Extraction Of Caffeine From Green And Black Tea

 The aim of the investigation was to determine the quantity of caffeine present in both green tea and black tea bags through extraction and make a comparison of the results. The samples were tested using mixed melting point and thin layer chromatography to ensure there was caffeine present. A pure caffeine control showed after losses the average yield was only 41%. The average content of caffeine in one black tea bag was 0.100g whereas the content in one green tea bag was 0.057g meaning that there is a higher caffeine content in Tetley’s black tea than Tetley’s green tea. To analyse the purity of the extracted caffeine mixed melting point was used. The results suggest Tetley’s black tea was purer than Tetley’s green tea as it had a lower range over which it melted (black tea, on average the range was 12°C which was closer to the pure caffeine which was 11°C, than the green tea caffeine which was not determined from the extraction samples.) Introduction Caffeine Caffeine is the more well-known name for trimethylxanthine (1,3,7-trimethylxanthine or 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione). The molecular formula for caffeine is C8H10N4O2. (ThoughtCo, n.d.) The structural formula of caffeine is as shown - (MolView, n.d.) This diagram shows the different bonds present in a caffeine molecule, these are both sigma and pi bonds. Covalent bonds when there is an end on end overlap are sigma bonds and pi bonds are where there is a side on overlap. Pi bonds arise when an atom forms multiple bonds, such as double or triple bonds. (Anon, n.d.). A double bond is made up of one sigma bond and one pi bond. A triple bond is made up of one sigma bond and two pi bonds. Pi bonds are weaker than sigma bonds and this explains why a c=c bond is not twice as strong as a c-c bond. (Gibb, 2015) 

The VSEPR (Valence Shell Electron Pair Repulsion) rules are a model for predicting the different shapes of atoms. As electron pairs are negatively charged they will repel each other and they will arrange themselves in such a way to reduce the repulsion and increase the distance between electron pairs. This then creates different angles between electron pairs. The nitrogen that is attached to three carbons gives a trigonal planar shape as the nitrogen has one lone pair, this makes repulsion between the lone pair and the bonded pair, this then gives it an angle of 109.5 degrees. The shape of the carbon double bonded to an oxygen and to either 2 nitrogens or nitrogen and a carbon gives a trigonal planar and gives a bond angle of 120 degrees. The nitrogen bonded to 2 carbons has an angle of 104.5 degrees due to the repulsion from the lone pair on the nitrogen atom. (Gibb, 2015) Caffeine is a naturally occurring compound and is produced by plants and it is called theine when it’s found in tea. It is a xanthine alkaloid. (Scienceofcooking.com, n.d.) The molecular weight of caffeine is 194.194g/mol. (Chemspider.com, n.d.) Caffeine is water soluble as it is a polar molecule and like dissolves like, this makes it easy to enter and affect the body. Caffeine has a similar structure to adenosine. Adenosine is made to slow down the speed that nerve cells are working at, as they have similar structure caffeine is able to bind to the adenosine receptors in the body and have the completely opposite effect resulting in increased nerve stimulation which is why caffeine makes people feel full of energy (American Chemical Society, n.d.) Caffeine also has characteristics that are shared with drugs such as amphetamines, cocaine and heroin. Caffeine is a competitive antagonist, this means that it binds more strongly to the receptor site and so the natural response is not triggered. (caffeine - receptor interactions) Solvent extraction Immiscible liquids are liquids that do not mix together, this is what solvent extraction uses to extract the product that is desired. The heavier of the two liquids that do not mix will sink to the bottom of the separating funnel, this is the easier to drain as there will be a distinct line of where one solution finishes and the other one starts.  When doing a caffeine extraction with, the caffeine is first dissolved in water to get the caffeine from the tea bags, and it dissolves in water as they are both polar substances. It is then separated using ethyl ethanoate, this is also a polar liquid which allows both the water and caffeine to mix with it. This makes two layers form. Some of the caffeine from the water moved into the ethyl ethanoate layer until equilibrium is reached and since the two layers are very clear the aqueous layer can be drained and the layer which the caffeine is in can be kept in another beaker. Any product that has been dissolved in an organic solvent is treated with an anhydrous salt, in this case, it is sodium sulphate and this removes any organic compounds from the sample. (partition coefficient - multiple extractions). 

 Thin layer chromatography is a fast way to determine if certain compounds are in a sample and to separate a sample. It is done using a chromatography plate, a solute and samples. There are a stationary phase and a mobile stage. The stationary phase is the water that the caffeine is dissolved in and the mobile phase which moved through the chromatography plate and carries the components of the mixture with it. The compounds in the sample travel at different speeds and this is how it separates, some compounds travel further than others and these can be compared against a pure sample to determine what the sample contains. The distance travelled relative to the solvent is the Rf value and is determined using Distance travelled by compoundDistance travelled by solvent. (Gibb, 2015) Mixed melting point analysis Mixed melting point analysis is used to determine the purity of a compound. A pure compound will have a melting point and the range over which it melts will be relatively short whereas if a compound is impure the melting point will be lower than the norms and will melt over a wider range.

Weigh 4 tea4 tea bags were weighed and placed in a 500cm3 beaker 250cm3 of water was added to the beaker with 5g of sodium carbonate and the mixture was heated to boiling where it was left to boil for 15 minutes. The mixture was left cool The tea bags were removed from the solution and squeezed out to ensure losses were reduced as much as possible 50cm3 of ethyl ethanoate was added to the tea solution. The mixture was then poured into a separating funnel. The glass stopper was placed into the top of the funnel and then the funnel was gently inverted. While the funnel was upside down the stopcock was opened to release the pressure build up (warning: this was not pointed at anyone some solution was to come out). The stop clock was then again closed and the funnel was placed back on the clamp stand. The layers then separated and the aqueous layer was collected in one beaker and the ethyl ethanoate upper layer was collected into another. The solvent layer was then separated into a 600cm3 beaker and set aside. Pour the aqueous layer back into the separating funnel. 50cm3 more ethyl ethanoate was added to the funnel. Then once again it was gently inverted as described above, vent it, and the organic layer was collected into the same 600cm3 beaker and then the organic layer was drained into its collection beaker. 5g of sodium sulphate was then added to remove any organic compound that was still present in the solution. After 5 minutes the solution appears clear (not brown and cloudy as before as all the water has been removed). A clean, dry, 250cm3 beaker was weighed with 2 boiling chips in it. Using a funnel and filter paper, the liquid was filtered into the preweighed beaker. The solvent was then boiled off using a hot plate. The ethyl ethanoate boiled off at a relatively low temperature (77c), so the hot plate was not used on a very high temperature. Do not heat completely dry, leave in the fume cupboard until the following lesson to allow the last of the ethyl ethanoate to evaporate off. The ethyl ethanoate was not completely boiled off. The beaker was left in the fume hood till the next lesson to allow the ethyl ethanoate to completely evaporate off. The mass of the beaker was then reweighed to calculate the mass off caffeine extracted. This process was then repeated twice for each of the types of tea investigated. 

 For the control experiment 0.2g of caffeine was added at the start of the process before the tea bags were boiled to see how much caffeine was lost during the extraction process. Risk assessment of caffeine extraction Significant Hazards Present. Steps that were taken for safety Ethyl ethanoate - Hazardous in case of ingestion, or inhalation. - Slightly hazardous in case of skin contact (irritant permeator)or eye contact (irritant). - Flammable - Wear goggles - Kept in the fume hood - Well ventilated room - Use a hot plate instead of a Bunsen flame as the chemical is easily flammable. Sodium Carbonate - Hazardous in case of; skin contact (irritant), of eye contact (irritant), of ingestion, or of inhalation (lung irritant) - Wear goggles. Thin layer chromatography The TLC plate was prepared by a line being drawn 1cm above the bottom of the plate and where the samples will go were labelled. 10mg was put into a small beaker with 4 ml of water to dissolve it. The capillary tube was then used to spot the samples on the Tlc plate in small but concentrated spots 0.5cm of water was put into the bottom of a jar, the jar was able to hold the plate upright TLC plate was placed in the jar upright. The solvent was left to move up the TLC plate until it was 2cm from the top. The plate was removed from the jar and the solvent front was marked. The plate was then left to dry. A UV lamp was used to see the spots and photos were taken to use for analysis The distance that the solvent front was travelled to the nearest mm was measured The distance to the centre of the spots was then measured to the nearest mm, this was used to calculate the Rf value MIxed melting point analysis A capillary tube with one sealed and one open end was collected. The sample was then ground together with the same weight of pure caffeine using a mortar and pestle. The mixed sample was put onto a watch glass. Then the capillary tube was filled to approximately 3mm high with the mixed caffeine crystals The capillary tubes were then placed in the melting point analysis apparatus where there was space for 3 samples so a pure caffeine sample, sample of green tea caffeine, and black tea caffeine were all placed in at the same time. The temperature that the samples started melting at was observed and recorded. The temperature that the sample was completely melted as also recorded. The pure sample of caffeine was used to see the accuracy of the instrument's measurements. 

nExtraction Caffeine Added (g) Mass of 4 tea bags (g) Mass of Beaker with Bumping Granules (g) Mass of Beaker with Bumping Granules and Caffeine (g) Mass of Caffeine extracted (g) Mass of caffeine per tea bag (g) Green Tea 1 0 8.691 107.518 107.577 0.059 0.01475 Green Tea 2 0 8.627 105.445 105.559 0.114 0.0285 Black Tea 1 0 13.525 120.043 120.172 0.129 0.03225 Black Tea 2 0 13.541 110.918 111.117 0.199 0.04975 Control/ Decaf Tea 1 0.2 13.211 116.504 116.589 0.085 0.02125 Control/ Decaf Tea 2 0.2 13.306 115.328 115.407 0.079 0.01975 Mean caffeine in one green tea bag - 0.021625g Mean caffeine in one black tea bag - 0.041g Percentage yield as shown by control experiments - (0.085+0.079)/20.2100 = 41% The actual mass of caffeine in one Tetley green tea bag - 0.02162541100 = 0.0527g The actual mass of caffeine in one Tetley black tea bag - 0.04141100 = 0.1g Thin layer chromatography First extraction samples - Caffeine sample Distance travelled by solvent front (mm) Distance travelled by sample (mm) Rf value (distance travelled by sample/ distance travelled by solvent front) Green tea 1 65 22 0.338 Black tea 1 65 22 0.338 Pure 65 25 0.385 Caffeine sample Distance travelled by solvent front (mm) Distance travelled by sample (mm) Rf value (distance travelled by sample/distance travelled by solvent front) Green tea 2 48 23 0.479 Black tea 2 48 23 0.479 Pure 48 24 0.5 Mixed melting point analysis Caffeine sample Start of melting point (°C) End of melting point (°C) Green tea 1 190 N/A* Black tea 1 213 237 Pure 217 232 Caffeine sample Start of melting point (°C) End of melting point (°C) Green tea 2 211 224 Black tea 2 218 228 Pure 223 230 *this is thought to be due to the inorganic material present in the green tea extract. This was to be expected due to the discolouration observed in the extracted caffeine sample from the green tea bag. The crystals in the black the samples were much closer to being pure white than that of the green tea samples, suggesting that there are more impurities in the green tea samples than that of the lack teas samples. 

During this procedure, the separation layer was often hard to determine accurately as there was not always a clear separation as shown in figure 1. This was fixed by draining the very bottom layer of the mixture and re-inverting the funnel and leaving it for longer on the clamp stand to let it separate, this was successful when done several times and made it easier to see the separation and drain the aqueous layer. The yield acquired may have been affected as the process takes a long time to complete from beginning to end so the mixtures were often left overnight as it could not be completed in a school day, this may mean that sometimes the tea bags had more time to dissolve the caffeine into the water than others when it was left. This difference is shown between the difference between the first and second green tea extraction as the second extraction was left to brew for much longer, overnight, compared to the first which was not. The control experiment done with a known amount of caffeine and a decaf tea bag was done to calculate losses during the extraction process. It was calculated that only 41% of the caffeine added to the decaf tea bag was actually extracted during the experiment. This means that there was a 59% loss during the process, but although this is a lot the process was kept the same (apart from the increased inversion) throughout the investigation to ensure the results were consistent and reliable. These losses may be due to the caffeine not dissolving fully in the ethyl acetate, this could have been improved by splitting the ethyl ethanoate into smaller volumes when adding it to the separation funnel and performing more smaller extractions to the tea mixture. This would have increased the yield and reduced loses. When measuring yields a three decimal point scale was used to reduce uncertainties when measuring such small amounts Mixed melting point analysis The mixed melting point analysis may have been improved by using more accurate equipment, this conclusion came from the pure caffeine sample that was used both times that the green and black tea samples were tested as a control. These results showed that the mixed melting point apparatus underestimates the temperature as the pure caffeine sample started melting at 220°C on average rather than 235 °C which it should. This means that the actual melting points of the caffeine extracted from the tea bags will be lower than the values in the table. The impurities in tea will reduce the value of the melting point. 

The control experiment done with a known amount of caffeine and a decaf tea bag was done to calculate losses during the extraction process. It was calculated that only 41% of the caffeine added to the decaf tea bag was actually extracted during the experiment. This suggests that the actual caffeine content in one Tetley's black tea bag is 0.1g. The actual amount of caffeine in a Tetley’s green tea bag is 0.0527g. Internet norms show that a black tea bag will normally have 60mg and green tea will normally contain 50mg. (Bigelowtea.com, n.d.) Thin layer chromatography As seen in figure 2 and 3 the thin layer chromatography showed that there is caffeine in the samples extracted from both green and black tea. This can be seen when looking at the spot made from the pure sample of caffeine as on both TLC plates there is direct correspondence from the spot to both the black and green tea samples. The TLC was visualised using UV light, so there may be some impurities on this which cannot be seen in visible or UV light which may have affected the yield and/or the melting point. Mixed melting point analysis These results showed that green tea was much less pure than black tea. This is shown by the mixed melting point analysis of the first green tea sample as it started melting then turned black and did not have an endpoint as it just turned black, this can be seen in the top and a middle capillary tube in figure 5. This may be due to the fact that green tea has several different components in its makeup such as impurities such as tannins, a variety of different vitamins and plant pigments (Ochadokoro.com, n.d.) These impurities were predicted to affect the result before the melting point analysis was performed as the extracted samples had a green colour to them, no white like pure caffeine. The impurity in the green tea can also be seen from when it did melt with the second extraction sample it started at 211°C and ended at 224°C much lower than the pure caffeine. Both the green and black tea extracted caffeine having a lower melting point than pure caffeine on average may be due to both types of tea containing flavonoids which are biochemical compounds found in tea. (Tetleyharris.com, n.d.). A lower melting point value over a wider range indicates that a sample is not pure. Conclusion Using solvent extraction it was found that on average a Tetley’s green tea bag contains 0.0527g of caffeine which is 2.165% of a tea bags total mass. In Tetley's black tea it was found that there was an average of 0.1g of caffeine per tea bag which is 2.956% of tea bags total mass. Overall it was found that Tetley’s black tea bags have a higher caffeine content than Tetley’s green tea bags on average. Black tea having more caffeine than green tea was confirmed by internet research (Bigelowtea.com, n.d.). Through analysis methods, thin layer chromatography and mixed melting point analysis, it can be concluded that there is was caffeine present in all of the extractions done. 

29 April 2022
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