JCBPS; Section A; November 2020 –January 2021, Vol. 11, No.

1 ; 043-053, E- ISSN: 2249 –1929

[DOI: 10.24214/jcbps.A.11.1.04353.]

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences
Available online atwww.jcbsc.org
Section A: Chemical Sciences

CODEN (USA): JCBPAT Research Article

Synthesis and crystallization of N-(4-nitrophenyl)

acetamides
Aida Smajlagić 1*, Majda Srabović1, Zahida Ademović2, Ekrem Pehlić3,
Melita Huremović1, Edina Huseinović1

1
University of Tuzla, Faculty of Natural Sciences and Mathematics, Department of
Chemistry, Bosnia and Herzegovina
2
University of Tuzla, Faculty of Technology, Bosnia and Herzegovina
3
University of Bihać, Faculty of Biotechnical Engineering, Bosnia and Herzegovina

Received: 01 November 2020; Revised: 10 November 2020; Accepted: 20 November 2020

Abstract: N-(4-nitrophenyl) acetamide (p-nitroacetanilide) is synthetic nitro

compound important for obtaining many colours, medicaments, explosive and other
compounds. Nitration reaction in organic synthesis is done according the electrophile
aromatic substitution mechanism, where one or more nitro (NO2) groups are
introduced in ortho and para position of organic compound molecule. Molecule of N-
(4-nitrophenly) acetamide is important semi-product to final synthesis of N-(4-
hydroxyphenyl) acetamide molecule. Pre-crystallization is the most frequent
procedure in purification of solid substances. Binary mixture (ethanol-water) of
different concentrations was used as a solvent in order to improve the output and
purity of synthetized product. For the purpose of identification and characterization of
N-(4-nitrophenyl) acetamide, the following methods were used: FTIR, UV, TLC and
MS spectroscopy.
Keywords: N-(4-nitrophenyl) acetamide, synthesis, pre-crystallization, FTIR, MS

INTRODUCTION

Aromatic nitro-compounds are of enormous synthetic and industrial importance, as they are
frequently used as intermediaries in various introductions of nitro functional groups into aromatic
compounds[1]. Molecule of N-(4-nitrophenyl) acetamide or p-nitroacetanilide is nitro derivative of
acetanilide that is used as semi-product in production of colours, pharmaceutical active compounds
and many other aromatic compounds. Molecular structure of N-(4-nitrophenyl) acetamide indicates
the presence of benzene core where acetamide (-NH-CO-CH3) and nitro (NO2) group is in para
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position (1,4) (Figure 1). It is the compound with molecular formula C8H8N2O3, mass 180,16 g/mol
and melting point from 212 to 216 0 C 2. N-(4-nitrophenyl) acetamide is partially soluble in water,
chloroform, and is soluble in ethanol[2]. Nitro compound is present in crystal form of yellow-green
and green-brown colour [3,4].

Figure 1: Molecular structure of N-(4-nitrophenyl) acetamide

Organic compound N-(4-nitrophenyl) acetamide is obtained by nitration reaction of N-
phenylacetamide molecule, which reacts with mixture of nitrate (HNO3) into sulphate acid (H2SO4).
Nitration of N-phenylacetamide is exothermic reaction whose temperature must continuously be kept
under 20°C by cooling and mixing. Nitration reaction gives possibility for occurrence of two isomers
of para and ortho product (Figure 2).

Figure 2 : Nitration reaction and formation of p-nitroacetanilide and o-nitroacetanilide

Important factor in the very beginning of the reaction is selection of polar solvent such as glacial
acetic acid (CH3COOH) for successful dissolving of N-phenylacetamide molecule, while acetate ion
is poor nucleophile, so that substitution reaction is not possible. For the purpose of selection and
prevention of undesired synthetic products formation (dinitro products), mixture for nitration is
gradually added (in small portions) to the solution of N-phenylacetamide. Monosubstituted aromatic
ring, which already has one substituent, directs in nitration reaction the arising nitro (NO2) group into
ortho, meta and para position. Acetamide group (-NH-CO-CH3) on benzene core is ortho-para
directing and steric limitations of this group prevent formation of ortho-product at large scale.

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Nitration reaction is done according to the electrophile aromatic substitution mechanism, where active
nitron ion (NO2 +) while reacting with π electrons of aromatic ring of N-phenylacetamide molecule
gives a semi-product with new C-N relation. Nitration mechanism of N-phenylacetamide molecule is
shown in the following steps: (Figure 3, 4).

1. Formation of electrophile (NO2 +)

Figure 3 :Formation of electrophile (NO2 +)

2. Attack by electrophiles to para carbon aromatic ring and formation of intermediary and
3. Deprotonation – loss H+ with presence of weak base (e.g. H2O, HSO4- ) and formation of para
isomer

Figure 4 : Mechanism of N-(4-nitrophenyl) acetamide synthesis reaction

As isomer o-nitroacetanilide is more soluble in more polar solvents, one of important ways to isolate
synthetically clean p-nitroacetanilide is crystallization procedure. Crystallization is thermal separation

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process which results with formation of new solid phase [5,6]. Selection of adequate solvent is the key
parameter in crystallization process and complies with the principle „similarities dissolve in similar
environments”. This task is not always easy, and mainly the used solvent is water, since it is
economically acceptable and available. Water is polar molecule and all hydrophile compounds
dissolve well in it. There are numerous organic compounds used in crystallization process, such as
lower alcohols e.g. methanol, ethanol, isopropanol, butanol. To achieve as good crystallization as
possible, the mixtures of solvents are used, mainly alcohol and water. Nature and type of solvent have
a significant impact to crystallization process, as well as cooling speed, presence of impurities, which
was the aim of the study in the specific case.
MATERIAL AND METHODS

For laboratory synthesis of N-(4 nitrophenyl) acetamide, the following chemicals were used:
 C8H9NO N-phenylacetamide, acetanilide 99% (Kemika)
 H2SO4 Sulphuric acid 96% (Semikem)
 HNO3 Nitric acid 65% (Semikem)
 C2H4O2 Glacial acetic acid 99,8% (Semikem)
 Distilled water, ice.

In synthetic procedure, in 10 ml of glacial acetic acid (CH3COOH) 6,75g N-phenylacetamide was

solved by mixing in the magnet mixer. After that, 10 ml of sulphuric acid (H2SO4) was added heating
it to complete dissolving. The flask was inserted into cold bath and cooled to 5 0C. The mixture for
nitration was prepared comprising 3,5 ml of concentrated HNO3 and 5 ml concentrated H2SO4, cooled
to 50C and gradually added (in small portions) to the solution of N-phenylacetamide. After that, the
mixture rested for 30 minutes at environmental temperature and was poured to 50 ml of water (H 2O)
and 30 g of ice for crystallization. Second part of the procedure included crystallization of the
obtained raw product N-(4-nitrophenyl) acetamide utilizing adequate solvents.
Utilizing pre-crystallization procedure method, the binary mixtures (ethanol-water) were used in
concentrations from 20 to 80%, water and pure ethanol in order to test the impact to the crystallization
output, where the synthetized N-(4-nitrophenyl) acetamide was dissolved in adequate quantity of
solvents with heating and filtering.
For the purpose of analysis and characterization of synthetized N-(4-nitrophenyl) acetamide, the
following instrumental methods were used:
Fouier transform infrared spectroscopy (FTIR): FTIR spectrum of synthetized samples of N-(4
nitrophenyl) acetamide was analysed on Perkin Elmer BX FT-IR spectrophotometer at resolution of
2 cm-1 and wave length range from 4000 to 450 cm-1.
Technique to determine melting point: Analysis was done on the device Melting-Point Meter-
KSPI, 360◦C. Melting point of samples was determined in interval from 212 to 216 0 C.
UV/VIS spectrophotometric analysis: was done on Perkin Elmer Lambda UV/VIS 25
spectrophotometer at wave length range from 200 to 400 nm. Spectrum N-(4-nitrophenyl) acetamide
has its peaks (λ max) at 315,78 nm, 223,45 nm and 201,39 nm.
LC-MS/MS mass spectrophotometric analysis: Mass spectrum N-(4-nitrophenyl) acetamide was
analysed at spectrometer LC-MS/MS Agilent Technologies.

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Thin-layer chromatography (TLC): As a stationary phase of TLC analysis of samples, jelly plates
(20x20) were used, and mobile phase is ethyl acetate-hexane (50:50). Visualization of samples was
done in Camag UV cabinet with the lamp at 254 nm.
Optical microscopy: Sample analysis was done on microscope Leica, model 2500D, working on the
principle of passed polarized light. When making microphotographs, Nikolov prisms were put in
vertical position (XPL). Samples were dissolved in water and DMSO, with previous waiting for about
4 to 6 hours.
RESULTS AND DISCUSSION

Synthesis of N-(4-nitrophenyl) acetamide as electrophile substitution reaction results with

replacement of nitro (NO2) group in para position compared to acetamide group due to its electron-
donating effect. Also, due to steric effect, orientation of nitronium electrophile (NO2+) in para
position compared to ortho (Figure 5) is favoured.

Figure 5: Synthesis of N-(4 nitrophenyl) acetamide.

Output of raw N-(4-nitrophenyl) acetamide synthesis is 49,74%. For the purpose of increase of purity
and acceptable output of synthetic product, crystallization with water was done, utilizing binary
mixture (ethanol-water) and 96% ethanol. As shown in the diagram (Figure 6), water partially
dissolves N-(4-nitrophenyl) acetamide, which indicates the output of crystallization phase of 40,42%,
which complies with the results of FTIR analysis and opens the possibility for assuming the existence
of certain share of impurities. On the other side, ethanol dissolves well the synthetic N-(4-nitrophenyl)
acetamide, which crystalizes harder in cooling process, and results with smaller output of
crystallization phase. Research results show that with use of binary mixture (ethanol-water) through
synergy effect of water during crystallization process, the increase of output in crystallization of N-(4-
nitrophenyl) acetamide is achieved.

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Figure 6: Impact of binary mixture (ethanol-water), water and 96% ethanol to the product of
crystallization phase of synthetized N-(4-nitrophenyl) acetamide

Qualitative analysis of FTIR spectrum in organic synthesis is of the biggest importance because it
enables determining presence or absence of functional groups in organic molecules. Comparing the
spectrum of synthetized N-(4-nitrophenyl) acetamide with spectrum of CRS standard, absorption
maximums range from 3275.52 cm-1 to 3903 cm-1 (amide N-H stretch is present), 1678,79 cm-1 (C=O
stretch). Single peaks at 1559,12 cm-1 and 1540,40 cm-1 comply with very strong absorption tape
(N=O) of nitro group, considering that it is para-substituted compound. Sharp single peaks at 847,55
cm-1 and 748,16 cm-1 comply with very strong aromatic C-H bending, moving another absorption tape
towards lower frequencies, where the mentioned values match the reference data from literature [7, 8].
In this part of the spectrum, NO2 group influences the absorption due to substitution on benzene.
Results of FTIR features of the obtained samples during crystallization process are given in the
Figure 7. The obtained results are compared with CRS standard N-(4-nitrophenyl) acetamide from
the database.

Figure 7: FTIR spectrum N-(4-nitrophenyl) acetamide

FTIR analysis of N-(4-nitrophenyl) acetamide samples pre-crystalized with binary solvents (20-80%
ethanol) shows the appropriate range of percentage match with CRS standard. The effect of

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purification process with given solvents is evident, where the results indicate increase of the matching
level with CRS standard ranging from 93,30 to 95,20% compared to the matching value of the raw N-
(4-nitrophenyl) acetamide of 74,14%.
On the other side, sample pre-crystalized with water shows lower percentage match with CRS
standard (70,51%), which complies with the values of melting point and indicates the need for
repeated purification procedure (Figure 8). Figure 9 shows the values of melting point of N-(4-
nitrophenyl) acetamide or p-nitroacetanilide samples after pre-crystallization ranging from 212,1 to
215,6◦ C, which comply with the reference data from literature 2 and indicate the increase towards the
upper limit of 216◦C.
Follow-up of the synthesis flow of N-(4-nitrophenyl) acetamide was done by thin-layer
chromatography (TLC) (Figure 10). The samples of N-phenylacetamide (Rf=0,39) were analysed,
which is the initial component in the synthesis, and then CRS standard of N-(4-nitrophenyl) acetamide
and synthetized N-(4-nitrophenyl) acetamide. Based on the obtained Rf values, the differences
between Rf values of initial compound of N-phenylacetamide, synthetized (0,74) and CRS standard
N-(4-nitrophenyl) acetamide (0,75) may be noticed [9].

Figure 8: FTIR characterization of synthetized samples of N-(4-nitrophenyl) acetamide

Figure 9: Melting point (°C) of pre-crystalized samples of N-(4-nitrophenyl) acetamide or p-

nitroacetanilide in different solvent ratio (ethanol-water), water and 96% ethanol

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Figure 10: a) TLC plate of Rf values for initial reactant (N-phenylacetamide), CRS standard N-(4-
nitrophenyl) acetamide and synthetized N-(4-nitrophenyl) acetamide

UV/VIS spectrum of synthetized molecule of N-(4-nitrophenyl) acetamide (Figure 11) shows

absorption maximum at λ max= 315,78 nm, λ max= 223,45 nm and λ max=201,39 nm, which complies
with the reference data from literature [10].

Figure 11 : UV/VIS spectrum of N-(4-nitrophenyl) acetamide or p-nitroacetanilide

The obtained mass spectrum of synthetized molecule of N-(4-nitrophenyl) acetamide complies with
fragmentation presented in the scheme (Figure 12a) and shows presence of molecule peak m/z
=181,1 and characteristic fragment peaks at m/z=43, m/z=123,1, m/z=107,1 and m/z =77,1
(Figure12b). Peak of smaller intensity m/z=138,1 characterizes presence of obtained para
isomer [11,12]. It is assumed that the peak at m/z=269,1 which is present in the spectrum, originates
from present impurities.

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Figure 12a: Mass spectrum N-(4-nitrophenyl) acetamide

Figure 12b: Fragmentation of N-(4-nitrophenyl) acetamide molecule

Physical–morphological characterization of N-(4-nitrophenyl) acetamide samples shows presence of

needle crystals, which complies with the reference data from literature [13] (Figure 13). The size of
crystal ranges from 0,03 to 0,25 mm. Morphology of analysed crystals indicates similarity of
morphological structure with CRS standard N-(4-nitrophenyl) acetamide.

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Figure 13: Morphological appearance of crystal N-(4-nitrophenyl) acetamide a) N-(4-nitrophenyl)

acetamide commercial (DMSO), b) N-(4-nitrophenyl) acetamide commercial (water), c) N-(4-
nitrophenyl) acetamide synthetized (DMSO) and d) c) N-(4-nitrophenyl) acetamide synthetized
(water)

CONCLUSION

Final product of acetanilide nitration reaction is very acceptable and useful in further researches of
important pharmaceutically active compounds such as molecules of N-(4-hidroxiphenyl) acetamide.
Utilizing analysis methods, synthetic N-(4-nitrophenyl) acetamide was identified and characterised.
The researchers showed that by using binary mixture (ethanol-water) in crystallization phase it is
possible to significantly influence the crystallization product and reaction in obtaining N-(4-
nitrophenyl) acetamide. FTIR analysis of pre-crystallized samples indicates the increased level of
compatibility with CRS standard compared to the compatibility values of raw N-(4-nitrophenyl)
acetamide.

ACKNOWLEDGEMENT

The authors are thankful to the pharmaceutical company “ZADA” for enabling them use the FTIR
machine in analysis of the obtained samples and providing necessary support during the research.

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Corresponding author: Aida Smajlagić,

Department of Chemistry, Faculty of Natural Sciences and Mathematics,
University of Tuzla, Univerzitetska 4, 75000 Tuzla, Bosnia and Herzegovina;
Email: aida.taletovic@untz.ba
Online publication Date: 20.11.2020

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