TLC is probably the most versatile technique for the analysis of complexes and their mixtures. TLC is quite efficient technique for separation and identification of synthesized soap, ligand and their complexes in sub microgram amounts using silica gel plates in various non-aqueous solvent systems. R_f of copper soap, ligands and the synthesized complexes in non-aqueous solvent systems has been determined. A thin layer chromatographic applicator was used for coating sorbent material on 20×3 cm glass plates to obtain absorbent layer of 0.25 mm thickness [13Sharma AK, Saxena M, Sharma R. Synthesis, spectroscopic and biocidal activities of environmentally safe agrochemicals. J Biochem Tech 2018; 7(3): 1139-47.]. The coated plates were developed in glass jars of 24×6 cm. A glass sprayer was used to spray chromogenic reagent on the plate to detect the spot. Silica gel G acted as stationary phase whereas the following solvent systems were used as mobile phase.

1. Acetone: Carbon tetrachloride
2. Acetone: Petroleum ether
3. Benzene: Acetone: Petroleum ether

Silica gel G and double distilled water are mixed in 1:3 (w/v) ratio with constant mechanical shaking until homogenous slurry was obtained to prepare the plates. Approx. 10μl of the test solution was applied on thin layer plates by using micropipette. The solvent ascent was fixed to 10-12 cm in all cases. When the development was completed, the plates are withdrawn from the jar and dried at room temperature. The spots were visible in day light.

The spectral measurements (IR and NMR) and elemental analysis were carried out at the “Regional Sophisticated Instrumentation Centre”, Central Drug Research Institute (RSIC, CDRI) Lucknow. The IR spectra of the complexes were obtained as KBr discs in the range 400-4000 cm^–1 on Perkin Elmer spectrophotometer. ¹H NMR spectra were recorded at CDRI, Lucknow using CDCl₃ as reference. ESR spectra of the complexes were recorded at SAIF, IIT, Mumbai in the X-band region at microwave frequency 9.1 GHz and microwave power 5 mW under the magnetic field of strength 2000G at room temperature. Tetracynoethylene (TCNE) was used as the standard. Magnetic susceptibility measurements were conducted at S.P. University, Vallabh Vidhyanagar, Gujrat.

Results were reproducible with in ±0.02 R_f unit values for various substituted compounds using procedure for three solvent systems – the main part of the chromatographic studies was the attempt to obtain the standard conditions for their resolution and identification. The best shaped spots given by the solvents were considered to be suitable, and are A = Acetone = carbon tetrachloride, B = Acetone = Petroleum ether, C = Benzene = Acetone = Petroleum ether. In pure acetone spots run along with the solvent front hence could not be used [16Mathur N, Sharma AK. Synthesis of hetero-cyclic compounds for bio-medical applications “ISBN 978-613-8-05861-8” 2018.].

Single spot was given by all compounds, uniform in shape in present investigation, so all of them are chromatographically pure. The results of copper palmitate, substituted benzothiazoles, phenylthiourea and complexes are given in Tables 5-7.

Table 5
Palmitate (C₁₅H₃₁COO)₂Cu.

Table 6
(BTA)A and (BTA)NA.

Table 7
Copper complexes.

Acetone = Carbontetrachloride, Acetone, Petroleum ether and Benzene, Acetone = Petroleum ether and Benzene, Acetone = Petroleum ether were selected as best solvent systems for synthesized compounds. In benzene, with or without nitrobenzene, the spots were diffused and comet likes having tail against the flow of solvent. We have observed that R_f values for the series of compounds under investigation increased as the amount of benzene increased in the solvent system. It is observed from the tables that for all the ligands as well as complexes; nitro derivatives gave higher R_f values as compared to methoxy derivatives [17Sharma AK, Sharma R, Gangwal A K. Antifungal activities and characterization of some new environmentally safe Cu (II) surfactants substituted 2-amino-6-methyl benzothiazole. Open Phar Sci J 2018; 5: 3-12.[http://dx.doi.org/10.2174/187484490180501] ].

As it is reported earlier, the presence of copper ion leads to a magnetic moment of 1.73 BM. Various scientists have reported subnormal values for the magnetic moment of copper soap and fatty acid, inferring that the soap exists as a binuclear molecule. This has been attributed to the formation of δ-bonds arising from inter-molecular exchange demagnetization through lateral overlapping of 3d_x2-y2 orbital on each copper ion.

The studies also reveal that these values of magnetic moments in the range 1.92-2.08 BM is due to the sub-dispersion unit of soap/complex which retains its binuclear configuration even in solution. It may be suggested that sub-dispersion units are bound together by weak polar forces resulting in the interaction between 3d_xy and 3d_xz orbital of two copper atoms [21Rai BK, Kumar A. Synthesis, characterization and biocidal activity of some Schiff base and its metal complexes of Co (II), Cu (II) and Ni (II). Oriental J Chem 2013; 29(3): 1187-91.].

The lower values of magnetic moments of copper ions in copper complex in benzene, as compared to those in solid state, reflect the terminals of binuclear molecules as [Cu₂(RCOO)₄L₂] Where L represents the solvent molecule in solution, but is replaced by the ligand on complexation.

The nature of the complexes is found to be non-electrolytic as the molar conductance values in benzene, lies in the range of 3.9-6.14 mhos cm²mol^–1 [22Mathur N, Bargotya S. Evaluation of novel N/S containing heterocyclic metal complexes as biologically potent agents. Res J Chem Sci 2018; 8(4): 30-4.].

The study aims to evaluate the bioactivity of synthesized complexes against test organisms. Antimicrobial sensitivity was performed for synthesized complexes (Compound S₃ and S₄) on Muller Hinton Agar against Candida abicans, Trichodermaharzianumby Kirbyethod [23Bargotya S, Mathur N, Sharma AK. Antimicrobial & DNA cleavage studies of heterocyclic metal complexes“ISBN978-613-8-34627-2 2018.]

Results obtained using Itraconazole (5 mg (w/v)) as antifungal agent control (C) and for negative reference Ethanol/Methanol (R) are mentioned in Table 12 and Fig. 3)

Table 12
Antifungal activity of the substituted benzothiazole ligands against test fungi.

Fig. (3) Plots presenting the comparative study of complex sensitivity against different fungi under study.

Activity index = zone of inhibition of sample[S]/zone of inhibition of reference [R] And the activity can be found out by [24Mathur N, Bargotya S, Manna B, Kasana A. Antifungal screening and oxidation behaviour of some microwave assisted benzothaizine drugs. Int J Chem Sci 2014; 12(2): 533-46.]:

If the Zone of Inhibition is

< 13 = it means that the extract is inactive,

13 – 18 = it means that the extract is bioactive,

> 18 = it means that the extract is highly active.

Table 12 data reveals that complexes show higher activity than ligands suggesting that complexes are more powerful anti- fungal agents and benzothiazole and other N, S, O etc. containing compounds are able to enhance the performance of copper soaps. The enhanced activity of newly synthesized complexes as compared to those of the ligand can possibly be explained on the basis of chelate formation, presence of donor atoms, basicity as well as the structural compatibility with molecular nature of the toxic moiety [25Sharma AK, Sharma R, Saxena M. Synthesis, spectroscopic and fungicidal studies of Cu (II) soaps derived from groundnut and sesame oils and their urea complexes. Bulletin Pure Appl Sci 2017; 36(2): 26-37.].

The results of ANOVA for the antifungal activities for all complexes are shown in Table 13. The predicted R² are in reasonable agreement and closer to 1.0. This confirms that the experimental data are well satisfactory. The descriptive static results of all complexes also confirm satisfactory results in triplet.

Table 13
ANOVA and descriptive statics results for antifungal activities of synthesized complexes.