1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58liquid chromatography (Reference method) ICS 67.100.01Anhydrous milk fat Determination of sterol co
2、mposition by gas BRITISH STANDARDBS ISO 12078:2006BS ISO 12078:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 15 February 2006 BSI 15 February 2006ISBN 0 580 47835 1Cross-referencesThe British Standards which implement international publ
3、ications 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 British Standards Online.This publication does not purport to include all the nec
4、essary 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.Summary of pagesThis document comprises a front cover, an inside front cover, the ISO title page, pages ii to v, a blank pa
5、ge, pages 1 to 16, an inside back cover 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 CommentsA list of organizations represented on this committee can be obtained on request to its se
6、cretary. present to 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.National forewordThis British Standard reproduces v
7、erbatim ISO 12078:2006 and 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: aid enquirers to understand the text;Reference numbersISO 12078:2006(E
8、)IDF 159:2006(E)INTERNATIONAL STANDARD ISO12078IDF159First edition2006-02-01Anhydrous milk fat Determination of sterol composition by gas liquid chromatography (Reference method) Matires grasses laitires anhydres Dtermination de la composition strolique par chromatographie en phase gazeuse (Mthode d
9、e rfrence) BS ISO 12078:2006ii iiiContents PageForeword iv Foreword. v 1 Scope 1 2 Normative references 1 3 Terms and definitions .1 4 Principle2 5 Reagents.2 6 Apparatus .3 7 Sampling.6 8 Preparation of test sample6 9 Procedure .6 9.1 Sterol standard solutions .6 9.1.1 Calibrating solution for the
10、determination of cholesterol response factor.6 9.1.2 Qualitative solution for the determination of sterol retention time 6 9.2 Test portion 6 9.3 Saponification 7 9.4 Extraction of the unsaponifiable matter7 9.5 Thin-layer chromatography (TLC)7 9.5.1 Preparation of TLC plates .7 9.5.2 Preparation of
11、 glass plate-developing tank 7 9.5.3 TLC of unsaponifiable matter.7 9.6 Preparation of silyl derivatives.8 9.7 Qualitative analysis .8 9.8 Quantitative analysis.8 9.8.1 Calculation of response factor.8 9.8.2 Determination of the test portion.9 9.8.3 Calculation and expression of results.9 10 Precisi
12、on.10 10.1 Interlaboratory test 10 10.2 Repeatability.10 10.3 Reproducibility.10 11 Test report 11 Annex A (informative) Examples of the gas-liquid chromatographic analysis.12 Annex B (informative) Results of an interlaboratory test14 Bibliography 16 BS ISO 12078:2006iv Foreword ISO (the Internation
13、al 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 member body interested in a subject for which a technical committee has been e
14、stablished 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 International Electrotechnical Commission (IEC) on all matters of electrotechnical standard
15、ization. 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 adopted by the technical committees are circulated to the member bodies for vo
16、ting. 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 patent rights. ISO shall not be held responsible for identifying any or all su
17、ch patent rights. ISO 12078IDF 159 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 ISO and IDF. BS ISO 12078:2006vForeword IDF (the International Dairy Federation)
18、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 collaborates with ISO in the development of standard methods of analysis and s
19、ampling 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 approval by at least 50 % of the IDF National Committees casting a vote. Atten
20、tion 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 12078IDF 159 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 3
21、4, 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 milk, under the aegis of its project leaders, Mrs M. Juarez (ES) and Mrs G. Con
22、tarini (IT). BS ISO 12078:2006blank1Anhydrous milk fat Determination of sterol composition by gas liquid chromatography (Reference method) WARNING The use of this International Standard may involve hazardous materials, operations and equipment. This standard does not purport to address all the safet
23、y 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 International Standard specifies a gas liquid chromatographic reference method for t
24、he determination of the sterol composition of anhydrous milk fat extracted from dairy products. 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 validated on milk fat samples c
25、ontaining approximately 28 % to 32 % of vegetable fat. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (includ
26、ing any amendments) applies. ISO 3696, Water for analytical use Specification and test methods. ISO 6799, Animal and vegetable fats and oils Determination of composition of the sterol fraction Method using gas-liquid chromatography. ISO 14156IDF 172, Milk and milk products Extraction methods for lip
27、ids and liposoluble compounds. 3 Terms 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 ei
28、ther as milligrams per 100 g of fat, or as percent of total sterol content. BS ISO 12078:20062 4 Principle A suitable standard solution (betulin) is added to the test portion. The fat is saponified with methanolic potassium hydroxide. The unsaponifiable matter is extracted by diethyl ether. The ster
29、ols in the unsaponifiable matter are separated by thin-layer chromatography. The sterols are converted into silyl derivatives. Gas-liquid chromatography is carried out using a capillary column. Individual sterols are identified by comparison with the retention time of a reference standard sample. St
30、erols are quantified by reference to an internal standard method. By using this International Standard, the adoption of thin-layer chromatography (TLC), as the purification procedure, guarantees the removal of the possible impurities included in the unsaponifiable matter. 5 Reagents Use only reagent
31、s of recognized analytical grade, unless otherwise specified. 5.1 Water, complying with grade 2 in accordance with ISO 3696. 5.2 Methanol (CH3OH), containing a mass fraction of water of u 0,5 %. 5.3 Saponification reagent, potassium hydroxide methanolic solution, c(KOH) = 2 mol/l. Dissolve 11,2 g of
32、 KOH in 100 ml of methanol (5.2) and mix well. 5.4 Sodium sulfate (Na2SO4), anhydrous. 5.5 n-Hexane CH3(CH2)4CH3. 5.6 Di-isopropyl ether, (CH3)2CH2O, free from peroxides and residues. 5.7 Betulin, of purity 99 %. 5.8 Betulin standard solution Accurately weigh about 60 mg of betulin (5.7) into a 100
33、ml one-mark volumetric flask (6.3). Add about 60 ml of di-isopropyl ether (5.6) and mix carefully until completely dissolved. To help the betulin dissolution, gently warm in a 50 C water bath. Wait for the temperature of the solution to decrease to room temperature. Dilute to the 100 ml mark with di
34、-isopropyl ether. The betulin standard solution may be stored in a refrigerator for one month. 5.9 Cholesterol, of purity 99 %. 5.10 Cholesterol standard solution Accurately weigh about 60 mg of cholesterol (5.9) into a 100 ml one-mark volumetric flask (6.3). Dilute to the 100 ml mark with n-hexane
35、(5.5) and mix. The cholesterol standard solution may be stored in a refrigerator for one month. 5.11 Campesterol, of purity 65 %. 5.12 Campesterol standard solution Accurately weigh about 10 mg of campesterol (5.11) into a 100 ml one-mark volumetric flask (6.3). Dilute to the 100 ml mark with n-hexa
36、ne (5.5) and mix. BS ISO 12078:20063The campesterol standard solution may be stored in a refrigerator for one month. 5.13 Stigmasterol, of purity 95 %. 5.14 Stigmasterol standard solution Accurately weigh about 10 mg of stigmasterol (5.13) into a 100 ml one-mark volumetric flask (6.3). Dilute to the
37、 100 ml mark with n-hexane (5.5) and mix. The stigmasterol standard solution may be stored in a refrigerator for one month. 5.15 -Sitosterol, of purity 95 %. 5.16 - Sitosterol standard solution Accurately weigh about 10 mg of -sitosterol (5.15) into a 100 ml one-mark volumetric flask (6.3). Dilute t
38、o the 100 ml mark with n-hexane (5.5) and mix. The -sitosterol standard solution may be stored in a refrigerator for one month. Since the phytosterol standard solutions (5.12, 5.14 and 5.16) are used only for a qualitative evaluation, they may be replaced by sterols prepared from soya oil having cam
39、pesterol, stigmasterol and -sitosterol as the major components. 5.17 Diethyl ether (C2H5OC2H5), free from peroxides and residues. 5.18 Chloroform (CHCl3). 5.19 Ethanol (C2H5OH), absolute. 5.20 Ammonia solution (NH3), containing a mass fraction of NH3of approximately 25 % (20(NH3) = 910 g/l). 5.21 Py
40、ridine (C5H5N3H2O), dry. 5.22 Silylating agents, such as trimethychlorosilane, hexamethyldisilazane or equivalent. 5.23 2,7-Dichlorofluorescein solution, with a concentration of 5 g/l absolute ethanol or, alternatively, rodamine G solution, with a concentration of 1,5 g/l absolute ethanol. 6 Apparat
41、us WARNING Since the determination involves the use of volatile flammable solvents, electrical apparatus employed shall comply with the legislation relating to the hazards in using such solvents. Usual laboratory apparatus and, in particular, the following. 6.1 Analytical balance, capable of weighin
42、g to the nearest 1 mg, with a readability of 0,1 mg. 6.2 Round-bottomed flasks, with ground neck, of capacity 200 ml to 250 ml. 6.3 One-mark volumetric flasks, of capacity 100 ml. 6.4 Reflux condenser, to fit the round-bottomed flasks (6.2). 6.5 Water bath, capable of being maintainied at 50 C 2 C a
43、nd of boiling. BS ISO 12078:20064 6.6 One-mark pipettes, of capacity 10 ml. 6.7 One-mark pipettes, of capacity 1 ml. 6.8 Separating funnels, of capacity 500 ml. 6.9 Glass funnels, of diameter 100 mm. 6.10 Dry filter paper, folded, fast grade, of diameter 250 mm. 6.11 Rotary vacuum evaporator, with w
44、ater bath capable of maintaining all temperatures up to boiling. 6.12 Graduated cylinders, of capacities 100 ml and 250 ml. 6.13 Nitrogen supply, with gas purity of at least 99 %. 6.14 Micropipette or microsyringe, capable of delivering drops of about 0,3 l to 0,4 l. Alternatively, an automatic drop
45、 applicator may be used. 6.15 Drying oven, capable of maintaining a temperature of 103 C 2 C. 6.16 Precoated silica plates1), for thin-layer chromatography, of size 200 mm 200 mm. 6.17 Glass plate-developing tank, with a ground-glass lid, suitably sized for the precoated silica plates (6.16). 6.18 S
46、praying device 6.19 Ultraviolet (UV) lamp, capable of working at wavelength 254 nm. 6.20 Micro-spatula 6.21 Round-bottomed flasks, with ground neck, of capacity 25 ml. 6.22 Water vacuum pump 6.23 Cylindrical filter funnels, with porous glass filter of diameter 40 m, to fit the 25 ml round-bottomed f
47、lask (6.21), with vacuum adapter. 6.24 Desiccator 6.25 Graduated cylinder, with ground neck, of capacity 100 ml. 6.26 Graduated pipette, of capacity 1 ml. 6.27 Vials, cone-shaped inside, of capacities 5 ml and 10 ml. 6.28 Gas-liquid chromatograph, equipped with flame ionization detector and capillar
48、y injection system (splitter, or on column). 6.28.1 Carrier gas, hydrogen or helium, of purity at least 99,999 %. 1) Machery Nagel Sirt 25 or Merck 5715 silica 60 are examples of suitable products available commercially. This information is given for the convenience of users of this International St
49、andard and does not constitute an endorsement by ISO or IDF of these products. BS ISO 12078:200656.28.2 Other gases, free from organic impurities (CnHmof below 1 part per million), nitrogen and hydrogen, of purity at least 99,995 %, and synthetic air. 6.28.3 Capillary column, with a stationary phase which has been successfully employed to perform separation of sterols that can have different polarities depending on their chemical composition (methylpolysiloxane, methylphenylpoly-siloxane, poly
