1、 STD-BSI BS EN IS0 bA00-ENGL 1778 Lb2qbb7 Ob7845Li BRITISH STANDARD Animal and vegetable fats and oils - Determination of the composition of fatty acids in the 2-position of the triglyceride molecules The European Standard EN IS0 800 : 1997 has the status of a British Standard. ICs 67.200.10 NO COPY
2、ING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW 741 m BS EN IS0 6800 : 1998 1998 BS 684-2-2.39 : STD*BSI BS EN IS0 b8OO-ENGL 1778 W 1b24bbS Ob78455 b88 AmdNo. BS EN IS0 6800 : 1998 Date Tex affected National foreword This British Standard is the English language version of EN IS0 680
3、0 : 1997. It is identicai with IS0 6800 : 1997. It supersedes BS 684-22.39 : 19% which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/11, Animal and vegetable fats and oils, which has the responsibility to: - aid enquirem to understand the text; - prese
4、nt to the responsible intemationai/European committee any enquiries on the interpretation, or proposais for change, and keep the UK interests informed; - monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can b
5、e obtained on request to its secretary. Cross-references Attention is drawn to the fact that CEN and CENELEC standards normaiiy include an annex which lists normative references to international publications with their corresponding European publications. The British Standards which implement intern
6、ational or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled International Standards Correspondence Index, or by using the Find facility of the BSI Standards Electronic Catalogue. Compliance with a British Standard does not of i
7、tself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN IS0 title page, the EN IS0 foreword page, the IS0 title page, page ii, paga 1 to 13 and a back cover. This British Standard, having been prepared under the direction of
8、 the Consumer products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 May 1998 O BSI 1998 ISBN O 680 29270 3 Amendments issued since publication STD*BSI BS EN IS0 b8OO-ENGL 1978 Lb29bbS Ob7895b 514 = EUROPEAN STANDARD NORME EUROPENNE E
9、UROPISCHE NORM EN IS0 6800 December 1997 ICs 67.200.00 Descriptors: see IS0 document English version Animal and vegetable fats and oils - Determination of the composition of fatty acids in the 2-position of the triglycerides molecules (IS0 6800: 1997) Corps gras dorigines animale et vgtale - Wtermin
10、alion de la composition des audes gras en posKion 2 dans les tnglydndes (IS0 6800:1997) Tierische und pflanzliche Fette und die - Bestimmung der Zusammensetzung von Fetsuren in der 2-Stellung von Triglyceridmoleklen (IS0 6800:1997) This European Standard was approved by CEN on 9 January 1998. CEN me
11、mbers are bound to comply with the CENICENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Upto-date lists and bibliographical references concerning such nationa! standards may be obtained on applicat
12、ion to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the
13、same status as the ocial versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France. Gemany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COWiITEE FOR STA
14、NDARDIZATION COMI78 EUROPEEN DE NORMALISATION EUROPISCHES KOMITEE FOR NORMUNG Centrai Secretariat: rue de Staaaart, 36 8-1050 Bruescl8 ID 1997 CEN All rights of exploilalion in any form and by any means resewed worldwide for CEN national Members. Ref. No. EN IC0 6800:1997 E STD-BSI BS EN IS0 b8DD-EN
15、GL 1778 LbZLtbb7 b78457 g5 m EN IS0 6800 : 1997 Foreword The text of the Intemational Standard IS0 6800:1997 has been prepared by Technical Committee, ISO/TC 34 “Agricultural food products“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their b
16、y-products - Methods of sampling and analysis“, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by June 1998, and conflicting national standards shall be wi
17、thdrawn at the latest by June 1998. According to the CENKENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, L
18、uxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of the International Standard IS0 6800:1997 was approved by CEN as a European Standard without any modification. STD-BSI BS EN IS0 b8OO-ENGL 1778 Lb2qbb7 Ob78Ll58 377 W EN IS0 680
19、0 : 1997 INTERNATIONAL STANDARD IS0 6800 Second edition 1997-1 2-1 5 Animal and vegetable fats and oils - Determination of the composition of fatty acids in the 2-position of the triglyceride molecules Corps gras dorigines animale et vgtale - Dtermination de la composition des acides gras en positio
20、n 2 dans les triglycrides Reference number IS0 6800: 1997( E) STD-BSI BS EN IS0 b8OO-ENGL 1778 lb2Vbb Ob78957 223 m EN IS0 6800 : 1997 Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing I
21、nternational Standards is normally carried out through IS0 technical committees. Each 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 liaiso
22、n with SO, also take part in the work. IS0 collaborates closely with the Intemational Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft Intemational Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as
23、an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard IS0 6800 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 11, Animal and vegetable fats and oils. This second edition cancels and replace
24、s the first edition (IS0 6800:1985), which has been technically revised. Annexes A to C of this International Standard are for information only. Descriptors: agricultural products, food products, animal fats, vegetable fats, animal Mis, vegetable oils, fatty acids, chemical analysis, determination,
25、chemical composition, gas phase chromatography. i STD.BS1 BS EN IS0 b8OO-ENGL 1998 Lb29bbS Ob784b0 TU = EN IS0 6800 : 1997 Animal and vegetable fats and oils - Determination of the composition of fatty acids in the 2-position of the triglyceride molecules 1 Scope This International Standard specifie
26、s a method for the determination of the composition of fatty acids which are esterified in the 2-position ( or internal position) of the triglyceride molecules in animal and vegetable fats and oils. Because of the nature of pancreatic lipase action, the method is applicable only to fats and oils wit
27、h a melting point below 45 OC. The method is not unreservedly applicable to all fats and oils, particularly those containing substantial amounts of - fatty acids with 12 or fewer carbon atoms (e.g. copra oil, palm kernel oil, butyric butter fats); - fatty acids with 20 and more carbon atoms and of a
28、 high degree of unsaturation (more than four double bonds) (e.g. fish oil and marine animal oil); - fatty acids which have secondary groups containing oxygen. NOTE - Fatty acids with double bonds in the (n-16) to (n-1 1) position (e.g. petroselinic acid) are converted only very slowly by pancreatic
29、lipase. This may lead to wrong results. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All standards are subject to revision,
30、 and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and IS0 maintain registers of currently valid International Standards. IS0 660:1996, Animal and vegetabl
31、e fats and oils - Determination of acid value and of acidity. IS0 661 :1989, Animal and vegetable fats and oils - Preparation of test sample. IS0 3696:1987, Water for analytical laboratory use - Specification and test methods. IS0 55083 990, Animal and vegetable fats and oils - Analysis by gas chrom
32、atography of methyl esters of fatty acids. 1 STD=BSI BS EN IS0 b8UU-ENGL 1998 Lb2Ybb9 Ob98qbL 981 D EN IS0 6800 : 1997 IS0 5509:-1) Animal and vegetable fats and oils - Preparation of methyl esters of fatty acids. 3 Principle After neutralization, where necessary, of any free fatty acids, purificati
33、on of the test portion by column chromatography. Partial enzymatic hydrolysis of the glycerides to yield 2-monoglycerides. Separation of lhe monoglycerides by thin-layer chromatography (TLC) and determination of their fatty acid composition by gas chromatography. 4 Reagents Use only reagents of reco
34、gnized analytical grade and water in accordance with grade 2 of IS0 3696. 4.1 Reagents for the purification of the test portion 4.1.1 2-Propanol. or ethanol, 95 YO (VN). 4.1.2 Hexane (if available) or light petroleum (boiling range 30 “C to 60 OC). 4.1.3 2-Propanol, 50 o/o (VN), or ethanol, 50 ? tri
35、c(hydroxymethy1)aminomethane. 2 STD.BS1 BS EN IS0 b8OO-ENGL 1778 = Lb24bb7 0b84b2 ALB m EN IS0 6800 : 1997 4.2.6 Pancreatic lipase, with an activity of between 8 and 20 units/mg. Store dry in a refrigerator. Before use, bring a portion of the powder to ambient temperature. NOTE - Lipase of suitable
36、activity is available commercially. If preferred, the lipase may be prepared and assayed in accordance with the procedure described in annex A. 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 Reagents for the isolation of the 2-monoglycerides Ethanol, 95 % ( WV). Hexane (if available) or light petroleum, boiling
37、range 30 OC to 60 “C. Acetone. Silica powder, with binder, for thin-layer Chromatography. Developing solvent, prepared as follows: hexane (if available) or light petroleum: 70 ml diethyl ether: 30 ml formic acid, 98 % (VN) min.: 1 ml - 4.3.6 2,7-Dichlorofluorescein, indicator solution, 2 g/l in etha
38、nol, rendered slightly alkaline by addition of a drop of 1 mol/l sodium hydroxide per 100 ml of the solution. 4.4 - E - ._ II - Reagents for the analysis of the 2-monoglycerides by gas chromatography * rn * See IS0 5508 and IS0 5509. 5 Apparatus Usual laboratoy equipment and, in particular, the foll
39、owing. 5.1 Apparatus for the purification of the test portion 5.1.1 Water bath, thermostatically controlled, and capable of being maintained at 30 OC to 40 OC. 5.1.2 Glass column, for chromatography, 13 mm internal diameter and 400 mm in length, equipped with a sintered glass plate and a tap. 5.1.3
40、Rotary evaporator, with 250 ml flask. 5.1.4 Tubing, for bubbling nitrogen. 5.1.5 Separating funnel, of 500 ml capacity. 5.1.6 Round-bottom flask, of 1 O0 ml capacity. 3 STD-BSI BS EN IS0 bAOO-ENGL 1778 = Lb2qbb7 Ob78Lib3 754 W EN IS0 6800 : 1997 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.3 5.3.1 Appa
41、ratus for the hydrolysis of the triglycerides Centrifuge. Centrifuge tube, glass, of 10 ml capacity, with ground stopper. Vibrating electric shaker, for vigorous agitation of the centrifuge tube. Water bath, thermostatically controlled, and capable of being maintained at 40 OC i 0,5 OC. Hypodermic s
42、yringe, of 1 ml capacity, with thin needle. Stopwatch. Apparatus for the isolation of the 2-monoglycerides Developing tank, for thin-layer chromatography, with ground glass lid, suitable for containing glass plates 200 mm x 200 mm. 5.3.2 Spreader and rack, for preparation of the plates. 5.3.3 Glass
43、plates, 200 mm x 200 mm. 5.3.4 Microsyringe, capable of dispensing drops of 3 pi to 4 pl. 5.3.5 Apparatus for spraying the indicator solution onto the plates 5.3.6 Microspatula. 5.3.7 Oven, capable of being maintained at 103 OC i 2 OC. 5.3.8 Ultraviolet lamp, for examining chromatographic plates, fo
44、r example with a wavelength of 254 nm. 5.3.9 Round-bottom flask, of 25 ml capacity, with air condenser of approximately 1 m length, with ground joint. 5.3.10 Conical flask, of 250 rnl capacity, with ground stopper. 5.3.1 1 Conical flask, of 50 ml capacity (if necessary). 5.3.12 Filter, of sintered g
45、lass, porosity P 40 (16 pm to 40 pm) (if necessary). 5.3.1 3 Desiccator, containing an efficient desiccant. 5.4 See IS0 5508 and IS0 5509. Apparatus for the analysis of the 2-monoglycerides by gas chromatography 6 Sampling It is important that the laboratory receive a sample which is truly represent
46、ative and has not been damaged or changed during transport and storage. 4 STD-BSI BS EN IS0 b8OO-ENGL 1778 m Lb24bb7 Ob784bY by0 m EN IS0 6800 : 1997 Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in IS0 5555 11. 7 Preparation of t
47、est sample Prepare the test sample from the laboratory sample in accordance with IS0 661. a Procedure NOTE - If it is required to check whether the repeatability requirement (see 10.2) is met, carry out two single determinations in accordance with 8.1 to 8.6. 8.1 Determination of the acidity of the
48、test sample Determine the acidity of the test sample in accordance with IS0 660. If the acidity is below 3 Oh (m/m), purify the sample through alumina in accordance with 8.3. If the acidity exceeds 3 Oh (mh), first neutralize the sample with sodium hydroxide in the presence of solvent in accordance
49、with 8.2 then purify through alumina in accordance with 8.3. 8.2 Neutralization with sodium hydroxide Dissolve about 10 g of the test sample in 100 ml of hexane (if available) or light petroleum (4.1 2) and transfer the solution to the separating funnel (5.1 5). Add 50 ml of 2-propanol or ethanol (4.1.1), a few drops of phenolphthalein solution (4.1.5) and a volume of sodium hydroxide solution (4.1.4) equivalent to the free acidity of the fat or oil, with an excess of 0,5 /O. Shake vigorously for 1 min,
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