THESIS

5. Determination of amylose-amylopectin content
5.1 Apparatus and reagent
5.1.1 Spectrophotometer (Hitachi 100-6 , Japan)
5.1.2 Hot plate
5.1.3 100 ml volumetric flasks
5.1.4 Glassware (Pipettes, beaker, cylinder)
5.1.5 Amylose, Type III from potato
5.1.6 Ethanol, 95 %
5.1.7 Sodium hydroxide, 1.0 N and 0.09 N
5.1.8 Acetic acid, 1.0 N
5.1.9 Iodine solution, .02 % I2 and 2 % KI in distilled water
5.2 Method determination
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Amylose and amylopectin content of tested samples was determined
using the colorimetric method, according to the Juliano et al. (1971) procedures.
Weigh duplicate 100 mg (db) starch samples and quantitatively transfer to 100 ml
volumetric flasks. Add L ml of 95 % ethanol, carefully washing down any sample
adhering to side of the flasks. Add 9 ml 1 N sodium hydroxide to each sample and
heat in a boiling water for 10 minutes and cool to room temperature. Make the
solutions to 100 ml volume with distilled water and votex vigorously and obtain 1
mg/ml solution. Let stand for at least 2 hours before continuing with the next steps.
Pipette 5 ml of the sample solutions (or else each of standard mixtures of amylose and
amylopectin prepared for working solutions) into 100 ml volumetric flasks,
containing about 50 ml distilled water. Add 1.0 ml N acetic acid and mix. Add 2 ml
iodine solution. Make up to 100 ml volume with distilled water, mix and let stand for
20 minutes. Read absorbance at 620 nm. For a blank, prepare using 5 ml 0.09 N
NaOH, instead of the sample solution in the previous step. Percent ash content was
calculated as follow:
% Amylose content = Amylose concentration from standard curve (mg/100 ml) ×10
5 × wt of dry rice starch (gram)