Rice husk-derived mesoporous biogenic silica nanoparticles for gravity chromatography

Abstract

Biogenic silica nanoparticle is a superb alternative to synthetic silica because of their highly active, polar, and porous nanostructure with a large interior area. Among the available agricultural bioresources, biogenic silica extracted from rice husks could be a simple, easily available, and cost-effective resource to use as the stationary phase for the column chromatographic technique. In the present study, highly pure amorphous biogenic silica nanoparticles (bSNPs) were synthesized using rice husk by a controlled combustion route followed by the sol-gel method. The bSNPs show better performance for the separation and isolation of ortho- and para-nitrophenol and nitroaniline. The outstanding performance of the as-synthesized bSNPs is attributed to the high surface area, high porosity, and presence of Si–OH polar bonds. These preliminary findings imply that rice husk, an agricultural waste, could be an alternative source of silica and applicable as a stationary phase in column chromatography.

Highlights

• •Silica extracted from rice husk is employed for the first time as the stationary phase in column chromatography.
• •Biogenic silica nanoparticles (bSNPs) were obtained from rice husk by controlled combustion followed by the sol-gel technique.
• •Highly pure mesoporous bSNPs shows better performance for gravity chromatography due to a high surface area (196.2 m²/g)
• •The presence of Si–OH bonds in RHS₆₀₀ offers a strong tendency toward the polar molecules.

1. Introduction

The rice processing industries produce a huge volume of Rice husk (RH) after the rice grain has been separated from mills as an undesirable agricultural mass residue. The silica concentration in Rice husk ash (RHA) varies with the fluctuation of geographical and environmental factors such as climate, harvesting seasons, soil types, and the amount of fertilizer used during cultivation [1]. The dry rice husk contains 70–85% organic matter (21.44% lignin, 32.24% cellulose, 21.34% hemicellulose), and the inorganic remainder (20–25%) that mainly consists of silica [2,3]. Nepal is an agricultural country where rice is an essential food crop for most of the population and ∼21% of the total agricultural domestic product is rice in Nepal. Most RH in Nepal is being utilized as fuel by small-scale industries generating a significant amount of Rice Husk Ash (RHA), but the high silica-enriched ash content (∼22% of total mass) makes their use problematic during co-firing [4]. The RHA thus formed after co-firing contains more than 90% silica with small fractions of other inorganic oxides like sodium, potassium, iron, and magnesium. The utilization of RHA as a source of silica is of great importance since it has wide applications. The silica is embedded in rice husks as nanoparticles [5]. The silica nanoparticles are concentrated in the protuberances and hairs on the outer and inner epidermis of the husk [6,7]. Because of the huge amount of Rice Husk (RH) as a waste product and the high percentage of silica concentration, extraction of silica from RH would be a cost-effective, value-added material from waste products. RHA represents a potential source for the synthesis of mesoporous silica. Synthesis of silica nanoparticles can be achieved simply by leaching rice husk with acid followed by pyrolysis. Amorphous silica with a purity of above 99% was produced from rice husk by hydrochloric or sulfuric acid leaching followed by controlled combustion at 600 °C for 2 h [8].

Biogenic silica can be obtained via several routes such as hydrothermal [9], biological [10], chemical [11], and thermochemical [12,13] methods. The highly reactive amorphous silica can be produced if RH is burnt under controlled conditions at lower temperatures (773–873 K) [14]. Silica is generally synthesized in two steps, i.e, leaching of the precursor followed by pyrolysis. Kalapathy et al. developed a simple method for the production of pure silica from rice husk ash based on alkaline extraction followed by acid precipitation with minimal mineral contamination [15]. Acid washing before extraction results in a lower concentration of metal impurities. However, the final water washing of the xerogel results in overall lower mineral content. RH-derived silica holds potential applications as an adsorbent for organic dye removal, wastewater treatment, thermal insulation, gas purification, ceramics processing, etc. due to its large interior surface area with highly active, polar, and porous adsorbent site [16]. Although the properties of mesoporous silica depend upon the extent of agglomeration, different characteristics of silica like the pore volume, pore size distribution, area, and surface chemistry can be modified by optimizing the synthesis parameters [17]. Various studies revealed that the silica prepared from the RH possessed properties like polarity, adsorption ability, micro-porosity, etc. [18]. These versatile properties enhanced the potential to use silica as appropriate packing material in column chromatography. From the different chromatographic experiments, it was found that the performance and chemical properties of rice husk silica were completely like those of conventional silica gels therefore RHS would be a promising candidate as stationary phase in gravity chromatography [19]. However, very few studies have been done related to the use of RHS as packing material in column chromatography.

Herein, we have synthesized RHS stationary phase from Rice husk using facile and eco-friendly controlled pyrolysis followed by the sol-gel technique to get better silica for the better performance of gravity column chromatographic separation. Silica is slightly acidic in nature hence it has a strong capacity to soak up basic contents present in the materials to be separated or purified. Due to its better adsorption properties silica gel is used as a stationary phase in gravity column chromatography. The presence of those hydroxyl groups renders the surface of colloids highly polar retains the polar component more strongly and is therefore eluted from the column last whereas the weakly adsorbed non-polar component eluted more quickly. The better surface morphology, active functional group, amorphous structure, large specific surface area, and suitable pore size distribution are the positive aspects of RHS.

2. Materials and methods

2.1. Materials

All the chemicals and reagents were purchased from Sigma Aldrich and are of analytical grade which is used as obtained without further purification. Methylene Blue (C₃₇H₂₇N₃Na₂O₉S₃), Hydrochloric acid (HCl), Sodium Hydroxide (NaOH), Sulfuric acid (H₂SO₄), o-nitrophenol (O₂NC₆H₄OH), p-nitrophenol (O₂NC₆H₄OH), o-nitroaniline (O₂NC₆H₄NH₂), p-nitroaniline (O₂NC₆H₄NH₂), acetone (CH₃COCH₃), n-hexane (C₆H₁₄), and ethyl acetate (CH₃COOC₂H₅), and Whatman Grade 41 filter paper are used. Rice Husk (RH) was sampled from Lamjung district, Gandaki province of Nepal.

2.2. Modification of rice husk (RH) into rice husk silica (RHS)

Rice husk (RH) was collected and washed with distilled water and dried in a hot air oven (Toshniwal, India) at 100 °C for 12 h and treated with 5% HCl to remove impurities for 5 h. The acid-treated RH was then washed and dried in a hot air oven at 100 °C for 6 h and then, the RH are charred and pyrolyzed in a muffle furnace (Vitco India, Sunvic in the U.K.) at temperature 600 °C for 4 h to achieve white ash. The white ash obtained was ground in the grinder (Signoracare, India) and sieved with a sieve of 250 μm. Thus, obtained rice husk ash was named Rice husk silica (RHS).

2.3. Synthesis of silica nanoparticles using the sol-gel method

10 g of washed and dried RHA was transferred in a round bottom flask and soaked with 30% (v/v) H₂SO₄ and 10% (v/v) HCl followed by reflux at 70 °C on a heating mantle fitted for 6 h with continuous stirring to remove metal impurities [20]. The acid-treated solution was then filtered, and the filtrate was discarded. The residue was then washed with distilled water until the pH of the last filtrate was neutral. Then acid-treated rice husk ash residue was treated with 1.0 M NaOH and refluxed at 80 °C on a heating mantle with continuous stirring for 8 h to solubilize silica present in RHS in the form of sodium silicate. The sodium silicate was filtered, and the residue was discarded. The filtrate consisting of sodium silicate was then titrated with 1 N HCl until the solution pH reached 7.0 whereby silica gel was formed. The silica gel thus formed was aged for 20 h and the gel is collected by centrifuging at 5000 rpm for 10 min [21]. The gel was repeatedly washed with distilled water to make it free from NaCl. The gel was then dried at 110 °C for 10 h and stored in a desiccator.

2.4. Characterization

The surface morphologies and elemental composition of RHS were characterized byfield emission scanning electron microscopy (FESEM; Carl Zeiss SUPRA 40VP, Germany) equipped with an energy dispersive spectroscopy (EDS). The multipurpose XRD (X'pert Pro, PANalytical, the Netherlands) with high-intensity monochromatic Cu–Kα radiation as an incident beam (λ = 1.54 Å) over a Bragg's angle ranging from 5 to 90° was used to elucidate the amorphous nature of synthesized silica. Furthermore, the absorption of infrared radiation by extracted RHS was analyzed by Fourier transmission infrared spectrometer (FTIR, FTIR, PerkinElmer, Spectrum GX, USA). The % transmission of the samples was recorded over 400–4000 cm⁻¹. Brunauer, Emmett, and Teller (BET, ASAP 2020, Micromeritics, USA)) measurement with nitrogen N₂ adsorbate was carried out to measure specific surface area and pore size analysis.

2.5. Adsorption study

Methylene blue Number (MB_N) was determined by adsorption of methylene blue by the batch adsorption method, in which the optimized weight of adsorbent (200 mg) RHS was taken in a reagent bottle and dispersed in 50 ml working MB solution followed by continuous shaking using a mechanical shaker at 150 rpm for 3 h to establish equilibrium. The effect of pH (2–8) of MB solution was achieved by, adjusting the pH using 0.01 M HCl and NaOH solutions. Finally, the absorption of MB solutions at a different initial concentration (10 mg⁻¹ – 120 mgL⁻¹) was analyzed before and after adsorption by each solid adsorbent and calculated MB_N by using Equation (1):

$$\mathrm{M}{\mathrm{B}}_{\mathrm{N}}=\frac{\left({\mathrm{C}}_{\mathrm{o}}-{\mathrm{C}}_{\mathrm{e}}\right)\times \mathrm{V}}{\mathrm{W}}\mathrm{m}\mathrm{g}/\mathrm{g}$$ (1)

where C_o and C_e are the initial and the equilibrium final concentration of the methylene blue in mg/L respectively, W is the weight of the adsorbent taken in g and V is the volume of the solution taken in L. The concentration of methylene blue was determined by spectrophotometric method using standard calibration curve.

2.6. RHS as stationary phase in column chromatography

To determine the suitable solvent system for the separation of the desired organic compound, a pre-coated TLC plate was taken and spotted with the organic compound. TLC was run with three different solvents i.e., acetone, hexane, and ethyl acetate as the mobile phase separately. The hexane and ethyl acetate were found to be better solvent systems. Different solvent systems with varying ratio of hexane, and ethyl acetate were then prepared. The retention factor (R_f) values of each system were determined. A solvent containing 60% hexane and 40% ethyl acetate was selected as a mobile phase for the separation of a mixture of o-nitroaniline and p-nitroaniline in column chromatography. A glass column with 1 cm diameter and 50 cm (1cm × 50 cm) height was taken and glass wool was inserted towards the nozzle of the column. Enough Rice Husk Silica obtained by combustion at 600 °C (RHS₆₀₀) was mixed in hexane in a beaker and the slurry was poured into the column up to a height of 41 cm. Hexane was allowed to run through the silica column until it settled down. When the column is perfectly packed, cotton plug is inserted at the top of the silica. 2 ml of mixture of o-nitroaniline and p-nitroaniline was loaded with the help of a pipette. 5 ml fraction of effluent was collected in a different labeled test tube from the column. The fractions collected were then subjected to TLC chromatography to determine the true purity of the products and the success of the column chromatography separation. Similarly, the process was repeated for the separation of a mixture of o-nitro phenol and p-nitro phenol using the solvent mixture of 85% hexane and 15% ethyl acetate. Parallelly, a column chromatography was run using commercial silica to compare the performance of RHS with the commercial silica (CS).

3. Results and discussion

3.1. Extraction, TGA, and morphological study of rice husk silica (RHS)

As displayed in Fig. S1, on treatment of rice husk with HCl, the color of the rice husk was changed from light brown to light yellow. Acid treatment of rice husk removes the metallic impurities present in the sample [4]. On heating, the rice husk in an open atmosphere, the volatile matter present in the rice husk was removed and the organic matter present in the rice husk was partially oxidized. The cellulose, hemicellulose, and lignin were decomposed at temperatures above 400 ^°C [22,23] and most of the pyrolysis products are driven away. The silica obtained at 400 and 500 ^°C was gray in color which is obviously due to the presence of incompletely burned carbon as impurities.

The silica obtained at 600 ^°C was white in color and was in fine powder form. Finally, shiny white powder of highly pure silica was obtained via sol-gel method and used as stationary phase in column chromatography. The Schematic diagram for synthesis and used of rice husk-based silica in gravity chromatography was shown in Fig. 1.

Fig. 1.

Schematic diagram for synthesis and application of rice husk-based silica in gravity chromatography.

Rice husk is composed of inorganic and organic materials which behave differently at high temperatures. The removal of organic components was necessary for transformation of RH into RHS to extract the silica. The weight changes that occurred during rice combustion are depicted by TGA curve in Fig. 2a which clearly displayed the mass changes. The inset Fig. 2a shows the corresponding DTGA curve with three stages of weight loss with two major weight loss at around 325.53 °C and 347.39 °C. The elimination of physically absorbed water by the porosity of rice husk; external water bounded by surface tension and light volatiles causes the first stage of weight loss of about 5.23% at 23–172 °C. The organic constituents started to decompose from 245 °C, the rapid weight loss of 42.32% at 245–332 °C. was a result of the decomposition of volatiles as carbon, oxygen, and hydrogen which are removed from the organic component during the decomposition of cellulose and hemicellulose; condensable vapors, and incondensable gas are released at this point. Subsequent weight loss of about 32.44% at 332–580 °C was mainly due to the decomposition of cellulose. The carbon backbone and Lignin decomposed and oxidized to carbon dioxide slowly over a wide range of temperatures up to 580 °C [7]. Finally, after 580 °C, there was no remarkable change in the TGA curves ensuring the complete elimination of organic components and the residual ash corresponded to 19.97% of the total weight which principally consists of silica SiO₂. Therefore, a calcination temperature of 600 °C was selected to produce the RHS. DTA curve (Fig. 2b) of the RH shows two endothermic peaks around 56.17 °C and 361.01 °C resulting in the distortion of water molecules and evaporation of residual moisture from the surface RH. The exothermic peak around 260.45 °C corresponds to the decomposition process of the residual organic materials. No further exothermic peak found in the DTA results confirms the complete decomposition of organic components and strong thermal stability of residual silica. The percentage of silica obtained is about 20.3% (Fig. S2) which is what we expect from thermogravimetric analysis [23]. The silica obtained by pyrolysis of RHA at 600 ^°C is further purified by the sol-gel method. The silica obtained by the sol-gel method is pure white. About 86.52% (of RHS) pure silica is obtained after the sol-gel purification process.

Fig. 2.

(a)TGA curve obtained by heating the rice husk at 10 °C/min in air atmosphere (inset figure shows the corresponding DTGA curve) and (b) simultaneous DTA curve for rice husk.

FESEM images of (a and b) Rice Husk (RH), (c and d) Charred Rice Husk, (e and f) Rice Husk Silica obtained at 600°C, and (g and h) Pure Rice Husk Silica obtained by sol gel method, at different magnification was shown in Fig. 3(a–h). The images were taken using a scanning electron microscope (FESEM; Carl Zeiss SUPRA 40VP, Germany) operated at an accelerating voltage of 2 kV. The FE-SEM images of rice husk exhibit uniquely bump-like arrays with rough and undulating surface texture. As presented in Fig. 3a the size of the bump is around 25 μM in which the silica nanoparticles are primarily present at the proboscis and tip of the RH (Fig. 3b). A significant and marked change in morphology can be observed after acid pretreatment of rice husk (Fig. 3c and d), because of the hydrolysis of the organic components [24]. After acid treatment silica particles are heterogeneous in shape with approximately about ∼50–60 nm. After the pyrolysis of RH at 600 °C for 4 h (Fig. 3e and f) the bulk like arrays of RH did not remarkably change, but the bump's size became smaller and broken into numberless pieces. As shown in Fig. 3g shows the mesoporous nature of thus obtained silica (RHS₆₀₀) and high resolution FESEM image (Fig. 3h) showed the mesoporous size distribution to be 10–30 nm. Such mesoporous structure with a lot of pores is highly distributed throughout the rice husk which is supposed to be an adsorption site for the absorption. More porosity of adsorbents causes more removal efficiency because of increased numbers of pores. Furthermore, the EDS (Fig. S3) confirms that RHS predominantly consist of Si and O with 62.00% of O and 38.00% of Si. According to the above results, pure SiO₂ fragments (RHS) with naturally nano porous configuration were formed during the controlled pyrolysis and acid leaching treatment process.

Fig. 3.

SEM images of (a and b) Rice Husk (RH), (c and d) Charred Rice Husk, (e and f) Rice Husk Silica obtained at 600 °C, and (g and h) Rice Husk Silica (RHS₆₀₀) obtained by sol gel method.

3.2. Spectroscopic, crystallography, and surface area characterization

Fourier-transform infrared spectroscopy analysis at wavenumber 4000–400 cm⁻¹ was carried out to examine the functional group of the silica extracted from RHS and the results was shown in Fig. 4a. Three characteristics bands centered at 1080.76 cm⁻¹, 794.23 cm⁻¹, 452.13 cm⁻¹ indicate the Si–O–Si structural framework of the siloxane, which are attributed to the asymmetric stretching vibration, symmetric stretching vibration, and bending vibration of the Si–O–Si bond, respectively. The broad band centered at around peak appears from 3450 cm⁻¹ region is attributed to hydroxyl (-OH) group of the silanol and adsorbed water molecules on the surface of silica [25]. All these peaks confirm the presence of pure silica in the isolated particle extracted from RH. The XRD pattern of the silica obtained at 600 °C was shown in Fig. 4b. A broad peak centered on 2θ of 22–24° without any sharp peak was observed clearly confirms the amorphous nature of silica [[26], [27], [28]].

Fig. 4.

(a) FT-IR spectra of RHS₆₀₀ (b) XRD of RHS₆₀₀, (c) N₂ adsorption-desorption isotherm of RHS₆₀₀, and (d) Pore size distribution of RHS₆₀₀.

The adsorption behavior of the rice husk ash was studied using Methylene Blue (MB) adsorption. In chromatography, porosity increases the surface area available for analyte interaction with the silica surface [29]. In order to measure the surface area, pore volume, and pore diameter of the RHS_600, we performed a BET measurement, with Nitrogen N₂ as adsorbent, the RHA₆₀₀ obtained after sol-gel method using RHS obtained at 600 °C by refluxed with 0.1 M sodium hydroxide followed by acidification using hydrochloric acid possess a high specific surface area and pore volume of 196.2 m²/g and 0.67 cm³/g, respectively. The high surface area of rice husk silica is due to the removal of organic matter and impurities during the reflux process with sodium hydroxide. Fig. 4c depicts the adsorption behaviors of RHS, the upper portion of loop is known as desorption, while the lower portion is known as adsorption. As shown in Fig. 4d the RHS₆₀₀ possesses pore diameter of about 14.4–25.6 nm, which confirms the mesoporous nature of silica nanoparticles [30,31].

Adsorption of Methylene blue (MB).

The adsorption behavior of the rice husk was studied using Methylene Blue (MB) adsorption. Fig. 5a shows the effect of contact time on the adsorption of methylene blue. At higher concentrations, lower adsorption is observed due to the saturation of the adsorption sites [32,33]. The MB adsorption is pH dependent, and the removal of MB was primarily determined by pH of the solution. Fig. 5b depicts the effect of pH for adsorption of MB dye by RHS. The percentage removal of methylene blue is quite high and remains constant in the pH range from pH 4 to pH 7. The above results demonstrate that the amorphous RHS obtained from RH is similar to commercial silica.

Fig. 5.

(a) Effect of contact time on the initial concentration (b) Effect of pH for adsorption of MB dye by Silica Gel.

RHS as stationary phase in gravity column chromatography.

The chemical properties of RHS were identical to those of conventional silica gel used in column chromatography. We investigated the separation of o-nitroaniline and p-nitroaniline using rice husk silica gel (RHS) as the packing material for column chromatography. Mixture containing, o-nitrophenol and p-nitrophenol was analyzed following same procedure. The proper selection of the solvent system is mandatory to separate components of organic mixture prior to run gravity chromatography [34]. For the selection of a solvent system for the separation of o-and p-nitrophenol, Thin Layer Chromatography (TLC) trials with three solvent systems i.e., acetone, hexane, and ethyl acetate were carried out. The results are shown in Table 1 and photographs of TLC plates are shown in Fig. S4. The only single spot was observed when hexane and acetone were used as mobile phases. Two distinct spots with different R_f values were observed with ethyl acetate. The results clearly indicate that a polar solvent is required for the separation of o-nitroaniline and p-nitroaniline. The acetone resulted in a single spot with a large R_f value, but the two compounds are not separated. So, acetone is not polar enough to separate the components. In pure ethyl acetate as a mobile phase, the separation of o-nitroaniline and p-nitroaniline was achieved. The R_f values are very close to each other and there is the smearing of the separated spots. So, a mixture of ethyl acetate and hexane in different volume ratios were tried for better separation of the components. The results were obtained by taking different percentage ratios of ethyl acetate and hexane as eluents for the separation of o-nitroaniline and p-nitroaniline as well as o-nitrophenol and p-nitrophenol were given in Table S1. The ratio of 60% hexane and 40% ethyl acetate was found to give the best results with the R_f values 0.75 and 0.26 respectively for o-nitroaniline and p-nitroaniline. Similarly, the ratio of 80% hexane and 20% ethyl acetate was found to give the best results with the R_f values 0.66 and 0.29 respectively for o-nitrophenol and p-nitrophenol (Table S2). On the basis of presence of highly electron donating alcohol group and highly electron withdrawing nitro group in ortho and para position, they exhibit different R_f values. The ortho product had the highest R_f value due to intramolecular hydrogen bonding between the alcohol and nitro group. The para product is more polar than ortho product because of ability to participate in intermolecular hydrogen bonding on the alcohol and nitro groups, therefore, eluted slowly with less R_f values.

Table 1.

R_f values of mixture of o-nitroaniline and p-nitroaniline in different solvents.

Trials	Mobile Phase	No. of spots and color	Rf values
1	Hexane	1, Orange	0
2	Acetone	1, Orange	0.69
3	Ethyl acetate	1, Orange	0.63
		1, yellow	0.58

Furthermore, the presence of -OH and -NO₂ groups in complimentary para position decreases the dissociation constant p-nitrophenol as with increase in the percentage of organic co-solvents [35]. More polar or ionogenic forms of para compounds have relatively high affinity to the polar stationary phase, and hence, the polar form of analyte has a higher retention as compared to the retention of the less polar form of the same compound, which is more soluble in organic phase.

Hence the solvent ratio of 80% hexane and 20% ethyl acetate was chosen as effluent in gravity chromatography for the separation of the mixture of o-nitrophenol and p-nitrophenol. For this, a mixture of ortho and para nitrophenols was run through a column chromatography. Two distinct yellow bands were observed, as expected (Fig. S5). Each band represented one of the nitrophenols, with the bottom band representing the ortho-nitrophenol because it is less polar and stayed within the solvent, allowing it to move quickly through the column. The para product remained at the top because it was polar like silica and could hydrogen bond to it. Once the bands were formed 12 different fractions from the yellow bands were collected in different test tube. The formed fractions were then subjected to TLC chromatography to determine the true purity of the products and the success of the column chromatography separation.

The R_f values of the components observed from fraction 1 to 12 for mixtures of organic compounds are tabulated in Table 2. In the case of a mixture of o- and p-nitroaniline (Fig. 6a), the first five fractions (1–5) and last five (8–12) resulted in a single spot when eluent is analyzed by TLC, whereas the first four fractions (1–4) and fractions (7–10) resulted in a single spot in case of CS (Fig. 6b). Fig. 6c shows the bar diagram for corresponding R_f values obtained from TLC for both RHS and CS. When H is bonded to an O, it forms Si–OH, which is one possibility for forming a Si–O:H bonds in a random fashion. This explains how the strongly stretching bonds of the H–O–H and OH were observed in the RHS film, as shown by the FTIR spectra in Fig. 4a. Thus, the nano porous RHS contains highly dense Si–O–Si and OH bonds, which are expected to repel electrons from their surface [36]. The intramolecular hydrogen bonding between the amino and nitro clusters in o-nitroaniline makes it less polar and interaction between the o-nitroaniline with the silica in the column is weak compared to that of p-nitroaniline and hence elutes the first. Conversely, in p-nitroaniline, intramolecular hydrogen bonding is not possible owing to the greater separation of nitro and amino groups. These results make p-nitroaniline more polar, therefore, when the mixture of o- and p-nitroaniline passes through the column, the NH₃ group of p-nitroaniline strongly interact with the polar group of RHS and makes strong H- bond with O–Si–O compared to that of o-nitroaniline and eluent at a later stage due to a higher percentage of non-polar hexane in the solvent mixture used as a mobile phase.

Table 2.

TLC assessment of eluents from column chromatography for the separation of mixture o- and p-nitrophenol & o- and p-nitroaniline (*Y=Yellow, *O=Orange, *LY = Light Orange, and *N= No spot).

Fractions	No. of spots on sample and color (Rf value)
	o-and p-nitrophenol		o- and p-nitroaniline
	RHS	CS	RHS	CS
1–4	1-Y (0.76)	1-Y (0.78)	1-O (0.72)	1-O (0.74)
5	1-Y (0.78)	N	1-O (0.72)	N
6	N	N	N	N
7	N	1-LY (0.32)	N	N
8	N	1-LY (0.32)	N	1-Y (0.39)
9–10	1-LY (0.28)	1-LY (0.32)	1-Y (0.35)	1-Y (0.39)
11–12	1-LY (0.29)	N	1-Y (0.35)	N

Fig. 6.

TLC of eluents (o- and p-nitrophenol) from column chromatography, using RHS (a) and commercial silica (b), (c) Bar diagram showing the corresponding R_f values for a mixture of o- and p-nitrophenol, TLC of eluents (o- and p-nitroaniline) from column chromatography using RHS (d) and commercial silica (e), and (f) Corresponding Rf values for a mixture of o- and p-nitroaniline.

Two clear bands of o-and p-nitroaniline were clearly observed in the column, from which we can say that the components in the mixture were completely separated in both RHS and CS. Similarly, in the case of a mixture of o- and p-nitrophenol (Fig. 6d), the first five fractions (1–5) and last four (9–12) resulted in a single spot when eluent is analyzed by TLC, whereas the first four fractions (1–4) and fractions (7–10) resulted in a single spot in case of CS (Fig. 6e). Fig. 6f shows the bar diagram for corresponding R_f values obtained for different fraction of eluent while separating the mixture of o- and p-nitroaniline using TLC for both RHS and CS. As in case of o-nitroaniline, the intramolecular hydrogen bonding between the nitro and hydroxyl clusters in o-nitrophenol makes it a less polar molecule. Thus, the formed bond reduces the interaction between the o-nitrophenol with the silicon dioxide within the glass column. Due to this, o-nitrophenol was eluted faster during column chromatographic separation. Conversely, in p-nitrophenol, the hydroxyl and nitro groups remain opposite to each other causing more polarity.

The polar molecule thus interacts strongly with silicon dioxide thereby causing slower elution. The time taken for separation gravity chromatography is about 2 h using RHS. The small particle size and high porosity of RHS result in stronger interaction between the RHS and mobile phase. This causes slower elution of the mobile phase. So longer time is required for proper separation of the organic compounds. However, the separation of the organic compounds by the RHS was satisfactory as compared to the CS, hence RHS would be better alternative to the stationary phase in gravity chromatography instead of commercial silica.

4. Conclusions

Rice husk-derived mesoporous silica was prepared from agricultural byproduct rice husk by hydrochloric acid leaching followed by pyrolysis at 600 °C. Highly pure amorphous silica was obtained by sol gel method that show XRD 2θ broad peak centered around 22-24° without any other sharp peak. Rice husk silica contained about 86.52% of amorphous silica nano particles of heterogeneous shape with particle size of about 50–60 nm with mesopores of size of 10–30 nm as revealed by FESEM. Fourier-transform infrared spectra showed a characteristic bands indicating the presence of Si–O–Si structural frame work of the siloxane and broad band at 3450 cm⁻¹ confirming the (-OH) group of the silanol and absorbed water molecules on the surface of silica. The mesoporous amorphous silica so obtained was successfully used as the stationary phase in gravity column chromatography. The results demonstrated the feasibility of environment-friendly extraction of mesoporous amorphous silica from RH with high adsorption capacity. The eluent of composition 80% hexane and 20% ethyl acetate was found to be most efficient for the separation of ortho and para -nitroanilines as well as ortho and para nitrophenols. This illustrated the conversion of waste material to a valuable product as an effective gravity chromatography stationary phase. Easy and cost effective extraction of silica from RH can minimize the laboratory expenses in developing nations.

Author contribution statement

Devendra Shrestha: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Tulsi Nayaju: Performed the experiments.

Mani Ram Kandel: Analyzed and interpreted the data.

Raja Ram Pradhananga: Conceived and designed the experiments.

Chan Hee Park, Cheol Sang Kim: Contributed reagents, materials, analysis tools or data.

Data availability statement

Data will be made available on request.

Declaration of interest's statement

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: