High-Resolution GC Analyses of Fatty Acid Methyl Esters (FAMEs)

High-Resolution GC Analyses of Fatty Acid Methyl Esters (s) Fatty acid methyl esters (s) analysis is an important tool both for characterizing fats and oils and for determining the total fat content in foods. Fats can be extracted from a matrix using a nonpolar solvent and saponified to produce salts of the free fatty acids. After derivatizing the free acids to form methyl esters, the mixture can readily be analyzed by gas chromatography (GC) due to the volatility and thermal stability of the s. Gas chromatography has become an important technique in fats and oils analysis because accurate results can be obtained for complex as well as simple sample matrices. s analyses were among the first applications for gas chromatography, so many of the GC methods originally written for analysis of fats and oils described packed column technology. However, capillary columns offer significant advantages, including more efficient separations. When analyzing fats and oils with complex fatty acid profiles such as the cis and trans forms of polyunsaturated fatty acids, higher efficiencies are needed to resolve the individual components. Capillary columns with Carbowax-type (polyethylene glycol) stationary phases are typically used for analyses of saturated and unsaturated fatty acid methyl esters (Figures and ), and biscyanopropyl phases are used to resolve cis and trans isomers of polyunsaturated components (Figure ). Creating s Lipids are normally extracted from matrices using a nonpolar solvent such as ether and saponified to produce the free fatty acid salts. The fatty acid salts then are derivatized to form the fatty acid methyl esters, to increase volatility, improve peak symmetry, and decrease sample activity, thus providing more accurate analytical data. The official methods of the Association of Official Agriculture Chemists (AOAC International) [] and the American Oil Chemists Society (AOCS) [] contain procedures for the derivatization reaction, as does the European Pharmacopoeia []. In general, the glycerides are saponified by refluxing with methanolic sodium hydroxide. The esterification is effected with a reagent such as boron trifluoride in methanol, and the s are extracted with a nonpolar solvent (e.g., heptane) for analysis by GC. Several groups of researchers have proposed simplified procedures for creating the methyl esters. For example, lipids can be transmethylated in situ. This option combines all of the conventional steps, except the drying and post-reaction workup, into one step. [] For some samples, trimethylsulfonium hydroxide (TMSH), sodium methoxide, or methanolic hydrochloric acid, which are alternative derivatization reagents, can be used for transesterification. A major advantage of this approach is that the derivatization can be performed in a single, fast reaction step. [,,]

Figure : A WAX column provides fast, efficient separations of s in a marine oil-based standard. Peaks. C:0. C:. C:0. C:. C8:0. C8: (oleate). C8: (vaccenate) 8. C8: n c 9. C8: n 0. C0:0. C0: n9. C0: n. C0: n. C0: n. C0: n. C:0. C: n9 8. C:0 9. C: n 0. C: 8 9 0 Column WAX, 0 m, 0. mm ID, 0. µm ( 98) Sample Marine oil mix ( 0) Conc.: 0 mg/ml total Inj. Vol.: 0. µl split (split ratio 0:) Inj. Temp.: 0 C Temp.: 9 C to 0 C at C/min (hold min) Carrier Gas H, constant flow Linear Velocity: cm/sec Detector FID @ 0 C 8 9 0 0 8 9 0 GC_FF00 Figure : Rapid, excellent resolution of ocean nutrition sample s, using a WAX column. Peaks. C:0. C:0. C:0. C:. C:. C:0. C: 8. C: 9. C8:0 0. C8: (oleate). C8: (vaccenate). C8: n cis. C8: n. C8: n. C8: n. C0:0. C0: n 8. C0: n9 9. C0: n 0. C0: n. C0: n. C: n. C: n. C:0 (IS). C: n. C: n. C: n 8. C: 8 9 0 FAST analysis of marine oil s 8 9 0 Column WAX, 0 m, 0. mm ID, 0. µm ( 98) Sample Ocean nutrition sample Conc.: mg/ml total s Inj. Vol.: 0. µl split (split ratio 0:) Liner: mm split w/ wool Inj. Temp.: 0 C Temp.: 9 C to 0 C at C/min (hold min) Carrier Gas H, constant flow Linear Velocity: cm/sec Detector FID @ 0 C 8 0 8 9 GC_FF008 www.restek.com

Figure : Near-complete resolution of a mix on an Rt-0 column. 8 9 0 8 8 9 0 9 0.0.0.0 8.0 0.0.0.0.0 8.0 0.0.0.0.0 8.0 0.0.0.0.0 8.0 0.0.0.0.0 8.0 0.0.0 GC_FF Conc. Structural Peaks t R (min) (µg/ml) Nomenclature. Methyl butyrate. 0 C:0. Methyl caproate. 0 C:0. Methyl octanoate 9. 0 C8:0. Methyl decanoate.0 0 C0:0. Methyl undecanoate.0 0 C:0. Methyl dodecanoate 9.8 0 C:0. Methyl tridecanoate. 0 C:0 8. Methyl myristate.9 0 C:0 9. Methyl myristoleate.0 0 C: (c9) 0. Methyl pentadecanoate. 0 C:0. Methyl pentadecenoate 9. 0 C: (c0). Methyl palmitate 9.8 0 C:0. Methyl palmitoleate.0 0 C: (c9). Methyl heptadecanoate.8 0 C:0. Methyl heptadecenoate. 0 C: (c0). Methyl stearate.9 0 C8:0. Methyl octadecenoate.9 0 C8: (t9) 8. Methyl oleate. 0 C8: (c9) 9. Methyl linolelaidate.9 0 C8: (t9,t) 0. Methyl linoleate. 0 C8: (c9,c). Methyl arachidate.9 0 C0:0. Methyl linolenate 8. 0 C8: (c,c9,c). Methyl eicosenoate 9.0 0 C0: (c). Methyl linolenate 9. 0 C8: (c9,c,c). Methyl heneicosanoate 9.8 0 C:0. Methyl eicosadienoate.09 0 C0: (c,c). Methyl behenate.0 0 C:0 8. Methyl eicosatrienoate. 0 C0: (c8,c,c) 9. Methyl erucate.88 0 C: (c) 0. Methyl eicosatrienoate.8 0 C0: (c,c,c). Methyl tricosanoate. 0 C:0. Methyl arachidonate. 0 C0: (c,c8,c,c). Methyl docosadienoate.8 0 C: (c,c). Methyl lignocerate. 0 C:0. Methyl eicosapentaenoate.09 0 C0: (c,c8,c,c,c). Methyl nervonate. 0 C: (C). Methyl docosahexaenoate. 0 C: (c,c,c0,c,c,c9) Column Rt-0, 00 m, 0. mm ID, 0.0 µm ( 98) Sample Food industry mix ( 0) Diluent: Hexane/dichloromethane Conc:,000 µg/ml Inj. Vol.: µl split (split ratio 0:) Liner: Premium mm Precision liner w/wool ( 0.) Inj. Temp: C Temp.: 00 C (hold min) to 0 C at C/min (hold min) Carrier Gas He, constant flow Flow Rate:.0 ml/min Detector FID @ 8 C Make-up Gas Flow Rate: ml/min Make-up Gas Type: N Hydrogen flow: 0 ml/min Air flow: 00 ml/min Data Rate: 0 Hz Instrument Agilent 890A GC www.restek.com

Analyzing Polyunsaturated s The WAX polyethylene glycol stationary phase is specially tested with a polyunsaturated s mix to ensure resolution of the omega- and omega- fatty acids of interest. s such as methyl eicosapentenoate (C0:) and methyl docosahexaenoate (C:), found in nutraceutical ingredients and products such as marine oils, also are resolved. WAX columns offer excellent resolution of polyunsaturated s with significantly reduced analysis times compared to traditional Carbowax stationary phases. In fact, analysis times of less than 0 minutes are possible! Figures and show analyses of a marine oil standard and ocean nutrition sample, respectively. Both analyses are characterized by fast, effective resolution and sharp, symmetric peaks. Like WAX columns, Stabilwax columns and Rtx-Wax columns provide excellent resolution of s derived from either plant or animal sources. When polyunsaturated s are analyzed on one of these Carbowax-type capillary columns, analysis times of -0 minutes are generally required to fully resolve the C: from the C:0 internal standard, and the C:0 from C:. Resolving cis and trans Isomers Individual cis and trans isomers are resolved on a 00-meter Rt-0 column, making this the column of choice for analyzing partially hydrogenated fats. The highly polar biscyanopropyl phase gives the selectivity needed for resolving isomers, such as the cis and trans forms of C8:. The trans isomers elute before the cis isomers on this phase, opposite of the elution order on Carbowaxbased phases such as WAX or Rtx-Wax. Figure shows the chromatographic separation of s typically encountered in vegetable, animal, or marine fats and oils using an Rt-0 column. AOAC method 99.0 [] describes the determination of total fat content based on the fatty acid content after conversion to methyl esters. This is the specified method for determining total fat content for nutritional labeling purposes. After quantifying the total s present in the derivatized sample, the amount of fat (as triglycerides) in the sample is calculated based on initial sample weight. The 00-meter Rt-0 column meets the requirements of this procedure (Figure ). This column also allows quantification of the total trans content. www.restek.com

Figure : Use this food industry mix to standardize fat-by-fatty acid composition methods, such as AOAC 99.0. 8 9 0 8 9 0 8 9 0.0 8.0 0.0.0.0.0 8.0 0.0.0.0.0 8.0 0.0.0.0.0 8.0 0.0.0.0.0 8.0 0.0.0.0.0 GC_FF Conc. Peaks t R (min) (µg/ml) Structural Nomenclature. Methyl caproate. 0 C:0. Methyl octanoate.88 0 C8:0. Methyl decanoate 8.8 0 C0:0. Methyl undecanoate 8.9 0 C:0. Methyl dodecanoate 9. 0 C:0. Methyl tridecanoate 0. 0 C:0. Methyl myristate. 0 C:0 8. Methyl myristoleate.8 0 C: (c9) 9. Methyl pentadecanoate. 0 C:0 0. Methyl pentadecenoate. 0 C: (C0). Methyl palmitate.8 0 C:0. Methyl palmitoleate.98 0 C: (c9). Methyl heptadecanoate. 0 C:0. Methyl heptadecenoate 8.8 0 C: (c0). Methyl stearate 9. 0 C8:0. Methyl octadecenoate.08 0 C8: (t9). Methyl oleate.8 0 C8: (c9) 8. Methyl linolelaidate.09 0 C8: (t9,t) 9. Methyl linoleate. 0 C8: (c9,c) 0. Methyl arachidate 8. 0 C0:0. Methyl linolenate 9.98 0 C8: (c,c9,c). Methyl eicosenoate.0 0 C0: (c). Methyl linolenate. 0 C8: (c9,c,c). Methyl heneicosanoate. 0 C:0. Methyl eicosadienoate. 0 C0: (c,c). Methyl behenate. 0 C:0. Methyl eicosatrienoate. 0 C0: (c8,c,c) 8. Methyl erucate 8. 0 C: (c) 9. Methyl eicosatrienoate 8. 0 C0: (c,c,c) 0. Methyl arachidonate 9. 0 C0: (c,c8,c,c,c). Methyl tricosanoate 9. 0 C:0. Methyl docosadienoate. 0 C: (c,c). Methyl eicosapentaenoate.0 0 C0: (c,c8,c,c,c). Methyl lignocerate. 0 C:0. Methyl nervonate. 0 C: (c). Methyl docosahexaenoate.0 0 C: (c,c,c0,c,c,c9) Column Rt-0, 00 m, 0. mm ID, 0.0 µm ( 98) Sample Food industry mix ( 0) Diluent: Hexane/dichloromethane Conc.:,000 µg/ml Inj. Vol.: µl split (split ratio 0:) Liner: Inj. Temp.: C Temp.: Premium mm Precision liner w/wool ( 0.) 80 C (hold min) to C at 0 C/min (hold. min) Carrier Gas He, constant flow Flow Rate:.0 ml/min Detector FID @ C Make-up Gas Flow Rate: ml/min Make-up Gas Type: N Hydrogen flow: 0 ml/min Air flow: 00 ml/min Data Rate: 0 Hz Instrument Agilent 890A GC Notes C:0 Methyl butyrate (--) elutes in the solvent front. www.restek.com

To calibrate the GC system for assays of this type, use a mixture such as our -component food industry mix (Figures and ), or our 8-component food industry mix. These standards include a gravimetric certificate of analysis to help ensure accurate quantification. To ensure correct identifications of the individual cis and trans isomers, use our cis/trans s mix as shown in Figure, or our trans fat mix shown in Figure. Rtx-0, a 90% biscyanopropyl phase, also resolves cis and trans isomers. These columns are slightly less polar than Rt-0 columns. Figure shows the analysis of an animal-based fat using an Rtx-0 column. As on Rt-0 columns, the trans forms of the s elute before the cis forms. Analyzing Botanical Products Gas chromatography can be used to analyze fatty acid marker compounds in some botanical products. Both Rtx-Wax and Stabilwax capillary columns provide the efficiency and selectivity needed to perform analysis and allow accurate identification of the individual fatty acids in products such as saw palmetto oil, olive oil, and palm oil (Figures 8-0). Figure : Resolve cis and trans isomers of unsaturated s on an Rt-0 column. 8 Excellent resolution of cis & trans isomers! 8 9 GC_FF Conc. Structural Peaks tr (min) mg/ml) Nomenclature. Methyl stearate.0.0 C8:0. Methyl petroselaidate.0 0.8 C8: (trans-). Methyl elaidate.88.0 C8: (trans-9). Methyl trans-vaccenate.0. C8: (trans-). Methyl petroselinate. 0.8 C8: (cis-). Methyl oleate.0.0 C8: (cis-9). Methyl vaccenate.88. C8: (cis-) 8. Methyl linoleate..0 C8: (cis-9,) Column Rt-0, 00 m, 0. mm ID, 0.0 µm ( 98) Sample cis/trans mix ( 09) Diluent: Methylene chloride Inj. Vol.: µl split (split ratio 00:) Liner: Topaz mm ID straight inlet liner w/ wool ( 00) Inj. Temp.: 0 C Temp.: 00 C (hold min) to 0 C at C/min (hold 0 min) Carrier Gas H, constant flow Flow Rate:. ml/min Detector FID @ 0 C Instrument Agilent 890A GC www.restek.com

Figure : Rt-0 columns provide excellent separation of trans fats. Conc. Structural Peaks tr (min) (wt.%) Nomenclature. Methyl myristelaidate.80 C: (trans-9). Methyl trans-pentandecen-0-oate.8 C: (trans-0). Methyl palmitelaidate.0 C: (trans-9). Methyl trans-heptadecen-0-oate 0.0 C: (trans-0). Methyl petroselaidate.0 C8: (trans-). Methyl elaidate. C8: (trans-9). Methyl trans-vaccenate.9 C8: (trans-) 8. Methyl linoelaidate.8 C8: (trans-9,) 9. Methyl trans-nonadecen--oate.0 C9: (trans-) 0. Methyl trans-nonadecen-0-oate. C9: (trans-0). Methyl trans-eicosan--oate 9.0 C0: (trans-). Methyl brassidate. C: (trans-). Methyl ricinelaidate.98 8 C8: (trans-9; OH-) 0 0 8 9 Column Rt-0, 00 m, 0. mm ID, 0.0 µm ( 98) Sample trans Fat reference standard ( 9) Diluent: Hexane Inj. Vol.: µl split (split ratio 00:) Liner: Topaz.0 mm ID straight inlet liner w/wool ( 00) Inj. Temp.: 0 C Temp.: 0 C (hold min) to 0 C at C/min (hold min) Carrier Gas H, constant flow Flow Rate: ml/min Detector FID @ 0 C Instrument Agilent 890A GC 8 9 0 0 0 0 GC_FF Figure : Animal fat-based s resolved, using an Rtx-0 column. Peaks. C:0. C:0. C:n. C8:0. C8:n9. C8:n. C8:n 8. C8:n 9. C8:n 0. C0:n. C0:n. C0:n. C:n. C:n. C:n Figure 8: Saw palmetto s resolved on an Rtx- WAX column. Conc. Peak List (mg/ml). methyl caproate (C:0) 0.. methyl caprylate (C8:0) 0.. methyl nonanoate (C9:0).0. methyl caprate (C0:0) 0.. methyl laurate (C:0).0. methyl myristate (C:0).0. methyl palmitate (C:0).0 8. methyl palmitoleate (C:) 0. 9. methyl stearate (C8:0) 0. 0. methyl oleate (C8:).0. methyl linoleate (C8:).0. methyl linolenate (C8:) 0. 0 8 9 GC_FF009 Column Rtx-0, 0 m, 0. mm ID, 0.0 µm ( 0) Sample PUFA mix Inj. Vol.: 0. µl split (split ratio 0:) Inj. Temp.: 0 C Temp: 0 C to 0 C at C/min (hold 0 min) Carrier Gas H, constant pressure Linear Velocity: 0 cm/sec Detector FID @ 0 C Notes FID sensitivity: 8 x 0 - AFS 8 9 0 GC_FF008 Column 0m, 0.mm, 0.µm Rtx-Wax ( ) Sample µl split injection of saw palmetto standard Conc.: see peak list Temp: 0 C (hold min.) to 0 C at 0 C/min. (hold min.) Inj./det. temp.: 0 C/00 C Carrier Gas helium Linear velocity: ml/min. ( cm/sec.) Split ratio: 00: www.restek.com

Figure 9: Olive oil s on a Stabilwax column. Conc. Structural Peaks tr (min) (wt.%) Nomenclature. Methyl palmitate..9 C:0. Methyl palmitoleate..0 C: (cis-9). Methyl stearate 8.. C8:0. Methyl oleate 8.. C8: (cis-9). Methyl linoleate 9. 8.9 C8: (cis-9,). Methyl linolenate 0. 0.8 C8: (cis-9,,). Methyl arachidate 0.98 0. C0:0 8. Methyl eicosenoate.8 0. C0: (cis-) Column Stabilwax, 0 m, 0. mm ID, 0. µm ( 0) Sample Olive oil reference standard (Sigma-Aldrich #8) Diluent: Hexane Inj. Vol.: µl split (split ratio 00:) Liner: Topaz mm ID straight inlet liner w/ wool ( 00) Inj. Temp.: C Temp.: 0 C (hold 0. min) to 0 C at C/min (hold min) Carrier Gas H, constant flow Flow Rate: ml/min Detector FID @ 0 C Instrument Agilent 890A GC Notes The sample was prepared using methanolic HCl. 8 8 9 0 GC_FF Figure 0: Palm oil s on a Stabilwax column. Conc. Structural Peaks tr (min) (wt.%) Nomenclature. Methyl laurate 8.9.0 C:0. Methyl myristate.0.0 C:0. Methyl palmitate. 0. C:0. Methyl palmitoleate.8. C: (cis-9). Methyl stearate 8.. C8:0. Methyl oleate 8.0. C8: (cis-9). Methyl linoleate 9.. C8: (cis-9,) 8. Methyl linolenate 0.8 0. C8: (cis-9,,) 9. Methyl arachidate.00 0. C0:0 Column Stabilwax, 0 m, 0. mm ID, 0. µm ( 0) Sample Palm oil standard (Supelco #9) Diluent: Hexane Inj. Vol.: µl split (split ratio 00:) Liner: Topaz mm ID straight inlet liner w/ wool ( 00) Inj. Temp.: C Temp.: 0 C (hold 0. min) to 0 C at C/min (hold min) Carrier Gas H, constant flow Flow Rate: ml/min Detector FID @ 0 C Instrument Agilent 890A GC Notes The sample was prepared using methanolic HCl. 8 9 0 8 0 GC_FF www.restek.com 8

Summary Capillary GC is especially useful for determining total fat content, trans fat content, and total omega- polyunsaturated fatty acid content in foods. The choice of capillary column depends on the information required. For polyunsaturated s analysis, a WAX column allows fast, accurate quantification. A more polar Rt-0 column is the column of choice when determining the total fat content, or the amount of trans fat, in an ingredient or end product. Whatever your fatty acid analysis requirements, Restek can provide the consistent-performance analytical columns and reference mixes that will help you to accurately characterize your materials. Composition of each mixture listed as a weight/weight % basis (minimum 0 mg/ampul) Compound Name # 00 # 0 # 0 # 0 # 0 # 0 # 0 Methyl caproate (:0) 0.0 0.0 Methyl heptanoate (:0) 0.0 Mix Cat. # Methyl caprylate (8:0) 0.0 0.0 0.0.0 Methyl nonanoate (9:0) 0.0 Methyl caprate (0:0) 0.0 0.0 0.0.0 #8 0 Methyl undecanoate (:0) 0.0 Methyl laurate (:0) 0.0 0.0 0.0 8.0 #9 08 # 0 Methyl tridecanoate (:0) 0.0 0.0 Methyl myristate (:0) 0.0 0.0 0.0 0..0.0.0 Methyl pentadecanoate (:0 0.0 0.0 Methyl palmitate (:0) 0.0 0.0 0.0 0.0.0. 0.0.0.0.0.0.0 0.0 Methyl palmitoleate (:) 0.0.0 0..0 # 0 Methyl heptadecanoate (:0) 0.0 0.0 0. Methyl stearate (8:0) 0.0 0.0 0.0.0..0.0.0.0.0.0.0 Methyl oleate (8:) 0.0 0.0 0.0. 0.0.0 8.0.0 80.0.0.0 Methyl linoleate (8:) 0.0.0. 0.0 0.0.0.0.0.0.0 Methyl linolenate (8:) 0.0 0.0 0..0.0.0.0 Methyl nonadecanoate (9:0) 0.0 0.0 Methyl arachidate (0:0) 0.0 0.0 0.0.0...0.0 # 0 Methyl eicosenoate (0:) 0.0 0.0 0.0 0.. Methyl eicosadienoate (0:) 0.0.0 Methyl homo gamma linolenate (0:) 0.0 Methyl arachidonate (0:) 0.0 Methyl heneicosanoate (:0) 0.0 Methyl behenate (:0).0.0 0..0.0 Methyl erucate (:) 0.0 0.0 0.0 Methyl docosadienoate (:) 0.0 Methyl lignocerate (:0) 0.0.0.0.0 Methyl nervonate (:) 0.0.0 # 0 # 0 # 0 #8 0 #9 0 #0 0 # 0 References [] M.M. Mossoba, J.K.G. Kramer, P. Delmonte, M.P. Yurawecz, J.I. Rader, Official methods for the determination of trans fat, AOCS Press, 00. http://www.aoac.org/aoac_prod_imis/ aoac/aoac_member/bs/00.aspx. [] AOCS Committee, Official methods and recommended practices of the AOCS, th edition, AOCS Press, 0. https://www.aocs.org/store/shop-aocs/shop-aocs?productid=09809. [] European pharmacopoeia (Ph. Eur.) 9th edition, European Pharmacopoeia, 0. Book, online, PDF. https://www.edqm.eu/en/european-pharmacopoeia-ph-eur-9th-edition. [] K. Liu, Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials, J Am Oil Chem Soc () (99) 9-8. https://onlinelibrary.wiley. com/doi/abs/0.00/bf00. [] K-D Müller, H.P. Nalik, E.N. Schmid, H. Husmann, G. Schomburg, Fast identification of mycobacterium species by GC analysis with trimethylsulfonium hydroxide (TMSH) for transesterification, J Sep Sci, () (99) -. https://onlinelibrary.wiley.com/doi/abs/0.00/jhrc.000. [] K. Ichihara, Y. Fukubayashi, Preparation of fatty acid methyl esters for gas-liquid chromatography, J Lipid Res, () (00) 0. https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC89. [] K. iichihara, C. Kohsaka, N. Tomari, T. Kiyono, J. Wada, K. Hirooka, Y. Yamamoto, Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography Anal Biochem 9 (0) -8. https://www.sciencedirect.com/science/article/pii/s000900 9 www.restek.com

Rtx-Wax Columns (fused silica) polar phase; Crossbond polyethylene glycol ID df temp. limits* -Meter 0-Meter 0-Meter 0. mm 0. µm 0 to 0 C 0 0.0 µm 0 to 0 C 8 0. mm 0. µm 0 to 0 C 0.0 µm 0 to 0 C 9.00 µm 0 to 0/0 C 0. mm 0. µm 0 to 0 C 0.0 µm 0 to 0 C 0.00 µm 0 to 0/0 C 8 *Maximum temperatures listed are for shorter length columns. Longer columns may have a different maximum temperature. Stabilwax Columns (fused silica) polar phase; Crossbond polyethylene glycol ID df temp. limits -Meter 0-Meter 0-Meter 0. mm 0.0 µm 0 to 0/0 C 00 008 0 0. µm 0 to 0/0 C 00 0 0 0.0 µm 0 to 0/0 C 0 08 0 0. mm 0. µm 0 to 0/0 C 0 0 0 0.0 µm 0 to 0/0 C 0 09 0.00 µm 0 to 0/0 C 0 0 0 0. mm 0. µm 0 to 0/0 C 0 0 08 0.0 µm 0 to 0/0 C 0 00 0.00 µm 0 to 0/0 C 0 0 08.0 µm 0 to 0/0 C 0 09 0.00 µm 0 to 0/0 C 0 00 ID df temp. limits 0-Meter 0-Meter 0. mm 0. µm 0 to 0/0 C 80 8 0.8 mm 0.8 µm 0 to 0 C 00 *Maximum temperatures listed are for shorter length columns. Longer columns may have a different maximum temperature. WAX Columns (USP G) (fused silica) polar phase; Crossbond polyethylene glycol ID df temp. limits 0-Meter 0. mm 0. µm 0 to 0/0 C 9 0. mm 0. µm 0 to 0/0 C 98 0. mm 0.0 µm 0 to 0 C 99 Rt-0 Column (fused silica) highly polar phase; biscyanopropyl polysiloxane not bonded 00 m* ID df temp. limits 0. mm 0.0 µm 0 to 0 C 98 *Nominal length = 00 m. The actual length is 0 m, which is equivalent to the length of the previous Rt-0 column ( 99). Rtx-0 Columns (fused silica) highly polar phase; biscyanopropyl cyanopropylphenyl polysiloxane ID df temp. limits* 0-Meter 0-Meter 0-Meter 0. mm 0.0 µm 0 to 0/ C 008 0 0 0.0 µm 0 to 0/ C 0 0 09 0. mm 0.0 µm 0 to 0/ C 0 0 00 0. mm 0.0 µm 0 to 0/ C 0 0-Meter 0-Meter 0-Meter ID df temp. limits 0.8 mm 0.0 µm 0 to 0 C 00 00 00 *Maximum temperatures listed are for shorter length columns. Longer columns may have a different maximum temperature..0 ml, mm Crimp Vial Convenience Kits (Vials, Caps, & Septa) Vials packaged in a clear-lid tray. Caps with septa packaged in a plastic bag. Description 00-pk.,000-pk. Clear.0 ml Vial, Deactivated, Silver Seal, PTFE/Natural Rubber Septa Amber.0 ml Vial, Deactivated, Silver Seal, PTFE/Natural Rubber Septa Clear.0 ml Vial, Untreated, Silver Seal, PTFE/Natural Rubber Septa 9 9 Amber.0 ml Vial, Untreated, Silver Seal, PTFE/Natural Rubber Septa 98 99 Clear.0 ml Vial, Untreated, Silver Seal, PTFE/Silicone Septa Amber.0 ml Vial, Untreated, Silver Seal, PTFE/Silicone Septa 8 9 www.restek.com 0 Inserts for.0 ml, mm Crimp-Top &.0 ml, 9 mm Short-Cap, Screw-Thread Vials Description Volume Material 00-pk.,000-pk. Big Mouth Insert w/bottom Spring 0 µl Glass 8 Big Mouth Insert w/bottom Spring 0 µl Glass Big Mouth Insert w/glass Flange (Step Design)* 0 µl Glass 9 Insert, Flat Bottom 0 µl Glass 80 Insert, Flat Bottom w/id Ring 0 µl Glass 9 9 Big Mouth Insert w/bottom Spring 0 µl Polypropylene 8 Big Mouth Insert w/bottom Spring & Graduated Markings 0 µl Polypropylene 8A Big Mouth Insert, Top Flange 0 µl Polypropylene 9 Big Mouth Insert, No Spring 0 µl Polypropylene 0 *Big Mouth insert w/glass flange (step design) not to be used with 9 mm screw-thread vials.

Marine Oil Mix (0 components) Chain, Compound (CAS#), % by Weight C:0 Methyl myristate (-0-), % C: Methyl myristoleate (9-0-8), % C:0 Methyl palmitate (-9-0), % C: Methyl palmitoleate (0--8), % C8:0 Methyl stearate (--8), 8% C8: Methyl oleate (--9), % C8: Methyl vaccenate (9--9), % C8: Methyl linoleate (--0), % C8: Methyl linolenate (0-00-8), % C0:0 Methyl arachidate (0-8-), % C0: Methyl -eicosenoate (90-09-), 9% C0: Methyl,-eicosadienoate (-0-), % C0: Methyl arachidonate (-89-), % C0: Methyl,,-eicosatrienoate (8-88-), % C0: Methyl eicosapentaenoate (--), 0% C:0 Methyl behenate (99--), % C: Methyl erucate (0--9), % C: Methyl docosahexaenoate (0-0-9), % C:0 Methyl lignocerate (-9-), % C: Methyl nervonate (-88-), % 0 (00 mg) Food Industry Mix ( components) Chain, Compound (CAS#), % by Weight C:0 Methyl butyrate (--), % C:0 Methyl caproate (0-0-), % C8:0 Methyl caprylate (--), % C0:0 Methyl decanoate (0--9), % C:0 Methyl undecanoate (-8-8), % C:0 Methyl dodecanoate (-8-0), % C:0 Methyl tridecanoate (-88-0), % C:0 Methyl myristate (-0-), % C: (cis-9) Methyl myristoleate (9-0-8), % C:0 Methyl pentadecanoate (--), % C: (cis-0) Methyl pentadecenoate (90--), % C:0 Methyl palmitate (-9-0), % C: (cis-9) Methyl palmitoleate (0--8), % C:0 Methyl heptadecanoate (-9-), % C: (cis-0) Methyl heptadecenoate (90-8-8), % C8:0 Methyl stearate (--8), % C8: (trans-9) Methyl octadecenoate (9--8), % C8: (cis-9) Methyl oleate (--9), % C8: (all-trans-9,) Methyl linolelaidate (-9-), % C8: (all-cis-9,) Methyl linoleate (--0), % C8: (all-cis-,9,) Methyl linolenate (--), % C8: (all-cis-9,,) Methyl linolenate (0-00-8), % C0:0 Methyl arachidate (0-8-), % C0: (cis-) Methyl eicosenoate (90-09-), % C0: (all-cis-,,) Methyl eicosadienoate (-0-), % C0: (all-cis-8,,) Methyl eicosatrienoate (0-0-9), % C0: (all-cis-,,) Methyl eicosatrienoate (8-88-), % C0: (all-cis-,8,,) Methyl arachidonate (-89-), % C0: (all-cis-,8,,,) Methyl eicosapentaenoate (--), % C:0 Methyl heneicosanoate (0-90-0), % C:0 Methyl behenate (99--), % C: (cis-) Methyl erucate (0--9), % C: (all-cis-,) Methyl docosadienoate (0--), % C: (all-cis-,,0,,,9) Methyl docosahexaenoate (-90-), % C:0 Methyl tricosanoate (-9-8), % C:0 Methyl lignocerate (-9-), % C: (cis-) Methyl nervonate (-88-), % 0 mg/ml total in methylene chloride, ml/ampul 0 (ea.) NLEA Mix (8 components) Chain, Compound (CAS#), % by Weight C:0 Methyl butyrate (--),.% C:0 Methyl hexanoate (0-0-),.% C8:0 Methyl octanoate (--), % C0:0 Methyl decanoate (0--9),.% C:0 Methyl undecanoate (-8-8),.% C:0 Methyl laurate (-8-0), % C:0 Methyl tridecanoate (-88-0),.% C:0 Methyl myristate (-0-),.% C: (cis-9) Methyl myristoleate (9-0-8),.% C:0 Methyl pentadecanoate (--),.% C:0 Methyl palmitate (-9-0), 0% C: (cis-9) Methyl palmitoleate (0--8), % C:0 Methyl heptadecanoate (-9-),.% C8:0 Methyl stearate (--8), % C8: (trans-9) Methyl elaidate (9--8),.% C8: (cis-9) Methyl oleate (--9), % C8: (all-trans-9,) Methyl linolelaidate (-9-),.% C8: (all-cis-9,) Methyl linoleate (--0), 0% C8: (all-cis-9,,) Methyl linolenate (0-00-8), % C0:0 Methyl arachidate (0-8-),.% C0: (cis-) Methyl eicosenoate (80-09-),.% C0: (all-cis-,8,,,) Methyl eicosapentaenoate (--),.% C:0 Methyl behenate (99--),.% C: (cis-) Methyl erucate (0--9),.% C: (all-cis-,,0,,,9) Methyl docosahexaenoate (80--),.% C:0 Methyl tricosanoate (-9-8),.% C:0 Methyl lgnocerate (-9-),.% C: (cis-) Methyl nervonate (-88-),.% 0 mg/ml total in methylene chloride, ml/ampul 08 (ea.) cis/trans Mix (8 components) Chain, Compound (CAS#), % by Weight C8: trans-9 Methyl elaidate (-8-), 0% C8: cis-9, Methyl linoleate (--0), 0% C8: cis-9 Methyl oleate (--9), 0% C8: cis- Methyl petroselinate (-8-), 8% C8: trans- Methyl petroselaidate (0--), 8% C8:0 Methyl stearate (--8), 0% C8: trans- Methyl transvaccenate (98-8-9), % C8: cis- Methyl vaccenate (9--9), % 0 mg/ml total in methylene chloride, ml/ampul 09 (ea.) www.restek.com

trans Fat Reference Standard ( components) For accurate determination of trans fat content in foods and edible oils. Pair with an Rt-0 GC column for reliable AOAC and AOCS method performance. Verified composition and stability. Neat Fatty Acid Methyl Esters Use these materials to prepare specific mixtures not commercially available. These products are of the highest purity available, typically 99% by GC-FID analysis. Each compound is packaged under a nitrogen blanket to ensure product stability. A certificate of analysis is provided with each ampul. Chain Description CAS # qty. C:0 Methyl caproate 0-0- ea. 0 C8:0 Methyl caprylate -- ea. 09 C0:0 Methyl caprate 0--9 ea. 0 C:0 Methyl undecanoate -8-8 ea. 0 C:0 Methyl laurate -8-0 ea. 0 C:0 Methyl tridecanoate -88-0 ea. 0 C:0 Methyl myristate -0- ea. 0 C:0 Methyl palmitate -9-0 ea. 08 C: D 9 cis Methyl palmitoleate 0--8 ea. 09 C:0 Methyl heptadecanoate -9- ea. 00 C8:0 Methyl stearate --8 ea. 0 C8: D 9 cis Methyl oleate --9 ea. 0 C8: D 9, cis Methyl linoleate --0 ea. 0 C8: D 9,, cis Methyl linolenate 0-00-8 ea. 0 C9:0 Methyl nonadecanoate -9-8 ea. 0 C0:0 Methyl arachidate 0-8- ea. 0 C0: D cis Methyl eicosenoate 90-09- ea. 0 C0: D,, cis Methyl eicosatrienoate 8-88- ea. 09 C:0 Methyl heneicosanoate 0-90-0 ea. 0 C:0 Methyl behenate 99-- ea. 0 C: D cis Methyl erucate 0--9 ea. 0 C:0 Methyl lignocerate -9- ea. 0 Chain, Compound, % by Weight C:T Methyl Myristelaidate, % C:T Methyl 0-Transpentadecenoate, % C:T Methyl Palmitelaidate, % C:T Methyl 0-Transheptadecenoate, % C8:T Methyl Elaidate, % C8:T Methyl Petroselaidate, % Neat blend,.0.0 wt/wt%, 00 mg/ampul Tritridecanoin Glyceride Tritridecanoin glyceride (--9) Neat, 00 mg/vial C8:T Methyl Transvaccenate, % C8:T Methyl Ricinelaidate, 8% C8:TT Methyl Linoelaidate, % C9:T Methyl -Transnonadecenoate, % C9:T Methyl-0-Transnonadecenoate, % C0:T Methyl -Transeicosenoate, % C:T Methyl Brassidate, % 9 (ea.) trans Fat Standards For accurate determination of trans fat content in foods and edible oils. Pair with an Rt-0 GC column for reliable AOAC and AOCS method performance. Verified composition and stability. Boron Trifluoride Boron trifluoride is very sensitive to moisture and should not be stored after opening. This is a singleuse container. Boron trifluoride (-0-) % in methanol, ml/bottle (ea.) (ea.) Triundecanoin Triundecanoin (-80-) Neat, 00 mg/ampul 8 (ea.) Questions? Contact us or your local Restek representative (www.restek.com/contact-us). Restek patents and trademarks are the property of Restek Corporation. (See www.restek.com/patents-trademarks for full list.) Other trademarks in Restek literature or on its website are the property of their respective owners. Restek registered trademarks are registered in the U.S. and may also be registered in other countries. To unsubscribe from future Restek communications or to update your preferences, visit www.restek.com/subscribe To update your status with an authorized Restek distributor or instrument channel partner, please contact them directly. 08 Restek Corporation. All rights reserved. Printed in the U.S.A. www.restek.com Lit. Cat.# FFAR9A-UNV