II. CHEMISTRY 593 



1. Autolysis to obtain a clear filtrate. 



2. Adsorption on fuller's earth to remove bases. 



3. Adsoiption of the vitamin on charcoal and its subsequent elution. 



4. Preparation of brucine derivatives by evaporation to dryness on 

 kieselguhr in the presence of brucine and brucine oxalate. 



5. Extraction of brucine pantothenate with chloroform. 



6. Fractional distribution of the brucine salts between chloroform and 

 water. 



7. Conversion of the pantothenic acid to its calcium salt. 



8. Fractionation of the calcium salts with various solvents and solvent 

 mixtures. 



The success of this procedure depended upon a rapid and accurate 

 quantitative assay method which was made possible by the use of yeast as 

 test organisms.*' ^ Without such a rapid test, this procedure would have 

 been hopeless. This work foreshadowed the wide use of microorganisms 

 for the assay of vitamins and amino acids. 



Pure pantothenic acid has never been isolated by this or any other pro- 

 cedure. Only a small quantity of pantothenic acid of 90 % purity has been 

 prepared. It was a colorless "varnish," which, when ground, yielded a 

 white amorphous hygroscopic powder which was about 11,000 times as 

 potent as their "standard" rice bran preparation. "' 



Long before pantothenic acid was isolated, Williams and his coworkers 

 obtained extensive preliminary information about it, made possible in 

 large part by the extensive use of a relatively new tool, i.e., fractional elec- 

 trical transport.^"' ^' ^ 



1. The compound was an acid with a molecular weight* of about 150 and 

 with an ionization constant* of about 3.9 X 10~^. 



2. It was unstable in hot acids and alkalies. 



3. It had a nitrogen atom with barely detectable basic properties.^ 



4. It had in its structure no olefin double bond, aldehyde, ketone, sulf- 

 hydryl, basic nitrogen, aromatic, or sugar group. 



5. It possessed several hydroxyl groups.* 



Snell ei al}^ were independently studying an essential nutrient for lactic 

 acid bacteria. They concluded that it was an acid, was adsorbed on char- 

 coal, lacked basic properties, and was unstable in hot acid or alkaline 

 solution. 



6 R. J. Williams, E. D. McAlister, andR. R. Roehm, J. Biol. Chem. 83, 315 (1929). 

 6 R. J. Williams and J. H. Truesdail, J. Am. Chem. Soc. 53, 4171 (1931). 

 ■> R. J. Williams, /. Biol. Chem. 110, 589 (1935). 

 8 R. J. Williams and R. J. Moser, J. Am.. Chem.. Soc. 56, 169 (1934). 

 s R. J. Williams, H. H. Weinstock, Jr., E. Rohrmann, J. IT. Truesdail, H. K. Mit- 

 chell, and C. E. Meyer, J. Am. Chem. Soc. 61, 454 (1939). 

 1" E. E. Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937). 



