Optical, dielectric, electronic and morphological study of biologically synthesized zinc sulphide nanoparticles using Moringa oleifera leaf extract and quantitative analysis of chemical components present in the leaf extract

Abstract

The biosynthesis of zinc sulphide nanoparticles with ∼30 nm diameter using the leaf extract of Moringa oleifera is reported here. The biosynthesized nanoparticles exhibit high stability for a period of three months as supported by the observed negative zeta potential values of 45–55 mV. Two peaks were observed at 427 and 560 nm in the photoluminescence spectrum of ZnS nanoparticles. The dielectrics as well as electronic properties of ZnS nanoparticles were systematically investigated. Thin-layer-chromatography densitometric technique was employed for the simultaneous quantification of the major chemical components present in the extract of M. oleifera leaves. The average amount of observed biomolecules such as crypto-chlorogenic acid, isoquercetin and astragalin were found to be 0.0423, 0.0467 and 0.0634% dry weight, respectively.

---

1. Introduction

II–VI semiconductors such as ZnS, CdS, CdSe etc., show immense potential as materials for the development of various optical as well as optoelectronic devices, mostly due to their large band gap. Zinc sulphide (ZnS) is a direct wide band gap optically transparent semiconductor, which can be widely used in photonics, optics and optoelectronics with diverse applications ranging from optical coatings, field effect transistors to sensors and transducers and in many other optoelectronic devices such as blue-emitting diodes, electroluminescence devices and solar cells.^1–3 Bulk ZnS can exhibit a wide band gap (E_g = 3.66 eV), which is responsible for its amazing properties and potential diverse applications in optical devices.⁴ It is important to mention that nanostructures of semiconducting compounds can display grain size dependent optoelectronic properties due to quantum size effects. ZnS nanostructure exhibits wider band gap compare to bulk semiconductor and also exhibits quantum size effect. ZnS nanostructures can be prepared via different synthetic protocols, including solid-state reaction, sol–gel process, wet-chemical synthesis, sputtering, microwave irradiation, ultrasonic and hydrothermal method.^5–7

Nanobiotechnology is a new branch of nanotechnology, which can provide an economically viable and environmental friendly green synthetic protocols alternative to commonly used chemical and physical methods of nanoparticle synthesis. Nanomaterials can be synthesized using microorganisms, including bacteria, viruses, fungi as well as plant, fruit and flower extracts. A wide variety of metal and semiconductor nanoparticles had been synthesized using microorganisms,⁸ as well as plant and fruit extracts.^9–16 The biosynthesized metal and semiconductor nanoparticles can be further utilized in versatile applications such as catalysis, sensing, SERS active substrates, diagnostics and biomedical purposes.^12–16 In a recent development, we have utilized biosynthesized silver nanoparticles for rapid detection of pathogens like bacteria¹⁵ and also reported the morphology dependent catalytic activity of biosynthesized gold and silver nanoparticles obtained from the plant extract of Piper betle.¹⁶ Silver nanoparticles synthesized from the leaf extract of Neolamarckia cadamba were used as SERS active substrate to detect bacteria within a very short period of 1–5 s with a limit of detection of 10³ CFU mL⁻¹ (CFU = colony-forming unit) for E. coli,¹⁵ while biosynthesized gold and silver nanoparticles obtained from the plant extract of Piper betle were utilized as an effective catalyst in the chemical reduction of 4-nitrophenol to 4-aminophenol.¹⁶

ZnS nanoparticles had been biogenically synthesized recently using edible mushroom Pleurotus ostreatus.¹⁷ Malarkodi and Annadurai¹⁸ had synthesized zinc sulfide nanoparticles using bacteria Serratia nematodiphila (S. nematodiphila), which was isolated from a chemical company effluent. However, the plant and fruit extracts can be used to eliminate the elaborate process of maintaining cell cultures and large-scale synthesis of nanocrystals at lower cost. On the other hand, there is no report on the biosynthesis of zinc sulphide nanoparticles using the plant extract as per our knowledge.

Moringa oleifera (M. oleifera) is recognized to be essential dietary ingredient of the Indian food since long time and nearly all parts of the plant have been utilized in the traditional system of medicine. The Moringa oleifera plant is widely available in different parts of Asia such as China, Thailand, India, Taiwan, Srilanka, Iran and also available in Africa, South America. It is well known fact that different parts of M. oleifera plant like roots, seeds and leaves have been used for various medicinal purposes such as treatment of bronchitis, diabetes and reduction of glandular swelling. Several groups have carried out systematic pharmacological investigations of M. oleifera leaves on anti-inflammation, anti-infection, antidiabetic, and antioxidant activities.^19,20 It contains biomolecules such as vitamin A, C, polyphenols, folic acid, beta-carotene and metals like calcium, iron etc. There are some reports on the biosynthesis of metal nanoparticles, especially silver nanoparticles^21,22 using the extract of M. oleifera plant as a potential source of reducing and stabilizing agents. However, synthesis of semiconductor nanoparticles like zinc sulphide using M. oleifera plant extract has not been reported so far as per our knowledge.

This paper describes the green biosynthesis of semiconductor zinc sulphide nanoparticles using the leaves extract of Moringa oleifera plant. The structural and morphological properties of zinc sulphide nanoparticles were systematically studied by X-ray diffraction, scanning electron microscopy, energy dispersive X-rays spectroscopy, transmission electron microscopy and Fourier transform infra-red spectroscopy. The biosynthesized semiconductor nanoparticles were further characterized by UV-visible absorption spectroscopy, photoluminescence spectroscopy to get insight about their optical properties. The dielectric properties of as synthesized ZnS nanostructures were studied at room temperature over the frequency range of 50 Hz to 5 MHz. The dielectric constants of the ZnS nanoparticles are high at low frequencies, and decrease rapidly when the frequency is increased. The electronic parameters for the biosynthesized ZnS nanoparticles were also determined in this study. We have determined the exact biomolecules present in the leaves extract of Moringa oleifera qualitatively and quantitatively by employing a comparatively cost efficient thin-layer-chromatography (TLC) densitometric method.

2. Experimental

2.1 Materials

All chemicals used in our studies (zinc chloride, sodium sulphide), were analytical grade and used without further purification. All aqueous solutions were prepared with Millipore water. TLC was carried out on precoated silica gel PF254 sheets (Merck, Germany).

2.2 Biosynthesis of zinc sulphide nanoparticles using the plant extract

Moringa oleifera leaves were collected from Botanical garden, Shibpur, India. The leaves were air dried for 15 days, and grinded to a fine powder after keeping them in the hot air oven at 60 °C for 48 h. 20 g of fine air-dried powder was placed in 100 mL of organic solvent (90% methanol) in a 250 mL conical flask, plugged with cotton and kept on a rotary shaker at 180 to 200 rpm for 24 h. After that, it was filtered through four-fold of muslin cloth and centrifuged at 5000 rpm for 10 min. The supernatant was collected and the solvent was evaporated. The crude extract was diluted with 5% of DMF and stored at 4 °C at refrigerator for the synthesis of zinc sulphide nanoparticles. Zinc chloride and sodium sulfide were used as the precursors and their aqueous solutions were mixed together using magnetic stirrer. 5 g of zinc chloride and 1 g of sodium sulphide were dissolved in 100 mL of Millipore water. The aqueous extract of M. oleifera leaves (20 mL) was added to 40 mL of aqueous mixture of zinc chloride and sodium sulphide at room temperature with vigorous and continues stirring for 4 hours. The solution was purged with high purity nitrogen gas before use. Subsequently, reduction proceeded in the presence of nitrogen to eliminate oxygen. The white precipitate of the ZnS nanoparticles is formed slowly in the solution. The obtained colloidal suspensions of zinc sulphide were subsequently centrifuged at 10 000 rpm for 30 minutes and washed five times to eliminate excess reagents and reaction by-products. The prepared ZnS nanoparticles were washed and dried, further. After drying, nanoparticles were grinded to obtain a fine powder for further characterization.

In addition to this, liquid broth of the plant extract can be also utilized for the synthesis of zinc sulphide nanoparticles. The broth used for the synthesis of zinc sulphide nanoparticles was prepared by taking 20 g of M. oleifera leaves in 500 mL Erlenmeyer flask with the addition of 100 mL of Millipore water and boiling in a water bath at 100 °C for 5 min. Next, it was filtered using four-fold muslin cloth and cooled down to room temperature. In the synthesis step, 5 mL of the broth was added to 10 mL of aqueous mixture of zinc chloride and sodium sulphide solution at room temperature. The whole process was completed after 4 h of reaction. Finally, nanoparticles were obtained by centrifugation at 10 000 rpm for 30 min followed by decanting of the supernatant and washing of the pellets with deionized water. The synthesis process can be summarized by the chemical equation given below.

2.3 Materials characterization and instrumentation

UV-visible spectroscopic studies were carried out on Shimadzu dual-beam spectrophotometer (model UV-1800, 240 V). The photoluminescence spectra were recorded using a (Shimadzu-2100) fluorescence spectrophotometer at room temperature with an excitation wavelength of 298 nm.

X-ray diffraction (XRD) measurements were carried out on Bruker axs (model D8 Advance) instrument operating at a radiation voltage of 40 kV and current of 40 mA with CuK_α radiation. SEM measurements were carried out on a JEOL model JSM-6360A instrument operated at an accelerating voltage 20 kV. Fourier transform infra-red (FTIR) spectroscopic studies were carried out on a Shimadzu (model FTIR 8400) instrument in the diffused reflectance mode operating at a resolution of 4 cm⁻¹. The zinc sulphide nanoparticles were dried and grinded with KBr pellets for further analysis. The size and morphology of the biosynthesized zinc sulphide nanoparticles were characterized by Transmission Electron Microscopy (TEM) instrument (JEOL, model JEM-2010) instrument operated at an accelerating voltage at 120 kV. Samples for TEM analysis were prepared by solution-casting the ZnS nanoparticles on a carbon-coated TEM grid.

The stability of the biosynthesized zinc sulphide nanoparticles was evaluated by zeta-potential measurement from the electrophoretic mobility value obtained using the well-known Smoluchowski equation with the help of a commercial dynamic light-scattering (DLS) experimental set up (DLS; Model DLS-nano ZS, Zetasizer, Nanoseries, Malvern Instruments). The hydrodynamic diameters, their dispersity and the particle size distributions of the biosynthesized ZnS nanoparticles were also determined employing the same DLS experimental set up. Both the zeta potential and hydrodynamic diameter were measured using aqueous dispersion of ZnS nanoparticles. The pH of the solution was measured by digital pH meter (brand: CHEMILINE, model: CL-110). Zeta potential was determined by varying the pH of the aqueous dispersion of biosynthesized ZnS nanoparticles ranging between 1 to 12.

The dielectric properties (dielectric constant and dielectric loss) of ZnS nanoparticles were determined using a HIOKI 3532-50 LCR HITESTER over the frequency range of 50 Hz to 5 MHz at room temperature to study the variation of dielectric constant and dielectric loss as a function of frequency.

In case of thin-layer-chromatography (TLC) densitometric method, we used a Linomat 4 automatic sample spotter (CAMAG, Switzerland) and a 50 μL syringe (Hamilton, Switzerland). In addition to these, a glass twin trough chamber (20 × 20 × 4 cm, CAMAG), TLC scanner 3 linked to winCATS software (CAMAG), and TLC plates of 20 × 20 cm with 0.2 mm layer thickness, precoated with silica gel PF254 were used in our experiment.

3. Results and discussion

Fig. 1 illustrates the schematic diagram of the synthetic protocol for the green synthesis of ZnS nanoparticles from leaves extract of Moringa oleifera plant. Although, ZnS nanoparticles can be synthesized employing the wet chemical method using zinc chloride and sodium sulfide as the starting materials at high temperature between 60–80 °C within a time period between 8 to 10 h. This may be due to presence of weak reducing agents like sodium sulfide. In addition to this, the synthesized nanoparticles were found to be very unstable due to absence of any stabilizing agent in the reaction mixture. In this work, we have employed the leaves extract of Moringa oleifera plant for the green biosynthesis of stable ZnS nanoparticles at room temperature. The biomolecules present in the leaves extract of Moringa oleifera plant can act as the reducing and stabilizing agents providing shorter reaction time of 4 h compared to chemical method of synthesis. The biosynthesized ZnS nanoparticles were stable up to a period of three months as observed from the zeta potential measurements. This shows the novelty of the biosynthetic protocol for the synthesis of ZnS nanoparticles.

Fig. 1 Schematic diagram of the synthetic protocol of green synthesis of ZnS from leaf extract of Moringa oleifera plant.

Negative zeta potential values of 45–55 mV were obtained from our experimental zeta potential measurement for the biosynthesized ZnS nanoparticles. The as synthesized ZnS nanoparticles exhibit more or less good stability as evident from the zeta potential values. There is no sign of aggregation of the synthesized ZnS nanoparticles yet after three months. Fig. 2 illustrates the dependence of pH on the experimental zeta potential value for biosynthesized zinc sulphide nanoparticles. The zeta potential measurements demonstrated that the isoelectric point for the ZnS nanoparticles lies between 4–5 and become negatively charged at a pH value of 6. The zeta potential values remain constant in the range between pH 9 to 12. The zeta potential would become positive (between 5 to 10 mV) below the pH 4, which indicates that ZnS nanoparticles become positively charged below pH 4 with extremely low stability.

Fig. 2 Variation of zeta potential as a function of pH measured for biosynthesized ZnS nanoparticles.

SEM and EDAX techniques were employed to evaluate the morphology and the structural composition of the biosynthesized ZnS obtained from the plant extract. Fig. 3(A and B) illustrate the SEM images of the biosynthesized ZnS nanoparticles, whereas Fig. 4 illustrates the EDAX spectrum of as synthesized ZnS sample. As shown from the SEM images in Fig. 3(A and B), the diameter of the synthesized nanoparticles was approximately around 30 nm. The EDAX spectrum shown in Fig. 4 indicates that there are clear peaks for zinc, sulphur, carbon and oxygen with the composition of the biosynthesized sample is mainly zinc (60.43 wt%), sulphur (12.38 wt%), carbon (8.29 wt%) and oxygen (18.91 wt%). We speculate that the presence of oxygen (18.91 wt%) and carbon (8.29 wt%) peaks in the EDAX spectrum may be due to the adsorption of different biomolecules which can act as the reducing and stabilizing agents coming from Moringa oleifera leaf extract. However, it is extremely difficult to identify the exact biomolecules presence in the leaves extract acting as the reducing and stabilizing agents. Therefore, the oxygen and carbon content data obtained from EDAX spectrum can not be used to estimate the weight fraction of the leaf extract present in the biosynthesized zinc sulphide nanoparticles as it is fully qualitative.

Fig. 3 The SEM images of the biosynthesized ZnS nanoparticles.

Fig. 4 The EDAX spectrum of as synthesized ZnS sample.

Transmission electron microscopy (TEM) technique was further employed to verify the morphology of the biosynthesized ZnS nanoparticles. Fig. 5 shows the TEM image of the biosynthesized ZnS nanostructures along with the histogram indicating their particle size distribution. As observed from the TEM image in Fig. 5(a), the biosynthesized ZnS nanoparticles are mostly spherical in shape with average diameter ∼30 nm with less monodisperse in nature. The histogram shown in Fig. 5(b) signifies that the biosynthesized ZnS nanoparticles have a wide size distribution. This was further supported by our DLS measurements for the determination of hydrodynamic diameters, dispersities and particle size distributions. The details of the observed experimental results obtained from DLS measurements are described in the later portion of this paper. The TEM results further support the dimension of ZnS nanoparticles measured by SEM technique. The selected-area diffraction pattern (SAED) of the synthesized ZnS nanoparticles has also been illustrated in Fig. 5(a). As monitored from the figure, the diffraction pattern consists of several concentric rings. The presence of concentric rings in the diffraction pattern clearly indicates that the biosynthesized ZnS nanocrystals are polycrystalline in nature.

Fig. 5 (a) The TEM image and SAED of biosynthesized ZnS nanoparticles. (b) The histogram of particle size distribution obtained from the TEM study.

The XRD pattern of our biosynthesized ZnS sample is demonstrated in Fig. 6. The prominent diffraction peaks were obtained with 2θ values 27.22°, 28.76°, 30.84°, 39.9°, 47.94°, 55.46°, 56.64°, 57.88°, 73.42°, 76.62° and 79.54° which corresponds to (100), (002), (101), (102), (110), (200), (112), (201), (203), (210) and (114) planes respectively. This signifies wurtzite structure of ZnS (JCPDS no. 75-1534). The 2θ values of the standard data obtained from the JCPDS powder diffraction file no. 75-1534 for ZnS are 27.074°, 28.634°, 30.701°, 39.815°, 47.835°, 55.827°, 56.701°, 57.919°, 73.351°, 76.529°, and 79.510°. This corresponds to the intensities 999, 616, 947, 324, 604, 86, 356, 110, 149, 97 and 17 respectively. Our experimental 2θ values obtained from the XRD results are almost a match with JCPDS powder diffraction file no. 75-1534 for ZnS. Fig. 6 also illustrates the experimental (black) and standard (red from JCPDS file no. 75-1534) XRD pattern of ZnS NPs with corresponding h, k, l planes along with their corresponding intensities to make a systematic comparison with the experimentally obtained XRD results.

Fig. 6 The experimental XRD pattern of biosynthesized ZnS nanoparticles (black) along with the standard (red from JCPDS file no. 75-1534) XRD pattern of ZnS NPs with corresponding h, k, l planes.

Our XRD results confirm the high purity of the ZnS sample and suggests that the sample is in the hexagonal wurtzite form.²³ The broad peak indicates the nanocrystalline behavior of the particles. The average size (D) of the ZnS sample was determined by using the classical Scherrer formula:²⁴

D = kλ/β cos θ (1)

where, the constant k is the shape factor usually equal to 1, λ is the wavelength of X-ray, θ is the Bragg's angle and β is the full width of the half maxima (FWHM). The average crystallite size of as synthesized ZnS sample was calculated to be 300 Å (30 nm), which is in good agreement with the reported value obtained from our TEM investigations.

Fig. 7 illustrate the UV-visible absorbance spectrum for the biosynthesized ZnS sample obtained from the plant extract. As demonstrated in the Fig. 7, an absorption peak is observed around 298 nm, which indicates to a large blue shift from the absorption peak value of the bulk ZnS sample at 345 nm. The band gap energy (E_g in eV) calculated from the absorption data was found to be 4.16 eV. This can be explained by the reduction of particle size with widening in the band gap energy of the ZnS nanostructure compare to bulk value of 3.66 eV. From the absorption peak, the band gap energy of the ZnS nanoparticles has been estimated.

Fig. 7 The UV-visible absorbance spectrum for the biosynthesized ZnS sample obtained from the plant extract.

The band gap energy (E_g) was also calculated from the absorption spectrum and optical absorption coefficient (α) near the absorption edge, which is given by the following formula,

αhν = A(hν − E_g)^1/2 (2)

where E_g corresponds to the optical band gap of the crystal and A is a constant. The plot of (αhν)² as a function of the photon energy (hν) at room temperature (Fig. 8) exhibits almost a linear behaviour, (α is absorption coefficient and h is Planck's constant, value 6.627 × 10⁻³⁴ J s) which can be considered as a proof of the indirect transition between valence and conduction band. The band gap energy (E_g) can also be estimated by the linear extrapolation of the curve to the point (αhν)² = 0. Using this method, the band gap of the ZnS nanoparticles was found to be 4.12 eV.

Fig. 8 Plot of (αhν)² against the photon energy (hν) at room temperature for synthesized ZnS nanoparticles to measure band gap.

Fig. 9 shows the photoluminescence spectra of ZnS sample is demonstrated. As demonstrated in the figure, the spectrum contains two peaks at 427 and 560 nm for ZnS sample. Appearance of a peak centered at 427 nm with high intensity can be ascribed by the presence of sulphur vacancies in the lattice.²⁵ Appearance of a peak at 560 nm which is much more broader with lower intensity can be attributed to the transition from the conduction band to the zinc vacancies VZn level (this localized vacancy level is over the valence band at 1.1 eV).²⁶ This is known as green emission, which may be due to some self activated defect centers associated to Zn-vacancies.²⁷

Fig. 9 The photoluminescence spectra of biosynthesized ZnS nanoparticles.

Fig. 10(a and b) illustrate the DLS profile of particle size distributions of biosynthesized ZnS nanoparticles in terms of intensity and number. The average particle size calculated from the DLS measurements for the biosynthesized ZnS nanoparticles was found to be 31.44 nm with polydispersity 0.337. The calculated average particle size obtained from the DLS measurements for the biosynthesized ZnS nanoparticles is in good agreement with the TEM result.

Fig. 10 The DLS profile of particle size distributions of biosynthesized ZnS nanoparticles in terms of (a) intensity (%) and (b) number (%).

The dielectric parameters such as the dielectric constant (ε_r) and dielectric loss (tan δ) are very important to understand both the electrical and dielectric properties of semiconductor nanomaterials like the ZnS nanoparticles. Fig. 11 and 12 show the variations of the dielectric constant and dielectric loss of the ZnS nanoparticles at frequencies between 50 Hz to 5 MHz. We have evaluated the dielectric constant using the relation: ε_r = Cd/ε_oA, where d is the thickness of the sample and A, is the area of the sample. The results indicate the strong dependency of dielectric constant and dielectric loss on the frequency of the a.c. signal of the ZnS nanoparticles. From the figure, we observe that dielectric constant has higher values in the lower-frequency (50 Hz) range and then it decreases up to the high frequency (5 MHz). This behaviour of dielectric constant can be explained due to the mixed contribution of the frequency dependence electronic, ionic, dipolar and space charge polarizations.²⁸ Fig. 12 demonstrates the variation of the dielectric loss with respect to the logarithm of frequency at room temperature. Dielectric loss also exhibits analogous tendency as shown by the dielectric constant. The decrease in the dielectric loss with the increase in frequency suggests that the dielectric loss is strongly dependent on the frequency of the applied field. The high values of dielectric loss at low frequencies can be explained by the charge lattice defects of the space charge polarization.²⁸

Fig. 11 Plot of dielectric constant of ZnS nanoparticles as a function of frequency.

Fig. 12 Plot of dielectric loss of ZnS nanoparticles as a function of frequency.

We have also studied the electronic properties of semiconductor nanoparticles like ZnS systematically from the high frequency dielectric constant value and some of the electronic parameters such as valence electron plasma energy; average energy gap or Penn gap and Fermi energy were evaluated. The valence electron plasma energy, ħω_p, can be calculated using the following relation proposed by Kumar and Sastry.²⁹

ħω_p = 28.8(Zρ/M)^1/2 (3)

The average energy gap, which is also known as Penn gap can be determined from the Penn model proposed by Penn in 1962.³⁰ The Penn gap was determined by proper fitting of the dielectric constant values with the plasmon energy. The average energy gap or Penn gap (E_p) for the ZnS nanoparticles can be determined from the following relation:

E_p = ħω_p/(ε_∞ − 1)^1/2 (4)

The Fermi energy (E_F) can be evaluated from the following relation:

E_F = 0.3(ħω_p)^4/3 (5)

Table 1 shows the evaluated electronic parameters for the biosynthesized ZnS nanoparticles.

Table 1 Estimated electronic parameters of ZnS nanostructures

Parameter	Value (eV)
Plasma energy	15.6
Penn gap	3.6
Fermi energy	12.1

---

FTIR spectroscopy was employed on the biosynthesized ZnS nanoparticles to detect the presence of different functional groups from the various biomolecules in the leaves extract of Moringa oleifera plant, which are responsible for the formation and stabilization of ZnS nanostructures. Fig. 13 shows the FTIR spectra in the range of 4000–400 cm⁻¹. The significant vibration peaks observed at 470, 630, 650 and 1022 cm⁻¹ are due to symmetric bending arising from Zn–S vibration.^31,32 Peaks obtained at 1390 and 2341 cm⁻¹ is due to adsorption of atmospheric CO₂ on the surface of the synthesized nanoparticles.²³ The peaks in the range of 3000–3600 cm⁻¹ can be attributed to O–H stretching frequency which indicates the presence of water adsorbed on the surface of the ZnS particles. A weak band at 1716 cm⁻¹ is characteristic of the C=O stretching mode of carboxylic acid. The IR bands around 1630 cm⁻¹ arise from amide I due to the stretching vibrations of the C=O and C–N groups and band at 1541 cm⁻¹ is from amide II mainly due to N–H bending. Peak observed at 1022 cm⁻¹ is from C–O–C symmetric stretching vibration.

Fig. 13 The FT-IR spectrum of ZnS nanoparticles obtained from leaf extract of Moringa oleifera plant.

The exact mechanism as well as the biomolecules responsible for the stabilization of ZnS nanoparticles with the M. oleifera leaves extract as well as identification of different chemical constituents present in the M. oleifera leaves extract is yet to be resolved. Although, FTIR studies had provided some data for the biosynthesized ZnS nanoparticles, these data are insufficient enough to identify the exact biomolecules responsible for the formation as well as stabilization of the biosynthesized ZnS nanoparticles. The available FTIR data are also insufficient enough to identify all the biomolecules present in the M. oleifera leaves extract. Sophisticated analytical techniques such as high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) were earlier employed for the quantitative analysis of the M. oleifera leaves extract.³³ These studies have demonstrated the presence of biomolecules such as isoquercetin, astragalin and crypto-chlorogenic acid in M. oleifera leaves extract.³⁴ However, both the HPLC and LC-MS techniques are very expensive as well as time consuming involving several tedious steps. Therefore, a much more simple, rapid, and cost-effective method for the quantitative analysis of leaves extract of M. oleifera plant is preferential. Thin-layer chromatography (TLC) densitometry, which has been popularly used for quality control of biological extracts due to its fast data acquisition, simplicity, and reliability can be employed for this purpose.³⁵ We have carried out thin-layer chromatography (TLC) densitometry to identify and quantify the presence of exact biomolecules in M. oleifera leaves extract, which are responsible for stabilization and formation of ZnS nanostructures. In our study, we have developed a thin-layer-chromatography (TLC) densitometric technique and simultaneously validated for real-time quantification of the major chemical components in the 90% ethanolic extracts of M. oleifera leaves. It was observed that the average amounts of crypto-chlorogenic acid, isoquercetin, and astragalin were found to be 0.0423, 0.0467, and 0.0634% dry weight, respectively. We obtained linearity in the range of 300–500 ng per spot with a correlation coefficient (r) over 0.9841. We have confirmed the accuracy of the above method from the determination of the recovery values. Table 2 illustrates the recovery values of each component from the extracts of M. oleifera leaves. The recovery values of each component from the extracts of M. oleifera leaves were in the range of 97.48 to 98.55%. Table 3 shows the robustness studies of crypto-chlorogenic acid, isoquercetin, and astragalin. The robustness of the TLC densitometric method was determined by introduction of minute modifications in definite chromatographic parameters at each standard concentration level of 300 ng per spot. The robustness test was carried out by calculating the standard deviation of peak areas for each parameter and the relative standard deviation was found to be less than 5% for all variations (Table 3). The alteration of mobile phase composition was the critical deviation of the method while the other parameters were relatively insignificant. The precision of the TLS densitometric technique was evaluated from the analysis of 400, 500, and 600 ng per spot of each standard solution after the use by the projected technique onto a TLC plate on the same day for intraday precision and on four successive days for interday precision. The precision was expressed in terms of percent relative standard deviation (RSD). Table 4 shows intraday and interday precisions of crypto-chlorogenic acid, isoquercetin, and astragalin. Fig. 14 shows the chemical structures of crypto-chlorogenic acid, astragalin and isoquercetin. The details of this study of the identification of biomolecules in M. oleifera leaves extract based on thin-layer chromatography densitometry technique will be communicated later.

Table 2 The recovery values of crypto-chlorogenic acid, astragalin, and isoquercetin, in M. oleifera leaf extract

Compound	Theoretical value (ng)	Amount found (ng)	Recovery (%)
Crypto-chlorogenic acid	394	392.6 ± 8.6	97.48
Astragalin	298	298.68 ± 2.7	98.28
Isoquercetin	325	317.5 ± 5.5	98.55

---

Table 3 Robustness values of crypto-chlorogenic acid, astragalin, and isoquercetin

Parameter	RSD^a (%)
	Crypto-chlorogenic acid	Astragalin	Isoquercetin
a Value from five determination.
Mobile phase composition ratio	3.10	1.30	2.55
The length of the chromatogram	1.7	1.3	0.95
Presaturation period	3.5	1.2	0.9

---

Table 4 The intraday and interday precisions of crypto-chlorogenic acid, isoquercetin, and astragalin

Compounds	Concentration (ng per spot)	Intraday precision^a (%)	Interday precision^a (%)
a Calculated in the form of RSD (n = 3).
Crypto-chlorogenic acid	400	3.2	4.5
	500	2.1	1.1
	600	3.9	3.2
Astragalin	400	1.5	1.9
	500	1.9	1.6
	600	1.8	2.2
Isoquercetin	400	3.5	3.4
	500	3.2	3.1
	600	2.9	2.2

---

Fig. 14 The chemical structures of (a) crypto-chlorogenic acid, (b) astragalin and (c) isoquercetin.

4. Conclusions

Biosynthesized ZnS nanoparticles using the leaves extract of Moringa oleifera were found to be mostly spherical in shape with average diameter ∼30 nm. Negative zeta potential values of 45–55 mV were obtained with isoelectric point which lies between 4–5 and become negatively charged at a pH value of 6. The band gap energy measured from the UV-visible absorption data was found to be 4.16 eV, which signifies the reduction of particle size with widening in the band gap energy compared to bulk value of 3.66 eV. The photoluminescence spectrum of ZnS contains two peaks at 427 and 560 nm ascribed by the presence of sulphur vacancies in the lattice and attributed to the transition from the conduction band to the zinc vacancies respectively. A thin-layer-chromatography densitometric technique revealed the major components present in the extract of M. oleifera leaves and it was observed that biomolecules such as crypto-chlorogenic acid, isoquercetin, and astragalin were present with average amounts of 0.0423, 0.0467, and 0.0634% dry weight, respectively.

Acknowledgements

UKS would like to acknowledge financial support from the projects funded by the UGC, New Delhi (grant no. PSW-045/13-14-ERO) and UGC-DAE CSR, Kolkata centre, Collaborative Research Schemes (UGC-DAE-CSR-KC/CRS/13/RC11/0984/0988). UKS would also like to thank INSA, New Delhi (SP/VF-9/2014-15/273 1^st April, 2014) for INSA visiting Scientist Fellowship for 2014–2015. BA thanks to the Board of College and University Development (BCUD), (BCUD, Finance/2013–14/1776/dated: 20/01/2014) University of Pune for provision of financial support and Advanced Instrumentation Center, Dept. of Chemistry, SPPU, Pune for materials characterization. UKS would like to thank Mr Pulak Das for helping to carry our some experiments and Professor Frank Marken for his valuable suggestions. UKS would like to thank Department of Forestry, Government of West Bengal for full support in carrying out this research work.

References

1. W. L. Davidson, Phys. Rev., 1948, 74, 116–117.
2. W. Q. Peng, G. W. Cong, S. C. Qu and Z. G. Wang, Opt. Mater., 2006, 29, 313–317.
3. X. Fang, T. Zhai, U. K. Gautam, L. Li, L. Wu, Y. Bando and D. Golberg, Prog. Mater. Sci., 2011, 56, 175–287.
4. E. Dutková, P. Baláž, P. Pourghahramani, S. Velumani, J. A. Ascencio and N. G. Kostova, J. Nanosci. Nanotechnol., 2009, 9, 6600–6605.
5. L. Chai, J. Du, S. Xiong, H. Li, Y. Zhu and Y. J. Qian, J. Phys. Chem. C, 2007, 111, 12658–12662.
6. S. S. Manoharan, S. Goyal, M. L. Rao, M. S. Nair and A. Pradhan, Mater. Res. Bull., 2001, 36, 1039–1047.
7. M. V. Limaye, S. Gokhale, S. A. Acharya and S. K. Kulkarni, Nanotechnology, 2008, 19, 415602.
8. M. Sastry, A. Ahmad, I. M. Khan and R. Kumar, Curr. Sci., 2003, 85, 162–170.
9. B. Ankamwar, G. Mandal, U. K. Sur and T. Ganguly, Dig. J. Nanomater. Biostruct., 2012, 7, 599–605.
10. S. Shiv Shankar, A. Rai, B. Ankamwar, A. Singh, A. Ahmad and M. Sastry, Nat. Mater., 2004, 3, 482–488.
11. S. Shivshankar, A. Rai, A. Ahmad and M. Sastry, J. Colloid Interface Sci., 2004, 275, 496–502.
12. B. Ankamwar, M. Gharge and U. K. Sur, Adv. Sci., Eng. Med., 2015, 7, 480–484.
13. B. Ankamwar, M. Gharge and U. K. Sur, Adv. Sci., Eng. Med., 2015, 7, 717–721.
14. B. Ankamwar, M. Chaudhary and M. Sastry, Synth. React. Inorg., Met.-Org., Nano-Met. Chem., 2005, 35, 19–26.
15. B. Ankamwar, U. K. Sur and P. Das, Anal. Methods, 2016, 8, 2335–2340.
16. B. Ankamwar, V. Kamble, U. K. Sur and C. Santra, Appl. Surf. Sci., 2016, 366, 275–283.
17. U. S. Senapati and D. Sarkar, Indian J. Phys., 2014, 88, 557–562.
18. C. Malarkodi and G. Annadurai, Appl. Nanosci., 2013, 3, 389–395.
19. A. R. Verma, M. Vijayakumar, C. S. Mathela and C. V. Rao, Food Chem. Toxicol., 2009, 47, 2196–2201.
20. B. N. Singh, B. R. Singh and R. L. Singh, Food Chem. Toxicol., 2009, 47, 1109–1116.
21. M. Shivashankar and G. Sisodia, Int. J. Life Sci. Biotechnol. Pharma. Res., 2012, 1, 182–185.
22. R. Sathyavathi, M. Bala Murali Krishna and D. Narayana Rao, J. Nanosci. Nanotechnol., 2011, 11, 2031–2035.
23. S. Ummartyotin, N. Bunnak, J. Juntaro, M. Sain and H. Manuspiya, Solid State Sci., 2012, 14, 299–304.
24. H. P. Klung and L. E. Alexander, X-ray Diffraction Procedures for Polycrystalline and Amorphous Materials, Wiley, New York, 2nd edn, 1974.
25. J. P. Borah, J. Barman and K. C. Sarma, Chalcogenide Lett., 2008, 5, 201–208.
26. N. Üzar and M. Arikan, Bull. Mater. Sci., 2011, 34, 287–292.
27. S. Biswas and S. Kar, Nanotechnology, 2008, 19, 045710.
28. D. Xue and K. Kitamura, Solid State Commun., 2002, 122, 537–541.
29. V. Kumar and B. S. R. Sastry, J. Phys. Chem. Solids, 2005, 66, 99–102.
30. D. R. Penn, Phys. Rev., 1962, 128, 2093–2097.
31. G. Murugadoss and M. R. Kumar, Appl. Nanosci., 2014, 4, 67–75.
32. B. S. R. Devi, R. Raveendran and A. V. Vaidyan, Pramana, 2007, 68, 679–687.
33. B. Vongsak, P. Sithisarn and W. Gritsanapan, Planta Med., 2012, 78, 1252–1256.
34. R. N. Bennett, F. A. Mellon and N. Foidl, J. Agric. Food Chem., 2003, 51, 3546–3553.
35. E. Bodoki, R. Oprean, L. Vlase, M. Tamas and R. Sandulescu, J. Pharm. Biomed. Anal., 2005, 37, 971–977.

---