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The methods for determining the content of vitamin C in fruits, vegetables and their products mainly include 2,4-dinitrobenzene colorimetry, 2,6-dichlorophenol titration, high-performance liquid chromatography and fluorescence methods
.
Among them, the 2,4-dinitrophenylhydrazine colorimetric method and the fluorescence method are used to determine the content of total ascorbic acid, which are also the determination methods specified in GB/T5009.
This training uses the 2,4-dinitrophenylhydrazine colorimetric method (according to the second method in GB/T5009.
86-2003)
.
1.
Purpose requirements
(1) Master the principle and method of 2,4-dinitrophenylhydrazine colorimetric method for the determination of total ascorbic acid
.
(2) Understand the working principle of the visible-ultraviolet spectrophotometer, and learn to use the visible-ultraviolet spectrophotometer correctly
.
(3) Master the method of colorimetric quantification and be able to draw standard curves proficiently
.
(4) Understand the content of vitamin C in different fruits and vegetables
.
2.
Principles of training
Total ascorbic acid includes reduced type, dehydrogenated type and diketogulonic acid.
The reduced type of ascorbic acid in the sample is oxidized to dehydroascorbic acid by activated carbon, and then reacts with 2,4-dinitrophenylhydrazine to produce red acetone.
Use H 2 The SO 4 dissolves, and the colorimetric quantification is carried out according to the content in the sulfuric acid solution which is proportional to the ascorbic acid content
.
3.
Training supplies
(1) Constant temperature box or super constant temperature water bath: (37±0.
5)℃
.
(2) Visible-ultraviolet spectrophotometer
.
(3) The masher
.
(4) Sulfuric acid (4.
5 mol/L): Slowly add 250 mL of concentrated sulfuric acid (relative density 1.
84) to 700 mL of water, and dilute to 1000 mL with water after cooling
.
(5) Sulfuric acid (9+1): slowly add 900 mL of concentrated sulfuric acid (relative density 1.
84) to 100 mL of water
.
(6) 2,4-Dinitrophenylhydrazine solution (20g/L): Dissolve 2g of 2,4-dinitrophenylhydrazine in 100mL of 4.
5mol/L sulfuric acid and filter
.
Store in the refrigerator when not in use, and filter before use
(7) Oxalic acid solution (20g/L): Dissolve 20g of oxalic acid (H 2 C 2 O 4 ) in 700 mL of water and dilute to 1000 mL
.
(8) Oxalic acid solution (10g/L): Dilute 500mL of 20g/L oxalic acid solution to 1000mL
.
(9) Thiourea solution (10g/L): Dissolve 5g of thiourea in 500mL of 10g/L oxalic acid solution
.
(10) Thiourea solution (20g/L): Dissolve 10g of thiourea in 500mL of 10g/L oxalic acid solution
.
(11) 1mol/L hydrochloric acid: Take 100mL of hydrochloric acid , add it to water, and dilute to 1200mL
.
(12) Ascorbic acid standard solution: Weigh 100mg pure ascorbic acid and dissolve it in 100mL 20g/L oxalic acid solution, this solution is equivalent to 1mg ascorbic acid per 1mL
.
(13) Activated carbon: need to be washed to no iron ions
.
(14) Different fruits and vegetables are fresh
.
4.
Safety reminder
(1) 2,4-Dinitrophenylhydrazine (DNPH) is a flammable solid, used in the manufacture of explosives and chemical reagents.
It will explode upon vibration and impact when it is dry.
It is also irritating to the eyes and skin.
During use Wear gloves and handle with care
.
(2) Concentrated sulfuric acid is extremely corrosive.
Avoid splashing on the skin and clothes, avoid inhaling vapor, and wear anti-corrosion gloves during the experiment
.
Sulfuric acid waste liquid must be placed in a designated hazardous waste container