292 TITRIMETRIC METHODS 



3. Set aside overnight at room temperature or heat to over 80° 

 for 10 min. to effect saponification. Keep the material at the one 

 end of the tube so that none will be lost when the other end is 

 subsequently cut off to introduce the next reagent. 



4. Centrifuge to drive down the droplets of toluol which con- 

 dense on the upper walls of the capillary, open the empty end of the 

 tube with a diamond point, and pipette an excess of 0.5 N hydro- 

 chloric acid down into the tube ( 13 fA. will suffice in most instances) . 



5. Reseal the open end of the capillary at once and thoroughly 

 mix the contents by shaking. The special centrifuging apparatus 

 which repeatedly turns the tubes (Fig. 63, page 178) has been 

 especially designed for mixing liquids in capillary tubes. 



6. Separate the toluol and water phases by centrifuging, open the 

 capillary, and pipette an aliquot of about 13 //,!. of the toluol layer 

 into a titration vessel with a standard ground mouth (Fig. 86) . 



7. Evaporate the toluol in a desiccator furnished with paraffin 

 chips at about 30 mm. mercury pressure. 



8. Dissolve the fatty acid in 100 fA. 0.01% alcoholic thymol 

 blue, add a stirring "flea," and titrate with the standard tetra- 

 methylammonium hydroxide using the set-up shown in Figure 86 

 to exclude atmospheric carbon dioxide. Match the color to that of 

 a faint green color standard prepared by titrating 100 jxl. alcoholic 

 thymol blue to a pure yellow color and then adding 5 /xl. 0.04% 

 bromophenol blue, which has been rendered blue with a little sodium 

 hydroxide. 



9. Run a blank titration on 100 fA. alcoholic thymol blue. 



EXTRACTION AND FRACTIONATION OF LIPIDS 



To enable the extraction and fractionation of the lipids in quanti- 

 ties of tissue of the order of 1 mg., so that analytical methods 

 might be applied to the material, Schmidt-Nielsen (1944b) elabo- 

 rated appropriate procedures. The tissue lipids are treated with 

 alkali and the unsaponifiable fraction is extracted with toluol. 

 Fatty acids are liberated from the aqueous portion by the addition 

 of mineral acid and then they are extracted with toluol. 



