bb4.png
U
A
[Hide] (342.1KB, 775x506) Reverse (35) Example I Milorganite was extracted with water to obtain an aqueous extract containing vitamin B12 active substances.
(36) [his aqueous extract was purified by treatment with an on exchange resin according to the following method:
(37) An aqueous extract of milorganite, 100 ml., containing X00 mcg. of vitamin 1B12 active substances and 4.5 gins.
(38) )f total solids, was combined with 0.5 gin. of sodium titrite and 0.4 gin. of potassium cyanide. The resulting olution was adjusted to pH 4.0 with hydrochloric acid nd heated to boiling. The boiled solution was filtered hrough a Super-Cel filter surface, and the filter was then washed with water. The filtrate was obtained in a total olume of 130 ml., including the washings.
(39) Amberlite XE-97, an ion exchange resin of the car- 'oxyl type manufactured by Robin and Haas, was classi- ed to an average wet particle size of 100-150 mesh. The lassified resin was utilized in the hydrogen form, and gas not buffered during the ion exchange fractionation.
(40) The classified resin, in the amount of 35 ml., was packed ito a glass column having a diameter of 25 mm. and a eight of 250, mm. The cyanide-treated aqueous extract f milorganite was infused gravitationally into the ion xchange bed at a rate of 3 ml. per minute. The effluent ras discarded, and the resin bed was then washed with le following solutions in the specified sequence:
(41) (1) 120 ml. of an aqueous 0.1 N hydrochloric acid )lution.
(42) (2) 75 ml. of an aqueous 85% acetone solution.
(43) (3) 70 ml. of an aqueous 0.1 N hydrochloric acid )lution.
(44) After washing, the resin bed was eluted with an aque- as 60% dioxane solution containing 0.1 N of hydro- dloric acid. In this elution, 8 ml. of colored eluate was )llected. 'Ibis portion of the eluate was found to con- tin 295 incg. of cyanocobalamin and 9 mng. of total solids.
(45) Example 11 An aqueous extract of milorganite was treated with an ,n exchange resin according to the following method:
(46) i1he aqueous extract, 100 ml., contained 300 mcg. of tamin B12 active substances and 4.5 gis. of total solids.
(47) his extract was infused into 35 ml. of an ion exchange -d prepared by the method of Example I. '!he eiiuent was discarded, and the resin bed was washed with the following solutions in the specified sequence:
(48) (1) 150 ml. of an aqueous 0.1 N sulfuric acid solution.
(49) (2) 75 ml. of an aqueous 85% acetone solution.
(50) (3) 75 ml. of an aqueous 0.1 N sulfuric acid solution.
(51) The washed resin bed was eluted with an aqueous 60% dioxane solution containing 0.1 N of sulfuric acid. The colored eluate portion, in the amount of 9 ml., was collected and subjected to analysis. The results indicated that this eluate portion contained vitamin B12 activity equivalent to 250 mcg. of cyanocobalamin and 8.5 mg.
(52) of total solids.
(53) Example III The following method was utilized in treating a feed supplement product manufactured by Pacific Yeast Prod- ucts, Inc., which contained 254 mcg. of vitamin B12 active substances per gin.
(54) To 1 gin. of the feed supplement product was added 30 ml. of water, 15,0 mg. of sodium nitrite, and 150 mg. of potassium cyanide. The resulting mixture was adjusted to pH 4 with hydrochloric acid, and then heated to boiling. The boiled preparation was filtered through a Super- Cel filter surface, and the filter cake was washed with water.
(55) Amberlite XE-97 ion exchange resin was classified to a wet particle size of 100-150 mesh. The classified resin, in the amount of 9 ml., was packed into a glass column having a diameter of 11 mm. and a height of 300 mm.
(56) 30The aforementioned filtrate and washings, totaling 50 ml., was infused into the packed resin bed at a rate of 2 ml.
(57) per minute. The effluent was discarded, and the resin bed was then washed with the following solutions in the specified sequence:
(58) (1) 35 ml. of an aqueous 0.1 N hydrochloric acid solution.
(59) (2) 25 ml. of an aqueous 85% acetone solution.
(60) (3) 20 ml. of an aqueous 0.1 N hydrochloric acid solution.
(61) The washed resin bed was eluted with an aqueous 60% dioxane solution containing 0.1 N of hydrochloric acid.
(62) The colored eluate portion, in the amount of 5 ml., was collected, and subjected to analysis. The results indicated that the eluate portion contained 250 mcg. of vitamin B12 and 6 mg. of total solids.
(63) It was ascertained from the solubility of this eluate portion in a mixture of cresol and carbon tetrachloride, upon acid washing, that the feed supplement product was substantially free from pseudo vitamin B12.
(64) Example IV The following method was utilized in treating an aqueous extract of mammalian liver:
(65) The aqueous liver extract, in the amount of 10 ml., contained 250 mcg. of vitamin B12 active substances and 1.1 gmn. of total solids. This aqueous solution was combined with 3 ml. of an aqueous 10% potassium cyanide solution, and adjusted to pH 7.5 with citric acid. After standing at room temperature for a period of 8 hours the solution was adjusted to pH 4 with citric acid, and the acidified solution infused into an Amberlite XE-97 ion exchange resin bed as described in Example III. The rate of infusion of this solution was 1.5 ml. per minute. The effluent was discarded, and the resin bed was then washed with the following solutions in the specified sequence:
(66) (1) 10 ml. of pH 4 citric acid-sodiumi citrate buffer solution.
(67) (2) 40 inl, of an aqueous 0.1 N hydrochloric acid solution.
(68) (3) 35 ml. of an aqueous 85% acetone solution.
(69) (4) 30 ml. of an aqueous 0.1 N hydrochloric acid solution.
(70) The washed resin bed was eluted with an aqueous 60% dioxane solution containing 0.1 N of hydrochloric acid.
(71) I1he colored portion of the eluate, in the amount of 6 nil., was collected, and subjected to analysis. The results indicated that such eluate portion contained 246 mg. of cyanocobalamin and 5.5 mg. of total solids.
(72) Examlple V5 An aqueous extract of liver tissue, in the amount of 15 ml., containing 213 mcg. of cobalamin and 915 mg. of total solids, was treated by the following method:
(73) This aqueous solution was combined with 200 mg. of potassium cyanide, and after standing at room temperature for a period of 3 hours, was adjusted to pH 4 with citric acid.
(74) Amberlite XE-97 ion exchange resin was classified to a wet particle size of 100-150 mesh. The classified resin, in the amount of 8 ml., was adjusted to pH 4 with a citric acid-sodium citrate buffer solution, and packed into a glass column having a diameter of it mm. and a height of 300 mm. The cyanide-treated aqueous liver solution was infused into the ion exchange bed at a rate of 2 ml. per minute. The effluent was discarded, and the resin bed washed with the following solutions in the specified sequence:
(75) (1) 35 ml. of an aqueous 0.1 N hydrochloric acid solution.
(76) (2) 30 ml. of an aqueous 85% acetone solution.
(77) (3) 25 ml. of an aqueous 0.1 N hydrochloric acid solution.
(78) The washed resin bed was eluted with an aqueous 60% acetone solution containing 0.1 N of hydrochloric acid.
(79) The colored portion of the eluate, in the amount of 16 ml., was collected, and subjected to analysis. The results indicated that such eluate portion contained 200 mc.g. of cyanocobalamin and 7 mg. of total solids.
(80) Example VI The following exemplifies the volume ratios employed in the ion exchange fractionation of vitamin B12 active substances utilizing a cationic exchange resin having a wet particle size of about 100-150 mesh:
(81) The ion exchange resin can be packed into a glass column having a diameter of 1.5 inches and a height of 24 inches. A column having these dimensions may contain about 2500 cc. of the ion exchange resin. An aqueous solution of vitamin B1,, active substances, in the amount of 3 1., containing about 7% of total solids and 2.6 mcg.
(82) of vitamin B,2 active substances per ml., can be infused 4 into the resin bed at a rate of about 6 ml. per minute.
(83) The effluent can be discarded, and the resin bed washed with the following solutions in the specified sequence:
(84) (1) 2 1. of a 0.1 N mineral acid solution.
(85) (2) 1.5 1. of an aqueous 85% acetone solution.
(86) (3) 1.51. of a 0.1 N mineral acid solution.
(87) The washed resin can be eluted with an aqueous 60% dioxane solution containing 0.1 N of mineral acid. The eluate portion in the amount of 250 ml., can he collected for further purification or packaged for commercial use.
(88) To achieve an eluate portion of 250 ml. the total eluant employed may be about 500 ml.
(89) By comparison, Amberlite IRC-50 ion exchange resin has an average wet particle size of from about 20 to 40 mesh, and substantially all of the particles contained therein are larger than 50 mesh.
Read the