Determination of Vitamin C Content by amber HPLC vials.

1. Determination of vitamin C by titration

1.1 Determination principle

The 2,6-dichlorohydrin method and the iodometric method are the more common methods for titrating vitamin C. Reduced ascorbic acid vat dye 2,6-dichloroindophenol, which is red in acidity and red after reduction. After reduction of ascorbic acid by reduced ascorbic acid, it is itself oxidized to dehydroascorbic acid. In the absence of impurities, a certain amount of sample extract reduces the amount of standard 2,6-dichlorophenol to be proportional to the amount of vitamin C contained in the sample.

Principle of iodometric method: Vitamin C includes three types: oxidized, reduced and diketogulonic acid. When titrating vitamin C with iodine, the titrated iodine is reduced to iodide by vitamin C, along with the vitamin during the titration. C is completely oxidized, and the dropped iodine will appear as iodine molecules. The iodine molecule can cause the solution containing the indicator (starch) to produce a blue color, which is the end point of the titration.

1.2 Determination operation

2,6-dichlorophenol method: Take an appropriate amount of the edible portion of the sample, add 100 mL of 2% oxalic acid solution, and make a homogenate. Take 10g of the same sample homogenate, add 20mL of 1% oxalic acid solution, shake well, filter with filter paper, take 5mL filter solution in Erlenmeyer flask, titrate with 2,6-dichloroindole sodium solution (1 mL ≈0.02 mgVitC), in the presence of light red for 30 s without fading as the end point of titration. The consumption of the 2,6-dichlorophenol indophenol sodium salt solution was recorded, and the vitamin C content (mg/100 g) in the sample was calculated based on the results.

Iodine method: Wash the fruits and vegetables, wipe the water attached to the outside with gauze, if the sample is clean, do not need to wash. The sample can be cut longitudinally into 4~8 parts, weigh 20g, but use the food part, put 2% Hcl 15~10ml in the mortar, grind to the slurry, transfer to 100ml volumetric flask, use 2% HCl Add to the mark line, mix, filter, and record the total volume of the filtrate. Determination of sample solution: In a 50ml beaker, pipette 10ml KI solution 0.5ml, 0.5% starch solution 2ml, sample solution 5ml, distilled water 2.5ml, use 0.001N KIO3 droplets, add one drop, Shake the beaker from time to time until the bluish color does not fade to the end point (it does not fade for a minute). Record the number of milliliters of KIO3 solution used to calculate the vitamin C content.

1.3 Evaluation of measurement methods

The 2,6-dichlorophenol indophenol titration method is simple, rapid, and accurate, and is suitable for analysis of many different types of samples. The disadvantage is that the content of dehydroascorbic acid and ascorbic acid in the sample cannot be directly measured, and is easily interfered by other reducing substances. If the sample contains a pigment substance, it will cause difficulty in observing the end point of the titration. Potassium iodate titration is cheaper. It is easier to determine the content of vitamin C in vegetables by potassium iodate titration. The 2,6-dichlorophenol method is relatively complicated. In general, the titration method is simple and fast, and does not require special equipment, but the accuracy and precision are poor when measuring dark samples.

2. Fluorimetric determination of vitamin C

2.1 Determination principle

Deutsch and Weeks have reported a fluorescent assay (OPDA) for the detection of vitamin C and have been designated as a classical fluorescence assay for vitamin C. In this method, vitamin C is first oxidized by activated carbon (Norit) to dehydroascorbic acid (DHAA), and DHAA is combined with the fluorescent substrate o-phenylenediamine (OPDA) to form a fluorescent product, and the vitamin product is detected to achieve vitamins. Quantitative analysis of C. Sun Zhenyan et al [1] proposed a new fluorescence analysis method for the determination of vitamin C. Based on the oxidation of vitamin C to DHAA by vitamin C, DHAA further synergistically sensitizes with benzoic acid and cetyltrimethylammonium bromide. The quantitative analysis of vitamin C is carried out by measuring the fluorescence intensity of the system.

2.2 Determination operation

Determination method of fluorescence analysis (OPDA): Weigh a certain amount of sample, soak it in water after grinding, add the appropriate amount of 1% oxalic acid solution, shake for about 3 minutes, add 0.2g of treated activated carbon and shake it for about 3min. After filtration, the filtrate was added to two 25 mL colorimetric tubes and then 5.0 mL of buffer solution was added. One of the tubes was added with 2.0 mL of boric acid solution (ie, blank) and shaken. After standing for 15 minutes, both tubes were added with 10 mL of o-phenylenediamine solution. Leave it in the dark for 30 minutes to be tested. The sample fluorescence intensity minus the blank fluorescence intensity value is the relative fluorescence intensity value of the sample.

The fluorescence analysis method of Sun Zhenyan et al.: 0. 6 mL CuSO4 solution, 2.0 mL of cetyltrimethylammonium bromide solution, 2.0 mL of benzoic acid solution, a certain volume in a 25 mL colorimetric tube. Vitamin C standard solution, 5.0 mL NaOH-potassium hydrogen phthalate buffer solution, make up to volume with distilled water, shake well. After heating in a constant temperature water bath at 35 ° C for 30 min, the solution was cooled to room temperature, the excitation wavelength was 308 nm, and the fluorescence intensity F was measured at an emission wavelength of 408 nm. The reagent blank containing no vitamin C was F0, and ΔF=F-F was calculated.

2.3 Evaluation of measurement methods

Fluorescence analysis has the advantages of simple operation, high precision and low detection limit. The method can be applied to the detection of vitamin C in fruits, vegetables and medicines, and is suitable for promotion.

3. Photometric analysis of vitamin C

3. 1 measurement principle

The 2,4-dinitrophenylhydrazine method and the molybdenum blue colorimetric method are a common photometric method for the determination of vitamin C. The principle of the 2,4-dinitrophenylhydrazine method is that the total vitamin C includes reduced, dehydrogenated and diketogulonic acid, and the reduced ascorbic acid in the sample is oxidized by activated carbon to dehydroascorbic acid, and then 2,4- The action of dinitrophenylhydrazine produces red hydrazine, and the content of hydrazine is directly proportional to the total ascorbic acid content, and colorimetric determination is performed. Molybdenum blue colorimetric method is a common method for determining the content of reduced vitamin C in fruits and vegetables. The ammonium phosphomolybdate formed by the reaction of metaphosphoric acid and ammonium molybdate is reduced by reduced vitamin C to form a bright blue complex. The content of reduced vitamin C in the sample can be determined by spectroscopic colorimetry.

3.2 Determination operation

2,4-Dinitrophenylhydrazine method: Take an appropriate amount of the edible portion of the sample, and add 100 mL of 2% oxalic acid solution to prepare a homogenate. Take 20 g of homogenate (containing 1~2 mg ascorbic acid) into a 100 mL volumetric flask, dilute with 1% oxalic acid solution, mix and filter. Take 25 mL of the filtrate into a 25 mL colorimetric tube with 2 g of activated carbon, shake for 1 min, and filter. Then take 10 mL of this oxidizing extract, add 10 mL of 2% thiourea solution, and mix. According to the color reaction method in GB12392-90, the colorimetric method was carried out with a spectrophotometer, and the ascorbic acid content in the sample was calculated based on the results. The content of Vc in the sample was calculated by the following formula: X = c · Vm × F × 1001000.

X—the total ascorbic acid content in the sample, mg/100g;

C—the concentration of total ascorbic acid in the “sample oxidizing solution” calculated by the standard curve or regression equation, μg/mL; V—the volume of the sample with 1% oxalic acid solution, mL; F—the dilution factor during the oxidation process of the sample M—sample quality, g.

Molybdenum blue colorimetric method: accurately weigh 100 g sample, add oxalic acid-EDTA solution, mash and transfer to 100 mL volumetric flask, dilute to volume, filter, and pipet 2 mL of supernatant into 50 mL volumetric flask, add 1 mL The metaphosphoric acid-acetic acid solution, 5% sulfuric acid 2.0 mL, shake well, add 4 mL of ammonium molybdate, dilute to 50 mL with deionized water, and measure the absorbance after 20 min.

3.3 Evaluation of measurement methods

Molybdenum blue colorimetric method for the determination of reduced vitamin C content in fruits and vegetables has good stability and accuracy. It is a fast, accurate and sensitive method for determination, and it is not affected by the color of the sample. Determination of total VitC (reduced and oxidized) by 2,4-dinitrophenylhydrazine colorimetric method with good specificity but complicated operation is a standard method for VitC determination in foods in China. This method is suitable for vegetables and fruits. Determination of total ascorbic acid in its products.

4. HPLC

4.1 Determination principle

HPLC (HPLC) is a method for determining the content of vitamin C in recent years. The determination of vitamin C content usually uses C18 column or C8 column. Because vitamin C absorbs ultraviolet light, the detector is commonly used as a UV detector.

4.2 Determination operation

Weigh the vitamin C standard sample 0.1000 g. Transfer to a 100 ml volumetric flask and dilute to volume with double distilled water to obtain a 1.0 mg·ml-1 vitamin C standard solution. Refer to the method of Nisperos-Carriedo et al. Accurately weigh 1.00 g of pulp, grind it with 5 ml 0.2% ammonium phosphate in ice bath, centrifuge at 10000 g for 15 min, add 4 ml of 0.2% metaphosphoric acid to the residue, extract the supernatant, dilute to 10 ml, filter through 0.45 μm. The membrane is filtered and tested. Each sample was repeated 5 times. Vitamin C has a maximum absorption peak at 240 nm, so 240 nm is used as the detection wavelength. 0.2% metaphosphoric acid was used as the mobile phase. Pipette the standard solution 1 ml, 2 ml, 4 ml, 6 ml, 8 m, l to a volume of 10 m, l from each sample 10.0 μl injection analysis, with peak area (mv) as the ordinate, standard concentration ( Mg·ml-1) is the abscissa, draw the standard solution curve, and calculate the regression coefficient and intercept of the linear regression equation. The sample solution was separately injected into 10.0 μl for liquid chromatography analysis, and the peak area of ??vitamin C was determined, and the vitamin C content was calculated by substituting a standard curve.

4.3 Evaluation of measurement methods

HPLC is characterized by high efficiency, rapidity, stability, accurate structure and easy operation. The method has short separation time and is particularly suitable for the structurally unstable vitamin C. It is also particularly suitable for the determination of darker color extract samples, and has become a popular vitamin C determination method in recent years. The disadvantage is that the instruments used are more expensive.