TITLE: Preparation of Nano particles from corn cobs.
FIELD OF INVENTION:
This invention relates to a process for the preparation of Nano particles from corn cobs.
BACKGROUND OF THE INVENTION:
Nanotechnology has rapidly become an interdisciplinary field and one exciting research area is the synthesis of cellulose-based nanoparticles (CNPs) from bioresources, especially from annual plants and agriculture crop residues such as corn cobs, corn grain, wheat stems, seed coats, and sugar cane stalks using top-down technologies. Corn cobs, central part of maize (Zea mays) are either thrown out as waste or burnt, an application with low added value, causing environmental impact. So it is an exciting research area to use corn cob for chemical processing to obtain end products with added values worldwide at very low price (Kumar et.al. 2010). Cellulose-based nanoparticles (CNPs) are expected to have a great prospective because cellulose materials can self-assemble into well-defined architectures in multiple scales, from micro to nanosize. Moreover, cellulose is not only renewable but also a multifunctional raw material and is expected to be able to replace many non-renewable materials (Wegner and Jones 2006). An important characteristic of cellulose-based nanoparticles is its ability to remain intact in the physiological stomach environment and small intestine. This property, together with the presence of specific enzymes produced by cellulytic bacteria (ruminococus identified by KOPECNY et. al 2004: KAREN et. al. 1988) for colon biodegradability, makes this polymer a suitable raw material for the medical field, particularly as a colon-specific drug carrier (Ratna et.al 2010).
The application of natural polysaccharides like cellulose, most abundant in nature and hemicellulose (mainly xylan), second most abundant in nature are increasing in pharmaceutical industries day by day. Agro waste (like corn cob) or other polysaccharides are extensively used to prepare the nanoparticles in narrow size distribution by various methods/techniques by researchers. Buckeve cellulose (Zhang et al. 2007) contain 100% of α-cellulose, was used to synthesized cellulose nanospheres with sizes ranging from 60 to over 570 nm. whereas in our investigations we observed that corn cob-based nanoparticles (treated with strong alkali) contain only around 72 % a-cellulose. However the difference has been reported in a-cellulose content owing to the sorption of xylan (McKinney 1946; Clayton and Phelps 1965). These nanoparticles can improve the pharmacokinetic properties of drugs and thereby reduce toxic side effects. In the recent past nanoscale particles of corn cob xylan have been reported, ranging from 120 to 1790 nm (Garcia et al. 2001). Owing to their controlled release properties and biodegradability, corn cob-based nanoparticles can exhibit the desired fine-tuned drug delivery characteristics and achieve a broad range of usefulness and minimal toxicity of powerful drugs. In view of the growing demand and modernisation of pharmaceutical industries, nanoparticles of reduced dimension can bring substantial changes in therapeutic systems by developing improved pharmaceutical grade biopolymer. Therefore, the objective of present study is focused on evaluating the use of corn cobs to prepare nanoparticles in narrow size distribution (~< 30nm size) with an aim to explore possible pharmaceutical applications.
OBJECTS OF THE INVENTION:
An object of this invention is to propose a process for the preparation of Nano particles from corn cobs;
Another object of this invention is to propose a process for the preparation of nanoparticles by chemical treatment;
Further object of this invention is to propose a process for preparing Nano particles to be as nanometric carriers to deliver drug or biomolecules.
DETAILED DESCRPTION OF THE INVENTION:
Locally collected corn cob sample was milled into powder in a laboratory Wiley mill, and fractions passing through 40 mesh (400um) screens but retained on 80 mesh (177pm) screens was collected, air-dried, homogenized in a single lot to avoid compositional differences among aliquots, and stored for compositional analysis and xylan extraction.
The other lot of corn cob sample was chopped manually into 1.5 to 2.0 cm long and 0.25 to 0.5 cm thick pieces. This sample was also air-dried, homogenized in a single lot to avoid compositional differences among aliquots, and stored for chemical analysis.
The compositional data of the corn cob samples used as a raw material (as the average of four replicate analyses) is shown in Table 1. The results are expressed as weight percent of holocellulose (α-, ß- and - Cellulose), pentosans, Klason lignin, ethanol-benzene solubles, hot water solubles and ashes by using TAPPI Standard Test Methods T 249 cm-00 (T 203 cm-99), T 223 cm-01, T 222 om-06, T 204 cm-07, T 207 cm-08, and T 211 om-85, respectively. The other fractions, including uronic acids, acetyl groups, etc., were not determined, owing to their minor importance for the purpose of this work.
Table 1. Compositions of Corn Cob (as the average of four replicate determinations)*
(Table Removed)
Concerning, specifically, the holocellulose from corn cobs, it has been demonstrated that such polymer presents a chemical composition of α-, ß-, and -cellulose in the proportion of 5.2:2.8:3.0 respectively. By this investigation it has been demonstrated that corn cobs contain a considerable amount of a-cellulose (47.27%) along with ß-cellulose, comprised as degraded cellulose (25.46%) and Y-cellulose, consists mainly of hemicelluloses (27.27%) is shown in Table 2.
Table 2. Chemical Composition of Holocellulose, oven dry basis
(Table Removed)
Alkaline Treatment of Raw Material
The objective of chemical treatment is to degrade and dissolve away the lignin and leave behind most of the cellulose and hemicellulose in the form of fiber or particles. Chopped Corn cobs were digested in Weverk electically heated rotatory digester of 0.02 m3/20 liter capacity having four bombs of 1 liter capacity each. In this process penetration of liquor occurs in two ways:
(1) By capillary movement along the lumina, pits.resin ducts etc.
(2) By diffusion through the cell walls.
Non-swelling liquids/ low swelling liquids penetrates 50 to 200 times more quickly in the longitudinal direction (i.e. by capillary action) than in the transverse direction (i.e. by diffusion). But swelling agent like NaOH, these differences are much less and it is only six times more in longitudinal direction than in transverse direction therefore, chip thickness is relatively more important. The chips of corn cobs were treated with sodium hydroxide in the range 0 (i.e. autohydrolysis) to 24% of sodium hydroxide on oven dry basis, at a temperature 165°C for 1.5 h at liquor to solid ratio of 4.5:1. After completion of the treatment, the samples were washed on a 270 mesh (53um) screen for the removal of residual chemicals. The samples were disintegrated and screened through 80 mesh screens (177µm), and the screened product was washed, pressed, and crumbled. The samples were analyzed for pentosans (T 223 cm-01), screening rejects, yield and lignin (T 22 om-88) by weight as per TAPPI Standard Test Methods (2007). The results are shown in Table 3.
Table 3. Effect of Chemical Treatment on Residual Lignin, Pentosan, Screened Yield, Rejects and Total Yield (results expressed as weight percent, oven dry basis of raw material* and screened yield*)
(Table Removed)
Effect of Autohydrolysis on Pentosans Content
Figure 1 shows that the percent of pentosans (7.49%) was less in auto-hydrolyzed corn cob samples than that of the raw material (28.23%). Since the hemicellulose fraction of corn cobs had a relatively high content of acetylated xylan (a polymer made up of xylose units with acetyl substituent), treated with water at 165°C, the hydronium ions (H3O+) generated from the water, i.e. auto-ionization, caused both xylan depolymerisation (to give xylooligomers and xylose) and cleavage of acetyl groups (to give acetic acid, which in turn increases the hydronium concentration in the reaction medium) (Garrote et al. 2002), leading to liquors with pH in the range 3-4 after treatment (Heitz et al. 1986). Thus in acidic environment, selective solubilisation of hemicelluloses and extractives takes place, which results, due to autohydrolysis, in less pentosans content than in the raw material and a solid phase enriched in cellulose and lignin.
Effect of Autohydrolysis on Lignin Content
Figure 1 also shows that the percent of lignin (13.21%) in an auto-hydrolyzed sample was slightly lower than in the raw material (14.01%) because of carbonium ion initiated repolymerisation takes place more or less simultaneously with lignin depolymerisation reaction (Jiebing and Goran 2008). An increase in hydronium ion concentrations (a large reduction in pH) during the auto-hydrolysis process (Garrote et al. 2002), and a large increase in phenolic hydroxyl groups due to degradation of lignin lead to the formation of carbonium ions from the benzylic position (Jiebing and Goran 2008).
In the presence of other electron-rich carbon atoms such as the C-2/C-6 present in guaiacyl and syringyl rings, condensation reactions may, however, compete with the acidolysis, leading to repolymerisation (Lora and Wayman 1979; Robert et al. 1986). The presence of repolymerised/condensed structures is also indicated by the colour change from almost colourless to brownish-yellow (Jiebing and Goran 2008). On the basis of the above explanation it was confirmed that lignin removal becomes almost negligible, whereas hemicelluloses and extractives removal becomes very large, leading to an approximately the equal percentage (as oven dry basis of raw material) of lignin during autohydrolysis.
Effect of Alkali Charge on Pentosans Content
From Fig. 1 it is observed that percent of pentosans increased first sharply with increase in alkali concentration from 0 (i.e. autohydrolysis) to 4% (owing to hinderance of the solubilisation of hemicelluloses), then remain almost practically constant up to 18%, and afterwards it decreased. The resistance of the residual pentosans in alkaline treatment is explained by McKinney (1946) by stating that
some of the wood hemicelluloses were originally chemically bound to lignin by a glycoside linkage and that during alkaline treatment a trans-glycosidation reaction takes place in which the hemicellulose is transferred to form a glycosidic link with the cellulose. Clayton and Phelps have also explained that the chemical bonds are formed between the cellulose and the sorbed hemicellulose because the activation energies for the sorption of both galactoglucomanan and xylan with a low uronic acid content was found to be less than 10 Kcal/mol (Clayton and Phelps 1965). But after 18% of sodium hydroxide the pentosans content decreased sharply, owing to hemicellulose degradation at the higher concentration of alkali. This explains the greater retention of pentosans when treatment was carried out in the presence of reagents that stabilize the polysaccharide against alkaline degradation. Hence 18% of sodium hydroxide concentration may be considered as an optimum dose of alkali for further processing.
Effect of Alkali Charge on Lignin Content
The objective of alkaline treatment is to degrade and dissolve away the lignin and leave behind most of the cellulose and hemicellulose in the form of particles. According to Jiebing and Goran (2008) sodium hydroxide suppresses the repolymerisation reactions by inhibiting the acid catalyzed reactions. On increasing the concentration of sodium hydroxide, the hydronium ions will be neutralised, and the medium shifts from acidic to alkaline. Hence, carbonium ion formation becomes less, and lignin depolymerisation dominates over lignin repolymerisation. From Fig. 1 it is seen that the percentage of lignin decreased sharply as the concentration of sodium hydroxide increased up to 18%, and afterward the percentage of lignin became almost constant. Therefore, on the basis of the above studies, 18% of sodium hydroxide concentration may be considered as the optimum level of alkali.
Effect of Alkali Charge on Yield
Figure 2 shows that the screened yield first decreased and reached a minimum (19.7%) and then increased up to a maximum (45.41%) and again decreased. Auto-hydrolysis of the raw material provided the hydronium ions to bring about the acidic condition. But alkali charge was given to the raw material, consuming the hydronium ions, leading to neutralization. Hence, screened yield was greater during auto-hydrolysis in comparison to treatment given at 2 to 6% of sodium hydroxide concentration. After that, the screened yield increased and rejects decreased with increased alkali charge, and the maximum yield was found at 18% of sodium hydroxide concentration. Therefore, an alkali dose of 18% sodium hydroxide concentration may again be considered as the optimum dose of alkali.
Figure 2 also shows that the rejects increased as sodium hydroxide concentration increased from 0 to 4% (owing to neutralization of medium) and then decreased continuously owing to alkaline treatment.
On the basis of the above studies, the process conditions were optimized to obtain maximum yield and minimum chemical consumption. Based on experimental studies an alkali dose of 18% of NaOH concentration was found to be optimum for chemical treatment of corn cobs raw material. The samples obtained at the optimized condition were delignified with acidified sodium chlorite solutions and dried in vacuum oven. The obtained powder after delignification and drying was further screened from 270 (53µm) mesh size for the determination of α-, ß-, and -cellulose by using TAPPI Standard Test Methods T 203 cm-99. The results are shown in Table 4.

Table 4. Chemical Composition of Alkali Treated Holocellulose, oven dry basis:
(Table Removed)
It may be concluded from Table 4 that almost all v-cellulose is removed and solid residue enriched in α-cellulose (72.75%) and ß-cellulose (25.19%) only. Powders thus obtained were further characterized by X-ray Diffraction (XRD), Fourier Transform Infrared (FT-IR), Scanning Electron Microscopy (SEM) and High-resolution Transmission Electron Microscopy (TEM) technique.
Characterisation of Nanoparticles
FT-IR spectroscopy was performed using a Nicolet spectrophotometer. Samples were oven dried at 105°C for 4 h, mixed with KBr in a ratio of 1:200 mg (corn cob-basednanoparticles: KBr) and pressed under vacuum to form pellets.
X-ray diffraction (XRD) of samples was recorded on a Bruker AXS D8 Advance diffractrometer with a scanning rate of 10 C/min with CuKa radiation source (A = 1.54060 A) operating at 40 kV and 30 mA. For that purposes finely powdered sample i.e. nanoparticles (NPs) placed in the central cavity of sample holder made up of polymethyl methacrylate (PMMA) were used for X-ray diffraction studies.
High-resolution Transmission Electron Microscopy (TEM) was carried out with an FEI Technai G2 F20 microscope at 200 kV; the samples were air dried before using the TEM to characterise the size and morphology of the dried particles. For TEM observation, the samples were prepared in methanol at 100 µg/mL concentration and dispersed in an ultrasonicator for ten minutes. The samples for TEM analysis were obtained by placing a drop of the colloidal
dispersion containing the corncob nanoparticles onto the carbon-coated copper grid. They were dried at room temperature and then examined using the TEM without any further modification. The particle sizes in morphology were measured using a scale bar in micrographs.
Scanning Electron Microscopy (SEM) was performed, using a. FEI Quanta 200 F microscope. The samples were mounted on Au coated stubs and observed at 20 kV.
Evidence for the preparation of corn cob cellulose-based nanoparticles
(CNPs)
Fourier Transform Infrared (FT-IR)
FT-IR spectroscopy is a powerful tool for studying the physico-chemical and conformational properties of polysaccharides. In addition to X-ray and electron diffraction studies, FT-IR spectroscopy makes it possible, in particular, to solve the problems of identification of polysaccharides, to confirm their purity, to carry out semi-quantitative functional analyses, to determine structure, and to investigate complexing and intermolecular interactions (Sun et al., 1998). The main peak of FT-IR spectra of the delignified raw material, holocellulose and alkali treated corn cob nanoparticles are shown in Fig. 3. The analysis of FT-IR data shows that the holocellulose has most significant absorption peak at 1734 cm-1 relates to C-O stretching of carbonyl group which was disappeared in alkali treated corn cob nanoparticles. The disappearance of peak in nanoparticles indicates that the acetyl group of acetylated xylan (i.e. hemicelluloses) is removed almost completely by alkali treatment (18% NaOH, on oven dry basis) at elevated temperature (165°C). A broad absorption band in the range of 3420-3424 cm-1 that can be attributed to the -OH stretching associated to polar groups
linked through intra- and intermolecular hydrogen bonding (Sun et al 2005b) and the symmetric C-H stretching vibration band is found at 2900- 2913 cm-1 owing to CH2 and CH3 group (Ren et al 2008). Furthermore, the band in the range 1160-1167 cm-1 is characteristic of glycosidic groups and attributed to C-O, C-O-C stretching and C-OH bending vibration in arabinoxylan structure (Sun et al., 1998). Also, there is an increase in the intensity of C—O—C ester stretching band at 1167 cm"1 as a result of the esterification. The IR band shifted to higher frequency (1167 cm"1) for nanoparticles (mainly cellulose) confirm that crystalline content increases in comparison to holocellulose (Colom et al 2003). In addition, a sharp absorption peak appeared at 1115 cm-1 in CNPs is further indicative of high cellulosic content. A sharp band at 1638-1642 cm-1 was also detected and attributed to H-O-H stretching, which occurs mainly in the amorphous state, and crystalline spectra measured in KBr which belongs to the absorbed water molecules associated with the cellulosic fibers (Oliveira et al, 2010). The band at 1430 cm-1, assigned to H-CH and -OCH in-plane bending vibrations in both holocellulose and CNPs (Satyamurthy et al, 2010). In addition, an absorption band near 1375 cm-1 is detected owing to the C-H bending vibration present in cellulose and hemicellulose chemical structures (Sun et al 1998). The prominent peak at 1046-1036 cm-1is attributed to the C-C, C-0 stretching vibration and C-OH bending vibration [(Oliveira et al, 2010] in both holocellulose and CNPs. Finally, a sharp band at 899 cm-1, which is typical of ßglycosidic linkages between the sugar units in hemicelluloses, was detected in the anomeric region [34] but this band is shifted to 896 cm-1in CNPs with increased intensity, may further be suggestive of increased crystallinity. These peaks match well with spectra of cellulose.
Although measurements of crystallinity index (CI) have a long history, it has been found that CI varies significantly depending on the choice of measurement method.
(Table Removed)
The crystallinity was found to increase owing to smaller particle size and removal of amorphous substances such as lignin, hemicelluloses, and extractives. The XRD pattern of corn cob-based nanoparticles showed several relatively strong reflection peaks. The main diffraction peaks were assigned: 26 = 16.230o (d = 5.60778), 22.2210 (d = 3.99738), and 34.787o (d = 2.57686). The average particle size may be estimated by using Scherrer's equation (1).
(Equation Removed)
where K is the shape factor, A is the X-ray wavelength (1.54060 A), (5 is the line broadening at half the maximum intensity (FWHM) in radians, 6 is the Bragg's angle, and D is the mean size of the ordered (crystalline) domains. The dimensionless shape factor has a typical value of about 0.93, but varies with the actual shape of the crystallite. The reflecting peaks at 20 = 22.221° and 34.787° are used to estimate the average size (~6 nm and ~13 nm respectively) of the corn cob-based nanoparticles as shown in (Fig. 4).
High-resolution Transmission Electron Microscopy (TEM)
TEM photographs (Fig. 5) show that the average particle size was 22 nm, and the corresponding electron diffraction pattern showed only diffused signals, as expected for amorphous materials.
Scanning Electron Microscopy (SEM) with EDAX
Many of these nanomaterials are made directly as dry powders, and it is a common myth that these powders will stay in the same state when stored. In fact, they will rapidly aggregate through a solid bridging mechanism in as little as a few seconds. Whether these aggregates are disadvantageous will depend entirely on the application of the nanomaterial. If the nanoparticles need to be kept separate, then they must be prepared and stored in a liquid medium designed to facilitate sufficient interparticle repulsion forces to prevent aggregation.
(www.malvern.com/labeng/industrv/.../nanoparticles definition.htm). So when nanoparticles were made by corncobs raw materials and it was stored then they aggregate with each other. Aggregation part was seen in SEM micrographs. These nanoparticles are made up of mainly cellulose and it has the ability to pass through the digestive tract unchanged so, this resistance to digestion makes it eligible as a potential excipient that could be used in the pharmaceutical industry (Olson AC, et al., 1983) shown in (Fig. 6a, b & c).

WE CLAIM:
1. A process for the nano-particles from corn cobs comprising:
digesting the chopped corn in non-swelling or low swelling liquids;
treating the chips of corn cobs with sodium hydroxide at liquor to solid
ratio is 4.5:1;
washing the treated corn chips to remove the residual chemicals; subjecting the washed products to the step of screening and washing.
2. The process as claimed in claim 1, wherein the corn cobs are treated with sodium hydroxide at 165°C for 1.5 hrs.
3. The process as claimed in claim 1, wherein the said step of digestion is preferred in weverk electrically heated rotary digester of 0.02 m3/20 liter capacity.
4. The process as claimed in claim 1, wherein the said step of digestion is preferred using 2 to 24% of sodium hydroxide.
5. The process as claimed in claim 4, wherein the preferable amount of sodium hydroxide used in 18%.
6. The process as claimed in claim 1, wherein the nano-particle size obtained is in the range of 10-40nm.

7. The composition of corn cob used is as follows:
Holocellulose 73.04
(a) α-Cellulose 34.45
(b) ß-Cellulose 18.73
(c) Y-Cellulose 19.84 Pentosans (Xylan) 28.23 Total lignin 16.03

(a) Klason Lignin (Acid insoluble) 14.01
(b) Acid soluble lignin 2.02 Ethanol-Benzene soluble 4.33 Others (Difference) 6.64
8. The nano-particles contains α, ß &  cellulose in the percentages of 72.75, 25.19 & 02.06 respectively.