Authors: Wei Zou
Abstract
SPE is an important technique to separate different classes of lipids after total lipid extraction.
Reagents
- 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
- 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
- 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 ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.246. Originally published online 28 July 2011.