Food & Beverage

Table 2. Summary of Fractionation (from AOCS Official Method Ce 1h-05)1. Condition Ag-Ion SPE tube with 4 mL of acetone, and allow

solvent to drip completely through the tube; discard eluant.2. Equilibrate by passing 4 mL of hexane completely through;

discard eluant.3. Load 1 mL of sample extract. Any eluant coming through may

be discarded. 4. Elute Fraction 1 with 6 mL of hexane:acetone (96:4), and collect

eluant. Be sure all solvent has passed through the tube.5. Elute Fraction 2 with 4 mL of hexane:acetone (90:10), and collect

eluant. Be sure all solvent has passed through the tube.6. Dry each fraction down at room temperature using nitrogen,

and reconstitute in 1 mL of hexane.Note: Gravity should be sufficient for each step with vacuum applied briefly only at the end of steps 4 and 5 to help draw the elution solvent completely out of the tube.

Table 1. Summary of Extraction and Derivatization (from AOCS Official Method Ce 1k-09)1. Homogenize sample and weigh 1 g into a 50 mL round

bottom flask. Add several boiling chips.2. Add 5 mL of 1.3 M methanolic hydrochloric acid (HCl).

Attach condenser and reflux for 15 min after mixture has started to boil.

3. Turn off heat, remove condenser and add 5 mL of 2.3 M methanolic sodium hydroxide (NaOH) to flask. Reattach condenser and reflux again for 15 min.

4. Turn off heat, remove condenser and add 14 mL of 10% BF3-methanol. Reattach condenser and reflux for 5 min.

5. Turn off heat, remove condenser and add 5 mL of hexane. Remove flask from mantle, and let it cool to room temperature.

6. Add saturated sodium chloride (NaCl) solution (aqueous) to flask until organic layer (top) is in neck.

7. Stopper the flask and shake. Remove top layer, pass through sodium sulfate bed and save.

Trans fats do not occur naturally in plant-based oils and animal fats. They can, however, be produced commercially through a hydrogenation process, which is used to prevent foods from spoiling and to produce specific structure effects. Concerns have been raised for several decades that the consumption of trans fats may contribute to coronary heart disease. In response, many government agencies established requirements that the trans fat content must be listed on the nutritional labels of foods and some dietary supplements.

ExperimentalA procedure used to determine trans fat levels in foods involves extraction, derivatization, fractionation and GC analysis. Extraction

(acid digestion followed by alkali hydrolysis) releases fats and oils from the sample matrix. Derivatization of fatty acids to fatty acid methyl esters (FAMEs) minimizes the influence of the active carboxyl groups. Silver-ion (Ag-ion) fractionation of FAMEs results in the physical separation of cis and trans monoene isomers. GC analysis requires specialized columns that are able to facilitate resolution of the cis and trans monoenes in the oleic (C18:1) region.

Table 1 and Table 2 summarize the sample preparation and fractionation procedures. Chromatograms obtained for the C18:1 region of fraction 1 and fraction 2 are shown in Figure 1. Isomers were identified using retention time and direct comparison to standards.

Using Silver-Ion SPE Fractionation, SP™-2560 and SLB®-IL111 GC Columns

Comprehensive Determination of Trans Fats in Cookies

Food & Beverage

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©2012 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA-ALDRICH and SUPELCO are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Solutions within is a trademark of Sigma-Aldrich Co. LLC. FLUKA is a trademark of Sigma-Aldrich GmbH, registered in the US and other countries. Discovery and SLB are registered trademarks of Sigma-Aldrich Co. LLC. Rezorian and SP are trademarks of Sigma-Aldrich Co. LLC. FocusLiner is a trademark of SGE International Pty Ltd. Supelco and Fluka brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.

OQZ11409-513821 / T412072

1082

Featured and Related ProductsDescription Qty. Cat. No.Extraction Glassware50 mL single neck, round bottom flask, 24/40 female 1 Z414484Modified Friedrichs condenser, 30 cm L x 54 mm O.D., 24/40 male 1 Z553654SPE TubesDiscovery® Ag-Ion, 750 mg/6 mL 30 54225-UDiscovery Ag-Ion, 750 mg/1 mL Rezorian™ cartridge 10 54226-UGC ColumnsSP-2560, 100 m x 0.25 mm I.D., 0.20 μm 1 24056SLB-IL111, 100 m x 0.25 mm I.D., 0.20 μm 1 29647-UAnalytical StandardsSupelco 37-Component FAME Mix,10 mg/mL (total wt.) in methylene chloride (visit sigma-aldrich.com/fame for composition details) 1 mL 47885-UAnalytical Reagents and SolventsSodium chloride, purum p.a., >99.5% 71381Sodium hydroxide, purum p.a., >97.0% 71692BF3-Methanol, 10% (w/w) 10 x 5 mL 33040-UMethanolic HCl, 3 N 10 x 3 mL 33051Acetone, for pesticide residue analysis 34480n-Hexane, for pesticide residue analysis 34484Methanol, for pesticide residue analysis 34485

Results and DiscussionThis application demonstrates the determination of trans fats from a cookie that listed partially hydrogenated vegetable oils as ingredients. Ag-Ion SPE was used to fractionate the extracted fats into two fractions. GC analysis of the monoene fractions on columns with different selectivity resulted in resolution of many of the C18:1 cis/trans geometric-positional isomers. When used together, Ag-Ion SPE, SP™-2560, and SLB®-IL111 provide more complete and accurate fatty acid identification and composition information.1

Reference1. Delmonte, P.; Kia, A-R.F.; Kramer, J.K.G.; Mossoba, M.M.; Sidisky, L.; Rader, J.I.

Separation Characteristics of Fatty Acid Methyl Esters Using the SLB-IL111, A New Ionic Liquid Coated Capillary Gas Chromatographic Column. J. Chrom. A 2011, 1218, 545-554.

Visit our Food and Beverage/Fats resources, sigma-aldrich.com/food-fats

Figure 1. GC Analysis inj. temp.: 250 °C detector: FID, 250 °C carrier gas: hydrogen, 1 mL/min injection: 1 μL, 10:1 split liner: 4 mm I.D., split type,

single taper wool packed FocusLiner™ design t4 t5

t6-8

t9t10

t11

t12

t13-14

t15t16

Fraction 1 = C18:1 trans isomers

17 18 19 20 21

Min

c4-5 c6-7

c9

c11

c10

c12

c13 c14 c15 c16

Fraction 2 = C18:1 cis isomers

SP-2560, 100 m x 0.25 mm I.D., 0.20 μm (24056) at 180 °C isothermal, the AOCS method-specified column and oven temperature

t4 t5

t7

t8-9

t10

t11

t12

t13-14

t15 t16

t6

Fraction 1 = C18:1 trans isomers

c4

c9

c11c10

c12

c13 c14c15 c16c5

c6-7

c8

14 15 16

Min

Fraction 2 = C18:1 cis isomers

SLB-IL111, 100 m x 0.25 mm I.D., 0.20 μm (29647-U) at 168 °C isothermal, the experimentally-determined optimal oven temperature

Including products from

©2012 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA-ALDRICH and SUPELCO are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. FLUKA is a registered trademark of Sigma-Aldrich GmbH. Supelco and Fluka brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.