Analysis of Fats (Fatty Acids, FAMEs, Glycerides) in Food and Beverages

Fats play an important part in the food nutrition and food chemistry areas of study. The compound classes, sample types, and analytical techniques of interest include:

• Short chain, volatile fatty acids, typically analyzed in their free acid form using GC
• Larger (C8-C24+) fatty acids (such as omega fats and trans fats), typically converted to fatty acid methyl esters (FAMEs) prior to GC analysis
• Edible oil characterization by GC
• Sterols by GC analysis

See our Lipid Standards

Free Fatty Acids

Short chain, volatile fatty acids are typically analyzed in the free form using specialized columns. This group of compounds may be referred to as free fatty acids (FFAs), volatile fatty acids (VFA), or carboxylic acids. The analysis of fatty acids in the free form instead of as fatty acid methyl esters results in easier and quicker sample preparation. Additionally, artifact formation that may result from a derivatization procedure is eliminated.

For the GC analysis of free fatty acids, a specialized column that will not allow the adsorption of active carboxyl groups is required. The Nukol™, with its acidic characteristic, is well-suited for this application, allowing chromatography with excellent peak shapes.

Derivatization of Fatty Acids to FAMEs

Larger (C8-C24+) fatty acids, such as omega fats and trans fats, are typically converted to fatty acid methyl esters.

See our Derivatization Reagents Under Chromatography & Spectroscopy Reagents

Unsaturated FAME Analysis by GC

Saturated, monounsaturated, polyunsaturated, and cis/trans configuration all refer to the structure of fatty acid moieties. Each group is believed to have the following effects on human health:

• Saturated fatty acids (no double bonds) raise LDL cholesterol (increases risk of cardiovascular disease).
• Mono- and poly-unsaturated cis fatty acids (≥1 cis double bond) lower LDL cholesterol (reduces risk of cardiovascular disease).
• Mono- and poly-unsaturated trans fatty acids (≥1 trans double bond) raise LDL cholesterol (increases risk of cardiovascular disease) and also lower HDL (increases risk of type II diabetes).
  

Because of this, it is important for food manufacturers to measure and report their levels so consumers have the chance to establish healthy dietary strategies. Nutritionally, saturated fats are of particular concern, because an excess in the diet leads to their accumulation in the cardiovascular system, resulting in several health-related problems. Due to this, food manufacturers typically report the saturated fat vs. unsaturated fat content on the nutritional panel, allowing consumers wishing to have a healthier diet to make food choices with less saturated fat.

Determining the degree of fatty acid unsaturation of a product is difficult because foods can contain a complex mixture of saturated, monounsaturated, and polyunsaturated fatty acids with a variety of carbon chain lengths.

• Milk and butter contain saturated C4 to C20, monounsaturated C16 and C18, and polyunsaturated C18 fatty acids.
• Vegetable oils contain saturated C6 to C24, monounsaturated C16, and monounsaturated cis C18, C20, and C22 fatty acids.
• Margarines contain the same fatty acids as vegetable oils plus monounsaturated trans C18, C20, and C22, and polyunsaturated C18 fatty acids.
• Fish and meat typically contain saturated and monounsaturated C12 to C24+ fatty acids, plus polyunsaturated omega 3 C18, C20, and C22, and polyunsaturated omega 6 C18 and C20 fatty acids.
• Fish tends to be richer in the polyunsaturated omega 3 fatty acids, whereas meats are richer in the polyunsaturated omega 6 fatty acids.

To confirm identification, very efficient capillary GC columns with the ability to resolve a large number of peaks are required. Our SPB-PUFA columns utilize a specially-developed phase for the analysis of polyunsaturated fatty acids (PUFAs) as FAMEs. It has a lower phase polarity than commonly used ′wax′ columns, resulting in a column with a slightly different selectivity. Another choice is our Omegawax^®, which provides highly reproducible analyses, being specially tested for reproducibility of FAME equivalent chain length (ECL) values and resolution of key components.

cis/trans FAME Isomers

Fatty acids in the cis configuration are the dominant form in nature. Correspondingly, enzymes have evolved to efficiently digest and metabolize them with a high degree of specificity. Conversely, trans fatty acids are relatively rare in nature. However, because they can increase the shelf life and flavor stability of foods containing them, they have become predominant synthetic additives to processed foods, especially fried foods and baked goods.

Unfortunately, trans fatty acids, formed by partial hydrogenation of vegetable oil, interfere with natural metabolic process, resulting in an imbalance of the LDL:HDL ratio, and also increasing lipoprotein(a) levels. Studies have linked their nutritional contribution to be similar to that of saturated fatty acids, possibly playing a role in the heightened risk of coronary artery disease.

Because trans fatty acids have adverse health consequences and no known nutritional benefits over other fats, consumer groups have pressured manufacturers and restaurants for their elimination. Many regulatory agencies worldwide now require content labeling to inform buyers of ′trans fat′ levels of foods and some dietary supplements.

The differences between cis isomer FAMEs and trans isomer FAMEs of the same carbon length and degree of unsaturation are very small. Therefore, very efficient capillary GC columns with highly polar phases are required.

• The highly polar SP-2560 column was specifically designed for the separation of geometric-positional (cis/trans) isomers of FAMEs, and is extremely effective for special FAME applications including the separation of FAMEs in hydrogenated vegetable oil samples. This well-established column is specified in many methods.
• The extremely polar SLB-IL111 column exhibits the highest polarity of any GC phase, providing an alternative selectivity for FAME applications typically performed on SP-2560 and SP-2380 columns. It is able to provide resolution of some key isomers that cannot be resolved on the SP-2560 or SP-2380 columns.
• The highly polar SP-2380 column allows the separation of geometric (cis/trans) isomers as a group. The phase is stabilized, providing a maximum temperature slightly higher than the popular SP-2560 column. It is available in shorter column lengths than the SP-2560, therefore, useful for short analyses where detailed resolution is not necessary.

See our Technical Articles and Protocols for FAME Analysis

Mono-, Di-, and Triglycerides

Triglycerides (also called triacylglycerol, triacylglyceride, or TAG) are the main constituent of vegetable oil and animal fat, and make up most of the fats digested by humans. They are important in that they allow the uptake and transport of fat-soluble vitamins. Plus, they play a role in metabolism (unused saturated or monounsaturated fatty acids are stored by the body as triglycerides). However, triglyceride intake should be monitored because high levels of triglycerides have been linked to an increased risk of heart disease and stroke. Mono-, di- and tri-glycerides are related to fatty acids:

• A monoglyceride is the condensation of one fatty acid and glycerol.
• A diglyceride is the condensation of two fatty acids and glycerol.
• A triglyceride is the condensation of three fatty acids and glycerol.
  

Efficient capillary GC columns with the ability to provide ample resolution are required for proper identification. Triglycerides are large compounds requiring a relatively high final oven temperature for elution in a reasonable time. Cool-on-column (COC) injection should be used. Use of a heated injection port can lead to sample discrimination and is not suggested. The syringe needle used must have a diameter small enough to fit inside the 0.53 mm I.D. guard column. Automated injection is highly recommended for consistency.

HPLC using a C18 phase is an alternative technique for the analysis of glycerides.

• For faster analysis on any HPLC system, turn to Ascentis^® Express C18 phase standard columns. These columns, based on Fused-Core^® technology, offer the performance of sub-2 μm particles but exhibit the backpressure of 3 μm particles.
• The Ascentis^® Express C18 phase is also available in a more traditional 5 μm particle. The Fused-Core^® technology results in increased efficiency, whereas the 5 μm particles exhibit limited backpressure, allowing longer column lengths than that possible with the standard 2.7 μm Ascentis Express particles.
• The Ascentis^® Express C30 provides exceptional efficiency and resolution for TAG separations.

See all our HPLC Columns

Sterols and Edible Oils

The edible oil industry boasts revenue measured in the tens of billions of dollars. As such, it can be subject to criminal acts of fraud aimed at increasing profits (adding a cheaper, inferior oil to boost the volume of a premium, higher priced oil). A GC fingerprinting technique can be used to monitor product for adulteration, and also to identify the source of oils in unknown samples. Following derivatization to convert the fatty acids to FAMEs, GC analysis is performed. This quick fingerprinting technique allows the oil type and purity to be identified, by comparison of the FAME ratios in sample oils to the FAME ratios in reference oil standards.

Olive oil, eggs, margarine, soybean oil, and chocolate are a few of the foods that contain sterols, which occur naturally in plants and animals, and are ingested as part of the diet. These compounds perform many roles in human, such as various cellular functions and as precursors to several hormones and vitamins. Sterols of interest include brassicasterol, campesterol, cholestanol, cholesterol, coprostanol, desmosterol, ergosterol, lanosterol, b-sitosterol, and stigmasterol.

Fats in a sample typically must be extracted and saponified to isolate the sterols, which will be in the non-saponifiable fraction, along with other large molecular weight alcohols, fat-soluble vitamins, and hydrocarbons. Vegetable oils, which are almost pure fat, do not require extraction prior to saponification. The general saponification procedure outlined by the AOAC Method 970.51 is useful for preparing most samples. Analysis by GC can typically be performed without derivatization.

See our SAC™-5 Capillary GC Column