1、Cheese Determination of propionic acid level by chromatography Part 1: Method by gas chromatography Fromages Dtermination de la teneur en acide propionique par chromatographie Partie 1: Mthode par chromatographie en phase gazeuse First edition 2017-05 ISO/TS 19046-1 TECHNICAL SPECIFICATION Reference
2、 numbers ISO/TS 19046-1:2017(E) IDF/RM 233-1:2017(E) ISO and IDF 2017 IDF/RM 233-1 ISO/TS 19046-1:2017(E) IDF/RM 233-1:2017(E) ii ISO and IDF 2017 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO and IDF 2017, Published in Switzerland All rights reserved. Unless otherwise specified, no part of t
3、his publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in
4、the country of the requester. ISO copyright office International Dairy Federation Ch. de Blandonnet 8 CP 401 Silver Building Bd Auguste Reyers 70/B B-1030 Brussels CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Tel. + 32 2 325 67 40 Fax +41 22 749 09 47 Fax + 32 2 325 67 41 copyrightiso.
5、org infofil-idf.org www.iso.org www.fil-idf.org ISO/TS 19046-1:2017(E) IDF/RM 233-1:2017(E) Forewords .iv 1 Scope . 1 2 Normative references 1 3 T erms and definitions . 1 4 Principle 1 5 Reagents 1 6 Apparatus . 2 7 Sampling 3 8 Preparation of test sample . 4 9 Procedure. 4 9.1 Extraction system .
6、4 9.2 Preparation of the soap . 4 9.3 Quantitative determination by gas chromatography . 4 9.3.1 Calculation of response factor 4 9.3.2 Determination of the test portion . 5 10 Calculation and expression of results . 5 10.1 Calculation of propionic acid in cheese . 5 10.2 Expression of results 5 11
7、Precision . 5 11.1 Interlaboratory test. 5 11.2 Repeatability . 5 12 Test report . 6 Annex A (informative) Example of extraction system 7 Annex B (informative) Example of GC chromatogram 8 Annex C (informative) Interlaboratory test. 9 Bibliography .10 ISO and IDF 2017 All rights reserved iii Content
8、s Page ISO/TS 19046-1:2017(E) IDF/RM 233-1:2017(E) Forewords 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. Ea
9、ch 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 collaborates closely with the Internatio
10、nal Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the differen
11、t types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www. iso. org/ directives). Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shal
12、l not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www. iso. org/ patents). Any trade name used in this documen
13、t is information given for the convenience of users and does not constitute an endorsement. For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the World Trade Orga
14、nization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: w ww . i s o. org/ iso/ foreword. html. This document 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
15、being published jointly by ISO and IDF. A list of all parts in the ISO/TS 19046 | IDF/RM 233 series can be found on the ISO website. iv ISO and IDF 2017 All rights reserved IDF (the International Dairy Federation) is a non-profit private sector organization representing the interests of various stak
16、eholders in dairying at the global level. IDF members are organized in National Committees, which are national associations composed of representatives of dairy-related national interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and governments/food contro
17、l authorities. ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International Standards using the logos and reference numbers of both organizations. Attention is dr
18、awn 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. Details of any patent rights identified during the development of the document will be in the Introduction and/or on
19、 the ISO list of patent declarations received (see www .iso .org/ patents). Any trade name used in this document is information given for the convenience of users and does not constitute and endorsement. ISO/TS 19046-1 | IDF/RM 233-1 was prepared by the IDF Standing Committee on Analytical Methods f
20、or Composition and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. The IDF Reviewed method is equal to an ISO Publicly Available Specification (ISO/PAS) or an ISO Technical Specification (ISO/TS) and is therefore published jointly under ISO conditions. Th
21、e work was carried out by the IDF/ISO Project Group on Propionic acid (C25) of the Standing Committee on Analytical Methods for Composition under the aegis of its project leader P. Trossat (FR). A list of all parts in the ISO/TS 19046 | IDF/RM 233 series can be found on the ISO website. ISO/TS 19046
22、-1:2017(E) IDF/RM 233-1:2017(E) ISO and IDF 2017 All rights reserved v Cheese Determination of propionic acid level by chromatography Part 1: Method by gas chromatography WARNING This document can involve the use of products and implementation of procedures and equipment of a hazardous nature. This
23、document does not aim to address all the risks related to its use. It is the responsibility of the user of this document to establish appropriate hygiene and safety practices before using it, and to determine the applicability of any other restrictions. 1 Scope This document specifies a method for t
24、he determination of propionic acid level in cheese, using gas chromatography. 2 Normative references There are no normative references in this document. 3 T erms a nd definiti ons For the purposes of this document, the following terms and definitions apply. ISO and IEC maintain terminological databa
25、ses for use in standardization at the following addresses: IEC Electropedia: available at h t t p :/ www .electropedia .org/ ISO Online browsing platform: available at h t t p :/ www .iso .org/ obp 3.1 level of propionic acid mass fraction of propionic acid determined following the procedure describ
26、ed in this document Note 1 to entry: The level of propionic acid is expressed in mg/100 g of cheese. 4 Principle Preparation of the test sample by addition of the internal standard and homogenization in the presence of sulfuric acid. Continuous extraction of the mixture in a liquid-liquid extractor
27、by a mixture of ethers. Separation of the volatile fatty acids from the fatty phase in the form of their sodium salts (soaps) after neutralization in the presence of phenolphthalein (or other equivalent indicator) and drying of the soaps recovered in the aqueous phase. Separation of the propionic ac
28、id using gas chromatography and quantification by reference to an internal standard. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. TECHNICAL SPECIFICATION ISO/TS 19046-1:2017(E) IDF/RM 233-
29、1:2017(E) ISO and IDF 2017 All rights reserved 1 5.1 Valeric acid (internal standard) CH 3 (CH 2 ) 3 COOH of purity 99 % mass fraction. 5.2 Sulfuric acid solution H 2 SO 4 containing a mass/volume fraction of 10 %. 5.3 Petroleum ether, with any boiling range between 45C and 65C. 5.4 Diethyl ether C
30、2 H 5 O C 2 H 5 . 5.5 Mixed solvent, prepared shortly before use by mixing equal volumes of diethyl ether (5.4) and petroleum ether (5.3). 5.6 Ethanol C 2 H 5 OH containing a volume fraction of ethanol of approximately 77 %. Into a 50 ml volumetric flask, add 40 ml 2 ml of ethanol 96 % not denaturat
31、ed and make up to the mark with water. 5.7 Sodium hydroxide, c(NaOH) around 1 mol/l. 5.8 Indicator solution. Phenolphthalein solution C 20 H 14 O 4 containing a mass fraction of 1 % (in ethanol 95 % to 96 %) or other indicator with equivalent turning zone. 5.9 T rifluor oac etic acid CF 3 COOH disso
32、lved in ethanol 95 % to 96 % (6,67 % in volume fraction). 5.10 Propionic acid CH 3CH 2 COOH of purity 99,5 % mass fraction. 5.11 Calibration solution . Weigh to the nearest 0,1 mg about 400 mg of propionic acid (5.10) and about 100 mg of valeric acid (5.1). Dissolve up to 100 g with ethanol (5.6). 6
33、 Apparatus W A R N I N G T h e d e t e r m i n a t i o n i n v o l v e s t h e u s e o f v o l a t i l e f l a m m a b l e s o l v e n t s . A l l e l e c t r i c a l apparatus employed shall be safe relating to the hazards in using such solvents. Usual laboratory equipment and, in particular, the f
34、ollowing. 6.1 Grinding or grating device. 6.2 Homogenizer/mixer (e.g. Ultraturrax type 251 1) ), equipped with a rotor. 6.3 Mixer type vortex. 6.4 Soxhlet-type extractor apparatus (for light solvents), equipped with flask heater. See Annex A. 6.5 Separating funnel, of capacity 500 ml. 6.6 Rotary eva
35、porator and controlled vacuum device. 1) Ultraturrax type T251 is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product. ISO/TS 19046-1:2017(E) IDF/RM 233-1
36、:2017(E) 2 ISO and IDF 2017 All rights reserved 6.7 Drying oven, capable of maintaining at a temperature around 60 C. 6.8 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg. 6.9 Measuring cylinders, of capacity 50 ml and 100 ml. 6.10 Volumetric flasks, of capac
37、ity 50 ml. 6.11 Glass flasks, of capacity around 125 ml. 6.12 Glass flasks, of capacity around 250 ml. 6.13 Dispensers, of capacity 2,5 ml and 10 ml. 6.14 Gas chromatograph, with the following equipment. 6.14.1 Carrier gas, hydrogen, helium or nitrogen, purity 99,999 7 %. 6.14.2 Semi-capillary colum
38、n, 30 m length, 0,53 mm internal diameter, 2 m film thickness, with a modified polyethylene glycols polar stationary phase phase free fatty acid phase (FFAP), Carbowax TM2) or equivalent. 6.14.3 Gas chromatographic conditions . The following conditions have been found suitable to obtain a correct se
39、paration of propionic acid: carrier gas flow: 7 ml/min; oven temperature program: initial temperature of 80 C, maintained for 0,5 min, raised at a rate of 20 C min 1up to 170 C, maintained at this temperature for 6,5 min. An example of a GC profile obtained with these conditions is shown in Annex B.
40、 6.14.4 Flame ionization detector, set at a temperature of 250 C. 6.14.5 On column injector, set at a temperature of 250 C. 6.14.6 Injection syringe, of capacity 10 l. 6.14.7 Integration system, preferably being computerized. 6.15 Glass tubes (with stoppers). 7 Sampling Sampling is not part of the m
41、ethod specified in this document. A recommended sampling method is given in ISO 707 | IDF 50. It is important that the laboratory receives a sample that is representative and has not been damaged or changed during transport or storage. 2) Carbowax TMis an example of a suitable product available comm
42、ercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product. ISO/TS 19046-1:2017(E) IDF/RM 233-1:2017(E) ISO and IDF 2017 All rights reserved 3 o o (1) where ISO/TS 19046-1:2017(E) IDF/RM 233-1:2017(E) 8 Prepa
43、ration of test sample Prior to analysis, grind or grate the test sample by using an appropriate grinding or grating device (6.1), if necessary, after removing the rind, the smear or the mouldy surface layer of the cheese. Weigh, to the nearest 0,1 mg, about 20 g of cheese in a 125 ml flask (6.11) an
44、d record the mass (m). Add about 50 mg of valeric acid to the test sample and record the mass of valeric acid to 0,000 1 g. Add 50 ml 2 ml of sulfuric acid solution (5.2) and crush for 1 min using the homogenizer (6.2). 9 Procedure 9.1 Extraction system Pour 100 ml of the mixed solvent (5.5) into th
45、e glass flask (6.12) and put the extraction unit (6.4) on the flask. Introduce the cheese prepared (see Clause 8) in the extraction unit (6.4) and rinse slightly with water. Place the adapter in the extraction unit (6.4), install the condenser and switch on the flask heater. Perform extraction for 6
46、 h (example of extraction system is presented in Annex A). Cool the flask 15 min in a beaker of cold water until ambient temperature approximately without disconnecting the assembly on the extraction unit (6.4). Transfer the contents of the flask into the separating funnel, containing 40 ml of ethan
47、ol solution (5.6), 10 ml of water, 2,5 ml of sodium hydroxide (5.7) and 2 or 3 drops of indicator solution (5.8). Add a sufficient quantity of sodium hydroxide (5.7) so that the solution turns pink. Mix and allow the separating funnel to stand for at least 15 min and collect the hydroalcoholic solut
48、ion lower phase of soaps in a glass flask (6.12). Evaporate in the rotary evaporator under controlled vacuum (water bath at 80 C to 90 C) and complete drying in the oven at 60 C. Scrape off the soaps using a spatula and keep them in tube (6.15) until determination. 9.2 Preparation of the soap Weigh
49、approximatively 30 mg of the soap prepared as in 9.1 in a tube (6.15) and add 1 ml 0,2 ml of the trifluoroacetic acid solution (5.9). Mix until dissolved using a mixer vortex (6.3). The solution thus prepared shall be injected immediately and shall not be stored. 9.3 Quantitative determination by gas chromatography 9.3.1 Calculation of response factor Inject into the gas chromatograph 1,0 l of the calibrating solution (5.11). Determine the area of t
