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Bulletin 907

595 North Harrison Road

Bellefonte, PA 16823-0048 USA

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email: supelco@sial.com

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most of the important saturated, monounsaturated, and polyunsatu-rated FAMEs (Table 1).

The Supelco 37 Component FAME Mix is designed to mimic the fattyacid composition of many food samples. For example, the saturated

series of fatty acids starting with C4, along with the unsaturated andpolyunsaturated C16 and C18 FAMEs, mimic milk fat. Representativecomponents in hydrogenated and nonhydrogenated vegetable oilsare a homologous series of even-carbon-numbered saturated FAMEsfrom C8 through C24, the monounsaturated C16:1, cisand transC18:1, C20:1, C22:1, and the polyunsaturated octadecenoic FAMEs.

The fatty acid composition of an average diet is a complexmixture of saturated, monounsaturated, and polyunsatu-rated fatty acids with a variety of carbon chain lengths. Toconfirm the identification of key fatty acids, several differ-ent standards and capillary columns are required. TheSupelco 37 Component FAME Mix can be used to identifykey FAMEs in many food products. Here, this mix isanalyzed on four capillary columns of different polarities:

Omegawax 250, PAG, SP-2380, and SP-2560, to compareelution patterns of the key FAMEs. A polyethylene glycoland cyanosilicone column combination provide the idealtool for best confirmational analyses of FAMEs.

Key Wordsfatty acids fatty acid methyl esters FAMEspolyunsaturates transfatty acids

With the implementation of the Nutrition Labeling and Education Act

of 1990 by the U.S. Food and Drug Administration (FDA), the total fatand saturated fat contents of a food must be listed on its label. An

optional listing of the cis-monounsaturated and cis,cis-methylene

interrupted polyunsaturated fatty acids can be stated on the label asmonounsaturated and polyunsaturated fatty acids. Unfortu-

nately for the food analyst, determining the fatty acid composition isdifficult because a food can contain many different fatty acids ofvarious carbon chain lengths. For example, milk contains fatty acidsranging from butyric acid (C4:0) to arachidic acid (C20:0). Included inthe fatty acid composition is a homologous series of even-carbon-numbered saturated fatty acids, monoenoic, hexadecenoic, andoctadecenoic fatty acids, and polyunsaturated C18 dienoic and

trienoic fatty acids. Milk and butter are also known to contain smallamounts of transfatty acids.

Some vegetable oils, included in many diets as a part of the foodprocessing or preparation step, contain fatty acids ranging from C6 to

C24 in carbon number, with varied amounts of saturated andunsaturated cisfatty acids. If the oils have been hydrogenated, as inmargarine, transfatty acids also will be present. In addition, meat andfish contribute saturated, monounsaturated, and cis,cis-methyleneinterrupted polyunsaturated fatty acids to the diet.

Fatty acids are typically analyzed as methyl esters, using capillary GC.

Multiple standards and capillary columns can be required to identifykey fatty acids from complex food samples, inefficiently expendinglaboratory time and money. The Supelco 37 Component FAME Mixcan be used to identify key fatty acid methyl esters (FAMEs) in manyfoods. This mix contains FAMEs ranging from C4:0 to C24:1, including

Table 1. Composition of Supelco 37 ComponentFAME Mix

Peak Component WeightID (acid methyl esters) (%)

1 C4:0 (Butryic) 42 C6:0 (Caproic) 43 C8:0 (Caprylic) 44 C10:0 (Capric) 45 C11:0 (Undecanoic) 26 C12:0 (Lauric) 47 C13:0 (Tridecanoic) 28 C14:0 (Myristic) 4

9 C14:1 (Myristoleic) 210 C15:0 (Pentadecanoic) 211 C15:1 (cis-10-Pentadecenoic) 212 C16:0 (Palmitic) 613 C16:1 (Palmitoleic) 214 C17:0 (Heptadecanoic) 215 C17:1 (cis-10-Heptadecenoic) 216 C18:0 (Stearic) 417 C18:1n9c (Oleic) 418 C18:1n9t (Elaidic) 219 C18:2n6c (Linoleic) 220 C18:2n6t (Linolelaidic) 221 C18:3n6 (-Linolenic) 222 C18:3n3 (-Linolenic) 223 C20:0 (Arachidic) 424 C20:1n9 (cis-11-Eicosenoic) 225 C20:2 (cis-11,14-Eicosadienoic) 2

26 C20:3n6 (cis-8,11,14-Eicosatrienoic) 227 C20:3n3 (cis-11,14,17-Eicosatrienoic) 228 C20:4n6 (Arachidonic) 229 C20:5n3 (cis-5,8,11,14,17-Eicosapentaenoic) 230 C21:0 (Henicosanoic) 231 C22:0 (Behenic) 432 C22:1n9 (Erucic) 233 C22:2 (cis-13,16-Docosadienoic) 234 C22:6n3 (cis-4,7,10,13,16,19-Docosahexaenoic) 235 C23:0 (Tricosanoic) 236 C24:0 (Lignoceric) 437 C24:1n9 (Nervonic) 2

Peak numbers in Figures AD.

Comparison of 37 Component FAME Standardon Four Capillary GC Columns

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FAMEs typically found in fish and meat include the previouslymentioned saturated and unsaturated FAME isomers, and the keyomega-3 and omega-6 polyunsaturated FAMEs. Fish contain largeamounts of the omega-3 fatty acids, such as C20:5n3 and C22:6n3,whereas meats are richer in the omega-6 fatty acids, such asarachidonic acid (C20:4n6). All of the omega-3 and omega-6 fattyacids are cis, cismethylene interrupted FAMEs. Also included in the37 component mix are some of the odd-carbon-numbered FAMEs

that are found in some foods.

Analysts evaluating food samples must choose from a number of

capillary columns. The sample type and the information beingsought determines the proper column choice. Routinely, polyethyl-ene glycol-based capillary columns are used for FAME analysis ofmarine fish oils and meat samples, as they will elute the FAMEisomers according to carbon chain length and degree of unsaturation.Cyanosilicone capillary columns are widely used for analyzingvegetable oils, as they provide resolution of cisand transFAMEs.

However, some of the carbon chain lengths usually overlap oncyanosilicone phases, causing problems in peak identification.

To compare columns for analyzing FAMEs, we evaluated theSupelco 37 Component FAME Mix on four capillary columns ofdifferent polarities: Omegawax 250, PAG, SP-2380, and SP-2560 (Figures A-D). Omegawax 250, a bonded polyethylene glycol-based phase, is used for analyses of polyunsaturated marine fishoils. For comparison, a phase of similar polarity, the slightly less polarpolyalkylene glycol (PAG) phase, was evaluated. This phase containsa percentage of propylene oxide in its polymer backbone, rather

than 100% polyethylene oxide (Omegawax 250). Two high-polaritycyanosilicone columns were evaluated: SP-2380 (95% cyanopropyl

5% phenyl polysiloxane) and SP-2560 (100% bis-cyanopropylpolysiloxane).

Injection and detection parameters for the analyses were identical,except for oven temperature and carrier gas linear velocity (Table 2).

To evaluate the figures, we compared the two lower polaritycolumns, then compared these results with observations for thehigher polarity columns. The Omegawax 250 and PAG column

analyses (Figures A and B) show slight differences in the elution

See Table 1 for peak IDs and Table 2 for conditions.

1

3

4

5

6

8

12

7

14

37

36

343533

32

31

2930

2726

25

24

23

21

2220

1819

16

17

605040Min

30200 10

2

11109

1315

28

794-0660

Figure A. Supelco 37 Component FAME Mix on Omegawax 250 Column

1

2 3

4

5

6

7

8

9 1011 13 14 15

16

12

17, 18

192021

22

23

2425

26

27

29

2831

323335

3734

36

605040Min

3020100

794-0661

30

Figure B. Supelco 37 Component FAME Mix on PAG Column

See Table 1 for peak IDs and Table 2 for conditions.

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Figure C. Supelco 37 Component FAME Mix on SP-2380 Column

lSee Table 1 for peak IDs and Table 2 for conditions.

Min

795-0781

1 2

3

4

5

6

7

8

1011

1513

22 3429

333527

26

21, 23

2019

24

30 25

403020100

16 17 31

36

3718

28, 32

914

12

Figure D. Supelco 37 Component FAME Mix on SP-2560 Column

795-0472

1

643

2

5 7

8 12

1110

9

131415 18

16 17

201921

23

24 22

30

2526

31

36

3227283533 29

37

34

403020Min

100

patterns for the polyunsaturated FAMEs. The most notable differ-

ences occur with the more highly unsaturated longer chain lengthFAMEs. The less polar PAG column provides a truer carbon chainlength separation. The even-carbon-numbered FAMEs elute strictlyaccording to carbon number and degree of unsaturation. For theOmegawax 250 column, there is some overlap, as C22:6n3 elutesafter C24:0.

The higher-polarity SP-2380 and SP-2560 columns show a much

different elution pattern. An advantage is that the cyanosiliconephases resolve cisand transFAMEs, with the transisomer elutingprior to the cis isomer. The SP-2560 column, in particular, isdesigned to resolve positional geometric FAME isomers. The reso-lution and elution patterns for the C18:1n9t and C18:1n9c andC18:2n6t and C18:2n6c isomers on all four columns show thedifferences in selectivity and resolving power. The polyethyleneglycol columns offer slight resolution of the cisand transisomers,with the cisisomer eluting first.

A disadvantage of using the high polarity cyanosilicone columns is

that there is significant carbon chain overlap in the elution patterns.Most of the trienoic and more polyunsaturated FAMEs elute afterthe next even-carbon-numbered saturated FAME. For example,C18:3n6 and C18:3n3 elute after the saturated C20 FAME. This canlead to peak identification problems. The polyalkylene glycol col-umns produce an elution pattern that more closely follows carbonchain length, and the higher polarity cyanosilicone columns pro-

duce better cisand transFAMEs resolutions.Given the differences in elution patterns of FAME isomers on thefour columns, we feel that using both a polyethylene glycol columnand a cyanosilicone column with the Supelco 37 Component FAMEMix provides the ideal tool for confirmation analyses of FAMEs.Columns of equal dimensions can be connected to a single injectionport and separate detectors and, by making a single injection, twoseparate elution profiles will be generated.

lSee Table 1 for peak IDs and Table 2 for conditions.

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