MODULE 1B: ANALYSIS OF FUNCTIONAL GROUPS

IN ORGANIC MOLECULES
(Carbonyl-Containing Organic Compounds)
Sion, Sofia Isabelle ; Velasco, Bea Katrina ; Wong, Aylla Patricia
Department of Psychology, College of Science
University of Santo Tomas, España Street, Manila 1051

ABSTRACT:
This experiment aimed to detect carbonyl-containing functional groups in organic molecules
using four specific tests (Tollen’s Test, DNPH Test, Iodoform Test, and Hydroxamic Acid Test)
on different organic compounds (n-butanol, tert-butanol, acetone, formaldehyde, and ethyl
acetate). In addition, the identification of an unknown compound was done through Infrared
Spectroscopy. Positive results were observed for formaldehyde in Tollen’s Test, acetone and
formaldehyde in the DNPH Test, acetone in the Iodoform Test, and ethyl acetate in the
Hydroxamic Acid Test. Furthermore, the characteristic peaks and wavelengths observed through
the IR Spectrophotometer showed that the unknown compound was, in fact, a phenol. The
experiment demonstrated the reactivity of carbonyl-containing compounds through various
reagents.

INTRODUCTION
The carbonyl group comprises a carbon atom bonded to two other atoms with a double
bond to oxygen, with the chemical formula C=O (Britannica, 2018). It is a key functional group
found in aldehydes, ketones, carboxylic acids, esters, and amides and is prevalent in organic
molecules derived from natural sources (Oullette & Rawn, 2014). In Experiment 1B: Analysis
of Functional Groups in Organic Molecules (Carbonyl-Containing Organic Compounds), a series
of experiments were conducted on four specific tests containing carbonyl compounds: Tollen’s
Test, DNPH Test, Iodoform Test, and Hydroxamate Test.

The Tollen's Test is widely utilized for identifying aldehydes, where ammonical silver
nitrate gets reduced by an aldehyde, leading to the formation of a silver mirror lining (Benet,
2011). The DNPH Test involves the use of dinitrophenylhydrazine, also known as Brady’s
reagent, which reacts with carbonyl compounds (including both aldehydes and ketones) to form a
colored precipitate (BYJU’s, n.d.). The Iodoform Test is employed to detect ketones through the
decomposition of the carbonyl compound (Isac-Garcia, 2016). Lastly, the Ferric Hydroxamate
Test is designed to identify the presence of esters, which react to produce a colored complex
(Solano, n.d.). These tests are crucial for differentiating various carbonyl compounds and
demonstrating their reactivity and structure via specific reactions.

By adjusting the variables within the experiment, the experimenters collected information
on compounds containing carbonyl groups, showcasing how particular reactions could identify
the presence of each compound of interest. Colors, precipitation, miscibility, and reactions are
demonstrated with each test for easier identification.

Accordingly, this experiment aims to identify the structure of each carbonyl-containing

compound, characterize the compounds based on the resulting reaction, derive the chemical
equations involved in each test, and identify an unknown compound utilizing the chemical tests
with standard IR spectroscopy.

METHODOLOGY
The experiment was conducted on the 19th of September 2024 by psychology students
from the University of Santo Tomas’ College of Science. Under the supervision of Asst. Prof.
Erlinda Dilan, MSc and Assoc. Prof. Cecilia Villaraza, MSc, safety precautions were ensured
throughout the experiment. The experiment aimed to detect carbonyl-containing functional
groups in organic molecules using four different tests: Tollen’s Test, DNPH Test, Iodoform Test,
and Hydroxamic Acid Test. The experimenters were also tasked to identify an unknown
substance through the use of IR Spectroscopy.

I. Materials and Equipment

To ensure the efficiency and success of the experiment, the following materials were
prepared:
● Test tubes w/ cork ● n-Butanol ● 1% Hydroxylamine Hydrochloride
● Dropper ● tert-Butanol ● 10% Sodium Hydroxide
● Beaker ● Acetone ● 10% Hydrochloric Acid
● Hot plate ● Formaldehyde ● 10% Ferric Chloride
● Timer ● Tollen’s Reagent ● 10% Potassium iodide
● Infrared (IR) ● DNPH Reagent ● 5% Sodium hypochlorite
Spectrophotometer ● Ethyl Acetate
II. Proper Set-up for Water Bath

Figure 1. Water Bath Set-up with Labels

 Three out of the five procedures involve heating the compound through a water
bath. For this, the hot plate was set to the desired temperature. Caution was practiced as
the hot plate might cause burns. A beaker was filled with enough water to submerge the
compound or mixture. After filling the beaker with the desired amount of water, it was
placed on the hot plate for it to boil. Once the water was heated, the test tube with the
compound was placed in the beaker and a timer was set depending on the instructions on
the procedure. Once finished, the hot plate was turned off and the water was properly
disposed of. Refer to Figure 1 for the proper set-up of the water bath.

III. Experiment Procedures

A. Tollen’s Test

Figure 2. Schematic Diagram of Tollen’s Test Procedures

Tollen’s test was utilized to detect the presence of an aldehyde group. In Tollen’s
Test, the organic compounds (n-butanol, tert-butanol, acetone, and formaldehyde) were
divided into four labeled test tubes with ten drops each. Next, 20 drops of Tollen’s
Reagent were added to each test tube. It was then shaken and heated in a boiling water
bath for five minutes. Lastly, changes in the solution were observed. The formation of a
silver mirror lining on the walls of the test tube indicated the presence of an aldehyde
group.

B. DNPH Test/Brady Test

 Figure 3. Schematic Diagram of DNPH Test Procedures

Aldehydes and ketones were detected through the use of the DNPH Test or Brady
Test. In the DNPH Test, ten drops of organic compounds (n-butanol, tert-butanol,
acetone, and formaldehyde) were placed into separate labeled test tubes. Next, five drops
of DNPH Reagent were added to each test tube. It was then shaken and observed. The
formation of a yellow or orange precipitate confirmed the presence of a carbonyl group
(aldehydes or ketones).

C. Iodoform Test

Figure 4. Schematic Diagram of Iodoform Test Procedures

The presence of methyl ketones were detected through the use of the Iodoform
Test. In the Iodoform Test, ten drops of organic compounds (n-butanol, tert-butanol,
acetone, and formaldehyde) were placed into separate labeled test tubes. Next, ten drops
of 10% Potassium Iodide and ten drops of 5% Sodium Hypochlorite were added. This
solution was then shaken and heated in a boiling water bath for ten minutes. Changes
 were observed. The formation of a yellow precipitate of iodoform (CHI₃) indicated the
presence of a methyl ketone.

D. Hydroxamic Acid Test

Figure 5. Schematic Diagram of Hydroxamic Acid Test Procedures

Hydroxamic Acid Test or Ferric Hydroxamate Test was utilized to detect the
presence of an ester group. In the Hydroxamic Acid Test, ten drops of organic
compounds (ethyl acetate and acetone) were placed into separate labeled test tubes. Next,
5 drops of 1% Hydroxylamine Hydrochloride and 10% Sodium Hydroxide were added. It
was then heated in a boiling water bath for five minutes. Once the time was up, the
compounds were set to cool to room temperature. After room temperature was achieved,
ten drops of 10% Hydrochloric Acid and two drops of 10% Ferric Chloride were added.
The product was observed. The appearance of a magenta color indicated the presence of
esters.

E. Infrared (IR) Spectroscopy

The infrared (IR) spectrum of an unknown organic compound was obtained to

identify its functional groups. A drop of an unknown compound was dropped on the IR
spectrophotometer which interpreted the characteristic absorption bands onto the
computer screen. The sample was scanned over the range of 4000–400 cm⁻¹; however,
only the bonds in the 4000–1500 cm⁻¹ will be analyzed. The peaks corresponding to
different functional groups were identified based on their specific wavenumbers, strength,
and shape.
RESULTS AND DISCUSSION
There are different tests used to observe the different reactions of the organic compounds
in each reagent and solution, namely the DNPH Test, Tollen’s Test, Iodoform Test, and the
Hydroxamate Test. Thus, in this section, the important observations and their causes will be
discussed, such as the reactions of the color, the act of precipitation, and whether they are
miscible and immiscible in the solutions made.

Tollen’s Test
Table 1. Tollen’s Test
Organic Compound Skeletal Structure Results

N-Butanol Clear Solution

Tert-Butanol Transparent White

to Brownish
Solution

Acetone Clear Solution

Formaldehyde Silver Mirror

Lining

Table 1 shows the reaction of the following organic compounds to Tollen’s reagent:
n-butanol, tert-butanol, acetone, and formaldehyde. Tollen’s reagent is known to be a test that
could only mildly oxidize aldehydes. The aldehyde exhibits an oxidation reaction with Tollen’s
test which transforms aldehydes into a carboxylic acid. Therefore, it only reacts with organic
compounds that contain R-C-H=O. This test does not have the ability to oxidize other organic
compounds such as alcohol (n-butanol and tert-butanol) or other carbonyl groups such as ketone
(acetone). It is also stated that in Tollen’s test, positive results contain a silver mirror lining while
negative results hold a clear solution.

Moreover, in the table, it is observed that only formaldehyde had a positive reaction to
Tollen’s test (silver mirror lining), while n-butanol, tert-butanol, and acetone contained negative
results (clear to white and brownish solutions). This is proven as formaldehyde is the only
aldehyde in the said organic compounds. Hence, with the said oxidation reaction formaldehyde
will turn into formic acid (HCOOH) as its product.
 DNPH Test
Table 2. DNPH Test
Organic Compound Skeletal Structure Results

N-Butanol Yellow to Orange

No Precipitate

Tert-Butanol Yellow to Orange

No Precipitate

Acetone Orange
Precipitate

Formaldehyde Yellow
Precipitate

Table 2 shows the reactions of the following organic compounds to the DNPH reagent:
n-butanol, tert-butanol, acetone, and formaldehyde. As observed in the table only two organic
compounds had solid colored precipitate with the DNPH reagent– namely, the acetone and
formaldehyde. This is because the aqueous solution, Brady reagent, reacts with organic
compounds that contain carbon-double bond oxygen (C=O), also known as the carbonyl group.
The DNPH test exhibits a condensation reaction where the water is eliminated as the molecules
join together. This reaction will result in positive results in which yellow, orange, or red
precipitates are shown. Moreover, both acetone (a methyl ketone) and formaldehyde (an
aldehyde) contained positive results as both have the presence of a carbonyl group. Hence, both
acetone and formaldehyde make products called hydrazone derivatives. To further elaborate, this
hydrazone derivative is formed when it reacts with the hydrazine from DNPH.

However, it is to be noted that the n-butanol and tert-butanol resulted in yellow-to-orange

solution with no precipitates. A transparent yellow-to-orange result is an indication that the
results reacted negatively. It indicates that the n-butanol and tert-butanol are incompatible with
the DNPH test; Thus, both n-butanol and tert-butanol do not contain a carbonyl group.

Iodoform Test
Table 3. Iodoform Test
Organic Compound Skeletal Structure Results
 N-Butanol No Yellow
Precipitate

Tert-Butanol No Yellow
Precipitate

Acetone Yellow
Precipitate

Formaldehyde Yellow
No Precipitate

Table 3 shows the reaction of the following organic compounds to 10% Potassium Iodide
(Kl) solution combined with 5% Sodium Hypochlorite (NaClO) solution: n-butanol, tert-butanol,
acetone, and formaldehyde. The Iodoform Test is used to detect the methyl ketones and
secondary alcohols in a methyl group. However, in this test, it is essential to note that it does not
result positively with all ketones and alcohols nor work well with water-insoluble compounds.
Moreover, cloudy yellow precipitates are the indications that the iodoform test shows positive
results, while a clear yellow or orange solution with no precipitates shows negative results.

The general reaction involves the halogenation of the methyl group adjacent to a carbonyl
group, followed by hydrolysis and oxidation steps. Acetone reacts with iodine and sodium
hydroxide to produce iodoform (CHI₃), which forms a yellow precipitate. Protonation in the
context of the iodoform reaction typically refers to the step where a base or a negatively charged
intermediate is neutralized by a proton (H⁺). Iodoform can be formed once three hydrogen atoms
have been attached to the carbon adjacent to the carbonyl group such as a methyl ketone.

Hence, it is exhibited in this table that only acetone reacted positively to the Iodoform
test— as it is the only organic compound that has shown a yellow precipitate. This is proven as
acetone is known to be a methyl ketone. However, n-butanol, tert-butanol, and formaldehyde
exhibited negative results as each embodied no precipitates.

Ferric Hydroxamate Test

Table 4. Ferric Hydroxamate Test
Organic Compound Skeletal Structure Results
 Acetone Yellow to Orange
Solution
Precipitate

Ethyl Acetate Magenta/ Reddish

Solution
No Precipitate

Table 4 shows the reaction of the following organic compounds to 1% Hydroxylamine

Hydrochloride (NH2OH · HCl) and 10% Sodium Hydroxide (NaOH) combined with 10%
Hydrochloric Acid (HCl) and 10% Ferric Chloride (FeCl3) reagent: Acetone and Ethyl Acetate.
This ferric hydroxamate test is used to look for the presence of the ester group in the organic
compounds. This test exhibits a complexation reaction where ester will become a hydroxamic
acid when it reacts with hydroxylamine hydrochloride, which later will turn into a ferric
hydroxamate.

A positive results indicate that the organic compound has magenta or deep red in color,
while negative results indicate any other color. Moreover, as indicated in the table the ethyl
acetate contains a positive result as this indicates a magenta or reddish solution, while acetone
holds a negative result as it shows a yellow to orange solution. It is proven that ethyl acetate is an
ester, while acetone is known to be a methyl ketone.

Infrared Spectroscopy
Figure 6. Infrared Spectroscopy of Unknown Compound
 Table 5. Functional Groups and Wavelengths

Functional Group Wavelength Unknown Compound

OH 3308.7 cm⁻¹

C–H 3048 cm⁻¹ and 3024 cm⁻¹

C=C 1596.7 cm⁻¹ and 1500.6 cm⁻¹ Phenol

The IR Spectrum of the unknown sample was identified as phenol. This is concluded
through the analysis of the peaks at different wavelengths that represent the different functional
groups. The broad peak at 3308.7 cm⁻¹ corresponds to the OH bond or hydroxy group which is
attached to the benzene ring. The peaks at 3048 cm⁻¹ and 3024 cm⁻¹ represent the C–H bonds in
the benzene ring. Meanwhile, the 1596.7 cm⁻¹ and 1500.6 cm⁻¹ are attributed to the C=C bonds in
the benzene ring. Ultimately, the spectrum aligns with the expected features and functional
groups of phenol

CONCLUSION

The tests conducted on carbonyl-containing compounds successfully demonstrated the

distinct reactivity and identification of aldehydes and ketones. The experiments were able to
identify the structures of each carbonyl compound. Moreover, the compounds were also
characterized based on their resulting reactions.

The experiment identified organic compounds as the focus of each test. Tollen’s test,
DNPH test, and Iodoform used n-butanol, tert- butanol, acetone, and formaldehyde; whereas the
Ferric Hydroxamate Test utilized only acetone and ethyl acetate.

Tollen's test showed clear solutions for n-butanol and acetone, a silver mirror lining for
formaldehyde, and transparent white to brown for tert-butanol. The DNPH test resulted in yellow
to orange color reactions with precipitation for n-butanol and tert-butanol, but no precipitation
for acetone and formaldehyde. The Iodoform test exhibited no yellow precipitates for both
n-butanol and tert-butanol, a yellow solution with precipitate for acetone, and yellow without
precipitate for formaldehyde. Lastly, the Ferric Hydroxamate test showed yellow to orange
solutions with precipitate for acetone and a magenta red color without precipitate for ethyl
acetate.
 In conclusion, these experiments allowed for the distinction and identification of
aldehydes, ketones, and other related groups under carbonyl by testing the structure and behavior
of carbonyl-containing compounds. An unknown IR was also discovered to be phenol. Within
the realm of organic chemistry, these tests could serve as crucial tools for distinguishing among
carbonyl compounds.

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