(Source: Laboratory instruction in biochemistry and molecular biology, Shao Xueling et al, Wuhan University Press, 2003)

Operation method

Phenol-sulfuric acid method

Principle

Phenol-sulfuric acid reagent can react with free or oligosaccharides, polysaccharides in hexose, glyoxalate (or toluene derivatives) to develop a color reaction, hexose at 490 nm (pentose and glyoxalate at 480 nm) has the maximum absorption, the absorption value and the sugar content is linear.

Materials and Instruments

Glucose
Concentrated sulfuric acid Phenol
Test tubes Test tube racks Pipettes Measuring cylinders Beakers Centrifuge tubes Glass mills Centrifuges Spectrophotometers

Move

1. Treatment of materials: 0.5-1 g of the measured material crushed, with the appropriate amount of distilled water, glass mill grinding and extracted in stages, the mixture centrifuged to obtain the extracted liquid that is the sample solution.

2. Standard curve production: take 50 μg/ml hexose standard solution 0 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml in a test tube, make up to 0.5 ml with water, add 0.3 ml 5% phenol solution, mix well and quickly add 1.8 ml of concentrated sulfuric acid, vibration and mixing, placed at room temperature for 20 min can appear orange-yellow color to the first tube zero point, can be at 490 nm at the same time. The OD value can be determined colorimetrically at 490 nm. Take the sugar content as the horizontal coordinate and the corresponding OD value as the vertical coordinate to draw the standard curve.

3. Determination of sample content: take 0.5 ml containing 2 ~ 25μg of sugar, also add 0.3 ml 5% phenol solution, mixing immediately after adding concentrated sulfuric acid along the wall of the tube 1.8 ml, vibration and mixing, up to room temperature, can be measured at 490 nm OD value, from the standard curve to check the corresponding sugar content.

Caveat

1. The colorants used in several hexose determination methods, such as anthrone and resorcinol, require heating in a boiling water bath, which is not required in this method, and the presence of proteins does not have much effect on the color reaction of this method, so it can also be used for the determination of hexose in glycoproteins. In addition, it can also be used for the determination of methylated derivatives of hexose and 6-deoxyribose, pentose.

2. The heat of this method comes from the mixture of concentrated sulfuric acid and water, so the reaction is fast when adding concentrated sulfuric acid and mixing immediately, and the test tube should be larger so as not to burn your hands.

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