Kail Brian W, Link Dirk D, Morreale Bryan D
United States Department of Energy, National Energy Technology Laboratory, Pittsburgh, PA 15236-0940, USA.
J Chromatogr Sci. 2012 Nov-Dec;50(10):934-9. doi: 10.1093/chromsci/bms093. Epub 2012 Jun 12.
A method for selectively determining both free fatty acids (FFA) and triacylglycerides (TAGs) in biological oils was investigated and optimized using gas chromatography after esterification of the target species to their corresponding fatty acid methyl esters (FAMEs). The method used acid catalyzed esterification in methanolic solutions under conditions of varying severity to achieve complete conversion of more reactive FFAs while preserving the concentration of TAGs. Complete conversion of both free acids and glycerides to corresponding FAMEs was found to require more rigorous reaction conditions involving heating to 120°C for up to 2 h. Method validation was provided using gas chromatography-flame ionization detection, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. The method improves on existing methods because it allows the total esterified lipid to be broken down by FAMEs contributed by FFA compared to FAMEs from both FFA and TAGs. Single and mixed-component solutions of pure fatty acids and triglycerides, as well as a sesame oil sample to simulate a complex biological oil, were used to optimize the methodologies. Key parameters that were investigated included: HCl-to-oil ratio, temperature and reaction time. Pure free fatty acids were found to esterify under reasonably mild conditions (10 min at 50°C with a 2.1:1 HCl to fatty acid ratio) with 97.6 ± 2.3% recovery as FAMEs, while triglycerides were largely unaffected under these reaction conditions. The optimized protocol demonstrated that it is possible to use esterification reactions to selectively determine the free acid content, total lipid content, and hence, glyceride content in biological oils. This protocol also allows gas chromatography analysis of FAMEs as a more ideal analyte than glyceride species in their native state.
研究并优化了一种通过气相色谱法选择性测定生物油中游离脂肪酸（FFA）和甘油三酯（TAG）的方法。该方法是先将目标物质酯化生成相应的脂肪酸甲酯（FAME），然后进行气相色谱分析。该方法采用酸催化甲醇溶液中的酯化反应，通过改变反应条件的严苛程度，使反应活性较高的FFA完全转化，同时保留TAG的浓度。结果发现，要使游离酸和甘油酯完全转化为相应的FAME，需要更严格的反应条件，即加热至120°C并持续2小时。采用气相色谱 - 火焰离子化检测、气相色谱 - 质谱和液相色谱 - 质谱对该方法进行了验证。该方法对现有方法进行了改进，因为与来自FFA和TAG的FAME相比，它能使总酯化脂质按FFA贡献的FAME进行分解。使用纯脂肪酸和甘油三酯的单组分和混合组分溶液，以及芝麻油样品模拟复杂生物油，对方法进行了优化。研究的关键参数包括：盐酸与油的比例、温度和反应时间。发现纯游离脂肪酸在相对温和的条件下（50°C下10分钟，盐酸与脂肪酸比例为2.1:1）酯化，FAME的回收率为97.6±2.3%，而在这些反应条件下甘油三酯基本不受影响。优化后的方案表明，利用酯化反应可以选择性地测定生物油中的游离酸含量、总脂质含量，进而测定甘油酯含量。该方案还使气相色谱分析FAME成为比天然状态下的甘油酯类更理想的分析物。
