- 學位論文
HPLC-UV檢測脂肪酸之方法建立
Development of HPLC-UV method for fatty acid detection
摘要
脂肪酸為油脂的主要組成分，在自然界以偶數碳的形式存在於動植物中。氣相層析法(Gas chromatography，GC)是分析脂肪酸最常用的技術，通常需經過衍生化使游離脂肪酸轉換為脂肪酸甲基酯，但酯化及分析過程中的高溫易造成脂肪酸結構發生降解。高效能液相層析法(High performance liquid chromatography，HPLC)相對於GC的主要優勢是分析過程中使用的溫度較低，降低了不飽和脂肪酸異構化的風險，且儀器的應用較廣泛，因此本研究目的在於開發一種以HPLC-UV檢測脂肪酸的方法。本實驗以HPLC-UV在波長242 nm下檢測在食用油脂中常見的六種脂肪酸，分別是月桂酸、肉豆蔻酸、棕櫚酸、硬脂酸、油酸及亞麻油酸。實驗以α-溴苯乙酮作為衍生試劑、三乙胺為催化劑，分別以不同衍生溫度及時間、衍生試劑濃度還有催化劑濃度，並找出最佳衍生條件。實驗結果顯示，脂肪酸與20 mg/mL之α-溴苯乙酮及15 mg/mL之三乙胺於 90℃反應40分鐘層析峰可獲得最高積分面積，以上述條件製作檢量線，各脂肪酸線性範圍，月桂酸、肉荳蔻酸、棕櫚酸及硬脂酸為1.5-300 μg/ mL，亞麻油酸及油酸為4.5-900 μg/ mL，R2皆大於0.99。確效分析方面，所有脂肪酸之偵測極限(Limit of detection，LOD)在0.2-0.6 μg/mL，定量極限(Limit of quantitation，LOQ)在0.5-1.5 μg/mL；intra-day及inter-day之變異係數分別為0.21-9.71%及1.50-11.21%；回收率介於85.24-114.21%，皆符合衛福部之確效規範。再來以HPLC及GC檢測已知濃度標準品，結果顯示HPLC之相對誤差低於GC，表示檢測結果較準確，且本研究之方法可用於市售產品之測定。綜合上述，本研究所使用的方法具有代替GC檢測脂肪酸的潛力。
並列摘要
Fatty acids are the main components of oils and fats. They exist in animals and plants in the form of even-numbered carbons in nature. Gas chromatography (GC) is the most commonly used technology for the analysis of fatty acids, it usually requires derivatization to convert free fatty acids into fatty acid methyl esters (FAME). However, the formation of by-products during the esterification process and thermal degradation caused by the analysis process have always been major problems. A major advantage of high performance liquid chromatography (HPLC) over GC is the lower temperature used during analysis, which reduces the risk of isomerization of unsaturated fatty acids and the application of the instrument is more extensive. Therefore, the purpose of this study is to develop a method to detect fatty acids by HPLC-UV. Six fatty acids were detected by HPLC-UV at a wavelength of 242 nm, respectively lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid. α-bromoacetophenone was used as the derivatization reagent and triethylamine was used as the catalyst. The reaction was performed by different conditions. Different derivatization temperature and time, derivatization reagent and catalyst concentration to find the best derivatization conditions. The experimental results show that the highest peak area was obtained by reacting fatty acids with 20 mg/mL α-bromoacetophenone and 15 mg/mL triethylamine at 90°C for 40 minutes. Next, the derivatization reaction was applied to standard mixture and make a standard curve. The rang of 1.5-300 μg/mL for lauric acid, myristic acid, palmitic acid, stearic acid and the rang of 4.5-900 μg/mL for oleic acid, linoleic acid, R2≧0.99. In terms of validation analysis, LOD and LOQ values for all fatty acids were in the range of 0.2-0.6 μg/mL and 0.5-1.5 μg/mL. The intra-day and inter-day coefficients of variation are 0.21-9.71% and 1.50-11.21% and the recovery rate is between 85.24-114.21%. The above validation experiments are in compliance with the specifications. Then, HPLC and GC were used to detect the known concentration standard. The results showed that the relative error of HPLC was lower than GC, indicating that the detection results were more accurate and the method in this study can be used for the determination of commercial products. According to the results, this study has the potential to replace the detection of fatty acids by gas chromatography.