Ascorbic acid, i.e. vitamin C, is widely found in fresh fruits and vegetables, and it is a highly active substance involved in many metabolic activities. Ascorbic acid is one of the members of the antioxidant system in organisms, therefore, ascorbic acid content can be used as an important physiological indicator of anti-aging and anti-adversity in plants, and can also be used as an indicator of the quality of fruits and the selection of good seeds. Through this experiment, we will master the method of determining the ascorbic acid content of plants by spectrophotometer.
Principle
The basic principle of the determination of ascorbic acid content is that reduced ascorbic acid (AsA) can reduce iron ions to ferrous ions, which react with red phenanthrene (BP) to form a red chelate. The absorption value of this substance at 534 nm is positively correlated with the content of AsA, so it can be measured by colorimetric method. Deoxyascorbic acid (DAsA) can be reduced to AsA by dithiothreitol (DTT), the total amount of AsA was determined, and the reduced AsA was subtracted from the total amount of AsA, that is, the amount of DAsA.
Operation method
Determination of ascorbic acid content
Principle
Reduced ascorbic acid (AsA) can reduce ferrous ions to ferrous ions, which react with red phenanthroline (4,7-diphenyl-1,10-phenanthroline, BP) to form a red chelate. The absorption value at 534nm is positively correlated with the AsA content, so it can be determined by colorimetric method. Deoxyascorbic acid (DAsA) can be reduced to AsA by dithiothreitol (DTT), the total amount of AsA was determined, and the reduced AsA was subtracted from the total amount of AsA, that is, the content of DAsA.
Materials and Instruments
Plant material Move 1. Prepare the standard curve to prepare the AsA series standard solution with the concentration of 2mg/L, 4mg/L, 6mg/L, 8mg/L, 10mg/L, 12mg/L and 14mg/L. Take 1.0 ml of each concentration standard solution in a test tube, add 1.0 ml of 5% TCA, 1.0 ml of ethanol and shake well, then add 0.5 ml of 0.4% H3PO4-ethanol, 1.0 ml of 0.5% BP-ethanol, 0.5 ml of 0.03% FeCl3- ethanol in a total volume of 5.0 ml. The solution was placed at 30°C for 90 min, and then the A534 was determined.A standard curve was plotted with AsA concentration as the horizontal coordinate and A534 as the vertical coordinate to find the linear equation. 2. Extract 1.0g of plant leaves, add 5% TCA grinding according to 1:5 (W/V), centrifuge at 4000r/min for 10min, and the supernatant was used for determination. 3. Determination (1) AsA determination Take 1.0 ml of sample extract in a test tube, determine it according to the same method as above, and calculate the AsA content according to the standard curve. (2) DAsA determination Add 0.5 ml of 60 mmol/L DTT-ethanol solution to 1.0 ml of sample solution with Na2HPO4-NaOH mixture, adjust the pH of the solution to 7~8, and place it at room temperature for 10 min, so as to reduce DAsA. Then 0.5 ml of 20% TCA was added, and the pH was adjusted to 1~2. Determination was carried out according to the same method for AsA, and the total AsA content was calculated, from which AsA was subtracted to obtain the DAsA content. Caveat The reaction should have a high selectivity, that is, the selected colorant should preferably react only with the component to be measured, but not with other interfering components or the interference of other components is very small. For more product details, please visit Aladdin Scientific website.
Trichloroacetic acid TCA Anhydrous ethanol solution Phosphoric acid-ethanol solution BP-ethanol solution FeCl3-ethanol solution DTT Na2HPO4-NaOH solution DTT-ethanol
Spectrophotometer Centrifuge Mortar Test tube