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

Operation method

thin-layer chromatography (TLC)

Principle

Silica G is a kind of silica gel powder with added binder, containing about 12 %-13 % gypsum, which can adsorb some substances from the solution on its own surface, using its different adsorption capacity for various substances, and then unfolding the layer with the appropriate solvent system, so as to enable the different substances to be separated, and using the color rendering agent to develop the color, and obtaining different Rf values. The speed of movement of sugar on a thin layer of silica gel G is related to the molecular weight and the number of hydroxyl groups of the sugar. After unfolding the layer with appropriate solvents, the speed of movement of sugar on a thin layer is Pentasaccharide > Hexasaccharide > Bisaccharide > Trisaccharide.

Materials and Instruments

Sugar standard solution Ethyl acetate Acetic acid Chloroform Methanol Sulfuric acid Diphenylamine Aniline Phosphoric acid Acetone
Polysaccharide
Chromatography cylinder Silica gel G plate Capillary tube Oven Hair dryer Test tubes Test tube racks

Move

Solution Preparation:

1 . Sugar standard solution: xylose, glucose, fructose and sucrose 10 mg/ml in water.

2 . Sample solution: Hydrolyzed solution of polysaccharides.

3 . Expanding agent: ① ethyl acetate:acetic acid:water=2:1:1; ② chloroform:methanol=60:40 (V/V).

4 . Color developer: ① 25% ~ 50% sulfuric acid; ② diphenylamine 1 g, aniline 1 ml and 85% phosphoric acid 5 ml dissolved in 50 ml acetone.

Operating Procedure:

1. The silica gel G plate in the 100 ℃ oven activation and cooled to room temperature, with a capillary tube spotting, spotting position from the lower end of the silica gel plate 1.5 cm, 2 ~ 3 cm on both sides, each sample point 1 ~ 1.5 cm apart, spot diameter 2 mm. spot a blow-drying once.

2. Equilibrate the spreading agent in the chromatography cylinder. After a few minutes, put the silica gel plate in.

3. When the spreading agent is moved to 2~3 cm from the upper end, take out the silica gel plate and blow dry, evenly spray the colorant on it and develop the color at 100 ℃.

4. Remove from the oven after 15～30 min and measure the Rf value.

Common Problems

1. What are the advantages of thin layer chromatography over other chromatographic methods?
2. What is the basis for selecting a spreading agent?
3. What are the other coloring agents for sugar?

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