How do organic functional group reactions contribute to

the qualitative assessment of paracetamol in widely used

analgesic medications?

Candidate code: ldj212

Session: May 2024
Word count: XXXX
Introduction 2
Background 3
Methodology 3
1. Phenol Nitration 4
2. Diazotization 5
Variables 7
Dependent Variables 7
Independent Variables 7
Controlled Variables 7
Apparatus 8
Experimental Procedure 9
Phenol Nitration 9
Diazotization 9
Ferric Chloride Test 9
Safety, environmental, and ethical considerations (сюда же их решение) 9
Hazard Identification and Risk Control 10
Results 10
Analysis 12
Evaluation 12
Weaknesses of the methodology + modification and explanation (in a table with strengths) 12
Conclusion 13
Works cited 13
Appendices 14

Introduction
In February our school experienced a spike in minor illnesses like colds and coughs, common
with the change of seasons. Curious about the remedies my peers relied upon during their
recovery process, I conducted an informal survey, identifying Theraflu, Pentalgin, and Citramon
P Renewal as popular choices. However, from personal experience and family preference, I
believed Paracetamol Renewal to be the most effective, always stocked in our medicine cabinet.
This sparked my interest to investigate the active ingredient in these medications further, aiming
to understand if Paracetamol Renewal truly stands out in efficacy, or if my preference is biased
towards the medicine we commonly use at home. My interest deepened in understanding the
science behind these choices, particularly focusing on paracetamol, known for its analgesic and
antipyretic properties.
Null hypothesis:
There is no detectable difference in the qualitative indicators for the presence or absence of
paracetamol in widely used analgesic medications using Phrnol Nitration, Diazotization, and
Ferric Chloride Tests. (i feel this null hypothesis is kinda irrelevant)

Alternative hypothesis:

Background
Paracetamol, also known as acetaminophen or para-hydroxyacetanilide, is a widely used
analgesic and antipyretic agent present as an active ingredient in various medications
(Anderson). Theraflu, Pentalgin, Citramon P Renewal, and Paracetamol Renewal all prominently
contain paracetamol. While each medication includes distinct auxiliary substances that slightly
alter their therapeutic effects, the common presence of paracetamol is central to their shared
analgesic properties. The efficacy of paracetamol stems from its ability to inhibit the synthesis of
prostaglandins in the brain, which are compounds associated with pain and fever responses
(Anderson).

Figure 1. The structural formula of paracetamol with functional groups labeled. Нужно ли
подписывать, что я нарисовала эту схему (eg “created by the author of the paper”)?

Methodology
When choosing tests for analyzing the presence of paracetamol in selected medications, I relied
on the functional groups of paracetamol. This approach ensures that the analysis specifically
targets the chemical properties inherent to the paracetamol molecule, enabling a focused
examination of its structure.
 1. Phenol Nitration
The phenol ring in paracetamol plays a crucial role in its function as a COX inhibitor, which
contributes to reducing prostaglandin synthesis in the brain, thereby alleviating pain and fever
(Anderson). It also makes the compound susceptible to nitration, an electrophilic aromatic
substitution reaction. This reaction, catalyzed by concentrated nitric acid (HNO3), introduces a
nitro group (NO2) to the phenol ring, serving as both a nitrating and oxidizing agent. However,
the presence of the acetaminophen group moderates the ring's reactivity, preventing excessive
oxidation and facilitating nitration at room temperature. This is due to the electron-donating
effect of the acetaminophen group, activating the ring for electrophilic attack.
The nitration process results in the formation of N-(2-nitro-4-hydroxyphenyl)acetamide, and
potentially N-(2,6-dinitro-4-hydroxyphenyl) acetamide, owing to the ring's susceptibility to
electrophilic attack at positions ortho and para to the phenolic OH group.

Figure 1. The nitration of paracetamol by concentrated nitric acid resulting in the

formation of N-(2-nitro-4-hydroxyphenyl)acetamide (I) and
N-(2,6-dinitro-4-hydroxyphenyl) acetamide (II)

Observable changes from this reaction include a color change due to nitro compound formation,
possible consistency alterations, and the release of gasses. These gasses are likely to be nitrogen
dioxide (NO2), a byproduct of the nitric acid reduction during the nitration process. This gas is
recognizable by its brown color and is indicative of the successful introduction of nitro groups
into the paracetamol molecule.

Figure X. The stoichiometric equation of nitric acid reduction with the formation of
nitrogen dioxide.
Важно заметить, что похожая качественная реакция проходит и на salicylic acid, which also
contains the phenol functional group, и который имеет схожие с парацетамолом (Paracetamol,
a widely used non-narcotic analgesic, has the same analgesic, and antipyretic efficacy as
acetylsalicylic acid - (Mburu et al.)) фармацевтические свойства, и поэтому важно провести
дополнительные качественные реакции на остальные группы, присущие парацетамолу,
чтобы подтвердить что это он.

2. Diazotization
The Diazotization Test is used for identifying the aromatic amine functional group. It begins with
formation of diazonium ions from the reaction between the sodium nitrite (NaNO₂) and the
hydrochloric acid (HCl):

NaNO₂ + HCl → HNO₂ + NaCl

In the acidic conditions provided by HCl, nitrous acid facilitates diazotization, forming
diazonium ions from paracetamol's amine group (is this accurate?). Upon heating, these
ions react with the phenol group in paracetamol, undergoing electrophilic substitution to
create an azo bond (–N=N–). The resultant azo compound exhibits a characteristic
orange-yellow hue in an alkaline medium (нужно ли дополнительно объяснять, почему
medium became alkaline?). , signifying the presence of paracetamol. The reaction is
summarized in Figure 2.

Figure 2. Mechanism of the diazotization of paracetamol resulting in the formation of an

orange-yellow azo compound. Как указать ресурс этой картинки? (A STATISTICAL
APPROACH BASED ON THE TOTAL ERROR CONCEPT FOR VALIDATION THE
BIOANALYTICAL METHOD)
Понять механизм - https://www.youtube.com/watch?app=desktop&v=cNBlYHmW_ok и
вставить его в appendices

3. Ferric Chloride Test

This test involves a reaction which is characterized by the formation of a coordination complex
between the ferric ion from FeCl3 and the phenol ring of the paracetamol, which dontaes a pair of
electrons. This reaction results in the formation of a colored complex, which is the observable
change that will indicate the presence of paracetamol.
The stochiometric equation from this reaction:

6C6H5OH + FeCl3 → [Fe(C6H5O)6]3 – (violet colour complex) + 3HCl

+ 3H+
Так как по-отдельности эти группы можно найти во множестве различных соединений,
проводится комплексный анализ выбранных веществ, чтобы убедиться в наличии
парацетамола
https://byjus.com/chemistry/test-for-phenolic-group/

Why are there 2 tests for the same functional group, phenol ring?

Two tests, Phenol Nitration and Ferric Chloride Test, analyze paracetamol's phenol group,
leveraging their complementary mechanisms—electrophilic reactivity and complex formation,
respectively. This approach not only offers a thorough analysis of the phenol ring but also
increases confidence in identifying paracetamol, distinguishing it from similar compounds like
salicylic acid. Such redundancy addresses the challenges of sensitivity, specificity, and potential
impurities, ensuring a more accurate and reliable confirmation of paracetamol's presence.

Why is there only one test for the aniline derivative then?

Utilizing just one test for paracetamol's aniline derivative is sufficient due to the
Diazotization Test's specificity and sensitivity towards aromatic amines. This test
uniquely identifies aromatic amines through diazonium salt formation and subsequent
color change upon coupling, providing clear, distinct evidence of the aniline derivative.
This singular, effective test aligns with practical analytical needs, balancing
thoroughness with resource and time constraints. Moreover, in a multi-component
analysis like paracetamol's, a strategy of one potent test per functional group optimizes
comprehensive molecular evaluation without unnecessary repetition.
(это можно в limitations/ weaknesses)
In a school laboratory setting, the choice of conducting only one test for the aniline
derivative in paracetamol, the Diazotization Test, was dictated by resource constraints.
More sophisticated qualitative tests, such as High-Performance Liquid Chromatography
(HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS), though offering higher
specificity and sensitivity, were not feasible. These methods require specialized
equipment and materials not typically available in a school lab environment, highlighting
the practical approach taken to confirm the presence of the aniline derivative efficiently.

Variables

Dependent Variables
The presence of paracetamol as indicated via qualitative chemical tests.

Independent Variables
The reactions for indication of functional groups of paracetamol

Controlled Variables

Controlled variable Explanation Method of control

The volume of reagents was

The amount of substance precisely measured with a
(chosen pipette to maintain uniformity
paracetamol-containing in the reactions.
medications) used in the tests

The concentration of iron(III)

chloride and nitric acid,
amount of sodium nitrite
(NaNO2).
 Temperature was controlled
The time, pressure, and by using an alcohol burner to
temperature of heating the ensure consistent heating
mixture. across all tests.

Apparatus

Materials/ apparatus Amount Uncertainty

Mortar and pestle 1 -

Retort stand 1 -

Alcohol burner 1 -

Spatula 2 -

Filter paper 10 -

Balancers 1 ±0.01 g

Stopwatch 1 ±0.2 sec

20 ml test tubes 8 ± 0.2 ml

3 ml graduated plastic Pasteur pipette 4 ±0.25 ml

250 cm3 volumetric flask 4 (?) ±0.3 cm³

100 ml evaporating dishes 4 ± 1 ml

Experimental Procedure

Phenol Nitration
1. Using a mortar and pestle, crush several tablets to obtain fine crystals. Transfer the
crystals into a 100 ml evaporating dish using a spatula.
2. Carefully add 3 ml of concentrated nitric acid using the pipette to the crushed tablet
crystals in the evaporating dish. Perform this step under a fume hood to ensure safety
from fumes.

Diazotization
1. Weigh out 0.06 g of the crushed tablets using balancers and place them into a 20 ml test
tube.
2. To the test tube, add 2 ml of distilled water, 1 ml of diluted hydrochloric acid, and 0.01 g
of crystalline sodium nitrite.
3. Secure the test tube in a retort stand and gently heat the mixture with an alcohol burner
for 30 seconds. Use a stopwatch to time the heating accurately.
​

Ferric Chloride Test

1. Dissolve 0.1 g of grated tablet powder in 20 ml of water in the volumetric flask to
achieve a homogeneous solution. Use a spatula for transferring the powder and a balancer
for accurate measurement.
2. Filter the solution through filter paper to remove any undissolved particles.
3. Add 1 ml of 10% FeCl₃ solution to 1.5 ml of the filtered medication solution in a clean 20
ml test tube using the pipette.

Нужно ли упоминать expected observable changes в Procedure? Или лучше оставить в

results

Safety, environmental, and ethical considerations (сюда же их решение)

In the experiment involving paracetamol analysis, adherence to established safety protocols was
strictly maintained. Safety Data Sheets were consulted, and chemicals were utilized in minimal
necessary amounts and labeled with GHS pictograms. All handling and disposal of chemical
waste were conducted per institutional guidelines, with appropriate waste containers clearly
labeled. Personal Protective Equipment, including gloves and safety goggles, was worn at all
times to mitigate exposure risks. Prior to experimentation, a thorough review of the procedures
and associated reagents was carried out, ensuring compliance with safety and exposure
standards.
Environmental - https://www.sciencedirect.com/science/article/abs/pii/S0147651319304609
Amines toxicity - https://doi.org/10.1021/acs.chas.3c00073 and ETD article

Hazard Identification and Risk Control

In the experimental procedures for paracetamol analysis, risks were effectively managed by
categorizing chemicals into corrosives (concentrated nitric acid, hydrochloric acid) and
irritants/toxics (sodium nitrite, iron(III) chloride). All were handled under a fume hood with full
personal protective equipment (PPE) including gloves and safety goggles, and in the case of
flammable substances, an alcohol burner was replaced with safer heating alternatives. Small
quantities were used to limit exposure and waste, adhering to safety and environmental
protocols.

Results
Table 1. Qualitative comparison of Paracetamol presence in Various Medications
FeCl3 Test Nitric Acid HCl and Labeled Dose Excipients
- (HNO3) Test NaNO2 Test Paracet mass
объяснить The change of amol (mg)
почему color to strong Content
Medi violet red-orange (mg)
catio indicates to…. per
n dose

Parac Violet Active Brown gas 500 545 -

etamo coloration emission of (NO2) release,
l reddish gas brown mixture
Rene and formation formation
wal of a reddish
mixture.
Citra Violet Active Orange-red 180 544 Acetylsalicylic
mon coloration emission of precipitate acid – 0.24 g,
P reddish gas formation; Paracetamol –
Rene and formation solution 0.18 g, Caffeine
wal of a reddish remains (as
mixture transparent monohydrate) –
0.03 g

Pental Violet Emission of Brown mixture 325 826 Naproxen –

gin coloration reddish gas formation 100.0 mg,
and formation Anhydrous
of a reddish caffeine – 50.0
mixture mg, Drotaverine
hydrochloride -
40.0 mg,
Pheniramine
maleate - 10.0
mg

Thera No color Formation of a No color 650 15000 Phenylephrine

flu change reddish change hydrochloride 10
mixture mg; Pheniramine
maleate 20 mg;
Ascorbic acid 50
mg

Explanation of the observed changes in the complexation test:

The color of an azo dye from a diazotization reaction varies with the coupling
component, leading to a spectrum from yellow to deep purple. This variation stems
from the structural differences of the coupling components that react with the
diazonium salt. In paracetamol analysis, a deep purple dye indicates the presence of
phenol or a phenol derivative as the coupling component. These compounds' structures
facilitate extended conjugation, allowing the dye to absorb at longer wavelengths and
display deep purple. This color change confirms the presence of paracetamol by
indicating the specific structure of the coupling component.

Table 2. (еще не решено как назвать) Сравнение количества действующего вещества

(парацетамол) в пересчете на один грамм и цены одного грамма препарата
 Medication concentration of paracetamol Price for 20 Tablets at a local
per g pharmacy (RUB)

Paracetamol Renewal 917.431193 86

Citramon P Renewal 330.882353 247

Pentalgin 393.46247 139

Theraflu 43.333333 1356

Analysis
Link your strengths and weaknesses directly to the independent/dependent variable and the
reliability of your data and therefore the certainty of your conclusion.Also, the strengths of the
experiment should also be explained clearly. I typically go for a 3-4 rule, explaining 3 strengths
and 4 weaknesses.
Explanations about teraflu (why is there not so much reactions)
Почему в цитромоне такая же реакция на нитро, при этом в тесте на железо не влияет

Evaluation

Weaknesses of the methodology + modification and explanation (in a

table with strengths)
The method relies on subjective measures such as color change, which can vary between
observers. Additionally, the presence of excipients in medication may interfere with the
reactions.

Limitations (что можно было бы улучшить)

Для верности дополнить методологию quantitative/ analytical analysis (examples of such)

Возможно стоило произвести негативные тесты для большей верности

#determine whether its phenol or phenol derivative

If you observed a deep purple color in the azo dye formed during the diazotization of
paracetamol, and you're considering whether the coupling component is phenol or a phenol
derivative, determining the exact nature of the compound based on color alone is challenging
without additional specific tests or information. However, the formation of a deep purple azo dye
typically suggests a complex structure that might be more indicative of a phenol derivative rather
than simple phenol itself.
Phenol derivatives often have additional substituents on the aromatic ring, which can affect the
electron distribution and, consequently, the color of the resultant azo dye. These substituents can
participate in extended conjugation with the azo group (-N=N-), leading to the deepening of the
dye color. In contrast, simple phenol, while capable of forming azo dyes, might not produce as
deep or as complex a coloration due to its simpler structure.
For a definitive identification, further analytical techniques such as mass spectrometry (MS),
nuclear magnetic resonance (NMR) spectroscopy, infrared spectroscopy (IR), or even
high-performance liquid chromatography (HPLC) would be required. These methods can
provide detailed structural information, distinguishing phenol from its derivatives based on
molecular weight, functional groups, and the presence of additional substituents on the aromatic
ring.

Conclusion
Paracetamol is the most cost-effective choice
This obtained results is not only of personal interest but also carries global importance, offering
insights into selecting effective, yet economical, treatments for common illnesses, thereby aiding
in informed healthcare decisions.

Works cited
https://www.ijpsonline.com/articles/novel-atomic-absorption-spectrometric-and-rapid-spectropho
tometric-methods-for-the-quantitation-of-paracetamol-in-saliva-applicati.html?view=mobile
https://scialert.net/fulltext/?doi=pjbs.2013.2050.2053
Appendices
Figure 2. The nitration of paracetamol scheme

Figure 3. The complexation of a phenol group with ferrum ion scheme. Res:
https://scialert.net/fulltext/fulltextpdf.php?pdf=ansinet/pjbs/2013/2050-2053.pdf
(переоформить)