VANILLIN AND 4-NITROANILLINE DERIVED SCHIFF-BASE AND ITS NICKEL(II) COMPLEX: SPECTRAL ANALYSIS AND ANTIBACTERIAL INVESTIGATION

. Ni(II) metal complex of Schiff base (SB) ligand 2-methoxy-4-[(4-nitrophenyl) iminomethyl]phenol (MNIMP); derived from vanillin and 4-nitroanilline was synthesized in 1:2 M:L by refluxing solution containing metal salt (NiCl 2 .6H 2 O, 97.0%) and SB in ethanol and characterized based on powder (XRD), NMR, FT-IR, UV-Vis spectrophotometer


INTRODUCTION
Transition metals can exhibit a wide variety of coordination properties and reactivity, which can be used to form complex with biologically active organic molecule as ligands.The structure, properties and biological importance of these complex is depends upon the central metal atom and surrounding ligand molecules they are connected to the metal atom by coordinate bond.In particular, metal complexes of N and O donor atom chelating ligands have attracted considerable attention because of their interesting biological activities.Those atoms play a key role in the coordination of metals at the active sites of numerous metallo-organic compounds and as donor atoms in the formation of dative bond with the corresponding ligand [1,2].
Coordination compounds combining Schiff base ligands with nitrogen and oxygen donor atoms are designed and synthesized extensively since the metal-ligand interaction controls and enhances the biological, chemical, and electrochemical activities of the metal centers.Metal complexes having bi-dentate Schiff base ligands with both nitrogen and oxygen donor atoms and containing halogen groups are interesting due to their antibacterial properties.Complex formation between transition metal and the Schiff Base ligand results in foundation of coordinate complexes and in recent studies, researchers have shown significant interest in various amino acid Schiff base ligands [3,4].
Vanillin is a phenolic organic compound with aldehyde and ether functional groups.Condensation product of vanillin (aldehyde as a center of reaction) and 4-nitroaniline (primary amines with -NH2 as a center of reaction) yields carbon-nitrogen double bonded organic compound called Schiff bases (SBs).The functional feature of SBs are represented by the azomethine group (-C=N-), where the substituents are alkyl, aryl, or heterocyclic groups [5,6].
A number of SB compounds are used as ligands by considering their metal chelating ability, physiological and pharmacological activities.Specifically, the highly stable complexes formed by SBs with transition metals often lead to compounds with strongly enhanced biological activities.It is known that the existence of metal ions bonded to biologically active compounds may enhance their activities and chelation; causes change in the biological properties of the ligands and also the metal moiety.It has been reported that chelation is the cause and cure of many diseases caused by microbial like bacteria and fungi [7,8].
The growing importance of the Schiff bases and their metal complexes in modern coordination chemistry is attributable to recent observations about their antimicrobial and oxygen carrier properties [9,10].Specifically, SBs derived from aromatic amines and aldehydes have a wide variety of applications in many fields.A search through literature reveals that vanillin Schiff confers good biological activities, have better complexation and possess polyvalent metal ions [11,12].However, there was no information on the combined ligand of Schiff base of vanillin and 4-nitroaniline with nickel(II) metal complex.In view of this, we herein report the derivatization, characterization and antibacterial investigation of Schiff base (

Characterization of the complex and physical measurements
The progress of the reaction and the purity was monitored by thin layer chromatography (TLC).Molar conductivity of the complex was measured at room temperature with synthesized solution in DMSO using a microprocessor based conductometer which gives valuable information about the coordination number, the nature of counter ions present in the complex and structure of the complexes.The specific conductance value recorded with EC-214 a microprocessor using this, the molar conductance of the metal complex was calculated using the following expression [13].
In test of chloride counter ion, the formation of white precipitate suggests the presence of chloride in the sample.Disappearance of the white precipitate on adding ammonia solution and regeneration of the white precipitate on dropping few drops of concentrated nitric acid confirms the presence of chlorides [14].Magnetic susceptibility measurement of the complex was recorded using a MSB-AUTO Magnetic balance (Sherwood Scientific) at 19 o C. It used to determine the extent of electron pairing, the stereochemistry and metal-ligand interactions in the complexes [15].The instruments generate gram susceptibility (χg) data for a given paramagnetic substance from which the magnetic moment was calculated using the formula given below [21].
where, μeff = effective magnetic moment, m = molar of magnetic susceptibility and T = Kelvin temperature.
Additionally, from the effective magnetic moment of the complex, the number of unpaired electrons present in the metal ions is calculated using the relation given below [16,17].
where, n = is number of unpaired. ( Elemental analyses (carbon, hydrogen, oxygen and nitrogen) were performed on a thermo finnigan Flash EA-1112 elemental analyzer.Elemental analysis of a compound enables one to determine the empirical formula of the compound [18].

Structural characterizations
The powder X-ray diffraction patterns were collected in the scan range 2θ between 10 o and 90 o using an advanced diffractometer equipped with source Cukα = 1.54 Å.This was used to elucidate the structure and crystallite size of the complex.FTIR spectra of the synthesized compounds were recorded on a Shimadzu 4000 instrument using KBr pellet within the range of 400±10 to 4000±10 cm −1 and applicable to examine the functional groups. 1 H-NMR and 13 C-NMR spectra of compounds were recorded on Bruker Avance II-400MHz.NMR spectrometer in deuterated DMSO solvents using TMS as internal standard.Chemical shift values are expressed in δ (ppm).It used to elucidate the structure and locating precisely the donor sites of the ligand.The UV-Vis spectrophotometer (electronic spectrum) was used to identify the geometry and charge transfer of the complex.The absorbance was recorded by the Sanyo UV-Vis spectrophotometer model (SP65, UK) [19].

Pharmacological reagents
Reagents for biochemical assays such as nutrient agar (NA, Micro master laboratories, India) and standard gentamicin (Research products international (RPI), India), were used for the antimicrobial studies.

Synthesis of MNIMP ligand
MNIMP Schiff base ligand was synthesized from the reaction (0.1521 g, 0.001 mol) of vanillin and (0.1381 g, 0.001 mol) of 4-nitroanilline with pure alcoholic medium of 15 mL ethanol with the addition of five drops of H2SO4 to the solution.The reaction mixture was left to reflux for 2 h to precipitate the product in a yellow color and the resulting precipitate filtered through suction filtration.Finally, the precipitate washed with distilled water, ethanol and with a 50:50 mixture of ethanol and petroleum ether then dried and stored in desiccators containing calcium chloride (Scheme 1) [20].

Synthesis of Ni(II) metal complex
A Schiff base of the metal complex was synthesized by adding NiCl2.6H2Ometal salt (0.2379 g, 0.001 mol) to an ethanolic solution (20 mL) of MNIMP Schiff base ligand (0. 5544 g, 0.002 mol) with stirring.The mixture was heated under reflux for 3 h at 70 ℃.Then a greenish yellow colored precipitate solution is obtained the solution were filtered while it is hot and washed with ethanol and recrystallized in methanol and diethyl ether.After that, the product was collected and dried over anhydrous CaCl2 (Scheme 2) [21].Scheme 1. Synthetic scheme of MNIMP SB ligand.Antibacterial test: The synthesized Nickel (II) complex at concentrations of 10µg/mL is tested for antibacterial activities against gram positive bacteria S. aureus and gram negative bacteria E. coli at 37℃ for 24 hours.The method used to evaluate the antimicrobial activity is disc diffusion method.The lowest concentration inhibiting growth of the organism is recorded as MIC to check the accuracy of the drug concentrations and have been compared standard; Gentamicin [22].

RESULTS AND DISCUSSION
The solubility of the Schiff base ligand and its Ni(II) complex are checked with different solvent polarities (Table 1).According to the result, the solubility behavior of Schiff base and the complex have polar nature characteristics (like dissolve like principle) [23].When a AgNO3 is added to the Ni(II) complex solution, a cruddy white precipitate (AgCl) is formed in the solution, which revealed the presence of chloride groups as counter ions (outside the complexes sphere) which also in good agreement with the results of molar conductance [24].The molar conductance of metal complex was measured and given in Table 1.
The elemental analysis data was applied to determine the percentage composition of C, H and N. The acceptable differences between the test results and certified values are presented in Table 1.The results revealed that the % C, H, and N are in good agreement with the proposed structures.From the data obtained it was found that the compounds analyzed as [M(L-L)2] indicating a 1:2 mole ratio of metal:ligand (M:L), which is in a good agreement with the proposed molecular formula [M(C14H12N2O4)2(H2O)2] [26].
The high melting point value of the complex than the ligand suggests that good chelating effect of the ligands; it is stable and not decomposed easily.Both decomposition temperatures and stability have direct relationship with melting point (determined using a melting point apparatus with sealed capillary tubes containing a few milligrams of sample) [27].The results of proposed molecular formula, color and melting point are summarized in Table 1.
Many transition metal salts and complexes are paramagnetic due the partially filled d-orbitals.The measured gram magnetic susceptibility of Ni(II) is 7.506 x10 -6 .The result indicates that the complex is paramagnetic subsequently the value is positive.The spin free nickel(II) complex is exhibiting magnetic moment in the range 2.82-3.4BM with specific value of 3.25 BM which corresponding to two unpaired electrons.This follows to conclude that the Ni(II) ion exist in d 8 (t2g 6 eg 2 ) octahedral environment in its complex.This is in a good agreement with literature data for Ni 2+ in octahedral environment [28,29].

Spectral characterization
FT-IR spectral.The FT-IR spectrum of ligand and their Ni(II) complex are given in Figure 1a.The FT-IR spectrum of ligand is found within the range of 400-4000 cm -1 .A strong band is appeared in the range of frequency 1600-1675 cm -1 which belongs to the C=N of azomethine group and conform the formation of the Schiff base.The absence of any frequency in 1700 cm -1 (frequency of C=O) and around 1300 cm -1 (frequency of N-H) in spectra of ligand indicated the completion of the reaction between aldehyde and amine [30,31].The band at 1458.5 and 1111.0 cm -1 indicate the presence of -NO2 and -OCH3 group.The peak appeared at 1587.4 cm -1 is also due to the presence of -C=C-stretching frequency of the aromatic group of the free ligand.The FT-IR spectrum of the metal complexes exhibit broad bands in the range of 3354-3423 cm -1 indicating the presence of coordinated water molecules.Frequency of (M-N) and (M-O) are observed in the far-infrared region.These bands are absent in the spectra of the ligand.Based on the data we could assign the bands at 408-443 and 524-599 cm −1 to M-N and M-O vibrations, respectively (Figure 1a and Table 2).
Due to the larger dipole moment change in the vibration of the M-O band in comparison to that of the -M-N-band, the band (M-O) usually occurs in the higher frequency region and the band is usually sharper and stronger than (M-N).There is no peak around 300-350 cm −1 indicating the absence M-Cl bond in the complexes.The spectra of complex show stretching vibration band at region between 1500-1700 cm -1 is shifted to lower frequencies compared with ligand and it is accepted for most complex (Figure 1a and Table 2).
The frequency corresponding to the azomethine group (C=N group (1632.0cm -1 )) in the ligand is shifted to higher frequency at 1647.5 cm -1 in the spectra of the complex (Table 2).This is due to the interaction of (C=N) nitrogen to the metal ion as a result of rear donation of π-electrons by the metals.This is attributed to the bonding affinity of nitrogen atom of the azomethine group of the Schiff base to the respective metal ions directly linked to the donation of electrons from nitrogen to the empty d-orbital's of the metal atom and decreasing the planar property after complexation [31].The spectra of ligand show band at 1458.5 cm -1 and 1111.0 cm -1 due to presence of -NO2 and -OCH3 group respectively, no appreciable change of signal is observed in the spectra of complex.It indicates that -NO2 and -OCH3 group do not participate in coordination with metals (Figure 1a and Table 2).
The broad band spectra for the ligand at 3485.0 cm -1 is related to the stretching vibration of -OH in phenol, the disappearance of this band at the spectrum for complex indicate the deprotonation of phenol group to coordination with metal ion.For the metal complex new band occurred at 566.2 cm -1 on the site of reaction (M-O).This supports the coordination mode of ligand through oxygen atom of the phenolic group.Also bands observed at frequency 408.0 cm -1 indicates the formation of M-N.All the FT-IR data suggested that the metal is bonded to the Schiff bases through the phenolic oxygen and the imino-nitrogen (Figure 1a and Table 2).The selected vibrational frequencies for the ligand and its metal complexes are summarized in Table 2.This occurrence indicates that there is coordination between the metal and the lone pairs of electron on the nitrogen atom of the ligand [32].Table 2. FT-IR spectral data and electronic spectral data of MNIMP ligand and [Ni(MNIMP)2(H2O)2]Cl2.
FT-IR spectral data () (where  is wavenumber in cm -1 ) Electronic spectral data

UV-Vis (electronic) spectral analysis
The UV-Vis spectra ligand and the complex are shown in Figure 1b and organized in Table 2.It can be shown that the compounds peaks are found within the range of 200-800 nm.There are two absorption bands in the electronic spectrum of the ligand which could be assigned to n→π* and π→π* transitions.These transitions are also found in the spectra of complex, but they appeared to be shifted towards lower and higher wavelength, confirming the coordination of ligands to the metal ions.The electronic spectra of the ligand showed three major bands at 239.1, 260.7 and 388.0 nm (Figure 1b and Table 2).The ligand transitions such as π→π* and n→π* are expected due to the presence of C=N, C=C and C-O groups in the ligand's structure.The band in the region of 239.1 and 260.7 nm can be recognized for possible π→π* transitions of phenyl ring (-C=C-) and C=N chromospheres.The spectral bands which was observed at 388.0 nm assigned possibly to n→π* transitions of C=N chromospheres.The complex shows three major bands at 275.8, 311.1 and 372.8 nm.However, there is a shift in the positions of the bands to a lower wavelength when compared to the ligand, from which formation of the complex can be confirmed.The expected d-d transition in the visible region for complexes of the ligand cannot be detected even

(a) (b)
with concentrated solution.It may be lost in the low energy tail of the charge transfer transition [33].
1 H-NMR and 13 C-NMR spectral analysis The 1 H and 13 C-NMR spectrum of the ligand is shown in Figure 2a and 2b, respectively.In the 1 H NMR spectrum of ligand (Figure 2a), a signal at δ = 9.8 ppm were attributed to the azomethine protons of the ligand suggesting the presence of C=N linkage.The proton which present next to the imine carbon shows more downfield when compare with the other protons in the aromatic ring.The signal due to aromatic ring protons appeared as multiplet in the range δ = 6.5-8.5 ppm, whereas signals due to methoxy protons appeared as singlet at δ 3.85 ppm.Another singlet corresponding to hydroxyl proton is observed at δ = 10.3 ppm.The free NH2 protons of 4nitroanilline usually show a broad singlet peak in a region at 4-6 ppm and thus signal are absent/disappeared in the observed spectra of Schiff bases which indicates the formation of the -C=N-bond.While the band at δ = 2.51 ppm to 2.54 ppm related to H2O in d-DMSO and in overall the 1 H-NMR confirms the presence of 12 protons (H12) within SB ligand (Figure 2a and Table 3).The 13 C NMR spectrum of the free ligand revealed peaks at δ = 156.5,153.5 and 148.7 ppm attributed to carbon atom of azomethine (-N=CH-) group, sp 2 oxygenated quaternary aromatic carbons where a hydroxyl and methoxy groups are attached, respectively [34,35].The signal appearing δ = 60 ppm belongs to quaternary aromatic carbon (C-N) group where the carbon is attached.The signal appearing at δ = 136 ppm belongs to quaternary aromatic carbon (C-N) group where the nitrogen is attached and the signal appearing δ =191 ppm belongs to quaternary aromatic carbon where the nitro group is attached.Seven aromatic methenes are clearly evident between δ =129-111 ppm.The multiplet and overlapping peak around δ = 40 ppm corresponds to carbon of methoxy group (-OCH3) in the Schiff base ligand and d6-DMSO/TMS (Figure 2b).In general, the 13 C-NMR confirms the presence of 14 carbons (C14) within Schiff base ligand (Table 3).Table 3. 1 H-NMR and 13 C-NMR spectral data of MNIMP ligand.base through the phenolic oxygen and the imino-nitrogen and the ligand behaves as bi-dentate towards the metal ion and coordinated through the azomethine-N and the phenolic-O center.From magnetic susceptibility analysis and electronic spectra; the complex was suggested to have octahedral geometry.According to powder (XRD) data, the particles of the examined complex were situated in Nano scale range and crystallite phase.The melting point datum showed high thermal stability and decomposition of the investigated metal complex.The electrical conductivity study confirmed that the complex had high conducting properties.The antimicrobial activity of the ligand and its complex was examined against a pathogenic bacterium E. coli and S. aureus and the data obtained reveals that the metal ion in the complex enhances the antibacterial activity compared to the free ligand, MNIMP.The obtained result showed the suggested structure of the complex under investigation, as demonstrated in Scheme 2.

Vanillin and 4 -Figure 1 .
Figure 1.(a) FT-IR spectra of Schiff base ligand and their Ni(II) complex and (b) UV-Vis spectra of Schiff base ligand and their Ni(II) complex.