Isolation of Caffeine From Tea

Isolation of caffein From TeaJames MaitlandXanthines are purine alkaloids synthesised in plants much(prenominal) as coffee, tea, and cacao, with the most known xanthine beingness caffein, theobromine and theophylline. Their harvestion is limited in the aforementioned plant species compared to different plant alkaloids such as morphine, nicotine and strychnine, which are widely expressed across the plant kingdom. Caffeine is the worlds most consumed psychoactive drug, with approximately 74% of it used as beverages, 25% utilised for pharmaceutical formulations and the remainder for other purposes.3 It is chemically known as 1,3,7-tri methyl groupxanthine, with a complex purine-based structure, chemically associated to adenine and guanine found in DNA and RNACaffeines as well found in human tissues as wholesome as heterogeneous organisms. It is appreciated for its role in stimulating the CNS, kidneys, bronchial smooth muscle and relaxing the cardiac muscle.2,3 As a therapeutic, caffein is administered as a powdered form, solely the psychoactive substance found in tea leaves constitutes 2-5% caffeine per weight. Caffeine is used to treat apnea of prematurity, fatigue, in analgesic preparations with aspirin and paracetamol, and in various other roles including night duties (promoting alerting effect by antagonising adenosine A1 and A2A receptors in the brain), and in sports as a performance-enhancer.Because of the prerequisites for its usage in beverages and its significant potential in the pharmaceutical perseverance, caffeine has been isolated primarily from coffee. However, the increase demand for naturally derived caffeine for consumption necessitates additional other sources, for its extraction.4Contrastive to its utilisation and because it is unregulated, in that location has been increasing c oncerns pertaining its connection to adverse side- effects on human physiology regarding its high consumptions by some, which is linked with tachycardia, arr hythmia, muscle tremors, headache, coma, or fatality.4,2,3 As such, alternative forms of beverages have been decaffeinated (e.g., green tea) to eliminate the set forth potential side-effects and the demand for alternative forms of beverages goes hand in hand with increasing environmental-friendly techniques used to decaffeinate the crude product.This article, therefore, projects at the water extracting method for the isolation of methylxanthines and victimisation thin-layer-chromatography and IR spectroscopy to characterise individual methylxanthines, as well as discussing their Rf values, the use of caffeine in cold and flu remedies, and a brief look into the caffeine market.MATERIALS AND METHODS Preparation of Tea origin200ml of water was placed into a beaker and the solution was allowed to boil using a Bunsen burner. Tea (10.3052g) was then added into the solution and boiled for approximately 15 minutes. The beaker was then removed from the heat and 50ml sulphuric blistering was then added to the hot solution and shake to ensure no emulsion occurs. The solution was later allowed to cool at room temperature and then filtered using Buchner system.Extraction of Caffeine20ml of 10% sulphuric acid was added to the filtrate and stirred. Sulphuric acid converts the tannins to their salts, therefore, making them insoluble in chloroform, though soluble in water. The solution was then extracted by 3 successive washes of 50ml chloroform using a separating funnel, pile up the organic layer for each consecutive wash. The mixture of the solution was frequently shaken with occasional venting to prevent pressure buildup. The extracts were collected and dried by adding the catalyst anhydrous sodium sulphate for 10-15mins, removing all the water, leaving behind a fine powder.Isolation of The CaffeineRemove the sodium sulphate by filtration using the Buchner system. The chloroform was then evaporated by the use of a rotary evaporator, leaving behind the methylxanthine crystals. The weight of caffeine was then measured and the yield calculated. skimpy Layer ChromatographyTLC was conducted at room temperature and was used to verify the presence of caffeine by obtaining the methylxanthines, which was re-dissolved in 2ml of chloroform, 1ml of which was examined under TLC along with bring up solutions and amaze liquor. The UV-absorbing methylxanthines absorb UV light and so fluoresce agent (dichloromethane) in the stationary phase was used for visualisation in the UV254nm.On the TLC house (Silica gel GF254 Dimension 5cm x 20cm), a 1cm line was drawn above the base of one end of the plate. Capillary micropipette to place a skin senses of the reference solution (Caffeine, theobromine, theophylline, and mother liquor) along the line drawn on the plate and tagged as appropriate to reduce confusion with the other samples, which were also labelled as appropriate. This step was repeated using different micropipettes for each sample, creating spots at ab out 1cm from each other. The TLC plate was then placed straightforward in the TLC chamber contacting the developing solution (Chloroform Acetone N-Butanol 30% Ammonia, at 30304010 ratio) at a direct below 0.5cm (the origin) and sealed with using a watch glass. The solvent was allowed to migrate along the TLC plate so it reaches at least 1cm from the top and once the solvent had evaporated, the plate subsequently visualised under UV light as most organic compounds are colourless(prenominal) in the naked eye, facilitated by the fact that the TLC plate contains chemical additives that fluorescent under UV-light.Infra-Red SpectroscopyThe remaining methylxanthine solution of caffeine obtained was used to generate an IR spectrum to deduce the operating(a) groups in the compound. The measured spectral range was between 600 4000 cm. The sample was placed on a sampling window and spectral data collected using a spectrum software (PerkinElmer Spectrum Express version 1.02.00, UK).10Tabl e 1 Weight of crude caffeineCaffeine0.1606gPercentage yield = Mass of crude caffeine / mass of tea bags x 100%= 0.1606g / 10g - 100%= 1.606%Table 2 Distance travelled by the standard solutions and Methylxanthines (mm)CTBTPML ensample362418Spot 1= 34 Spot 2= 26 Spot 3= 1634Note C=caffeine, TB = Theobromine, TP = Theophylline, ML = Mother Liquor, S = SampleRf value= Distance travelled by the compound (mm)Distance travelled by the solvent (mm)Caffeine = 36mm/38mm = 0.95Theobromine =24mm/38mm = 0.63Theophylline =18mm/38mm = 0.47Mother Liquor spot 1= 34mm/38mm = 0.89ML spot 2 = 26mm/38mm = 0.68ML spot 3 = 15mm/38mm = 0.39S= 34mm/38mm = 0.89Table 3 Rf values of the standard solutions and Methylxanthines (mm)CTBTPMLSample0.950.630.47Spot 1= 0.89 Spot 2= 0.68 Spot 3= 0.420.89Note C=caffeine, TB = Theobromine, TP = Theophylline, ML = Mother Liquor, S = SampleFigure 1 IR spectra of the caffeine. Peak 3000 cm-1 represents amides and amines, and peaks between the regions 1700 -1600cm-1 indicate s the presence of alkene in caffeine. A and B shows the carboxyl group groups C=O bands of carbon-2 and 6 in region 1700-1659 cm-1.Figure 2 IR spectrum of the standard. The peak slightly 3000 is ascribable(p) to amides and amines. Peaks 1700-1600 is alkene in the caffeine scintillaThe structure of caffeine (Figure 3) is a function of how it behaves and interacts with other molecules and defines its properties such as solubility (e.g., due(p) to the presence of nitrogen atoms), boiling point, as well as the melting point. The tertiary purine-based caffeine constitutes an amine, amide and alkene function group, all containing lone pairs of electrons on the nitrogen atom. The achiral molecule is polar in nature due to the electronegativity difference between carbon-oxygen and carbon-nitrogen covalent bonds due to dipole-dipole fundamental interactions, capital of the United Kingdom dispersion forces, and hydrogen bonding once in water. The higher melting point of this molecule i s as a result of these strong intermolecular forces and would necessitate high energy to break the associated bonds.1,2Figure 3 Structures of caffeine, theophylline and theobromineFigure 3 indicates that the methylxanthines helping similar structures (purine ring), merely the slight molecular difference in structure results in the differences in properties. The difference in structure is based on the location of the methyl groups. Caffeine has ternion methyl group in carbon-1, 3 and 7 theobromine has two methyl groups on carbon-3 and 7and theophylline has two methyl group on carbon-1 and 3, and is deficit of methyl group at position 7 and so has only a proton that can be donated, the self very(prenominal)(prenominal) for theobromine having a proton on carbon-1, making them a weakly amphoteric compared to caffeine (a base compound).2 The TLC result shows the distance travelled by caffeine, theobromine and theophylline were 0.95, 0.63 and 0.47mm, respectively (Table 2). This imp lies that caffeine is more of a solvent compared to theobromine and theophylline. Mother Liquor contains three substances as shown by spot 1 to spot 3 in Table 3, with spot 1, 2 and 3 having Rf values of 0.89, 0.68 and 0.42, respectively. This means that spot 1 and caffeine are more attracted to the mobile phase, interacting less with the polar adsorbent (thats interaction with the silanol group of the silica gel, with the most prevailing interactive force being dipole-dipole) as the compound is less polar, restricted to form hydrogen bonds by its three methyl groups (these contain electrophilic sites and the compound possess electrophilic and nucleophilic function groups, but caffeine is not a proton donor so less polar to adsorb) and so having walloping Rf values, consequently spent less time travelling towards the solvent front as it is more soluble in the mobile phase. Theoretically, compounds that are less polar characteristically dissolve in the solvent, thus migrate debauch eder, and that the silanol group of the silica surface is highly polarised and is capable of forming dipole-dipole and hydrogen bonds. The more polar compounds such as theophylline (spot 3) strongly binds to the silanol group of the adsorbent or the stationary phase as theyre both capable of hydrogen bonding (theophylline is more of a proton donor thus has more electronegative substituents than for example, caffeine, so binds tightly to the stationary phase), and so moved slower than both spot 1 and 2, but also spends more time closer the origin, held by the resistive force of the sorbent.5,28 Spot 1 is likely to be containing samples of caffeine as they travel almost the same distance. Spot 2 is likely to contain samples of theobromine and spot 3 contains samples of theophylline as they have almost similar Rf values. IR spectroscopy was apply to elucidate the types of the functional groups in an unknown sample. The results from the IR (Figure 1) shows the presence of a compound in dicated by the energy speaks. The spectrometer produced a graph based on the measurements of the photon within 600 4000 cm-1 frequencies. Comparison between the IR spectrum of the standard solution (Figure 2) and that of Figure 1 confirms the likeliness in functional groups shared between these solutions. Figure 1 indicates photon energy peak visible at 3000 cm-1, representing amides and aliphatic amines6, and peaks between the regions 1700 -1600cm-1 are due to the presence of alkene in caffeine molecule9. A and B specifies the carboxyl groups (C=O) of carbon-2 and carbond-6 in region 1700-1659cm-1, indicating the most intense bands.17Opinion on the use of caffeine in cold and flu remediesThe effects of caffeine vary around the body and are dependent on the dose limit (400mg), at which beyond this parameter will elicit a range of physiological effects including muscle tremors, stomach upset, urinary incompetence, and a possible death. Below this dose limit, however, its effect is l ess detrimental to health.11 Due to it being readily available in foodstuffs and medicines, some may not be aware of the imposing dangers of caffeine, coupled with the fact that the FDA and the European guidelines consider caffeine not being a nutrient, but a natural ingredient found in beverages and so does not require identification in labelling of caffeinated product unless theres added caffeine in the product.12 This makes it a frighten away task for those tracking their caffeine intake especially those that are more vulnerable to its side-effects.Caffeine is issued both as a prescription and as an over-the-counter medication treating various conditions from lethargy to being used as an adjuvant in analgesic, as well as in flu or cold remedies.12 flu causes rhinorrhea, resulting in loss of fluid, which is counter to sustaining the bodys fluid remainder needed for healthy wellbeing. The elderly are the most at risk if not hydrated and the job exacerbates with the consumption of diuretic substances including any of the methylxanthines.14 A literature review by R. J. Maughan and colleague of caffeine ingestion and its effects on fluid balance assessed various age groups (adults) and the elderly of both sexes. Robertson et al. (1978) reported that R. J. Maughan and colleague administered a single dose of caffeine (250mg) and a placebo to the subjects and urine was accumulated for 3hrs. The result produced an increase in urine output from 366 30 mL (mean SD) on the placebo trial to 469 43 mL on the caffeine trial, accompanied by an increase in urinary sodium excretion. However, in the same report by Robertson et al., other studies indicated that the diuretic effect of small doses of caffeine had minimal effects, which may be in the same dose range in these flue/cold remedies. It was also reported that long-term caffeine users are not susceptible to this diuretic effect and may not lose water via urine output14, but those that are may be disposed to elect rolyte abnormalities (e.g., natriuresis) to kidney dysfunction. The mechanism in which caffeine induces diuresis is not yet clear, but it is believed that the compound acts as a phosphodiesterases inhibitor in the kidneys, along with its antagonistic effect on adenosine receptors.15 Another complication that may arise using caffeine remedies is the possibility of drug-drug interactions such as in the case of taking tizanidine(muscle relaxant), causing low blood pressure and dizziness16, or its inhibitory effect on the antipsychotic medications clozapine and olanzapine, metabolised by CYP1A2. Caffeine may also pose as a competitive inhibitor of CYP1A2 if metabolised at a slower rate compared to an administered drug, thus minimising the drugs plasma tightness with the likelihood of toxicity.20The compounding benefits of caffeine, when consumed within physiological limits, cannot be contested. Studies have shown that the groups most at risk of caffeine overdose are young people/childr en and adolescents due to the lack of sentience and incorrect social perception regarding the benefits versus harmful effects.17 It was reported by the American National Poison Data System that 6,309 cases related to caffeine overdose. A recent article by the telegraph newspaper reported that some students came close to fatality upon accidentally overdosing on caffeine (consumed 30000mg) and were placed on dialysis to remove the intoxication from the kidneys.19 So, in support of it still being a legal stimulant, not only does the benefits outweighs the adverse effects, the compounds plasma half-life is approximately 5hrs. This fast pharmacokinetics or elimination via urinary excretion entails that its dumbness in the blood will always be regulated, adverse side-effects occurring or at least pass its effects.20Due to caffeine being readily available, there are no age limits for their purchase and are not costly either as beverages. The FDA and EMA must assume responsibilities in engaging and communicating with the most at-risk groups, using public education campaign, and firmly addressing the potential risks of overdosing, especially when using multiple caffeine products in combination, and also labelling of such products, indicating the caffeine concentration, as well as targeting the medium used by these at-risk groups such as social media, the internet, and television. The European legislation, however, has taken the inducement in labelling beverages containing caffeine equal to or over 150mg caffeine per litre, affirming in their statement, High caffeine subject area. Not recommended for children or pregnant or breastfeeding women.17 Additionally, self-monitoring of caffeine concentration by providing device similar to those used by diabetic patients may assist with staying within physiological limits, but also restricting accessibility (age-dependent) top executive just be one way to control the likelihood of abuse.As a commodity, caffeine can be ob tained in various forms for many applications (cosmetics, medical, etc.) and there is always a huge demand,which are popular amongst young people, particularly with the emergence of caffeine-fuelled energy drinks that are used to mix alcohols in social venues.21 Compared with other drugs, the worldwide medicate Survey 2014 (Figure 4) reported that caffeinated energy drinks were the fourth most drug bought (45.9% prevalence use) subsequently alcohol, tobacco, and cannabis.This illustrates caffeines importance both at physiological level, and as a big earner for the industrys major players in global caffeine market (Pfizer, Boehringer Ingelheim, CSPC Pharma, BASF, and Cocam) as it is consumed by 90% of the worlds population.24,25 One of the drivers of the industry is the production of coffee, which is forecasted to produce 156.6 million bags in 2016/17, and global consumption to be 153.3 million bags.26 This points out the growing demand for caffeinated products, predominantly in t raditional markets including Canada, EU, USA, Japan, Norway and Switzerland, but also in emerging markets Turkey, Algeria, Russia.27Figure 4 Prevalence of top 20 drug use. Caffeinated drinks was among the most drugs bought in the year 2014.21It is concluded by the study that the amount of caffeine extracted in tea was almost consistent with the theoretical constituents per weight, 2-5%. This research yields 0.1606g (1.606% ) from 10kg of tea bag and so was impossible to recover 100% of caffeine, greatly impacted by the fact that the reaction was never at completion, not all the caffeine was extracted through the funnel separation, loss of product may have occurred due to emulsions, discrepancies with the instruments due to factors affecting calibration, and steaming during brewing affects the mass of the extracted caffeine. One way to improve the percentage yield may be to explore different organic solvents.Although caffeine has numerous health benefits within physiological optima, it is also detrimental and causes death if these limits are breached. As such, healthcare authorities, as well as the caffeine industry must put in place measures so it is better regulated, and may mean being transparent about the health benefits/risk factors, and spread this awareness in all media used by their target users, especially young people as this is the group that are less aware of the risks.References Caffeine The Molecule. 2017 Feb 9. open from http//caffeinethemolecule.weebly.com/physical-properties.htmlPreedy V.R, editor. Caffeine Chemistry, Analysis, Function and Effect. UK The Royal Society of Chemistry 2012, p.3 -6,44Vuong Q.V, dress circle P.D. Caffeine in Green Tea Its Removal and Isolation.2017 Feb 16. Available from http//www.tandfonline.com/doi/full/10.1080/15422119.2013.771127Vuong Q.V, Bowyer M.C, and Roach P.D. L-Theanine properties, synthesis and isolation from tea. J. Sci. Food Agric. 2011 91 1931-1939Analyzing a Mixture by Thin Layer Chromatography (TL C). 2017 Feb 17. Available from http//cactus.dixie.edu/smblack/chemlabs/analyzing_a_mixture_by_thin_layer_chromatography.pdfGunasekarana S, Sankarib G, Ponnusam S. Vibrational spectral investigation on xanthine and its derivatives-theophylline, caffeine and theobromine. Spectrochimica Acta Part A. 2005Caffeine. 2017 Feb 16. Available from https//tinyurl.com/hsbppmnAl-Hitti IK, Ibrahim SS. EXTRACTION, IDENTIFICATION AND DETERMINATION OF CAFFEINE AND TRACE METALS IN THREE TYPES OF TEA LEAVES. J. of al-anbar university for pure science. 2009Sing BM et al., Determination of caffeine content in coffee using Fourier transform infra-red spectroscopy in combination with attenuated total reflectance technique a bioanalytical chemistry experiment for biochemists. biochemical Education. 1998Palo M et al., Quantification of caffeine and loperamide in printed formulations by infrared spectroscopy. JDDST. 2016Behind the hype Caffeine. 2017 Feb 12. Available from http//nutritionandactivity.govt.nz /sites/default/files/2%202%20NPA161%20Behind%20the%20Hype-Caffeine_5.pdfWhy isnt the amount of caffeine a product contains required on a food label?. cited 2017 Feb 12. Available from http//www.fda.gov/aboutfda/transparency/basics/ucm194317.htmCaffeine and Your Body.Cited 2017 Feb 13. Available from https//www.ctclearinghouse.org/Topics/Customer-Files/Caffeine_And_Your_Body_052113.pdfMaughan RJ, Griffin J. Caffeine ingestion and fluid balance a review. J Hum Nutr Diet. 2003 Dec16(6)411-20.Zhang Y et al., Caffeine and diuresis during rest and exercise A meta-analysis. J Sci Med Sport. 2015 18(5) 569-57425 Most Severe Caffeine and Drug Interactions. 2017 Feb 14. Available from http//www.caffeineinformer.com/caffeine-drug-interactionsCaballero B, Finglas PM, Toldra F, editors. Encyclopedia Of Food And Health. Oxford Elsevier 2016S. Jain, et al., Caffeine addiction Need for awareness and research and regulatory measures. 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