Grasp the basic principles of quantitative determination of reducing sugar and total sugar, learn the basic operation of colorimetric determination of sugar method, and be familiar with the use of 721 type spectrophotometer
Operation method
Experiments for the determination of reducing sugars and total sugars
Principle
Various monosaccharides and maltose are reducing sugars, sucrose and starch are non-reducing sugars. Monosaccharides, disaccharides and polysaccharides in plant samples can be extracted separately by taking advantage of differences in solubility, and then acid hydrolysis is used to completely hydrolyze the non-reducing disaccharides and polysaccharides to the reducing monosaccharides. Under alkaline conditions, reducing sugar will reduce 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid (brown-red substance), reducing sugar is oxidized into glycolic acid and other products, within a certain range, the amount of reducing sugar and the degree of brown-red substance is a certain proportion, in the determination of the extinction value of brown-red substance at a wavelength of 540 nm, check the standard curve and calculations, you can be derived respectively. The content of reducing sugar and total sugar in the sample can be determined by checking the standard curve and calculating. When the polysaccharide is hydrolyzed, a molecule of water is added to the monosaccharide residue, so the amount of water added must be deducted from the calculation, and the total amount of sugar obtained from the measurement is multiplied by 0.9 to be the actual total amount of sugar. Move I. Experimental materials, apparatus and reagents Common Problems 1. It is easier to use a blood glucose tube than a large test tube. For more product details, please visit Aladdin Scientific website.
1, experimental materials: flour.
2. Apparatus: (1) 25 ml blood glucose tube or graduated test tube × 11; (2) large centrifuge tube or glass funnel × 2; (3) beaker: 100 ml × 1; (4) triangular flask: 100 ml × 1; (5) volumetric flasks: 100 ml × 3; (6) Graduated pipettes: 1 ml × 11, 2 ml × 4, 10 ml × 1; (7) Constant temperature water bath; (8) Boiling water bath; (9) Centrifuge (this equipment was not used for the filtration method); (10) Electronic balance; (11) Spectrophotometer;
3. reagents:
(1) 1mg/ml glucose standard solution: accurately weigh 100 mg of analytically pure glucose (pre-baked at 80 ℃ to weigh), placed in a small beaker, with a small amount of distilled water to dissolve, quantitatively transferred to 100 ml volumetric flasks, to distilled water to the gradient of the volume, shaking, and stored in the refrigerator for spare.
(2) 3,5-dinitrosalicylic acid reagent: add 6.3g 3,5-dinitrosalicylic acid and 262ml 2NaOH solution to 500ml hot water solution containing 185g methyl sodium tartrate, then add 5g crystallized phenol and 5g sodium sulfite, stir to dissolve. After cooling, add distilled water to volume to 1000 ml, stored in a brown bottle for spare.
(3) Iodine-potassium iodide solution: weigh 5g iodine and 10g potassium iodide, dissolve in 100ml distilled water.
(4) Phenolphthalein indicator: weigh 0.1g phenolphthalein, dissolve in 250ml 70% ethanol.
(5) 6NHCl
(6) 6NNaOH
II. Procedure
1、Make glucose standard curve:
Take 7 blood glucose tubes or test tubes with 25 ml graduations, number them, and accurately add glucose standard solution with a concentration of 1 mg/ml and 3,5-dinitrosalicylic acid reagent in the amounts shown in the table below. 
Shake the tubes well, heat them in a boiling water bath for 5 minutes, remove them and immediately put them into a beaker containing cold water to cool to room temperature, then fix the volume with distilled water to the 25 ml mark, plug the mouth of the tubes with a rubber stopper, and mix upside down (if large test tubes are used, add 21.5 ml of distilled water to each tube and mix well). At the wavelength of 540nm, adjust the zero with tube 0 and read the extinction value of tubes 1-6 respectively. Take the extinction value as the vertical coordinate and the mg of glucose as the horizontal coordinate to plot the standard curve.
2、Determination of reducing sugar and total sugar in the sample:
(1) Extraction of reducing sugar in the sample:
Accurately weigh 2g of edible flour, put it in a 100ml beaker, first make a paste with a small amount of distilled water, then add 50ml of distilled water, stir well, and put it in a 50℃ constant temperature water bath for 20 minutes to keep warm, so that the reducing sugar is leached out. Centrifugation or filtration, with 20ml distilled water to wash the residue, and then centrifugation or filtration, the two centrifugation of the supernatant or filtrate all collected in a 100ml volumetric flask, with distilled water to the graduation, mixing, as reducing sugar to be measured.
(2) Hydrolysis and extraction of non-reducing sugar in the sample:
Accurately weigh 1 g of edible flour, put it in a 100 ml triangular flask, add 10 ml of 6NHCl and 15 ml of distilled water, and put it in a boiling water bath to be heated and hydrolyzed for 30 minutes. Take 1~2 drops of the hydrolyzed solution on a white porcelain plate, add 1 drop of iodine-potassium iodide solution and check whether the hydrolysis is complete. If the hydrolysis is complete, it does not show blue color. After the hydrolysate in the triangular flask is cooled, add one drop of phenolphthalein indicator, neutralize with 6NaOH until slightly red, filter, then rinse the triangular flask and filter paper with a small amount of distilled water, collect all the filtrate in a 100ml volumetric flask, and mix it well with distilled water to the scale. Accurately aspirate 10 ml of the hydrolyzed solution, transferred to another 100 ml volumetric flask, fixed volume, mixed well, as the total sugar to be measured solution.
(3) Color development and colorimetry:
Take five 25 ml graduated blood sugar or graduated test tubes, number them, and add the solution to be measured and reagents accurately in the amounts shown in the table below. 
2、Centrifugation is easier than filtration to get clear liquid.
3、Standard curve and sample sugar content determination should be carried out at the same time, together with the color development and colorimetry.