Starches are used in large quantities in various industrial applications to provide body and consistency to solutions (Ragheb et al., 1995). However, natural starches often do not match the properties required for a particular application (BeMiller, 1997). The inherent objectionable characteristics of native starches include poor aqueous dispersion and poor freeze thaw stability.
Carboxymethylation is used to improve aqueous dispersibility and cold storage stability of starch pastes. In carboxymethylation, the hydroxyl groups of linear amylose and branched amylopectin molecules of starch granule are derivatized to form carboxymethyl starch ethers. This derivatization interferes with the side-by-side alignment of starch hydroxyl groups such that when the starch is pasted in water, the granules disrupt and the carboxymethyl groups stabilize the aqueous dispersion through improved starch-water interaction. In addition, since carboxymethylation occurs without degradation of starch molecules, they are excellent thickeners and have found application in many industrial formulations (Nabais et al., 2007; Kittipongpatana and Sirithunyalug, 2006; Kim and Lim, 1999; Ragheb et al., 1997). The numerous applications of carboxymethyl starch require large-scale production of it to match demand. Hence the need to study conditions which optimize the carboxymethylation process. A few of these works have been reported by Khalil et al. (1990), Raval et al. (1994) and Tijsen et al. (2001). This study was carried out in continuation of the search for the best medium and the reaction conditions which favor carboxymethylation process.
MATERIALS AND METHODS
Materials: The cassava starch sample used in the carboxymethylation reactions was prepared in our laboratory as described elsewhere (Ogunmola et al., 2001). The sodium monochloroacetate was prepared from monochloroacetic acid according to the method of Green (1963). All other chemicals used were products of British Drug House and of laboratory grade.
Carboxymethylation of starch in different solvent-water mixtures: Starch
(5.0 g (dry basis, d,b)) was weighed into large heating tubes, to each was added
10 mL of appropriate solvent-water mixtures (0-80% solvent range) containing
28 mmol of sodium hydroxide. This was followed by addition of 1.08 g of sodium
monochloroacetate and the dispersions thoroughly mixed. The dispersions were
set up in a thermostatic Clifton water bath maintained at 45°C for 1 h.
The dispersions were kept agitated throughout the reaction. At the end of reaction
time, the samples were brought down and recovered by washing with excess aqueous
80% methanol and finally with methanol. Plasticized samples were granulated
by grinding in excess aqueous 80% methanol and finally washed with methanol.
The modified starch materials were dried in the oven at 100°C for 1 h and
stored. The solvents used were xylene, n-propanol, methanol and water.
Carboxymethylation of starch in aqueous 80% n-propanol Effect of Starch-Liquor Ratio (SLR): Starch (5.0 g d,b) was weighed into large heating tubes, each containing 28 mmol of sodium hydroxide in different volumes of aqueous 80% n-propanol to give different SLRs. This was followed by the addition of 1.08 g of sodium monochloroacetate and the dispersions thoroughly mixed. The samples were set up in a thermostatic Clifton water bath maintained at 45°C for 1 h. At the end of reaction time, the modified starches were processed as described earlier.
Effect of sodium hydroxide-reagent molar ratio (NaOH/RMR): The effect of NaOH/RMRs at SLR of 1:3 was investigated by treating starch (5.0 g, d.b.) in different large heating tubes with 15 mL of aqueous 80% n-propanol containing different amounts of sodium hydroxide (9.25-65.25 mmol). This was followed by the addition of 1.08 g sodium monochloroacetate and the dispersions thoroughly mixed. The samples were kept in a thermostatic Clifton water bath maintained at 45°C for 1 h. At the end of reaction time, the modified starch samples were processed as earlier described and stored.
Effect of Reagent-Starch Molar Ratio (RSMR): The effect of RSMR was investigated at NaOH/RMR of 4.0 and SLR of 1:3 in aqueous 80% n-propanol containing 37.25 mmol of sodium hydroxide. Thus to 5.0 g starch (d,b) in large heating tubes containing the stated reaction mixture were added different amounts of sodium monochloroacetate to give different RSMRs (0.1- 0.7). The dispersions were kept in a Clifton water bath maintained at 45°C for 1 h and resulting product processed as earlier described.
Effect of duration: The effect of duration was investigated at NaOH/RMR of 4.0, RSMR of 0.35 and SLR of 1:3, in aqueous 80% n-propanol at different reaction times of 0.5-5.0 h in a thermostatic Clifton water bath at 45°C. The modified starch samples were processed as earlier described and stored.
Effect of temperature: Starch was treated at the same reaction conditions as used in effect of duration except that the reaction temperature was maintained at 55°C. The resulting modified starch samples were processed as earlier described and stored.
Analysis by reaction parameters
Determination of percentage substitution: The percentage substitution was determined according to the method of Wing (1996) with some modification. 1.0 g of carboxymethyl starch was converted to the H-form by treating with excess 0.1 M aqueous 80% methanolic HCl in a 100 mL beaker with occasional stirring for 1 h. This was filtered and washed under suction in a sintered glass funnel with aqueous 80% methanol until free from acid (filtrate had no effect on blue litmus paper). The resulting sample was dried in the oven at 100°C for 1 h and cooled in a desiccator. 0.25 g of the sample was weighed into a 250 mL conical flask and 100 mL distilled water added, followed by 10 mL of standard NaOH solution. This was heated over a boiling water bath for 20 min when a clear solution resulted. The hot solution was titrated with standard HCl solution to a phenolphthalein endpoint. Native starch processed similarly as above was used as correction factor for the blank. Each sample analysis was carried out in triplicate and values averaged.
The percentage carboxyl was calculated as follows:
Determination of degree of substitution and Reaction Efficiency (RE): The DS was calculated according to the method of Khalil et al. (1990) and the RE by the method of Kweon et al. (1996).
Statistical analysis: One-way Analysis of Variance (ANOVA) was conducted on each of the variables. Duncan multiple range test at p<0.05 was performed using SPSS/11 software for windows to compare the differences among means. The results were expressed as the means±standard deviation of three separate readings. Non-linear regression was performed using Microsoft Excel 2003 software to generate the regression models and the corresponding R2 values.
RESULTS AND DISCUSSION
Sodium monochloroacetate reacts with starch in the presence of sodium hydroxide to form carboxymethyl starch. The process involves two main reactions:
||The carboxymethylation reaction which proceeds through the
||The hydrolysis reaction leads to loss of sodium monochloroacetate
in side reactions with formation of sodium glycolate.
The extent of carboxymethylation depends on which of the two reactions-carboxymethylation and hydrolysis- prevails under a set of reaction conditions.
Since the main objective of this study was to establish the conditions which favor carboxymethylation, the effect of nature of reaction medium, SLR, NaOH/RMR, RSMR, duration and temperature were monitored by following the DS and RE to optimize the reaction conditions.
Effect of nature of reaction medium: Table 1 shows
that DS and RE varied significantly (p<0.05) with percentage solvent in solvent-water
mixtures. With a given solvent,the highest DS was obtained in a reaction medium
consisting aqueous 80% solvent. Khalil et al. (1990) have reported similar
observation. Aqueous 80% n-propanol gave the highest DS and therefore provides
the best medium for carboxymethylation reaction. Figure 1
shows the effect of solvent dielectric constant on the carboxymethylation reaction.
The DS was not significantly different in xylene, methanol and water, but different
in n-propanol. Among the hydroxy solvents, that is, n-propanol, methanol and
water, the DS decreased with increase in solvent dielectric constant (Marsden
and Mann, 1963), hence hydrolytic loss of monochloroacetate was favored by increasing
solvent polarity. The low DS in xylene may be attributed to its large molecular
size (Patel et al., 1993).
Since aqueous 80% n-propanol provided the best medium for reaction, further carboxymethylation studies were carried out in it.
Optimization of carboxymethylation reaction in aqueous 80% n-propanol
of Starch-Liquor Ratio (SLR) on extent of carboxymethylation of starch:
Figure 2 shows the effect of SLR on extent of carboxymethylation.
An increase in DS was obtained from SLR 1:1 to 1:3 and decreased again as SLR
increased to 1:5. It is worth noting the limiting effect of SLR on extent of
carboxymethylation. The results show that there is a critical amount (volume)
of liquor required for swelling of the starch granule and dissolving the reagents
as well as facilitating dissociation, diffusion and adsorption of the etherifying
agent on starch granule for reaction to occur.
||Effect of solvent dielectric constant on degree of substitution
||Effect of percentage solvent in solvent-water mixture on extent
of carboxymethylation of starch
|a-e: Means in the same column with different superscripts
are significantly different at p<0.05, Values are means of triplicates±standard
The SLR 1:3 constitute that optimum for aqueous 80% n-propanol. Khalil et
al. (1990) have reported maximum DS at SLR 1:2.5 in water. The gradual decrease
in DS above SLR 1:3 shows the effect of increasing dilution on extent of carboxymethylation.
Effect of sodium hydroxide-reagent molar ratio (NaOH/RMR): The result in Fig. 3 shows that the DS and RE increased to a maximum at NaOH/RMR of 4.0 and then decreased sharply again with increasing NaOH/RMR. This observation points to the role of sodium hydroxide in the carboxymethylation reaction. Sodium hydroxide is needed to swell the starch granule to facilitate the carboxymethylation process. Sodium hydroxide attacks the starch molecule to form sodium starchate, which then reacts with the sodium monochloroacetate in a nucleophilic attack to form the carboxymethyl starch (Eq. 1 and 2). For a high DS to take place, sufficient alkali must be present to generate enough starchate ions for the reactions. This optimal alkali concentration was obtained at NaOH/RMR of 4.0. This is why the DS and RE increased gradually up till NaOH/RMR of 4.0. The highest DS obtained was 0.205 and the corresponding RE was 68.3%. Above this NaOH/RMR, excess alkali was present in the reaction medium, the resultant effect was the hydrolytic loss of the sodium monochloroacetate in side reactions and the formation of sodium glycolate (Eq. 4). Thus carboxymethylation and hydrolysis were competing reactions in the process. Carboxymethylation prevailed up till NaOH/RMR of 4.0 but above this point hydrolysis of the monochloroacetate predominated. This explains why both the DS and RE decreased at higher NaOH/RMR.
Effect of Reagent-Starch Molar Ratio (RSMR): From Fig.
4, the increase in DS against RSMR obeyed a second-order polynomial (R2
= 0.94), conversely the RE decreased polynomially (R2 = 0.79) against
RSMR. The highest RE was 98.0% at RSMR of 0.1. The intercept of the R.E and
DS curves was at RSMR of 0.35. Thus RSMR of 0.35 corresponds to the optimum
RSMR for carboxymethylation of starch with best DS and RE under the stated conditions
in aqueous 80% n-propanol. The DS under this condition was 0.230 and its corresponding
RE was 65.0%. The enhancement of DS by increasing the RSMR is equivocally due
to greater availability of the sodium monochloroacetate in the proximity of
the starch molecules, since the sites of carboxymethylation are the starch hydroxyl
groups, which are immobile. On the other hand, the decrease in RE is attributed
to the increasing concentration of sodium monochloroacetate in the alkaline
reaction medium, which favored glycolate formation (Khalil et al., 1990).
||Effect of starch-liquor ratio on extent of degree of substitution
in aqueous 80% n-propanol
||Effect of NaOH/reagent molar ratio on degree of substitution
in aqueous 80% n-propanol
||Effect of reagent starch molar ratio on degree of substitution
in aqueous 80% n-propanol
Effect of time and temperature: From Fig. 5, the DS
and RE varied polynomially with time of reaction at both 45°C and 55°C.
There was better correlation between DS and RE with reaction time at 45°C
(R2 = 0.98) than at 55°C (R2 = 0.78). At 45°C,
the maximum DS and RE of 0.297 and 84.8%, respectively were achieved after 3
h treatment, whereas at 55°C under the same conditions, the highest DS and
RE of 0.296 and 84.6%, respectively were obtained after 0.5 h.
||Effect of duration and temperature on degree of substitution
in aqueous 80% n-propanol
This shows that the rate of carboxymethylation is greatly increased with rise
Cassava starch was carboxymethylated in different solvents and solvent-water mixture. The results showed that the extent of carboxymethylation is a function of nature of solvent used. In all reaction media, aqueous 80% solvent gave the highest degree of carboxymethylation, with aqueous 80% n-propanol providing the best medium for caboxymethylation.
Optimization studies carried out in aqueous 80% n-propanol showed that SLR of 1:3, NaOH/RMR of 4.0 and RSMR of 0.35 gave the best result in terms of DS and RE. A higher temperature was required to effect the reaction at shorter time as indicated by the highest values of DS and RE being achieved at 55°C in 0.5 h against 3.0 h required to attain the same values of DS and RE at 45°C.