Ultra High-Speed Analysis of Free Fatty Acids in Vegetable Oil using ADAM Derivatization by Ultra High-performance Liquid Chromatography

By DJ Tognarelli

Download PDF August 18, 2022

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Introduction

In fatty acid analysis, UV detection at a short wavelength, refractive index detection and evaporative light scattering detection (ELSD) are generally employed. However, in trace analysis, a derivatization method is preferred because it offers much higher sensitivity. There are several derivatization methods for the detection of free fatty acids that require either UV or fluorescence detection. Among them, 9-Anthryldiazomethane (ADAM) is most commonly used because it reacts with fatty acids easily at room temperature and enables a highly sensitive and selective analysis.

Here, the separation and fluorescence detection of free fatty acids in vegetable oil is demonstrated using ADAM derivatization by Ultra High-performance Liquid Chromatography (UHPLC)

JASCO X-LC system

Experimental

Results

Figure 1 shows the procedure for ADAM derivatization and figure 2 shows a reaction mechanism of ADAM reagent.

Figure 1. Procedure for ADAM derivatization.
Figure 2. Reaction mechanism of ADAM reagent with fatty acid

Figure 3 shows the chromatogram of the standard mixture of fatty acids derivatized with ADAM. 11 different fatty acids were separated within 7 minutes.

Figure 3. Chromatogram of the standard mixture of fatty acid derivatized by ADAM. 1: Caprylic acid (C8), 2: Capric acid (C10), 3: Lauric acid (C12), 4: Linolenic acid (C18:3), 5: Myristic acid (C14), 6: Linoleic acid (C18:2), 7: Palmitic acid (C16), 8: Oleic acid (C18:1), 9: Stearic acid (C18), 10: Arachidic acid (C20), 11: Behenic acid (C22) (0.05 mg/mL each in ethyl acetate)

Figures 4 and 5 show the chromatograms of fatty acids in rice oil and coconut oil derivatized with ADAM, respectively.

Figure 4. Chromatogram of the ADAM derivative of rice oil. The peak numbers and corresponding compounds are the same as in figure 3.

Preparation. Dissolve 1.0 g of rice oil in 10 mL of ethyl acetate and then follow the procedure for ADAM derivatization as in figure 1.

Figure 5. Chromatogram of the ADAM derivative of coconut oil. The peak numbers are the same as in figure 3.
Preparation : Dissolve 1.0g of coconut oil in 10mL of ethyl acetate and then follow the procedure of ADAM derivatization as in figure 1.
Table 1 shows quantitative value of rice oil and coconut oil.

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About the Author

DJ Tognarelli received his B.S. from the University of Richmond where he studied Analytical Chemistry. He is a Chromatography Applications Scientist at JASCO.