University of Maryland Researchers Investigate HPLC-PDA Method for Fatty Acid Analysis

Researchers from the University of Maryland in College Park, Maryland, led by Esther A. Olonimoyo and Naresh Kumar Amradi, created a high-performance liquid chromatography (HPLC)-based method for analyzing short-chain fatty acids. The team published their findings in the Journal of Chromatography A (1).

In this study, a fast analytical method was created for quantifying fatty acids, consisting of a high-performance liquid chromatograph coupled with a photodiode array detector (HPLC-PDA). Six short-chain fatty acids were searched for formic, acetic, propionic, butyric, isovaleric, and valeric acids.

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Fatty acids (FAs) are an important type of compound classified by carbon chain length and degree of saturation. These compounds consist of one hydrocarbon chain, with a methyl group at one end (the omega end) and a carboxyl group at the other (alpha) end (2). Short-chain fatty acids (SCFAs), also referred to as volatile fatty acids (VFAs), have gained recognition as essential components for biofuel production, including ethanol and butanol. SCFAs are generated through fermentation, which involves the breakdown of complex carbohydrates under specific conditions with the assistance of microbial activity.

Fatty organic acids naturally occur in biological systems and are frequently studied in environmental science, including atmospheric sciences. Fatty organic acids are found in various foods, vegetation, and biological systems. These acids are frequently used for renewable energy generation from biological waste materials. There is a need for analytical methods that can detect various FAs within complex matrices. These methods need to be adaptable, especially for environmental samples, to ensure that they do not alter a sample’s chemical composition or introduce extraneous artifacts, the scientists wrote. Moreover, the method should feature low detection limits consistent with the compounds’ natural abundance.

The researcher’s method using HPLC-PDA was compared to existing literature. The researchers found that the new method does not require derivatization and produces rapid results with relatively low chromatographic cost. The optimized method can be characterized by a gradient elution mode with flow rates ranging from 1–2.5 mL min^-1 and a short analysis time of 7.6 mins. The limits of detection for all six acids (LOD) ranged from 0.0003 to 0.068 mM, while the limits of quantification (LOQ) ranged from 0.001 to 0.226 mM. Validation and quantification were tested using fermentation broth samples. Elution modes, column temperature, mobile phase buffer pH, and mobile phase flow rates were investigated to resolve overlapping peaks and optimize retention time. A time-efficient method (approximately 8 minutes long) was created for the chromatographic determination of six short-chain fatty acids. The optimization method reported in this study showed that more SCDAs can be analyzed in a complex mixture in a shorter run time. Compared to previous literature from the past decade, the scientists concluded their method to be beneficial, as it does not require a derivatization step, is cost-effective, less cumbersome to operate, and is time efficient.

The optimized method was validated and applied to the analysis of fermentation broth samples. Low LOD and LOQ values were determined for the optimized HPLC method to represent excellent dynamic range and instrument sensitivity. Distinct peak separation in the optimized method allowed for better analyte quantification, which is applicable to a wide range of samples, notably samples in aqueous media, such as food, waste, and environmental samples. The short analysis times and low detection limits are applicable for time-dependent studies, such as atmospheric samples and industrial applications that require many samples. Overall, this method presented researchers with a cost-effective and time-efficient method for analyzing the six SCFAs in question.

References

(1) Olonimoyo, E. A.; Amradi, N. K.; Lansing, S.; et al. An Improved Underivatized, Cost-Effective, Validated Method for Six Short-Chain Fatty Organic Acids by High-Performance Liquid Chromatography. J. Chromatogr. Open 2025, 7, 100193. DOI: 10.1016/j.jcoa.2024.100193

(2) Fatty Acid. ScienceDirect 2017. https://www.sciencedirect.com/topics/medicine-and-dentistry/fatty-acid (accessed 2025-4-11)