LIPID EXTRACTION AND FRACTIONATION 293 



Schmidt-Nielsen Method for Extraction 

 and Fractionation of Lipids 



SPECIAL REAGENTS 



Pure Toluol. 



1 N Potassium Hydroxide in Absolute Alcohol (aldehyde free). 



4 N Hydrochloric Acid ( chlorine free) . 



PROCEDURE 



A. Unsaponifiable Fraction 



1. Place the tissue in the bottom of a capillary tube (1.8-2 mm. 

 internal diameter, 50 mm. long, and sealed at one end). Two micro- 

 tome sections each 25 /x thick and 5 mm. in diameter weighing 

 approximately 1 mg. may be used. It is convenient to employ the 

 cryostat microtoming technique (page 427), in which the sections 

 are allowed to curl up on the knife edge and are transferred into 

 the capillary tube kept cold in the cryostat. 



2. Pipette 2.5 fA. alcoholic potassium hydroxide into the tube 

 with the sample and follow with the addition of 20 /xl. toluol. Seal 

 the open end of the tube at once. Follow pipetting directions on page 

 291. 



3. Saponify by heating the tube to 80-100° for 2 min. Centrifuge 

 to throw down droplets of toluol that condense along the tube. 



4. Open the tube, introduce 16 fA. water, and reseal. 



5. Mix the liquids thoroughly by placing in the mixing centri- 

 fuge (page 178). 



6. Separate the emulsion into two layers by centrifuging at 

 3500 R.P.M. or faster, after heating the capillary tube, the ordinary 

 centrifuge tube into which it is placed, and the metal centrifuge 

 tube holder in an oven at 110°. If the separation is incomplete, 

 reheat and recentrifuge. 



7. Open the tube and pipette a 16 fA. aliquot of the toluol phase 

 into a paraffined reaction tube (page 169). Evaporate off the toluol 

 in a desiccator over paraffin shavings at a pressure of 20 mm. 

 mercury. 



