THESIS

1) Standard curve procedure
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Using the glucose standard solution (100 µg/ml) and distilled water as
indicated in the Appendix Table A2. Pipette aliquots of the glucose standard into
clean test tubes (triplicates for each concentration) such that the tubes contain 0, 20,
40, 60, 80 and 100 µg/ml. The 0 µg/ml sample tube was used to prepare the reagent
blank. Pipette 1 ml of each glucose concentration into test tube. Add 1 ml of 5%
Phenol solution into each tube from Part 2.4.2 and immediately mix on a Vortex test
tube mixer. Add 5 ml H2SO4, concentrated to each tube from Part 2.4.3. The sulphuric
acid reagent should be added rapidly to the test tube. Direct the stream of acid against
the liquid surface rather than against the side of the test tube in order to obtain good
mixing. Mix on a Vortex test tube mixer. Let the tube 4 to stand for 30 min in hooder.
Read absorbance at 485 nm by using Spectrophotometer. Plot absorbance at 485 nm
against the assigned glucose content for the standard curve (Appendix Figure A 2).
2) Analysis of total sugar in samples
The total sugar for the native starches, debranched products and RS III
sample were purified and destructured by dissolving in DMSO prior dilution. In
sample tubes, the sample tested was completely solution and contained 20 – 100
µg/ml by dilution. After dilution as indicated, pipette 1.0 ml of sample into a test tube
and analysis following step as in standard curve procedure. Analyze each diluted
sample in duplicate. Calculate the concentration of glucose in the samples in term of
glucose (µg/ml) as following equation:
Total sugar (µg/ml) = Absorbance of sample × Dilution
Slope