9
1. Chemical Analysis
Chemical analysis in this research including amylose analysis and proximate analysis. Proximate analysis were done for moisture, ash, fat, protein, and carbohydrate.
a. Moisture by Air Oven Method AOAC 1995
Dry metal dish and cover at 100
o
C, cool in desiccators, and weigh soon after reaching room temperature. Accurrately weigh 2 g well-mixed sample. Uncover sample. Put dry dish, cover, and
sample in oven provided with opening for ventilation and maintained at 100
o
C for 1 h. Cover dish while still in oven, transfer to desiccators and weigh soon after reaching room temperature. Repeat
until consistent wight is obtained. Loss in weight is reported as moisture indirect method.
b. Ash by Direct Method AOAC 1995
Weigh 3-5 g well mixed sample into ashing dish that has been ignited, cooled into desiccators and weighed soon after reaching room temperature. Ignite in furnace at 550
o
C dull red until light gray ash result, or to constant weight. Cool in desiccators and weigh soon after reaching room
temperature. The remaining weight of sample is ash weight.
c. Fat by Soxhlet Extraction Method AOAC 1995
Transfer 5 g sample to defatted extraction thimble and dry 6-18 hours in small beaker at 100
o
C. Place cotton plug over paper. Add few defatted antibumping chips to 250 ml Erlenmeyer and dry 1
hour at 100
o
C. Cool to room temperature in dessicator and weigh. Place thimble containig dried sample in soxhlet, supporting it with spiral or glass beads. Add 150 ml hexane to the soxhlet or until
thimble is fully submerged. Reflux digested sample 4 hours adjusting heat so that extractor siphon 30 times. Remove flask, and evaporate solvent on steam bath. Dry flask at 100-101
o
C to constant weight 1.5-2 hours. Cool in desiccator to room temperature and weigh. Constant weight is attained when
successive 1 hour drying periods show additional loss of 0.05 fat. Duplicate determinations should agree within 0.1 fat.
Fat = g fat x 100g sample
= x 100
Notes : a = flask and final sample mass g
b = empty flask mass g c = initial sample mass g
d. Protein by Kjeldahl Method AOAC 1995
Sample of 100-250 mg was put into Kjeldahl flask. Then 1.0 ± 0.1 g K
2
SO
4
, 40 ± 10 mg HgO, and 2.0 ± 0.1 ml H
2
SO
4
were added. Boiling stones around 2-3 items were put and boil the solution for 1-1.5 hours. At distilattion stage, a little amount of water is transferred step by stage through flask
wall and shaken carefully to resform the crystal. Solution was transferred to the destillation ware, rinsed 5-6 times with 1-2 distilled water, followed by adding rinsing water to distillation ware and 8-
10 ml of 60 NaOH - 5 Na
2
S
2
O
3
. Erlenmeyer was placed under the condencer with 5 ml H
3
BO
3
and 2-4 drops of red-methylene blue added.end of condenser must be soaked in H
3
BO
3
solution. A
10 Amylose = [Amylose] x V x FP x 100
Sample weight titration stage, sample solution was diluted into 50 ml, and then titrated with 0.02 N standardized HCl
until grey color appears. Volume of HCl for titration was then reported. N =
e. Carbohydrate Content by difference AOAC 1995
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