Recently Osborne and Wakeman have proposed a modification which yields a concentrate of high potency. Their method is to add fresh yeast to slightly acidified boiling water and continue the boiling for about five minutes. This process coagulates the proteins that are present and permits their removal by filtration. The protein-free filtrate appears to contain all of the vitamine originally present in the yeast but attempts to precipitate the vitamine fractionally from the evaporated filtrate by means of increasing concentration of added alcohol has been only partially successful. The method however yields a concentrated extract, and Harris has made use of this process to prepare tablets for medicinal purposes.
Seidell and Williams some time ago devised a procedure which seemed to give promise of good results. Their discovery was that when a filtrate from autolysed yeast is prepared, rich in the vitamine, and is shaken with a specially activated fuller's earth (the preparation produced by Lloyd and known as Lloyd's reagent has this power) in a proportion of 50 grams to the liter of extract the vitamine is absorbed by the earth and when the latter is filtered off it carries the vitamine with it. In their process they shake the mixture for about one-half hour and then remove the earth by filtration. Analysis of the yeast liquor after the extraction shows it to contain practically the same solids as originally present but to have lost practically all its vitamine. The latter is firmly attached to the earth and repeated washing with water fails to remove any appreciable amount of vitamine from it. Furthermore the vitamine-activated fuller's earth retains its active vitamine properties for at least a period of two years. Large amounts of the vitamine can be accumulated in this way and when fed to animals or infants the vitamine is liberated physiologically and produces the usual effects of a vitamine extract. When this discovery was made the discoverers thought that in the fuller's earth they had a means for arriving at the identification of the substance but attempts to recover the vitamine from the earth developed unexpected difficulties. Acids were found to split it off but they also split off aluminium compounds and left an impure mixture little better than the original extract for study. By using a dilute alkali they were able to obtain the substance without aluminium contaminations and by this method they actually obtained some microscopic fibrous needles which were curative. These needles however on recrystallization resulted in the production of a compound contaminated with adenin or rather in adenin contaminated with the curative substance and on standing for some time the adenin crystals gradually lost their curative power. These results led Williams to suggest an interesting hypothesis. By experiments conducted with the hydroxy- pyridines he believed that he had demonstrated a relation between tautomerism or changed space relations in these sort of substances and curative properties. He states his view as follows: