Available at: http://publications.ictp.it IC/2008/030

United Nations Educational, Scientific and Cultural Organization and

International Atomic Energy Agency

THE ABDUS SALAM INTERNATIONAL CENTRE FOR THEORETICAL PHYSICS

THE SYNTHESIS CONDITIONS, CHARACTERIZATIONS AND THERMAL DEGRADATION STUDIES OF AN ETHERIFIED STARCH FROM

AN UNCONVENTIONAL SOURCE

O.S. Lawal1 Department of Chemical Sciences, Olabisi Onabanjo University,

P.M.B 2002, Ago-Iwoye, Ogun State, Nigeria, Institute of Technical and Macromolecular Chemistry, University of Hamburg,

Bundestrasse 45, D-20146, Hamburg, Germany and

The Abdus Salam International Centre for Theoretical Physics, Trieste, Italy,

M.D. Lechner

Institute of Physical Chemistry, University of Osnabrück, Babarastrasse 7, 49069, Osnabrück, Germany

and

W.M. Kulicke

Institute of Technical and Macromolecular Chemistry, University of Hamburg, Bundestrasse 45, D-20146, Hamburg, Germany.

MIRAMARE – TRIESTE

May 2008

1 Junior Associate of ICTP. laidelawal2@yahoo.com

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Abstract

Starch isolated from an under-utilized legume plant (pigeon pea) was carboxymethylated. Influences of reaction parameters were investigated on the degree of substitution (DS) and the reaction efficiency (RE). Studies showed that optimal DS of 1.12 could be reached at reaction efficiency of 80.6 % in isopropanol-water reaction medium (40 oC, 3h). The scanning electron microscopy showed that after carboxymethylation, the granular appearance of the native starch was distorted. Wide-angle X-ray diffractometry revealed that crystallinity was reduced significantly after carboxymethylation. The infrared spectra revealed new bands in the carboxymethyl starch at ν =1600, 1426 and 1324 cm-1 and they were attributed to carbonyl functional groups vibration, –CH2 scissoring and OH bending vibration respectively. Broad band 13C NMR of carboxymethyl starch showed intense peak at δ = 180.3 ppm and it was assigned for carbonyl carbon on the carboxymethyl substituent on the AGU (Anhydroglucose Unit). DEPT (Distortionless Enhancement by Polarization Transfer) 135 NMR showed negative signals which correspond to methylene carbons on the AGU. The differential scanning calorimetry (DSC) suggests loss of crystallinity after carboxymethylation. Thermogravimetry (TG), Derivative Thermogravimetry (DTG) and Differential Thermal Analysis (DTA) show that thermal stability improved after carboxymethylation. The study provides information on the preparation and characterization of a biomaterial from a new source which could be used alone or in the preparation of other functional polymers for diverse polymer applications.

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Introduction Starch derivatives play vital roles in the burgeoning biopolymers industries. This is because they are cheap, non-toxic, renewable and compatible with many other materials for industrial applications. Diverse polymer applications employ the use of starch derivatives directly or after they have been combined with other synthetic polymers. Applications in food [1, 2] environmental management [3–5], agriculture [6], pharmacy [7], biomedical engineering [8] and textile [9] have been reported widely in the literature. Unfortunately, using starch in its native form is often limited by certain undesirous characteristics such as poor solubility, low mechanical properties and instability at high temperature, pH and shear during processing. Hence it is always reasonable to modify it to suit specific industrial process. Chemical modification of starch concerns reaction of the hydroxyl groups on the AGU and these have been used to produce starch derivatives based on oxidation [10], acetylation [11], hydroxypropylation [12], carboxymethylation [13] and cross-linking [14]. Among starch derivatives, etherified starch derivative such as carboxymethyl starch (CMS) have attracted a lot of attention in recent years. Carboxymethyl starches are usually synthesized by the reaction of starch with monochloroacetic acid or its sodium salt after activation of the polymer with aqueous NaOH in slurry of an aqueous organic solvent, in most cases an alcohol. Different approaches have also been reported in the literature for the synthesis of carboxymethyl starch. This include induced phase separation method using dimethyl sulphoxide [15], synthesis of 2,3-di-O-carboxymethyl starch via 6-O-triphenylmethyl [16], carboxymethylation after γ irradiation [17], carboxymethylation after starch oxidation [18] and using of various organic solvents such as ethanol, methanol, isopropanol, butanol, acetone [19]. The aim of all these approaches is to synthesize carboxymethyl starch at optimized conditions such as high product yield, high reaction efficiency as well as high DS. The DS is an indication of the amount of carboxymethyl group formed on the starch molecule. Technically, the DS is defined as the average number of substituent per AGU. The functional properties of CMS are dependent on the DS. Such properties include the viscosity of the solution, film forming properties, interaction with cations, formation of supramolecular aggregates and rheological properties. In our previous publications, we reported on the synthesis of CMS using cocoyam starch [20]. Also, in a recent work, we reported the synthesis of CMS using water yam starch [13]. In the literature, starches from other origins have also been used for the synthesis of carboxymethyl starch and these include potato starch [21], rice starch [22], corn starch, amaranth starch [19], cassava starch [23] and mung bean starch [24]. However, the increasing demand for starch and derivatised starches in the ever-increasing biopolymer based industries justifies the need for the exploration of new resources for starch, particularly cheaper alternatives to conventional sources such as potato and maize. In the present investigation, pigeon pea was used as the source of starch for carboxymethylation. Pigeon pea is a woody perennial legume crop belonging to the family Fabaceae. It is cultivated throughout the world in both tropical and sub-tropical regions. In Nigeria and India, it is used both as a food and as

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cover crop. Studies have revealed that pigeon pea contains between 57.3 % carbohydrate, 19.2 % protein, 1.5% fat and 8.1% fiber [25]. The rich carbohydrate content of pigeon pea suggests that it is a reasonable source of starch. The authors are cognizant of the fact that CMS is a vital raw material for the synthesis of other functional bio-based polymers. Hence, the concern of the present investigation is to explore the preparation of a valuable biomaterial from a relatively cheap and under-utilized source of starch (pigeon pea). The authors are not aware of any previous publication concerning preparation and the polymer characterization of carboxymethyl starch from pigeon pea. Hence, preparation, characterization and polymer stability studies of CMS based on pigeon pea are presented herein.

Materials and Methods Materials Pigeon pea was donated by the International Institute of Tropical Agriculture, Ibadan, Nigeria. Sodium hydroxide and sodium monochloroacetate were purchased from Merck Schuchardt Germany. Isopropanol, methanol, ethanol, t-butanol, and acetone were technical grade. All other reagents were analytical grade.

Isolation and purification of pigeon pea starch Starch isolation was carried out using the method described by Adebowale and Lawal [26]. The yield of the starch obtained was 22.5% and the result of the analyses of the composition using AOAC methods [27] was moisture, 8.2%; ash, 0.7%; fat, 0.5%; crude fibre, 0.8%; protein, 2.5%; carbohydrate, 87.3%; pH, 7.9. It was washed twice before drying in the air for 48 h at 30 ± 2 oC. The native pigeon pea starch obtained (NPPS) was stored in polythene bag at 30 ± 2 oC until use.

Preparation of carboxymethyl starch For the preparation of carboxymethyl pigeon pea starch (CMP). NaOH of different quantities (8-16 g) were added to water in a 2 L three-necked round bottom flask equipped with motor driven stirrer and the mixture was stirred at 250 rpm until complete dissolution of sodium hydroxide was observed. Organic solvent (500 mL) was added to the solution in each case and the temperature was raised to between 30 and 60 OC. Organic solvents used in this experiment were isopropanol, methanol, ethanol, and t-butanol. The water content in the reaction mixture was varied between 40 and 80 mL (The moisture content of NPPS was predetermined). Starch (40 g dry wt) was added to the mixture and it was stirred at 400 rpm while nitrogen gas was flushed through the reaction mixture to maximize the reaction of NaOH with starch. After stirring for 1 h, SMCA (20-50 g) was added to the mixture and the reaction time was varied between 1-4 h following which the mixture was filtered, suspended in methanol and neutralized with acetic acid. Following filtration, the slurry was dispersed again in 80 % methanol and it was washed several times until the filtrate gave negative response to silver nitrate test

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of the chloride. The slurry obtained was suspended in acetone, stirred for 20 min, and dried in an oven at 40 OC for 48 h.

Determination of degree of substitution Titrimetry was used for the determination of the DS. CMS (10 g) was dispersed in acetone (300 ml) and 5 M HCl (30 ml) was added to the dispersion which was stirred for 30 min. During this process, the CMS which was in sodium form was converted to the H-CMS (carboxymethyl starch in hydrogen form). H-CMS was washed four times with 80 % (v/v) methanol until the solution became neutral with pH test. The neutral dispersion was filtered again, suspended in acetone and it was stirred for another 15 min, following which it was filtered, and dried for 24 h in a desiccator over silica gel. 2 g of H-CMS was dissolved in 1% (w/v) NaCl solution and it was titrated with 1 M NaOH. The DS was determined as follows: nNaOH × Mo DS = (1) mc − nNaOH × MR mp × F mc = mp − (2) 100

Mo = molar mass of the anhydroglucose unit = 162 g/ mol MR = molar mass of carboxymethyl residue = 58 g/ mol nNaOH = quantity of sodium hydroxide used (mol) mp = weight of polymer taken (g) mc = corrected weight of polymer (g) F = moisture (%) The DSt is the theoretical degree of substitution. It is the maximal degree of substitution when the limiting reactant either SMCA or NaOH is totally used. nSMCA,0 DSt = if nNaOH,0 ≥ nSMCA,0 (3)

nAGU,0

nNaOH,0 DSt = if nNaOH,0 < nSMCA,0 (4)

nAGU,0

nSMCA = Number of moles of sodium monochloroacetate nAGU = Number of moles of anhydroglucose unit nNaOH = Number of moles of sodium hydroxide

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The RE is a measure of the amount of carboxymethyl group bonded to the starch. The RE is defined as: DS *100 RE = (5) DSt

The molar mass of the unsubstituted anhydroglucose unit = 162 g/mol The molar mass of a substituted anhydroglucose unit = 242 g/mol

Starch granule morphology Starch granule morphology was examined with a Leo 1550 ultra scanning electron microscope. The samples were mounted on studs, sputter-coated with gold (Balzers, SCD-040; Norderstedt, Germany) and examined under the scanning electron microscope.

X-ray Diffraction (XRD) The X-ray diffraction pattern of native pigeon pea starch and its carboxymethylated derivatives were recorded with X´Pert pro X-ray diffractometer equipped with X´celerator as detector. The diffractograms were registered at Bragg angle (2Ө) = 5 – 60 o at a scan rate of 5o min-1. Multi-peak fitting was performed to get the integrated area of crystalline peaks and amorphous peak, and the degree of crystallinity [Xc (%)] was determined by Xc (%) = Ac/ Ac+Aa X 100%.

FT-IR spectroscopy The IR spectra of starches were run as KBr pellets on impact 410 Nicolet FTIR spectrometer in the frequency range 4000 – 500 cm-1.

13C and DEPT 135 NMR Spectroscopy The broad band 13C and DEPT 135 NMR spectra were obtained on a Bruker Avance 400 MHz spectrometer. The internal reference used was 3-(Trimethylsilyl) propionic 2,2,3,3-d4 acid, sodium salt. The spectra were obtained in D2O as a solvent. The chemical shifts are given in ppm. Prior to the measurements, the samples were degraded by ultrasonic degradation with a sonifier W-450 ultrasonic degradation device (Branson Schallkraft GmbH, Heusenstamm, Germany) equipped with ¾” titanium resonator. For this experiment, 300 mL of 0.5 % w/w solution was used. The sound frequency of the device was 20 kHz, max output was 400 W and a thermostat was used to cool the solution below 0OC during degradation. The density of the ultrasonic output was approximately 80 W/cm2. Following degradation for 3 h, ultracentrifugation (10, 000 rpm, 1 h) was carried out to remove abraded metal fines from the ultrasonic resonator. The clear solutions obtained were lyophilized (Betta 1-16, Christ, Osterode, Germany).

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Differential Scanning Calorimetry (DSC) Measurements for the differential scanning calorimetry were performed with Dupont 2000 Thermal Analysis DSC. 10 mg samples sealed in aluminum pans with 5.0μL of water were scanned from 30 oC to 130 oC at a scanning rate of 10 oC/ min while sealed empty pans were used as reference. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and the enthalpy of gelatinization were determined.

Thermal analysis TG, DTG and DTA were measured with Netzsch STA 409 instrument. The measurements were recorded in an air atmosphere (flow rate: 50 ml/min). The sample mass was 20 mg and it was heated from room temperature to 600 oC at the heating rate of 5 K/min.

Results and Discussion

Effect of various molar ratio of SMCA to starch Keeping other parameters constant, studies showed progressive increases in the DS as the amount of SMCA added to the reaction mixture increased (Fig.1). Contrarily, the reaction efficiency declined with increases in molar ratio of SMCA to starch. Increase in the DS could reasonably be attributed to increased contact between the starch molecules and the etherifying agent as the concentration of SMCA increased. The reduction of RE with higher amount of SMCA could be explained by understanding the stages and the products involved in the carboxymethylation process. The first step is an alkalization reaction where the hydroxyl groups of the starch molecules are activated and changed into the more reactive alkoxide form (St-O-).

St-OH + NaOH St-ONa + H2O (6) The second step is the etherification:

St-ONa + Cl-CH2-CO-ONa St-O-CH2-COONa + NaCl (7)

Unfortunately, a side reaction also occurs which competes with the production process of carboxymethyl starch. In this side reaction, sodium gylocate is produced as a bye product.

NaOH +ClCH2COONa HOCH2COONa + NaCl (8)

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The decrease in the reaction efficiency could be attributed to the formation of products in the bye reaction or steric hindrance resulting from higher SMCA concentration. Similar observation has also been reported in the literature [20].

Effect of various molar ratio of NaOH to starch The effect of various molar ratio of NaOH to starch is presented in Figure 2. Both the DS and the RE increased as the molar ratio of NaOH to starch increased from 0.81–1.62. Thereafter, reductions were observed in both the DS and the RE. As indicated in Eq. 7, the NaOH is used in the formation of starch alkoxide and it also facilitates the swelling of starch for enhanced larger surface area for the etherification process. This accounts for the increase in the DS and RE at the initial stage. However, there is tendency for alkaline gelatinization as the amount of NaOH in the reaction mixture increases. This means inhibition of contact between starch etherifying agents within the reaction mixture, this development could account for the reduction in the DS and the RE at higher ratio of NaOH to Starch. It is also reasonable that higher amount of NaOH enhances the reaction for the formation of sodium glycolate. Similar observation was reported for corn and amaranth starches [19].

Effect of water in the reaction medium The effect of water in the reaction medium is presented in Fig. 3. The result shows increases in the RE and the DS as the ratio of water to organic solvent increased from 0.8–0.16 after which a sharp decline was observed when H2O/IPA was 0.2. Dissolution of etherifying reagents and absorption of the reagents are dependent on water content of the reaction mixture. In addition, Swelling is also dependent on water and this would increase the surface area of the reaction. These factors are responsible for the initial increase in the DS and the RE. However, at higher concentration beyond H2O/IPA = 0.16, starch agglomeration and gelation occurred, stirring of the reaction mixture was impaired and this limited the contact of etherifying agent with starch molecules. Based on this, it is reasonable to say that a critical ratio for water-organic solvent mixture should be maintained in the preparation carboxymethyl starch. Optimal water fraction of 0.1 in isopropanol has been reported for the preparation of carboxymethyl potato starch [21].

Effect of temperature Both the DS and the RE increased as the temperature of the reaction increased from 30 – 40 OC (Fig. 4). However, it is instructive that increases in the DS and RE were not pronounced after 50 OC. Higher temperature increases the solubility and diffusion of the etherifying reagents and swelling of starch molecules. The proportion of molecules with higher energy than the activation energy increased as the temperature increased and this also contributes to higher rate of reaction. However, it is reasonable to work at temperatures below the gelatinization temperature of the starch. This becomes necessary to avoid poor product recovery and as well as limitation of the contact between the starch molecules and the etherifying reagents. Previously, it was reported that preparing carboxymethyl cocoyam starch at 70

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OC caused starch gelatinization [13]. Increase in the DS with temperature as observed here is consistent with the carboxymethylation of mungbean starch [7].

Effect of duration of reaction The effect of duration of reaction is presented in Fig. 5. The study revealed that both the DS and the RE increased as the reaction time increased. Longer duration of reaction enhanced dissolution and diffusion of the reagents hence carboxymethylation was enhanced. Also, it is reasonable that swelling of starch molecules increased with time of reaction because of longer stay in reaction medium. When swelling increases, absorption of reagent by the starch molecules is improved. In the present investigation, the increase in both the DS and the RE were not remarkable after 3 h. The DS and the RE after 3 h were 0.58 and 41.7 % respectively whereas prolonging the reaction for another 1 h only increased the DS and the RE to 0.59 and 42.4 respectively. It may be reasonable to keep the reaction at 3 h, to safe time and the cost of production.

Effect of various organic solvents in the reaction media Various organic solvents were investigated for use in the water-organic solvent reaction media for the starch carboxymethylation process. The solvents used were methanol, ethanol, isopropanol and butanol. When other parameters were kept constant, studies indicate that optimal the DS and the RE were obtained in isopropanol-water reaction medium (Fig. 6). The organic solvent for carboxymethylation process is expected to have good miscibility with water to prevent phase separation, good solubility for the etherifying agents and good selectivity for carboxymethylation and not the formation of sodium glycolate. The results obtained here showed that optimal performance was obtained when isopropanol – water reaction medium was used. In the literature, investigations on the carboxymethylation of starch in various organic media have been reported. Optimal performance was reported for carboxymethylation of potato starch in isopropanol-water reaction medium [21]. A similar result was reported for maize starch in isopropanol-water reaction medium [28]

Scanning Electron Microscopy Scanning electron microscopy was used to investigate the granule morphology of both the NPPS as well as the CMP. The results of the investigation are presented in Figs. 7 and 8 for NPPS and a representative carboxymethyl starch (CMP-1, DS 0.72) respectively. The NPPS granules appeared oval or elliptical in shape width sizes ranging from 7 − 40 μm in width and 10 − 30 μm in length (Averages of 40 measurements were taken). The granules appeared smooth with very minimal damage; this suggests that the method of extraction and drying did not cause significant damage to the starch. In previous investigations on legume starches, oval and round shapes were reported for mucuna bean starches with sizes ranging from 12-22 and 14-31 μm for width and length respectively [29]. After carboxymethylation, the granular appearance of the native starch was distorted. This observation suggests that carboxymethylation affects the structural arrangement of the starch. It is reasonable that

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the strong alkaline condition used for the synthesis caused granular disintegration. Similar observations have been reported for cassava carboxymethyl starches in the literature [23, 30].

Wide angle X- ray diffractometry The wide angle X-ray diffractograms obtained for both native pigeon pea starch and the representative carboxymethylated derivative (CMP-1, DS 0.72) are presented in Fig. 9. The NPPS shows the “C” pattern characteristic of legumes. Prominent peaks were centered on 2θ = 15o, 17o and 23o. Previously, it was reported that the ‘C’ crystalline polymorphs of starches is not a true crystalline polymorph but mixtures of ‘A’ and ‘B’ polymorphs observed in cereals and tuber starches respectively [31]. Also, both ‘A’ and ‘B’ type starches are based on the parallel stranded double helices, in which the double helices are closely packed in the ‘A’ type starch but loosely packed in the ‘B’ type starch. It has been shown that starches with amylopectin of short chain length (

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carboxymethyl substituent on C-3. The C-4 peaks are not well resolved compared with C-1 peaks possibly because distribution of carboxymethyl substituent on C-2 is more pronounced compared with C-3. The reason for this is because the hydroxyl function of the C-2 position forms the basis of the electronic effect of the adjacent glycoside bond and of the electron-attracting properties of the ring oxygen. Consequently, C-2 substitution takes place before C-3 and C-6. C-2U and C-3U were assigned peaks at δ = 74.48 ppm and δ = 75.25 ppm respectively while C-2s and C-3s appear at δ = 77.76 ppm and δ = 80.28 ppm respectively. This observation indicates that down field shifts of ~ 3-5 ppm occurred on C-2 and C-3 after carboxymethylation. C-5 and C-6U are assigned to the signals at δ = 76.07 ppm and δ = 63.49 ppm respectively. Carboxymethyl substitution causes a downfield shift of ~ 8 ppm on C-6 as indicated by the resonance at δ = 71.96 ppm which is assigned to C-6s. A plausible explanation for these observations is that the electron withdrawing effect imposed by oxygen in the carboxymethyl group accounts for the down field shifts after modification. The methylene carbon atoms of the carboxymethyl substituents (C-7) are assigned with peaks at δ = 72.64 ppm, 73.41 ppm and 74.18 ppm. As expected, three peaks appear here because of the three different positions possible for the carboxymethyl substituent on the AGU. To further elucidate the structure, DEPT 135 NMR measurement of CMP-1 is presented in Fig. 12. The DEPT 135 NMR measurements indicate whether a carbon carries a proton, and the degree of protonation (CH, CH2 or CH3). In the present investigation, the negative signals are due to the CH2 groups on the carboxymethyl substituents and the CH2 corresponding to C-6 and C-6s assigned as indicated.

Differential Scanning Calorimetry (DSC) The differential scanning calorimetry thermograms of NPPS and a representative CMP is presented in Fig.13. The To, Tp and the Te for the NPPS starch are 75.2, 84.2 and 88.7 OC respectively. The gelatinization enthalpy (ΔH) was 9.12 J/g. This value is consistent with details of gelatinization reported for other legume starches [11, 29]. Starch gelatinization breaks down the intermolecular bonds of starch molecules in the presence of water and heat and it allows the hydrogen bonding sites to absorb more water. Penetration of water increases randomness in the general structure and decreases the number and size of crystalline regions since crystalline regions do not allow water entry. The presence of heat causes crystalline regions to be diffused, so that the chains begin to separate into an amorphous form. In this regard, the significance of amorphous region in water absorbtion is underscored. As the result of the DSC indicated, gelatinization enthalpy could not be determined for the CMP-1, this is indicative that amorphous region increased after carboxymethylation. Previous characterization techniques reported earlier in this work, particularly the X-ray diffraction studies also indicate increase in amorphous region.This would make carboxymethyl starch enjoy wide technical application in areas where water absorption is vital.

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Thermal stability The results of TG, DTG and DTA for the NPPS and its carboxymethyl derivative are presented in Figs. 14 and 15 respectively. Thermophysical parameters provide vital information about the thermal stability of polymeric materials. Such information are needed for reasonable industrial applications of polymers [34]. In this result, three step thermograms were noticed in the NPPS and the progressive decompositions were -11.87%, -61.81% and -24.08% successively. The studies indicate that the maximum degradation occurred within the range 263 – 358 OC and the peak as indicated by the DTG (DTGmax) was at 300 OC. Also, the representative carboxymethyl starch showed a three-stage decomposition process of -7.33%, -39.50 % and -12.99 %. In addition, the range of maximum decomposition was within 240 – 326 OC (DTGmax= 286 OC). The DTA curves also show intense exothermic transitions around 300 OC for carboxymethyl starch and 335 OC for the native starch. This intense exothermic peak is observed for several natural and modified polysaccharides such as gum arabic, xanthan gum, sodium carboxymethyl cellulose [35]. It is an important degradation process yielding mainly volatile products [36]. The second prominent exothermic transition observed in the DTA of native starch around 500 OC is due to its crystalline component and it is not pronounced in the DTA of carboxymethyl starch. This also justifies the increased amorphous component of the starch after carboxymethylation. The early minor weight loss (TG) in both samples is attributed to the desorption of moisture as hydrogen bond water to the polysaccharide structure. It is instructive that initial decomposition temperature (IDT) of carboxymethyl starch is lower than the IDT of the native. However, the result shows that the native starch rapidly decomposed as soon as it reached the IDT. Only 39.5 % of carboxymethyl starch decomposed within the similar range where 61.81% of the native starch decomposed. The reason for the lower IDT of the carboxymethyl starch is the heterogeneity that results after modification. Increase in thermal stability after carboxymethylation has been reported for other polysaccharide such as hemicelluloses [37]. The reason for this development is the substitution of the hydroxyl groups on the native starch with carboxymethyl group after carboxymethylation. The main decomposition mechanism of starch is the dehydration reaction between starch hydroxyls; this suggests that the smaller the amount of hydroxyl group left on the starch, the more stable it is. This position was also corroborated in the higher thermal stability of methylcellulose compared with the ummodified cellulose [38].

Conclusion The first preparation of carboxymethyl derivative of pigeon pea starch is reported in this work. Optimal DS of 1.12 was achieved at a reaction efficiency of 80.6% after 3h in isopropanol-water reaction medium. Thermal stability increased after modification and the tendency for increased water absorption was also observed because of enhanced amorphous component. For technical applications, such as the preparation of super-absorbent hydrogels, preparation of biopolymer based flocculants, drag-reduction biomaterials, drug-release and other applications where bio-based polymers are relevant, carboxymethyl pigeon starch could be strategic because the source material is reasonably cheaper than

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other conventional sources of starch and it has a reasonable wide distribution globally. This investigation would provide relevant information to bio-polymer based industries.

Acknowledgments OSL is grateful to the Alexander von Humboldt Foundation of Germany for the award of postdoctoral fellowship with Professor Dr. W-M Kulicke. This work was done within the framework of the Associateship Scheme of the Abdus Salaam International Centre for Theoretical Physics, Trieste, Italy.

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Table 1: The compilation of reaction conditions used for the synthesis of carboxymethylated pigeon pea starches. DS, the degree of substitution; RE , the reaction efficiency. nNaOH, moles of sodium hydroxide. nAGU, moles of anhydroglucose unit. nsmca, moles of sodium monochloroacetate.

Sample Organic Solvent

Time (Hr)

Temperature (OC)

H2O/ Solvent nNaOH/nAGU(s) nSMCA/nAGU(s) DS RE (%)

CMP-1 Isopropanol 3 60 0.08 1.62 1.39 0.72 51.8 CMP-2 Isopropanol 3 50 0.08 1.62 1.39 0.71 51.1 CMP-3 Isopropanol 3 30 0.08 1.62 1.39 0.15 10.8 CMP-4 Isopropanol 3 40 0.08 1.62 1.74 0.65 40.1 CMP-5 Isopropanol 3 40 0.08 1.62 1.39 0.58 41.7 CMP-6 Isopropanol 3 40 0.12 1.62 1.39 0.62 44.6 CMP-7 Isopropanol 3 40 0.16 1.62 1.39 1.12 80.6 CMP-8 Isopropanol 3 40 0.08 1.21 1.39 0.46 38.0 CMP 9 Isopropanol 3 40 0.08 0.81 1.39 0.17 21.0 CMP-10 Isopropanol 3 40 0.08 1.62 1.04 0.44 42.3 CMP-11 Isopropanol 3 40 0.08 1.62 0.69 0.34 49.3 CMP-12 Isopropanol 3 40 0.20 1.62 1.39 0.36 25.9 CMP-13 Methanol 3 40 0.08 1.62 1.39 0.18 13.0 CMP-14 Ethanol 3 40 0.08 1.62 1.39 0.30 21.6 CMP-15 t-Butanol 3 40 0.08 1.62 1.39 0.52 37.4 CMP-16 Isopropanol 3 40 0.08 1.82 1.39 0.22 15.8 CMP-17 Isopropanol 3 40 0.08 2.02 1.39 0.20 14.4 CMP-18 Isopropanol 2 40 0.08 1.62 1.39 0.42 30.2 CMP-19 Isopropanol 1 40 0.08 1.62 1.39 0.26 18.7 CMP-20 Isopropanol 4 40 0.08 1.62 1.39 0.59 42.4

15

0,6 0,8 1,0 1,2 1,4 1,6 1,80,30

0,35

0,40

0,45

0,50

0,55

0,60

0,65

DS RE

nSMCA/nAGU

DS

40

42

44

46

48

50

RE

(%)

Fig.1 The effect of various molar ratio of SMCA to starch (AGU) on the DS and the RE for the carboxymethylation of pigeon pea starch. nsmca, moles of sodium monochloroacetate. nAGU, moles of anhydroglucose unit. DS, degree of substitution, RE, reaction efficiency. The reaction conditions are given in Tab.1.

0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,20,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

DS RE

nNaOH/nAGU

DS

10

15

20

25

30

35

40

45

50

RE

(%)

Fig.2. The effect of various molar ratio of NaOH to starch (AGU) on the DS and the RE for the carboxymethylation of pigeon pea starch. nNaOH, moles of sodium hydroxide. nAGU, moles of anhydroglucose unit. DS, degree of substitution, RE, reaction efficiency. The reaction conditions are given in Tab.1.

16

0,08 0,10 0,12 0,14 0,16 0,18 0,200,00,10,20,30,40,50,60,70,80,91,01,11,2

DS RE

H2O/IPA

DS

20

30

40

50

60

70

80

RE (%

)

Fig.3 The effect of various ratio of H2O to organic solvent in the reaction medium on the DS and the RE for the carboxymethylation of pigeon pea starch. IPA, isopropyl alcohol, DS, degree of substitution, RE, reaction efficiency. The reaction conditions are given in Tab.1.

30 35 40 45 50 55 600,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

DS RE

Temperature (OC)

DS

10

20

30

40

50

60

RE (%

)

Fig.4 The effect of temperature on the DS and the RE for the carboxymethylation of pigeon pea starch. DS, degree of substitution, RE, reaction efficiency. The reaction conditions are given in Tab.1.

17

0 1 2 3 4 50,0

0,1

0,2

0,3

0,4

0,5

0,6 DS RE

Time (h)

DS

20

25

30

35

40

45

RE (%

)

Fig. 5 .The effect of reaction time on the DS and the RE for the carboxymethylation of pigeon pea starch. DS, degree of substitution, RE, reaction efficiency. The reaction conditions are given in Tab.1.

0.1

0.2

0.3

0.4

0.5

0.6

DS RE

DS

Ethanol Butanol Methanol Isopropanol

10

20

30

40

RE (%

)

Fig.6. The effect of various organic solvent on the DS and the RE for the carboxymethylation of pigeon pea starch. DS, degree of substitution, RE, reaction efficiency. The reaction conditions are given in Tab.1.

18

Fig.7. Scanning electron micrograph of native pigeon pea starches. Magnification X 1000.

Fig.8. Scanning electron micrograph of carboxymethyl starch (CMP-1, DS 0.72) Magnification X 1000.

19

10 20 30 40 50 60Diffraction angle 2 Θ

NPPS

CMP-1

Inte

nsity

Fig. 9 The wide-angle X-ray diffractograms of native pigeon pea starch (NPPS, moisture content, 8.2%) and a carboxymethylated pigeon pea starch (CMP-1, DS 0.72, moisture content 7.2 %).

4000 3500 3000 2500 2000 1500 1000 5000

5

10

15

20

25

30

35

40

Wavenumber (cm-1)

16001426

1324 1017

1643

2931

Tran

smitt

ance

(%)

NPPS

CMP-1

Fig. 10 The Infrared spectra of native pigeon pea starch (NPPS) and a carboxymethylated pigeon pea starch (CMP-1, DS 0.72).

20

Fig.11. The broad band 13C NMR spectrum (D2O, 5000 scans) of ultrasonically degraded carboxymethylated starch (CMP-1, DS 0.72). Inset: The enlarged range of 65 – 85 ppm. R = –CH2COONa or H depending on the DS. S= Carbon with a carboxymethyl substituent; U = Carbon without a carboxymethyl substituent.

Fig.12. The DEPT 135 NMR (D2O, 5000 scans) spectrum of ultrasonically degraded carboxymethylated pigeon pea starch (CMP-1, DS 0.72). Inset, the enlarged range of 71 – 73 ppm. R = –CH2COONa or H depending on the DS. DEPT, Distortionless Enhancement by Polarization Transfer. S= Carbon with a carboxymethyl substituent; U = Carbon without a carboxymethyl substituent.

180 160 140 120 100 80 60

80 78 76 74 72 70

ppm

ppm

C-8

C-6u

C-1C-4

C-3s C-2s

C-5C-3u

C-2u

C-6s

C-7

O

OCH2COONa

ORO

RO7 8

5

1

23

4 6

O

OCH2COONa

ORO

RO7 8

5

1

23

4 6

1 8 0 1 6 0 1 4 0 1 20 1 0 0 8 0 6 0

73 72 71

ppm

C -6 u

C -7

C -6s

21

Temperature (OC)

Endo

ther

mic

hea

t flo

w

TO

TPTCΔ H = 9.12 J/g

NPPS

CMP-1

70 75 80 85 90 95

Fig.13. The differential scanning calorimetry thermograms of native pigeon pea starch (NPPS) and a carboxymethylated pigeon pea starch (CMP-1, DS 0.72).

0 100 200 300 400 500 600

0

20

40

60

80

100

-12

-10

-8

-6

-4

-2

0

2

-4

-3

-2

-1

0

1

TG(%

)

Temperature (OC)

TG DTG DTA

DTG

(%/m

in)

DTA

(uV

)

Fig. 14. Thermogravimetry (TG), Derivative Thermogravimetry (DTG) and Differential Thermal Analysis (DTA) for native pigeon pea starch.

22

0 100 200 300 400 500 600

0

20

40

60

80

100

-12

-10

-8

-6

-4

-2

0

2

-4

-3

-2

-1

0

1

TG(%

)

Temperature (OC)

TG DTG DTA

DTG

(%/m

in)

DTA

(uV

)

Fig. 15. Thermogravimetry (TG), Derivative Thermogravimetry (DTG) and Differential Thermal Analysis (DTA) for carboxymethylated pigeon pea starch (CMP-1, DS 0.72).

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