Determination of total vitamin C - 2,4 - dinitrobenzene color method.

related topics first, experimental purposes
1. Understand the structural function of natural vitamin C and natural forms of existence.
2. Master the principles and methods for determining total vitamin C., experimental principle
total astrophic acid includes also prototype Vc, dehydrogenated Vc and dyloxone glucosaccharide. The sample also prototyped asbic acid oxidized from activated carbon to dehydrogenated astrophic acid, and then produced a red blood cell with 2,4-dinitrobenzene .the content in the sulfuric acid solution is directly related to the total astrophic acid content, so that the total assachic acid in the sample can be quantified by color rationing., instrument and
reagents
instrument ringer thermostat
box: 37±0.5 degrees C, visible - ultraviolet
termosometer
,
tissue
masher.
reagents
the experimental water are distilled water, reagent purity are pure analysis.
1.4.5mol/L sulphuric acid: Carefully add 250mL sulphuric acid (specific gravity 1.84) to 700mL of water and dilute the water to 1000mL after cooling.
2.85% sulphuric acid: carefully add 90mL sulphuric acid (specific gravity 1.84) to 100mL water and stir well.
3.2% 2,4 - Dinitrobenzene Solution: Dissolved 2g2,4 - Dinitrobenzene in 100mL 4.5mol/L sulphuric acid,
filtered
. Store in the refrigerator when not in use, before each use must filter
4.2% herbal acid solution: dissolved 20g of herbal acid (H2C2O4) in 700mL of water, and then diluted to 1000mL of water.
5.1% achloric solution: dilute 500mL 2% grass acid solution to 1000mL.
6.1% sulfonate solution: Dissolve 5g thiolyculfan in 500mL 1% herbal acid solution.
7.2% sulfonate solution: Dissolve 10g thiolyculfan in 500mL 1% herbal acid solution.
8.1mol/L hydrochloric acid: take 100mL hydrochloric acid, add it to water and dilute to 1200mL.
9. Astroc acid standard solution (1mg/mL): Dissolve 100mg pure astrophic acid in 100mL 1% astic acid.
10. Activated carbon: 100g activated carbon added to 750mL1mol/L hydrochloric acid, reflow 1 to 2h, filter, wash with water several times, until the filter is free of iron ions (Fe 3 plus), and then put in 110 degrees C oven drying.
test iron ion method: using Prussus blue reaction. Mix 2% potassium iron cyanide with 1% hydrochloric acid equivalent, and the above-mentioned washing-out filter drops in, and if there are iron ions, produce blue precipitation. , operation steps (i) sample preparation
all experimental processes should be avoided.
1. Immersion:
fresh sample preparation: 100g fresh sample and 100g 2% grass acid solution, poured into the masher to make a smooth slurry, take 10 to 40g slurry (containing 1 to 2 mg astrophic acid) poured into 100mL
capacity bottle
, diluted with 1% asphalt solution to the scale, mixed.
dry sample preparation: said 1 to 4g dry sample (containing 1 to 2 mg astrophic acid) into the milk, add 1% herbal acid solution grinding into a homogenous slurry, poured into a 100mL capacity bottle, diluted to the scale with 1% ascoic acid solution, mixed.
the leachate is filtered and the filter is backed up. Non-filterable samples
can be
centrifuges, after precipitation, pour out liquid, filtration, spare.
2. Oxidation treatment:
take 25mL of the above filter, add 2g activated carbon, shake 1min, filter, discard the initial few milliliters of filter. Take 10mL of this oxidation extract, add 10mL 2% sulfur solution, mix well.
3. Coloration reaction
4mL oxidation extract mixed with thiophenidate was added to each of the three
tive tubes
As a blank, add 1.0mL 2% 2,4 - dinitrobenzene solution to the remaining test tubes and place all test tubes in a 37±0.5C thermostat or water bath to keep warm by 3h. After 3h, remove, except for the blank tube, put all the test tubes in ice water, the blank tube removed to make it cold to room temperature. Then add 1.0mL 2%2,4 - dinitrobenzene solution, place 10 to 15min at room temperature and put in ice water. When the test tube is put into ice water, add 5mL 85% sulphuric acid to each test tube, the drip time needs at least 1min, and the test tube needs to be shaken side by side. The sulphuric acid droplets are added, the test tube is removed from the ice water, and 30min color is accurately placed at room temperature.
4. The color
is measured at a wavelength of 500nm by using a 1cm color cup, which is zeroed with blank liquid. (ii) standard curve drawing
plus 2g activated carbon in a 50mL standard solution, shaking 1 min, filtration. Take 10mL filter liquid into a 500mL capacity bottle, add 5.0g thiolyculfan, dilute with 1% herbic acid solution to scale, astic acid concentration of 20 mg/mL.
5,10,20,25,40,50,60 dilutions were placed in seven 100mL bottles and diluted to the scale with a 1% thioly solution, so that the concentration of assaclemic acid in the final dilution was 1,2,4,5,8,10,12?g/mL. According to the sample determination step to and color.
is based on the absorbance value as the ordinate, and the astro-astic acid concentration (sg/mL) is used to draw the standard curve for the horizontal coordinates. , the calculation
: X-sample total astrocertic acid content, mg/100g;
c - by the standard curve or by the regression equation to calculate the "sample oxidation solution" in the total astrophic acid concentration, sg/mL;
V - sample with 1% oxalic acid solution to determine the volume, mL;
F - sample oxidation treatment process dilution multiplication;
m - sample mass, g.
parallel or repeated assays in the same laboratory, the absolute value of the relative deviation ≤ 10%.
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