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1、BRITISH STANDARD BS 684-2.46: 1998 ISO 9936:1997 Methods of analysis of fats and fatty oils Part 2: Other methods Section 2.46 Determination of tocopherols and tocotrienols contents ICS 67.200.10 Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) B
2、SI BS 684-2.46:1998 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 March 1998 BSI 04-1999 ISBN 0 580 29339 4 National foreword This British Standa
3、rd reproduces verbatim ISO 9936:1997 and implements it as the UK national standard. 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; present to the responsibl
4、e international/European committee any enquiries on the interpretation, or proposals 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 be obtained on request
5、 to its secretary. Cross-references The British Standards which implement international 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
6、BSI Standards Electronic Catalogue. 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 from legal obligations. Summa
7、ry of pages This document comprises a front cover, an inside front cover, pages i and ii, the ISO title page, pages ii to iv, pages 1 to 6, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in t
8、he amendment table on the inside front cover. Amendments issued since publication Amd. No.DateComments Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.46:1998 BSI 04-1999i Contents Page National forewordInside front cover Forewordii
9、i Text of ISO 99361 Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii blank Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13
10、:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.46:1998 ii BSI 04-1999 Contents Page Forewordiii 1Scope1 2Normative reference1 3Definition1 4Principle1 5Reagents1 6Apparatus1 7Sampling2 8Preparation of test sample2 9Procedure2 10Expression of results3 11Precision4 12Test report4 Annex A (
11、informative) Saponification5 Annex B (informative) Results of interlaboratory tests6 Annex C (informative) BibliographyInside back cover Table 1 Division factors2 Table 2 Repeatability limit (r) and reproducibility limit (R)4 Table B.1 Statistical results6 Descriptors: Agricultural products, food pr
12、oducts, animal fats, vegetable fats, animal oils, vegetable oils, chemical analysis, determination of content, vitamins, tocols, high performance liquid chromatography. Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.46:1998 BSI 04-
13、1999iii 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 member body interested in a subject for w
14、hich 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 International Electrotechnical Commission (IEC) on all
15、 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 approval by at least 75 % of the member bodies casting a vote. International Standard ISO
16、9936 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 11, Animal and vegetable fats and oils. Annex A to Annex C of this International Standard are for information only. Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontro
17、lled Copy, (c) BSI iv blank Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.46:1998 BSI 04-19991 1 Scope This International Standard specifies a method for the determination of the contents of free !-, “-, *- and $- tocopherols and
18、tocotrienols (referred to jointly as tocols) in animal and vegetable fats and oils (referred to hereinafter as fats) by high-performance liquid chromatography (HPLC). For products containing tocopherol or tocotrienol esters, it is necessary to prepare the unsaponifiable matter. NOTEA suitable method
19、 involving a cold saponification procedure is described in Annex A for information only. 2 Normative reference The following standard contains provisions which, through reference in this text, constitute provisions of this International Standard. At the time of the publication, the edition indicated
20、 was valid. All standards are subject to revision, and parties to agreements based on the International Standard are encouraged to investigate the possibility of applying the most recent edition of the standard indicated below. Members of IEC and ISO maintain registers of currently valid Internation
21、al Standards. ISO 661:1989, Animal and vegetable fats and oils Preparation of test sample. 3 Definition For the purposes of this International Standard, the following definition applies. 3.1 tocols contents mass fractions of the individual tocols, determined using the method specified in this Intern
22、ational Standard NOTEThey are expressed in micrograms per gram. 4 Principle A test portion is dissolved and the individual tocols are separated by high-performance liquid chromatography. The content of each tocol is calculated using calibration factors determined from calibration solutions. 5 Reagen
23、ts All reagents shall be of HPLC grade or equivalent. 5.1 !-, “-, *- and $- tocopherol and tocotrienol standards 5.1.1 If tocopherol standards are not available, a blend of wheat germ and soya bean oil can be used to obtain chromatograms which contain !-, “-, *- and $-tocopherols. 5.1.2 If tocotrien
24、ol standards are not available, palm oil can be used to identify !- and *-tocotrienols. The chromatograms obtained can be used to assist peak identification in test sample chromatograms, in which case the calibration factors for the corresponding tocotrienols should be used. NOTE“-, *- and $-tocophe
25、rol standards can be obtained from Merck; !-tocopherol can be obtained from various suppliers. It has been reported that the purity of some commercially available tocopherol standards may vary between 85 % and 100 %. Thus it is important to determine the concentration of prepared calibration solutio
26、ns by UV spectrometry. 5.2 Methanol. 5.3 Dichloromethane. 5.4 n-Hexane. 5.5 Propan-2-ol. 5.6 HPLC mobile phase, propan-2-ol, 0,5 % (V/V) solution in n-hexane. 6 Apparatus All glassware shall be of low actinic activity. No apparatus shall give an alkaline reaction. This requirement is not applicable
27、when solutions are protected by pyrogallol. Usual laboratory apparatus and, in particular, the following. 6.1 HPLC system, consisting of a high-pressure pump, a sample injection device, a fluorescence detector with the excitation wavelength set at 290 nm and emission wavelength at 330 nm, and a char
28、t recorder or recording integrator. NOTEAn ultraviolet (UV) detector may be used if a fluorescence detector is not available but it is not recommended. However, if a UV detector is used, the wavelength should be set at 292 nm. 6.2 HPLC analytical column, 250 mm 4 mm, packed with microparticulate sil
29、ica having a mean particle size of about 5 4m. NOTE 1Suitable silica column packing materials available commercially are 5 4m LiChrosob SI 60 and Spherisorb S5W1). NOTE 2The length and the diameter of the column may be varied according to the HPLC technique used. 1) This information is given for the
30、 convenience of users of this International Standard and does not constitute an endorsement by ISO of these products. Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.46:1998 2 BSI 04-1999 NOTE 3Depending on the silica HPLC column st
31、atus (prehistory, dry or deactivation by traces of water, etc.) a triacylglycerol peak could overlap the !-tocopherol peak. If this happens, different results may be obtained from a fluorescence detector (possibility of quenching) and a UV detector (peak disturbance). This will be most problematic i
32、f a calibration is carried out with a calibrant that does not contain fat for the analysis of fat-containing samples. 6.3 UV spectrometer, capable of absolute measurement of absorbance at precisely defined wavelengths. 6.4 Rotary evaporator. 7 Sampling It is important that the laboratory receive a s
33、ample which is truly representative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 5555 2. 8 Preparation of test sample In the case of liquid laboratory sample
34、s, prepare the test sample by homogenization as described in ISO 661, except that filtration should be avoided. In the case of solid samples, transfer a representative portion (i.e. not less than 10 % by mass of the laboratory sample) to a glass beaker and carefully homogenize by melting, with gentl
35、e mixing, in a water bath at a temperature not exceeding 40 C. Preparation of the test samples should be carried out, as far as is practicable, in subdued light and in any case out of direct sunlight. 9 Procedure NOTEIn general, the oxidation of tocols during the analysis may lead to low results. In
36、 the presence of oxygen they are oxidized more quickly in the presence of alkalis, or under the influence of heat or light, and measures should be taken to guard against these influences. 9.1 Preparation of calibration solutions 9.1.1 Stock calibration solutions Prepare a stock solution of each toco
37、l by weighing 10 mg 1 mg of the standard (5.1) into a 100 ml one-mark volumetric flask and diluting to the mark with hexane (5.4). Pipette 10 ml of this solution into an amber glass round-bottomed flask and remove all hexane on a rotary evaporator (6.4) under vacuum at a temperature not greater than
38、 40 C. Restore atmospheric pressure with nitrogen and remove the flask from the evaporator as soon as all the solvent has been removed. Pipette into the flask 10 ml of methanol (5.2) and swirl to dissolve the residue. Measure the absorbance of this solution at the appropriate wavelength using the UV
39、 spectrometer (6.3). Calculate the concentration (in micrograms per millilitre) by dividing the absorbance value by the appropriate factor given in Table 1. Table 1 Division factors 9.1.2 Working calibration solution Mix appropriate volumes of the stock calibration solutions (9.1.1) to obtain a mixe
40、d tocol working calibration solution, and dilute with n-hexane to give a solution containing between 1 4g and 5 4g of each standard per millilitre. The working calibration solution shall be freshly prepared each working day. Protect all solutions from light and store them at between 0 C and 4 C. NOT
41、E 1Stock standard solutions can be satisfactorily stored in amber low-actinic glassware for up to 1 week if refrigerated. Flasks may be wrapped in aluminium foil. NOTE 2If a UV detector is used, a more concentrated solution may be needed. 9.2 Optimization of working parameters 9.2.1 If the column (6
42、.2) is new or of unknown history, or if for any other reason it is necessary to condition it, wash and condition it for about 10 min with methanol, then dichloromethane, followed by n-hexane at a flow rate of about 1 ml/min. Pump the HPLC mobile phase (5.6) through the column at a flow rate of 1 ml/
43、min for at least 30 min. WARNING Methanol and dichloromethane are hazardous to human subjects and to the environment. Handle them with care. Wavelength nm TocopherolDivision factor 292 296 298 298 !-tocopherol “-tocopherol *-tocopherol $-tocopherol 0,007 6 0,008 9 0,009 1 0,008 7 NOTEThe factors quo
44、ted are derived from the E values (1 %/1 cm) of the tocopherols. For example, the E value (1 %/1 cm) of -tocopherol is 76 at 292 nm (in methanol); therefore a 1 4g/ml solution of -tocopherol will have an absorbance of 0,007 6 at 292 nm. Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:00
45、 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.46:1998 BSI 04-19993 9.2.2 Inject about 20 4l of the working calibration solution (9.1.2) into the column and, if necessary, adjust the propan-2-ol content of the mobile phase and the flow rate to achieve the following conditions: a) !-tocopherol
46、retention time of not less than 5 min; b) resolution factor RF for the separation of “- and *-tocopherols of not less than 1,0; i.e. almost baseline separation, where RF is calculated using the following formula: where 9.2.3 Select the optimum settings for the detector and integrator sensitivity and
47、 the chart speed. Inject about 20 4l of the working calibration solution (9.1.2). Repeat the injection and check that reproducible chromatograms are obtained. NOTE 1Mobile-phase flow rates in the range 0,7 ml/min to 1,5 ml/min have been found to be satisfactory. Higher flow rates can result in poor
48、chromatography, and are to be avoided if UV detection is used. NOTE 2It should be possible to achieve an efficiency of 10 000 plates per metre calculated on the $-tocopherol peak. The efficiency n, in plates per metre, may be calculated using the formula: n = 5,54dr(III)/b0,5(III) where 9.3 Preparat
49、ion of test solution Depending on the tocol concentration (9.1.2), weigh, to the nearest 1 mg, 2 g 0,1 g of the test sample (clause 8) into a 25 ml one-mark volumetric flask. Add a quantity of hexane (5.4), swirling to dissolve the test portion, and dilute to the mark with the same solvent. NOTEIt may be necessary to dilute this solution further prior to chromatography. It is important that the test solutions be protected from light prior to analysis, and analysed on the day of preparation. 9.4 Determination 9.4.1 Inject 20 4l of the working