Terckian,Theraplix,France,1972
The following is taken from US Patent 3,057,851. Milorganite was extracted
with water to obtain an aqueous extract containing vitamin B12 active
substances. This aqueous extract was purified by treatment with an ion
exchange resin according to the following method. An aqueous extract of
milorganite, 100 ml containing 300 μg of vitamin B12 active substances and
4.5 grams of total solids, was combined with 0.5 gram of sodium nitrite and
0.4 gram of potassium cyanide. The resulting solution was adjusted to pH 4.0
with hydrochloric acid and heated to boiling. The boiled solution was filtered
through a Super-Cel filter surface, and the filter was then washed with water.
The filtrate was obtained in a total volume of 130 ml including the washings.
Amerlite XE-97, an ion exchange resin of the carboxyl type (Rohm and Haas),
was classified to an average wet particle size of 100 to 150 mesh. The
classified resin was utilized in the hydrogen form, and was not buffered during
the ion exchange fractionation. The classified resin, in the amount of 35 ml,
was packed into a glass column having a diameter of 25 mm and a height of
250 mm. The cyanide-treated aqueous extract of milorganite was infused
gravitationally into the ion exchange bed at a rate of 3 ml per minute.
The effluent was discarded and the resin bed was then washed with the
following solutions in the specified sequence: (1) 120 ml of an aqueous 0.1 N
hydrochloric acid solution; (2) 75 ml of an aqueous 85% acetone solution;
and (3) 70 ml of an aqueous 0.1 N hydrochloric acid solution. After washing,
the resin bed was eluted with an aqueous 60% dioxane solution containing
0.1 N of hydrochloric acid. In this elution, 8 ml of colored eluate was
collected. This portion of the eluate was found to contain 295 μg of
cyanocobalamin and 9 mg of total solids.
