Department of Chemistry & Biochemistry, The University of Southern Mississippi, 118 College Drive, Box # 5043, Hattiesburg, MS 39406, USA.
Department of Chemistry & Biochemistry, The University of Southern Mississippi, 118 College Drive, Box # 5043, Hattiesburg, MS 39406, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Dec 1;1102-1103:52-59. doi: 10.1016/j.jchromb.2018.10.012. Epub 2018 Oct 15.
In this paper, we report a method for the separation of hydroxy fatty acid and non-hydroxy fatty acid containing neutral lipid classes via normal phase HPLC with UV detection on a PVA-Sil column. The hexane/isopropanol/methanol/water based method separates all the neutral lipids in 21 min, and subsequently flushes through the polar lipids by 27 min such that prefractionation of neutral and polar lipids are not required, and the column is re-equilibrated for the next run in 15 min, for a total run time of 45 min per sample. The separation was demonstrated at both 1.0 mL/min and 1.5 mL/min for added applicability for fraction collection or inline analysis. Separation of various hydroxy fatty acid containing lipids was demonstrated from three different plant species Ricinus communis, Physaria fendleri, and engineered Arabidopsis thaliana. Additionally, we have combined this method with an in-line liquid scintillation counter for the separation and quantification of C labeled lipids obtained from in vivo metabolic flux experiments conducted in the developing seeds of Arabidopsis thaliana.
本文报道了一种通过 PVA-Sil 柱正相 HPLC 结合 UV 检测分离含羟基脂肪酸和非羟基脂肪酸的中性脂质类别的方法。基于正己烷/异丙醇/甲醇/水的方法可在 21 分钟内分离所有中性脂质，随后在 27 分钟内冲洗极性脂质，因此不需要对中性和极性脂质进行预分离，并且在 15 分钟内重新平衡柱子，以便下一次运行，每个样品的总运行时间为 45 分钟。在 1.0 mL/min 和 1.5 mL/min 两种流速下进行了分离，以增加用于馏分收集或在线分析的适用性。从三种不同的植物物种蓖麻、鬼针草和工程拟南芥中分离出了各种含羟基脂肪酸的脂质。此外，我们还将该方法与在线液闪计数器相结合，用于分离和定量从拟南芥发育种子中进行的体内代谢通量实验获得的 C 标记脂质。
