11 Chapter 3

3. Materials & Methods

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Chapter 3

MATERIALS AND METHODS

Materials

Good quality starches of high purity were extracted from cassava, Dioscorea alata,

Amorphophallus, and Xanthosoma tubers harvested from CTCRI Farm, Thiruvananthapuram.

Commercial grade arrowroot starch was purchased from Kovalam Starch Industries,

Kovalam, Thiruvananthapuram, Kerala, India, All the chemicals used for the modification

processes were of analytical grade. Paracetamol, Magnessium stearate and talc of IP grade

were procured from Pandia chemicals, Chennai.

3. I. Experimental Methods for modified starch synthesis

3.1.1 Heat-Moisture Treatment (HMT) of Starch

Heat moisture treatment is a process that involves treatment of starch granules at low

moisture level for a certain periods at a temperature above glass transition points. HMT was

carried out by first adjusting the moisture content of starch to a level up to 20% by adding

distilled water to the starch having 13% moisture level. The equilibrated sample was then

air-dried to allow the moisture content to drop to the desired level. The samples were taken

in large glass petridishes and covered with aluminum foil and placed inside the autoclave (

Remi,India Ltd) and treated at 121 lbs for 3hrs. After cooling the jar was opened and the

starch samples were air dried to a moisture content of 10% and stored in polythene bags for

analysis.

3.1.2 Acid modified starch

Starch samples were weighed in a conical flask, to which 2.2M HCl was added. The flask

was kept at 350C in water bath for 48 hours. The zero hour samples were not placed in water

bath, the conical flask was slightly agitated at definite intervals. After removing from the

water bath, the pH of the residue was adjusted to 7.0, filtered, dried in vacuum oven and

stored free from moisture.


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3.1.3 Hydroxypropyl starch

Hydroxypropylation of starch was accomplished by the modified method of Kim et al (1992).

Dried starch sample (100g) was suspended in distilled water (200ml) containing sodium

sulphate (20g). The pH was adjusted to 11.3 using 1N NaOH. The mixture was placed in

screw cap jars in water bath equipped with shaker. The temperature was maintained at 350C.

Propylene oxide (30ml) was added into the jars and sealed immediately and vigorously

shaken and subsequently returned to shaker bath. The reaction was allowed to proceed for 24

h by continuous shaking at 120 rpm. The reaction was terminated by adjusting the pH to 5.5.

The residue was washed with distilled water, centrifuged and dried.

3.1.4 Cross linked starch

Cross linked starch was prepared by the modified method of Wurzburg (1986). 100 g of

starch was weighed and slurried in 200 ml of 0.5% NaOH. Epichlorohydrin (0.5% v/w) was

slowly added with agitating using magnetic agitator. After agitating for 5 h at room

temperature, reaction was terminated by adjustment of pH of suspension to 5.0 with 1 M

HCl. The slurry was washed with distilled water, filtered, and dried in the oven at 400C.

3.1.5 Hydroxypropylated-cross linked starch

Hydroxypropylated cum cross linking was done by the combining the method of Kim et al

(1992) and Wurzburg (1986) with slight modification. Here the modification process was

carried out through hydroxypropylation followed by cross linking. The product obtained after

hydroxypropylation was slurried again in distilled water and the cross linking process was

done as described above. The pH of the final product was adjusted to 7.0, dried and stored.

3.1.6 Starch acetate

Starch acetate was prepared by slight alteration of the method described by Sodhi and Singh

(2005). Starch (100 g) was dispersed in distilled water (225 ml) and stirred for 1 h at 30 0C.

The pH of the slurry was adjusted to 8.0 using sodium hydroxide (3%) solution. Acetic

anhydride (8 g) was added drop-wise to the stirred slurry, while maintaining the pH within

the range 8.08.4 using 3% NaOH solution. This reaction was allowed to proceed for 10 min


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after the completion of acetic anhydride addition. The slurry was then adjusted to pH 4.5 with

0.5 M HCl. The slurry was washed twice with distilled water and once with 95% ethanol,

filtered, and oven-dried at 400C.

3.1.7 Starch succinate

Starch succinate was prepared by the method of Trubiano (1987) with some minor

modifications. Cassava starch was finely powdered, weighed out into a beaker, and distilled

water was added to make 40% slurry (w/v). The pH of the slurry was adjusted using 3%

sodium hydroxide solution. To this slurry, weighed quantity of succinic anhydride (1, 3, and

5% w/v) was added in small portions, while simultaneously adding sodium hydroxide

solution to maintain the ph of the medium to desired level. The slurry was stirred for the

required reaction time. After the reaction was over, the pH of the medium containing the

products was adjusted to 6.5 using 0.5N hydrochloric acid. The product was recovered by

filtration, washed with water and re filtered. The washing was repeated and the product was

collected and dried overnight at 550C in an oven. The product was powdered and stored.

3.1.8 Starch octenyl succinte

Starch octenyl succinate was prepared by the method described by Ruan Hui et al., (2009).

For the preparation of octenyl succinate derivative, both water and dimethyl sulfamide were

used as the solvent systems. Starch (100 g, dry weight) was suspended with agitation in

distilled water or DMF to get a concentration of 40%. The pH of the suspension was adjusted

by adding 3% NaOH solution. A weighed quantity of octenyl succinic anhydride was added

(diluted five times with absolute alcohol, v/v) slowly over 1 h. The reaction was continued

for the 2 hours. After reaction, the pH was adjusted to 6.5 with 3% HCl solution, the mixture

was centrifuged, washed twice with distilled water, filtered and dried.

3.1.9 Starch phosphate

Starch phosphate having low degree of substitution was prepared by the method of Eugene(

1964). Starch phosphate was prepared by accurately weighing 12.6 gram of sodium

tripolyphospahte in 167 ml water, and 100gm of starch was slurried in this salt solution and

the mixture was stirred for 10 minutes. The slurry was filtered with suction on a fritted glass


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funnel. The filter cake was crumbled and then dried overnight at 40-500C. The starch-salt

mixture was powdered in a Waring blender and was dried at 650C for 90 minutes in an oven.

The dried mixture was transferred to a stainless steel beaker. The beaker was placed in an oil

bath pre-heated to 1250C and heated for 3 h with sufficient agitation (with a half-moon

stirrer), to give continuous movement of the entire mass. The mixture was cooled and

slurried in 750ml of 50% aqueous methanol, and stirred for 30 minutes. Filtering and

washing three times with 50% methanol recovered the product. The product was re-slurried

in 50% methanol and washing step was repeated. The filter cake was dehydrated by washing

with absolute ethanol. The product was dried in oven and packed in bottles

3.1.10 Carboxymethyl starch

Carboxymethyl starch was prepared by dissolving chloroacetic acid (94.5g) in isopropyl

alcohol (700ml), and neutralized with aqueous sodium hydroxide. The mixture was stirred

vigorously. Subsequently air-dried starch (91.3g) and dry NaOH were added. The mixture

was kept at 40 0C for 4h. After carboxymethylation, the mixture was neutralized with glacial

acetic acid and was washed several times with 80% aqueous methanol, and was dried at 60 0C.

3.1.11 Enzyme modified starch

Enzyme modification of starch was achieved using the enzyme -amylase (Porcelain

pancreatic amylase). 40% starch slurry was prepared by adding 40gm of starch to 100ml of

distilled water, and enzyme (100l) was added. The samples were allowed to stand at room

temperature for 6 h. The flasks were then kept in the incubator at 50 0C for 3h. After

incubation, the supernatant was decanted and the starch was filtered and dried in the oven at

500C.

3.2 Determination of degree of substitution of starch derivatives.

3.2.1 DS of starch succinates

The level of succinylation of the modified starches was determined using the titrimetric

method described by Wurzburg (1964). Accurately weighed quantity (0.5 g) of the starch


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sample was taken in a conical flask (100 mL) and dispersed in distilled water (5 mL). The

sample was mixed thoroughly and added sodium hydroxide solution (0.5 N, 20 mL). The

solution was kept for 72 h with occasional swirling of the flask. The excess alkali was back

titrated with hydrochloric acid (0.5 N). A blank was simultaneously titrated with native starch

as a sample.

For the high DS succinate derivatives, DS was determined according to the method described

by Wurzburg (1964). Accurately weighed quantity of starch (0.5 g) was dispersed in aqueous

ethanol (75 %, z25 mL) in a conical flask. The flask was loosely stoppered, warmed to 500C

for 30 min, cooled and sodium hydroxide solution (20 mL, 0.5N) was added. The flask was

stoppered and allowed to stand for 72 h with occasional swirling. The excess alkali was back

titrated with standard hydrochloric acid solution (0.5 N).A blankwas titrated using the native

starch.

Degree of Substitution (DS) and Reaction Efficiency (RE) were calculated according to

Wurzburg (1964).

Where, 162 = molecular weight of glucose unit

10,000 = 100 molecular weight of succinyl group and

99 = molecular weight of succinyl group1.


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3.2.2 Degree of Substitution of Starch Phosphates

The phosphorus contents in the native and phosphorylated starch products were determined

colourimetrically by the reaction with ammonium molybdate, according to the standard

procedure (Jackson, 1967). The starch sample (1 g) was digested with triacid mixture (nitric

acid : perchloric acid : sulphuric acid = 10 : 4 : 1) (15 mL). When the contents became clear,

transferred to a standard flask (50 mL) and diluted with distilled water (10 mL). Ammonium

molybdate reagent (5 mL) was then added and made up to the mark. A blank was prepared

with molybdate reagent (5 mL). The absorbance of the sample was measured at 490 nm using

a uv-visible spectrophotometer (Systronics, India). A standard curve was constructed by

determining the absorbance values of different concentrations of a standard solution of

potassium dihydrogen orthophosphate. The DS was calculated using the equation of Paschall

(1964) as follows:

Where, P = % phosphorus content (dry basis) of the phosphorylated starch.

3.2.3 Degree of Cross-linking

The degree of cross-linking of the starches treated with epiclohydrin was determined

according to the method of Chatakanonda et al., (2000) from the viscosity values recorded as

follows: a 10 % starch suspension (2.5 g in 25 g distilled water) was heated from 500C to

950C and then cooled back to 500C in a Rapid Visco Analyzer using a built in profile. The

peak viscosity of the starch (the maximum viscosity attained by the starch paste during the

heating cycle) was recorded. The degree of cross-linking was calculated using the following

equation,

Where, A is the peak viscosity in RV units of the native starch and B is that of the cross

linked starch.


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3.2.4 Molar Substitution of Hydroxypropyl Derivatives

In the case of hydroxypropyl groups, which can react further with the reagent to form a

polymeric substituent, the term molar substitution (MS) is used to denote the level of

substitution in modified starches, which represents the moles of monomeric units in the

substituent per mole of AGU (Wurzburg, 1986a). The % hydroxypropyl groups and MS of

the modified starches were determined spectrophotometrically by the method of Johnson

(1969) with slight modifications as reported in

http://www.fao.org/docrep/W6355E/w6355e0o.htm. Starch (100 mg) was weighed accurately

into volumetric flasks (50 mL) and dilute H2SO4 (25 mL, 1 M) was added to each. The

mixture was heated in a boiling water bath to dissolve the starch, cooled and made up to

volume with distilled water. An aliquot of each (1 mL) was transferred to graduated test tube

(25 mL) with glass stopper. The tubes were immersed in cold water and conc. H2SO4 (8 mL)

was added drop wise to each tubeand mixed well. The tubes were then placed in boiling

water for exactly 3 min and then transferred to an ice bath until the solution was chilled and

the ninhydrin reagent (3 % ninhydrin in 5 % sodium bisulphite, 0.6 mL) was added by

carefully allowing the reagent to run down the wall of the tube. The solutions were shaken

thoroughly and placed in a water bath at 250C for 100 min. The solutions were then made up

to volume with conc. H2SO4 and the tubes were inverted several times without shaking. The

absorbance of the solutions at 590 nm was measured after 5 min with a solution based on

unmodified starch as the reference. An average of three readings for each sample was used

for calculations. A calibration curve was constructed using aqueous solutions of propylene

glycol (10- 50 g/mL). A factor of 0.7763 was used to convert weight of propylene glycol

into that of hydroxypropyl group (HPG).

The % HPG was calculated using the following equation:

HPG (%) = C 0.7763 50

W 10

where, C = amount of propylene glycol in the sample solution read from the calibration curve

(g),


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W = weight of the sample (mg).

The number of moles of propylene oxide per AGU (MS) was calculated using the following

equation (Moser, 1986):

3.2.5 DS of OSA starches

The DS was determined by alkali saponification followed by back titration of excess alkali.

Octenyl succinylation level of the modified starches was determined using the titrimetric

method of Whistler and Paschall (1967). 25 ml of a 0.5 N aqueous NaOH solution was added

to the suspension of the OSA starch (5 g of starch in 50 ml distilled water) and then shaken

for 24 h. Excess of alkali was titrated with 0.5 N HCl, using phenolphthalein as an indicator.

A blank was simultaneously titrated with native unmodified starch. DS was determined from

% OSA substitution. The calculation was as follows:

% OSA substitution = Vblank - Vsample x 0.1xNx100

W

Whereas Vblank the volume of HCl requires for the blank titration

Vsample the volume of HCl required for the sample titration, W weight of sample taken (g), N

is the normality of the HCl solution.

DS = 162x % OSA substitution

2100 - (209 x % substitution)

Whereas 162 = molecular weight of glucose unit;

2100 = 100 x molecular weight of octenyl succinte group


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209 = molecular weight of octenyl succinte group

3.2.6 DS of starch acetate

Determination of DS of acetylated starch by titration involved complete basic hydrolysis of

the ester linkages and titration of the excess alkali (Hui Chi a et al, 2008). Native and

acetylated starches (0.50 g) were weighed accurately and added into the sodium hydroxide

solution (25 mL, 0.5 N). The mixture was stirred for 72 h at50 rpm/min, at room temperature.

After indicator (3 to 5drops of 1% thymolphthalein) was added the solution was immediately

titrated with 0.5 N hydrochloric acid to the thymolphthalein endpoint. Reference sample was

treated in a similar way. Acetyl content (x) was calculated according to the following

equation.

Where: v1 the volume of 0.5 N HCl in mL used for titration of 0.50 g native starch;

v2 the volume of 0.5 N HCl in mL used for titration of 0.50 g sample;

N the normality of HCl solution; m the weight of the sample;

43 the molecular weight of the acetyl group;

3.2.7 DS of carboxymethyl starch

Titrimetry was used for the determination of the DS of Carboxymethyl starch (Xia Li et al.,

2010) CMS(10g) was dispersed in acetone(300ml) and 5 M HCl was added to the dispersion

which was stirred for 30 minutes. 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 filtered and dried for

another 24hours in a desiccators over silica gel. 2g of H-CMS was dissolved in 1% (w/v)


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NaCl solution and it was titrated with 1M NaOH solution. The DS was determined using the

following calculation

DS = nNaOH x M0

mc_ nNaOH X MR

mc = mp mpF

1000

M0: molar mass of the anhydrous glucose units = 162g/mol

MR molar mass of carboxymethyl residue = 58g/mol

nNaOH The quantity of sodium hydroxide used(mol)

mpWeight of polymer taken (g)

mc Corrected weight of polymer (g)

F moisture (%)

3.3 Characterisation Techniques

3.3.1 FTIR

The FTIR spectra of starch samples were recorded using an FTIR spectrometer (ABB FTLA

2000, ABB Inc. Analytical and advanced solutions, Canada) by pelletisation with KBr in the

range of 4500-400 cm-1. Each interferogram was generated by signal averaging 32 scans and

the spectra were obtained as percentage transmittance versus wave number


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3.4 Analytical Procedure

3.4.1 Swelling volume and Solubility

The swelling volume and solubility of the starch complexes were determined according to the

standard procedures (Schoch, 1964, Crosbie, 1991). The sample (400mg) was weighed out

into a conical flask (100mL), and dstilled water (40 mL) was added. The samples were kept

in a boiling water bath for 20 minutes with continuous swirling of the flask. After cooling the

samples were transferred to graduated centrifuge tubes and centrifuged at 2000 rpm for 20

minutes. The height of the gel was noted and recorded as swelling volume in mL. From the

supernatant, 10 mL was pipetted out carefully into a preweighed petridish and evaporated in

an air oven at 65C over night. The weight of the residue was measured for calculating

solubility.

3.4.2 Total amylose content

Total amylose contents of the derivatives were determined iodimetrically using the standard

procedure described by Morrison and Laignelet (1983) using pure amylose (sigma) as

standard and I2-KI solution (iodine-potassium iodide solution). Starch sample (40 mg) was

weighed out into a conical flask (50 mL), 10 mL U-DMSO soluion (6M urea 10 mL +90 mL

DMSO) was added and heated for 15 minutes in a boiling water bath with continuous

shaking. The flasks were kept in an air oven at 100o C for 1h. After cooling, an aliquot

(1mL) was pipetted out into a standard flask (100 mL) and was made up using 2 mL I+-KI

solution and distilled water. The absorbance was noted after 15 minutes at 635 nm. Total

amylose was estimated by pipetting 1 mL of the starch DMSO solution into a test tube,

adding 9 mL absolute ethanol and keeping over night wrapped with aluminum foil. The tubes

were centrifuged at 2000 rpm, decanted and an aliquot (1mL) of the U-DMSO solution was

added to the residue. The tubes were kept in boiling water bath for 10 minutes. The clear

solution obtained was carefully washed into a standard flask (100mL) using distilled water

and made up using 2 mL I2-KI solution and distilled water. The absorbance was noted after

15 minutes at 635 nm. A standard curve was constructed by determining the absorbance of

aqueous solutions of amylose (20-80 g/mL) at 635 nm


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3.4.3 Water binding capacity (WBC)

The water binding capacity of the native starch and starch complexes was determined using

the standard procedure of Medcalf and Gilles (1965). A suspension of starch (1g) in distilled

water (15 mL) was agitated using a mechanical stirrer for 1 h and centrifuged (3000 x g) for

10 minutes. The water layer was decanted from the wet starch and the residue was weighed.

The water binding capacity was calculated as percentage from the weight of the water bound

by the starch sample.

3.4.4 In vitro enzyme digestibility

In vitro enzyme digestibility of the starch samples was estimated by the method of Padmaja

et al (2005) using pancreatic - amylase [pancreatin 3X (100units/mg/min) (SRL, Mumbai,

India)]. The sample (100 mg) was weighed, sodium phosphate buffer (10 mL, 0.02 M, pH-

6.9) was added and the solution was heated in a boiling water bath. After cooling, the volume

was made up to 20 mL using the buffer; pancreatic amylase solution (0.5 mL of a solution of

25 mg of enzyme dissolved in 25 mL phosphate buffer) was added and incubated at 30oC for

1 h. After the incubation period, the samples were heated in a boiling water bath to deactivate

the enzyme. The reducing sugar formed was estimated by the Nelsons method (1944).The

digestibility was calculated as percentage.

3.5. Retrogradation Studies

3.5.1 Light transmittance

Light transmittance was measured as transmittance percentage (%T) described by Craig et al

(1989). Starch samples (1% w/v) were heated in a water bath at 90 C for 1h with constant

stirring. The suspension was cooled and held at room temperature. The sample was then

stored for 4 days at 4C and transmittance was measured every 24 h at 640 nm against a

distilled water blank.


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3.5.2 Least concentration for gelation (LCG)

The least concentration for gelation of starches was determined by the method modified by

Mishra and Rai (2006). Five milliliters of starch solutions (1-10 % w/v) in test tubes was

heated at 85 C in a water bath for 15 minutes, cooled immediately in ice chilled water bath

and kept overnight at 4C. The gelation was confirmed by inverting the test tubes.

3.6 Thermal Properties

The thermal properties of the complexes and native starch were determined using a

Differential Scanning Calorimeter (Mettler Toledo DSC 822e, Schwerzenbach, Switzerland).

The sample (5mg) was weighed into an aluminium pan, deionised water (10l) was added

and hermetically sealed and transferred to the heating chamber of the DSC instrument. The

sample was heated from 25C to 125C at a rate of 10C/ min. An empty pan was used as the

reference. The information regarding gelatinization temperatures, the onset, (To) peak (Tp),

endset (Te) and enthalpy of gelatinization (H) were recorded from the thermograms.

3.7 Pasting Properties (Viscometry)

The viscosity parameters (peak viscosity, breakdown, setback viscosity, and pasting

temperature) of the complexes and the native starch were obtained using a Rapid Visco

Analyzer (RVA-4, Newport Scientific, Warriewood, Australia) controlled by thermocline for

Windows software. A fixed starch concentration (10%) was used for the study. Standard 1

measurement profile was chosen. The temperature programme was as follows: heating from

50C to 95C at 12C/min, holding at 95C for 2 minutes, cooling to 50C at 12C/min and

holding at 50C for 2 minutes. The sample+ water was stirred in an RVA canister at 960 rpm

for 10s, then at 160 rpm for the remainder of the test. Determination was done in triplicate.

The viscosity was recorded in centipoises (cP) (1cP= 1 mPas). The viscosity profile recorded

by the RVA reflects the peak viscosity (PV), breakdown (BD), final viscosity (FV), setback

(SB) and pasting temperature.


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3.8. Rheological Analysis

Rheological properties of the starch suspensions and gel were analyzed using a Physica VT2

Rheometer (Anton Paar MCR 51, GmbH, Ostfildern, Germany) equipped with a Parallel

plate system (PP20, dia: 19.957 mm, gap: 1 mm).

Different rheological analyses done were listed below:-

3.8.1 Rotational test:

a. Flow curves

Starch gels for flow curves was prepared in distilled water by placing 10% (w/v) of

suspension in boiling water bath for 20 minutes, cooled and placed in the rheometer plate.

Shear rate was increased from 0.1 to 100/s logerithmetically in a stepped ramp mode. Flow

curve test was run at 30oC for samples and plots were made of shear rate vs. viscosity and

shear rate vs. shear stress. The yield stress and infinite shear viscosity was determined using

the Casson model (Vincent et al., 2001)

b. Thixotropy / shear/ structural decomposition/recovery test

The thixotropy test was done in the 10% starch gel and the experiment was preceded by the

loop test which was carried out in three different intervals. Starch was first subjected to

constant shear rate of 1/s for 10 seconds followed by increasing the shear rate to 100/s for 10

seconds. In the last interval the shear rate was release and the recovery of the starch sample

was measured for 300 seconds. The recovery was measured using the 3 Interval Thixotropy

Test (3ITT)

3.8.2 Dynamic oscillatory measurements.

a. Frequency sweep measurements

The viscoelastic properties were measured using oscillatory test in the same system as in the

rotational test. Before starting the test the linear viscoelstic region (LVR) of the starch gels

were measured by the strain amplitude sweep test with strain ranging from 0.1 to 5%, and a


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frequency of 0.1 to 20Hz. Once the LVR was determined the rheometer was programmed for

running frequency sweep (0.1 to 10Hz) and strain of 1%. The temperature was fixed to 30oC

for the analysis. The three main parameters determined in the dynamic oscillation tests were

storage modulus G, loss modulus G and loss tangent tan .

b. Temperature sweep test

For the temperature sweep measurements, 20% (w/v) of starch suspension was prepared by

taking 20g dry starch sample in 100ml distilled water. During the temperature sweep

measurements the strain was fixed to 1% and frequency to 10Hz. The temperature was

increased from 30 to 90oC using the peltier system of rheometer. The storage modulus G,

loss modulus G and loss tangent tan . values of the starch samples were determined during

the heating process.

3.9 Tablet manufacturing and evaluations

3.9.1 Powder flow and compression properties

a. Loss on drying (LOD)

The sample (5 g) was accurately weighed into a petridish. It was then placed in an oven at

105C for 3 hours. The petridish was removed from the oven and the weight was noted and

the weight loss was recorded.

b. True density

The particle density of each starch samples was determined using the specific gravity bottle

with xylene as the displacement fluid. An empty 50-ml specific gravity bottle was weighed (

W1), filled with xylene and the excess wiped off and the weight was noted(W2). 2 grams of

sample was weighed (W3) and quantitatively transferred into the specific gravity bottle.

The excess solvent was wiped off and the bottle with starch was weighed again (W4). The

particle density rt (g/cm3) was calculated from the following equation:

rt = (W2 x W3) 50 (W3 W4 + W2 + W1)


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c. Bulk density and tapped density

The bulk density of each starch samples at zero pressure (bulk density) was determined by

pouring the starch powders at an angle of 45 through a funnel into a 50 ml glass measuring

cylinder. The weight of starch powder and the volume it occupied was noted. The relative

density (D0) of each starch was obtained from the ratio of its bulk density to its particle

density. The tapped density was determined by tapping the measuring cylinder containing

powder on to a wooden surface from a height of 3cm at a rate of 2 tapping per second. The

tappings were continued until a constant volume (300 tappings ) was obtained for the starch

samples .Tapped density was measured as the mass/ tap volume of sample after tapping.

d. Angle of repose

The flow properties were evaluated by determining the angle of repose The angle of repose

was measured by gently pouring a weighed amount of powder through a glass funnel to a

pipe (4 cm diameter and 8 cm length) fixed at a ground surface and open at both the ends.

Once the pipe is filled completely with powder the pipe is slowly removed to make a free-

standing cone of the powder. The height and diameter of the cone was measured and the

angle of repose was calculated using the following equation:

q = Tan-1 (2 h/D)

Where h is the height of the cone, and D is the diameter of the cone.

The Hausner ratio and Carrs index were used as measurement of interparticle friction

and the potential powder arch or bridge strength and stability, respectively. They have also

been also used to estimate the flow properties of powders. Hausner ratio was calculated as

the ratio of tapped density (TD) to bulk density (BD)

The Carrs compressibility index was calculated using the following equation:

(TD BD/TD) x 100

Where TD is the final tapped density, BD is the initial bulk density of the starch powder


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3.10 Formulation of paracetamol tablets using different modified starch as binder

3.10.1 Preparation of starch mucilage (paste)

Starch mucilage was prepared by weighing amounts of modified starch powder that produces

various concentrations of 2.5% w/w, 5% w/w, 7.5%w/w and 10% w/w of the modified starch

paste as binders in the tablet formulation. Each weighed quantity of starch was suspended in

a small amount of water which resulted in starch slurry and this slurry was transferred to the

required amount of boiling water on a water bath. A translucent paste was formed which was

used as the binding agent for preparing the tablets.

3.10.2 Preparation of granules

Batches (50 g) of a basic formulation of paracetamol (83.33% w/w), maize starch

(6.67%w/w), were dry mixed for 5 minutes in a mortar with a thick glass rod, and then

moistened with about 10 ml of distilled water or appropriate amounts of binder solution

(different strength of starch pastes) as binding agent. Mixing was continued for 5 minutes

and the wet masses were granulated by passing them manually through a mesh 10 sieve

(1700 mm), dried in a hot air oven for 3 hours and 50C. Dried granules were sieved through

a mesh 14 sieve and then stored in air tight containers.

3.10.3 Preparation of tablets

The dried granules were taken and mixed thoroughly with maize starch (5% w/w), talc

(1.67% w/w) and magnesium stearate (0.83%w/w) for 5 minutes in a mortar using the

spatula. The total tablet weight was fixed as 620 mg which contained 500 mg of paracetamol

along with the other excipients. 620 mg quantities of 500 1000 mm size fractions of the

granules from each formulations were weighed , and compressed for 1 second using a

Cadmach Rotary Tabletting machine at a constant pressure of 3 Tons. The punches and dies

used were flat faced with the die diameter (13 mm). After compression, the tablets were

stored over silica gel for 24 hours to allow for elastic recovery and hardening and to prevent

falsely low yield values.


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3.11 Formulation of paracetamol tablets using different modified starch as

disintegrant

3.11.1 Preparation of starch mucilage (paste)

Starch mucilages wee prepared by weighing suitable amounts of maize starch that produce

concentrations of, 5% w/w, as binder in the tablet formulation. Each weighed quantity of

starch then was suspended in a small amount of water which resulted in starch slurry and this

slurry was transferred to the required amount of boiling water on a water bath. A translucent

paste was formed which was used as the binding agent for preparing the tablets.

3.11.2 Preparation of granules

Batches (50 g) of a basic formulation of paracetamol (83.33% w/w), maize starch

(6.67%w/w), were dry mixed for 5 minutes in a mortar with a thick glass rod, and then

moistened with about 10 ml of distilled water or appropriate amounts of binder solution

(different strength of starch pastes) as binding agent. Mixing was continued for 5 minutes

and the wet masses were granulated by passing them manually through mesh 10 sieves (1700

mm), dried in a hot air oven for 3 hours and 50C. Dried granules were sieved through a

mesh 14 sieve and then stored in air tight containers.

3.11.3 Preparation of tablets

The powder (Emcompress) was mixed with different concentrations (5, 10, 15% w/w) of

starch and starch derivatives to be tried as tablet disintegrants for a period of 5 minutes in a

vessel using a spatula. The total tablet weight was fixed as 500 mg. From each formulation,

the powder was weighed and compressed using a Carver Hydraulic Press machine at a

compression load of 3 tons, resulting in a placebo tablet. The punches and dies used were flat

faced with the die diameter of 13 mm. After compression, the tablets were stored in a closed

container for 24 hours to allow for elastic recovery & hardening and to prevent falsely low

yield values.


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3.12 Evaluation of prepared tablets

The prepared paracetamol tablets were evaluated as per the Indian Pharmacopoeia tests.

Hardness

The tablets were placed horizontally in contact with the lower plunger of the Monsanto

hardness tester and the zero reading was taken. The tablet was then compressed by forcing

the upper plunger until fracture of the tablet was observed. The force of the fracture was

noted.

Friability

Ten tablets were weighed and then placed in the plastic chamber of the Roche friabilator,

which was adjusted to revolve at a speed of 25 rpm. After 100 revolutions, the tablets were

taken out, dusted and reweighed. The percentage change of weight for the tablets was

calculated.

Disintegration test

Tablet disintegration time was determined in distilled water at 370.5C in an IP

disintegration test unit. Six tablets were tested from each batch. Disintegration is considered

to be achieved when no tablet fragments remain on the screen.

Dissolution testing

The dissolution testing was carried out on the tablets using a 6 station USP standard rotating

basket type dissolution apparatus. The dissolution apparatus was rotated at 50 rpm in 900 ml

of distilled water, maintained at 37 0.5C. Samples (5ml) were withdrawn at different time

intervals and replaced with equal amounts of fresh medium. The samples withdrawn were

filtered using a What man filter paper and then diluted to a suitable proportion. The amount

of paracetamol in each sample was analyzed spectrophotometrically at 249 nm with a UV-

Visible Spectrophotometer (Systronics, India).


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3.13. Film preparation from native and modified starches

Filmogenic solution was prepared by mixing starch with gum acacia, and glycerol as follows.

Cassava starch (6 g) was dispersed in 100 ml distilled water by moderately stirring by using a

mechanical stirrer for 15 min at room temperature. Gum, 0.90 g (15% (w/v) of starch) and

glycerol 1.2 g (30% (w/v) of starch) were taken in another beaker containing 50 ml water and

stirred for 15 min. The starch suspension and gum- glycerol mixture was directly mixed and

make up the suspension to a total volume of 200 ml. hence the starch concentration in the

suspension was 3% (w/v). The mixture was heated to boiling to ensure that starch was

gelatinized fully under controlled magnetic stirring. Heating and stirring was continued for 2

min and a viscous transparent solution was obtained. Air bubbles formed during boiling were

removed by keeping the gelatinized solution in a water bath at 70 C for 30 min. Modified

starch based film was prepared by casting method in which the hot suspension at 70C was

immediately transferred to a leveled non stick teflon coated plate (25 x 25 cm) through a

cheese cloth to remove the air bubbles, if any. After drying, the films were peeled off from

the plate and used for characterization of physical and mechanical propertie

3.14 Physico-mechanical properties starch film

Moisture content

The films were cut into small strips of 1x1cm and known weights of these pieces (w1) were

taken in previously weighed petri-dish. It was kept in a convection oven at 103C. The

weight of the samples were taken at every half an hour till there was no change in weight

and the final weight of the film after drying was taken as w2. The moisture content was

expressed on wet basis, as the ratio of the moisture evaporated (w2 - w1) to the initial weight

of the sample (w1). The experiments were replicated twice and average values were reported.

Conditioning of films.

The films were conditioned at 50% RH for about one week by keeping over 45%

concentrated sulphuric acid solution taken in a dedicator. Conditioned films were used for

thickness, color and mechanical strength analysis.


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Thickness of films.

Thickness of the films was measured using a screw gauge of 0.01 mm accuracy. It was

measured at 10 different locations and average was reported.

Colour of films

The colour of the films was analyzed by measuring the colour coordinates L, a and b

using a spectrophotometer (CM 2600 D, Konica Minolta, Japan) with UV excluded (spin)

calibration mode and lens position kept as MAV. From these primary color coordinates, the

total color difference and whiteness index were calculated using standard equations.

Mechanical properties:

Mechanical properties viz, tensile force and elongation at break were measured

using food texture analyzer (TA HDi, Stable Micro Systems, Surrey, UK) with Texture

Expert Exceed software under the following conditions: mode- measure force in tension, pre

and post test speed-10 mm/sec, test speed- 2 mm/sec and distance 200 mm using a tensile

grip (A/TG). The upper tensile grip was attached to the load cell carrier and the lower grip

was secured to the base of the machine. The tensile grip was calibrated to start from a set

distance apart for each test of 50 mm. Calibration is done by lowering the grips so that they

are very close together, click on TA, then calibrate probe and specify the distance for the grip

to start apart from each for each test is 50 mm. A conditioned strip of the film (10 mm wide

and 100 mm long) was clamped in a vertical manner and test was started. When the test

commenced, the film was pulled by the upper grip upwards till the film breaks into two

pieces From the force-deformation (time) curve, the maximum peak force was noted as

tensile force and the distance to which it elongates before it breaks into two pieces is

designated as elongation at break.

Water Solubility of Films

Solubility is defined as the percentage of film dry matter solubilized after 24 hour

of immersion in distilled water and is calculated by the method described by Gontard et al

(1992). The films were cut into pieces and about one gm of film strips was weighed in a pre


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weighed conical flask. The initial percentage of dry matter was determined by drying the film

in an oven set at 103C for 24 h. After 24 h, the flask was reweighed. 20 ml distilled water

was added to the flask with periodic stirring kept for 24 hour at room temperature.

Subsequently, it was filtered to obtain undissolved film and final dry weight of undissolved

film was determined by drying again in an oven at 103C for 24h. The percentage of total

soluble matter (% solubility) was determined as follows.

Percentage water solubility= [(Initial dry weight- Final dry weight)/ Initial dry weight] 100

3.15 Statistical Analysis

All measurements were replicated three times and the results were subjected to analysis of

variance, ANOVA using GENSTAT Discovery Edition 3 package. Duncans multiple range

test was performed to determine any significant differences (p< 0.05) between native and

treatments. The mean values obtained from ANOVA were subjected to DMRT for testing

pairwise difference. The probability value (p-value) of a statistical hypothesis test is the

probability of getting a value of the test statistic as extreme as or more extreme than that

observed by chance alone, if null hypothesis is true.

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11 Chapter 3