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Hydrophobically modified starch using long-chain fatty acids for preparation of nanosized starch particles
Published Date June 2011, Vol.18(3):439–445,doi:10.1016/j.scient.2011.05.006 Open Access, Creative Commons license, Funding information
Author
H. Namazi a,b,,
F. Fathi a
A. Dadkhah a
aResearch Laboratory of Dendrimers and Nanopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, P.O. Box 51666, Iran
bResearch Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Science, Tabriz, Iran
Received 10 September 2010. Revised 13 January 2011. Accepted 28 February 2011. Available online 27 May 2011.
Abstract
An efficient method for synthesis of hydrophobically modified starch without using organic solvents is described. The esterification of starch was performed with long chain fatty acid chlorides ( , , ), in two steps. In the first step, native starch was dispersed in an alkali reaction medium, and in the second step, it was treated for esterification. Finally, hydrophobic starch esters were obtained with moderate degrees of substitution (DS-values < 0.45). The reactivity of corn and potato starches under the same reaction conditions was also studied. The chemical structural determinations of the obtained products were investigated using common spectroscopy methods, such as FTIR and 1H NMR spectra. Starch nanoparticles and nanodispersion solutions were prepared via a dialysis method. The particle size distribution of the nanoparticles was determined using a laser diffraction particle size analyzer in an aqueous dispersion. The morphology investigation of the starch and the grafted starch nanoparticles was performed using SEM after freeze drying.
Keywords
Esterification
Hydrophobically modified starch
Nanoparticle
Dialysis method
1 Introduction
The chemical modification of starch has been widely studied for producing hydrophobic and water-resistant materials, by way of a chemical reaction with hydroxyl groups in the starch molecule. Starch esters are a kind of modified starch, which is synthesized with various reactants, such as acid anhydrides, Octenyl Succinic Anhydride (OSA), dodecenyl succinic anhydride (DDSA) fatty acids and fatty acid chlorides [1], [2], [3], [4] and [5]. Hydroxyethyl starch was esterified with long chain fatty acids under mild reaction conditions, using DCC and DMAP [6]. The synthesis of modified hydrophobic starch using fatty acids was performed by means of using potassium persullphate as a catalyst in DMSO [7]. Several substituted starches were prepared through an acylation reaction, using fatty acid chlorides in organic solvents, such as pyridine or dimethylacetamide [8] and [9].
However, the utilization of an organic solvent is prohibited for industrial applications. There are also economical, environmental and safety problems involved. In some reactions, the problems of waste, corrosive atmospheres, and purification of products created difficulties [10] and [11]. Recently, the hydrophobically modification of polysaccharides has received increased attention, because they can form self-assembled nanoparticles for biomedical uses. Chitosan, dextran and pullulan are polysaccharides that have been hydrophobically modified with various reactants. After the modification step, the self-assembled nanoparticles, based on hydrophobically modified polysaccharides, were prepared using the dialysis method [12], [13], [14], [15], [16] and [17]. The dialysis method is a simple and effective preparation method for small and narrow size distribution of nanoparticles in using modified biopolymers and amphiphilic materials. Their utilization for the preparation of drug carriers, such as liposomes and polymeric micelles has been developed [18] and [19]. Biodegradable nanoparticles of starch were prepared by an acid treatment process. The obtained starch nanoparticles were hydrophobically modified in the aqueous media under mild conditions [20]. Nanoparticles are defined as particulate dispersions or solid particles with sizes in the range of 10–1000 nm. Depending upon the method of preparation, nanoparticles, nanocapsules or nanospheres can be obtained. Usually, the drug is dissolved, entrapped, adsorbed, attached or encapsulated into the nanoparticle matrix [21], [22], [23], [24], [25], [26], [27], [28], [29] and [30]. Modified starches are known for their suitablity in biomedical applications, because in the aqueous phase, the hydrophobic cores of polymeric nanoparticles are surrounded by hydrophilic outer shells. Thus the inner core can serve as a nano-container for hydrophobic drugs [31] and [32]. Also, hydrophobically modified polysaccharides are emerging as novel carriers of drugs, because of their controlled solublity and biocompatibility in vivo. In addition, their improved properties, as compared to original starch, could be used as a thickener or an emulsifier [33], [34] and [35].
Here, the modification of potato and corn starches, using different long-chain fatty acid chlorides through a simple and convenient method, is reported. The esterification of starches using fatty acid chlorides, has several advantages in comparison to previous classic methods, including: (a) utilizing water as a green solvent instead of organic solvents, (b) the reaction time is very short and is completed just over several minutes, and (c) the hydrophobically modified products are precipitated in water and are separated without any need for a nonsolvent. The chemically modified starch, through this method, has an amphiphilic character and could be used for the preparation of starch nanoparticles utilizing a dialysis method. The stability of nanoparticles in aqueous dispersion after freeze drying was investigated using a Scanning Electron Microscope (SEM).
2 Experimental
2.1 Materials
Potato starch and waxy maize (corn starch) were purchased from Fluka and dried at 110°C for about 10 h to remove absorbed moisture. Octanoyl, lauroyl and palmitoyl chloride, as the reagent grade, were purchased from Sigma Chemicals. The dialysis membranes, with a molecular weight cutoff (MWCO) of 12,000 g/mol, were purchased from spectra/ProTM membranes. All other chemicals and solvents were reagent grade and used as received, except dimethyl sulfoxide (DMSO), which was stored over 3 Å molecular sieves for drying.
2.2 Methods
2.2.1 Preparation of esterified potato and corn starches
The starch esterification was carried out in two steps. In the first step, native starch was dispersed in an alkali reaction medium, and in the second step, it was treated for esterification. Finally, hydrophobic starch esters were obtained. The starch was dried at 100 °C for 2 h before reaction was accomplished. Starch (1 g) was added to the NaOH solution (10 ml, 0.25 M) at room temperature, then starch and water alkaline were mixed under low speed conditions for approximately 10 min. The selected amount of fatty acid chlorides (see Table 1) was added dropwise to the reaction container, under stirring at 300 rmp and room temperature. The reaction was allowed to stir until the modified products precipitated in water and was ended by filtering the precipitated esterified starch. Finally, it was extracted by MeOH in a soxhlet extractor for 1 day to completely remove unreacted fatty acid chlorides from the esterified starch. The esterified starch was then washed with water and ethanol and dried under vacuum at 40 °C. Precipitation time was very short and completed over just several minutes (3–5 min). Table 1 includes a list of the materials, used during synthesis, and their amounts. The recuperation yield of the modified starches was calculated according to Eq. (1). This formula is for 1 gram of starch.
equation1
Table 1. Reaction conditions for starch esterification, calculated degree of substitution (DS) and yields of modified starches.
2.2.2 Preparation of hydrophobically modified starch nanoparticles
The nanoparticles were prepared by the dialysis method. An appropriate amount of esterified starch (20–25 mg) was dissolved in 10 mL DMSO. The solution was stirred at room temperature and completely solubilized. The solution was introduced into the dialysis tube (molecular cutoff 12,000 g/mol), dialyzed 3 times against 1.0 L of distilled water for 3 h, and then the distilled water was exchanged at intervals of 3–4 h over 24 h to remove the organic solvent. The resulting suspension was used for immediate analysis or freeze-dried.
2.2.3 Determining the degree of substitution (DS)
Each glucose unit in a polymer chain of starch contains three free hydroxyl groups that can be substituted. The average degree of substitution (DS) can range from 0 up to 3.0. The samples were dried at 105 °C for 2 h before analysis. The Degree of Substitution (DS) was determined by proton NMR (1HNMR). The peaks between 4.58 and 5.50 ppm corresponded to the signals from the four protons of the glycoside structure. The three protons of the CH3 terminal of the acyl chain were observed as a triplet at 0.86 ppm. The DS was obtained from the ratio of the area of the proton peak at 0.86 ppm to that of the proton peak between 4.40 and 5.10.
2.2.4 Proton nuclear magnetic resonance (1H NMR) spectra
NMR spectra analysis was recorded on a Bruker 400 MHz for a carbon 13 isotope. The sample was dissolved in DMSO at 60 °C and the solution concentration was 15% w/v. The spectra were obtained at 60 °C with a pulse angle of 30°, a delay time of 10 s and an acquisition time of 2 s. All chemical shifts are reported in parts per million (ppm) using HMDS as references, which is usually used as an internal standard for NMR measurements at elevated temperature.
2.2.5 Fourier transformation infra red (FTIR) spectra
The FTIR analysis was performed using a FTIR Bruker-Tensor 270 spectrometer. The modified starches were mixed with analytical grade KBr at a weight ratio of 5/200 mg.
2.2.6 Particle size distribution measurement
The particle size of polymeric micelles was measured with a SALD-2101 Laser Diffraction Particle Size Analyzer. The particle size is calculated by measuring the angle of light scattered by the particles as they pass through a laser beam. This technique allows for continuous measurement of bulk material across a wide size range (10 nm–3 mm). A sample solution prepared by the dialysis method was used for particle size measurement (concentration: 0.2 wt.%).
2.2.7 Scanning electron microscopy (SEM) observation
Scanning electron micrographs were obtained with a LEO 440i scanning electron microscope, under vacuum, at an operating voltage of 10 kV. The morphology of the starch, grafted with fatty acid chlorides and polymeric nanoparticles (after freeze drying), was observed using a scanning electron microscope. Dried modified starch samples were gold coated by sputtering for 15 s.
2.2.8 Freeze-drying of nanoparticles
The resulting solution of nanoparticles was freeze-dried using a freeze dryer, Christ alpha 1–4. Freezing is the first step of freeze-drying. During this step, the liquid suspension of modified starch nanoparticles is cooled at −70 °C for 24 h and ice crystals of pure water form. The primary drying stage involves sublimation of ice from the frozen product at 0.07 mbar pressure at −25 °C for 24 h, and secondary drying involves the removal of absorbed water from the product at 25 °C for 2 h.
3 Results and discussion
3.1 Esterification of starch
In order to prepare hydrophobically modified starches, esterified starch was prepared in a NaOH solution (0.25 M) at room temperature in the presence of fatty acid chlorides (as shown in Figure 1). A hydrophobically modified starch polymer was chemically modified using long-chain fatty acids as the hydrophobic functionalities. When starch, as a polysaccharide, is modified through a hydrophobic reagent, such as fatty acids, some of its free hydroxyl groups are substituted, but not all of them. Therefore, after the hydrophobic modification of starch, it is still soluble in water and the solubility behavior of hyrophobically modified starch can vary, depending on the Degree of Substitution (DS). The DS of the products could be controlled by varying reaction conditions, such as reaction temperature, concentration of reactants and the reaction solvent. In other words, native starch is highly soluble in water, however, after hydrophobic modification, starch is still soluble, but its solubility is decreased. For this reason, Hydrophobically Modified starch polymers (HM polymers) are amphiphilic macromolecules that are mainly constituted of a hydrophilic backbone and hydrophobic side chains. Therefore, to archive the designed compounds, starch and fatty acid chloride were added in the molar ratio 1:1, respectively. Indeed, recent work by Fang [36] has demonstrated the suitability of the method for the aqueous esterification of four starches (Corn, Hylon VII, Hylon, Amioca). They reported the successful esterification reaction of starch with fatty acids, which was limited to acid chlorides containing 6–10 carbon chains, because outside that range, the acyl chlorides were hydrolyzed under reaction conditions and converted to their salt. But we succeeded in carrying out the esterification reaction of potato starch and corn starch for fatty acids, even with longer carbon chain acid chlorides with some modifications, such as decreasing NaOH concentration, reaction, temperature and reaction time, in this method. The reaction was completed just over several minutes, and the hydrophobically modified products precipitated from water and separated without any need for a non solvent, because of their low solubility in water, unlike starch. However, the same conditions applied to the shorter fatty acid chloride (C1, C3, C5) led to no reaction. It is clear that competition between the acyl group substitution and acid chloride hydrolysis occurred, particularly for those acyl chlorides (propionyl, pentanoyl) that were water-miscible. Also this reaction cannot be performed for higher degrees of substitution (2, 3), choosing starch and fatty acid chloride in the molar ratio 1:2 and 1:3, and no precipitated products have been observed. Only unreacted starches (no reaction products) were isolated when acetyl, propionyl and pentanoyl chlorides were used as acylating agents.
Figure 1. Scheme for reaction of starch with fatty acid chlorides (R: C8, C12, C16).
3.2 FTIR measurements
The FTIR analysis of modified potato starch and corn starch was performed using a FTIR Bruker-Tensor spectrometer. The absorption bands of esterified starches are summarized in Table 2. FTIR spectra of esterified starches with octanoyl, lauroyl, and palmitoyl chloride showed some new absorption bands at 1738–1742 cm−1. These new absorptions suggested that the esterified starch products were formed during the esterification process, because the vibrations of the carbonyl group in ester used to reside in this region. The band at 2850 cm−1 was assigned to aliphatic C–H stretching vibrations. The C–H stretching absorbance at 2926 cm−1 is increased in intensity upon grafting.
Table 2. The FTIR analysis data of esterified starches.
Absorption band (cm−1)
CO
CH2
CH3
CO
CH2
CH3
Acyle chain
Potato starch
Corn starch
C8
1738.5
2928
2857
1742
2928
2859
C12
1739
2925
2853
1741
2925
2853
C16
1741
2928
2859
1740
2926
2857
3.3 1H NMR measurements
A typical 1H NMR spectrum of native starch (Figure 2(a)), modified potato starch (Figure 2(b)) and modified corn starch (Figure 2(c)) dissolved in -DMSO is shown in Figure 2. We assigned 1H-chemical shifts of the protons at 3.15–3.64 ppm connecting to the proton at 3.36 ppm to H-4, 3.64 ppm to H-3, 3.31 ppm to H-2, 3.57 ppm to H-5, and 3.15 ppm to H-4 (end group). The chemical shifts of H-1 and OH-2, 3, 6 were possible to assign peaks between 4.58 and 5.50 ppm to four protons (Figure 2(a)). With the esterification process, aceyl groups were introduced into the starch, and proton resonances of the anhydroglucose unit showed some changes, compared with native starch (1H NMR spectra of aceylated starches are presented in Figure 2(b)). The 1H NMR spectra of the esterified starch showed three protons of the terminal methyl group of the acyl chain, as a triplet, around 0.85 ppm (peak e in Figure 2(b)). The peak at 2.10–2.25 ppm (peak a) is related to the methylene group, beside the carbonyl group, and the one at 1.45 ppm (peak b) is the methylene group directly before it. All other methylene groups have a peak at 1.22 ppm (peak d). The clear broadening of the peaks for the methylene groups close to the ester bond (at 2.1 and 1.45 ppm) indicates successful esterification. Thus the DS could be determined from the ratio of the normalized, integrated intensities of the signals of three protons of the terminal methyl group of the acyl chain to four protons of the anhydroglucose units, according to Eq. (2):
equation2
where 3 is the number of protons from the signal of the methyl proton, and IAGU is the integral for the 4 protons of the AGU between 4.58 and 5.50 ppm.
3.4 Influence of acyl chain length and starch source upon DSn and yield of modified starch
The degree of substitution diminishes with increasing the length of the acyl chain, due to the steric hindrance effect (Figure 3(a)). Changing the shorter chain of octanoyl chloride (C8) to longer chain palmitoyl chloride (C16) led to a higher size of the corresponding acylium ion and consequently a more pronounced steric hindrance, which provokes a decrease in the esterification reaction. A decreasing degree of substitution, by increasing the acyl chain length, was observed for both types of corn starch and potato starch. But in comparison to potato starch and corn starch, the degree of substitution corn starch was slightly higher than potato starch, as a result of different amounts of amylase and amylopectin.
Figure 3. Influence of the length of the fatty acid chloride chain and type of starch on (a) degree of substitution (DS) and (b) product yields.
Grafting efficiency or the percentage graft-yield depends on the acyl chain length and the source of the starch. By altering these variables, the percentage graft yield can be improved. Product yields were determined from the weight of the recovered starch product and the obtained results are shown in Figure 3(b). The product yields increase with increasing acyl chain length, because with a lower Degree of Substitution (DS), the final modified products remain soluble in water and do not precipitate during the separation step. However, the solubility of long chain modified starch in water becomes lower (hidrophobicity is higher) than those of short chains. Consequently, the precipitation of the product and the yield of the product increased. Meanwhile, the long chain modified starches usually precipitated better than the short chain ones. This increase was observed for both corn starch and potato starch, but corn starch had higher yield in comparison to potato starch.
3.5 Particle size analysis
Hydrophobically modified starch, as natural polymers (HM polymers), are amphiphilic macromolecules mainly constituted of a hydrophilic backbone and hydrophobic side groups. The preparation of nanoparticles from polysaccharides, such as dextran, chitosan and pullulan, has been previously performed using the dialysis method. Their nanoparticles have been produced after hydrophobically modified polysaccharides.
In order to prepare nanoparticles, hydrophobically modified starch was dissolved in DMSO, the nanoparticles were prepared by the dialysis method against water and the particle size was evaluated by a laser diffraction particle size analyzer. A laser diffraction particle size analyzer gives us two types of data that include surface diameter ( ) and volume diameter ( ). In the dialysis method, solvent systems to make nanoparticles are limited to water-miscible solvents, because water-immiscible solvents, such as dichloromethane or chloroform, cannot diffuse out or evaporate from the dialysis membrane to the outer aqueous environment. Knowledge and control of the size and the size range of particles is of profound importance in pharmacy. The size of a sphere is readily expressed in terms of its diameter. A sphere has minimum surface area per unit volume. The surface diameter, , is the diameter of a sphere having the same surface area as the particle. The diameter of a sphere having the same volume as the particle is the volume diameter, .
The particle size distribution diagrams have been presented in Figure 4. As indicated, in these diagrams, the volumetric diameter of the particle size has been changed with increasing the acyl chain of the grafted starch. For modified potato starch with acyl chain C16 (DS: 0.1), the median volumetric diameter of the particle size ( ) was 490 nm, and the median volumetric diameters of the particle size ( ) for modified starch with acyl chain C12 (DS: 0.21) and C8 (DS: 0.41) were 360 nm and 400 nm, respectively (Figure 4(a)). For modified starch with acyl chains C16 (DS: 0.1), C12 (DS: 0.21) and C8 (DS: 0.41), the median surface diameters of the particle size ( ) were 420, 316 and 320 nm, respectively.
Figure 4. The particle size distribution. (a) Modified potato starch nanoparticles with acyl chains C16 (DS: 0.1), C12 (DS: 0.21) and C8 (DS: 0.41); and (b) modified corn starch nanoparticles with acyl chains C16 (DS: 0.11), C12 (DS: 0.32) and C8 (DS: 0.45).
For modified corn starch with acyl chain C16 (DS: 0.11), the median volumetric diameter of the particle size ( ) was 505 nm, and median volumetric diameters of the particle size ( ) for modified starch with acyl chains C12 (DS: 0.32) and C8 (DS: 0.45) were 410 and 480 nm, respectively (Figure 4(b)). For modified starch with acyl chains C16 (DS: 0.11), C12 (DS: 0.32) and C8 (DS: 0.45) the median surface diameters of particle size ( ) were 410, 350 and 405 nm, respectively.
The closer the size of volumetric diameter and surface diameter, the more spherical the shape of the particle will be. These results indicated that hydrophobically modified starch was associated in the aqueous solution by the hydrophobic properties of the acyl chain domain. It is expected that hydrophobically modified starch nanoparticles will be formed by a self-assembling process in an aqueous environment.
3.6 Effect of dialysis time upon nanoparticles size
The effect of dialysis time upon nanoparticle size was discovered on one synthesized sample of modified starch (modified potato starch with lauroyl chloride). The modified starch was dissolved in DMSO and was dialyzed for 3 h and 20 h, respectively. Measurements of the laser diffraction particle size analyzer showed that particle size reduces with increasing dialysis time. As the particle size distribution shows in Figure 5, the particle size of the sample (dialyzed for 3 h) was 450 nm, and the particle size of the same sample (dialyzed for 24 h) was 360 nm; this is probably because of reducing the concentration of polymer solution inside the dialysis membrane during dialysis time.
Figure 5. Impact of dialysis time upon nanoparticle size.
3.7 Morphological investigation
The scanning electron micrographs of native potato starch were shown in Figure 6(a). The SEM of native potato starch showed typical granules of spheroid forms of size 10–20 μm. Smaller particles of damaged starch granules were also seen. Figure 6(b) shows SEM micrographs of starch nanoparticles prepared by dialysis, after freeze-drying, for the modified potato starch (acyl chain: c8, DS: 0.41). The granular structure of potato starch was completely destroyed and, also, we can see particles of modified potato starch ranging between 500–800 nm, which is similar to the particle size analyzer. Images suggested that the particles somewhat gather together after freeze drying. But, an overall difference has been created in the structure and size of the granules after preparation of nanoparticles in the dialysis method.
Figure 6. Scanning Electron Microscopic (SEM) micrographs of the: (a) native potato starch; (b) nanoparticles of modified potato starch with (C8, DS: 0.41); (c) native corn starch; and (d) nanoparticles of modified corn starch with (C8, DS: 0.45).
Scanning electron micrographs of native corn starch, and nanoparticles of modified corn starch (acyl chain: c8, DS: 0.45) were shown in Figure 6(c, d). The SEM image of corn starch is completely different from potato starch nanoparticles, because nanoparticles of corn starch have not been able to stabilize after freeze drying.
4 Conclusion
In summary, hydrophobic modification of potato and corn starch was successfully carried out in a mild esterification reaction using acyl chlorides (octanoyl, lauroyl and palmitoyl chloride) in aqueous media, which resulted in good yields. The evidence for the formation of chemical modification was confirmed by using common spectroscopy methods, such as FT-IR and 1H NMR. Corn starch grafted with acyl chlorides has a good yield in comparison to potato starch. Hydrophobically modified starch was able to form nanoparticles using the dialysis method. The formation and size of hydrophobically modified starch nanoparticles were confirmed by the laser diffraction particle size analyzer measurement, and their particle size was in the range of 360–500 nm. Study of the effect of dialysis time upon nanoparticle size indicated that particle size reduces with increasing dialysis time. SEM investigations of starch and grafted starch nanoparticles showed that hydrophobically modified starch nanoparticles were aggregated after freeze-drying.
Acknowledgments
We would like to thank the Department of Material Engineering for SEM Images and the Faculty of Pharmacology, Medical University of Tabriz, for particle size analysis. The authors also gratefully acknowledge the Center of Excellence for New Materials and Clean Chemistry, Tabriz University, for financial support of this work.
New intercalated layer silicate nanocomposites based on synthesized starch-g-PCL prepared via solution intercalation and in situ polymerization methods: as a comparative study
Hassan Namazi received his B.S. and M.S. degrees in Pure Chemistry and Polymer Chemistry from the University of Tabriz, in 1985 and 1988, respectively, and his Ph.D. degree in Natural Polymer Chemistry from the University of Dalhousie, Canada, in 1995, with a minor in Dendrimer and Carbohydrate Chemistry. Since then, he has served in the University of Tabriz as a Faculty Member and now as Full Professor. He has published more than 4 books in his related field, more than 56 scientific papers in highly ranked, respected international journals, and more than 120 conference proceedings. In 2010, he was honored to receive an Award and Honorary Diploma from the Iranian Academy of Medical Sciences for being one of the top three distinguished researchers in Iran. In 2006, he also was honored to receive an award from the Iranian Nanotechnology Initiative for being one of the top ten distinguished researchers in Iran. Prof. Namazi is the referee of more than 15 ISI ranked, International Journals in the fields of Polymer Chemistry, Dendrimers and Nanostructure Compounds. He has won many academic awards and, also, the national medal of merit for outstanding research activities (2004, 2003, 2001 and 2000).
Farzaneh Fathi received B.S. and M.S. degrees in Pure Chemistry and Organic Chemistry from Tabriz University in 2006 and 2009, respectively. She has been working since 2006 in the Laboratory of Dendrimers and Nanopolymers, on a project in ‘Modified Starch’. She has published more than 4 papers at conference proceedings.
Abbas Dadkhah received a B.S. degree in Pure Chemistry from the Faculty of Chemistry, Yazd University, in 2001, and an M.S. degree in Organic Chemistry from the University of Tabriz in 2005. He has published eight scientific papers in highly ranked, respected international journals. His research interests include Starch, Characteristic, Properties and Nanotechnology, and the Chemical Modification of Starch. He received his Ph.D. in Polymer Chemistry from the Department of Organic Chemistry in Tabriz University in 2010.
NMR spectra analysis was recorded on a Bruker 400 MHz for a carbon 13 isotope. The sample was dissolved in DMSO at 60 °C and the solution concentration was 15% w/v. The spectra were obtained at 60 °C with a pulse angle of 30°, a delay time of 10 s and an acquisition time of 2 s. All chemical shifts are reported in parts per million (ppm) using HMDS as references, which is usually used as an internal standard for NMR measurements at elevated temperature.
, 
, 
), in two steps. In the first step, native starch was dispersed in an alkali reaction medium, and in the second step, it was treated for esterification. Finally, hydrophobic starch esters were obtained with moderate degrees of substitution (DS-values < 0.45). The reactivity of corn and potato starches under the same reaction conditions was also studied. The chemical structural determinations of the obtained products were investigated using common spectroscopy methods, such as FTIR and 1H NMR spectra. Starch nanoparticles and nanodispersion solutions were prepared via a dialysis method. The particle size distribution of the nanoparticles was determined using a laser diffraction particle size analyzer in an aqueous dispersion. The morphology investigation of the starch and the grafted starch nanoparticles was performed using SEM after freeze drying.
O
CH2
-DMSO is shown in Figure 2. We assigned 1H-chemical shifts of the protons at 3.15–3.64 ppm connecting to the proton at 3.36 ppm to H-4, 3.64 ppm to H-3, 3.31 ppm to H-2, 3.57 ppm to H-5, and 3.15 ppm to H-4 (end group). The chemical shifts of H-1 and OH-2, 3, 6 were possible to assign peaks between 4.58 and 5.50 ppm to four protons (Figure 2(a)). With the esterification process, aceyl groups were introduced into the starch, and proton resonances of the anhydroglucose unit showed some changes, compared with native starch (1H NMR spectra of aceylated starches are presented in Figure 2(b)). The 1H NMR spectra of the esterified starch showed three protons of the terminal methyl group of the acyl chain, as a triplet, around 0.85 ppm (peak e in Figure 2(b)). The peak at 2.10–2.25 ppm (peak a) is related to the methylene group, beside the carbonyl group, and the one at 1.45 ppm (peak b) is the methylene group directly before it. All other methylene groups have a peak at 1.22 ppm (peak d). The clear broadening of the peaks for the methylene groups close to the ester bond (at 2.1 and 1.45 ppm) indicates successful esterification. Thus the DS could be determined from the ratio of the normalized, integrated intensities of the signals of three protons of the terminal methyl group of the acyl chain to four protons of the anhydroglucose units, according to Eq. (2): 
) and volume diameter ( 
). In the dialysis method, solvent systems to make nanoparticles are limited to water-miscible solvents, because water-immiscible solvents, such as dichloromethane or chloroform, cannot diffuse out or evaporate from the dialysis membrane to the outer aqueous environment. Knowledge and control of the size and the size range of particles is of profound importance in pharmacy. The size of a sphere is readily expressed in terms of its diameter. A sphere has minimum surface area per unit volume. The surface diameter, 
, is the diameter of a sphere having the same surface area as the particle. The diameter of a sphere having the same volume as the particle is the volume diameter, 
.
) was 490 nm, and the median volumetric diameters of the particle size ( 
) for modified starch with acyl chain C12 (DS: 0.21) and C8 (DS: 0.41) were 360 nm and 400 nm, respectively (Figure 4(a)). For modified starch with acyl chains C16 (DS: 0.1), C12 (DS: 0.21) and C8 (DS: 0.41), the median surface diameters of the particle size ( 
) were 420, 316 and 320 nm, respectively.
) was 505 nm, and median volumetric diameters of the particle size ( 
) for modified starch with acyl chains C12 (DS: 0.32) and C8 (DS: 0.45) were 410 and 480 nm, respectively (Figure 4(b)). For modified starch with acyl chains C16 (DS: 0.11), C12 (DS: 0.32) and C8 (DS: 0.45) the median surface diameters of particle size ( 
) were 410, 350 and 405 nm, respectively.-butanol
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