荧光标记C_(12)～C_(20)等脂肪酸在反相高效液相色谱系统中的超微量分析

Ultramicro Analysis of C 12 C 20 Fatty Acids by Reversed Phase High Performance Liquid Chromatography(RP HPLC) with Florescence Labelling

对Ｃ１２～Ｃ２０等脂肪酸在反相高效液相色谱（ＨＰＬＣ）系统中的超微量分析进行了研究。月桂酸、肉豆蔻酸、棕榈酸、硬脂酸、花生酸等有机酸在所选择的色谱条件下都能得到有效的分离，采用Ｃ１８键合相的色谱柱，流动相采用甲醇∶水＝９７∶３，流速为１．５ｍＬ／ｍｉｎ，荧光检测波长为λｅｘ＝３２５ｎｍ，λｅｍ＝３９７ｎｍ。在实验中采用４－溴甲基－７－甲氧基香豆素作为Ｃ１２～Ｃ２０等脂肪酸的荧光标记物，以丙酮为溶剂，以１８－冠醚－６为催化剂，以碳酸钾为碱试剂，通过相转移催化完成衍生化反应。对方法的准确度、精密度及最小检出限进行了验证。

High performence liquid chromatographic ultramicro analysis of C 12 C 20 fatty acids with 4 bromomethyl 7 methoxycoumarin as a fluorescent label was studied. A mild condition is needed for the fluorescence derivatization reaction. The reaction was based on the nucleophilic substitution of halide and liquid solid phase transfer and completed in acetone solvent. Anhydrous K 2CO 3 powder and 18 crown 6 were used as an alkali reagent and catalyst respectively. The lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid were separated excellently. A C 18 bonded phase column (φ4 6mm×15cm), MeOH/H 2O=97/3( V/V ) as the mobile phase with flow rate of 1 5mL/min and the detection wavelength at λ ex =325nm and λ em =397nm were adopted in HPLC analysis. The accuracy, reproducibility and detection limit of the proposed method were tested and confirmed by using synthesized samples. The minimum detectable level was 10 -7 g/L.