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

6. Glass bottle

7. Ice (for cooling down the reaction)

Preparation of Reagent and Standard Solution

1. 2% anthrone in ethyl acetate in a glass bottle. [SAFETY NOTES: Ethyl acetate is highly flammable in presence of open flames and sparks, of heat. Be careful when using it.] (Prepare this reagent as much as you need. Use it within 12 hours.)

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.

3.(In chemical hood) Use glass pipette to add 5mL concentrated sulfuric acid into the tube gently. Put the tip of pipette into the bottom of lower layer. Concentrated sulfuric acid will vaporize ethyl acetate immediately if you drop this sulfuric acid on it. [SAFETY NOTES: sulfuric acid is highly hazardous (corrosive, irritant). Be sure to wear gloves and prevent direct contact.]

(The test tube will be very hot after sulfuric acid is added. Hold the higher part of test tube to prevent burning your fingers)

4.(In chemical hood) Swirl gently and you’ll see the vaporization of ethyl acetate.

5.(In chemical hood) After ethyl acetate has been vaporized, anthrone will crystallize in the solution. Then swirl violently to dissolve anthrone, and color will produce after anthrone has been dissolved.

6.(In chemical hood) Put the test tube into ice to cool down. You can proceed with another test tube and leave this to cool down on the ice

7.(In chemical hood) After all the test tubes are finished cooling, take 1mL of each test tube into an individual cuvette.

8.Turn spectrometer to 620nm and get the readers from the cuvettes.

(Use water as the refernece)

Revised Protocol (The aim of this revised protocol is to reduce the usage of sulfuric acid and other chemicals.)

Preparation of Reagent and Standard Solution

1. Anthrone reagent: Use suitable glass bottle to prepare 2% anthrone in sulfuric acid (w/v). [No ethyl acetate is needed. Prepare anthrone reagent as much as you need and use it within 12 hours. Each sample needs 1mL anthrone reagent and standard curve needs five glucose samples, so prepare at least 10ml of anthrone reagent.][SAFETY NOTES: sulfuric acid is highly hazardous (corrosive, irritant). Be sure to wear gloves and prevent direct contact.]

2. Glucose standard solution: Prepare 2, 10, 20, 50, 100µg/mL glucose solution for standard curve. [In this revised protocol, only 0.5mL of test solution is needed. Prepare suitable volume for your experiment. For example: If you want triplicate data of standard curve, then 1.5mL of each concentration of glucose solution is needed.]

Procedure

1.Add 0.5mL sample or standard [2, 10, 20, 50, 100µg/mL] solution into all prepared test tubes.

2.(In chemical hood) Add 1mL of anthrone reagent into each of the test tube. Shake the tube by hand until the solution becomes clear. [SAFETY NOTES: sulfuric acid is highly hazardous (corrosive, irritant). Be sure to wear gloves and prevent direct contact.]

3.(In chemical hood) Put the test tube into ice to cool down. You can proceed with another test tube and leave this to cool down on the ice

4.(In chemical hood) After all the test tubes are finished cooling, take 1mL of each test tube into an individual cuvette.

5.Turn spectrometer to 620nm and get the readers from the cuvettes.

(Use water as the refernece)