Separation of the Colored Pigments Found in Malunggay

Classifying Compounds containing Hydroxyl- and Carbonyl groups using specified tests Abraham, K.D., Acebedo, D.M.A., Alap-ap, M.L.M., Asprec, W.A.A., Balaaldia, K.C.A. and Basilio, Z.A.S. 2B-PH, Group No. 1, Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines ABSTRACT Hydroxyl- or carbonyl- containing samples was given for this experiment for analysis. Hydroxyl group refers to a functional group containing OH- when it is a substituent in an organic compound whereas carbonyl group refers to a divalent chemical unit consisting of a carbon and an oxygen atom connected by a double bond [1]. Hydroxyl group is the characteristic functional group of alcohols and phenols while carbonyl group is the characteristic functional group of aldehydes and ketones [2]. In this experiment, several differentiating tests were conducted with samples ethanol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, benzyl alcohol, nbutyraldehyde, benzaldehyde, acetone, acetophenone, isopropyl alcohol, and acetaldehyde. These tests include solubility of alcohols in water, Lucas test, Chromic Acid test, 2,4Dinitrophenylhydrazone test, Fehling’s test, Tollens’ Silver Mirror test, and Iodoform test. The first three test done helped in the identification of the structure of the alcohols and tell if they are Primary (the alpha carbon atom where OH group is attach is attached to only one alkyl group), secondary (the alpha carbon is attached to two alkyl group), or tertiary (the alpha carbon is attached to three alkyl groups). The fourth test, The 2,4-Dinitrophenylhydrazon test was used to identify the presence of carbonyl groups-, aldehydes and ketones. 2,4-Dinitrophenylhydrazone was also used to distinguish its aromaticity. A positive result of red-orange precipitate indicates a carbonyl group, while a yellow precipitate indicates a presence of aldehydes and ketones. The fifth and sixth test, the Fehling’s and Tollens’ Silver Mirror test were used to identify the presence of an aldehyde and distinguishing an aldehyde from a ketone. In this experiment, only an aldehyde would yield a positive result while ketones won’t. And lastly, the Iodoform test was used to ensure the presence of CH3CO group or methyl carbonyl group in the samples given. INTRODUCTION An alcohol is a compound that has a hydroxyl group bonded to a sp3-hybridized carbon atom, R-OH. They are also classified as primary (1”), secondary (2”) or tertiary (3”), depending on the number of carbon substituents bonded to the hydroxyl-bearing carbon. Their most important physical property is the polarity of their –OH groups. Due to the large difference in the electronegativity of C-O and O-H, both of their bond of an alcohol are polar covalent and alcohols are polar molecules. Hydroxyl group is used to describe the functional

group –OH when it is a substituent in an organic compound. Hydroxyl groups are known for their tendency to form hydrogen bonds either as a donor or as an acceptor. This is also related to their ability to increase hydrophilicity and water solubility [3]. The functional group of an aldehyde is a carbonyl group bonded to a hydrogen atom. Another carbonyl group is a ketone bonded to two carbon atoms. Due to the polarity of a carbonyl group, aldehydes and ketones are polar compounds and interact in the liquid state by dipole-dipole interaction. The result is that aldehyde and ketones have higher

boiling points than those of nonpolar compounds with comparable molecular weight [4]. The Lucas test is performed with the use of the Lucas Reagent, where Lucas reagent is a solution of anhydrous zinc chloride in concentrated hydrochloric acid. This reagent is used to classify alcohols of low molecular weights. This reaction shows a substitution, which the chloride replaces a hydroxyl group. A positive result indicated the formation of a chloroalkane which is based on the difference in reactivity of the three classes of alcohols with hydrogen halides. The difference in reactivity shows the different ease of formation of the carbocations. Tertiary carbons are the most stable and Primary carbons are the least stable. Tertiary alcohols react immediately to with the Lucas Reagent, Secondary alcohols react within a few minutes while Primary alcohols do not react with Lucas Reagent at room temperature. Meaning that the time taken for a positive effect to be visible is the measure of the reactivity of the class of alcohol, and it is also the way of determining the class of a alcohol the solution is. Here is the reaction mechanism in Lucas Test [5].

primary and secondary resulting in the reduction of the orange chromium Cr6+ ion to a blue-green Cr3+ ion. Carboxylic acids and their derivatives are the most abundant of all organic compounds in living organisms and in the laboratory. The most common derivatives of carboxylic acids are acyl halides, acid anhydrides, esters, and amides. They contain acyl group attached to a nucleophilic molecule that replaced the –OH group of a carboxylic acid. Meaning that Jones test is used to check for the presence of a primary and secondary alcohol both of which shows a positive result of the formation of a green color. Here is the reaction mechanism in Chromic Acid Test [6].

Figure 2 : Jones’ test Rxn Mechanism

Figure 1 : Lucas Test Rxn Mechanism The Chromic acid test, also known as Jones Oxidation test, differentiates primary and secondary alcohols from tertiary alcohols. A primary alcohol is oxidized to an aldehyde or to a carboxylic acid, while a secondary alcohol to a ketone. Tertiary alcohols in the other hand do not react. The OH-bearing carbon must have a hydrogen atom attached since that carbon atom is being oxidized in

The 2,4-Dinitrophenylhydrazone (2,4-DNP) test serves as to form a derivative. The aldehydes and ketones react with 2,4-DNP to form a solid 2,4-DNP Derivative. If the solid is yellow, this means that the carbonyl group in the unknown is unconjugated while a reddish-orange color most likely means that the carbonyl group is conjugated. Meaning this test help determine whether the carbonyl group is alipathic or aromatic. The reddish-orange color means that the substance is aromatic and the yellow color

means that the substance is alipathic. Here is the reaction mechanism in 2,4-DNP [7].

Figure 3 : 2,4-DNP Rxn Mechanism

The Tollens’ Test, also known as Silver Mirror Test, is used to distinguish between an aldehyde and a ketone. This test also has the same function as the Fehling’s Test. This test uses a reagent known as Tollens’ reagent, which is a colorless, basic, aqueous solution containing solver ions coordinated to ammonia [Ag(NH3)2+]. Tollens’ reagent oxidizes an aldehyde into the corresponding carboxylic acid. A positive result would make the reagent metallic silver in color and shows a mirror-like expression in the test tube. Ketones are not reactive with the Tollens’ reagent, so it would not show a mirror-like impression in the test tube. Here is the reaction mechanism in Tollens’ Test [9].

The Fehling’s test is used to determine the presence of an aldehyde in the solution and it doesn’t react with ketones. Aldehydes reduces copper to make the deep blue solution of copper (II) to muddy green solution, and then forms a brick-red precipitate of insoluble cuprous oxide (Cu2O). Meaning this test is usually used for reducing sugars but us known to be not specific with aldehydes. Here is the reaction mechanism in Fehling’s Test [8].

Figure 5 : Tollens’ Test Rn Mechanism

Figure 4 : Fehling’s Test Rxn Mechanism

Lastly, the Iodoform test used to see the presence of CH3CO in aldehydes and ketones. The sample is allowed to react with a mixture of iodine and base. The alpha hydrogens of the carbonyl group are acidic and will react with a base to form the anion, which then reacts with iodine and forms triiodo compound which then reacts with another base to form the carboxylic acid salt plus a iodoform. A positive result would produce a yellow precipitate, and if there is no CH3CO group in the sample being tested, then it will get different result. Here is the reaction mechanism in Iodoform Test [10].

alcohol was added. The test tube was covered and shaken vigorously and was allowed to stand at room temperature. The 2 other test tubes were placed with 2-3 drops of sec-butyl alcohol and tert-butyl alcohol. The test tubes were shaken vigorously and the mixtures were allowed to stand. Note the time of the formation of the cloudy suspension or the formation of two layers. Figure 6 : Iodoform Test Rxn Mechanism METHODOLOGY The materials needed for this experiment in this experiment are the following: Lucas Reagent, Chromic Acid reagent, 95% ethanol, Fehling’s A and B, Tollens’ reagent, 5% NaOCl solution, Iodoform test reagent, 2,4-dinitrophenylhydrazine, Pasteur pipette, test tubes and vials and some beakers. While the sample compounds needed are the following: ethanol, n-butyl alcohol, secbutyl alcohol, tert-butyl alcohol, benzyl alcohol, n-butyraldehyde, benzaldehyde, acetone, acetophenone, isopropyl alcohol, acetaldehyde. A. Solubility of Alcohols in Water Five test tubes were labelled and ten drops each of ethanol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, and benzyl alcohol were placed on the test tubes with the use of a Pasteur pipette. Followed by addition of 1mL of water and shaking of the test tube. If cloudiness occurs, continue adding a quarter of an mL of water with vigorous shaking, until a homogenous dispersion results. If no cloudiness results after the addition of 2mL of water, the alcohol is water soluble in water. B. Lucas Test Three test tubes were prepared and 1mL of Lucas Reagent was dropped in each of them. One the first test tube, 2-3 drops of n-butyl

C. Chromic acid test (Jones’ Oxidation) Six test tubes were prepared as well as six samples. The samples are n-butyl alcohol, isopropyl alcohol, tert-butyl alcohol, acetaldehyde, benzaldehyde, and acetone. One drop of each sample was placed on different test tubes and was dissolved in 1mL of acetone followed by the addition of 5 drops of the reagent dropwise with continuous shaking. Place the test tubes in 60oC water bath for 5 minutes and note the color of each solution. D. 2,4-Dinitrophenylhydrazone Test Four test tubes were labelled with the samples to be used. The samples used were acetone, acetaldehyde, benzaldehyde, and acetophenone. One drop of each sample was placed in different test tubes followed by the addition of 5 drops of 95% ethanol with continuous shaking. Then add 3 drops of 2,4-DNP. If no yellow or orange-red precipitation forms, allow the solution to stand for 15 minutes. Note the results. E. Fehling’s Test Into each test tube, 1mL of freshly prepared Fehling’s reagent (made by mixing equal amounts of Fehling’s A and Fehling’s B) was placed. 3 drops of the sample to be tested were added and the test tubes were placed into a beaker of boiling water. Changes were observed that occurred within 10-15 minutes. The samples to be used are acetaldehyde, acetone, benzaldehyde, and acetophenone.

F. Tollens’ Silver Mirror Test Four test tubes were prepared that contains 1mL of freshly prepared Tollens’ reagent and two drops of each sample was placed into separate test tubes. The samples were acetaldehyde, benzaldehyde, acetone, acetophenone. Shake the mixture well and allow it to stand for 10 minutes. If no reaction has occurred, place the test tubes in a beaker with warm water for 5 minutes. Record any observation. I. Iodoform Test 2 drops of each sample (acetaldehyde, acetone, acetophenone, benzaldehyde, and isopropyl alcohol) were placed in different test tubes. 10 drops of 10% KI solution were added. Followed by the addition of 20 drops of fresh chlorine bleach (5% sodium hypochlorite) were added slowly to each test tube with continuous shaking. The formation of a yellow precipitate was noted. G. Hydrolysis of Acid Derivatives Acid Halides and Acid anhydrides In a test tube, 1mL of water was placed, and then 10 drops of the sample was cautiously added dropwise. The resulting mixture was divided into two portions. 1mL of 2% AgNO was added to the first test tube while 1mL of saturated NaHCO3 was added to the 2nd portion. Esters 2mL of 25% NaOH solution was added to 1mL of ethyl acetate. The mouth of the test tube was covered with a marble then the test tube was heated in a boiling water bath for 5 minutes. Afterwards, the mixture was neutralized with 10% HCl solution. The test tube was wafted to take note of the odor.

Amides

Here 1 mL of benzamide was treated with 5mL of NaOH solution and was got immersed in a boiling water bath. During heating, a moist red litmus paper was placed over the test tube and any change in the litmus paper was noted. H. Alcoholysis: Schotten-Baumann Rxn Acetic Acid In this procedure, 10 drops of acetic acid, 1mL ethanol, and 5 drops of concentrated H2SO4 was warmed on a water bath for 2 minutes and then note the odor of the ester formed. Acyl halides and Acid anhydrides In another test tube, we placed 0.5 mL ethanol, 1mL water, and 0.2mL of acyl halide or acid anhydride sample. Then add 2mL of 20% NaOH solution. Plug the test tube using a cork stopper or parafilm and shake the mixture for several minutes. Afterwards, note the odor and the layers of the ester formed. I. Aminolysis: Anilide Formation Acyl halides and acid anhydrides A few drops of either acetyl chloride or acetic anhydride were added to 0.5mL aniline. The mixture was then transferred to a new test tube containing 5mL of water. Formation of precipitation was then observed. J. Hydroxamic Acid Test Preliminary test was done by mixing 1mL of 95% ethanol and 1M HCl to separate test tubes that contained drops of the sample to be used. If upon addition of 1 drop of 5% FeCl3, a color other than yellow will make the test futile. 2mL of alcoholic NH2OH∙HCl and 1mL of 1M KOH was added to separate test tubes of 2 drops ethyl acetate and acetamide. Immersion in a boiling water bath for 2 minutes was done; afterwards the mixture was allowed to cool.

1mL of 5% FeCl3 was introduced in each test tube. Then the color of the formed precipitate was noted.

true for organic compounds that have the same number of carbon atoms present.

RESULTS AND DISCUSSION

The solubility of alcohols decreases as the length of the hydrocarbon chain of the alcohol increases.

For the test of the solubility of alcohols in water, the turbidity of the solution was observed and it was the basis if the specific alcohol is soluble to water. Also, the amount of water needed to produce homogenous dispersion was observed. Table 1 shows the data gathered from the test.

The next test was conducted with the use of the Lucas Reagent and it differentiated 1”, 2” and 3” alcohols. Alkyl chloride formation was observed and caused turbidity or cloudiness. Also, the rate of the reaction was observed. Table 2 shows the result of Lucas Test.

Alcohol

ethanol n-butyl alcohol sec-butyl alcohol Tert-butyl alcohol Benzyl alcohol

Amount of water needed to produce homogenous dispersion 1mL 1.50mL 1mL

Solubility to water

Sample n-butyl alcohol Sec-butyl alcohol Tert-butyl alcohol

Observation Homogenous solution Homogenous solution Formation of 2 layers

Table 2 : Lucas Test results Soluble Soluble Soluble

1mL

Soluble

2mL

Insoluble

Table 1 : Solubility of Alcohols in Water Table 1 shows the solubility of alcohols to water and the amount of water that is needed to produce a homogenous dispersion.Out of the 5 alcohol samples, only Benzyl Alcohol is insoluble. The principle behind this is that “like dissolves like”. It can be said that the other alcohols are soluble to water because they exhibit a polar bond and water is also known to have a polar bond as well. There are factors that affect the solubility of alcohols to water. One of these factors is the number of carbon atoms present. The lower the number of carbon atoms present, the more soluble or miscible a substance is. Another factor is the branching of carbon chains. The more branching present, the more soluble a compound is. This is only

According to the table above, n-butyl alcohol and sec-butyl alcohol are both soluble in the Lucas Reagent while tertbutyl alcohol formed a cloudy layer. The absence of visible reaction at room temperature and the appearance of the cloudy layer on the application of heating, represents that it is a primary alcohol. A delayed formation of a cloudy layer; around 3-5 minutes tells us that it is a secondary alcohol. And lastly, if the substance turn cloudy immediately after the addition of Lucas reagent and the phases separate means that it is a tertiary alcohol. Basing on the table above, tert-butyl alcohol is a tertiary alcohol due to the immediate clouding of the alcohol and the separation of layers. Chromic Acid test (Jones Oxidation) permits the conversion of primary alcohols to aldehydes and secondary alcohols to ketones through oxidation. Table 3 shows the results of Chromic Acid test.

Sample n-butyl alcohol Tert-butyl alcohol Acetaldehyde Benzaldehyde Acetone Acetophenone Isopropyl Alcohol

Observation Blue-green solution Dirt yellow solution Blue-green solution Blue-green solution Green solution Dirty yellow solution Blue-green solution

Table 3 : Chromic Acid test results In the data gathered, n-butyl alcohol, acetaldehyde, benzaldehyde, and isopropyl alcohol gave a positive result of a blue-green solution while tert-butyl alcohol and acetophenone both gave a dirty yellow solution while acetone gave a green solution. Chromic test or Jones Oxidation involved reduction-oxidation or redox reaction. 1” and 2” alcohols and aldehydes went through oxidation and chromium underwent reduction from Cr6+ to Cr3+. The reduction of the chromium ion which is colored orange resulted to a blue-green solution telling us that the 1” and 2” alcohols were oxidized. Ketones are the result of the reaction of 2” alcohols with chromic acid, which do not oxidize further. Tertiary alcohols are nonreactive and aldehydes got oxidized to carboxylic acids. The next test, which is 2,4Dinitrophenylhydrazne test / 2,4-DNP is the test used for distinguishing the presence of an aldehyde or ketone in a compound. Table 4 shows the data gathered from the test. Sample Acetaldehyde Benzaldehyde Acetone Acetophenone

Observation Yellow-orange ppt. Yellow-orange ppt. Canary yellow ppt. Orange-red ppt.

Table 4 : 2,4-DNP test results

In the test, acetophenone was the only sample to give an orange-red ppt. while the other samples gave a yellow-orange and canary yellow precipitates. The orange-red precipitate represents the presence of a conjugated carbonyl compound and the appearance of the yellow precipitate indicates the presence of unconjugated carbonyl compound. Fehling’s Test is another differentiating test for aldehydes and ketones. In this test aldehydes reacted in this test while ketones did not produce any reaction. Sample Acetaldehyde Benzaldehyde Acetone Acetophenone

Observation Brick-red ppt. Brick-red ppt. Dark blue solution Royal blue solution

Table 5 : Fehling’s test results As shown on the table below above, acetaldehyde and benzaldehyde both produced a brick-red precipitate indicating that they are both aldehydes. Meanwhile acetone and acetophenone did not produce a reaction what so ever. Fehling’s test involved reduction-oxidation or redox reaction. Aldehydes got oxidized while ketones did not undergo oxidation. Tollens’ Silver Mirror test differentiated aldehydes from ketones wherein aldehydes were expected to be oxidized while ketones did not undergo any oxidation. Table 6 shows the results on Tollens’ Silver Mirror test. Sample Acetaldehyde Benzaldehyde Acetone Acetophenone

Observation Silver mirror Gray sol’n w/ globules Colorless sol’n Turbid gray sol’n

Table 7 : Tollens’ Silver Mirror test results On the table above, only acetaldehyde formed a silver mirror. Benzaldehyde, even

though it is an aldehyde did not form any silver mirror. Acetone formed a colorless solution and acetophenone produced a turbid gray solution. This test is similar to Fehling’s test making it undergo redox reaction as well. Aldehydes got oxidized while ketones did not. Iodoform test was used to detect the presence of an aldehyde or a ketone in a substance where methyl groups are attached directly to the carbonyl carbon. Sample Acetaldehyde n-butylraldehyde Benzaldehyde Acetone Acetophenone Isopropyl Alcohol

Observation Yellow ppt. Yellow solution Red ppt. w/ globules Yellow ppt. Yellow ppt. Yellow crystal ppt.

Table 7 : Iodoform test results As shown in the table above, acetaldehyde, acetone, and acetophenone produced a yellow precipitate. Benzaldehyde resulted produced a red precipitate with globules. Isopropyl alcohol resulted in a yellow crystalline precipitate and n-butyraldehyde resulted in a yellow solution. The appearance of a yellow precipitate or a yellow crystal indicates a positive result. It also tells us that the carbonyl group of the sample has a methyl group attached directly to it. REFERENCES From books 1. Brown, W., Poon, T. (2011). Introduction to organic chemistry international student version (5th edition). NJ, USA: John Wiley & Sons, Inc. 2. Zumdahl, S., Zumdahl, S. (2012). Chemistry: An Atoms First Approach (International edition). USA: Brooks/Cole, Cengage Learning.

From the Internet 3. Clark, J. (2003). An Introduction to Alcohols. Retrieved from http://www.chemguide.co.uk/organic props/alcohols/background.html 4. Clark, J. (2003). Aldehydes and Ketones. Retrieved from http://www.chemguide.co.uk/organic props/carbonyls/background.html 5. Lucas Reagent (n.d.) Retrieved from http://www.chemistrylearner.com/luc as-reagent.html 6. Test for Aldehydes and Ketones (n.d.) Retrieved from http://academics.wellesley.edu/Chem istry/chem211lab/Orgo_Lab_Manual /Appendix/ClassificationTests/aldehy de_ketone.html 7. 2,4-Dinitrophenylhydrazine test (n.d.) Retrieved from http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/qual/ dnp.html 8. Fehling’s Test (n.d.) Retrieved from https://fenix.tecnico.ulisboa.pt/downl oadFile/3779571247498/Testes %20de%20a%C3%A7ucaresalunos.pdf 9. Tollens’ Test (n.d.) Retrieved from http://chemwiki.ucdavis.edu/? title=Organic_Chemistry/Aldehydes _and_Ketones/Reactivity_of_Aldehy des_%26_Ketones/Tollens %E2%80%99_Test 10. Clark, J. (2004). Iodoform Reaction with Aldehydes and Ketones. Retrieved from http://www.chemguide.co.uk/organic props/carbonyls/iodoform.html