Synthesis and Characterization of Highly Substituted Acetylated Moth Bean Starch

AKHILESH V. SINGH*1, LILA K NATH1, MANISHA GUHA2

1

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh 786004 Assam 2 Central Food Technological Research Institute, Mysore 570020 Karnataka

ABSTRACT This study was carried out to understand and establish the changes in physicochemical parameter of moth bean starch (MBS) after acetylation. Highly substituted starch acetate was prepared by reaction with native MBS with acetic anhydride using pyridine as organic solvent. Their formation was confirmed by the titrimetric analysis and Fourier transforms infra red spectroscopy (FT-IR). The presence of absorption band in FT-IR Spectra at 1748 cm-1 confirmed the carbonyl group attachment. The thermal behavior of native and acetyl substituted MBS was investigated using thermo gravimetric method (TG-DTA). The results reveal that highly substituted starch acetate was more thermally stable as compared to native form. The x-ray diffraction (XRD) patterns showed loss of crystalline nature and its transformation into amorphous form. The scanning electron microscopy (SEM) suggested that the smooth surfaces of starch granules were changed into fibrous form after acetylation. KEYWORDS: Moth bean starch, acetylation, XRD, FT-IR, SEM INTRODUCTION Starch is one of the most important reserve polysaccharides of higher plants. Native starches are readily available from different botanical origins like maize, rice, potato, cassava etc. The starch granule mainly consists of linear amylose molecules and branched amylopectin. In all the natural biopolymers, starch has received the most attention because of its low cost, ready availability, and total degradation[1]. Native starches are utilized in different forms in various commercial purposes like food pharmaceutical, textile, paper and packaging industry. However, starch has disadvantages of being hydrophilic, having poor mechanical properties, and having poor dimensional stability, especially in aqueous environments. Acetylation of starches is an important modification that has been applied to

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the native starches to impart thickening known for more than a century. In the acetylation, parts of the hydroxyl groups of the -D-glucopyranose units have been converted by esterification to acetyl groups. Highly acetylated starch with a degree of substitution (DS) of 2-3 was of research interest because of their solubility into acetone and chloroform and for their thermoplasticity[2]. The demand for modified starches in developing countries like China, India is growing annually with a growth rate of 8- 9%. So, there is a market demand to explore other tropical crops to fulfill this demand[3]. The moth bean is an edible bean belonging to the Fabaceae family. Moth bean starch is obtained from Phaseolus aconitifolius Jacq. and grown well in Southeast Asian countries as a tropical crop. Moth bean starch was earlier evaluated as the pharmaceutical raw material in our laboratory, because it is relatively cheap and widely available in south Asian region[4,5,6]. The objective of this study was to synthesize reaction parameter optimization and evaluate

physicochemical properties of highly substituted moth bean starch acetate. MATERIALS AND METHODS
Moth bean seeds were purchased from Indian Institute of Public Research Kanpur, India. Acetic anhydride and pyridine extra pure were purchased from Qualigens, India. All other chemicals used were of AR grade. Double distilled water was used in the study. Isolation of Starch from raw moth bean seeds The starch was isolated by method described by Singh et al [7]. Synthesis of acetylated moth bean starch Native MBS was firstly grinded and passed through a sieve # 120. The uniform native MBS was pregelatinized by heating it with water at 70C. The pregelatinized starch was dried and sieved through mesh # 60. The acetylation of 25g pregelatinized starch was done with 100g acetic anhydride in a medium of 200g pyridine[7]. The reaction was carried out at varying temperature (50C-150C)and time (1-6 h).The final product was precipitated with ethanol filtered and dried in a vacuum oven and passed through a sieve # 60 and stored till further study.

acetic anhydride OH starch hydroxyl group Pyridine

O O C CH3 starch acetate (1)

Determination of degree of substitution (DS) DS was determined using the method elsewhere[8]. 1 g of grounded sample accurately weighted was added to the aqueous solution of ethanol (75%). Slurry was kept in the water bath (50C) for 30 min and after the slurry was cooled down an exact amount of aqueous solution of potassium hydroxide (0.5N, 30 mL) was added and the solution was stirred for 72 h. After

indicator (phenolphthalein) was added, the excess of alkali was titrated with 0.5N hydrochloride acid. The acetyl content (%A) was calculated according to following equation: (Value for blank value for sample) (mL) (Sampling weight)(g)

(2)

%Acetyl group=

X normality of HCI X 0.043 X 100

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Synthesis and Characterization of Highly Substituted Acetylated Moth Bean Starch

The acetyl content was used to calculate the degree of substitution, (DS), according to following equation DS = 162 X % Acetyl group 4300(42 X % Acetyl) (3)

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Scanning electron microscopy of starches The morphological features of the native MBS and highest substituted starch acetate were observed with a scanning electron microscope (JEOL JSM-6100, JEOL Ltd., Tokyo, Japan). The dried samples were coated with gold to make the sample conductive. Micrographs were recorded at different magnification. Swelling study Swelling power was determined in triplicate using the method described by Liu et al [9]. Starch (1.0g dry basis) was weighed into a centrifuge tube to which 40 mL distilled water was added. The tube was heated at temperatures of 30C, 60 C and 90C in a shaking water bath for 30 min. The tubes were cooled to room temperature and centrifuged for 20 min. The supernatant was carefully poured; swelling power was determined as a ratio of sediment weight to dry starch (g/g). Swelling index = Sediment weight (g) Dry weight (g) (4)

FT-IR analysis of starches The FT-IR spectra of the native MBS and starch acetate were recorded using spectrophotometer (Perkin Elmer, USA). Potassium bromide (KBr) discs were used to prepare samples, which is mixed with Starch Samples in the ratio of 1:20. X-ray diffraction (XRD) analysis of starches X-Ray diffraction patterns of the native MBS and starch acetate were analyzed using x-ray powder diffractometer (JADE, USA) at a voltage of 40kV and a current of 200mA. The scattered ratio was detected in the angular range of 5-60 (2), with a scanning speed of 8 (2)/min and a step of 0.06 (2). Thermogravimetric analysis (TGA) of starch Thermogravimetric analysis of native and highly substituted acetylated starch was performed using thermal analyzer (Model TGA-400, Perkin Elmer, USA). Samples of approximately 5 mg were heated in an aluminum cell between 30C to 500C temperature at a heating rate of 10C/min. 2.5
Degree of Substitution (DS)

RESULTS AND DISCUSSIONS The starch acetate with high degree of substitution (DS=2.35) was synthesized by acetylation using pyridine as catalyst. The optimized temperature (Fig. 1) for synthesizing

2 1.5 1

0.5 0 0 1 2 3 Time (hr) 4 5 6

Fig. 1. Effect of varying reaction duration on degree of substitution

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2.5

Degree of Substitution (DS)

2 1.5

0.5

0 0 25 50 75 100 125 150 175 Temperature (C) Fig. 2. Effect of varying temperature on degree of substitution

(A)
% Transmittance

3430.2

1646.15

995.42

1747.6 (B) 985.8

3445.6 4000 3000 1500 Wavenumber (cm-1) 1000

500

Fig. 3. FT-IR spectrum of A) Moth bean starch, B) Starch acetate with DS 2.35.

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highly substituted acetylated moth bean starch was found to be 100C, whereas increasing the temperature caused destruction of the starch helical. The reaction duration for synthesizing the highly substituted starch was found to be 4 h (Fig. 2), and increasing the duration also caused starch granule destruction which restricted esterification of moth bean starch. The preliminary estimation of degree of substitution was done by titrimetric analysis and was found to be 2.35. FT-IR spectroscopy The formation of starch acetate was confirmed by the FT-IR spectroscopy (Fig. 3). Esterification resulted in the presence of

carbonyl absorption band peak at 1747.6 cm-1. The hydrogen bonded hydroxyl group contribute to an extremely absorption band peak at 3445.6 cm-1. The intensity of absorption peak decreased as the degree of substitution increased. XRD analysis The native MBS powder had crystalline structure, as shown in (Fig.4) the diffraction pattern. The degree of crystallinity decreased after achieving higher substitution in the starch granules because intra and intermolecular hydrogen bonds are responsible for the highly ordered crystalline structure. On achieving the highest esterification, the acetyl group replaces

MBS

Intensity (counts)

Acetylated MBS

2 (Theta)
Fig. 4. XRD patterns of Moth bean starch and Starch acetate with DS 2.35

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Fig. 5. Scanning electron micrograph of native moth bean starch (A) Highly substituted starch acetate (B)

some of the hydrogen groups on the starch backbone, which reduced the formation of intermolecular hydrogen bonding and thereby reducing the orderly crystalline structure. As the degree of substitution of starch acetate is increased the crystalline pattern is changed into the amorphous form[10]. Morphological study

Native MBS granules were of oval in shape. The starch granules lost their smooth surface texture after esterification as a result of substitution of hydroxyl groups. After reaching the highest substitution (DS=2.35), the granular structure is lost. Once the reaction completed, the starch became fibrous in nature and break

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into small fragments (Fig. 5). TGA analysis The thermogravimetric analysis was used to examine the thermal stability of MBS and starch acetate. The native MBS showed thermal decomposition at 236.5C and highest decomposition occurs at around 398.5C. The
30.93C 99.475%

modified starch acetate showed shifting of peaks i.e. onset at 246.2C and highest decomposition at 403.2C (shown in Fig 6). Shifting of the peak was observed after acetylation that confirmed its higher thermal stability; this might be due to replacement of hydroxyl groups in starch molecules after

236.54C 88.884%

(A)

Weight (%)

Endotherm (up)

398.56C 17.703%

Temperature 24.48C 99.341% 246.21C 88.228%

(B)

Endotherm (up)

Weight (%)

403.26C 13.464%

Temperature (C) Fig. 6. TGA thermogram of A) Moth bean starch, B) Starch acetate with DS 2.35

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acetylation[9,11]. Swelling study Swelling power indirectly indicates the water holding capacity of starch and it depends upon
5 4.5 4 3.5

the nature of starch and extent of modification. The swelling power of acetylated starch showed slight increase in the swelling (Fig.7) that might be due to increase in the volume due to substitution of bulky acetyl groups. Increasing

Moth bean starch Moth bean starch acetate

Swelling Index

3 2.5 2 1.5 1 0.5 0

30

60 Temperature (C)

90

Fig. 7. Comparative swelling index at various temperatures

the temperature also increased further swelling due to increase in hydration[12]. CONCLUSION Moth bean starch acetate with high degree of substitution (DS=2.35) were synthesized by the reaction with acetic anhydride in pyridine. The DS value can be controlled by varying the temperature and reaction duration. Chemical
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modification with acetic anhydride resulted in significant changes of the physicochemical property of native moth bean starch. The titrimetric analysis and IR spectra confirmed acetylation. The XRD pattern showed conversion of crystalline MBS into amorphous after high substitution. It has also been shown that acetyl substitution increased the thermal stability which is confirmed by TG and DSC

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studies. The swelling power significantly studies improved after acetylation. This modified starch could be used for different commercial application like pharmaceutical, food industry, textile, paper industry etc. REFERENCES
1. 2. H. Roper, Carbohydr. Eur. 15 (1996) 14 W. Jarowenko, In O. B. Wurzburg (Ed.) Modified Starches: Properties and Uses CRCPress Boca Raton, 1986 A.V. Singh, L.K. Nath and A. Singh, Electron. J. Environ. Agric. Food Chem. 9 (2010) 1214 A.V. Singh and L.K. Nath, Int. J. Pharm. Tech. Res.1 (2010) 365

5. 6. 7. 8.

A.V Singh, L.K. Nath, R.D. Pandey and A. Das, J. Polym. Mater. 27 (2010) 173 A.V.Singh and L.K.Nath, Lat.Am.J. Pharm.29 (2010) 279 A.V. Singh and L.K. Nath, Electron. J. Environ. Agric. Food Chem. 8 (2009) 984 K. Ogawa, I.Hirai, C. Shimasaki, T. Yoshimura, S. Ono, S. Rengakuji, Y. Nakamura and I.Yamazaki, Bull. Chem. Soc. Jpn. 72 (1999) 2785 H. Liu, L. Ramsden and H. Corke, Starch/Starke. 51 (1999) 249 Y. Xu, V. Miladinov and M.A. Hanna, Cereal Chem. 81(6) (2004) 735 E.Rudanik, G. Matuscheck and N. Milanov, Thermochimica Acta. 427 (2005) 163 N. Onanong and K. Eakphan, In: Proceedings of the first national conference on starch technology, Bangkok, Thailand, 2002

9. 10. 11. 12.

3. 4.

RECEIVED : 18 February 2011 ACCEPTED : 22 April 2011

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