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ARS Home » Southeast Area » Miami, Florida » Subtropical Horticulture Research » Research » Publications at this Location » Publication #420317
Research Project: Mitigation of Invasive Pest Threats to U.S. Subtropical Agriculture

Location: Subtropical Horticulture Research

Title: Development of a combined 2D-MGD TLC/HPTLC method for the separation of Terpinen-4-ol and a-Terpineol from tea tree, Melaleuca alternifolia, essential oil

Author
item Vazquez, Aime
item Tabanca, Nurhayat

Submitted to: Biomolecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2025
Publication Date: 1/18/2025
Citation: Vazquez, A., Tabanca, N. 2025. Development of a combined 2D-MGD TLC/HPTLC method for the separation of Terpinen-4-ol and a-Terpineol from tea tree, Melaleuca alternifolia, essential oil. Biomolecules. 15,147. https://doi.org/10.3390/biom15010147.
DOI: https://doi.org/10.3390/biom15010147

Interpretive Summary: Plant volatiles mediate the interactions between plants and pests, influencing pest population and dynamics. There is a resurgence of interest in natural products for integrated pest management (IPM) strategies because of concerns over undesirable health and environmental effects of frequent use of conventional pesticides. Advances in analytical instrumentation and bioassays have facilitated the discovery of new chemicals from natural sources. A more recent study involving manual thin-layer chromatography (TLC) and parallel bioassays revealed new kairomones from tea tree (Melaleuca alternifolia) oil (TTO). TTO is a popular essential oil widely used in cosmetic and medicinal products. Its antimicrobial properties have been attributed to its high terpinen-4-ol content. More so, there is a distinct enantiomeric ratio between (+) and (-) terpinen-4-ol, as well as between (+) and (-) a-terpineol. Though native of Australia, tea tree it is now cultivated and extracted in several locations to satisfy the demand. This has caused variations of the oil due to differences in geography, environment and extraction procedures. Regrettably, with high demand and costs of operation, adulteration has also increased. Quality control protocols generally involve the use of sophisticated, expensive equipment not easily available across laboratories. Planar chromatography offers a viable alternative, yet traditional TLC methods may not allow full separation of complex samples such as TTO. Therefore, ARS scientists in Miami, FL developed a semi-automated, two-dimensional, multi-gradient thin layer chromatography method (2D-MGD TLC/HPTLC) to isolate target compounds. This method combines the simplicity of planar chromatography with the improved chromatographic resolution of 2D, multi-gradient and HPTLC techniques. These combined features make the 2D-MGD TLC/HPTLC method an alternative eluent system for co-migrating compounds such as terpinen-4-ol and a-terpineol.

Technical Abstract: Tea tree oil (TTO), acquired from Melaleuca alternifolia (Maiden & Betche) Cheel, Myrtaceae, is a widely utilized essential oil (EO) due to its bioactive properties. Many personal care and aromatherapy products contain TTO, making it a valuable commodity. Originally from Australia, Melaleuca alternifolia is now grown in other countries to mitigate the demand. On account of differences in geographical location, field conditions and extraction methods, strict ISO criteria, including enantiomeric distribution of main ingredients such as terpinene-4-ol and a-terpineol, have been established to certify the oil for commercial and medical purposes. Chemical analysis and identification of TTO ingredients is generally performed by GC-MS, which provides the most accurate results. However, in some cases, cost and time of analysis may be a constraint. Thin layer chromatography (TLC) and High-performance thin-layer chromatography (HPTLC) offer a simpler, faster, cost-effective alternative capable of simultaneously analyzing and quantifying multiple samples. For more complex oils, two-dimensional (2D) or multigradient development (MGD) TLC provide better separation of comigrating components. This study showcases a combined 2D-MGD TLC/HPTLC method for the successful separation of TTO producing highly pure terpinene-4-ol (100 %) and a-terpineol (= 94 %), confirmed by GC-MSD. Enantiomeric distribution of both major TTO components, evaluated by GC-FID, was not altered during the separation and extraction process.