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Physical Sciences Department

Laboratory Activity Sheet No. 1

Qualitative Tests for the Identification of Functional Groups in Organic Compounds

Group Members: Abcede, Cheda Trisha D. Laboratory Schedule: June 27, 2022
Anico, Joshua G.
Aporbo, Albraiell Paul Justin U.
Antony, Johanny Paula O.
Aquino, Joshua Keifer Q.

Program and Year: BSMT - 1A Score: _______________________

I. Objectives:
1. Identify the different functional groups present on the endemic plant structure; and
2. Determine the necessary qualitative tests used for identifying various functional groups.

II. Scenario
Your team is working in a pharmaceutical company and has isolated a new compound from an endemic plant
in Cebu that has a potential antimicrobial property. The structure of the compound was suspected to be the
same as the one shown below based on previous studies of the plant. Using qualitative tests, create a method
that would identify the different functional groups in the said structure.

III. Hypothesis-Testing Method Design

A. Problem:
The structure of a new compound isolated from an endemic plant having potential antibacterial properties is
believed to be the same as the structure based on previous studies of the plant. To identify the various functional
groups contained in the chemical, a series of qualitative tests must be performed.

B. Theory:
Alcohols are compounds containing a hydroxyl group (–OH) attached to a saturated carbon. An alcohol has a
generic formula of R – OH, to denote that the carbon directly attached to OH is saturated. Alcohols can be classified
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as primary, secondary, or tertiary according to the number of carbons attached to the carbon directly attached to the –
OH group. one carbon directly attached to the (-OH) indicates a primary alcohol, two carbons attached indicates a
secondary alcohol, while three carbons attached indicates a tertiary alcohol.

Phenols are compounds containing hydroxyl group (-OH) attached to a benzene ring. Moreover, it is not
considered an alcohol.

Carboxylic acids are compounds containing carboxyl groups. When a carbonyl group is attached directly to a
hydroxyl group, it becomes a supra- functional group known as carboxyl group. The reason this is considered to be a
new functional group is because this combination results in acidic properties. A carbon (C) atom is bonded to an oxygen
(O) atom by a double bond and to a hydroxyl group (―OH) by a single bond. A fourth bond links the carbon atom to a
hydrogen (H) atom or to some other univalent combining group.

Ketone is a functional group with the structure R2C=O, where R can be a variety of carbon-containing substituents.
Ketones contain a carbonyl group (a carbon-oxygen double bond). In the case of ketones, there are two carbon atoms
bonded to the carbonyl carbon and no hydrogen. If the carbonyl group is present in both carbons, the compound is
classified as a ketone.

Lucas Test is a differentiating test among primary, secondary, and tertiary alcohols. Reagents used include a
mixture of concentrated hydrochloric and zinc chloride that make up the Lucas reagent. The Lucas reagent converts
alcohols into alkyl chlorides, this is evident through the formation of a cloudy layer after the reaction. The speed of the
reaction is what differentiates the types of alcohols wherein tertiary alcohol has the fastest reaction while primary
alcohol has the slowest reaction.

Ferric Chloride Test is a specific test for phenolic group detection. Ferric Chloride is the reagent used and this test
is based on the fact that phenolic groups can form purple colored complex with ferric ions which cannot be seen in
other compounds containing hydroxyl group. The formation of a violet color complex indicates a positive test for the
phenolic group.

2,4-DNP or 2,4-Dinitrophenylhydrazine Test is a general test for the presence of a carbonyl group. This means
that both aldehydes and ketones will give a positive test result. The positive test result indicates a yellow precipitate
which is the DNP product produced from the reaction between the 2,4-DNP and carbonyl compound. Heating the
mixture after adding 2,4-DNP solution triggers a reaction to make yellow precipitate from the mixture. Positive result
indicates a yellow color precipitate and a confirmatory presence of a carbonyl group.

Tollen’s Test is used as a differentiating test between aldehydes and ketones. The test is based on oxidation
reaction. Between aldehydes and ketones, only aldehydes can be oxidized and can only be the one to give a positive
result. Positive result indicates a formation of a silver mirror or precipitate which is due to the conversion of reagent
into silver metal. Positive result indicates a silver mirror or precipitate and a confirmatory presence of an aldehyde,
differentiating aldehyde from ketone.
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Blue Litmus Paper Test is a test wherein a blue litmus paper will turn red upon contact with an acid. Thus, the
same reaction is expected for carboxylic acids having acidic properties. Litmus paper is widely used to determine if a
substance is an acid or a base. There are 2 types of litmus paper, the blue and the red. A change of blue litmus paper
to red indicates that the substance being tested is an acid. Like most acids, carboxylic acid turns blue litmus red.
Moreover, blue litmus paper confirms a basic solution, while red litmus paper confirms an acidic solution or acidic
group.

Sodium Bicarbonate Test is a test wherein the acidic group will react with sodium bicarbonate ions producing
carbon dioxide and is seen in the formation of brisk effervescence or bubbling indicating a positive result. The reagent
used for this test is the saturated sodium bicarbonate solution. It is the confirmatory test for the presence of a carboxylic
acid.

C. Hypothesis:
Alcohol, phenol, carboxylic acid, and ketone are the functional groups that are believed to be present in the
compound isolated from the endemic plant.

D. Procedure:

Qualitative Tests used for Functional Group Identification

1. Lucas Test (Differentiating 1st to 3rd Alcohol)

The first test that was conducted to confirm whether the collected aqueous sample from the plant is an alcohol
that could be primary, secondary, or tertiary. The acquired result that showed positive result when mixed with the Lucas
reagent would imply that the alcohol is one of the three types of Alcohol.
Lucas test was conducted by mixing 0.4 mL of the unknown sample with 6 mL of the hydrochloric acid-zinc chloride
reagent along with the other control samples labeled as Primary, Secondary, and Tertiary in a test tube. The test tubes
showed result before it was placed in a hot water bath. However, to confirm the sample's result the test tube underwent
the hot water bath treatment. Compared with the result from the control samples, the unknown solution showed an
emulsion even before the bath and the result was taken at the same time.

2. Ferric Chloride Test (For Phenolic Group)

The 2nd test that was conducted to detect Phenols was Ferric Chloride test. 1mL of Ethanol, isopropyl, alcohol,
tert-butyl alcohol and 3% phenol in their respective tubes was used and 1 drop of 2% Ferric Chloride solution was
added as its reagent. The positive result that showed the presence of Phenolic group when mixed was a violet color
that cannot be seen in any other hydroxyl group therefore besides the 3% Phenol, the rest stayed in the color orange.

Test for Aldehydes and Ketones

3. 2,4-DNP (Dinitrophenylhydrazine) Test (General Test for Carbonyl Group)

The first test that was conducted to detect the presence of Carbonyl Group was 2,4-Dinitrophenylhydrazine (2,4-
DNP). 1 mL of Acetaldehyde was used to represent Aldehydes, Acetone for Ketone and ethanol in their respective test
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tubes was used. 1 mL of 2,4-DNP reagent was added to each of the test tubes and was then heated in a boiling water
bath for 5 minutes. While it was boiling, yellow precipitate was seen in both acetaldehyde and acetone which indicates
a positive result while ethanol did not although this test is not used to distinguish either aldehydes or ketones.

4. Tollen’s Test (Differentiating Test between Aldehydes and Ketones

The 2nd test conducted was Tollen’s Test to distinguish aldehydes and ketones. From the experiment, only
aldehydes were oxidized in normal condition while ketones did not. 10 drops of each Acetaldehyde, Acetone and
ethanol was used and was placed in their respective tubes. After, 1mL of Tollen's reagent was added in each tube and
was mixed or shaken vigorously and was placed in boiling water for 2 minutes. A precipitate was seen coming from
Acetaldehyde which was a grey precipitate that indicates a positive result from this test.

Test for Carboxylic Acids

5. Blue Litmus Paper Test
The first conducted to test for Carboxylic acid was the Blue Litmus paper test. The representative of Carboxylic
Acid used in this test was Acetic Acid, Ethanol, and acetaldehyde to compare results. A drop of each was added in
their respective prepared litmus paper in a watch glass. A positive result was seen in this test through a change of color
of the litmus paper into a red color litmus paper. After adding each drop, only the litmus paper that has a drop of Acetic
acid turned red while the rest stayed blue. This indicated that only the Acetic acid has a presence of a carboxylic acid.

6. Sodium Bicarbonate Test

The Acids undergo neutralization reactions with bases, such as hydroxides, carbonates, and bicarbonates. The
product is usually salt and water, with addition of other products depending on the type of base that is used in the
reaction. Carboxylic acid reacts with sodium bicarbonate solution producing carbon dioxide as evidence by a brisk
effervescence with concomitant formation of carboxylate salt. The effervescence indicates positive test.

Procedure:
1. Add 1 mL each of ethanol, acetaldehyde, and acetic acid on 3 different test tubes.
2. A 1 mL of saturated sodium bicarbonate solution in each test tube. Note any production of effervescence.
Record your results.

E. Results and Discussion:

Qualitative Test Used for
Functional Functional Group Rationale Results
Groups Present Identification
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Alcohol Lucas Test The speed of cloudy layer
(Tertiary Alcohol) reaction made by Lucas reagent
will differentiate the types of
alcohols (primary, secondary,
tertiary).
Tertiary Alcohol has the
fastest reaction while
Primary Alcohol has the
slowest reaction.

The Lucas reagent converts

alcohols into alkyl chlorides, this
is evident through the formation
of a cloudy layer after the
reaction.

Phenol Ferric Chloride Test It is a specific test for phenolic The formation of a violet
group detection. color complex indicates a
positive test for the
Ferric Chloride is the reagent phenolic group.
used and this test is based on
the fact that phenolic groups can
form purple colored complex
with ferric ions which cannot be
seen in other compounds
containing hydroxyl group.

2,4-DNP or 2,4- It is a general test for the Positive result indicates a

Dinitrophenylhydrazine presence of a carbonyl group. yellow color precipitate and
Test This means that both aldehydes a confirmatory presence of
and ketones will give a positive a carbonyl group.
test result.

Ketone The positive test result indicates

a yellow precipitate which is the
DNP product produced from the
reaction between the 2,4-DNP
and carbonyl compound.
Heating the mixture after adding
2,4-DNP solution triggers a
reaction to make yellow
precipitate from the mixture.

Tollen’s Test It is used as a differentiating test A silver mirror or

between aldehydes and precipitate indicates a
ketones. positive result and a
confirmatory presence of
The test is based on oxidation an aldehyde,
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reaction. Between aldehydes differentiating aldehyde

and ketones, only aldehydes from ketone.
can be oxidized and can only be
the one to give a positive result.

Blue Litmus Paper Test
Carboxylic acid
Sodium Bicarbonate Test
It is a test wherein a blue litmus Blue litmus paper confirms
paper will turn red upon contact a basic group or solution.
with an acid. Thus, the same
reaction is expected for Red litmus paper confirms
carboxylic acids having acidic an acidic group or solution
properties. which is the same reaction
expected for carboxylic
Litmus paper is widely used to acids having acidic
determine if a substance is an properties.
acid (red) or a base (blue).
It is a test wherein the acidic Positive result indicates a
group will react with sodium formation of brisk
bicarbonate ions producing effervescence or bubbling
carbon dioxide and is seen in the and a confirmatory
formation of brisk effervescence presence of a carboxylic
or bubbling indicating a positive acid.
result.

Based on the table, corresponding qualitative tests was used as a confirmatory presence of certain functional
group. Each qualitative test has its rationale or the explanation and also a corresponding result indicated per test.
To determine the presence of alcohol (tertiary alcohol), Lucas test was used to determine the speed of cloudy
layer reaction made by Lucas reagent which can differentiate the types of alcohol. Based on the results, tertiary alcohol
has the fastest reaction while primary alcohol has the slowest reaction. Hence, the determination of certain type of
alcohol is observed in Lucas test.
To determine the presence of phenol, Ferric Chloride test was used to confirm the presence of phenolic
functional group. Phenolic groups can form purple colored complex with ferric ions which cannot be seen in other
compounds containing hydroxyl group. The formation of a violet color complex indicates a positive test for the phenolic
group.
To determine the presence of Ketone, a 2,4-DNP or 2,4-Dinitrophenylhydrazine Test was conducted first to
detect the presence of a carbonyl group since ketone also belongs to a carbonyl group. The positive test result indicates
a yellow precipitate which is the DNP product produced from the reaction between the 2,4-DNP and carbonyl
compound. Heating the mixture after adding 2,4-DNP solution triggers a reaction to make yellow precipitate from the
mixture. Second test conducted was the Tollen’s test wherein it is used as a differentiating test between aldehydes and
ketones.The test is based on oxidation reaction. Between aldehydes and ketones, only aldehydes can be oxidized and
can only be the one to give a positive result. A silver mirror or precipitate indicates a positive result and a confirmatory
presence of an aldehyde. Hence, it can be inferred that the negative test result confirms a ketone functional group.
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To determine the presence of carboxylic acid, a blue litmus paper test was conducted first to determine if a
solution is acidic, turning the blue litmus paper into a red litmus paper. Thus, the same reaction is expected for
carboxylic acids having acidic properties. Hence, the Red litmus paper confirms an acidic group. Second Test
conducted was the Sodium Bicarbonate Test, the acidic group will react with sodium bicarbonate ions producing carbon
dioxide and is seen in the formation of brisk effervescence or bubbling indicating a positive result. Hence, it is a
confirmatory presence of a carboxylic acid.

In summary, the table shows the different types of qualitative tests used to determine its certain confirmatory
presence of functional groups found in the endemic plant structure.

F. Conclusion:
The presence of a tertiary alcohol group in the compound was established using the Lucas tests. Cloudiness
appeared quickly when it interacted with hydrochloric acid (HCl) and zinc chloride (ZnCl2). The compound also had a
phenol group, since it produced a violet color when reacting with ferric ions (Fe3+).
The compound also had a ketone functional group, as shown by the 2,4-DNP Test and Tollens’ Test. When
the 2,4-DNP reagent was added, it produced a yellow precipitate, demonstrating the presence of a carbonyl group.
The compound, however, did not react with Tollens’ reagent and remained colorless. As a result, it was determined
that it was a ketone rather than an aldehyde. The compound also contained carboxylic acid, as shown by the positive
outcomes from the Sodium Bicarbonate Test and Blue Litmus Paper Test. It changed the blue litmus paper from blue
to red, confirming the presence of an acid. Because of the effervescence, the Sodium Bicarbonate Test verified it was
carboxylic acid.
Therefore, the functional groups present in the endemic plant compound are: alcohol, phenol, ketone, and
carboxylic acid.

III. Laboratory Safety Supplementary Activity

1. During the conduct of your method, you accidentally splash some hydrochloric in your eyes. How would you handle
this situation?
Answer:
When accidentally splashing some hydrochloric acid in the eyes, the first thing to do when handling this kind of situation
is to immediately rush into a faucet and flush the eyes with running lukewarm tap water. For about 15–30 minutes,
keep your eyes open while flushing with water. Rubbing the eyes must be avoided, as this may cause further damage.
Acids usually damage the front of the eyes. However, they can cause damage to the cornea that may result in
blindness. To ensure safety, seek emergency medical assistance immediately by taking it to the nearest eye specialist,
such as an ophthalmologist, or dial the emergency local number.

2. You are using a lot of chemicals in your method. Some of them have identical appearance. Describe a way that will
prevent you from using these chemicals interchangeably.
Answer:
Chemicals when used incorrectly may cause different effects on the experiment, which could be hazardous. To avoid
mixing the chemicals, the best thing to do is to label the containers of every chemical. After labeling, the chemicals
must be stored according to their compatibility. Incompatible chemicals must be segregated from different storages to
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prevent uncontrolled mixing or contact that will usually happen in circumstances such as spills, leaking containers, or
incompletely closed containers that will result in a reaction that will produce a hazard. When incompatible chemicals
are mixed together, they may react to produce heat, pressure, fire, explosion, or violent reactions, which must be
avoided in the laboratory. A simple labeling and organization of the various chemicals will create a safe environment,
reducing some of the accidents in the laboratory.

3. In every experiment, there’s always an excess of reagents. You are not allowed to put them back to their original
containers. Some of these chemicals are hazardous. Classify the hazardous chemicals in your method based from the
waste classification provided.
Answer:
Excess reagents will always be present in every experiment if right amount of chemicals are not premeasured prior to
the experiment. However, these excess chemicals are not to be returned to their original containers to avoid
incompatible mixing. Since chemicals are never cheap, using just the right amount of chemicals in the laboratory can
be beneficial. Chemicals will not only be saved, but also teach the researchers to be responsible with regard to chemical
conservation. In this experiment, the chemicals used with their corresponding waste classification are as follows:

Qualitative Tests Reagents used Waste Classification

-HCl Receptacle no. 1

Lucas test
ZnCl2 Receptacle no. 1

Ferric Chloride Test FeCl3 Receptacle no. 5

2, 4-DNP test 2,4-dinitrophenylhydrazine Receptacle no. 3

AgNO3 Receptacle no. 5

Tollen’s Test
NH3 Receptacle no. 3

Blue Litmus Paper Test no reagent N/A

Sodium Bicarbonate Test NaHCO3 Receptacle no. 3

Legend: Receptacle 1 (Inorganic Acids) Receptacle 4 (Mercury and Mercuric Compounds)

Receptacle 2 (Organic Acids) Receptacle 5 (Oxidizing Agents)
Receptacle 3 (Bases) Receptacle 6 (Aromatic and Phenolics)

IV. Real-Life Connection

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1. Qualitative tests are no longer very useful in the industry now due to the development of elegant instrumentations.
Discuss at least 1 modern instrumental methodology that can be used to determine the functional groups of a
compound without using the qualitative tests.
Answer:
For the past couple of years, qualitative analysis has been ignored due to the ineffeciency of the classical method as
it takes too much time in determining the functional groups present in the substance. However, with the emergence of
technology, the industry has taken a leap towards a more accurate and efficient method. An example of which is; IR
(infrared) spectroscopy. IR Spectroscopy was first invented in the year 1881 by Abney and Festing using photographic
plates, and was later on developed in the mids of 1940s. The IR spectroscopy works through analyzing a substance
by sending infrared light through a sample at several frequencies that would interact with the molecule and quantifying
the absorptions caused by each type of bond in the compound. This generates a spectrum, which is often a 'plot' of
percent transmittance against wavenumber.

2. What is the significance of functional groups in drug design?

Answer:
The significance of the functional groups in drug design are the section of a molecule susceptible to distinctive reactions
and is responsible for the overall characteristics of the molecule, which dictate the properties and chemistry of many
organic molecules. Since each functional group has an electronic impact, a solubility effect, and a steric effect, the
entire pharmacological and pharmacokinetic features of any specific drug molecule must be addressed. Each functional
group has an electronic effect, a solubility effect, and a steric effect that needs to be considered when evaluating the
overall pharmacodynamic and pharmacokinetic properties of any given drug molecule. The concept of functional
groups (FGs), sets of connected atoms that can determine the intrinsic reactivity of the parent molecule and in part
are responsible for the overall properties of the molecule, form a foundation within modern medicinal chemistry.