Principle of determination of soluble sugar by phenol method: Soluble sugar in plants mainly refers to monosaccharides and oligosaccharides dissolved in water and ethanol. The principle of determination of soluble sugar by phenol method is that furfural or hydroxymethylfurfural (molecular formula is shown in anthrone method) generated by dehydration of sugar under the action of concentrated sulfuric acid can condense with phenol to generate orange-red compounds. In the range of 10 ~ 100 mg, the color depth is directly proportional to the sugar content, and there is a maximum absorption peak at the wavelength of 485nm, so it can be determined by colorimetry at this wavelength. Phenol method can be used to determine methylated sugars, pentoses and polysaccharides. The method is simple, the reagent is cheap and the sensitivity is high. The experiment is basically unaffected by the presence of protein, and the color produced can be stable above 160min.

Second, materials, equipment and reagents

(1) material: fresh plant leaves.

(2) Instruments and equipment: 1. Spectrometer; 2. water bath pot; 3. Calibrate the test tube; 4. Fish scale straws.

(3) Reagent: weigh 90g phenol (AR) from 1.90% phenol solution, dissolve it in 10ml distilled water, and keep it for several months at room temperature; 2.9% phenol solution Take 3 ml of 90% phenol solution, add distilled water to 30 ml, and you can use it. 3. Concentrated sulfuric acid (specific gravity1.84); 4. 1% sucrose standard solution Bake analytically pure sucrose at 80℃ to constant weight, accurately weigh 1.000g ... add a small amount of water to dissolve, transfer to 100ml volumetric flask, add 0.5ml concentrated sulfuric acid, and adjust the volume to scale with distilled water; 5. Accurately suck 100μ g/L sucrose standard solution 1% sucrose standard solution 1ml, add it into the 100ml volumetric flask, and add water to the scale.

Third, the experimental steps

1. Preparation of standard curve: Add1ml9% phenol solution to the test tube and shake it evenly, then add 5ml of concentrated sulfuric acid from the front end of the test tube solution for 5 ~ 20s and shake it evenly. The total volume of colorimetric solution is 8ml, and it is kept at room temperature for 30min. Then, with blank as reference, at the wavelength of 485nm, with sugar content as abscissa and absorbance as ordinate, a standard curve is drawn to obtain a standard linear equation.

2. Extraction of soluble sugar: take fresh plant leaves, wipe off surface dirt, cut into pieces, mix well, weigh 0. 10 ~ 0.30g, ***3 parts (or dry materials), put them in three graduated test tubes, add 5 ~10.25ml distilled water, seal them with plastic film, and extract them in boiling water.

3. Determination: Absorb 0.5ml sample solution in the test tube (repeated twice), add 1.5ml distilled water, add phenol and concentrated sulfuric acid solution in turn according to the steps of making standard curve, develop color, and determine absorbance. Find the sugar content from the standard curve.

Fourth, the result calculation

Calculate the sugar content of the sample according to the following formula:

Soluble sugar content (%) = the amount of sugar found on the standard curve (μg)× the volume (mL) of the extraction solution× the dilution multiple/[the volume (mL) of the sample solution for determination× the sample weight (g)× 106]× 100.

Fresh cut flowers are neither medicine nor food, so there is no national standard.