Difference (caffeine mass)

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Petri dish without caffeine 37.121 46.286 40.494 34.451

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3 Petri dish with caffeine 37.146 46.308 40.516 34.475

Difference (caffeine mass) 0.025 0.022 0.022 0.024

Petri dish without caffeine 37.121 46.286 40.494 34.451

4 Petri dish with caffeine 37.147 46.314 40.518 34.476

Difference (caffeine mass) 0.026 0.028 0.024 0.025

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Data processing

Formula 1. Obtaining the mass of caffeine

𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑚𝑎𝑠𝑠 = 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑃𝑒𝑡𝑟𝑖 𝑑𝑖𝑠ℎ 𝑤𝑖𝑡ℎ 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 − 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑃𝑒𝑡𝑟𝑖 𝑑𝑖𝑠ℎ
𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑚𝑎𝑠𝑠 (𝑓𝑜𝑟 𝑡𝑟𝑖𝑎𝑙 1 𝑜𝑓 𝐿𝑖𝑝𝑡𝑜𝑛 𝐸𝑎𝑟𝑙 𝐺𝑟𝑒𝑦 = 37.148 − 37.121 = 0.027

Formula 2. Calculating the average caffeine mass per trial

𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑚𝑎𝑠𝑠 𝑓𝑜𝑟 𝑡𝑟𝑖𝑎𝑙 1 + . . . + 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑚𝑎𝑠𝑠 𝑓𝑜𝑟 𝑡𝑟𝑖𝑎𝑙 4

𝑎𝑣𝑒𝑟𝑎𝑔𝑒 =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡𝑟𝑖𝑎𝑙𝑠 (4)
0.027+. . . +0.026
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑜𝑟 𝐿𝑖𝑝𝑡𝑜𝑛 𝐸𝑎𝑟𝑙 𝐺𝑟𝑒𝑦 = = 0.027
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Formula 3. Calculating the standard deviation of the caffeine mass per trial

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𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 (𝑆𝐷) 𝑝𝑒𝑟 𝑡𝑒𝑎 𝑡𝑦𝑝𝑒

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∑ (𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑚𝑎𝑠𝑠 𝑝𝑒𝑟 𝑡𝑟𝑖𝑎𝑙 − 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑝𝑒𝑟 𝑡𝑒𝑎 𝑡𝑦𝑝𝑒)2
=G

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𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡𝑟𝑖𝑎𝑙𝑠 𝑝𝑒𝑟 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛
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𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 (𝑆𝐷)𝑓𝑜𝑟 Lipton Earl Grey
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(0.027 − 0.027)! + (0.028 − 0.027)! + (0.025 − 0.027)! + (0.026 − 0.027)!

= G
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= 0.0013 (2 𝑠. 𝑓)
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Table 3. Average and standard deviation

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Tea type Average [g] 2 s.f. Standard deviation 2 s.f.

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Lipton Earl Grey 0.027 0.0013

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Lipton Yellow Label 0.025 0.0013

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Remsey Earl Grey 0.023 0.00076

Milton Earl Grey Classic 0.024 0.00035

This data was then used to construct a bar graph presented below.

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Graph 1. Caffeine content (in grams) per tea type. Error bars represent standard deviation.

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gm
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Uncertainty calculations
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Formula 3. Percentage uncertainty of measurement

𝑎𝑏𝑜𝑠𝑙𝑢𝑡𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦
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𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 = × 100%

𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑚𝑒𝑛𝑡
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0.001
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𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑓𝑜𝑟 𝐿𝑖𝑝𝑡𝑜𝑛 𝐸𝑎𝑟𝑙 𝐺𝑟𝑒𝑦 = × 100% = 3.8%

0.027
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Table 4. Percentage uncertainty

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Tea type Percentage uncertainty [%]

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Lipton Earl Grey 3.8

Lipton Yellow Label 4.0

Remsey Earl Grey 4.4

Milton Earl Grey Classic 4.2

Analysis and discussion

The study was successful in determining the caffeine content of the set volumes of various tea
type infusions. The highest caffeine content (on average 0.027 g) was demonstrated in Lipton
Earl Grey, whereas the lowest was seen in Remsey Earl Grey solution (0.023 g). It must be

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noted that the differences in the caffeine content proved to be relatively small, not exceeding
20% and 0.004 g in absolute values. This shows that although the values differ slightly, it is
debatable whether such differences have any perceptible impact on the drinking experience of
the consumer. Another interesting observation was that both the highest and lowest content of
caffeine was found in Earl Grey type of tea, which differed however quite significantly in price.
This may provide an insight into the heterogenous nature of the quality tea products available
on the market, measured in this case by the caffeine content of the leaves, which might have
been impacted by the differing processing techniques.

Although no producer data could have been obtained on the declared caffeine content of these
types of tea, the found values lay within the range demonstrated by other research papers,
namely between 0.014 and 0.061 grams per 200 ml (Chin et al., 2008). This, alongside the low
values for both percentage uncertainty (<4.2%) and standard deviation (<0.001291), supports

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the reliability of the produced results.

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Conclusion

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To conclude, the results of the experiment determined the caffeine content of all studied types
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of tea infusions (5 g of leaves per 200 ml of water, 5 minutes of infusion), respectively Lipton
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Earl Grey (0.027 g), Lipton Yellow Label (0.025 g), Remsey Earl Grey (0.023 g), Milton Earl
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Grey Classic (0.024 g). The differences between the tea varieties proved to be relatively small,
demonstrating relative uniformness of the caffeine content across different brands and types of
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tea leaves. Low values for both the percentage uncertainty and standard deviation allow for the
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acceptance of this conclusion.

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Evaluation
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Strengths
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● Repetitions - for every condition, 4 repetitions of the experiment were conducted. This
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design allowed for the gathering of larger amounts of data, greatly increasing the
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reliability of the results, especially as the tea tended to escape the funnel, which could
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have impacted the amount of caffeine noted per single trial.

● Conducting the preliminary experiment - the results of the preliminary experiment
allowed for improvements in the design of the work, including the prevention of the
leakage of tea from the funnel.
● The employment of a precise balance - a digital balance with uncertainty of ±0.001g
was used. This allowed for the precise determination of the mass of caffeine in each tea
sample.

Weaknesses [W] and Limitations [L]

● Incomplete evaporation of dichloromethane [W] - it was difficult to assess whether
DCM has managed to evaporate completely from the solution, which could have
potentially increased the mass of caffeine. This problem could have been potentially
alleviated if the evaporation time was increased, for instance to several hours.
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● Leakage of tea [W] - particularly in the preliminary investigation, tea would sometimes
leak through the tap during the drainage of DCM, which could have impacted the
composition of this solution and hence also the final caffeine mass. Instead, the pouring
of dichloromethane could have been performed at a slower rate, minimizing the chance
of spillage.
● Substances dissolved in dichloromethane [L] - as DCM is a potent solvent for a variety
of widely identified organic compounds, it is possible that the solids weighed at the
ended of the experiment also included substances like nucleotides or alkaloids. To
improve this, further purification of caffeine could have been performed or the samples
could have been analyzed using techniques like infrared spectroscopy to determine the
purity of the sample.

Extensions

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● Studying the caffeine content of other beverages - there are numerous drinks with
varying contents of caffeine available, including coffee, cocoa, and energy drinks. Their

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caffeine content could be measured and compared, to find the most caffeinated one.

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● Measuring the impact of temperature on caffeine content - although caffeine appears to
be relatively thermally stable, heating beverages may contribute to its decomposition.
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This could be tested by measuring caffeine mass in drinks like tea heated up to
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subsequent intervals of temperature.

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Chin, J.M., Merves, M.L., Goldberger, B.A., Sampson-Cone, A. and Cone, E.J. (2008).
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doi:https://doi.org/10.1093/jat/32.8.702.

Dews, P.B. (1982). Caffeine. Annual Review of Nutrition, 2(1), pp.323–341.

doi:https://doi.org/10.1146/annurev.nu.02.070182.001543.

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Spriet, L.L. (1995). Caffeine and Performance. International Journal of Sport Nutrition, 5(s1),
pp.S84–S99. doi:https://doi.org/10.1123/ijsn.5.s1.s84. 7@
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Zhao, L., Wei, J., Hu, Y., Pi, D., Jiang, M. and Lang, T. (2023). Caffeine Synthesis and Its
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Mechanism and Application by Microbial Degradation, A Review. Foods, 12(14), pp.2721–

2721. doi:https://doi.org/10.3390/foods12142721.
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www.amli.com. (2024). Different Teas and How They’re Made. [online] Available at:
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https://www.amli.com/blog/different-teas-and-how-theyre-made.
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