Synthesis of novel metal complexes of 2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid formazan dyes: Characterization, antimicrobial and optical properties studies on leather

Article abstract of DOI:10.1016/j.molstruc.2018.07.081

We have developed novel formazan dyes (18–23) of enhanced fastness properties with cost-effectiveness in an aqueous system, without employing any Buffers and organic solvents. Their development was entailed of the synthesis of 2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid followed by the diazotization of 2-aminobenzoic acid that further reacted with the 4-[(2Z)-2-benzylidenehydrazinyl] benzene sulfonic acid. The multi-chromic (1:1 and 2:1) metal complexes of 2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid had been developed with the salts of Cr, Fe, Co, Cu and Ni; and they were characterized by elemental analysis, powder X-ray crystallography, Ultraviolet–visible, Fourier transforms infrared, Proton nuclear magnetic resonance and C¹³-nuclear magnetic resonance spectroscopic techniques. Density functional theory (DFT) studies of all dyes (18–23) were performed to evaluate the most stable geometries and structural parameters as well. The synthesized formazan dyes were evaluated for their different fastness (light, wash, perspiration), exhaustion and fixation properties on goat leather fabric and were revealed to have virtuous fastness properties (3–5, 4–5, 3–5, and 4–5) with high percentage value of exhaustion and fixation ranged from 91 to 97% and 90–98% respectively. The synthesized formazan dyes (18–23) were developed different colors such as Red, Brown, Green, Black, and Blue on leather. Synthesized formazan dyes were also evaluated for their antibacterial propensity on leather substrate and in solution by agar well diffusion method. The metal complex formazan dye (21) was demonstrated the significant bactericidal propensity against E. coli, S. aureus, Klebsiella and B. subtilis in solution and on leather by exhibiting maximum ZOIs (19 ± 0.05 mm, 25 ± 0.07 mm, 23 ± 0.09 mm, 27 ± 0.03 mm) and percentage reduction in bacterial growth (60 ± 0.03%, 69 ± 0.07%, 80 ± 0.05% and 86 ± 0.08%) respectively. Hence, newly synthesized formazan dyes (18–23) have efficient optical and antibacterial properties that would be proved valuable for the development of new industrial products to be commercialize.

Full text of DOI:10.1016/j.molstruc.2018.07.081

Accepted Manuscript
Synthesis of novel metal complexes of 2-((phenyl (2-(4-sulfophenyl) hydrazono)
methyl) diazenyl) benzoic acid formazan dyes: Characterization, antimicrobial and
optical properties studies on leather
Shakeel Ahmad Khan, Sammia Shahid, Sadia Kanwal, Komal Rizwan, Tariq
Mahmood, Khurshid Ayub
PII:
S0022-2860(18)30900-1
10.1016/j.molstruc.2018.07.081
MOLSTR 25486
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 19 May 2018
Revised Date: 24 July 2018
Accepted Date: 24 July 2018
Please cite this article as: S.A. Khan, S. Shahid, S. Kanwal, K. Rizwan, T. Mahmood, K. Ayub, Synthesis
of novel metal complexes of 2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid
formazan dyes: Characterization, antimicrobial and optical properties studies on leather, Journal of
Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.07.081.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of Novel Metal Complexes of 2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl)
diazenyl) benzoic acid Formazan Dyes: Characterization, Antimicrobial and Optical
Properties Studies on Leather
Shakeel Ahmad Khana*, Sammia Shahida, Sadia Kanwalb, Komal Rizwanc, Tariq Mahmoodd,
Khurshid Ayubd
aDepartment of Chemistry, School of Science, University of Management & Technology,
Lahore-54770, Pakistan
bDepartment of Biochemistry, University of Agriculture, Faisalabad-38000, Pakistan
cDepartment of Chemistry, Government College Women University Faisalabad-3800 Pakistan
dDepartment of Chemistry, COMSATS University, Abbottabad Campus-22060, Pakistan
*
Corresponding Author: Email: shakilahmadkhan56@gmail.com; Tel: +92-3441865064
Abstract: We have developed novel formazan dyes (18-23) of enhanced fastness properties with
cost-effectiveness in an aqueous system, without employing any Buffers and organic solvents.
Their development was entailed of the synthesis of 2-((phenyl (2-(4-sulfophenyl) hydrazono)
methyl) diazenyl) benzoic acid followed by the diazotization of 2-aminobenzoic acid that further
reacted with the 4-[(2Z)-2-benzylidenehydrazinyl] benzene sulfonic acid. The multi-chromic (1:1
and 2:1) metal complexes of 2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic
acid had been developed with the salts of Cr, Fe, Co, Cu and Ni; and they were characterized by
elemental analysis, powder X-ray crystallography, Ultraviolet-visible, Fourier transforms
infrared, Proton nuclear magnetic resonance and C13-nuclear magnetic resonance spectroscopic
techniques. Density functional theory (DFT) studies of all dyes (18-23) were performed to
evaluate the most stable geometries and structural parameters as well. The synthesized formazan
dyes were evaluated for their different fastness (light, wash, perspiration), exhaustion and
fixation properties on goat leather fabric and were revealed to have virtuous fastness properties
(3-5, 4-5, 3-5, and 4-5) with high percentage value of exhaustion and fixation ranged from 91-97
% and 90-98 % respectively. The synthesized formazan dyes (18-23) were developed different
colors such as Red, Brown, Green, Black, and Blue on leather. Synthesized formazan dyes were
also evaluated for their antibacterial propensity on leather substrate and in solution by agar well
diffusion method. The metal complex formazan dye (21) was demonstrated the significant
bactericidal propensity against E. coli, S. aureus, Klebsiella and B. subtilis in solution and on
leather by exhibiting maximum ZOIs (19±0.05 mm, 25±0.07 mm, 23±0.09 mm, 27±0.03 mm)
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and percentage reduction in bacterial growth (60±0.03%, 69±0.07%, 80±0.05% and 86±0.08%)
respectively. Hence, newly synthesized formazan dyes (18-23) have efficient optical and
antibacterial properties that would be proved valuable for the development of new industrial
products to be commercialize.
Keywords: Synthesis; Formazan dyes; Characterization; Optical properties; Antimicrobial
propensity
1. Introduction
Both the formazan and azo dyes have formal resemblance with each other, because of the
presence of azo group in their structural molecule; however, formazan dyes molecules have
enough dissimilarities in their structures than azo dyes to be considered as a separate class [1].
Because of the π-π* transitions of π electrons in formazan skeleton, the formazan/tetrazolium
and their derived metal complexes dyes i.e. Cr, Fe, Cu, Ni, Co etc. impart color and they have (-
N=N–C=N–NH-) formazan skeleton in their molecules [2]. Moreover, the formazans dyes have
propensity to form metal complexes upon metallization process with the salts of different metals
such as FeSO4.7H2O to form Fe-complex, CrCl3.6H2O to form Cr-complex and CuSO4.5H2O to
from Cu-complex etc. This is because of fact that the formazan dyes are comprise of the
characteristics features of multidentate ligands with donor atoms. The non-metal complex as well
as metal complex formazan dyes of various kinds of metals involve in producing the intense
color of different shades i.e. Red, Blue, and Brown etc., which are directly depend on the
structure either what type of auxochromes as well as chromospheres are attached to color
imparting molecule [3]. Ciba was the pioneer who used the first molecule of tetradentate
formazan metal complexes as a colorant material for the dyeing purposes of wool in 1947 [4].
Subsequently, numerous tetradentate formazans metal complexes had been invented and they
were become significant class of dye, specifically in the sectors of acid and reactive dyes even
though additional applications have also been reported for them [5]. The formazan dyes have
become important reactive dyes for cotton as well as for leather fabric [6]. In respect of
investigation on their use as an end-product, research studies were focused to improve their
synthetic routes and more deeper studies were done to understand the relationships of chemical
constitutions of the formazan dyes to the color properties [4]. The plentiful literature related to
formazans dyes is available which displays their synthesis, structural features, photochromic
transitions, tautomer formation, redox potentials [7,8] as well as the synthesis of crown
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formazans [9,10]. Beside of their use as a colorant material, the formazan and tetrazolium salts
were also employed for the identification of the existence of Brucella species in Brucella ring
test in milk [11,12]. The Brucellosis is a bacteriological contamination which blowouts from
animals to human beings most frequently through un-pasteurized milk, cheese, and other dairy
products [13]. In normal and neoplastic tissues, the formazan systems and blue tetrazolium salts
are employed to unveil enzymes activity [14]. The formazan dyes system is also comparatively
more beneficial in the identification of efficiency of anti-cancer drugs [15,16]. The un-metallized
and metal complex formazan dyes are also employed in analytical chemistry because of the
development of more intense color of different shades upon combining with the substrate to be
analyzed [17]. Because of their profligate and intense color reaction, non-metal and metal
complex formazans dyes can be employed for the spectroscopic identification of variety of metal
ions through fluorescence mechanism [18]. The reported literature are also disclosed that un-
metalized and metal complex formazan dyes also have potential to act as antimicrobial to inhibit
the growth of microbial agents. Moreover, they also have capacity to perform their action as an
antioxidant agent by inhibiting the growth of free radicals that are produced due to oxidative
stress and hydrolysis. The presence of different functionality such as (-OH, -SO3H, -NO2, -CH3)
in their molecules empower them to spectacle improve biological applications. Because of their
extraordinary and substantial features towards colorant material, antimicrobial and antioxidants,
they must be have commercial worth but in actual practice, it is not like that because of their
high production cost. Various literature reported their synthesis through different methods and
strategies with the use of different type of Buffers and organic based solvents, which make their
cost high, environmentally un-friendly and eventually non-commercializable. The aims of the
present research study were involved to (i) develop novel un-metallized and metal complex
formazan dyes of enhanced fastness properties with cost-effectiveness in an aqueous system,
without employing any Buffers and organic solvents (ii) evaluation of their antimicrobial
activities.
2. Experimental Work
The current research work was performed at the R&D Lab, Shafi Reso-Chem, Lahore Pakistan
and research laboratory, Department of Chemistry, University of Management and Technology,
Lahore Pakistan. The used chemicals and reagents in present research were of analytical grade as
well as all of them were purchased from BASF (Germany) and Sigma Aldrich, USA.
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2.1. Physical measurement
The functional group recognition of the synthesized formazan dyes (18-23) was accomplished by
using Agilent Cary 630, Agilent Technologies, USA, FTIR spectrophotometer in the range of
450-4000 cm-1. By employing UV-visible spectrophotometer = Spectra Flash SF 550, Data color
Inc., USA, the electronic absorption spectral studies of the synthesized formazan dyes (18-23)
were performed in the water at the range of 200-800 nm. Through NMR Bruker DPX 400-
Operating at 300/75-13C MHz spectrophotometer, the H1-NMR spectral investigations were
recorded in a CDCl3 solvent for the synthesized formazan dyes (18-23). By employing the
PANalytical X’Pert diffractometer PRO instrument with Cu–Kα radiation (wavelength 0.154
nm) operating at 40 kV and 30 mA, the powder X-ray di raction analysis was executed to
explore the crystallinity of the synthesized formazan dyes (18-23). The measurements were
scanned for di raction angles (2θ) ranging from 20o to 90◦ with a step size of 0.02◦ and a time
per step of 1 s. By using Flash EA 1112 elemental analyzer, the elemental analysis (C, H, N, S)
of the synthesized formazan dyes (18-23) was performed to compare the %age composition
theoretically vs. practically. The melting points determination of the synthesized formazan dyes
(18-23) was performed employing the Melting Point apparatus, Gallenkamp, UK. The Portable
pH Meter Model PHB4pH was used to monitor the pH of the reaction and synthesized formazan
dyes. The Leather Dying Drum Machine = Jiangsu Lianyungang Leather Machinery Factory
China, Model# R-350-6 was employed for the application of dyes on goat leather fabric. The
Xenon Fad-o-meter Model# XF-15N, Shimudzu Corporation Kyoto Japan Light was used for the
assessment of fastness properties of synthesized formazan dyes (18-23) on leather fabric. The
progress of the complete synthesis of formazan dyes (18-23) was monitored through thin layer
chromatography (TLC) in this research work and TLC was executed on aluminum sheets of 2 ×
5 cm dimensions, which were pre-loaded with silica gel 60F254 of the thickness of 0.25 mm
(Sigma Aldrich). The visualization of the developed chromatograms were implemented under the
ultraviolet light at the wavelength range of 254-366 nm or with the iodine vapors.
2.3. General methodology for the preparation of 4-[(2Z)-2-benzylidenehydrazinyl] benzene
sulfonic acid (5)
The preparation of coupler such as 4-[(2Z)-2-benzylidenehydrazinyl] benzene sulfonic acid (5)
was entailed of the transformation of 4-aminobenzenesulphonic acid (1) into diazonium salt (2)
which was yielded 4-hydrazinylbenzenesulfonic acid (3) on reduction in the presence of sodium
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sulfite and HCl at 0-5 °C temperature and lastly, required product (5) was obtained through the
condensation reaction of benzaldehyde (4) with compound (3) [19].
Scheme 1: The synthesis of 4-[(2Z)-2-benzylidenehydrazinyl] benzene sulfonic acid (5)
2.3.1. The diazotization of p-Aminobenzenesulfonic acid (1)
The 173 g (1 mole) of p-Aminobenzenesulfonic acid (Sulfanilic acid) (1) having 100 per cent
purity was added in 1500 ml water in 3 L beaker and addition of 65 g of Na2CO3 was made
slowly due to the foam formation along with mixing on multiple agitator to complete dissolution.
Then the solution was sifted to confiscate un-dissolved particles and scums. Then ice jacket was
made around the beaker to accomplish temperature 0-5 °C. Then during constant agitation, the
400 ml HCl was added progressively again because of the foam formation (foam was formed due
to the evaluation of CO2 from the reactant solution). Then the 100 g of ice flakes was added into
the solution to sustain temperature 0-5 °C. At that temperature, solution of Sodium nitrite (73 g
of NaNO2 in 150 ml water) was added into the reactant beaker drop-wise in 15 minutes and the
congo-iodo paper was checked which was positive by giving blue coloration. After that, the
reaction mixture was permitted to complete reaction at continuous stirring for 1:30 hr.
Subsequently, the diazonium salt was sifted through the sanction unit linked with the vacuum-
pump [20]. The preparation of diazonium salt (2) of 4-aminobenzenesulphonic acid (1) were
presented in scheme 1.
2.3.2. The preparation of 4-hydrazinylbenzenesulfonic acid (3)
For the reduction of diazonium salt, 565 g of crystalline Na2SO3.7H2O was added into a beaker
of 3 L containing 830 ml of water and provided the stirring until a clear solution of it was not
achieved. The Sodium sulfite solution was cooled in an ice bath to accomplish 0-5 °C
temperature and on this temperature, the diazonium salt wet paste (2) was transferred into the
beaker of Sodium sulfite solution in 1 hr and orange coloration was started to appear in reaction
solution without turbidity. Afterwards, a constant stirring was provided to the reaction solution
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for 1 hr. Subsequently, the reaction solution was heated to boil-age on constant stirring and 664
ml of concentrated HCl was added in 30 min. The color of reaction solution was become lightens
and lastly very light yellow color was seemed at the end of reaction. The reaction mixture was
cooled down to room temperature over a period of over-night and after the formation of
precipitate formation, reaction mixture was sifted through sanction unit, and cold water was
employed to wash the precipitates. The precipitates of 4-hydrazinylbenzenesulfonic acid (3)
dried out in an electric-oven at the temperature of 100 °C. Further purification of the 4-
hydrazinylbenzenesulfonic acid (3) was performed following the method [19]. The percentage
yield of the product was 96 % of the calculated amount. The preparation of 4-
hydrazinylbenzenesulfonic acid (3) was denoted in scheme 1.
2.3.3. The preparation of 4-[(2Z)-2-benzylidenehydrazinyl] benzene sulfonic acid (5)
The 198.75 g (1 mole) quantity (considering 95% pure) of 4-hydrazinylbenzenesulfonic acid (3)
was measured accurately on analytical-balance and was added into a beaker of 3 L having 2508
ml H2O and 99.99 ml sodium hydroxide was transferred to accomplish pH~10. The reaction
mixture was heated on constant stirring at the temperature of 45 °C for 30 min to complete
dissolution. Subsequently, the reaction mixture was cooled down to room temperature and was
sifted to confiscate contamination and un-dissolved materials. Then, the filtered reaction solution
was transferred into the reactor of 3 L capacity and was heated up to the temperature of 55-60
°C. At that temperature, the condensation reaction was performed by the addition of 106.2 g
(1mole) of benzaldehyde (4) dropwise in 30 min on constant stirring and heating (55-60 °C) with
constant mixing was provided to the reaction mixture until the reaction was completed. The
reaction was completed in 3 hr and the precipitation of the required product was accomplished
through salting out and acidification process at pH~2.5 at room temperature (30 °C). The 20 %
of NaCl of the total volume of the solution and 25 ml HCl were employed for precipitation.
Afterwards, the precipitates was filtrated off at ambient temperature and the filtered cake of the
product was dried out in an electric-oven at the temperature of 60 °C until constant weight
obtained. The percentage yield was 97 % of the theoretical amount and the product was 4-[(2Z)-
2-benzylidenehydrazinyl] benzene sulfonic acid (5) which was coupler reagent used for the
prepartion of formazan dyes (18-23) through the azo-coupling reaction. This product (5) was
purified through the procedure of [20,21] and the prepartion of 4-[(2Z)-2-benzylidenehydrazinyl]
benzene sulfonic acid (5) was denoted in Scheme 1.
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2.4. General methodology for the preparation of formazan dyes (18-23)
The 4-[(2Z)-2-benzylidenehydrazinyl] benzene sulfonic acid (5) which were acted as coupler
reagent were prepared in first step (Scheme 1). The 2-((phenyl (2-(4-sulfophenyl) hydrazono)
methyl) diazenyl) benzoic acid (18) were prepared in the second step and was non-metal
complex formazan dye which was transformed into metal complexes with the salts of Iron (19),
Copper (20), Chromium (21), Nickel (22) and Cobalt (23) and were characterized them through
different spectroscopic-techniques. Scheme 2 were presented the synthesis of un-metallized and
metal complex formazan dyes (18-23).
Scheme 2: The preparation of non-metal complex formazan dye (18) and metal complex
formazan dyes of Iron (19), Copper (20), Chromium (21), Nickel (22) and Cobalt (23)
2.4.1. Synthesis of un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono)
methyl) diazenyl) benzoic acid] (18)
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The 13.714 g (0.1 mole) on 100 % pure bases of anthranilic acid (2-aminobenzoic acid) (10.1)
was added into 500 ml beaker containing 150 ml water and the constant stirring was provided
through multiple agitators. Afterward, the 40 ml of HCl was transferred into the reaction mixture
and permitted for 15 min to complete dissolution of the reaction mixture. Then the reaction
solution was sifted off through the suction unit to eliminate impure and un-dissolved substances.
The filtered reaction solution was ice-jacketed in beaker and 30 g of ice flakes were transferred
into the beaker to accomplish the zero centigrade temperature. Subsequently, the solution of
NaNO2 /water (7.3 g/l5 ml) was transferred into the reaction beaker in four portions. The
positivity of Congo red and Iodo paper was tested for hydrochloric acid and sodium nitrite
respectively and the reaction solution was agitated at the temperature of 0-5 ºC for 1 hr. The
Congo-iodo= +ve was patterned after every fifteen min throughout the stirring for 1 hr. After
that, excess amount of Iodo was made negative through the addition of 5 g of Sulfamic acid
(H3NSO3) which finally yielded the diazonium-salt (11.1) and its purification was performed
through filtration-process. The diazonium-salt (11.1) was ready for azo coupler reaction. In
which diazonium salt (11.1) of anthranilic acid (2-aminobenzoic acid) (10.1) was coupled with
28.48 g (0.1 mole) (97 % percent pure basis) of 276.31 g/mole of 4-[(2Z)-2-
benzylidenehydrazinyl] benzene sulfonic acid (5) that was accomplished by the dissolution of (5)
into 400 ml H2O at highly alkaline pH~10-11 at the temperature of 0-5 ºC. The alkaline-pH
attained with the transfer of 35 g Na2CO3 and the diazonium-salt (11.1) transfer to coupler
reagent (5) was completed within 1 hr. Azo-coupling reaction was completed in 3 hr and yielded
the un-metallized formazan dye (18) which cooled down to room temperature. After that, the un-
metallized formazan dye (18) was isolated by 35 ml HCl (pH~4.75) and 22 % NaCl of the total
volume of dye. The synthesized formazan dye (18) was sifted off and dried out at the
o
temperature of 60-70 C until the constant weight obtained. The obtained yield of the product
(18) was 86 % over theoretical calculation and scheme 2 were presented the synthesis of
formazan dye (18). Reddish Brown (Solid). Yield (86%). Melting Point (380ºC). λmax in nm (log
υ
ε
): λmax1 550 (0.490), λmax2 360 (0.698), λmax3 230 (0.25). FTIR (KBr, Cm-1) max: 1535 (Ar-
C=N, str), 1365 (Ar-N=N, str), 1600 (aromatic C
=
C), 820 (-CN=NC-, str), 3380 (Ar-N-H, str),
1
1430 (Ar-SO3H, str), 1690 (Ar-C=O, str), 2930 (Ar-OH-carboxylic acid, str). H-NMR (CDCl3,
400 MHz)
δ: 11.30, (s 1H, Aromatic-COOH), 1.498 (s 1H, NH), 12.18 (s 1H, SO3H), 7.60-7.91
(m 13H, Ar-H). 13C-NMR (CDCl3, 75 MHz)
δ
: 155 (C=N), 166.5 (-COOH), 117.6, 123.8, 125.4,
8

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125.6, 126.9, 127.3, 128.7, 128.8, 129.9, 131.0, 133.9, 134.7, 139.2, 146.8, (Aromatic carbons).
Anal. Calc. for C20H16N4O5S (MW: 424.429 g/mol): C, 56.60; H, 3.80; O, 18.85; N, 13.20; S,
7.55% and Found: C, 56.55; H, 3.75; O, 18.80; N, 13.15; S, 7.51%.
2.4.2. Detailed procedure for the synthesis of metal complex formazan dyes (19-23)
For the preparation of metal complex formazan dyes (19-23), the un-metallized formazan dye [2-
((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid] (18) which was
tridentate-ligand had been synthesized following the procedure ascribed in scheme 2. After that,
1:1 metal complex formazan dyes (19, 20, 22 and 23) were synthesized by the metallization
reaction of 0.1 mole (42.44 g) un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl)
hydrazono) methyl) diazenyl) benzoic acid] (18) with the equimolar concentration (0.1 mole) of
FeSO4.7H2O (27.80 g), CuSO4.5H2O (24.97 g), NiSO4.7H2O (28.09 g) and Co(CH CO ) ·4
H O (24.91 g) at pH~6.40-6.50 and at temperature 65-70 ºC respectively. While, 2:1 metal
complex formazan dye (21) were synthesized by the metallization reaction of 0.1 mole (42.44 g)
un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl)
benzoic acid] (18) with 0.05 mole (13.32 g) concentration of CrCl3.6H2O (26.64 g, 0.1 mole) at
pH~6.70 at elevated temperature 90-100 ºC. The metallization reaction was proceeded in 2-liter
glass reactor with heating mantle and slightly acidic pH of the metallization reaction for each
metal complex formazan dye (19-23) was adjusted with HCl (35-40 ml). In detail, 0.1 mole un-
metallized formazan dye (18) (ligand) was dissolved in 150 ml H2O in glass reactor and pH was
adjusted with HCl accordingly to the synthesis of above stated metal complex formazan dyes. At
required pH level, the heating such as (65-70 ºC for 1:1 and 90-100 ºC for 2:1) was provided to
the ligand dye solution through heating mantle. At required temperature level as stated above, the
metal salts was transferred into the ligand dye solution in 30 min. Subsequently, heating was
provided continuously to the reaction mixture of ligand and metal salts until completion of
metallization according to the synthesis of required metal complex formazan dye, as each of
them required different time and temperature for the completion of metallization reaction. The
metallization reaction time for the synthesized metal complex formazan dyes (19-23) was 3.5 hr,
4.5 hr, 5hr, 2.5 hr and 3 hr respectively and the resulted metal complex formazan dyes solution
was cool down to room temperature for their isolation process. The each metal complex
formazan dye (19-23) was isolated through acidification and salting out process for which HCl
with different amounts was added for pH (4.0, 1.75, 0.80, 1.30 and 1.10) and the NaCl (15 %, 23
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%, 18 %, 25 % and 19 %) of the total volume of the respective dye solution was added
respectively. After that, respective metal complex dyes solution was stirred for 2-3 hr for
complete isolation from the solution and then they were filtered off employing filtration unit
o
linked with vacuum pump and dried out them in an electric oven at the temperature 60-70 C
until the constant weight of the dye. Over the theoretical calculation, the obtained yield for the
synthesized metal complex formazan dyes (19-23) were 91 %, 91 %, 93 %, 84 %, and 84 %
respectively. The scheme 2 is denoted the reaction for the synthesis of metal complex formazan
dyes (19-23).
2.4.2.1. Synthesis of [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic
acid] Fe II complex (1:1) (19)
For the preparation of metal complex formazan dye (19), the un-metallized formazan dye [2-
((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid] (18) which was
tridentate-ligand had been synthesized following the procedure ascribed in scheme 2. After that,
1:1 metal complex formazan dye (19) was synthesized by the metallization reaction of 0.1 mole
(42.44 g) un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl)
diazenyl) benzoic acid] (18) with the equimolar concentration (0.1 mole) of FeSO4.7H2O (27.80
g) at pH~6.40-6.50 and at temperature 65-70 ºC respectively according to the above mentioned
procedure (Scheme 2). Dark Brown (Solid). Yield (91%). Melting Point (418ºC). λmax in nm (log
ε
): λmax1 490 (0.860), λmax2 350 (1.10), λmax3 225 (0.30). FTIR (KBr, Cm-1) υmax: 1530 (Ar-C=N,
str), 1420 (Ar-N=N, str), 1590 (aromatic C
=
C), 840 (-CN=NC-, str), 1390 (Ar-SO3H, str), 1670
1
(Ar-C=O str), 675 (Metal-O, str), 3420 (O-H, str). H-NMR (CDCl3, 400 MHz)
δ
: 11.15, (s 1H,
SO3H), 8.26-8.70 (m 13H Ar-H). 13C-NMR (CDCl3, 75 MHz)
δ: 155.2 (C=N), 169.4 (-COOH),
117.6, 125.3, 125.4, 127.3, 128.6, 128.8, 129.7, 130.3, 131.0, 134.7, 136.3, 139.2, 145.5, 150.5,
(Aromatic carbons). Anal. Calc. for C20H20FeN4O8S (MW: 532.305 g/mol): C, 45.13; H, 3.79, O,
24.05; N, 10.53; S, 6.02; Fe, 10.49%. Found: C, 45.09; H, 3.76; O, 24.01; N, 10.49; S, 6.01; Fe,
10.45%.
2.4.2.2. Synthesis of [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic
acid] Cu II complex (1:1) (20)
For the preparation of metal complex formazan dye (20), the un-metallized formazan dye [2-
((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid] (18) which was
tridentate-ligand had been synthesized following the procedure ascribed in scheme 2. After that,
10

ACCEPTED MANUSCRIPT
1:1 metal complex formazan dye (20) was synthesized by the metallization reaction of 0.1 mole
(42.44 g) un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl)
diazenyl) benzoic acid] (18) with the equimolar concentration (0.1 mole) of CuSO4.5H2O (24.97
g), at pH~6.40-6.50 and at temperature 65-70 ºC respectively according to the above mentioned
procedure (Scheme 2). Reddish Brown (Solid), Yield (91%), Melting Point (408ºC). λmax in nm
(log
C=N, str), 1475 (Ar-N=N, str), 1590 (aromatic C
1610 (Ar-C=O, str), 690 (Metal-O, str), 3460 (O-H, str). 1H-NMR (CDCl3, 400 MHz)
1H, SO3H), 8.30-8.80 (m 13H, Ar-H). 13C-NMR (CDCl3, 75 MHz)
: 155.9 (C=N), 168.9 (-
ε
): λmax1 460 (0.985), λmax2 345 (0.89), λmax3 225 (0.30). FTIR (KBr, Cm-1) υmax: 1520 (Ar-
C), 850 (-CN=NC-, str), 1470 (Ar-SO3H, str),
: 11.70 (s
=
δ
δ
COOH), 117.8, 125.2, 125.5, 127.7, 128.3, 128.9, 129.7, 130.2, 131.1, 134.8, 136.6, 139.5,
145.1, 150.7, (Aromatic carbons). Anal. Calc. for C20H20CuN4O8S (MW: 540.006 g/mol): C,
44.48; H, 3.73; O, 23.70; N, 10.38; S, 5.49; Cu, 11.77% and Found: C, 44.44; H, 3.69; O, 23.65,
N, 10.35; S, 5.44; Cu, 11.74%.
2.4.2.3. Synthesis of Bis-[2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl)
benzoic acid] Cr III complex (2:1) (21)
The 2:1 metal complex formazan dye (21) were synthesized by the metallization reaction of 0.1
mole (42.44 g) un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl)
diazenyl) benzoic acid] (18) with 0.05 mole (13.32 g) concentration of CrCl3.6H2O (26.64 g, 0.1
mole) at pH~6.70 at elevated temperature 90-100 ºC according to the above mentioned
procedure (Scheme 2). Chocolate Brown (Solid), Yield (93%), Melting Point (479ºC). λmax in nm
υ
(log
ε
): λmax1 430 (1.05), λmax2 340 (0.89), λmax3 225 (0.35). FTIR (KBr, Cm-1) max: 1540 (Ar-
C=N, str), 1465 (N=N, str), 1600 (aromatic C=C), 860 (-CN=NC-, str), 1430 (Ar-SO3H, str),
1
1715 (Ar-C=O, str), 650 (Metal-O, str). H-NMR (CDCl3, 400 MHz)
δ
: 11.45 (s 2H, SO3H),
13
8.25-8.75 (m 26H, Ar-H). C-NMR (CDCl3, 75 MHz)
δ
: 155.4 (C=N), 169.6 (-COOH), 117.2,
125.6, 125.8, 127.5, 128.1, 128.7, 129.8, 130.9, 131.5, 134.3, 136.8, 139.7, 145.3, 150.9,
(Aromatic carbons). Anal. Calc. for C40H28CrN8O10S2 (MW: 896.824 g/mol): C, 53.57; H, 3.15;
O, 17.48; N, 12.49; S, 7.15; Cr, 5.80% and Found: C, 53.56; H, 3.14; O, 17.45; N, 12.45; S, 7.11;
Cr, 5.76%.
2.4.2.4. Synthesis of [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic
acid] Ni II complex (1:1) (22)
11

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For the preparation of metal complex formazan dye (22), the un-metallized formazan dye [2-
((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid] (18) which was
tridentate-ligand had been synthesized following the procedure ascribed in scheme 2. After that,
1:1 metal complex formazan dye (22) was synthesized by the metallization reaction of 0.1 mole
(42.44 g) un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl)
diazenyl) benzoic acid] (18) with the equimolar concentration (0.1 mole) of NiSO4.7H2O (28.09
g) at pH~6.40-6.50 and at temperature 65-70 ºC respectively according to the above mentioned
procedure (Scheme 2). Greyish Black (Solid), Yield (84%), Melting Point (436ºC). λmax in nm
υ
(log
C=N, str), 1450 (Ar-N=N, str), 1570 (aromatic C
1615 (Ar-C=O, str), 680 (Metal-O, str), 3445 (O-H, str). 1H-NMR (CDCl3, 400 MHz)
1H, SO3H), 8.15-8.65 (m 13H, Ar-H). 13C-NMR (CDCl3, 75 MHz)
: 155.5 (C=N), 169.8 (-
ε
): λmax1 420 (1.30), λmax2 335 (1.15), λmax3 215 (0.25). FTIR (KBr, Cm-1) max: 1530 (Ar-
=
C), 835 (-CN=NC-, str), 1295 (Ar-SO3H, str),
: 11.85 (s
δ
δ
COOH), 117.1, 125.1, 125.7, 127.9, 128.5, 128.6, 129.3, 130.5, 131.6, 134.6, 136.5, 139.8,
145.4, 150.8, (Aromatic carbons). C20H20NiN4O8S (MW: 535.153 g/mol): C, 44.89; H, 3.77; O,
23.92; N, 10.47; S, 5.99; Ni, 10.97% and Found: C, 44.85; H, 3.74; O, 23.89; N, 10.45; S, 5.96;
Ni, 10.95%.
2.4.2.5. Synthesis of [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic
acid] Co III complex (1:1) (23)
For the preparation of metal complex formazan dye (23), the un-metallized formazan dye [2-
((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid] (18) which was
tridentate-ligand had been synthesized following the procedure ascribed in scheme 2. After that,
1:1 metal complex formazan dye (23) was synthesized by the metallization reaction of 0.1 mole
(42.44 g) un-metallized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl)
diazenyl) benzoic acid] (18) with the equimolar concentration (0.1 mole) of Co(CH CO ) ·4
H O (24.91 g) at pH~6.40-6.50 and at temperature 65-70 ºC respectively according to the
above mentioned procedure (Scheme 2). Dark Reddish Brown (Solid), Yield (84%), Melting
Point (441ºC). λmax in nm (log ε): λmax1 410 (1.10), λmax2 330 (1.29), λmax3 210 (0.45). FTIR (KBr,
υ
Cm-1) max: 1560 (Ar-C=N, str), 1460 (Ar-N=N, str), 1615 (aromatic C
=
C), 855 (-CN=NC-,
1
str), 1320 (Ar-SO3H, str), 1680 (Ar-C=O, str), 660 (Metal-O, str), 3410 (O-H, str). H-NMR
(CDCl3, 400 MHz)
MHz)
δ
: 11.95 (s 1H, SO3H), 8.30-8.75 (m 13H, Ar-H). 13C-NMR (CDCl3, 75
δ
: 156 (C=N), 169.0 (-COOH), 116.9, 124.9, 125.9, 126.9, 128.9, 129.0, 129.5, 130.4,
12

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131.3, 134.9, 136.9, 139.4, 145.9, 160.1, (Aromatic carbons). Anal. Calc. for C20H20CoN4O8S
(MW: 535.393 g/mol): C, 44.87; H, 3.77; O, 23.91; N, 10.46; S, 5.99; Co, 11.02% and Found: C,
44.86; H, 3.75; O, 23.90; N, 10.44; S, 5.96; Co, 11.01%.
2.5. Computational methods
Energy minima structures were obtained by using the Gaussian 09 software [22], and geometries
and structural parameters were extracted with the help of GaussView05 [23]. Optimization was
performed by using the B3LYP/6-311G(d,p) level of theory. Frequency analysis was performed
at the same level to confirm the true optimization (no imaginary value for any frequency).
2.6. Dyeing method for valuation of color shade development on leather
The synthesized un-metallized and metal complex formazan dyes (18-23) had been evaluated for
color shade development features by their application on goat leather fabric at 2 % dyeing rate
through standard procedure stated by [21,24]. Accordingly, the dye solution (250 ml, 1.0 g dye
equivalent to 2 % of goat crust weight) was transferred into the dyeing-drum. The dye bath
pH~5.5 had been accustomed through the transfer of (5.0 ml. 10% w/v) of acetic acid solution.
Afterwards, the dye bath volume had been attuned to 500 ml through the addition of required
water amount and then, on constant agitation at 24 rpm, 50 g leather pieces were transferred into
º
the dye bath. The dye-bath contents had been agitated at room temperature (30 C) for 1 hr and
º
its temperature 50-55 C was up-raised progressively over a period of 35 min and continued
stirring for 1 hr at that temperature. While on the rotation of dyeing drum, the addition of 5.0 ml
of formic acid was accomplished to attain the pH~3.6 and again rotation of 30 min more was
provided to the dyeing drum until the complete fixation of the applied formazan dye molecules
(18-23) onto the leather. Afterwards, the dye-liquor was transferred in volumetric flask (500 ml
and the leather fabric pieces was washed through the cold-water and combined solution of the
dye liquor and washings was then further diluted to 500 ml with water. From this diluted
solution, 2.0 ml was further diluted to 100 ml with water and the absorbance of this solution was
recorded to estimate the exhaustion of the applied dyes (18-23) on goat leather fabric. The dyed
leather piece was piled up over-night to dry; provided the chukrum and toggle and lastly,
mounted on shade card. A weighed amount of leather piece was stirred in boiling acidified
13

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pyridine which dissolves the unfixed dye from fabric and from the absorbance of this solution
percentage fixation was determined.
2.7. Assessment of fastness properties on leather
The synthesized un-metallized and metal complex formazan dyes (18-23) were assessed for their
fastness properties like color change, staining on adjacent fiber, light, wash and perspiration on
goat leather fabric at 2 % dyeing rate following the standard procedure ascribed by [25].
2.8. Antibacterial potential determination
The synthesized formazan dyes (18-23) was scrutinized for their antibacterial potential against
different pathological strains such as Bacillus subtilis (ATCC 9637), Staphylococcus aureus
(ATCC 25923), Escherichia coli (ATCC 25922) and Klebsiella (ATCC 10031) on substrate such
as goat leather fabric and in solution [26]. The synthesized formazan dyes (18-23) for their
antibacterial propensity on agar plates were examined through well-known agar well-diffusion
procedure [27,28] and the standard antibiotic drug such as “Cefradine” was cast off as a positive
control for antibacterial comparative studies. The bacteriological strains were attained from
Pakistan Chemical Scientific Industrial Research (PCSIR) laboratories complex, Lahore,
Pakistan.
2.8.1. Antibacterial potential of synthesized formazan dyes (18-23) in solution
In the first step, by using distilled water, the solutions of 1 M strength of standard drug and the
synthesized formazan dyes (18-23) in 1L volumetric flask were prepared separately [29]. In the
second step, by dissolving 28 g of nutrient agar into 1000 ml distilled water in 1 L volumetric
flask, the solution of nutrient agar was prepared and for complete and even dissolution of
dissolved nutrient agar, the constant stirring was provided for 15 min. After that, flask mouth had
been sealed off through cotton plug for the preservation of cultured media. Then, cultured media
was pasteurized in an autoclave at 120-121 °C temperature by maintaining pressure such as 15
LD/ sq inch for 20-25 min. After pasteurization, cultured media was cooled down to room
temperature and was cast off for the development of inoculum of bacteriological strains [30].
In the third step, already cultured slants of each frozen bacteria had been taken and a loop full of
cultured microbes were transferred into the test-tube containing pasteurized nutrient agar and
preserved for future use. Afterwards, from stock slants, E. coli, B. subtilis, S. aureus, and
Klebsiella were transferred into four conical flasks containing 25 ml of nutrient agar and they
14

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were hatched in a shaker at 37 °C temperature for 24 hr; and these cultures were cast off as a
inoculum [31].
In the fourth step, as a basal layer, the newly prepared 200 ml of molten nutrient agar was shifted
into twenty-eight pasteurized petri-dishes. Then, 1 ml inoculum of microbial strains such as E.
coli, B. subtilis, S. aureus, and Klebsiella were transferred into twenty-eight petri-dishes and
covered them through their lids and permitted them for a while to solidify and cool down to
room temperature. After that, at three peripheral positions, the 2 mm diameter of solidify agar
core was bored through sanitized hollow iron rod. Then the hovels were packed through the
standard drug and already prepared samples and the petri-dishes were permitted for 1 hr and then
in order to complete the reaction for bio-activity, the petri-dishes was hatched at 37 °C
temperature for 24 hr in an incubator. Finally, clear zones diameter were calculated through the
Vernier calipers [32].
2.8.2. Antibacterial potential of synthesized formazan dyes (18-23) on substrate (Leather
fiber)
The synthesized formazan dyes (18-23) and standard drug were examined for their antibacterial
potential on the goat leather fabric, which was dyed by them accordingly the procedure stated in
dyeing section. The leather fabric specimens of 1 inch2 dyed with synthesized formazan dyes
(18-23) and standard drug were individually transferred into the nutrient agar of 200 ml
inoculated with E. coli, B. subtilis, S. aureus, and Klebsiella. After that, they were hatched for
the time-period of overnight such as 16 hr at the temperate of 37 °C. The similar experiment as
performed with the dyed leather fabric was also accomplished for the non-dyed leather fabric
[33-35]. The percentage-reductions in bacterial growth by the standard drug (Cefradine) and the
synthesized formazan dyes (18-23) were expressed as follows:
R= B-A/A×l00
Here, R= % reduction in bacterial population; B= absorbance (660 nm) of the media inoculated
with microbial strains and undyed fabric; A= absorbance (660 nm) of the media inoculated with
microbial strains and dyed fabric.
2.9. Antifungal potential evaluation of synthesized formazan dyes (18-23)
15

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The synthesized formazan dyes (18-23) were assessed for their antifungal potential following the
standard procedure stated by [36-39]. The fungal strains such as Trichoderma harzianum (ATCC
20846), Aspergillus chevalieri, (ATCC 9908), Aspergillus candidus (ATCC 34330), Aspergillus
flavus (ATCC 9643), Penicillium stipitatum (ATCC 10500) and Aspergillus niger (ATCC
16404) were cast off for the determination of their antifungal potential. The fungous microbes
were obtained from the microbiology laboratory of University of Veterinary and Animal Science
Lahore, Pakistan. The standard drugs “Fluconazole and Terbinafine” were employed as a
positive control for the comparative study of antifungal potential of the synthesized formazan
dyes (18-23) against different fungal strains.
3. Results and discussion
In present research studies, the un-metallized formazan dye (18) and metal complex formazan
dyes were manufactured by using metal salts of Iron (Fe) (19), Copper (Cu) (20), Chromium (Cr)
(21), Nickle (Ni) (22), and Cobalt (Co) (23). The synthesized formazan dyes (18-23) were
characterized by using elemental analysis, 1H-NMR, 13C-NMR, powder XRD, FT-IR and UV-
visible spectroscopy. The assessment of their performance as a colorant material was carried out
through leather-processing drums on the leather fabric of goat.
3.1. Theoretical structural studies
Since last decade quantum chemical methods have attained the attention of scientific community
because of their wide range applications in different fields [40-42]. Among quantum chemical
methods, DFT is more popular due to its accuracy with low computational cost [43-45].
Nowadays chemists are widely using the DFT methods for studying the energy minima
conformations and their geometrical parameters as well [46-49]. The most stable structures of all
synthesized dyes (18-23) are optimized at B3LYP/6-311G(d,p) level of DFT and shown in Fig.
1. The important structural parameters (bond lengths and bond angles) of all dyes are given in
the Table 1.
The important bond lengths in free ligand i.e. un-metallized formazan dye (18), such as N3-N5,
N5-C7, N2-N6, N6-C7, N2-C8, O4-C9 and O5-C9 are 1.31, 1.31, 1.26, 1.39, 1.41, 1.35 and
1.20Å, respectively. The important bond angles such as N3-N5-C7, N5-C7-N6 and C7-N6-N2
are 121.6, 128.1 and 116.9o, respectively.
All metal complex formazan dyes (18, 19, 20, 22, 23) except (21) have distorted octahedron
geometry mainly due to the presence of a tridentate ligand. The tridentate ligands, due to its
16

ACCEPTED MANUSCRIPT
geometric constraints, forces the other three ligands (water molecules) to adopts position where
the inter-ligand angles deviate from 90o. The Fe-metal complex formazan dye (19) has N-M-N
bond angle of 87.81 degrees, which, in turn, results in N-M-O bond angles 93.18 and 100.40
degrees. Three water ligands make angles of 99.50, 168.75 and 86.76 degree with N3. Some
other important bond angles in (19), such as N2-Fe1-N3, O4-Fe1-N2, N3-N5-C7, Fe1-N3-N5,
Fe1-N2-N6, N5-C7-N6, C7-N6-N2, Fe1-O4-C9 and Fe1-N2-C8 are 87.8, 91.1, 121.1, 121.7,
121.7, 126.4, 119.7, 214.8 and 117.9o, respectively. The important bond lengths such as Fe1-N2,
Fe1-N3, Fe1-O4, N3-N5, N5-C7, N2-N6, N6-C7, N2-C8, O4-C9 and O5-C9 (atomic labels are
in accordance with Fig. 1) in (19) are 1.86, 1.93, 1.91, 1.30, 1.33, 1.28, 1.36, 1.42, 1.30 and
1.21Å, respectively. After complexation with Fe, there is change in geometric parameters of
ligand. N2-N6 bond length is changed form 1.26 to 1.28Å, N6-C7 is changed from 1.39 to 1.36Å
and O4-C9 is changed from 1.35 to 1.30Å. These all changes are due to reorientation and
involvement of ligand in the complex formation with metal.
In Cu-metal complex formazan dye (20), the geometry is very much comparable to that of (19).
For example, the important bond lengths around Cu, such as Cu1-N2, Cu1-N3, Cu1-O4 are 1.96,
1.93 and 1.90Å, respectively. These bond lengths are slightly lower than those of (19) which
may be attributed to smaller van der Waals radius of Cu compared to Fe. The rest of bond
lengths are in the similar range as are observed in the Fe-metal complex formazan dye (19). The
important bond angles involving the Cu, such are N2-Cu1-N3, O4-Cu1-N2, Cu1-N3-N5, Cu1-
N2-N6, Cu1-O4-C9, Cu1-N2-C8 and O4-Cu1-N2 are 89.4, 163.5, 122.7, 124.1, 122.8, 122.8 and
93.7o, respectively. The Cr-metal complex formazan dye (21) is different from the others because
in this, the central Cr atom is surrounded by two molecules of un-metallized formazan dye (18).
This structural change results in geometry very similar to a regular octahedron, which angles are
close to 90 degrees. The important bond lengths around Cr, such as Cr1-N2, Cr1-N3, Cr1-O4,
O10-Cr1 and Cr1-N11 in 21 are 1.91, 1.98, 1.88, 1.88 and 1.91Å, respectively. The rest of
important bond lengths are in the similar as are observed in the previous two metallized dyes
(19) and (20). The important bond angles in (21), as N2-Cr1-N3, O4-Cr1-N2, Cr1-N3-N5, Cr1-
N2-N6, Cr1-O4-C9, Cr1-N2-C8, O10-Cr1-O4 and O10-Cr1-N11 are 86.1, 91.3, 120.6, 124.3,
127.4, 120.2, 89.7 and 91.2o, respectively. The important bond lengths and bond angles in Nickle
(22) and Cobalt (23) metal complex formazan dyes are comparable to those of (19) and (20) (for
detailed values see Table 1).
17

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Dye 18
Dye 19
Dye 20
Dye 21
18

ACCEPTED MANUSCRIPT
Dye 22
Dye 23
Fig. 1. The optimized geometries of all dyes (18-23), at B3LYP/6-311G(d,p) level of DFT
Table 1. The theoretical structural parameters (Bond lengths and bond angles) of all dyes (18-
23) (atomic labelling is in accordance with the labels shown in the Fig. 1)
Dye 18
Bond angle (o)
Dye 19
Bond angle
Bond
(Å)
Bond
(Å)
(o)
length
length
---
---
---
----
Fe1-N2
Fe1-N3
Fe1-O4
N3-N5
N5-C7
N2-N6
N6-C7
1.86
1.93
1.91
1.30
1.33
1.28
1.36
N2-Fe1-N3
O4-Fe1-N2
N3-N5-C7
Fe1-N3-N5
Fe1-N2-N6
N5-C7-N6
C7-N6-N2
87.8
----
---
----
---
91.1
----
----
N3-N5-C7
----
121.6
121.1
121.7
121.7
126.4
119.7
N3-N5
N5-C7
N2-N6
N6-C7
1.31
1.31
1.26
1.39
-----
N5-C7-N6
C7-N6-N2
128.1
116.9
19

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N2-C8
O4-C9
O5-C9
1.41
1.35
1.20
----
-----
N2-C8
O4-C9
O5-C9
1.42
1.30
1.21
Fe1-O4-C9
Fe1-N2-C8
124.8
117.9
-----
Dye 20
Dye 21
Cu1-N2 1.96
Cu2-N3 1.93
Cu1-O4 1.90
N2-Cu1-N3 89.4
O4-Cu1-N2 163.5
N3-N5-C7 122.2
Cr1-N2
Cr1-N3
Cr1-O4
N3-N5
N5-C7
N2-N6
N6-C7
N2-C8
O4-C9
O5-C9
1.91 N2-Cr1-N3
1.98 O4-Cr1-N2
1.88 N3-N5-C7
1.28 Cr1-N3-N5
1.34 Cr1-N2-N6
1.27 N5-C7-N6
1.35 C7-N6-N2
1.41 Cr1-O4-C9
1.30 Cr1-N2-C8
1.21 O10-Cr1-O4
86.1
91.3
122.6
120.6
124.3
129.2
121.4
127.4
120.2
89.7
N3-N5
N5-C7
N2-N6
N6-C7
N2-C8
O4-C9
O5-C9
1.29
1.33
1.28
1.35
1.42
1.28
1.23
Cu1-N3-N5 122.7
Cu1-N2-N6 124.1
N5-C7-N6
C7-N6-N2
127.7
123.8
Cu1-O4-C9 122.8
Cu1-N2-C8 122.8
O4-Cu1-N2 93.7
O10-Cr1 1.88 O10-Cr1-N11 91.2
Cr1-N11 1.91
Dye 22
N2-Ni1-N3
Dye 23
Ni1-N2 1.84
Ni1-N3 1.85
Ni1-O4 1.83
N3-N5 1.28
N5-C7 1.33
N2-N6 1.28
N6-C7 1.34
N2-C8 1.43
O4-C9 1.28
91.2
Co1-N2
Co1-N3
Co1-O4
N3-N5
N5-C7
N2-N6
N6-C7
N2-C8
O4-C9
1.85 N2-Co1-N3
1.88 O4-Co1-N2
1.85 N3-N5-C7
1.30 Co1-N3-N5
1.33 Co1-N2-N6
1.28 N5-C7-N6
1.35 C7-N6-N2
1.43 Co1-O4-C9
1.29 Co1-N2-C8
91.1
O4-Ni1-N2
N3-N5-C7
Ni1-N3-N5
Ni1-N2-N6
N5-C7-N6
Ni1-O4-C9
Ni1-N2-C8
C8-N2-N6
94.5
95.7
120.7
126.1
123.7
125.9
129.5
123.1
112.8
120.7
126.0
124.8
127.0
123.5
125.3
122.9
20

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O5-C9 1.23
C9-O5
1.23 C8-N2-N6
112.1
3.2. FTIR spectral analysis
The fourier-transform infrared spectroscopy (FT-IR) considered as a valuable basis for
accomplishing the structural information of organic nature based compounds, therefore the
recognition and identification of different functional groups of the synthesized formazan dyes
(18-23) was carried out by employing this analytical technique. The FT-IR data of the
synthesized formazan dyes (18-23) is presented in Fig. 2. The results of FT-IR spectra were
demonstrated that the sharp peaks at 1570-1615 cm-1 were exhibited by the synthesized formazan
dyes (18-23) which were indicted the manifestation of C=C (Aromatic stretching). The small
peaks at 1520-1560 cm-1 in the spectra of the synthesized formazan dyes (18-23) were designated
the presence of C=N (Stretching) which affirm the formation of formazan dye as shown in Fig. 2
(a). Further, the small peaks were perceived at 1365-1475 cm-1 in their spectra, that were
demonstrating the existence of N=N (Stretching). The broad peak in the spectra of synthesized
un-metallized formazan dye (18) at 3380 cm-1 revealed the existence of N—H stretching. The
sharp peaks experimental at 820-860 cm-1 in their spectra illustrated the presence of C-N=N-C
skeleton, which were also acknowledged and confirmed the formation of desired formazan dyes
(18-23) successfully. The peaks that were perceived at 1230-1470 cm-1, 1610-1715 cm-1, 2930
cm-1 in their spectra were found to acknowledge the existence of -S3OH, Ar-C=O, and OH
(Carboxylic acid stretching) respectively. A broad and sharp peak detected at 3410-3460 cm-1
that was attributed to OH functional group. The IR peaks for OH (Carboxylic acid stretching)
and N-H were only detected before metallization in the spectra of dye (18) that performances as
ligand as presented in Fig. 2 and both IR peaks vanish after the metallization as in the
synthesized formazan dyes (19-23). The IR peaks of M-O were found to perceived only after the
metallization of dye (18) with metal like Fe, Cu, Cr, Ni, and Co in their respective metal
complex formazan dyes (19-23). The formation and manifestation of metal-oxygen bond in
terms of metal ligand bond to form metal complex formazan dyes (19-23) was confirmed from
the small peaks observed at 650-690 cm-1 in their spectra as presented in Fig. 2 (b). Furthermore,
the IR peaks of OH (stretching) were only experimental in metal complex formazan dyes (19, 20,
22 and 23) as these are (1:1) metal complexes of Fe, Cu, Ni and Co respectively. Whereas that
IR peak of OH (stretching) were not observed the in spectra of synthesized formazan dye (21) as,
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this dye was (2:1) metal complex of Cr. Hence, FT-IR spectra outcomes were magnificently
confirmed the synthesis of formazan dyes (18-23) and were founded in close agreement with the
[3].
Fig. 2. The FT-IR spectra of the synthesized un-metallized formazan dye (18) (a) and metal
complex formazan dye (19) (b)
3.3. 1H-NMR spectral analysis
The 1H-NMR spectroscopy is most expedient and viable analytical tool for the identification and
structure elucidation of a molecule, with respect to hydrogen-1 nuclei inside the molecule of a
substance to be analyzed; therefore, we further employed the 1H-NMR spectroscopy to get more-
insight structural information and confirmation of our desired synthesized formazan dyes (18-
1
23). The results of H-NMR spectral studies of the synthesized formazan dyes (18-23) were
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demonstrated that the chemical shift values (δ: 7.60-7.91) were attributed for the (m 13H, Ar-H)
of the synthesized un-metalized formazan dye (18) as presented in Fig. 3 (a). While, the chemical
shift values (δ: 8.26-8.70), (δ: 8.30-8.80), (δ: 8.15-8.65), (δ: 8.30-8.75) for the (m 13H, Ar-H)
and ( : 8.25-8.75) for the (m 26H, Ar-H) were designated for the synthesized metal complex
δ
formazan dyes Fe (19), Cu (20), Ni (22), Co (23) and Cr (21) 2:1 respectively (Fig. 3 b). The
results were displayed that the chemical shift values of multiplet of aromatic hydrogens (m 13H,
26H, Ar-H) in the metal complex formazan dyes (19-23) were shifted towards lower field than
the synthesized un-metallized formazan dyes (18). This is because of the fact that an electron-
donating group amine (N-H) was substituted to the aromatic ring of the synthesized formazan
dye (18) that was responsible for shifting the chemical shift (δ) value to higher field. The
literature studies indicate that the electron-withdrawing group (-SO3H, -COOH, -NO2) are
promising for shifting the chemical shift values to lower field while the electron-donating groups
(-NH2, CH3, -OH) are liable for shifting the chemical shift (δ) value to higher field. Conversely,
amine (N-H) group were absent in all other synthesized metal complex formazan dyes (19-23).
Moreover, the chemical shift peaks value for (s 1H, SO3H) was designated at (11.15-12.18) in all
1
synthesized formazan dyes (18-23). The H-NMR spectral investigations were also illustrated
and inveterate the formation of metal complexes with the un-metallized formazan dye (18), were
acting as a ligand. As two functional groups such as amine (N-H) and carboxylic acid attached to
the aromatic ring (Ar-COOH) were presenting the chemical shift (δ) values for their hydrogen
nuclei at (1.498 and 11.30) only in synthesized un-metallized formazan dye (18) respectively but
were not showing their chemical shift peaks in the synthesized metal complex formazan dyes
(19-23). This is because of the fact that the amine (N-H) and carboxylic acid attached to the
aromatic ring (Ar-COOH) were involved in the formation of metal-oxygen and metal nitride
coordinate covalent bond with the metals (Fe, Cu, Cr, Ni and Co) with the elimination of their
hydrogen to form their respective metal complex. This observation evidently and admissibly has
inveterate the formation of coordinate covalent bond among non-metal complex formazan dye
ligand (18) and metals (Fe, Cu, Cr, Ni, and Co) in metal complex formazan dyes 19, 20, 21, 22,
1
and 23 respectively. Hence, H-NMR and FT-IR spectral studies were successfully avowed the
synthesis of our desired formazan dyes and current research outcomes were found in agreement
with the earlier reported studies [3].
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Fig. 3. The 1H-NMR spectra of synthesized un-metallized (18) and metal complex (19) formazan
dyes (a and b) and 13C-NMR spectrum of synthesized un-metallized (18) (c) recorded in CDCl3
at 25 °C
3.4. UV-visible spectral investigation
The absorption maxima for the synthesized un-metallized formazan dye (18) and metal complex
formazan dyes (19, 20, 21, 22 and 23) was observed in distill H2O for their UV-visible spectral
investigation. Results were demonstrated that the distinctive three peaks of absorption maxima
were perceived in the UV-vis spectrum of synthesized formazan dyes (18-23). It has been
observed from the spectrum pattern that value of λmax1 was assigned and specific for the
framework (-C-N=N-C-) of formazan dyes (18-23) and because of that fact, current research
studies relates to the λmax1 value. The formazan peak value of λmax1 is perceived typically at the
wavelength range of 410-500 nm. Sometimes these peaks could be redirected probably at the
wavelength range of 350-600 nm, based on the molecular structure of the formazan and their
peak absorption is resulted due to two different types of electronic transition in the framework of
the formazan such as π→π* and n→π* transition. The values of λmax2 are generally give their
impression at the wavelength range of 300 nm to 350 nm, which are assigned for the electronic
transitions of azo group (-N=N-), is present in the molecule of formazan dyes. Furthermore, the
values of λmax3 are typically observed at the wavelength range of 270 nm to 300 nm that are
resulted because of n→π* transition of azomethine (-C=N-) functional group in the molecules of
formazan dyes. Results of UV-visible spectral investigation were disclosed that the synthesized
formazan dyes (18-23) consisted of stated identical chromophoric functionalities. However, the
large change in the value of λmax were perceived in the form of their bathochromic and
hypochromic shift when un-metallized complex formazan dye (18) were converted to metal
complex formazan dyes (19-23) through metallization process. The results were exhibited that
the absorption maxima values of λmax1 for the synthesized formazan dyes (18-23) were detected
at 550 nm, 490 nm, 460 nm, 430 nm, 420 nm and 410 nm respectively. While the absorption
maxima values of λmax2 and λmax3 for the synthesized formazan dyes (18-23) observed at the
wavelength of (360 nm, 350 nm, 345 nm, 340 nm, 335 nm and 330 nm) and (230 nm, 225 nm,
220 nm, 225 nm, 215 nm and 210 nm) respectively and these observations were designated that
there was found a shift of longer wavelength to shorter wavelength (Hypsochromic effect) as
presented in Fig. 4. The reason behind the blue shift mechanism of metal complex formazan dyes
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(19-23) than non-metal complex formazan dye (18), the metal behave as Lewis acid and pull out
the electron density towards itself from the ligands that ultimately lead the transformation of
longer wavelength to shorter wavelength. Results were revealed that for the metal complex
formazan dye (19), a blue shift of 60 nm was experimental with the color transformation from
yellowish brown to dark reddish brown from its non-metal complex formazan dye (18). Similar
hypsochromic effect was also observed in all synthesized metal complex formazan dyes i.e. Cu
(20), Cr (21), Ni (22) and Co (23) with the blue shift of 90 nm, 130 nm, 160 nm and 205 nm
respectively with color transformation from yellowish brown to cyan, greyish beige, indigo and
violet respectively.
Fig. 4. The UV-Visible spectra for the synthesized un-metallized and metal complex formazan
dyes (18-23)
3.5. Elemental analysis
The elemental analysis technique implicates to determine mass of different fractions associate
with Carbon, Hydrogen, Nitrogen, heteroatoms such as (F, Cl, Br, I, At), Sulfur and metal atoms
of the analyzed compound. The elemental analysis technique also known as CHNS analysis
technique, which provide valuable and significant knowledge in structure elucidation of an
unknown molecule. Moreover, by employing CHNS analysis technique, purity level of newly
synthesized compound can also be verified [3]. Therefore, we employed elemental analysis
technique to get more deep knowledge and confirmation about the structure of our synthesized
formazan dyes (18-23); their outcomes presented in spectral studies section evidently revealed
the correlation between the calculated and experimentally founded mass of different fractions
associate with the synthesized formazan dyes (18-23). Henceforth, elemental analysis data and
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spectral investigates were successfully corroborated the structures of our synthesized formazan
dyes (18-23) demonstrated in scheme 2.
3.6. Powder-XRD investigations
The synthesized formazan dye [2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl)
benzoic acid] (18) and its respective synthesized metal complex formazan dyes (19-23) were
assessed for single crystal growth in different kinds of solvents such as dimethyl formamide
(DMF), ethyl alcohol and chloroform but remained fruitless. Therefore, powder X-ray diffraction
was employed to find out more they are either amorphous or crystalline in nature. The nature of
lattice parameters, crystal system and cell volume of the synthesized formazan dyes (18-23) was
determined through X-ray powder di raction analysis. The results of XRD pattern i.e. sharp and
intense peaks with no broadening were evidently exhibited that the synthesized formazan dyes
(18-23) were crystalline in nature (Fig. 5). Further, a High Score Plus software was employed to
execute their di raction indexing patterns and unit cell parameters were determined from the
indexed data enlisted in Table 2. Moreover, it has been observed from the powder XRD
investigations that XRD patterns of synthesized metal complex formazan dyes (19-23) and un-
metallized formazan dye (18) were entirely di erent from each other, which were authenticating
the formation of coordination compounds successfully. From the results, it has been found that
the monoclinic structure was demonstrated by synthesized formazan dyes (18, 19, 20, 22 and 23)
however, orthorhombic structure was presented by dye (21); the monoclinic and orthorhombic
crystal structures were reported for similar type of compounds by [50-52,33]. Furthermore, the
mean crystallite sizes “D” of the synthesized formazan dyes (18-23) were calculated from
di raction data. According to the Scherrer equation (D = 0.9
λ/ (β cosθ), where λ is X-ray
wavelength (1.5406 ˚A), is Bragg di raction angle, and is the full width at half maximum of
θ
β
the di raction peak) [53,54]. The average crystallite size 39 nm to 80 nm were observed for all
the synthesized formazan dyes (18-23) (Table 2).
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Fig. 5. The XRD-spectra of synthesized un-metallized and metal complex formazan dyes (18-23)
Table 2. The XRD data of the synthesized formazan dyes (18-23).
Dye#
Lattice Parameters
Volume Crystallite
Crystal
system
(A3)
size D
(nm)
63
a (A)
b (A)
c (A)
β (º)
11.0421 17.9426 9.2121 101.2034 1189.727
Monoclinic
Monoclinic
Monoclinic
18
19
20
13.2111 6.1211 10.0991 100.0103
14.8888 11.8583 9.8097 90.098
490.901
70
1401.872
52
15.9812 13.1231 10.0821 110.0873 1503.087
17.1212 8.2332 14.0987 112.3120 1531.312
18.1101 8.8981 16.2003 114.0863 1940.051
39
80
49
Orthorhombic
Monoclinic
Monoclinic
21
22
23
Values are mean ± SD triplicate assays.
3.7. Assessment of color properties of synthesized formazan dyes (18-23)
3.7.1. Fastness properties
The synthesized formazan dyes (18-23) were evaluated for their color shade development along
with the assesmet of different kinds of fastness properties such as color change, staining on
adjacent fibre, light, wash and perspiration on leather fabric of goat. The results of their fastness
properties are presented in Table 3. Results shows that the synthesized formazan dyes (18, 19,
20, 21, 22 and 23) developed different kinds of color shades such as Red, Brown, Green, Black,
and Blue with different types of color tones like Bright brick, Reddish, Bluish, Greyish and
Greenish on leather respectively (Fig. 6). Results were demonstrated that average wash fastness
(3-4) were accounted for the synthesized un-metallized formazan dye (18) while good wash
28