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Chapter 11 Vitamins Analysis

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1 Chapter 11 Vitamins Analysis

2 Introduction 1. What is vitamins? VITAMIN: VITA (LIFE)-AMINE
2. Vitamins classification Hydrosoluble vitamins: B, C, Liposoluble Vitamins: A, D, E, K 3. Vitamins and health

3 Vitamins determination
The general techniques for determining vitamins can be used for biological liquids and plant or animal tissues. The general principles are the same, but the extraction methods differ depending on the matter being analyzed. For many decades, the determination of vitamins has been based on the evolution of diverse technologies from microbiological methods to more sophisticated techniques like liquid or gaseous phase chromatography coupled with specific detection systems(ultraviolet or fluorimetric). The real problem at present is no longer the methods of determination; rather, it is extraction procedures. The current change in legislation that permits vitamin supplement food substances necessitates constant analytical control, and underlines the importance of the methods for determining these vitamins.

4 VA1 VA2 β-胡萝卜素 生育三烯酚 生育酚 麦角固醇 7-脱氢胆固醇 维生素D3(胆钙化醇)

5 Global Extraction Tech for Liposoluble Vitamins
Weigh proper amount of the food substance and transfer to a 150 ml round flask Add 10 ml of 33% KOH and 40 ml ethanol Cool very rapidly, then add 17 ml of 25% HCl, and cool again Boil at reflux for 30 mins Add 50 ml of petroleum ether and shake vigorously for 3 mins Wait to obtain complete separation of the two phases Evaporate to dry in a vacuum at a temperature of 35 degree C Remove and filter the organic phase through anhydrous sodium phases Redissolve the residue with a know volume of hexane for HPLC analysis

6 Extraction of Vitamin A
1. Weigh 5-10 g of the previously crushed food substance into a 1 L round flask. 2. Add 20 ml of a 50% NaOH solution and warm the mixture in a water bath. 3. Then, add 100 ml of diethyl alcohol and 2 ml of a hydroquinone solution that was obtained by dissolving 20 g in 100 ml of pure alcohol. 4. Maintain the water bath at 90℃ for 30 minutes. 5. Pour the contents of the round flask into a decanting vial and add 100 ml of water. 6. Add 50 ml of ethylic ether and shake. 7. Add 50 ml of petroleum ether. Shake and allow it to decant. 8. Extract once or twice with 50 ml of petrol ether. 9. Wash the ether phase three times with 100 ml of water. 10. Filter, evaporate, and concentrate until 1 ml is obtained. It must be noted that all of these steps are conducted away from light. Moreover, saponification with NaOH is not useful with nonfatty products.

7 Extraction of Vitamin D2 or D3
1. Weigh between 5 and 10 g of the sample food substance. 2. Add 1 g of pyropanol, 90 ml of a mixture of 60 ml absolute ethanol, and 30 ml of a 50% potash solution. 3. Extract three times, each time with 50 ml of petroleum ether. 4. Wash the ether extract and material three times with water. 5. Filter, evaporate, and concentrate until 1 ml is obtained. Saponification with the alcoholic potash mixture is not necessary if the products to be analyzed do not contain fats.

8 Extract of Vitamin E 1. Weigh between 5 and 10 g of the food substance that you crush. 2. Add 100 ml of ascorbic acid methanol solution obtained by mixing 0.5 g of ascorbic acid, 4 ml of water, and 20 ml of ethanol brought to 100 ml with methanol. 3. Keep in boiling water for minutes. Add 15 ml of a 70% KOH solution Place again in the water bath for 40 minutes. 4. Decant the contents of the flask into a separation flask vial, washing the flask with 50 ml of water. 5. Add 120 ml of ethylic ether and stir the mixture. Decant and filter on Na2SO4. 6. Extract again with 120 ml of ethyl ether. 7. Filter, evaporate, and concentrate to 1 ml. Saponification with potash is not necessary for nonfat products.

9 Other Extraction Techniques
Other Extraction Techniques Specific to Each Liposoluble Vitamin and the Product Being Analyzed These techniques are detailed in the official analysis methods proposed by the Official Association of Analytical Chemists (OAAC).

10 Determination Methods
Numerous determination techniques can be proposed: ● determination by fluorimetry and by colorimetry; ● determination by liquid phase chromatography, with ultraviolet or fluorimetric detection; ● determination by gas phase chromatography (vitamin E); ● microbiological determination.

11 Determination of Vitamin A
After extraction, the determination is carried out on the solvent of the liquid extraction. Colorimetric Determination Through this method, carotenoids and vitamin A are determined. Determination of carotenoids. Carotenoids are determined at 450 nm. After evaporating the ether phase of the extracted solution, dissolved the extraction with 1 ml of hexane. Determine the O.D. of this phase at 450 nm. Determination of vitamin A. The hexane phase obtained earlier is taken again and concentrated in a vacuum. Redissolve the extract in a chloroform. Then, to the volume of chloroform, add four volumes of the trifluoroacetic acid reagent prepared by mixing 1 v of trifluoroacetic acid with three volumes of chloroform. Then, observe the DO at 620 nm

12 Determination of Vitamins D
Determination of Vitamins D2,D3,and Their Metabolites If the sample contains all the vitamian D metabolites, then you can carry out a liquid chromatography under the following conditions: Column : fatty acid analysis column Solvent : MeCN (acetonitrile), 55% Mixture of water/acetic acid (4 ml of acetic per liter) 45% Flow rate : 1 ml/min Wavelength : 265 nm Solvent temperature : 25℃ Temperature of oven : 40℃

13 Determination of Vitamin E
A number of methods can be used to determine this vitamin. 1. Colorimetric Determination After extraction and evaporation, re-dissolve the residue using n-heptane. Add 1 ml of dipyridil solution, then determine the absorbance at 460 nm. Methods derived from this one have been recommended for use with ferric chloride with a reading at 510 nm. 2. Determination by Liquid Chromatography Using the extract prepared as described earlier, proceed with an HPLC determination under the following conditions: Column : Lichrosorb R P 8,25 cm ×, 4.6 mm, 5 µm Solvent : methanol/water (92:8) Flow rate : 1.5 ml/min Wavelength : 288 nm Solvent temperature : 25℃ Temperature of oven : 40℃ *For some foods, all three vitamins (A, D, E) can be determined, or only vitamins A and E simultaneously.

14 Extraction of Hydrosoluble Vitamins
1. General principle is that the sample must be crashed as finely as possible. 2. Enzymatic extraction is conducted with amylolytic or proteolytic enzymes.

15 Tech of extracting the ensemble of hydrosoluble vitamins
homogenize the sample after crushing it finely and rapidly (if necessary) Weigh 5-10 g sample into a 250 ml flask and add 65 ml 0.1 M HCl ►► Heat at 100 deg C for 30 mins in a water bath Cool and adjust to pH4.5 with 2.5 M NaOAc ►► Add 50mg β-amylase and 50 mg takadiastase, incubate at 37 oven for overnight Decant quantitatively into a 100 ml volumetric flask and adjust volume with water ►► Filter the supernatant and do further treatment to the filtrate if necessary

16 Ascorbic Acid Extraction
1. In a breaker weigh to about 0.1 mg of a certain product quantity as a function of the assumed vitamin C content of the sample food substance. 2. Decant into a 50 ml volumetric flask using 0.4% metaphosphoric acid. 3. Bring the volume to 50 ml with this solution. *. For the analysis of a liquid, pippet the sample directly into the flask and adjust the volume to 50 ml with the 0.4% metaphosphoric acid solution. 4. Filter the solution an a cellulose acetate membrane (0.2 µg), then pass the filtered substance through a SEP-PAK C18 cartridge (supplied by Waters, a division of Millipore). Eliminate the first 2 ml, then collect 5 ml for analysis by RP-HPLC. (Note: It must be pointed out that some authors may propose a determination of ascorbic and dehydro-L-sacorbic acids for which the extraction technique is different from the preceding one).

17 Determination of Water Soluble Vitamins
1. Determination of Vitamin B1 Thiamine 1.1 Fluorimetric Determination After the action of potassium ferricyanide in the presence of potash, determine fluorescence using a nm primary filter and a nm secondary filter. 1.2 Microbiological Determination A number of lactobacilli can be used, such as Lactobacillus fermentum and Lactobacillus viridiceus ATCC 1270 C, depending on the chemical methods of determination. The statistical calculation of activity is based on the six-point method. 1.3 Determination by Liquid Chromatography Chromatographic conditions Column : C 18, 25 cm × 4.6 mm, 5µm Solvent : methanol 0.005 M sodium acetate adjusted to 4.5 pH (30-70) Flow rate : 1 ml/min Detection : excitation: 366 nm emission: 435 nm

18 * Principle of NP-C and RP-C
流动相: 正己烷 固定相: 普通硅胶 极性较大的物质 弱极性 物质 流动相: 乙氰/水 流动相:C18改性硅胶 非极性 Reverse phase chromatography Normal phase chromatography

19 So much for this lesson and see you next time !

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