EXP 7 V1 - To conduct the experiment related to the extraction, isolation and purification techniques PDF

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To conduct the experiment related to the extraction, isolation and purification
techniques in natural product chemistry...

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TITLE NATURAL PRODUCT ISOLATION OF CAFFEINE FROM TEA LEAVES OBJECTIVES 1. To conduct the experiment related to the extraction, isolation and purification techniques in natural product chemistry. 2. To study the extraction method of caffeine from tea leaves 3. To learn the conversion of caffeine to caffeine salicylate.

INTRODUCTION Tea is one of the most commonly used caffeinated beverages in the world. The caffeine (C8H10O2N4) found in tea is a bitter, white, crystalline methylxanthine and a member of a class of compounds known as alkaloids (Wang, 2011). Alkaloids are basic nitrogen containing compounds present in plants. The structure of caffeine affects the functions it performs.

Figure 1: Structure of caffeine Retrieved from https://www.sigmaaldrich.com/catalog/product/aldrich/c53? lang=en®ion=MY

Alkaloids, such as caffeine, are often physiologically active in humans and are known central nervous system stimulants and diuretics (Wang, 2011). Caffeine also causes an increase in respiration and heart rate, as well as nervousness and insomnia. Though caffeine has

demonstrated to have physical dependence, it is also capable of improving alertness, learning capacity, and exercise performance (NCBI, 2013). Tea leaves, in which caffeine is found, also contain acidic tannins, undecomposed chlorophyll, cellulose, and pigments. In order to extract caffeine from tea leaves, caffeine must be present as the free base (Amrita, 2013). In order to do so, the above-mentioned acidic substances must remain water-soluble. In order to extract caffeine from tea, several methods are used. First, a solid/liquid extraction must take place in order to get the solid natural product into the liquid solvent. This can be done by using a Soxhelet extractor, or by simply brewing a cup of tea. In order to isolate the desired reaction compounds from the natural product, liquid/liquid extractions are used. Neutral and acid/base are two forms of liquid/liquid extractions (Williamson, 2011). Caffeine extraction from tea leaves involves an acid/base liquid/liquid extraction (Oneota, 2003). The reaction involves a homogenous mixture of an organic and aqueous layer. The ideal solvent in the extraction should have a low boiling point, not react with the solute or other solvents, not be toxic or highly flammable, not miscible with water, be inexpensive, and should readily dissolve caffeine at room temperature. A common liquid/liquid solvent pair for the extraction of caffeine is water-dichloromethane (Williamson, 2011). Because water is present in the pairing, it possible to separate inorganic compounds from organic compounds due to the fact that organic substances are immiscible in water (Amrita, 2013). When mixing the liquid pairs, the density of the both solvents predict which solvent is the top and which is the bottom layer. Caffeine, which was present in the organic layer, was located below the aqueous layer (Williamson, 2011). The product that is collected after extraction still has many impurities. Sublimation is one way to purify the sample, because caffeine has the ability to pass directly from the solid to vapor and reverse to form a solid all without undergoing the liquid phase. Caffeine has the ability to undergo sublimation under different conditions than the impurities, and can thus be isolated (Tello, 2011). A series of techniques were used to extract pure caffeine from tea leaves. The percent error and percent recovery were calculated to assess how much pure caffeine was obtained, and to account for errors that may have occurred that led to a loss of product.

PROCEDURE 1. The mixture of 25.0 g dried tea leaves, 250 ml water and 25 g of carbonate power in 500 ml were refluxed on an electrical hot plate for 20 minutes. 2. The hot mixture then filtered by suction on a Buchner funnel and the water was pressed out as much as possible from the solid. 3. The filtrate was cooled to room temperature and the compounds contained in it was extracted using two 25 ml portions of chloroform, twice. 4. The extracts was then placed in a 100 ml round bottom flask and the solvent is distilled off in a boiling water bath. Similarly, the rotary evaporator can be used. 5. The solid was crystallized by dissolving it in a minimum amount of benzene. 6. The product was filtered and recrystallized using same solvent. 7. A salicylate derivative was prepared by dissolving 0.2 g of caffeine obtained and 0.15 g of salicylic acid in 15 ml of hot benzene. 8. The mixture is then cooled. The formation of crystal should be slow. 9. In order to speed up the crystallization process, the mixture was cooled in water bath. 10. The product was filtered, air dried and the melting point of caffeine salicylate was measured produced. (The pure compound should melt at 137 °C)

RESULTS AND CALCULATION Table 1: The results for the preparation of caffeine salicylate experiment Weight of caffeine extracted

0.540 gram

Filter paper

0.306 gram

Filter paper + product (caffeine salicylate)

1.188 gram

Product (experimental yield)

0.982 gram

Theoretical yield of caffeine salicylate

14.995 gram

Melting point caffeine salicylate

130 °C

Reaction equation: C8H10O2N4 + C7H6O3

C15H16O2N4

1. Theoretical yield of caffeine salicylate No. of mole of caffeine =

=

Mass Molar mass 0.2 g 194.19 g/mol

= 1.03 x 10-3 mole No. of mole of salicylic acid =

=

Mass Molar mass 0.15 g 138.121 g/mol

= 1.09 x 10-3 mole Thus, the limiting reactant is caffeine, C8H10O2N4 Based on the equation of reaction, 1 mole of C8H10O2N4, produced 1 mole of C15H16O2N4, Thus, 1.03 x 10-3 mole of C8H10O2N4, produced 1.03 x 10-3 mole of C15H16O2N4, Theoretical yield of caffeine salicylate = 1.03 x 10-3 mole x 332.316 g/mole = 0.36 gram

2. Percentage yield of caffeine salicylate Percentage yield =

=

Experimental yield Theoretical yield

0.982 gram 0.34 gram

x 100 %

x 100 %

= 288.8 % 3. Percentage error of the product Percentage error =

=

Theoretical yield−Experimental yield Theoretical Yield

0.34 g−0.982 g x 100 % 0.34 g

= 188.82 %

x 100 %

Figure 2: IR Spectrum for caffeine salicylate

Table 2: IR interpretation based on functional group present in the structure Functional Group Ketone Aromatic Alcohol (OH)

DISCUSSION

Wave number (cm-1) 1693.50 1643.35 3113.11 (Weak)

The structure of the caffeine extracted from the tea leaves deeply impacts the functions it performs (Wang, 2011). Caffeine is a purine with three functional groups: an amine, amide, and an alkene. Caffeine is also a polar molecule; this is evident because of the London dispersion forces, dipole-dipole interactions, and hydrogen bonding present when it is in water. It also has a very hydrophobic region (NCBI, 2013). Solubility controlled by the nitrogen present in caffeine. It is soluble in water at approximately 2.2 mg/ml at 25°C, 180 mg/ml at 80° C, and 670 mg/ml at 100°C (Williamson, 2011). Caffeine is also an organic molecule that has the properties of an organic amine base (Tello, 2011) .When extracting caffeine, the water was kept at a high temperature in order to increase solubility of caffeine in water to about 670 mg/ml at 100°C. Boiling chips were added to the solution in order to prevent “bumping” and enable the smooth formation of bubbles when boiling occurs. The solution was later cooled to a lower temperature in order to impact the solubility once more and to minimize the attraction to the aqueous layer while in the separatory funnel (Williamson, 2011). Extraction the solid insoluble material such as cellulose is separated from caffeine and tannins, which are water soluble during the solid/liquid. A difference in solubility must occur in order to isolate caffeine, to separate the tannins into the aqueous layer. Sodium carbonate is added to the extraction medium to ensure that the acidic components in the tea leaves remain water soluble and that caffeine is the free base. Sodium carbonate is basic. Tannins are acidic compounds with a high molecular weight that have an –OH directly bound to an aromatic ring. Adding something basic to caffeine will make it more neutral, and the “like dissolves like” idea can be applied. In this situation, the sodium carbonate acts as a nucleophile and the tannin is an electrophile. Nucleophile attacks electrophile. It is basically an acid/base reaction. The aqueous layer (density of 1 g/ml) contained dissolved tannin salts and chlophyll. Dipole dipole interactions, london dispersion forces, hydrogen bonding, and ionic bonding with the salts took place. When chloroform was added to extract caffeine from the aqueous solution, two immiscible layers formed: an organic and aqueous layer. In this instance, caffeine is usually a polar substance, but it becomes significantly less polar when it is in a basic solution. Therefore, it is soluble in

chloroform and suspends in the organic layer. The concentration of the solutes in the organic layer also contributes to the fact that it is located below the aqueous layer. There is a high concentration of caffeine, reactants (because the reaction does not go to 100% completion), and small amounts of water. The intermolecular forces in the organic layer are van der walls interactions, dipole dipole moments, and london forces. Caffeine was extracted with chloroform in order to “wash” it three separate times to obtain as much of the pure sample. Emulsions are small droplets of the organic layer that are suspended in the aqueous that are a result of vigorous shaking of the separatory funnel (Williamson, 2011). There are numerous ways to remove emulsions, though the best form is prevention. However, they can the emulsions may break after a sufficient amount of time. The aqueous layer can also be made more ionic, and centrifugation works very well especially on a microscale level. A drying agent was added to the organic layer because dichloromethane dissolved not only the caffeine, but water as well. The drying agent, anhydrous CaCl2 should be added to remove excess water so that a pure sample of caffeine could be obtained after the solvent evaporated at room temperature (Williamson, 2011). Anhydrous calcium chloride has a high affinity for water, and then reverses back to the hydrous form after it has absorbed the water. In order to remove the chloroform, rotary evaporator is used so that the solvent would evaporate and leave a pure sample. Sublimation is a technique that may have been used to produce a purer caffeine sample, but it could have led to a higher loss of product. Liquidliquid extractions were used to transfer a solute from one solvent to another and isolate desired product. The weight of the caffeine extract was approximately around 0.540 g. The second part of the experiment is preparation of salicylate derivative by dissolving a little amount of caffeine obtained with salicylic acid. The mixture then cooled and it can be observed that the formation of the crystal. However, the formation is a bit slower. In order to speed up the crystallization process, it was cooled in ice bath. The calculated percent recovery was 288.8 %. This was the amount of caffeine extracted from the crude caffeine in the tea leaves. This demonstrates that there was a significant amount of product lost

throughout the procedure. It is also important to consider that the reaction cannot go to completion, so 100% yield is not possible. A loss of product could have occurred due to emulsions and due to not thoroughly “washing” with chloroform to extract as much caffeine as possible. There was a lot of transfer throughout the procedure, which presented many opportunities to loose product. It is also possible that the concentration of caffeine was not height enough because too much water was added. A systematic error with the scales was observed due to a lack of calibration, this could have affected the measurement of the final product. Another source of error could be the theoretical amount of caffeine in the tea leaves, if it was more or less due to random error, the percent recovery would be calculated differently. The overall percent error was about 188.82 %. This number could be skewed due to measurement errors of the crude product. In order to reduce sources of error in the future, some precaution could be taken. 1. All the apparatus need to be cleaned completely in order to avoid any contamination with the samples. 2. Weight the mass of the chemical needed for several time and calculated the average to reduce the zero error. 3. Dissolve in minimum amount of hot solvent to ensure solution is saturated (watch carefully). If adding solvent fails to dissolve any more solid, it is likely that insoluble impurities are present. 4. To prevent superheating (heating solution above its boiling point w/o actually boiling, which occurs w/ explosive violence), add boiling stick or boiling chip. 5. Cool slowly; if cooled too fast, solid will come “crashing out” of solution as powder precipitating impurities along with it.

CONCLUSION

Overall, a total of 0.540 g was obtained from a possible amount of 25.0 g of dried tea leaves. The total percent recovery was 288.8 % for the synthesis of caffeine salicylate. This number reflects on how accurate the procedure was performed. It was not possible to obtain 100% recovery because the reaction never goes 100 % to completion and because of material loss through transfer during the procedure.

REFERENCES 1. Amrita University. Extraction of Caffeine from Tea. 2013. Retrieved from http://amrita.vlab.co.in/? sub=3&brch=64&sim=169&cnt=1 2. National Center for Biotechnology Information, U.S. National Library of Medicine. Caffeine

Compound

Summary.

Retrived

from

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=2519 3. Wang, H. Dynamic microwave-assisted extraction coupled on-line with clean-up for determination of caffeine in tea. [Online] 2011, 1490-1495. Retrieved from www.elservier.com/locate/lwt. 4. Williamson, K and Katherine Masters. Macroscale and Microscale Organic Experiments, 6th ed.; Brooks/Cole, 2011....