Phenolic substances in plant tissues are oxidized to Da by polyphenol oxidase, and Da can automatically polymerize into colorful substances and browning occurs. Therefore, the quantitative determination of phenolics in plant tissues is of certain significance and is often used as an index in the physiological study of plant resistance. The purpose of this experiment is to learn the principle and method of determining the content of phenolic substances.
Principle
Folin-phenol method is often used for the determination of phenolic substances. Under alkaline conditions, phenolics can react with phosphoribustic acid and phosphorus key acid in the Folin-phenol reagent to form bustard blue and key blue compounds, which have the maximum light absorption at 700 nm, and the size of their absorbance is proportional to the content of phenolics. The absorbance is directly proportional to the phenolic content. Therefore, the content of phenolic substances in plant tissues can be determined by measuring the absorbance.
Operation method
Determination of coolant content in plant tissues
Principle
Folin-phenol method is often used for the determination of phenolic substances. Under alkaline conditions, phenolics can react with phosphoribustic acid and phosphorus key acid in the Folin-phenol reagent to form bustard blue and key blue compounds, which have the maximum light absorption at 700 nm, and the size of their absorbance is proportional to the content of phenolics. The absorbance is directly proportional to the phenolic content. Therefore, the content of phenolic substances in plant tissues can be determined by measuring the absorbance.
Materials and Instruments
1. Material: Various plant tissues. Move 1 . Extraction of phenolic substances: weigh 1 g of plant material, add a small amount of water and grind into a homogenate, wash the mortar thoroughly and dilute to 100 mL.2. Standard curve: add all kinds of reagents according to Table 56, mix well, add 10% Na2CO3 2 mL after 3 min, shake, and then measure the absorbance at 700 nm after standing at room temperature for 1 h. Take the concentration of phenol solution as the horizontal coordinate. The absorbance at 700 nm was measured after 1 h of standing at room temperature. The concentration of the phenol solution was taken as the horizontal coordinate, and the absorbance as the vertical coordinate, and the standard curve was plotted.Table 56-1 Standard curve spiking table Caveat Phenolics are extracted to avoid being oxidized. Common Problems What is the significance of determining the content of phenolics in plant tissues? For more product details, please visit Aladdin Scientific website.
2. Reagents:
(1) Folin-phenol reagent: weigh 25 g of sodium bustinate and 5 g of phosphoaluminate, put them into a reflux flask, add 12.5 mL of phosphoric acid and 188 mL of steam-gluttonous water, and boil them together at reflux for 2 h. Cool them down, and then condense them with steam-gluttonous water to 1 000 mL.
(2)10% Na
2
CC)
3
: Weigh 10 g of Na
2
CO
3
Dissolve 10 g of Na 2 CO 3 in 100 mL of evaporated water.
(3) Standard phenol solution: 0.45 mmol - catechol solution. 3.
3. Apparatus: spectrophotometer, centrifuge, mortar, test tubes, test tube racks, pipettes, etc.
3. Determination of phenol content of the sample: Dilute the extract appropriately, put 2 mL in a test tube, add 2 mL of Folin-phenol reagent, after 3 min, add 10% Na2CO3 2 mL, shaking, and let it stand for 1 h at room temperature to determine its absorbance at 700 nm, and then find out the corresponding phenol concentration of the plant on the standard curve.4. Calculation of results:c X VPhenol content (Mmol- g-,) = - -, where: c i is the concentration of phenol from the standard.Where: c I is the phenol concentration from the standard curve, 卩 mol・mL 1;V - is the sample volume, mL;W - for the sample mass "go Reagent Tube No. 1 2 3 4 5 6 Standard phenol solution/mL 0 0.2 0.4 0.6 0.8 1.0 Evaporated pyruvic water/mL 2.0 1.8 1.6 1.4 1.2 1.0 Final Concentration/(mol - mL-1) 0 45 90 135 180 225 Folin-phenol reagent/mL 2 2 2 2 2 2