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595 North Harrison Road, Bellefonte, PA 16823-0048 USA Tel: (800) 247-6628 (814) 359-3441 Fax: (800) 447-3044 (814) 359-3044 Technical Report SLB-IL111 for Methyl Ester (FAME) Applications Michael D. Buchanan, Katherine K. Stenerson, and Leonard M. Sidisky

The extremely polar SLB™-IL111 column Derivatization of Fatty Acids to Methyl Esters exhibits the highest polarity of any GC GC can be used to analyze fatty acids, either as free fatty acids or as fatty phase, allowing it to resolve key cis/trans acid methyl esters. The primary reasons to analyze fatty acids as fatty acid fatty acid methyl ester (FAME) isomers that methyl esters include: cannot be resolved on other columns. It ● also provides an alternative selectivity for In their free, underivatized form, fatty acids may be diffi cult to analyze, FAME applications typically performed on because these highly polar compounds tend to form hydrogen bonds, highly polar cyanopropyl siloxane columns. leading to adsorption issues. Reducing their polarity may make them Its maximum temperature of 270 °C is very more amenable for analysis. impressive for such an extremely polar ● To distinguish between the very slight diff erences exhibited by column. The chromatograms shown here demonstrate the utility of the unsaturated fatty acids, the polar carboxyl functional groups must SLB-IL111 as a great column choice for the analysis of FAMEs. fi rst be neutralized. This then allows column chemistry to perform separations by degree of unsaturation, position of unsaturation, and even the cis vs. trans confi guration of unsaturation. Fatty Acid Chemistry The esterifi cation of fatty acids to FAMEs is performed using an alkylation The most commonly analyzed fatty acids consist of C4-C24+ derivatization reagent. Methyl esters off er excellent stability, and provide hydrocarbon chains with a carboxyl group (-COOH) at one end. Many quick and quantitative samples for GC analysis. As shown in Table 1, the types of fatty acids exist, including: esterifi cation reaction involves the condensation of the carboxyl group ● Saturated = all single bonds between carbon atoms. of the fatty acid and the hydroxyl group of an alcohol. Esterifi cation is best done in the presence of an acidic catalyst (such as boron ● Unsaturated = at least one double bond. trichloride). The catalyst protonates the oxygen atom of the carboxyl ● Polyunsaturated = at least two double bonds. group, making the acid much more reactive. Nucleophilic attack by ● cis = bonds on either side of the double bond are on the same side. an alcohol then yields an ester with the loss of water. The catalyst is ● trans = bonds on either side of the double bond are on opposite sides. removed with the water. The alcohol used determines the alkyl chain length of the resulting esters; the use of methanol will result in the ● Omega 3 = initial double bond located directly after the third carbon formation of methyl esters whereas the use of ethanol will result in ethyl atom as measured from the methyl end. esters. (1,2) ● Omega 6 = initial double bond located directly after the sixth carbon atom as measured from the methyl end. Table 1. Typical Esterifi cation Procedure It is important for food manufacturers to report the levels of each type of fatty acid, as each has known or suspected health eff ects. Figure 1 shows 1. Weigh 1-25 mg of sample into a 5-10 mL Micro Reaction Vessel. the structures of several varieties of fatty acids. 2. Add 2 mL BCl3-Methanol, 12% w/w. A water scavenger (such as 2,2-dimethoxypropane) can be added at this point. Figure 1. Fatty Acid Structures 3. Heat at 60 °C for 5-10 minutes. Derivatization times may vary, depending on the specifi c compound(s) being derivatized. 4. Cool, then add 1 mL water and 1 mL hexane. 5. Shake the reaction vessel (it is critical to get the esters into the non- polar solvent). Saturated Fatty Acid (C5:0) 6. After allowing the layers to settle, carefully transfer the upper (organic) layer to a clean vial. Dry the organic layer either by passing through a bed of anhydrous sodium sulfate during the transfer step to the clean vial, or by adding anhydrous sodium sulfate to the clean vial and then cis Unsaturated Fatty Acid (C9:1n5c) shaking. 7. Proceed to the appropriate GC analysis procedure. trans Unsaturated Fatty Acid (C5:1n3t) An Omega 3 Fatty Acid

sigma-aldrich.com GC Column Polarity Scale Standard: 37-Component FAME Mix A visual depiction of our GC column polarity scale is shown in Figure 2, To properly identify fatty acid composition, standards of known reference showing the relationship of several columns to one another. The must be used. One such standard is the Supelco 37-Component FAME positions/maximum temperatures of several non-ionic liquid capillary Mix (47885-U). This standard contains methyl esters of fatty acids ranging GC columns are shown to the left of the scale. Listed to the right of the from C4 to C24, including key monounsaturated and polyunsaturated scale are the positions/maximum temperatures of Supelco® ionic liquid fatty acids; making this standard very useful to food analysts since it can capillary GC columns. All polarity number values are relative to both be used to identify fatty acids in many diff erent types of foods. This mix squalane (0 on the scale) and SLB-IL100 (100 on the scale). This simple was analyzed on the SP™-2560, a popular cyanopropyl siloxane column, but useful scale allows multiple columns to be quickly compared. and the SLB-IL111. Optimized chromatograms are shown in Figure 3. Detailed information concerning the scientifi c basis used to generate Observations are: this scale can be found at sigma-aldrich.com/il-gc ● Baseline and Peak Shape. Both columns produced chromatograms with minimal bleed and sharp peaks. Signal-to-noise ratios for the later Figure 2. GC Column Polarity Scale, Positions/Maximum eluting peaks are slightly better with the SLB-IL111 column. Temperatures of Columns ● Run Time. Dispersive interaction is weaker on the SLB-IL111; therefore, overall analysis time is shorter than on the SP-2560 (22 minutes compared to 26 minutes). ● Selectivity. Dipole-induced dipole interactions are stronger on the SLB-IL111 than on the SP-2560. These interactions result in increased retention of polarizable analytes (those with double bonds) relative to non-polarizable analytes. In fact, the higher the degree of unsaturation (the greater the number of double bonds), the stronger the retention is. Overall, this results in alternative selectivity to the SP-2560. A comparison of elution orders and elution locations are summarized in Table 2 and Table 3, respectively.

Table 2. Comparison of Elution Orders

Peak ID Isomer SP-2560* SLB-IL111* 19 C18:2n6c C18:0 C20:0 21 C18:3n6 C20:0 C21:0 22 C18:3n3 C20:0 C21:0 25 C20:2 C21:0 C22:0 26 C20:3n6 C22:0 C23:0 27 C20:3n3 C22:0 C23:0 33 C22:2 C23:0 C24:0 * On this column, this isomer elutes after this saturated FAME.

Table 3. Comparison of Elution Locations

Peak ID Isomer SP-2560 SLB-IL111 28 C20:4n6 Right after C23:0 Right before C24:0 29 C20:5n3 Right after C24:0 >1 min. after C24:0 32 C22:1n9 Between C22:0 and C23:0 Right before C23:0 34 C22:6n3 ~2.5 min. after C24:0 ~3.2 min. after C24:0

2 sigma-aldrich.com/il-gc Figure 3. 37-Component FAME Mix

SP-2560 Conditions column: SP-2560, 100 m x 0.25 mm I.D., 0.20 μm (24056) 19. Methyl Ester (C18:2n6c) at 2 wt % oven: 140 °C (5 min.), 8 °C/min. to 180 °C, 4 °C/min. to 210 °C, 20 °C/min. to 250 °C (7 min.) 20. Linolelaidic Acid Methyl Ester (C18:2n6t) at 2 wt % inj.: 250 °C 21. γ-Linolenic Acid Methyl Ester (C18:3n6) at 2 wt % det.: FID, 250 °C 22. -Linolenic Acid Methyl Ester (C18:3n3) at 2 wt % carrier gas: hydrogen, 40 cm/sec 23. Methyl Ester (C20:0) at 4 wt % injection: 1 μL, 100:1 split 24. cis-11-Eicosenoic Acid Methyl Ester (C20:1n9) at 2 wt % liner: 4 mm I.D., cup design 25. cis-11,14-Eicosadienoic Acid Methyl Ester (C20:2) at 2 wt % sample: Supelco 37-Component FAME Mix (47885-U), 26. cis-8,11,14-Eicosatrienoic Acid Methyl Ester (C20:3n6) analytes at concentrations indicated in methylene chloride at 2 wt % SLB-IL111 Conditions 27. cis-11,14,17-Eicosatrienoic Acid Methyl Ester (C20:3n3) column: SLB-IL111, 100 m x 0.25 mm I.D., 0.20 μm (29647-U) at 2 wt % oven: 140 °C (5 min.), 8 °C/min. to 180 °C, 5 °C/min. to 260 °C 28. Methyl Ester (C20:4n6) at 2 wt % det.: FID, 260 °C 29. cis-5,8,11,14,17- Methyl Ester (C20:5n3) at 2 wt % All other conditions the same as those used for the SP-2560 30. Heneicosanoic Acid Methyl Ester (C21:0) at 2 wt % 1. Methyl Ester (C4:0) at 4 wt % 10. Pentadecanoic Acid Methyl Ester (C15:0) at 2 wt % 31. Methyl Ester (C22:0) at 4 wt % 2. Methyl Ester (C6:0) at 4 wt % 11. cis-10-Pentadecenoic Acid Methyl Ester (C15:1) at 2 wt % 32. Methyl Ester (C22:1n9) at 2 wt % 3. Methyl Ester (C8:0) at 4 wt % 12. Methyl Ester (C16:0) at 6 wt % 33. cis-13,16-Docosadienoic Acid Methyl Ester (C22:2) 4. Methyl Ester (C10:0) at 4 wt % 13. Methyl Ester (C16:1) at 2 wt % at 2 wt % 5. Undecanoic Acid Methyl Ester (C11:0) at 2 wt % 14. Heptadecanoic Acid Methyl Ester (C17:0) at 2 wt % 34. cis-4,7,10,13,16,19- Methyl Ester 6. Methyl Ester (C12:0) at 4 wt % 15. cis-10-Heptadecenoic Acid Methyl Ester (C17:1) at 2 wt % (C22:6n3) at 2 wt % 7. Tridecanoic Acid Methyl Ester (C13:0) at 2 wt % 16. Methyl Ester (C18:0) at 4 wt % 35. Tricosanoic Acid Methyl Ester (C23:0) at 2 wt % 8. Methyl Ester (C14:0) at 4 wt % 17. Methyl Ester (C18:1n9c) at 4 wt % 36. Methyl Ester (C24:0) at 4 wt % 9. Methyl Ester (C14:1) at 2 wt % 18. Methyl Ester (C18:1n9t) at 2 wt % 37. Methyl Ester (C24:1n9) at 2 wt %

SP-2560

12 31 36 23

2 3 4 1 6 8 16 17 22 27 30 35 24 32 37 25 33 26 21 5 7 10 20 19 28 9 11 13 15 18 29 14 34

10 20 Min SLB-IL111

12

3 4 6 2 8 16 28 17 23 31 36 1 27

5 30 35 7 10 9 11 18 24 32 37 14 13 15 20 19 25 33 21 22 26 29 34

10 20 Min

3 Application: Trans Fatty acids in the cis confi guration are the dominant form in nature. Once optimal conditions were established, the PHVO, trans fraction, Correspondingly, enzymes have evolved to effi ciently digest and and cis fraction extracts were sequentially analyzed on each column. metabolize them with a high degree of specifi city. Conversely, trans Figure 4 fatty acids are relatively rare in nature. However, they have become shows the resulting chromatograms; on the SP-2560 at 180 °C predominant synthetic additives to processed foods, especially fried isothermal, the method-specifi ed oven temperature, and the SLB-IL111 foods and baked goods, because they can increase the shelf life and at 168 °C isothermal, the experimentally-determined optimal oven fl avor stability of foods containing them. It is now known that trans temperature. Following the completion of these analyses and a review fatty acids, formed by partial hydrogenation of , interfere of Reference 6, several observations were made. with natural metabolic process. Studies have linked their nutritional ● Elution Temperature. Even at a lower oven temperature, analytes eluted contribution to be similar to that of saturated fatty acids, possibly faster from the SLB-IL111 than the SP-2560. This is due to the higher playing a role in the heightened risk of coronary artery disease. (3-5) polarity of the SLB-IL111 phase. Because of this, consumer groups have pressured manufacturers and ● restaurants for the elimination of trans fats. Many regulatory agencies Elution Order. The SLB-IL111 resulted in a diff erent elution order than worldwide now require content labeling, to inform buyers of ‘trans ’ that obtained with the SP-2560. This was predicted due to the diff erent levels of foods and some dietary supplements. selectivities of the columns observed during the analysis of the 37-component FAME mix, and based on the data used to generate our The analysis of nutritionally signifi cant C18 cis and trans fatty acids, GC column polarity scale. as their methyl esters requires the use of a highly polar column to resolve the geometric positional isomers. The 100 m SP-2560 column is ● C18:1 Isomers. The SLB-IL111 was able to provide resolution of extremely useful for this type of analysis. Based on the results seen with C18:115t from C18:19c, a separation not possible with the SP-2560. analysis of the 37-component FAME mix, the SLB-IL111 was expected Additionally, the SLB-IL111 off ered improved resolution of some to off er selectivity for this application that is complementary to the SP- isomers that cannot be completely resolved with the SP-2560, such 2560. To compare the two columns, a partially hydrogenated vegetable as C18:110t from C18:111t, and the pair C18:113t/C18:114t oil (PHVO) sample containing various C18:1 geometric positional isomers from other isomers. was analyzed on both. The PHVO extract was graciously provided by Dr. ● C18:2 Isomers. Delmonte et al. reported that the SLB-IL111 is able Pierluigi Delmonte at the United States Food and Drug Administration to resolve C18:29c,11t from C18:27t,9c. These are the two most (US FDA). A trans fraction extract and a cis fraction extract, both prepared abundant conjugated linoleic acid (CLA) isomers found in ruminant from PHVO using an Ag-ion fractionation procedure, were also supplied fats. These two important isomers cannot be resolved using any by Dr. Delmonte. The PHVO extract contains thirteen C18:1 trans FAME other GC column without fi rst performing time-consuming Ag-ion isomers (from C18:14t to C18:116t), and thirteen C18:1 cis FAME fractionation. (6) isomers (from C18:14c to C18:116c). Complete details of the PHVO extract preparation and Ag-ion fractionation can be found in Reference 6. Based on the data presented here and the work reported by The established AOCS method for determining fat content requires Delmonte et al., it appears the SP-2560 and SLB-IL111 can be used in an SP-2560 column to be operated with a 180 °C isothermal oven a complementary fashion, to provide more complete and accurate temperature. (7) To determine the optimal oven temperature for these fatty acid identifi cation and composition information than currently analytes on the SLB-IL111, the PHVO extract was analyzed at several possible. It requires less time and labor to inject one extract on two diff erent oven temperatures. We determined that 168 °C isothermal diff erent columns, than to perform Ag-ion fractionation of an extract was optimal, the same temperature reported by Delmonte et al. as prior to injecting multiple fractions on a single column. being optimal for these analytes on this column. (6)

4 sigma-aldrich.com/il-gc Figure 4. PHVO Sample and cis/trans Fractions on SP-2560 and SLB-IL111

SP-2560 Conditions SLB-IL111 Conditions column: SP-2560, 100 m x 0.25 mm I.D., 0.20 μm (24056) column: SLB-IL111, 100 m x 0.25 mm I.D., 0.20 μm (29647-U) oven: 180 °C isothermal oven: 168 °C isothermal inj.: 250 °C All other conditions the same as those used for the SP-2560. All peak IDs as labeled det.: FID, 250 °C carrier gas: hydrogen, 1 mL/min. injection: 1 μL, 100:1 split liner: 4 mm I.D., split liner with cup (2051001)

t15, c9 c9 t10 t10 t8-9 t13-14 t11 c6-8 t12,c6-7 t11 t6-8 t9 t15,c8 c4-5 t13-14 c10 t12 PHVO total PHVO total c12 c10 c12 c11 c11 t16 t6,c5 t7 c13 c14 c13 t16 t4 t5 c14 c15 c16 t4 c4t5 c15 c16

t10 t10 t8-9 t11 t11 t13-14 t6-8 t9 t13-14 t12 PHVO (trans fraction) t12 PHVO (trans fraction)

t6 t7 t15 t15 t16 t4 t5 t16 t4 t5

c9 c9

c10 PHVO (cis fraction) PHVO (cis fraction) c12 c10 c12 c11 c8 c11 c6-8 c14 c13 c14 c6-7 c13 c4-5 c15 c16 c4 c5 c15 c16

16 17 18 19 13 14 15 Min Min

5 Application: Edible Oils Figure 5. Characterized Reference Oils The edible oil industry boasts revenue measured in the tens of billions column: SLB-IL111, 30 m x 0.25 mm I.D., 0.20 μm (28927-U) of dollars. As such, it can be subject to criminal acts of fraud aimed oven: 180 °C inj.: 250 °C at increasing profi ts. A GC fi ngerprinting technique can be used to det.: FID, 260 °C monitor product for adulteration (adding a cheaper, inferior oil to carrier gas: helium, 25 cm/sec boost the volume of a premium, higher priced oil), and also identify injection: 1 μL, 50:1 split liner: 4 mm I.D., split type, cup design the source of oils in unknown samples. samples: characterized reference oils, methylated using BF3-Methanol To properly identify an edible oil through pattern recognition, it prior to analysis is necessary to have standards of known reference; such as, our 1. Lauric Acid Methyl Ester (C12:0) Characterized Reference Oils. These can be used as part of a quality 2. Myristic Acid Methyl Ester (C14:0) 3. Palmitic Acid Methyl Ester (C16:0) control program, providing an excellent means of standardizing 4. Palmitoleic Acid Methyl Ester (C16:1) procedures and comparing results between facilities. 5. Stearic Acid Methyl Ester (C18:0) 6. Oleic Acid Methyl Ester (C18:1n9c) The esterifi cation of fatty acids to FAMEs is performed using an alkylation 7. Linoleic Acid Methyl Ester (C18:2n6c) derivatization reagent. Methyl esters off er excellent stability, and provide 8. Linolenic Acid Methyl Ester (C18:3n3) quick and quantitative samples for GC analysis. The esterifi cation reaction 9. Arachidic Acid Methyl Ester (C20:0) 10. cis-11-Eicosenoic Acid Methyl Ester (C20:1) involves the condensation of the carboxyl group of the fatty acid and the hydroxyl group of an alcohol. Esterifi cation is best done in the presence 6 of an acidic catalyst (such as boron trichloride). The catalyst protonates Canola Oil the oxygen atom of the carboxyl group, making the acid much more 7 reactive. Nucleophilic attack by an alcohol then yields an ester with the loss of water. The catalyst is removed with the water. The alcohol used 3 8 determines the alkyl chain length of the resulting esters; the use of 5 9 10 methanol will result in the formation of methyl esters, whereas the use of ethanol will result in ethyl esters. (6,7) 6 Following derivatization, fi ve of our Characterized Reference Oils were 3 Lard Oil

analyzed on the SLB-IL111. The resulting chromatograms are shown in 7 Figure 5. A 30 m column was selected because the higher resolution provided by a longer column was not necessary for this application. 4 5 Additionally, the shorter column resulted in shorter run times. The % 2 8 compositions, based on peak areas, agree with those listed on the

Certifi cate of Analysis for each mix. 3 Palm Oil 6

7 1 2 5

7 Soybean Oil

6 3 5 8

7 Sunfl ower Seed Oil

6

3 5

3 4 5 6 Min

6 sigma-aldrich.com/il-gc Conclusion Ordering Information The extreme polarity of the SLB-IL111 phase, in combination with a Description Cat. No. 270 °C maximum temperature, makes the SLB-IL111 useful for several SLB-IL111 Column FAME applications. Specifi cally: 15 m x 0.10 mm I.D., 0.08 μm 28925-U 30 m x 0.25 mm I.D., 0.20 μm 28927-U ● cis/trans FAME isomers: when paired with the SP-2560, provides 60 m x 0.25 mm I.D., 0.20 μm 28928-U more complete and accurate fatty acid identifi cation/composition 100 m x 0.25 mm I.D., 0.20 μm 29647-U information than currently possible Supelco 37-Component FAME Mix ● FAME profi le fi ngerprinting of edible oils:provides great resolution and 10 mg/mL (total wt.) in methylene chloride, 1 mL 47885-U peak shapes in a quick analysis See Figure 3 for list of analytes and concentrations Characterized Reference Oils Additionally, the SLB-IL111 provides alternative selectivity to Canola Oil, 1 g 46961 cyanopropyl siloxane columns for other applications. The extremely Coconut Oil, 1 g 46949 polar SLB-IL111 exhibits weaker dispersive interactions than less polar Corn Oil, 1 g 47112-U phases; which, in the applications shown here, results in lower elution Cottonseed Oil, 1 g 47113 temperatures and shorter analysis times. Lard Oil, 1 g 47115-U Linseed (Flaxseed) Oil, 1 g 47559-U References Menhaden Fish Oil, 1 g 47116 Olive Oil, 1 g 47118 1. W. W. Christie, “Gas Chromatography and ” The Library, http:// Palm Oil, 1 g 46962 www.lipidlibrary.co.uk/GC_lipid/gc_lip.html (accessed Jun 26, 2008). Peanut Oil, 1 g 47119 2. W. W. Christie, “Why I Dislike Boron Trifl uoride-Methanol” Lipid Technology Saffl ower Oil, 1 g 47120-U (1994) 6, 66-68. Soybean Oil, 1 g 47122 3. A. Ascherio and W. Willett, “Health Eff ects of Trans Fatty Acids” Am. J. Clin. Sunfl ower Seed Oil, 1 g 47123 Nutr. (1997) 66 (supplement), p. 1006S. Derivatization Reagents 4. S. Stender and J. Dyerberg, “Infl uence of Trans Fatty Acids on Health” An- BCl -Methanol, 12% w/w, 20 x 1 mL 33353 nals of Nutrition and Metabolism (2004) 48 (2), p. 61. 3 BCl3-Methanol, 12% w/w, 20 x 2 mL 33089-U 5. American Heart Association Web Page, http://www.americanheart.org/ BCl3-Methanol, 12% w/w, 400 mL 33033 presenter.jhtml?identifi er= 1728 (accessed Jan. 4, 2006). BF3-Methanol, 10% w/w, 20 × 1 mL 33356 6. P. Delmonte, A-R.F. Kia, J.K.G. Kramer, M.M. Mossoba, L. Sidisky, and J.I. BF3-Methanol, 10% w/w, 19 × 2 mL 33020-U Rader, “Separation Characteristics of Fatty Acid Methyl Esters Using SLB- BF3-Methanol, 10% w/w, 10 × 5 mL 33040-U IL111, A New Ionic Liquid Coated Capillary Gas Chromatographic Column” BF3-Methanol, 10% w/w, 400 mL 33021 Journal of Chromatography A 1218 (2011) p. 545. Micro Reaction Vessels and Caps 7. AOCS Method Ce 1h-05, “Determination of cis-, trans-, Saturated, Monoun- 5 mL Clear, with Hole Caps, 12 ea 33299 saturated and Polyunsaturated Fatty Acids in Vegetable or Non-ruminant 5 mL Clear, with Solid Caps, 12 ea 27039 Animal Oils and Fats by Capillary GLC” AOCS Offi cial Methods (2005) Ameri- 5 mL Amber, with Hole Caps, 12 ea 27478-U can Oil Chemists Society. 10 mL Clear, with Hole Caps, 12 ea 27479 Water Scavenger SLB-IL111 2,2-Dimethoxypropane, 98%, 25 mL D136808-25ML Sodium Sulfate, Anhydrous, >99.0% ● Application: This extremely polar ionic liquid column was the Granular, 500 g 239313-500G world’s fi rst commercial column to rate over 100 on our GC col- Granular, 1 Kg 239313-1KG umn polarity scale. As such, it has the most orthogonal selectivity Granular, 2.5 Kg 239313-2.5KG compared to commonly used non-polar and intermediate polar columns, providing increased selectivity for polar and polarizable Trademarks analytes. Its temperature limit of 270 °C is very impressive for such SLB, SP, Supelco – Sigma-Aldrich Co. LLC an extremely polar column. The 60 m version is excellent at resolv- ing benzene and other aromatics in gasoline samples. The 100 m version is suitable for detailed cis/trans FAME isomer analysis, and is a great complementary column to the SP-2560. Launched in 2010. ● USP Code: None ● Phase: Non-bonded; proprietary ● Temperature Limits: 50 °C to 270 °C (isothermal or programmed)

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