Isolation and Characterization of Nicotine From Tobacco Leaves

ISOLATION AND QUALITATIVE TESTS OF CINNAMALDEHYDE FROM CINNAMON POWDER Jack D. Acedera, Jonathan Michael I. Acha, Rachellyn M. Alba, Kathleene Anne V. Andaluz, Jan Vincent V. Arafiles Group 1 2A-Biochemistry Organic Analysis Laboratory

ABSTRACT 3-phenyl-2-propenal or cinnamaldehyde is isolated from cinnamon powder using heating under reflux, extracted using dichloromethane and dried using sodium sulfate. The isolate was brown crystalline solid with cinnamon-like odor, soluble in dichloromethane and ethanol but slightly insoluble in water. Percent yield of the extraction was 0.458% Tollen’s test gave a positive result of silver mirror and phenylhydrazine test yields yellow solution.

INTRODUCTION Cinnamon id the dried bark of the Cinnamomum zeylanicum which grows in Sri Lanka and south India. The bark of this tree is also known as Ceylon Cinnamon or True Cinnamon. There are other kinds of cinnamon present but the primary use of the substance is for spicing purposes. Cinnamon was regarded as a high valued spice by many different civilizations. It has been extensively used as a spicing agent, oils and even medicine. The Chinese regarded the cinnamon as a medicine for various diseases like diarrhea and arthritis. The main component of the cinnamon is the cinnamaldehyde. Cinnamic aldehyde, 3-phenyl-2-propenal, structure shown below, is a oily yellow liquid with strong odor of cinnamon.

Figure 1: Cinnamaldehyde This compound is the main component of cinnamon oil, a volatile oil used as a flavoring agent for pharmaceuticals. Cinnamaldehyde is obtained from the distillation with steam from the leaves and twigs of Cinnamomum. It can be prepared from the synthesis from related compounds like cinnamyl alcohol and from the condensation of benzaldehyde and acetaldehyde. Cinnamic aldehyde is a benzene ring substituent arylic aldehyde having carbon-carbon double bond. The conjugated double bond makes geometry of the compound planar. Though there are cis and trans isomers, cinnamic aldehyde usually refers to the latter, which the terminal carbonyl is on the opposite side of the benzene ring over the rigid double bond.It is non-toxic but can irritate the skin in prolonged contact. As with many components of essential oils it exhibit

antiviral, antibacterial and antifungal properties. It has been also reported as a good pesticide. Minor component of Cinnamon oil are eugenol, 4Allyl-2-methoxyphenol, structure shown below.

Figure 2: Eugenol This component makes up 10% of the oil and displays antiseptic and analgesic properties, which may contribute to cinnamon’s soothing effect. A different kind of extraction was utilized in the experiment, heating under reflux. Temperature is one of the variables that one can easily control in the laboratory to speed or slow down a reaction. Most of the organic reactions are performed in the liquid phase using a suitable solvent. A convenient way of carrying out a reaction at high temperature and keeping it constant is by using a reflux apparatus. The set-up consists of: a round bottom flask attached to the condenser at vertical position. The round bottom flask is heated to make the solvent boil. The solvent vaporizes and ascends through the flask into the condenser. When the vapor reaches the cool surface of the condenser they drip down back. It is because the water circulates through the outer tube, which removes the heat from the vapor and condenses them. This process allows little liquid to be lost in the atmosphere by evaporation, as the mixture is being kept at a constant temperature. Cinnamaldehyde contans the formyl group that can be tested using the Tollen’s test and the Phenylhydrazine test. The formyl group contributes to the reaction towards this test and will yield a positive result. Both tests are qualitative tests for the formyl group of the aldehyde.

The experiment aimed to isolate the cinnamaldehyde from commercial cinnamon powder using heating under reflux then separation using an organic solvent and observe the physical properties of the isolate including the solubility of the isolate. The isolate was then qualitatively tested with Tollen’s test and phenylhydrazine test.

EXPERIMENTAL A. Compound Tested Cinnamon powder, distilled water, dichloromethane, anhydrous sodium sulfate, absolute ethanol, 10% silver nitrate solution, 10% sodium hydroxide solution, 6M ammonium hydroxide solution, phenylhydrazine solution, B. Procedure Isolation of Cinnamaldehyde Twenty-five grams of cinnamon powder was weighted in a tarred Erlenmeyer flask and 100 ml of distilled water was added into the flask. A reflux setup was installed by placing a bored cork on the mouth of the Erlenmeyer flask. A condenser was connected to the opening of the flask by the cork and another cork topped the opening of the condenser. The flask was immersed into a water bath and was heated for 30 minutes. The mixture was left inside the locker. The mixture was decanted into a separatory funnel and the first portion of 10 ml of dichloromethane was introduced and the lower layer was collected and another round of separation was done with the other portion of 10 ml dichloromethane. The two lower layers were combined and added with sodium sulfate. The liquid was decanted into a tarred evaporating dish, was covered with a perforated bond paper and left in the locker. The physical states and solubility was observed. Tollen’s Test Dissolve the cinnamaldehyde in absolute ethanol. The tollen’s reagent was prepared in a test tube by adding 1.0ml of 10% silver nitrate to 1.0ml of sodium hydroxide. The brown precipitate was dissolved with the addition of just enough ammonium hydroxide. 1.0ml of the isolated cinnamaldehyde was added into the tollen’s reagent and was heated in a water bath. The result was observed. Phenylhydrazine test Dissolve the cinnamaldehyde in absolute ethanol. One milliliter of phenylhydrazine solution and 1ml of acetic acid was added to 0.5ml of the cinnamaldehyde-ethanl solution. The result was observed.

RESULTS AND DISCUSSIONS Refluxing is the process of returning the liquid evaporated to the flask. This technique was used to prevent the loss of the solvent during the vaporization phase of the solvent. The condenser above the flask is used to condense any vapor before it escapes the set up. The cork on top of the condenser serves as a lock for the vapor to not escape. Presented below are the physical properties of the isolated cinnamaldehyde: Isolate

Color

Cinnamaldehyde

brown

Appearance Turbid

Odor Cinna monlike odor

Solubility property Soluble in DCM

Cinnamaldehyde is slightly soluble in water but soluble in Dichloromethane. The cinnamon powder was boiled in an Erlenmeyer flask for 30 minutes. The temperature of the inside solution is at constant 100oC because of the reflux setup. The condenser condenses the vapor as soon as it enters the tube and reenters the solution in this method; the temperature is remained constant due to the recycling of the solvent. The next step was the fractional extraction with Dichloromethane. Since the cinnamaldehyde is soluble in dichloromethane and only slightly soluble in water, the cinnamaldehyde molecules will be dissolved in the extraction solvent once introduced. The cinnamaldehyde is present in the lower organic layer of the separatory funnel because cinnamaldehyde is more soluble to dichloromethane compared to water. The extraction method used is a fractional extraction. This method utilize the use of 2 portions of the extraction solvent, in the case of the experiment, 2 portions of 10ml dichloromethane. The fractional extraction assures that the cinnamaldehyde is fully extracted from the aqueous layer. After the extraction, the organic layer was added with a drying agent, sodium sulfate. The organic solvent used for extraction dissolved not only compounds being extracted but also water. Evaporation of the solvent then leaves the desired compound contaminated by water. Solvent such as dichloromethane does not dissolve nearly as much water and are therefore dried over a chemical drying agent. Many choices of chemical drying agents are available for this purpose, and the choice one which one to use is governed by the following factors: [1] the possibility of reaction with the substance being extracted, [2] the speed with which it removes water from the solvent, [3] the efficiency of the process, and [4] the ease of recovery from the drying agent. Sodium sulfate is a very poor

drying agent. It has a very high capacity for water but is slow and not very efficient in the removal of water. Like calcium chloride pellets, it clumps together when wet, and the solutions are easily removed from it using a Pasteur pipette. Sodium sulfate has often used in the past and should still be used for compounds that react with calcium chloride. The extracted layer was added with sodium sulfate until the powder clumped together to assure the total elimination of water. Afterwards the solution was decanted into tarred evaporating dish, covered with the perforated white bond paper and left in the locker overnight. Cinnamaldehyde observed was brown crystalline solid with a cinnamon-like odor, soluble in dichloromethane and ethanol but slightly insoluble in water. Compared to the observed physical states above, the researched physical state of cinnamaldehyde is a yellow oily liquid with a cinnamon-like odor. The percent yield was calculated and yielded 0.458% the very small percent yield may be due to an inefficient heating under reflux. The insolubility of the cinnamaldehyde in water may have contributed to the percent yield of the experiment. The isolate was dissolved in absolute ethanol as preparation for the qualitative test. Below are the results for the qualitative tests.

The aldehyde on cinnamaldehyde condenses with the hydrazine reagent forming the product of yellow crystals in the form of hydrazone. This test is also used to detect the presence of the aldehyde group. Although, the group was able to obtain only yellow solution, this may be due to poor extraction of the cinnamaldehyde or the decreased number of isolates which may affect on the reaction towards the test.

Table 1: Qualitative test results Test Result Tollen’s test Silver mirror Phenylhydrazine test Yellow solution

[2]Cinnamaldehyde.http://www.chm.bris.ac.uk/ motm/cinnamaldehyde/cinnc.htm. taken March 7, 2010.

One milliliter of the ethanol-cinnamaldehyde solution was added to the prepared Tollen’s reagen and was subjected into a water bath for several minutes. A silver mirror appeared as a result of the test. This confirms the presence of an aldehyde in the solution in theform of the cinnamaldehyde. The reaction mechanism is shown below. -CHO + 2Ag(NH3)2OH 2Ag(S)+ -COO-NH4+ +H2O+ 3NH3 The aldehyde served as the reducing agent and the Tollen’s reagent served as the oxidizing agent. The reduction of the silver ion, results to the formation of the silver mirror upon heating of the solution. The last test was the phenylhydrazine test. Most aldehydes and ketones yield hydrazone crystals which are insoluble solids. The precipitate may be oily at first and become crystalline ion standing. The reaction of an aldehyde is shown below. NH2NH2 + -CHO  -CONH2NH2

REFERENCES From book: [1] Shriner R.L. & et. Al. The Systematic Identification of Organic Compounds: a laboratory Manual. (1956) [2] Shriner R.L. & et.al. The Systematic Identification of Organic Compounds. (2004) [3] Williamson K.L., Minard R.D., & Masters K.M. Macroscale and Microscale Organic Experiments. 5th ed. (2007) From the internet: [1] Cinnamaldehyde. www.chemicalland21.com. taken March 8, 2010