自制氨基哌嗪功能化丹磺酰氯(DNS-CL)荧光衍生试剂(DNS-Pi-NH₂),并用其衍生化小球藻中碳原子数为10~20的典型脂肪酸[十二烷酸、十四烷酸、顺-9,12-十八(碳)烯酸、顺-7,10,13-十六(碳)烯酸、十六烷酸、顺-9-十八(碳)烯酸、十八烷酸、二十烷酸],用高效液相色谱法(HPLC)测定8种脂肪酸的含量。将小球藻脂肪酸甲酯样品用含氢氧化钾的乙醇溶液于79℃皂化60 min,滴加盐酸溶液,直至酚酞指示剂由红色变为无色。蒸发除去乙醇,所得固体经水清洗、离心、振荡、过滤,即制得小球藻脂肪酸样品。以DNS-CL和N-(2-氨基乙基)哌嗪为原料制备DNS-Pi-NH₂,并用红外光谱、核磁共振碳谱、质谱对其结构进行表征。取制备好的DNS-Pi-NH₂、二丙基二硫醚、三苯基磷的乙腈溶液和1.0 mg小球藻脂肪酸样品在室温下振荡衍生10 min,所得溶液用90%(体积分数,下同)乙腈溶液稀释后,用HPLC测定其中脂肪酸衍生物的含量。以Eclipse XDB C₈色谱柱为固定相,以90%乙腈溶液为流动相进行等度洗脱,分离得到的目标物用荧光检测器检测。结果显示:8种脂肪酸衍生物在30 min内可实现完全分离;各脂肪酸的浓度在2.0×10^-10~2.0×10^-4mol·L^-1内与其对应的峰面积呈线性关系,检出限(3s/k)为21~65 pmol·L^-1;对微藻培养液样品进行加标回收试验,所得回收率为95.3%~102%,测定值的相对标准偏差(n=5)为1.7%~2.6%;方法用于小球藻脂肪酸甲酯样品的分析,检出了十六烷酸、顺-9,12-十八(碳)烯酸、顺-7,10,13-十六(碳)烯酸、顺-9-十八(碳)烯酸,检出量分别为3.08,0.64,0.83,2.57 mg·g^-1。

Aminopiperazine functionalized dansyl chloride (DNS-CL) fluorescent derivatization reagent (DNS-Pi-NH₂) was self-made, and used for derivatizing typical fatty acids containing 10-20 carbon atoms (dodecanoic acid, tetradecanoic acid, cis-9,12-octadecenoic acid, cis-7,10,13-hexadecenoic acid, hexadecanoic acid, cis-9-octadecenoic acid, octadecane acid, eicosanoic acid) in Chlorella, and the 8 fatty acids were determined by high performance liquid chromatography (HPLC). Chlorella fatty acid methyl ester sample was saponified with ethanol solution containing potassium hydroxide at 79℃ for 60 min, and hydrochloric acid solution was added dropwise until the phenolphthalein indicator changed from red to colorless. The ethanol was removed by evaporation, and the solid obtained was washed with water, centrifuged, vibrated and filtered to prepare Chlorella fatty acid samples. DNS-Pi-NH₂ was prepared from DNS-CL and N-(2-aminoethyl) piperazine, and its structure was verified by infrared spectroscopy, nuclear magnetic resonance carbon spectroscopy and mass spectrometry. Dipropyldisulfide, acetonitrile solutions of DNS-Pi-NH₂ and triphenylphosphorus were mixed with 1.0 mg Chlorella fatty acid sample, and the mixture was shaken at room temperature for 10 min to derivatize. The obtained solution was diluted with 90% (volume fraction, the same below) acetonitrile solution, and derivatives of fatty acids in which was determined by HPLC. Eclipse XDB C₈ column was used as stationary phase, 90% acetonitrile solution was used as mobile phase for isocratic elution, and fluorescence detector was used for detection. As found by the results, the derivatives of 8 fatty acids could be completely separated within 30 min. The concentration of each fatty acid was linearly related with its corresponding peak area in the range of 2.0×10^-10-2.0×10^-4mol·L^-1, with detection limits (3s/k) of 21-65 pmol·L^-1. Test for the spiked recovery was made on microalgae culture medium, giving recoveries in the range of 95.3%-102%, and RSDs (n=5) of the determined values ranged from 1.7% to 2.6%. The proposed method was applied to the analysis of Chlorella fatty acid methyl ester sample, and hexadecanoic acid, cis-9,12-octadecanoic acid, cis-7,10,13-hexadecenoic acid and cis-9-octadecanoic acid were detected, with detection amounts of 3.18, 0.64, 0.83, 2.57 mg·g^-1, respectively.