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1、Reference number ISO 14378:2000(E) ISO 2000 INTERNATIONAL STANDARD ISO 14378 First edition 2000-06-01 Milk and dried milk Determination of iodide content Method using high-performance liquid chromatography Lait et lait en poudre Dtermination de la teneur en iodure Mthode par chromatographie en phase
2、 liquide haute performance Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:18:47 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14378:2000(E) PD
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7、ry of the requester. ISO copyright office Case postale 56 ? CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 734 10 79 E-mail copyrightiso.ch Web www.iso.ch Printed in Switzerland ii ISO 2000 All rights reserved Copyright International Organization for Standardization Provided by IHS under licen
8、se with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:18:47 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14378:2000(E) ISO 2000 All rights reservediii Foreword ISO (the International Organization for Standardization) is a worldwide
9、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 established has the right to be represented on that c
10、ommittee. 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 standardization. International Standards are drafted in acco
11、rdance with the rules given in the ISO/IEC Directives, Part 3. 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. Attention
12、is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 14378 was prepared by Technical Comm
13、ittee ISO/TC 34,Agricultural food products, Subcommittee SC 5,Milk and milk products, in collaboration with the International Dairy Federation (IDF) and AOAC International, and will also be published by these organizations. Copyright International Organization for Standardization Provided by IHS und
14、er license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:18:47 MDTNo reproduction or networking permitted without license from IHS -,-,- Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Sta
15、ndards 1/9972545001 Not for Resale, 04/22/2007 09:18:47 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARDISO 14378:2000(E) ISO 2000 All rights reserved1 Milk and dried milk Determination of iodide content Method using high-performance liquid chromatogra
16、phy WARNING The use of this International Standard may involve hazardous materials, operations 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
17、determine the applicability of regulatory limitations prior to use. 1Scope This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the iodide content of pasteurized whole milk and dried skim milk, when present at levels from 0,03 ?g/g to
18、 1 ?g/g and 0,3 ?g/g to 10,0 ?g/g respectively. NOTE 1The method has been collaboratively studied with samples of liquid whole milk and dried skim milk. There are no reasons to expect that the method would not be applicable to skimmed or partially skimmed milk as well as to dried whole milk. NOTE 2T
19、he method measures free (ionic) iodide. However, the total iodide content of fresh milk and good quality milk powder, in which no microbial growth has occurred, may contain a mass fraction of 5 % to 10 % of organically bound iodide. More iodide might be bound organically in milk where microbiologica
20、l deterioration has been occurred. 2Normative reference The following normative document contains provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, this publication do not apply. Ho
21、wever, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the normative document indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and
22、IEC maintain registers of currently valid International Standards. ISO 3696,Water for analytical laboratory use Specification and test methods. 3Term and definition For the purposes of this International Standard, the following term and definition applies. 3.1 iodide content of pasteurized whole mil
23、k or dried skim milk mass fraction of substances determined by the procedure specified in this International Standard NOTEThe iodide content is conventionally expressed in micrograms per gram. 4Principle A test portion is diluted with water. Insoluble and high-molecular-mass material is removed by f
24、iltration through a 25 000 D cut-off membrane. Iodide ions are separated by reverse-phase ion-pair HPLC with an electrochemical detector and a silver working electrode at 0 mV to 50 mV. The iodide content is calculated by means of a calibration graph. Copyright International Organization for Standar
25、dization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:18:47 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14378:2000(E) 2 ISO 2000 All rights reserved 5Reagents Use only reagents of recognized anal
26、ytical grade or, if appropriate, of special HPLC grade. 5.1Water, complying with grade 2 of ISO 3696. 5.2Iodide standard solutions WARNING: Aqueous solutions of iodides are unstable when exposed to light and shall be protected from light. 5.2.1Iodide stock solution, corresponding to 100 mg of iodide
27、 per litre. Dissolve 130,8 mg of potassium iodide (KI) in water in a 1 000 ml one-mark volumetric flask (6.2). Dilute to the mark with water and mix. The iodide stock solution may be kept for 1 month if stored in the dark at room temperature. 5.2.2Iodide working standard solutions, corresponding to
28、20 ?g, 50 ?g, 150 ?g and 250 ?g of iodide per litre respectively. Pipette 20 ?l, 50 ?l, 150 ?l and 250 ?l of the iodide stock solution (5.2.1) into four separate 100 ml one-mark volumetric flasks (6.2). Dilute each solution to the mark with water and mix. The iodide working standard solutions may be
29、 kept for 1 week if stored in the dark at room temperature. 5.3Acetonitrile (CH3CN), HPLC grade. 5.4Hexadecyltrimethylammonium chloride solution CH3(CH2)15N(CH3)3Cl, 25 % (by mass) solution in water, ion-pair chromatography grade. 5.5HPLC eluent: mixture of disodium hydrogen phosphate and hexadecylt
30、rimethylammonium chloride in a mixture of acetonitrile and water (68:32 by volume), pH = 6,8. Dissolve 1,42 g of disodium hydrogen phosphate (Na2HPO4) in about 600 ml of water in a 1 000 ml one-mark volumetric flask (6.2). Add 1,3 ml of hexadecyltrimethylammonium chloride solution (5.4) and mix well
31、. Then add 320 ml of acetonitrile (5.3) and mix again. Adjust the pH to 6,8 with concentrated orthophosphoric acid (H3PO4). Dilute to the mark with water and mix well. Clarify the solution by filtering first through a 1,2 ?m membrane filter and then through a 0,5 ?m membrane filter. Swirl the soluti
32、on to mix it and simultaneously degas by means of a vacuum or sonification for 2 min before initial use. The eluent can be modified by the addition of small amounts of water or acetonitrile to achieve minor adjustments in the retention time of iodide. The eluent may be kept for 1 year, if stored in
33、a tightly closed container. 5.6n-Pentanol 6Apparatus Usual laboratory equipment and, in particular, the following. 6.1Analytical balance, capable of weighing to the nearest 0,01 g, with a readability of three decimal places. 6.2One-mark volumetric flasks, of capacities 100 ml and 1 000 ml. 6.3Microp
34、ipettes, capable of delivering 20 ?l, 50 ?l, 150 ?l and 250 ?l, respectively. 6.4Graduated pipette, of capacity 2 ml, with 0,1 ml graduations. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale
35、, 04/22/2007 09:18:47 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14378:2000(E) ISO 2000 All rights reserved3 6.5Graduated cylinder, of capacity 500 ml. 6.6pH-meter, with combined glass electrode. 6.7Membrane filters, 1,2 ?m and 0,5 ?m, Nylon-6-61?, or equivalent, w
36、ith filter equipment to clarify HPLC eluent. 6.8Centrifuge, capable of holding 50 ml centrifuge tubes and capable of producing a radial acceleration of 1 000 g. 6.9Centrifuge tubes, of capacity 50 ml, internal diameter 27 mm, conical, made of disposable plastic, with screw caps. 6.10Conical membrane
37、 supports, to support membrane filter cone (6.11) in centrifuge tubes (6.9) (Amicon CS1A 1? , or equivalent). 6.11Membrane filter cones, 25 000 D to 30 000 D cut-off (Amicon Centreflo1?CF-25, or equivalent). Prepare new membrane filter cones before use as follows. Soak in a mixture of ethanol and wa
38、ter (2:8 by volume) for 1 h. Remove the cone and drain. Mount it in a conical membrane support (6.10) and place it in a 50 ml centrifuge tube. Centrifuge the cone at a radial acceleration of 900 g to 1 000 g for 5 min to 10 min. Invert the new membrane filter cone to drain any remaining solvent. Pla
39、ce the prepared cones into supports in clean, labelled centrifuge tubes (6.9) for sample analysis. After each use, soak the cones immediately in hot water, flush well with hot water and store them in a mixture of ethanol and water (1:5 by volume). Remove the solvent before the next use of the cones
40、as described above for new cones. NOTEAlternatively, Millipore Ultrafre-PF1?(UFP1, 10 000 D cut-off) filtration units may be used. These disposable filters do not need any pretreatment, and filtering can be carried out by slight pressure or vacuum; no centrifuge is needed. 6.12HPLC equipment, consis
41、ting of the following. 6.12.1Pump, capable of delivering a volume flow rate of 2 ml/min. 6.12.2Injector, manual or automatic, with injection capacities of 50 ?l to 200 ?l. 6.12.3Analytical column, PARTISPHERE C-182?, 5 ?m, internal diameter 4,7 mm, length 110 mm, or equivalent. 6.12.4Guard column (o
42、ptional), SPHERI-5 C-18 cartridge,3?internal diameter 3,2 mm, length 15 mm, or equivalent. 6.12.5Electrochemical detector, to be used in the d.c.-mode or pulsed amperometric mode, with a silver working electrode at 0 mV to + 50 mV potential. 6.12.6Strip chart recorder or integrator, capable of peak
43、area measurement; preferably using an electronic integrator having so-called “negative peak“ function (for example, Spectra Physics4)is suitable). 1? Nylon 6-6, Amicon CS1A, Amicon Centreflo and Millipore Ultrafre-PF are examples of suitable products available commercially. 2? PARTISPHERE C-18 is an
44、 example of a suitable product available from Whatman Inc. 3? SPHERI-5 C-18 cartridge is an example of a suitable product available from Brownlee. 4)Spectra Physics is an example of a suitable product available commercially. This information is given for the convenience of users of this Internationa
45、l Standard and does not consitute an endorsement by ISO of these products. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:18:47 MDTNo reproduction or networking permitted wit
46、hout license from IHS -,-,- ISO 14378:2000(E) 4 ISO 2000 All rights reserved 7Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707. It is important that the laboratory receive a sample which is truly representative an
47、d has not been damaged or changed during transport or storage. Store the sample in such a way that deterioration and change in its composition are prevented. 8Preparation of test sample 8.1General Avoid any bacterial contamination during preparation of the sample. 8.2Milk Bring the test sample to 20
48、 C ? 2 C and mix carefully. If a homogeneous dispersion of the fat is not obtained, heat the sample slowly to 40 C and mix gently by inversion. Then cool the sample to 20 C ? 2 C. 8.3Dried milk Transfer the sample to a container of capacity about twice the volume of the sample, provided with an airtight lid. Close the container immediately and mix the sample thoroughly by repeated shaking and inverting the container. 9Procedure 9.1Test portion 9.1.1Milk Weigh, to the nearest 0,1 g, 45 g ? 5 g of the test sample into a 100 ml one-mar