1、BRITISH STANDARDBS ISO 18252:2006Anhydrous milk fat Determination of sterol composition by gas liquid chromatography (Routine method)ICS 67.100.01g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3
2、g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 18252:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 15 February 2006 BSI 15 February 2006ISBN 0 580 47836 XNational forewordThis British Standard reproduces verbatim ISO 18252:2006 an
3、d implements it as the UK national standard.The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secreta
4、ry.Cross-referencesThe British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of Britis
5、h Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present t
6、o the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.Summary of pagesThis document comprises a front cover, an inside fron
7、t cover, the ISO title page, pages ii to v, a blank page, pages 1 to 13 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsReference numbersISO 18252:2006(E)IDF 200:2006(E)INTERNATI
8、ONAL STANDARD ISO18252IDF200First edition2006-02-01Anhydrous milk fat Determination of sterol composition by gas liquid chromatography (Routine method) Matire grasse anhydre du lait Dtermination de la composition strolique par chromatographie liquide en phase gazeuse (Mthode de routine) BS ISO 18252
9、:2006ii iiiContents Page Foreword iv 1 Scope 1 2 Normative references 1 3 Terms and definitions .1 4 Principle2 5 Reagents.2 6 Apparatus .3 7 Sampling.5 8 Preparation of test sample5 9 Procedure .5 9.1 Sterol standard solutions .5 9.2 Test portion 6 9.3 Saponification 6 9.4 Extraction of the unsapon
10、ifiable matter6 9.5 Qualitative analysis .6 9.6 Quantitative analysis.7 10 Precision.8 10.1 Interlaboratory test 8 10.2 Repeatability.8 10.3 Reproducibility.8 11 Test report 9 Annex A (informative) Example of the gas-liquid chromatographic analysis.10 Annex B (informative) Results of an interlaborat
11、ory trial .11 Bibliography 13 BS ISO 18252:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each
12、 member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the Internationa
13、l Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards
14、 adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of
15、 patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 18252IDF 200 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by IS
16、O and IDF. BS ISO 18252:2006vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF
17、 collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires
18、 approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 18252IDF 200 was prepar
19、ed by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team on Fat, of the Standing Committee on Main components in
20、milk, under the aegis of its project leaders, Mrs. M. Juarez (ES) and Mrs. G. Contarini (IT). BS ISO 18252:2006blank1Anhydrous milk fat Determination of sterol composition by gas liquid chromatography (Routine method) WARNING The use of this International Standard may involve hazardous materials, op
21、erations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish safety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This Inter
22、national Standard specifies a routine gas liquid chromatographic method for the determination of the sterol composition in anhydrous milk fat extracted from dairy products directly on the unsaponifiable matter, without purification and derivatization. The first goal of this International Standard is
23、 the quantitative evaluation of cholesterol, which represents about 98 % of the sterol fraction of pure milk fat. Moreover, in the case of analysis of milk fat in a mixture of vegetable fats, the specified procedure allows the evaluation of the most important phytosterols. The procedure has been val
24、idated on milk fat samples containing approximately 28 % to 32 % of vegetable fat. Due to the absence of the purification step, which allows the complete removal of interfering compounds from the unsaponifiable matter, particular care should be taken when applying this method to the verification of
25、the purity of milk fat of unknown origin. In the case of suspicious results, the reference method described in ISO 12078IDF 159 can be used. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited
26、applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 14156IDF 172, Milk and milk products Extraction methods for lipids and liposoluble compounds 3 Term
27、s and definitions For the purposes of this document, the following terms and definitions apply. 3.1 sterol composition mass fraction of substances determined by the procedure specified in this International Standard NOTE The sterol composition can be expressed either as milligrams per 100 g of fat,
28、or as percent of total sterol content. BS ISO 18252:20062 4 Principle 5cholestane is added to the test sample as the internal standard. The fat is saponified with methanolic potassium hydroxide. Unsaponifiable matter is extracted by diethyl ether. It is concentrated and determined by capillary gas-l
29、iquid chromatography. Individual sterols are identified by comparison with the retention time of the reference standard sample. Sterols are quantified by reference to the internal standard. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 5.1 Water, complying
30、with grade 2 in accordance with ISO 3696:1987. 5.2 Ethanol (C2H5OH), absolute. 5.3 Methanol (CH3OH), containing a mass fraction of water of u 0,5 %. 5.4 Saponification reagent, potassium hydroxide methanolic solution, c(KOH) = 2 mol/l. Dissolve 11,2 g of KOH in 100 ml of methanol (5.3) and mix well.
31、 5.5 Sodium sulfate (Na2SO4), anhydrous. 5.6 Diethyl ether (C2H5OC2H5), free from peroxides. 5.7 n-Hexane CH3(CH2)4CH3. 5.8 5-Cholestane, of purity 99 %. 5.9 5-Cholestane standard solution. Accurately weigh about 60 mg of 5-cholestane (5.8) into a 100 ml one-mark volumetric flask (6.4). Dilute to th
32、e 100 ml mark with n-hexane( 5.7)/ethanol (5.2) with a ratio of 1:10 or n-hexane (5.7) and mix. The 5-cholestane standard solution may be stored in a refrigerator for one month. 5.10 Cholesterol, of purity 99 %. 5.11 Cholesterol standard solution. Accurately weigh about 60 mg of cholesterol (5.10) i
33、nto a 100 ml one-mark volumetric flask (6.4). Dilute to the 100 ml mark with ethanol (5.2) or n-hexane (5.7) and mix. The cholesterol standard solution may be stored in a refrigerator for one month. 5.12 Campesterol, of purity 65 %. 5.13 Campesterol standard solution. Accurately weigh about 10 mg of
34、 campesterol (5.12) into a 100 ml one-mark volumetric flask (6.4). Dilute to the 100 ml mark with n-hexane (5.7) and mix. The campesterol standard solution may be stored in a refrigerator for one month. 5.14 Stigmasterol, of purity 95 %. BS ISO 18252:200635.15 Stigmasterol standard solution. Accurat
35、ely weigh about 10 mg of stigmasterol (5.14) into a 100 ml one-mark volumetric flask (6.4). Dilute to the 100 ml mark with n-hexane (5.7) and mix. The stigmasterol standard solution may be stored in a refrigerator for one month. 5.16 -Sitosterol, of purity 95 %. 5.17 -Sitosterol standard solution. A
36、ccurately weigh about 10 mg of -sitosterol (5.16) into a 100 ml one-mark volumetric flask (6.4). Dilute to the 100 ml mark with n-hexane (5.7) and mix. The -sitosterol standard solution may be stored in a refrigerator for one month. NOTE Since the phytosterol standard solutions (5.13, 5.15 and 5.17)
37、 are used only for a qualitative evaluation, they may be replaced by sterols prepared from soya oil having campesterol, stigmasterol and -sitosterol as major components. 6 Apparatus WARNING Since the determination involves the use of volatile flammable solvents, electrical apparatus employed shall c
38、omply with the legislation relating to the hazards in using such solvents. Usual laboratory apparatus and, in particular, the following. 6.1 Drying oven, capable of being maintained at a temperature of between 50 C and 70 C. 6.2 Analytical balance, capable of weighing to the nearest 1 mg, with a rea
39、dability of 0,1 mg. 6.3 Round-bottomed flasks, with ground neck, of capacity 100 ml. 6.4 One-mark volumetric flasks, of capacity 100 ml. 6.5 Graduated pipettes, of capacity 10 ml. 6.5 One-mark pipettes, of capacity 1 ml. 6.7 Water bath, capable of being maintained at 50 C 2 C and of boiling. 6.8 Ref
40、lux condenser, to fit the round-bottomed flask (6.3). 6.9 Separating funnels, of capacity 100 ml. 6.10 Solvent dispenser or graduated cylinders, of capacities 10 ml and 20 ml. 6.11 Distillation or evaporation apparatus (e.g. rotary vacuum evaporator), for distilling or evaporating solvents, maintain
41、ing all temperatures up to boiling. 6.12 Glass funnels, of diameter 100 mm. 6.13 Dry filter paper, folded, fast grade, of diameter 200 mm. 6.14 Vials, cone shaped inside, of capacities 5 ml and 10 ml. 6.15 Nitrogen supply, with gas purity of at least 99 %. BS ISO 18252:20064 6.16 Test tube, with PTF
42、E-lined screw cap, of capacity 50 ml. 6.17 Gas liquid chromatography. 6.17.1 Injector. Maintain the vaporizing type injector (split/splitless), which is equipped with a thermo-stable septum, at a temperature of 30 C above the maximum oven temperature. In the case of a cold on-column injector, mainta
43、in the injector at a temperature several degrees below the boiling point of the solvent. 6.17.2 Oven, capable of being run with the temperature programmed between 50 C and 320 C. 6.17.3 Column, fused silica capillary column. Different types of stationary phase, film thickness, column length, and dia
44、meter may be used to successfully obtain sterol separation. In any case, the selected column shall produce both a complete separation between the solvent peak and 5-cholestane (i.e. 5-cholestane shall not elute in the broadening of the solvent) and a baseline resolution among the cholesterol, campes
45、terol, stigmasterol and -sitosterol peaks. Moreover, no baseline bleeding shall appear during the whole gas chromatographic (GC) run. An example of a correct GC profile, obtained by using the operative conditions listed in 6.18.2, is shown in Figure A.1. NOTE Commercial stationary phases containing
46、dimethylpolysiloxane or dimethylpolysiloxane, together with different percentages of phenyl/cyanopropyl-polysiloxane, are suitable. 6.17.4 Flame ionization detector, capable of being heated to a temperature of 30 C above the final temperature of the column oven. 6.17.5 Carrier gas, nitrogen, helium
47、or hydrogen, of purity at least 99,999 % 6.17.6 Other gases, free from organic impurities (CnHmof below 1 parts per million), nitrogen and hydrogen, of purity at least 99,995 %, and synthetic air. 6.17.7 Injection syringe, of capacity 1 l to 10 l. 6.17.8 Integration system, preferably computerized.
48、6.18 Gas chromatographic conditions. Follow the manufacturers instructions for the instrument set-up. The oven temperature and the carrier gas flow depend on the column selected and on the injection system adopted. The examples listed below report applicable conditions for split and on-column inject
49、ion systems. 6.18.1 Split injector. An example of applicable conditions using a split injector is: a) Carrier gas: helium; b) Column head pressure: 90 kPa; c) Column: fused silica capillary column, of length 25 m, of internal diameter 0,32 mm, of film thickness 0,25 m; d) Stationary phase: 5 % phenyl and 95 % dimethylpolysiloxane; e) Column temperature: isothermal, set at 280 C; BS ISO 18252:20065f) Detector temperature: set at 310 C; g) Injector temperature: set at 310 C; h) Split ratio: of ratio 1:40; i) Sample injected: 0,5 l. 6.
