1、BRITISH STANDARD BS EN ISO 6800:1998 BS684-2-2.39: 1998 Animal and vegetable fats and oils Determination of the composition of fatty acids in the2-position of the triglyceride molecules The European Standard EN ISO6800:1997 has the status of a British Standard. ICS 67.200.10BSENISO6800:1998 This Bri
2、tish Standard, having been prepared under the directionof the Consumer Products and Services Sector Board, was published under the authority of the Standards Boardand comes into effect on 15 May1998 BSI04-1999 ISBN 0 580 29270 3 National foreword This British Standard is the English language version
3、 of EN ISO6800:1997. It is identical with ISO6800:1997. It supersedes BS684-2-2.39:1986 which is withdrawn.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 enquirers to understand the text; p
4、resent to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UKinterests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can
5、 be obtained on request to its secretary. Cross-references Attention is drawn to the fact that CEN and CENELEC standards normally include an annex which lists normative references to international publications with their corresponding European publications. The British Standards which implement inte
6、rnational 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. A British Standard does not purport to inc
7、lude 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 from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, page
8、s i and ii, theEN ISO title page, page 2, the ISO title page, pages ii to iv, pages1 to7 and aback 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 front cover. Amendments issued since pub
9、lication Amd. No. Date CommentsBSENISO6800:1998 BSI 04-1999 i Contents Page National foreword Inside front cover Foreword 2 Foreword iii Text of ISO 6800 1ii blankEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO6800 December 1997 ICS 67.200.00 Descriptors: See ISO document English version An
10、imal and vegetable fats and oils Determination of the composition of fatty acids in the2-position of the triglycerides molecules (ISO6800:1997) Corps gras dorigines animale et vgtale Dtermination de la composition des acides gras en position2 dans les triglycrides (ISO6800:1997) Tierische und pflanz
11、liche Fette und le Bestimmung der Zusammensetzung von Fettsuren in der2-Stellung von Triglyceridmoleklen (ISO6800:1997) This European Standard was approved by CEN on9 January1998. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
12、 European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (En
13、glish, 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 same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, CzechRep
14、ublic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de St
15、assart 36, B-1050 Brussels 1997CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO6800:1997 EENISO6800:1997 BSI 04-1999 2 Foreword The text of the International Standard ISO6800:1997 has been prepared by Technical Committee ISO/TC3
16、4 “Agricultural food products” in collaboration with Technical Committee CEN/TC307 “Oilseeds, vegetable and animal fats and oils and their by-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
17、, either by publication of an identical text or by endorsement, at the latest by June1998, and conflicting national standards shall be withdrawn at the latest by June1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to
18、implement this European Standard: Austria, Belgium, CzechRepublic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UnitedKingdom. Endorsement notice The text of the International Standard ISO6800:1997 w
19、as approved by CEN as a European Standard without any modification.ENISO6800:1997 ii BSI 04-1999 Contents Page Foreword iii 1 Scope 1 2 Normative references 1 3 Principle 1 4 Reagents 1 5 Apparatus 2 6 Sampling 2 7 Preparation of test sample 2 8 Procedure 2 9 Expression of results 4 10 Precision 4 1
20、1 Test report 4 Annex A (normative) Preparation of the lipase and checking its activity 5 Annex B (informative) Results of interlaboratory tests 5 Annex C (informative) Bibliography 7 Table B.1 Statistical results of study on pork and beef fats using a packed column 6 Table B.2 Statistical results o
21、f study on olive oil using the capillary column GLC method 7ENISO6800:1997 BSI 04-1999 iii 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 o
22、ut through ISO 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 liaison with ISO, also take part in the work. ISO c
23、ollaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approva
24、l by at least75% of the member bodies casting a vote. International Standard ISO6800 was prepared by Technical Committee ISO/TC34, Agricultural food products, Subcommittee SC11, Animal and vegetable fats and oils. This second edition cancels and replaces the first edition (ISO6800:1985), which has b
25、een technically revised. Annex A to Annex C of this International Standard are for information only. Descriptors: Agricultural products, food products, animal fats, vegetable fats, animal oils, vegetable oils, fatty acids, chemical analysis, determination, chemical composition, gas phase chromatogra
26、phy.iv blankENISO6800:1997 BSI 04-1999 1 1 Scope This International Standard specifies a method for the determination of the composition of fatty acids which are esterified in the2-position ( or internal position) of the triglyceride molecules in animal and vegetable fats and oils. Because of the na
27、ture of pancreatic lipase action, the method is applicable only to fats and oils with a melting point below45C. The method is not unreservedly applicable to all fats and oils, particularly those containing substantial amounts of fatty acids with12 or fewer carbon atoms (e.g.copra oil, palm kernel oi
28、l, butyric butter fats); fatty acids with20 and more carbon atoms and of a 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. NOTEFatty acids with double bonds in the (n-16) to (n-11) position (e.
29、g.petroselinic acid) are converted only very slowly by pancreatic 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 edi
30、tions indicated were valid. All standards are subject to revision, 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 ISO maintain registers of currentl
31、y valid International Standards. ISO660:1996, Animal and vegetable fats and oils Determination of acid value and of acidity. ISO661:1989, Animal and vegetable fats and oils Preparation of test sample. ISO3696:1987, Water for analytical laboratory use Specification and test methods. ISO5508:1990, Ani
32、mal and vegetable fats and oils Analysis by gas chromatography of methyl esters of fatty acids. ISO5509: 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, purification of the test portion b
33、y column chromatography. Partial enzymatic hydrolysis of the glycerides to yield2-monoglycerides. Separation of the monoglycerides by thin-layer chromatography (TLC) and determination of their fatty acid composition by gas chromatography. 4 Reagents Use only reagents of recognized analytical grade a
34、nd water in accordance with grade2 of ISO3696. 4.1 Reagents for the purification of the test portion 4.1.1 2-Propanol, or ethanol, 95%(V/V). 4.1.2 Hexane (if available) or light petroleum (boiling range30C to60C). 4.1.3 2-Propanol, 50%(V/V), or ethanol,50% (V/V). 4.1.4 Sodium hydroxide, 0,5mol/l sol
35、ution. 4.1.5 Phenolphthalein solution, 1g per100ml of ethanol,95% (V/V). 4.1.6 Activated neutral alumina, for chromatography, Brockmann activity l, recently activated for2h at260C and kept in a desiccator. 4.1.7 Nitrogen. 4.2 Reagents for hydrolysis of the triglycerides 4.2.1 Diethyl ether, free fro
36、m peroxides. 4.2.2 Hydrochloric acid, 6mol/l solution. 4.2.3 Sodium cholate, 1g/l solution of enzymatic quality. 4.2.4 Calcium chloride, 220g/l solution. 4.2.5 Buffer solution, 2-amino-2-(hydroxymethyl)propan-1,3-diol 2) , 1mol/l, adjusted to pH8 with hydrochloric acid(4.2.2) using a pH-meter. Store
37、 this solution at between0C and4C and use within14 days. 4.2.6 Pancreatic lipase, with an activity of between8and20units/mg. Store dry in a refrigerator. Before use, bring a portion of the powder to ambient temperature. NOTELipase of suitable activity is available commercially. If preferred, the lip
38、ase may be prepared and assayed in accordance with the procedure described in Annex A. 4.3 Reagents for the isolation of the2-monoglycerides 4.3.1 Ethanol, 95% (V/V). 1) To be published. (Revision of ISO5509:1978) 2) Alternative names are: tris(hydroxymethyl)methylamine; tris(hydroxymethyl)aminometh
39、ane.ENISO6800:1997 2 BSI 04-1999 4.3.2 Hexane (if available) or light petroleum, boiling range30C to60C. 4.3.3 Acetone. 4.3.4 Silica powder, with binder, for thin-layer chromatography. 4.3.5 Developing solvent, prepared as follows: 4.3.6 29,79-Dichlorofluorescein, indicator solution,2g/l in ethanol,
40、 rendered slightly alkaline by addition of a drop of1mol/l sodium hydroxide per100ml of the solution. 4.4 Reagents for the analysis of the2-monoglycerides by gas chromatography See ISO5508 and ISO5509. 5 Apparatus Usual laboratoy equipment and, in particular, the following. 5.1 Apparatus for the pur
41、ification of the test portion 5.1.1 Water bath, thermostatically controlled, and capable of being maintained at30C to40C. 5.1.2 Glass column, for chromatography,13mm internal diameter and400mm in length, equipped with a sintered glass plate and a tap. 5.1.3 Rotary evaporator, with250ml flask. 5.1.4
42、Tubing, for bubbling nitrogen. 5.1.5 Separating funnel, of500ml capacity. 5.1.6 Round-bottom flask, of100ml capacity. 5.2 Apparatus for the hydrolysis of the triglycerides 5.2.1 Centrifuge. 5.2.2 Centrifuge tube, glass, of10ml capacity, with ground stopper. 5.2.3 Vibrating electric shaker, for vigor
43、ous agitation of the centrifuge tube. 5.2.4 Water bath, thermostatically controlled, and capable of being maintained at40C 0,5C. 5.2.5 Hypodermic syringe, of1ml capacity, with thin needle. 5.2.6 Stopwatch. 5.3 Apparatus for the isolation of the2-monoglycerides 5.3.1 Developing tank, for thin-layer c
44、hromatography, with ground glass lid, suitable for containing glass plates200mm 200mm. 5.3.2 Spreader and rack, for preparation of the plates. 5.3.3 Glass plates, 200mm 200mm. 5.3.4 Microsyringe, capable of dispensing drops of34l to44l. 5.3.5 Apparatus for spraying the indicator solution onto the pl
45、ates 5.3.6 Microspatula. 5.3.7 Oven, capable of being maintained at103C2C. 5.3.8 Ultraviolet lamp, for examining chromatographic plates, for example with a wavelength of254nm. 5.3.9 Round-bottom flask, of25ml capacity, with, air condenser of approximately1m length, with ground joint. 5.3.10 Conical
46、flask, of250ml capacity, with ground stopper. 5.3.11 Conical flask, of50ml capacity (if necessary). 5.3.12 Filter, of sintered glass, porosity P40(164m to404m) (if necessary). 5.3.13 Desiccator, containing an efficient desiccant. 5.4 Apparatus for the analysis of the2-monoglycerides by gas chromatog
47、raphy See ISO5508 and ISO5509. 6 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport and storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is
48、 given in ISO55551. 7 Preparation of test sample Prepare the test sample from the laboratory sample in accordance with ISO661. 8 Procedure NOTEIf it is required to check whether the repeatability requirement (see10.2) is met, carry out two single determinations in accordance with8.1 to8.6. 8.1 Deter
49、mination of the acidity of the test sample Determine the acidity of the test sample in accordance with ISO660. If the acidity is below3% (m/m), purify the sample through alumina in accordance with8.3. If the acidity exceeds3% (m/m), first neutralize the sample with sodium hydroxide in the presence of solvent in accordance with8.2 then purify through alumina in accordance with8.3. hexane (if available) or light petroleum: 70ml diethyl ether: 30 ml formic acid,98% (V/V) min.: 1 mlENISO6800:1997 BSI 04-1999 3
