Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters. Part 4: Determination by capillary gas chromatography

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1 Provläsnngsexemplar / Prevew INTERNTIONL STNDRD ISO Frst edton nmal and vegetable fats and ols Gas chromatography of fatty acd methyl esters Part 4: Determnaton by capllary gas chromatography Corps gras d orgnes anmale et végétale Chromatographe en phase gazeuse des esters méthylques d acdes gras Parte 4: Détermnaton par chromatographe capllare en phase gazeuse Reference number ISO 2015

2 Provläsnngsexemplar / Prevew COPYRIGHT PROTECTED DOCUMENT ISO 2015, Publshed n Swtzerland ll rghts reserved. Unless otherwse specfed, no part of ths publcaton may be reproduced or utlzed otherwse n any form or by any means, electronc or mechancal, ncludng photocopyng, or postng on the nternet or an ntranet, wthout pror wrtten permsson. Permsson can be requested from ether ISO at the address below or ISO s member body n the country of the requester. ISO copyrght offce Ch. de Blandonnet 8 CP 401 CH-1214 Verner, Geneva, Swtzerland Tel Fax copyrght@so.org ISO 2015 ll rghts reserved

3 Provläsnngsexemplar / Prevew Contents Page Foreword...v 1 Scope Normatve references Prncple Reagents and materals Reference fatty acd methyl esters (FMEs) Internal standards pparatus Samplng Preparaton of test sample Preparaton of methyl esters from fats, ols, and fatty acds Procedure General GC condtons Performance check Calculatons Qualtatve analyss and peak dentfcaton Quanttatve analyss Calculaton of the composton of fatty acd methyl esters Calculaton of the composton of fatty acd methyl esters usng correcton factors Calculaton of the composton of fatty acd methyl esters usng an nternal standard Precson Results of nterlaboratory test Repeatablty Reproducblty Test report... 7 nnex (nformatve) Theoretcal flame onzaton detector correcton factor (TCF) for fatty acd methyl esters (FMEs)... 9 nnex B (nformatve) Examples of chromatograms...10 nnex C (nformatve) Comparson of FME composton wth two dfferent GC columns...12 nnex D (nformatve) Results of an nterlaboratory tral...14 Bblography...20 ISO 2015 ll rghts reserved

4 Provläsnngsexemplar / Prevew Foreword ISO (the Internatonal Organzaton for Standardzaton) s a worldwde federaton of natonal standards bodes (ISO member bodes). The work of preparng Internatonal Standards s normally carred out through ISO techncal commttees. Each member body nterested n a subject for whch a techncal commttee has been establshed has the rght to be represented on that commttee. Internatonal organzatons, governmental and non-governmental, n lason wth ISO, also take part n the work. ISO collaborates closely wth the Internatonal Electrotechncal Commsson (IEC) on all matters of electrotechncal standardzaton. The procedures used to develop ths document and those ntended for ts further mantenance are descrbed n the ISO/IEC Drectves, Part 1. In partcular the dfferent approval crtera needed for the dfferent types of ISO documents should be noted. Ths document was drafted n accordance wth the edtoral rules of the ISO/IEC Drectves, Part 2 (see ttenton s drawn to the possblty that some of the elements of ths document may be the subject of patent rghts. ISO shall not be held responsble for dentfyng any or all such patent rghts. Detals of any patent rghts dentfed durng the development of the document wll be n the Introducton and/or on the ISO lst of patent declaratons receved (see ny trade name used n ths document s nformaton gven for the convenence of users and does not consttute an endorsement. For an explanaton on the meanng of ISO specfc terms and expressons related to conformty assessment, as well as nformaton about ISO s adherence to the WTO prncples n the Techncal Barrers to Trade (TBT) see the followng URL: Foreword - Supplementary Informaton The commttee responsble for ths document s ISO/TC 34, Food products, Subcommttee SC 11, nmal and vegetable fats and ols. Ths frst edton cancels and replaces ISO 5508:1990 and ISO 15304:2002, whch have been techncally revsed. ISO conssts of the followng parts, under the general ttle nmal and vegetable fats and ols Gas chromatography of fatty acd methyl esters: Part 1: Gudelnes on modern gas chromatography of fatty acd methyl esters Part 2: Preparaton of methyl esters of fatty acds Part 3: Preparaton of methyl esters usng trmethylsulfonum hydroxde (TMSH) Part 4: Determnaton by capllary gas chromatography v ISO 2015 ll rghts reserved

5 Provläsnngsexemplar / Prevew INTERNTIONL STNDRD nmal and vegetable fats and ols Gas chromatography of fatty acd methyl esters Part 4: Determnaton by capllary gas chromatography 1 Scope Ths part of ISO specfes a method for the determnaton of fatty acd methyl esters (FMEs) derved by transesterfcaton or esterfcaton from fats, ols, and fatty acds by capllary gas chromatography (GLC). Fatty acd methyl esters from C8 to C24 can be separated usng ths part of ISO ncludng saturated fatty acd methyl esters, cs- and trans-monounsaturated fatty acd methyl esters, and cs- and trans-polyunsaturated fatty acd methyl esters. The method s applcable to crude, refned, partally hydrogenated, or fully hydrogenated fats, ols, and fatty acds derved from anmal and vegetable sources. Ths method s not sutable for the analyss of dary, rumnant fats and ols, or products supplemented wth conjugated lnolec acd (CL). Mlk and mlk products (or fat comng from mlk and mlk products) are excluded from the scope of ths part of ISO Ths part of ISO s not applcable to d-, tr-, polymerzed and oxdzed fatty acds, and fats and ols. 2 Normatve references The followng documents, n whole or n part, are normatvely referenced n ths document and are ndspensable for ts applcaton. For dated references, only the edton cted apples. For undated references, the latest edton of the referenced document (ncludng any amendments) apples. ISO 661, nmal and vegetable fats and ols Preparaton of test sample ISO 3696, Water for analytcal laboratory use Specfcaton and test methods ISO 6353, Reagents for chemcal analyss ISO , nmal and vegetable fats and ols Gas chromatography of fatty acd methyl esters Part 2: Preparaton of methyl esters of fatty acds ISO , nmal and vegetable fats and ols Gas chromatography of fatty acd methyl esters Part 3: Preparaton of methyl esters usng trmethylsulfonum hydroxde (TMSH) 3 Prncple Usng capllary gas chromatography, FMEs are separated on a hghly polar statonary phase wth respect to ther chan length, degree of (un)saturaton, and geometry and poston of the double bonds. 4 Reagents and materals Unless otherwse stated, use only reagents as specfed n ISO and ISO (f lsted there). If not, then use reagents of recognzed analytcal grade and water of at least grade 3, as defned n ISO ISO 2015 ll rghts reserved 1

6 Provläsnngsexemplar / Prevew WRNING ttenton s drawn to the regulatons whch specfy the handlng of dangerous matter. Techncal, organzatonal, and personal safety measures shall be followed. 4.1 Reference fatty acd methyl esters (FMEs) Reference mxtures of pure FMEs and/or ols wth known fatty acd composton should be used for the dentfcaton of fatty acds analysed under the test condtons of ths method Fats and ols wth certfed fatty acd composton, e.g. certfed reference materal BCR Reference fatty acd methyl esters (FMEs) - Methyl esters of pure fatty acds, n partcular, cs- and trans-somers of octadecenoc (olec), trans-somers of octadecadenoc (lnolec), and octadecatrenoc (α-lnolenc) acds. Wde ranges of cs- and trans-octadecenoc methyl ester somers are avalable on the market. Trans-geometrcal somers of lnolec and α-lnolenc acds can be prepared n the laboratory wth the ad of p-toluenesulfonc acd. In addton to pure compounds, convenent mxtures of FMEs are also commercally avalable. 4.2 Internal standards For the quantfcaton of the fatty acds, n grams per 100 g, the use of a FME as an nternal standard (IS) s necessary. n external calbraton wth mxtures of dfferent fatty acds s also possble. NOTE If t s necessary to check the recovery and the effectveness of the dervatzaton method, then ether or both a TG and a FME nternal standard should be used. Whle the TG-IS s added to the sample pror to the FME preparaton, the FME-IS s added before or after the FME preparaton. The FME-IS s used to calculate the recovery of the FME from the TG-IS and therefore, the effcency of the dervatsaton procedure. In ths case, a dfferent chan length of the standards s requred. Dependng on the type of fat, dfferent nternal standards can be used (C11:0 FME, C17:0 FME, C19:0 FME, C21:0 FME, C23:0 FME, etc.). n external calbraton wth mxtures of dfferent fatty acds s also possble. It s recommended to carry out further analyss of the sample wthout the addton of the nternal standard to check the natural content of the fatty acd whch s used as the nternal standard. The content shall be consdered n the calculaton. IMPORTNT If the TG-IS (4.2.2) s hard to dssolve n the cold, a hot methylaton procedure, as specfed n ISO :2011, 4.3, 4.4, and 4.5, shall be used. The nternal standard solutons are stable f precautons are taken to elmnate the loss of solvent and therefore, a change n the concentraton of the IS. For example, store the soluton n a refrgerator n a well-sealed amber bottle when not n use. Pure standards are avalable on the market. Purty of the IS shall be confrmed by thn-layer chromatography, hgh-performance lqud chromatography, gas chromatography analyss, or by any other approprate technque. The followng are examples of sutable standards (as FME and TG): Fatty acd methyl ester (FME) as nternal standard (IS) soluton: C21:0 FME henecosanoc acd methyl ester (purty >99 %), mass concentraton 5,0 mg/ml n sooctane or MTBE should be used as the nternal standard Tracylglycerol (TG) nternal standard (IS) soluton: C21:0 TG - trhenecosanon (purty >99 %), mass concentraton 5,0 mg/ml n chloroform. The TG nternal standard soluton s stable f precautons are taken to elmnate the loss of solvent and therefore, a change n the concentraton of the IS. For example, store the soluton n a refrgerator n a well-sealed amber bottle when not n use. Pure trhenecosanon s avalable on the market. Purty of the IS shall be confrmed by thn-layer chromatography, hgh-performance lqud chromatography, gas chromatography analyss, or by any other approprate technque. 2 ISO 2015 ll rghts reserved

7 Provläsnngsexemplar / Prevew Toluene can be used n place of chloroform wth the followng consderatons. Trhenecosanon s not as soluble n toluene as t s n chloroform. soluton wth a mass concentraton of 2 mg/ml can be prepared n toluene. It s necessary to warm the soluton slghtly to get t dssolved, but once n soluton, t wll stay dssolved f kept at room temperature. If the soluton s stored n a refrgerator, t wll crystallze out, but can be dssolved agan by slght warmng of the soluton. Care has to be taken so none of the toluene s evaporated durng ths warmng procedure. lso, care has to be taken to prevent the loss of toluene durng storage. Solvents other than so-octane (.e. chloroform or toluene) have to be removed after the addton of the TG-IS as these solvents are not used n the dervatzaton accordng to ISO Iso-octane (2,2,4-trmethyl pentane). 4.4 Methyl tert-butyl ether (MTBE) (2-Methoxy-2-methylpropane). 4.5 Chloroform. SFETY PRECUTIONS Chloroform s classed as a carcnogenc solvent (Category 3). 4.6 n-hexane. 4.7 n-heptane. SFETY PRECUTIONS Prolonged exposure through nhalaton and swallowng could cause serous damage to health despte lmted evdence on the carcnogenc effect (Category 3). 4.8 Toluene. 5 pparatus Usual laboratory equpment and, n partcular, the followng. 5.1 Gas chromatograph, equpped wth flame onzaton detector, splt or spltless njector, and data acquston system. NOTE The use of on-column and programmable temperature vaporzer (PTV) njectors are also possble. 5.2 Capllary column, fused slca capllary 100 m and 0,25 mm.d. coated wth SP-2560 or CP-Sl 88 1), 100 % cyanopropylslcone statonary phase to a thckness of 0,20 µm. Commercally prepared columns are avalable from dfferent supplers. NOTE The use of 100 m, 0,25 mm ID, 0,20 µm flm thckness columns wth SP-2560 or CP-Sl 88 as the statonary phase are recommended as the separaton capacty of these columns s suffcent to separate most C18:1 trans- and cs-somers. If ths separaton s not requred, a 50 m or 60 m column can also be used. However, some 50 m or 60 m long columns mght also acheve ths separaton mostly for vegetable ols. Other types of columns (BPX70, DB-23, HP-23, Rtx-2330, SP-2330, SP-2380, etc.) are also possble, but a shft n the eluton order s possble. For fast GC analyss, short columns are also possble (10 m to 15 m), but wth lmted nformaton whch n certan cases, wll not be a problem. 5.3 Mcro syrnge, for gas chromatography, 10 μl delvery wth a hardened needle. 5.4 Carrer gas, hydrogen (recommended) or helum, 99,999 5 % pure or better, gas chromatography qualty, dred, oxygen removed by sutable flters (<0,1 mg/kg), free from organc mpurtes. NOTE Ntrogen gas s not acceptable as a carrer gas for ths method. 1) Examples of sutable products avalable commercally. Ths nformaton s gven for the convenence of users of ths document and does not consttute an endorsement by ISO of these products. Equvalent products may be used f they can be shown to lead to the same results. ISO 2015 ll rghts reserved 3

8 Provläsnngsexemplar / Prevew WRNING Hydrogen, whch s used wth capllary columns, can double the speed of the analyss (n comparson wth helum), but s hazardous. Hydrogen generators and safety devces are avalable and t s essental that a sutable devce be ncorporated nto the apparatus. 5.5 Flame gases, hydrogen and ar, gas chromatography qualty, free from organc mpurtes. 5.6 Make-up gas, ntrogen or helum, gas chromatography qualty, free from organc mpurtes. 6 Samplng representatve sample should be sent to the laboratory. It should not be damaged or changed durng transport or storage. Samplng s not part of the method specfed n ths part of ISO recommended samplng method s gven n ISO Preparaton of test sample Prepare the test sample n accordance wth ISO Preparaton of methyl esters from fats, ols, and fatty acds The fatty acd methyl esters shall be prepared n accordance wth ISO or ISO NOTE Pror to methylaton, the nternal standard soluton, f requred, s added to the reacton flask so that after the ol or fat s added, the mass fracton s between 0,05 and 0,10 mg IS/mg ol or fat. Snce a solvent s used n the IS, t shall be evaporated from the flask pror to the methylaton procedure. Dssolve the prepared FMEs n n-heptane, n-hexane, or so-octane. The mass concentraton should be approxmately 15 mg/ml to 20 mg/ml for splt njecton. For on-column njecton, the mass concentraton should be adapted. 9 Procedure WRNING Due to the toxc character of some solvents, a ventlated hood shall be used. 9.1 General The frst sample n an analyss batch shall always be a blank FME dssoluton solvent. No peaks shall be detected n ths blank run. 9.2 GC condtons dapt the temperatures and GC condtons consderng the type of fat, ol, or fatty acd analysed and the apparatus used. The followng condtons have been proven to be sutable for the separaton of FMEs (C4 to C24) on 100 m columns. However, other condtons are also possble and can be used. Injector temperature 250 C Detector temperature: 250 C Oven temperature: 120 C to 240 C wth 4 C/mn, hold for further 7 mn at 240 C Carrer gas hydrogen: column head pressure, 220 kpa lnear velocty; (30 to 40) cm/s, flow rate approx. 1,0 ml/mn 4 ISO 2015 ll rghts reserved

9 Provläsnngsexemplar / Prevew splt rato, 1:100 Injecton volume: 1 µl (equvalent to 15 µg to 20 μg FME) Examples of chromatograms and alternatve condtons are shown n nnex B and nnex C: NOTE For the analyss of anmal fats, the complete eluton of all FMEs can be checked wth certfed reference standards. 9.3 Performance check Column performance s checked usng a sutable mxture of fatty acd methyl esters coverng the range of fatty acds under nvestgaton. Snce commercal GC desgns are dfferent and the separaton obtaned s not dentcal to the example chromatograms, small changes n the sample sze, sample concentraton, or oven temperature may be requred. If so, adjust the sample sze, sample concentraton, or oven temperature untl the best separaton results are obtaned. If the column oven temperature needs to be adjusted, t should be adjusted by small ncrements, preferably n steps of 1 C. NOTE On all cyanopropylslcone capllary columns, the column temperature has a major effect on the eluton pattern of 13t- and 14t-C18:1, 16t-C18:1, 14c-C18:1, 9c,12c,15t-C18:3, 11c-C20:1 and 9c,12c,15c-C18:3. 10 Calculatons 10.1 Qualtatve analyss and peak dentfcaton The ndvdual FMEs are dentfed by ther retenton tmes and n comparson wth FME reference standards and reference hydrogenated ol samples. When unknown peaks are observed, they should be dentfed usng approprate procedures such as GC- MS, FTIR, slver-on chromatography, and classcal chemcal methods. Peaks of unknown dentty should not be ncluded n the summaton of peak areas when calculatng the fatty acd composton, unless they have been confrmed to be fatty acds. It s also possble to summarze unknown peaks as such. NOTE There s mnor co-eluton of cs- and trans-fatty acd somers, partcularly n the C18:1 (cs-9-olec acd) regon usng ths technque. Durng (hgh temperature) refnng (deacdfcaton and deodorzaton), only geometrcal somers are formed of the mono- and poly-unsaturated fatty acds,.e. the double bond(s) reman(s) at the same natural poston. Durng hydrogenaton, both postonal and geometrcal somers are formed Quanttatve analyss Calculaton of the composton of fatty acd methyl esters Calculate the area fracton, x, of the ndvdual fatty acd methyl esters, expressed as a percentage by area of methyl esters, as gven by Formula (1): x where = 100 (1) s the area of the ndvdual fatty acd methyl ester ; Σ s the sum of areas under all peaks of all ndvdual fatty acd methyl ester. For most fats and ols, the area fracton of the fatty acd methyl esters s equal to the area fracton of tracylglycerols n grams per 100 g (for certan cases, see ). ccordng to the method OCS Ce 1h-05, the factors for the converson of FMEs to TG equvalents are between 0,9114 (C8:0) and 0,9965 (C24:1) and are therefore neglgble. If the chromatographc system ISO 2015 ll rghts reserved 5

10 Provläsnngsexemplar / Prevew obeys these factors, t can be assumed that the rato of the peak areas of the FMEs s dentcal to the rato of the mass fractons. The results are expressed n grams per 100 g wth one decmal place for values Calculaton of the composton of fatty acd methyl esters usng correcton factors In certan cases, for example, when fatty acds wth fewer than 16 carbon atoms are present (laurc fats and ols wth C10, C12, and C14), the areas should be corrected wth specfc correcton factors (F). These factors should be determned for each sngle nstrument. For ths purpose, sutable reference materals wth certfed fatty acd composton n the correspondng range should be used. ccordng to the requrements of the clents, the correcton factor mght not be used. However, the utlzaton (or not) of the correcton factor shall be specfed on the analyss report. These correcton factors are not dentcal wth theoretcal FID correcton factors, whch are gven n nnex, as they also nclude the performance of the njecton system, etc. However, n the case of bgger dfferences, the whole system shall be checked for performance. For the reference mxture, the mass fracton w, n grams per 100 g of FME,, s gven by Formula (2): w where m = 100 (2) m m Σm s the mass of the FME,, n the reference mxture; s the total of the masses of the varous components, as FMEs of the reference mxture. From the chromatogram of the reference mxture, calculate the percentage by area for the FME,, as follows: x where = 100 (3) Σ s the area of the FME,, n the reference mxture; s the sum of all areas of all FMEs of the reference mxture. The correcton factor, F, s then: F m = m For the sample, the mass fracton, w, n grams per 100 g of each FME,, s as gven by Formula (5): w F = Σ ( F ) NOTE The calculated value corresponds to the percentage of mass of the ndvdual fatty acd calculated as tracylglycerol per 100 g fat Calculaton of the composton of fatty acd methyl esters usng an nternal standard In certan analyses (for example, where not all of the fatty acds are quantfed, such as when acds wth four and sx carbons are present, alongsde acds wth 16 and 18 carbons, or when t s necessary to determne the absolute amount of a fatty acd n a sample), t s necessary to use an nternal standard. (4) (5) 6 ISO 2015 ll rghts reserved

11 Provläsnngsexemplar / Prevew Fatty acds wth 15, 17, 19, or 21 carbons are frequently used. The correcton factor (f any) for the nternal standard should be determned. The mass fracton n grams per 100 g, of the fatty acd,, expressed as methyl ester, s then gven by Formula (6): where w m F = IS m F IS IS (6) s the area the FME, ; IS F F IS m s the area of the nternal standard; s the correcton factor of the fatty acd,, expressed as FME; s the correcton factor of the nternal standard; s the mass of the test porton, n mllgrams; m IS s the mass of the nternal standard, n mllgrams, corrected by ts purty (usually 0,99). The results are expressed wth one decmal place. 11 Precson 11.1 Results of nterlaboratory test Detals of an nterlaboratory test on the precson of the method are summarzed n nnex D. The values derved from ths nterlaboratory test mght not be applcable to concentraton ranges and matrces other than those gven Repeatablty The absolute dfference between two ndependent sngle test results obtaned usng the same method on dentcal test materal n the same laboratory, by the same operator, usng the same equpment wthn a short nterval of tme, wll, n not more than 5 % of cases, be greater than r gven n Tables D.1 to D Reproducblty The absolute dfference between two sngle test results, obtaned usng the same method on dentcal test materal, n dfferent laboratores, wth dfferent operators, usng dfferent equpment, wll, n not more than 5 % of cases, be greater than R gven n Tables D.1 to D Test report The test report shall specfy: a) all nformaton necessary for the complete dentfcaton of the sample; b) the samplng method used, f known, wth reference to ths part of ISO 12966,.e ISO ; c) the test method used wth reference to ths part of ISO 12966; d) all operatng detals not specfed n ths part of ISO 12966, or regarded as optonal, together wth detals of any ncdents whch mght have nfluenced the result; e) the test result(s) obtaned; ISO 2015 ll rghts reserved 7