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Procedure

Prepare DNS reagent


•Solution A = 10 g DNS + 200 mL 2M NaOH.
•Solution B = 300 g sodium potassium tartrate tetrahydrate + distilled water (500 mL).
•Solution A + B → Make 1 L with distilled water.

Sample preparation.
•(1.) Solid sample
• 3 g solid (in beaker) + 50 mL distilled water (warm and stir-5 minutes).
• Filter in 100 mL volumetric flask, wash residue. Make 100 mL. (mix and invert)
• 10 mL sample solution, dilute up to 250 mL with distilled water in volumetric flask).
• Mix and invert. Dilute further if necessary.
•(2.) Liquid sample
• Pipette 5 mL sample, filter in 100 mL volumetric flask.
• Wash residue with small amount of distilled water, make up 100 mL. Mix and invert
well.
• 10 mL sample solution dilute up to 100 mL (with distilled water in volumetric flask).
• Mix and invert. Dilute further if necessary.

Prepare glucose standard solution.


•Glucose stock solution 100 mg/mL concentration.
• 10 g glucose standard + 50 mL distilled water in beaker, transfer into 100 mL volumetric flask.
• Wash residue and make up to 100 mL volume. Mix and invert well.
•Repeat above step to prepare 2 mg/mL, 4 mg/mL, 6 mg/mL, 8 mg/mL, 10 mg/mL and 15 mg/mL.
•Mix well and invert it repeatedly.

Absorbance measurement
- Blank: 4 mL distilled water (pipette) + 1 mL DNS (pipette). All pipette into test tube and
labelled.
- Standard: 1 mL of each concentration of glucose standard solution + 1 mL DNS + 3 ml
distilled water (all pipette into test tube, labelled).
- Sample: 1 mL of sample solution prepared + 1 mL DNS + 3 mL distilled water (all pipette into
test tube, labelled).
- All blank, standards and sample are prepared in triplicate. After prepare, mix well using a
vortex mixture.
- All test tubes arrange in a test tube rack and place in water bath for exactly 5 minutes. Then,
transfer into a container of iced water. Add 10 mL of distilled water in each test tube and mix
well using a vortex mixture.
- Using a double-beam spectrophotometer, determine maximum wavelength by measuring the
absorbance spectrum of 4 mg/mL glucose standard solution (within range of 400 to 600 nm).
Set the spectrophotometer wavelength at the highest absorbance obatined.
- Measure the absorbance for each standard solution and the sample solution.

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