Cfrench:anthrone

Assaying polysaccharides with the anthrone assay

Equipment and Instrument

• 1. 19×150mm glass test tube

• 2. Cuvettes

• 3. Glass pipette and pipette aid

• 4. Spectrometer

• 5. Chemical hood

Preparation of Reagent and Standard Solution

• 1. 2% anthrone in ethyl acetate [SAFETY NOTES: Ethyl acetate is highly flammable in presence of open flames and sparks, of heat. Be careful when using it.]

• 2. Glucose standard solution (0~80µg):

• • SolA: 1mg/2mL std. (100mL): 50mg glucose + 100mL H2O (50mg glucose in total)

• • SolB: 100µg/2mL std. (50mL): 5mL SolA + 45mL H2O (2.5mg glucose in total)

  1) 80µg/2mL std. (10mL): 8mL SolB + 2mL H2O (400µg glucose in total)

  2) 50µg/2mL std. (20mL): 10mL SolB + 10mL H2O (500µg glucose in total)

  3) 25µg/2mL std. (20mL): 5mL SolB + 15mL H2O (250µg glucose in total)

  4) 10µg/2mL std. (20mL): 2mL SolB + 18mL H2O (100µg glucose in total)

  5) 5µg/2mL std, (20mL): 1mL SolB + 19mL H2O (50µg glucose in total)

  (All the standard solutions can store in refrigerator or freezer to re-use.)

Procedure

1.Add 2mL samples or standards [0(pure water), 5, 10, 25, 50, 80µg/2mL] solution into all prepared test tubes.

2.(In chemical hood) Use glass pipette and gently add 0.5mL 2% anthrone solution into each test tube. There will be two layers of this solution. The upper layer is anthrone and the lower layer is the sample/standard. Try not to disturb solution, because ethyl acetate is easy to vaporize.