Separation of Lipids by Solid Phase Extraction (SPE)

Authors: Wei Zou ### Abstract SPE is an important technique to separate different classes of lipids after total lipid extraction. ### Reagents 1. Chloroform: methanol W/BHT 2:1 = 2 L: 1 L:100 mg - Acetone: water (ACW) = 7:1 as 700 mL:100 mL (v/v) - Hexane - Hexane:methyl tert-butyl ether:acetic acid (HBA) = 100:3:0.3 (v/v/v) as 1000 mL hexane:30 mL methyl butylether:3 mL acetic acid - Hexane: chloroform:ethyl acetate (HCA) = 100:5:5 (v/v/v) as 800 mL hexane:40 mL chloroform:40 mL ethyl acetate - Chloroform:2-propanol (CIP) = 2:1 (v/v/v) as 600 mL chloroform:300 mL 2-propanol - Chloroform:methanol:acetic acid (CMA) = 100:2:2 (v/v/v) as 750 mL chloroform:15 mL methanol:15 mL acetic acid - Methanol:chloroform:water (MCW) = 10:5:4 (v/v/v) as 500 mL methanol:250 mL chloroform:200 mL water - Bond Elut NH2-Aminopropyl column (Varian Sample Preparation Products, Harbor City, CA USA) - 1g capacity columns are for liver samples, while 500 mg capacity columns are for bile samples. - Test tube with Teflon cap (16 mm x 100 mm) - Vac Elut SPS 24TM Vacuum elution apparatus (Varian Sample Preparation Products, Harbor City, CA USA) ### Procedure 1. Determine the total lipid concentration in liver lipid extraction or bile. - SPE method allows lipid concentration <1.46 mg/100µl. - Prepare new solvent in dispenser from stock solution. - Throw away the old solvent because it is easy to evaporate in dispenser than in stock bottle. - Take proper amount of liver lipid extraction or bile. Evaporate under N2. Add 900µl HBA into the tube. - Connect the SPE vacuum apparatus to the negative water pressure system. Switch on the pressure control. (Don’t exceed 2) - Switch the upper part of SPE vacuum apparatus to “Waste” position. - Use 1000 µL automatic pipette to add 600 µL ACW twice to wash the SPE column. - Change the switch on the SPE vacuum apparatus from WASTE to COLLECTIVE. - Use 1000 µL automatic pipette to add 1000 µL hexane twice. - Apply 300 µL lipid HBA (LHBA) into the column. - Set up the tubes of CE fraction into the SPE vacuum apparatus. - Use dispenser to add 5 mL hexane into the column to elute CE. Wait until all the solution is eluted. - Change the tubes to TG. Use dispenser to add 5 mL HCA. - Change the tubes to MDG. Use dispenser to add 5 mL CIP. - Change the tubes to FFA. Use dispenser to add 5 mL CMA. - Change the tubes to PL. Use dispenser to add 5 mL MCW. - For PL fraction, add 2 mL water + 1 mL chloroform to separate water in the PL fraction. Shake upside down 3 times. Siphon off the upper layer. Cap tightly and seal it with Teflon tape. Evaporate the lower layer for FA analysis. ### References 1. Zou, W., Noh, S.K., Owen, K.O., & Koo, S.I. (2005) Dietary carnitine enhances the lymphatic absorption of fat and a-tocopherol in ovariectomized rats. *J. Nutr*. 135: 753-756. http://jn.nutrition.org/content/135/4/753.full - Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. *J. Biol. Chem*. 226:497-509. - Agren, J.J., Julkunen, A., and Penttila, I. (1992) Rapid separation of serum lipids for fatty acid analysis by a single aminopropyl column. *J. Lipid Res*. 33:1871-1876. ### Acknowledgements Thanks to Koo, S.I. for mentoring and financial supporting efforts. ### Author information **Wei Zou**, Zou's lab, DTSC, California State Government Correspondence to: Wei Zou (wzou@dtsc.ca.gov) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2156) (2011) doi:10.1038/protex.2011.246. Originally published online 28 July 2011*.