Separation of neutral lipids by high-performance liquid chromatography: quantification by ultraviolet, light scattering and fluorescence detection.
The recent increased use of cell cultures to model physiological events, in particular signal transduction and traumatic injury, has produced a need for a quantitative, high-performance liquid chromatographic separation of neutral lipid classes with a high degree of resolution and reproducibility. We report an isocratic separation using a Phenomenex Selectosil silica column (5 microns). Two solvents were used, 1.2% 2-propanol in n-hexane containing 0.1% acetic acid (90%) and n-hexane (10%) at a flow-rate of 0.6 ml/min. Column temperature was maintained at 55 degrees C and this temperature was critical for baseline resolution of 1,3-diacylglycerol and cholesterol. The use of 10% n-hexane permitted the resolution of low polarity compounds such as butylated hydroxytoluene, triacylglycerols and cholesteryl esters. All of the detectors used produced standard curves with linearity over a wide concentration range.
Murphy
E J
EJ
Department of Medical Biochemistry, Ohio State University, Columbus 43210, USA.
Rosenberger
T A
TA
Horrocks
L A
LA
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D008027:Light; D008055:Lipids / Q000302:isolation & purification*; D051381:Rats; D012542:Scattering, Radiation; D013696:Temperature; D014466:Ultraviolet Rays
D008055:Lipids