1、BRITISH STANDARD CONFIRMED NOVEMBER1992 BS684-2.33: 1983 ISO5558:1982 Methods of analysis of Fats and fatty oils Part 2: Other methods Section 2.33 Detection and identification of antioxidants ISO title: Animal and vegetable fats and oilsDetection and identification of antioxidantsThin-layer chromat
2、ographic method IMPORTANT NOTE. It is essential that this Section be read in conjunction with the information inBS 684-0 which is published separately. UDC 665.1.014:543.544.061:661.7.094.382BS684-2.33:1983 This British Standard, having been prepared under the directionof the Food and Agriculture St
3、andards Committee,was published underthe authority of the BoardofBSI and comes intoeffecton 29April1983 BSI 11-1999 The following BSI references relate to the work on this standard: Committee reference FAC/18 Draft for comment 81/51638 DC ISBN 0 580 11907 6 National foreword This Section of BS684 wa
4、s prepared under the direction of the Food and Agriculture Standards Committee and is identical with ISO 5558:1982 “Animal and vegetable fats and oilsDetection and identification of antioxidantsThin-layer chromatographic method”, published by the International Organization for Standardization (ISO).
5、 Terminology and conventions. The text of the International Standard has been approved as suitable for publication as a British Standard without deviation. Some terminology and certain conventions are not identical with those used in British Standards; attention is especially drawn to the following.
6、 The comma has been used as a decimal marker. In British Standards it is current practice to use a full point on the baseline as the decimal marker. Wherever the words “International Standard” appear, referring to this standard, they should be read as “British Standard”. Additional information. In c
7、lause1, the antioxidant listed first has the lowest R fvalue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity fro
8、m legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pagesi andii, pages1 to3 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside fro
9、nt cover. Amendments issued since publication Amd. No. Date of issue CommentsBS684-2.33:1983 BSI 11-1999 i Contents Page National foreword Inside front cover 1 Scope and field of application 1 2 Principle 1 3 Reagents 1 4 Apparatus 1 5 Procedure 2 6 Test report 3ii blankBS684-2.33:1983 BSI 11-1999 1
10、 1 Scope and field of application This International Standard specifies a thin-layer chromatographic method for the detection and identification of the following antioxidants in animal and vegetable fats and oils: nordihydroguairetic acid (NDGA) propylgallate (PG) octylgallate (OG) dodecylgallate (D
11、G) tertiobutylhydroquinone (TBHQ) butylhydroxyanisole (tert-butyl-4-methoxyphenol) (BHA) -tocopherol butylhydroxytoluene (2,6-di-tert-butyl-4-methylphenol) (BHT) The antioxidants are listed in the order of their R fvalues. The detection of -tocopherol is sometimes difficult as the spots of this anti
12、oxidant are of a generally diffuse aspect. The method has limits of detection of20 mg/kg(ppm) for BHT, and 10mg/kg (ppm) for the other antioxidants. It should be noted that BHT is not completely isolated by this method. 2 Principle Dissolution of a test portion in n-hexane and extraction of the anti
13、oxidants with acetonitrile. Identification of the antioxidants by thin-layer chromatography. 3 Reagents All reagents shall be of recognized analytical quality. The water used shall be distilled water or water of at least equivalent purity. 3.1 Silica powder with binder, thin-layer chromatographic qu
14、ality, of particle size less than304m. 3.2 Methanol, containing not more than0,5%(m/m) of water. 3.3 Ethanol, 96% (V/V). 3.4 n-hexane or, failing this, light petroleum having a distillation range from 30 to 60 C. 3.5 Acetonitrile saturated with n-hexane Pour 900ml of acetonitrile into a 1000ml flask
15、 and add100ml of the n-hexane(3.4) and a little anhydrous sodium sulphate or anhydrous calcium chloride. Shake, stopper the flask and allow to stand overnight. 3.6 n-hexane saturated with acetonitrile Pour 900ml of the n-hexane (3.4) into a 1000ml flask and add100ml of acetonitrile and a little anhy
16、drous sodium sulphate or anhydrous calcium chloride. Shake, stopper the flask and allow to stand overnight. 3.7 Indicator solution: 2,6-dichloro-p-benzoquinone-4-chloroimide, 10g/l solution in96%(V/V) ethanol. Prepare, just before use, by dissolving 500mg of2,6-dichloro-p-benzoquinone-4-chloroimide
17、in50ml of the ethanol (3.3). 3.8 Developing solvent mixture Prepare, just before use, a mixture of two volumes of the n-hexane (3.4), two volumes of benzene and one volume of glacial acetic acid. WARNINGBenzene is toxic and flammable and care should be exercised in using it. 3.9 Antioxidant standard
18、 solutions, containing1g of antioxidant per litre of methanol. In a series of eight beakers, dissolve separately100mg of each of the antioxidants (seeclause1) in the methanol (3.2), transfer to a series of eight100ml volumetric flasks, make up to the mark with the methanol and mix. 4 Apparatus Usual
19、 laboratory equipment, and in particular: 4.1 Developing tank for thin-layer chromatography, made of glass, suitable for glass plates of dimensions200mm 200mm, fitted with a ground glass lid. 4.2 Spreader and platform, for preparation of the plates. 4.3 Glass plates, 200mm 200mm, coated with a layer
20、 of silica gel of thickness 0,40mm. Use commercially available prepared plates, or prepare as follows. Clean the plates thoroughly with ethanol, n-hexane and acetone until all traces of fat are removed. Weigh 30g of the silica gel (3.1) into a250ml conical flask (4.8) and add 60ml of water. Stopper
21、the flask and thoroughly mix the contents by shaking vigorously for 1min. Immediately apply a 0,4mm layer of the silica gel slurry on to the surface of the plates using the spreader (4.2). Dry the plates at ambient temperature for15min, and then place them in the oven (4.6), controlled at103 2 C, fo
22、r 1h. Allow to cool in the dessicator(4.13) and store in the desiccator until required.BS684-2.33:1983 2 BSI 11-1999 4.4 Rotary evaporator 4.5 Separating funnels, of capacity 250ml. 4.6 Electrically heated drying oven, preferably well ventilated, capable of being controlled at103 2 C. 4.7 Electrical
23、ly heated drying oven, preferably well ventilated, capable of being controlled at60 2 C. 4.8 Conical flask, of capacity 250ml, with a ground glass stopper. 4.9 Volumetric flasks, of capacity 100ml, with ground necks and ground glass stoppers. 4.10 Beaker, tall form, of capacity150ml. 4.11 Round-bott
24、omed flask, of capacity250ml, suitable for use with the rotary evaporator (4.4). 4.12 Syringe, of capacity 204l, graduated in microlitres. 4.13 Desiccator 4.14 Apparatus for spraying the indicator solution on to the plates 5 Procedure 5.1 Test portion Take a test portion of 7,5 to10g from the labora
25、tory sample. 5.2 Extraction of antioxidants Dissolve the test portion in 100ml of the n-hexane(3.4) in the150ml beaker (4.10), heating gently, if necessary, and transfer the solution into one of the 250ml separating funnels (4.5), taking care that no insoluble particles pass into the separating funn
26、el. Rinse the beaker with 25ml of the n-hexane(3.4) and transfer the rinsing liquid into the separating funnel, again taking care that no insoluble particles pass into the separating funnel. Add 25ml of the acetonitrile saturated with n-hexane(3.5) and shake for1min. Collect the acetonitrile phase (
27、lower layer) in a second250ml separating funnel (4.5). NOTEIf an emulsion forms, turn the separating funnel carefully under a stream of water at about 50 C until clear phases are obtained. Carry out three further extractions of the n-hexane phase using 25ml of acetonitrile saturated with n-hexane (3
28、.5) each time. Combine the acetonitrile extracts and wash twice using 25ml of the n-hexane saturated with acetonitrile (3.6) each time. Transfer the acetonitrile phase to the250ml round-bottomed flask(4.11) and evaporate the solvent under reduced pressure in the rotary evaporator(4.4) at as low a te
29、mperature as possible. It is essential that the temperature of the water bath does not exceed 40 C. Dissolve the residue in 2ml of the methanol(3.2) and pour the solution into a small flask. If the residue is not completely dissolved, filter the solution. NOTEIf an antioxidant content of less than10
30、0mg/kg(ppm) is expected, dissolution of the residue in only1ml of methanol is recommended. 5.3 Chromatography and detection Coat the walls of the developing tank (4.1) with filter paper. Pour the developing solvent mixture (3.8) into the developing tank to a depth of about1cm and put on the lid. All
31、ow to stand in the dark for1to2h, in order to saturate the atmosphere inside the tank with the solvent vapours. Using the syringe(4.12), transfer104l and204l portions of the methanolic solution of extracted antioxidants, prepared as described in 5.2, to two distinctive points20mm apart and 20mm from
32、 the edge of the plate(4.3), previously activated for1h at a temperature of 60 C in the oven(4.7). Transfer44l of each of the antioxidant standard solutions(3.9) to distinctive points20mm apart and20mm from the same edge of the plate. Mark a line parallel to the edge of the plate and150mm from the p
33、oints of application. Place the plate in the developing tank and leave in the dark until the solvent front reaches the marked line. Remove the plate from the tank and allow to dry under a fume hood. Spray the indicator solution (3.7) on to the plate, using the apparatus (4.14), and place the plate i
34、n the oven (4.6), controlled at103 2 C, for10 to15min. Remove the plate from the oven, and compare the R fvalues of the spots resulting from the standard solutions with those of the extracted antioxidants. Further information on the identification of antioxidants can be obtained by placing the plate
35、, for about30s, at laboratory temperature, in a developing tank saturated with ammonia and by comparing the resulting colours for the various antioxidants.BS684-2.33:1983 BSI 11-1999 3 6 Test report The test report shall show the method used and the results obtained. It shall also mention all operat
36、ing conditions not specified in this International Standard, or regarded as optional, as well as any circumstances that may have influenced the results. The test report shall include all the information necessary for the complete identification of the sample.BS684-2.33: 1983 ISO5558:1982 BSI 389 Chi
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