Fats are essential to life and we consume fats in our diets every single day to assist in maintaining brain function, providing energy to our bodies, and to be the backbone of various cellular structures. Often we associate fats with good or bad, healthy or unhealthy, cis or trans, but the basis of these assumptions are the chemical structures. In order to understand something that we take in on a daily basis, and is an essential part of us, fatty acid structures must be analyzed for nutritional health research as well as for food quality purposes.

Fats have a carboxylic acid end followed by a long carbon tail and can largely be placed into two categories: saturated and unsaturated. In addition, when three fatty acids are bonded with glycerol, triglycerides are formed. Saturated fats (Figure 1) usually come from animal sources (meat, dairy, etc.) and tend to be solid at room temperatures. These fatty acids have a carbon chain that contains only single bonds. The other primary category of fatty acids is unsaturated fats (Figure 2). Unsaturated fats are commonly found in vegetables, nuts, and marine animals. These are commonly thought of as the ‘good fats.’ Unsaturated fatty acids contain at least one double bond, with some containing multiple double bonds.

stearic acid Saturated fat analysis

Figure 1.  Stearic Acid, a Saturated Fat
Public Domain, https://commons.wikimedia.org/w/index.php?curid=2839649

oleic acid analysis of unsaturated fats

Figure 2.  Oleic Acid, an Unsaturated Fat
Ben Mills – Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=14951692


Digging Deeper into Unsaturated Fatty Acids

Since unsaturated fatty acids contain a double bond, the bond orientation can be cis or trans configurations. Natural fats favor the cis configuration.  Due to the placement of the cis double bond, they tend to have a lower melting temperature, making them liquid at room temperature.  Another popular type of unsaturated fats are polyunsaturated fatty acids (PUFA), which have multiple double bonds. An example of these are Omega-3 and omega-6, which have been popular in recent years due to the potential health benefits, and have the double bond at the third or sixth carbon from the tail of the carbon chain, respectively (for the omega position we count backwards from the end of the carbon chain). Two common essential omega-3 fatty acids are Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA). A common omega-6 essential fatty acid is Linoleic acid (LA).

So, as nature tends to favor cis configuration. What about trans?

Trans Fats

Since trans fats are found in limited quantities in nature, they typically result from human engineering by hydrogenation of unsaturated fats to yield Partially Hydrogenated Oils (PHO). These PHOs, Figure 3, have been used in various food products such as margarine, shortening, creams, and frostings. A biological issue with trans PHOs is that they are poorly metabolized by the body, and research indicates that these fats have a deleterious effect on human health. As such, organizations across the globe, including the US FDA, have made moves to reduce the human intake of these products.

Elaidic Acid, a Trans Unsaturated Fat

Figure 3. Elaidic Acid, a Trans Unsaturated Fat
By Ben Mills – Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=5406427

From Fats to FAME

In order to determine the chemical structure and isomeric composition of these fats by GC, the fats need to be derivatized. The most popular method by which to do this is methyl esterification. This is done with an acid or base in methanol, where the methyl group of the alcohol exchanges with the hydrogen on the carboxylic acid of the fatty acid, creating a methyl ester, Figure 4. Common catalytic reagents for this are boron trifluoride (BF3) or potassium hydroxide (KOH). Several procedures for this method exists in extant literature. This esterification makes the fatty acids into Fatty Acid Methyl Esters (FAMEs), amenable to analysis by GC.

Fatty Acid Methyl Esters FAMEs

Figure 4. Methyl Esterification

FAME Analysis

The general method of analysis is GC/FID. Mass spectrometry offers minimal value as these are isobaric compounds and the resulting fragments are the same regardless of cis or trans configuration or the number of carbons in the FAME compounds. The difficulty arises from many of these compounds eluting close to each other and many scientists find that they need greater resolution between various peaks for their projects.

A commonly used standard 37 Fame mix, often requires a long GC capillary column to obtain baseline resolution. The Phenomenex Zebron™ ZB-FAME column is comparable to traditional GC columns used for the 37 FAME mixture analysis, and provide cis/trans separation on a shorter 30 meter column (Figure 5) or even a 20 meter column (Figure 6) with a reduced run time.

App ID 23821

37 FAMEs on 30 meter Zebron ZB FAMEs

Figure 5. 37 FAMEs on 30 meter Zebron™ ZB-FAME

App ID  23839

37 fames in 11 minutes on Zebron ZB-fame 20 m analysis

Figure 6. 37 FAMEs on 20 meter Zebron™ ZB-FAME


Fatty Acids are commonly analyzed components in the food testing industry, as well as clinical and biofuels. Global regulatory changes and research has resulted in an increase interest in the analysis of FAMEs to determine the cis/trans structures of fatty acids.

A reliable FAME GC column, such as the Zebron ZB-FAME column, can be used to achieve chromatography with separation of common cis/trans isomers to help determine the composition of the fats. Laboratories seeking to increase sample analysis throughput can realize significant gains by considering columns of similar improved phase chemistry and dimensions optimized for faster runtimes.



Federal Register – 68 FR 41433 July 11, 2003: Food Labeling: Trans Fatty Acids in Nutrition Labeling, Nutrient Content Claims, and Health Claims

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