Experiment 9 Classification Tests for Hydroxyl- & Carbonyl-Containing Compounds

Experiment 9 Classification Tests for Hydroxyl- & Carbonyl- Containing Compounds Sunglao, A., Supan, E., Tan, C., Tayag, P., Tuason, A. Group # 9, 2G – Medical Technology, Faculty of Pharmacy, University of Santo Tomas

Abstract In organic chemistry, functional groups are specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules and examples of functional groups are hydroxyl and carbonyl groups. Hydroxyl groups have one hydrogen paired with one oxygen atom (symbolized as –OH) and are usually seen in alcohols while carbonyl groups have one oxygen atom double-bonded to a carbon atom (symbolized as C=O) and are usually seen in aldehydes and ketones. In this experiment, several differentiating tests were conducted with the samples ethanol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, benzyl alcohol, n-butyraldehyde, benzaldehyde, acetone, acetophenone, isopropyl alcohol and acetaldehyde. These tests include solubility of alcohols in water, Lucas test, Chromic Acid test (Jones oxidation), 2,4-Dinitrophenylhydrazone (2,4-DNP/ 2,4-DNPH) test, Fehling’s test, Tollens’ Silver Mirror test and Iodoform test. A positive result on solubility was seen on alcohols under six (6) carbon atoms while the Lucas test differentiates 1˚, 2˚ and 3˚ alcohols, gives a positive result of turbidity (alkyl chloride formation), and the rate of the reaction was observed. The Chromic Acid test/Jones Test tests for oxidizable or any compounds that possess reducing property (has an alpha acidic hydrogen) and 1˚, 2˚ and 3˚ alcohols and aldehydes give a positive visible result of a bluegreen suspension. On the other hand, the 2,4-Dinitrophenylhydrazone (2,4-DNP/2,4-DNPH) test is used as confirmatory for carbonyl groups and therefore, gives a positive result of red-orange precipitate or yellow precipitate for aldehydes and ketones. Fehling’s test and Tollens’ Silver Mirror test are both used to identify aldehydes wherein a positive result of brick-red precipitate can be seen through the Fehling’s test and silver mirror for the Tollens’ test. Iodoform test is used as confirmatory for methyl carbinol (2˚ alcohol with adjacent methyl group) and methyl carbonyl groups and shows a positive result of yellow crystals or precipitate. In conclusion, n-butyraldehyde, benzaldehyde and acetaldehyde are aldehydes while acetone and acetophenone are ketones and all of these contain carbonyl groups. Ethanol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol and benzyl alcohol are all alcohols containing hydroxyl groups wherein 3˚ alcohols are the most stable while 1˚ alcohols are the most polar among the three.

Introduction An alcohol is a compound that has a hydroxyl group bonded to a saturated, spᶟhybridized carbon atom, R-OH (Figure 1). Alcohols are classified as primary (1˚), secondary (2˚), or tertiary (3˚), depending on the

number of carbon substituents bonded to the hydroxyl-bearing carbon (McMurry, 2010). The most important physical property of alcohols is the polarity of their –OH groups. Because of the large difference in electronegativity between oxygen and carbon and between oxygen and hydrogen, both the C-O and O-H bonds of an

alcohol are polar covalent, and alcohols are polar molecules (Brown & Poon, 2011). Alcohols usually have much higher boiling points than might be expected from their molar masses. This difference can be understood if we consider the types of intermolecular attractions that occur in these liquids (Zumdahl & Zumdahl, 2012).

carbocation, which in turn reacts with the chloride ion (nucleophile) to generate an alkyl halide product. Figure 4 shows the general mechanism for this SN1 reaction.

Figure 1. Structure of Alcohol The functional group of an aldehyde is a carbonyl group bonded to a hydrogen atom (Figure 2). The functional group of a ketone is a carbonyl group bonded to two carbon atoms (Figure 3). Because of the polarity of the carbonyl group, aldehydes and ketones are polar compounds and interact in the liquid state by dipole-dipole interactions. As a result, aldehydes and ketones have higher boiling points than those of nonpolar compounds with comparable molecular weight (Brown & Poon, 2011).

Figure 4. General Mechanism of Lucas test The Chromic acid test (Jones oxidation) distinguishes primary and secondary alcohols from tertiary. Chromic acid will oxidize a primary alcohol first to an aldehyde and then to a carboxylic acid and it will oxidize a secondary alcohol to a ketone. Tertiary alcohols do not react. The OH-bearing carbon must have a hydrogen atom attached. Since the carbon atom is being oxidized in primary and secondary, the orange chromium Cr6+ ion is being reduced to the blue-green Cr3+ ion. The reactions involved are shown in Figure 5.

Figure 2. Structure of Aldehyde

Figure 3. Structure of Ketone The Lucas reagent is an aqueous solution of strong acid (HCl) and zinc chloride (ZnCl₂). The alcohol starting material must be sufficiently soluble in aqueous environments for the reaction to take place. The reaction that occurs in the Lucas test is an SN1 nucleophilic substitution. The acid catalyst activates the OH group of the alcohol by protonating the oxygen atom. The C-OH₂+ bond breaks to generate the

Figure 5. General Mechanism of Jones Oxidation The 2,4-Dinitrophenylhydrazone test serves as a derivative formation. Both aldehydes and ketones react with 2,4dinitrophenylhydrazine to form a solid 2,4dinitrophenylhydrazone (DNP) derivative. The color of this derivative can also provide useful structural information. If the solid is yellow, this most often means that the carbonyl group in the

unknown is unconjugated. A reddish-orange color most likely means that the carbonyl group is conjugated. In a few cases, compounds in which the carbonyl group is not conjugated produce orange precipitates. Simply having a double bon or phenyl group somewhere in an aldehyde or ketone does not necessarily mean that the carbonyl group is conjugated. The double bond must be separated from the carbonyl by one single bond only. If the double bond is further away, it is isolated from the carbonyl and not conjugated with the carbonyl. Figure 6 illustrates the reaction occurring in the 2,4-DNP/2,4-DNPH test.

oxidized, whereas ketones are not. Tollens’ test uses a reagent known as Tollens’ reagent, which is a colorless, basic, aqueous solution containing silver ions coordinated to ammonia [Ag(NH3)2+]. Tollens’ reagent oxidizes an aldehyde into the corresponding carboxylic acid. The reaction is accompanied by the reduction of silver ions in Tollens’ reagent into metallic silver, which, if the test is carried out in a clean glass test tube, forms a mirror on the test tube. Ketones are not oxidized by the Tollens’ reagent, so the treatment of a ketone with Tollens’ reagent in a glass test tube does not result in a silver mirror. The reactions involved in the silver mirror test are shown in Figure 8.

Figure 8. General Mechanism of Tollens’ Silver Mirror test Figure 6. General Mechanism of 2,4Dinitrophenylhydrazone test In Fehling’s test, the presence of aldehydes and not ketones is detected by reduction of the deep blue solution of copper (II) to muddy green solution, and then form a brickred precipitate of insoluble cuprous oxide (Cu2O). This test is commonly used for reducing sugars but is known to be not specific for aldehydes. Figure 7 shows the general mechanism undergone in the Fehling’s test.

Figure 7. General Mechanism of Fehling’s test Tollens’ test, also known as silvermirror test, is a qualitative laboratory test used to distinguish between and aldehyde and a ketone. It exploits the fact that aldehydes are readily

The Iodoform test indicates the presence of an aldehyde or ketone in which one of the groups directly attached to the carbonyl carbon is a methyl group. Such a ketone is called a methyl ketone. In the Iodoform test, the sample is allowed to react with a mixture of iodine and base. Hydrogens alpha to a carbony group are acidic and will react with base to form the anion, which then reacts with iodine in this way to form the triiodo compound, which the reacts with more base to form the carboxylic acid salt plus iodoform, a yellow precipitate. Formation of a yellow precipitate therefore indicates the presence of a methyl group directly attached to the carbonyl. The mechanism of the iodoform reaction is that of alpha-halogenation of a carbonyl compound under basic conditions, followed by nucleophilic displacement of the resulting triiodomethyl group by hydroxide. Figure 9 illustrates the reaction involved in the Iodoform test.

Figure 9. Genral Mechanism of Iodoform test One of the objectives of this experiment is to distinguish whether a compound is hydroxyl- or carbonyl-containing. Other objectives include differentiating the three types of alcohols, differentiating aldehydes from ketones, and explaining the mechanisms involved in the differentiating tests. Materials and Methods A. Materials In this experiment, the materials needed are Lucas reagent, chromic acid reagent, 95% ethanol, Fehling’s A and B, Tollens’ reagent, 5% NaOCl solution, iodoform test reagent, 2,4dinitrophenylhydrazine, Pasteur pipette, test tubes, beaker, and the sample compounds ethanol, n-butyl alcohol, sec-butyl alcohol, tertbutyl alcohol, benzyl alcohol, n-butyraldehyde, benzaldehyde, acetone, acetophenone, isopropyl alcohol and acetaldehyde. B. Methods 1. Solubility of Alcohols in Water In the five test tubes, 10 drops each of ethanol, n-butyl alcohol, sec-butyl alcohol, tertbutyl alcohol and benzyl alcohol were placed using a Pasteur pipette. 1 mL of water was added dropwise in each of the test tubes containing alcohol while shaking the mixture after each addition. When cloudiness resulted, 0.25 mL of water was added at a time, with vigorous shaking, until a homogenous dispersion appeared. The total volume of water added was noted. When no cloudiness resulted after the addition of 2.0 mL water, alcohol was noted down to be soluble in water. 2. Lucas test

Three test tubes were prepared and 1 mL of Lucas reagent was dropped in each of the test tubes. On the first test tube, 2-3 drops of n-butyl alcohol was added. The test tube was shook vigorously for a few seconds and the mixture was allowed to stand at room temperature. The two other test tubes were placed with 2-3 drops of sec-butyl alcohol and tert-butyl alcohol, respectively, shook vigorously and the mixtures were allowed to stand. The rate of formation of cloudy suspension or the formation of two layers was observed. 3. Chromic Acid test (Jones Oxidation) Using the samples n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, n-butyraldehyde, benzaldehyde, acetone, and acetophenone, one drop of each liquid or small amount of the solid sample was dissolved in 1 mL of acetone in different test tubes. 2 drops of 10% aqueous K2Cr2O7 solution and 5 drops of 6 M H2SO4 were added in each of the test tubes. 4. 2,4-Dinitrophenylhydrazone DNP/2,4-DNPH) test

(2,4-

One drop of a liquid sample (acetone, acetaldehyde, n-butylraldehyde, benzaldehyde and acetophenone) was placed in a test tube and 5 drops of 95% ethanol were added. After shaking well, 3 drops of 2,4dinitrophenylhydrazine were added. The solution was allowed to stand for at least 15 minutes when no yellow or orange-red precipitate forms. 5. Fehling’s test Into each test tube, 1 mL 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 in a beaker of boiling water. Changes were observed that occurred within 10-15 minutes. The test was performed on acetaldehyde, n-butyraldehyde, acetone, benzaldehyde and acetophenone. 6. Tollens’ Silver Mirror test

Five test tubes with 1 mL each of freshly prepared Tollens’ reagent were prepared. In each of the test tubes, 2 drops of each of the samples (acetaldehyde, benzaldehyde, acetone, nbutyraldehyde and acetophenone) were added separately. The mixtures were shaken and allowed to stand for 10 minutes. When no reaction occurred, the test tubes were placed in a beaker with warm water for 5 minutes. Observations were recorded. 7. Iodoform test 2 drops of each sample (acetaldehyde, acetone, acetophenone, benzaldehyde and isopropyl alcohol) were placed in different test tubes. 20 drops of 10% KI solution were added. While shaking, 20 drops of fresh chlorine bleach (5% sodium hypochlorite) were added slowly to each tube and mixed. The formation of a yellow precipitate was noted. Results and Discussion For the test for solubility of alcohols in water, turbidity of the solution was to be observed. Cloudiness of the solution indicated insolubility of that specific alcohol to water. The amount of water needed to produce homogenous dispersion was also observed. Table 1 shows the data gathered from the test.

butyl alcohol, sec-butyl alcohol and tert-butyl alcohol were all soluble in water. This follows the principle “like dissolves like” and therefore, it can be said that the alcohols that were soluble in water are polar compounds since water is polar. Of the alcohols that were soluble in water, ethanol, sec-butyl alcohol and tert-butyl alcohol all required only 1 mL of water to be added in order to be considered soluble. This indicates that there are certain factors affecting solubility. One of these is the presence of number of carbon atoms. The lower the number of carbon atoms present, the more soluble or more miscible a substance is. Branching of carbon chains also affect solubility. The more branching present, the more soluble a compound is. This is only true for organic compounds that have the same number of carbon atoms present. The Lucas test differentiated 1˚, 2˚ and 3˚ alcohols. Alkyl chloride formation was observed and caused turbidity or cloudiness. The rate of reaction was also observed. Table 2 presents the results of the Lucas test. Table 2. Lucas test Sample n-butyl alcohol sec-butyl alcohol tert-butyl alcohol

Reaction observed colorless slightly turbid turbid

Table 1. Solubility of Alcohols in Water Alcohol

ethanol n-butyl alcohol sec-butyl alcohol tert-butyl alcohol benzyl alcohol

Amount of water (in mL) needed to produce a homogenous dispersion 1 mL 1.50 mL

Solubility to water

1 mL

soluble

1 mL

soluble

2 mL

insoluble

soluble soluble

As indicated in the table, only benzyl alcohol was insoluble in water, while ethanol, n-

According to the table above, n-butyl alcohol was soluble in Lucas reagent while secbutyl alcohol and tert-butyl alcohol were observed to have a formation of cloudy layer. Tert-butyl alcohol took the shortest time to form the layer while sec-butyl alcohol took the longest time. The reaction mechanism involved in the Lucas test is based on SN1 reaction, which depends on the formation of stable carbocations. Reactivity of alcohols in SN1 reaction is 3˚ > 2˚ > 1˚. 3˚ alcohols formed the second layer in less than a minute. 2˚ alcohols required 5-10 minutes while 1˚ alcohols were usually unreactive. The presence of ZnCl2, a good Lewis acid, made the reaction mixture even more acidic; thus, it enhanced the formation of carbocations.

The Chromic Acid test (Jones Oxidation) tested for oxidizables or any compounds that possess reducing property (has an alpha acidic hydrogen). Table 3 shows the results gathered from the said test. Table 3. Chromic Acid test (Jones Oxidation) Sample n-butyl alcohol sec-butyl alcohol tert-butyl alcohol n-butyraldehyde benzaldehyde acetone acetophenone

Reaction observed blue-green solution blue-green solution orange solution blue-green solution blue-green solution orange solution orange solution

According to Table 3, n-butyl alcohol, sec-butyl alcohol, n-butyraldehyde and bezaldehyde gave a positive result of blue-green solution while tert-butyl alcohol, acetone and acetophenone gave a result of orange solution. Chromic Acid test / Jones Oxidation involved reduction-oxidation or redox reaction. 1˚ and 2˚ alcohols and aldehydes underwent oxidation and chromium underwent reduction from Cr6+ to Cr3+. 1˚ and 2˚ alcohols and aldehydes reduced the orange-red chromic acid/sulphuric acid reagent to an opaque green or blue suspension of Cr (III) salts in 2-5 seconds. 1˚ alcohols reacted with chromic acid to yield aldehydes, which are further oxidized to carboxylic acids. 2˚ alcohols reacted with chromic acid to yield ketones, which do not oxidize further. 3˚ alcohols were usually unreactive and aldehydes were oxidized to carboxylic acids. The 2,4-Dinitrophenylhydrazone (2,4DNP/2,4-DNPH) test detected the presence of carbonyl groups and tests positive for aldehydes and ketones. Table 4 shows the results from the test. Table 4. 2,4-Ditrophenylhydrazone (2,4DNP/2,4-DNPH) test Sample acetaldehyde n-butyraldehyde benzaldehyde

Reaction observed yellow ppt yellow ppt yellow ppt

acetone acetophenone

yellow ppt red-orange ppt

As indicated in the table, only acetophenone gave a result of red-orange precipitate while the rest of the samples gave a result of yellow precipitate. A result of redorange precipitate indicated the presence of conjugated carbonyl compounds while a result of yellow precipitate indicateed the presence of unconjugated carbonyl compounds. The reaction of 2,4-DNPH with aldehydes and ketones in an acidic solution is a dependable and sensitive test. Its reaction mechanism involved condensation or nucleophilic addition of NH2 to C=O and elimination of H2O. Some high molecular weight ketones may fail to react or may yield oils. Most aromatic aldehydes and ketones produce red dinitrophenylhydrazone while many nonaromatic aldehydes and ketones produced yellow products. Fehling’s test was another differentiating test for aldehydes and ketones. In this test, aldehydes gave a positive result of brick-red precipitate while ketones did not produce any reaction. Table 5 presents the results of the test. Table 5. Fehling’s test Sample acetaldehyde n-butyraldehyde benzaldehyde acetone acetophenone

Reaction observed brick-red ppt brick-red ppt brick-red ppt blue solution blue solution

As shown in the given table, only acetone and acetophenone did not react to form a precipitate while the rest gave a positive result of brick-red precipitate. Fehling’s test involved reduction-oxidation or redox reaction. Aldehydes were oxidized to carboxylic acids while ketones did not undergo oxidation. In here, copper was reduced from Cu2+ to Cu1+. 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 from the said test. Table 6. Tollens’ Silver Mirror test Sample acetaldehyde n-butyraldehyde benzaldehyde acetone acetophenone

Result observed silver mirror flesh solution light yellow solution with globules dark-gray solution turbid gray solution

According to Table 6, only acetaldehyde formed a silver mirror. The samples nbutyraldehyde and benzaldehyde, although they are aldehydes, did not form any silver mirror. The ketones acetone and acetophenone formed dark-gray solution and turbid gray solution, respectively. The preparation of Tollens’ reagent was based on the formation of a silver diamine complex that is water soluble in basic solution. The Tollens’ Silver Mirror test involved reduction-oxidation or redox reaction. Aldehydes were oxidized to carboxylic acids while ketones did not undergo oxidation except alpha-hydroxyketone. Silver was reduced from Ag1+ to Ag0. Iodoform test was used to detect the presence of methyl carbinol (2˚ alcohol with adjacent methyl group) and methyl carbonyl groups. Table 7 shows the results from the said test. Table 7. Iodoform test Sample acetaldehyde n-butyraldehyde benzaldehyde acetone acetophenone isopropyl alcohol

Reaction observed yellow ppt no reaction red ppt with globules yellow ppt yellow ppt yellow crystal ppt

According to the give table, acetaldehyde, acetone and acetophenone gave aresult of yellow precipitate. Benzaldehyde gave

a result of red precipitate with globules while isopropyl alcohol gave a result of yellow crystal precipitates. No reaction was observed from nbutyraldehyde. In this test, yellow crystals or precipitate gave a positive result. An alkaline solution of sodium hypoiodite, formed from sodium hydroxide and iodine, converted acetaldehyde and aliphatic methyl ketones into iodoform (haloform reaction). Since the reagent was also an oxidizing agent, alcohols which are readily oxidized to acetaldehydes or methyl ketones also gave a positive reaction. The mechanism of iodoform synthesis occurred through a series of enolate anions, which are iodinated; hydroxide displaced the Cl3- anion through an addition/elimination pathway. References From books: Brown, W., Poon, T. (2011). Introduction to organic chemistry international student version (5th edition). NJ, USA: John Wiley & Sons, Inc. McMurry, J. (2010). Foundations of organic chemistry (Philippine edition). USA: Cengage Learning Asia Pte. Ltd. Zumdahl, S., Zumdahl, S. (2012). Chemistry: An Atoms First Appraoch (International edition). USA: Brooks/Cole, Cengage Learning. From the internet: http://swc2.hccs.edu/pahlavan/2425L6.pdf http://www.as.utexas.edu/astronomy/education/s pring07/scalo/secure/AbioFunctionalGrpsVollIR spect.pdf http://www.phschool.com/science/biology_place /biocoach/biokit/hydroxyl.html http://www.organicchem.org/oc2web/lab/exp/ox id/lucas.pdf http://myweb.brooklyn.liu.edu/swatson/Site/Lab oratory_Manuals_files/Exp6.pdf https://dspace.ist.utl.pt/bitstream/2295/50905/1/ Testes%20de%20a%C3%A7ucares-alunos.pdf

http://science.uvu.edu/ochem/index.php/alphabet ical/s-t/tollens-test/ http://www.chem.umass.edu/~samal/269/aak.pdf