AGRICULTURAL A N D FOOD CHEMISTRY

DIVISION O F AGRICULTURAL A N D FOOD CHEM8STRY

B12 Symposium Emphasizes Need For More Research in the Field

• Fermentation program is dedicated to Prof. W . H. Peterson of Wisconsin

• Cooperation of industry on fundamental re­search on antibiotics urged by academician

ΤΓΉΕ chemistry and functions of vitamin •*- B12 and the numerous vitamin Be­

like compounds appear to offer a most fertile territory for some intensive and fun­damental research effort. Judging from the tone of prepared papers and presented questions, one of the most urgent needs is for a completely satisfactory method of assay. These points were discussed in a special symposium on Β\2 presented before the Division of Agricultural and Food Chemistry.

A revie^w of the current status of the knowledge of the vitamin was outlined by Edward A. Kaczka, Merck & Co. Vita­min Bia has an empirical formula which approaches Cei-wHse-wN^OwPCo, giving a molecular weight of approximately 1300, Kaczka said.

Microbiological activity, according to Kaczka, is not good as a method of dif­ferentiating between B12 and Bis*. The presence of the cyano group is the best criterion for the confirmation of Bis, he said. I n the case of B^», the cyano has been replaced by a hydroxo group. Kaczka discussed the nomenclature which has been adopted explaining that the term "cobalamin" is used to designate all of the vitamin Β12 molecule except the cyano group. Prefixes are supplied to this root to indicate the group in the cyano posi­tion. For example, B12 is called cyanoco-balamin and Biaa is called hydroxocobala-min. This hydroxocobalamin has been sho-wn to react with the chloride, bromide, nitrite, and cyanate ions* to form the corre­sponding cobalamins, he said.

The u s e of sodium cyanide in the micro­biological assay procedure was described in a paper by Helen R. Skeggs, Charlotte A. Driscoll, Jesse Charney, and Lemuel Wright, Sharp & Dohme, Inc. In her com­parison of the stability of pure vitamin B12 with the stability of the crude samples, Dr. Skeggs pointed out that treatment with reducing agents increases the stability of the crude to a greater degree. How­ever, a pretreatment "with cyanide elimi­nates the need for a reducing agent, she said.

T h e Sharp & Dohme group reported that the pretreatment of a variety of ma­terials "with cyanide increases the apparent BM content as deterniined microbiologi-cally. They concluded that this pretreat­ment w i t h cyanide converted naturally oc­

curring substituted forms of the vitamin to the relatively heat stable cyanocobala-min complex. Dr. Skeggs emphasized the need for further study to establish a quan­titative relationship for this conversion.

Improvements in a plate assay method for vitamin B12 which had been described previously were reported in a paper by I. Roy Cohen, J. M. Pensack, and R. E. Bennett, Commercial Solvents Corp. T h e medium which is used is a modification of that suggested by Skeggs and her co­workers for the tube assay. According to Mr. Cohen, additions are made consisting of small amounts of thioglycollic acid, potassium sulfate, and ribonucleic acid.

During the round-table discussion which followed these first papers in the sym­posium, the question of assay methods seemed to be of general interest. Mr. Cohen remarked that a clear perspective must be maintained and that the objective is not necessarily a method which gives the highest assay, but rather one wHich will give a value comparable to the true growth promoting activity of the sub­stance.

Soil samples which had been brought to this country from Japan before the last war were the source of an organism which has been found to produce vitamin B12. Harlow H. Hall, J. C. Benjamin, C . F.

Wiesen, and Η . Μ. Tsuchiya, Northern Regional Research Laboratory, found that Streptomyces olix>aceusy produced the vita­min in yields as high as 2.0 micrograms per milliliter. Growth was in submerged aerated culture media and the fermenta­tion was carried out in 2 0 liter stainless steel fermentors, Dr. Hall explained. An unidentified antibiotic effective against Gram-positive bacteria is also produced in the liquid culture, he said. Work is being continued in an effort to identify the anti­biotic.

The importance of trace amounts of co­balt to the vitamin B12 fermentation was indicated in a report by J. C. Garey, J. F. Downing, Pennsylvania State College, and W. H. Stark, Vulcan Copper and Supply Co. Using an organism which, to date, has been identified only as a species of Streptomyces they produced Bu» in syn­thetic culture media and in media supple­mented with natural materials such as ani­mal stick liquor, distillers* solubles, corn steep liquor, and soybean oil meal. In order to obtain respectable yields, ac­cording to Garey, it was necessary to pro­vide the cobalt ion in concentrations as high as δ parts per million. However, a certain amount of toxicity from the cobalt was observed and it was necessary to make the additions on an incremental basis, he said. The additions, in the form of the cobalt chloride, were started after the first few hours of the fermentation, Garey ex­plained.

• F e r m e n t a t i o n P r o g r a m A full day's program of fermentation

papers was dedicated to W . H. Peterson, University of Wisconsin. In his opening remarks, Robert D . Coghill, Abbott Lab­oratories, pointed out that with only one or two exceptions, all of the papers had as one of its authors a former student or associate of Professor Peterson. "The program—more than anything I can say—

A. F. Langlykke, E . R. Squibb & Sons; W. H. Peterson, University of Wis­consin, chairman of the Fermentation Subdivision; and R. D . Coghill, Abbott Laboratories, get together for a discussion of the papers on fermentation

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119TH NATIONAL ACS MEETING

reveals the breadth of his interest and the thoroughness of his teaching," Dr. Coghill said.

A soil sample from the back yard of the lulxmitory provided an organism which produced the antibiotic, hydroxystrepto-mycin, studied by workers at Abbott Lab­oratories. Merlin H. Peterson described the scale-up operations from shake flasks to 30-liter and then to 400-gallon stirred fermentors. A relatively simple medium containing soybean meal, Oerelose, sodium chloride, and calcium carbonate was found to give good yields of hydroxystreptomy-cin, Peterson said. Sugar utilization and loss in mycelium volume follows in the same pattern as observed in streptomycin fermentations, he said. Marjorie E. Hanes and John Sylvester collaborated on the investigations.

The effects of various types of dilution water in the operation of pilot-plant vine­gar generators were outlined by R. J. Alîgeier. R. T. Wisthoff. and F. M. Hildc-brandt, U. S. Industrial Chemicals. Dr. Alîgeier explained how water samples had K m collected from all parts of the coun­try and used for extended tests in the laboratory generators. Although conclu­sive evidence is still lacking, there are in­dications that better vinegar yields are obtained from treated waters and that water from the eastern seaboard areas is slightly better for the fermentation, Alî­geier said. He stressed the need for fur­ther investigations to bring out the reasons for the variations observed.

Fundamental Research On Antibiotics Urged

Pointing out that industrial organiza­tions are too apt to discard an antibiotic if it does not show immediate promise, H. E. Carter, University of Illinois, urged a closer cooperation between universities and industry. He said that the universi­ties are willing to take over the funda­mental type of research required to obtain more information relating structure to anti­biotic activity. These were the conclud­ing remarks on his paper describing re­search on the chemistry of streptothricin.

DIVISION O F ANALYTICAL CHEMISTRY

Div is ional Repor ts

Agr i cu l t u ra l and Food Chemis t ry

Ana l y t i ca l Chemist ry B io log ica l Chemist ry C o l l o i d Chemis t ry Gas and Fuel Chemist ry . . H i g h Polymer Chemist ry Industr ia l and Engineering

Chemis t ry M e d i c i n a l Chemis t ry . . . .

1 O r g a n i c Chemist ry Petro leum Chemis t ry . . . .

1 Physical and Inorganic 1 Chemis t ry 1 W a t e r , Sewage, and Sani-1 t a t i o n Chemis t ry

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Uses of Nonaqueous Titrations Broader Now Than Those in Water

• High-frequency methods create great interest as a new analytical tool

• Procedures in nitrogen and sulfur in steel de­terminations varied to meet specific needs of analyst

*TpHE field of titrations in nonaqueous •*• solvents is now even broader in scope

than that of titrations performed in water, believes John A. Riddiclc, head of the ana­lytical research division of Commercial Solvents Corp. Reviewing developments in this field during a round tahle discus­sion at sessions of the Division of Ana­lytical Chemistry, Dr. Riddiclc said he thought that, in view o f its advantages, the method was used by too few labora­tories.

He said that the technique was being used successfully in Commercial Solvents' control labs and that in some cases it had reduced costs from between $5.00 and $15 to as low as 4 0 cents per run. The rea­sons for this amazing reduction, he indi­cated, were the method's speed, accuracy, and the elimination of high-cost equip­ment necessary previously.

James S. Fritz of Wayne University dis­cussed the solvents and titrants useful in nonaqueous titrations ancl proposed several which he thought were best for certain types of compounds to b e analyzed. The best all around solvent for titrating acids right now, he thinks, is dimethylform-amide or DMF, because it is cheap and easy to handle.

For redox titrations, Dr . Fritz suggested carbon tetrachloride, acetic acid, iodine in methanol, and tetrahydrofuran. As titrants he mentioned particularly sodium ethoxide in ethanol, benzene and methanol in a six-to-one proportion, triphenylmethyl-sodium, and pyrrole ( wnich acts as its own indicator) in toluene or toluene-ether.

The problem of indicators themselves was taken up separately by Marion Mac-Lean-Davis of the National Bureau of Standards. Dr. Davis discussed both indicator dyes and electrodes. She and several other members of the panel seemed to be of the opinion that dyes were very important in this work, be­cause they necessitate less complicated apparatus and are in some cases just as accurate as potentiometric methods.

She said that in going over a list of the known indicator dyes she had found that over a third of them are of the triphenyl-methane type. Another third fall into the azo type. She provided slides with the

structural formulas of many of these types and the section of the molecule which is thought to provide the characteristic color was tinted appropriately on the slide. She said she hopes the idea that ions must be present for titration will be dispelled "once and for all."

Method Used in Drug Analysis

A paper which preceded the round table offered a method of analyzing many physiologically active compounds by ti­trations in solvents other than water-Charles W. Pifer and Ernest G. Wollish, working at Hoffmann-LaRoche, developed the technique which is applicable to salts of amines, basic heterocyclic compounds, and quaternary ammonium compounds, which includes many antihistamines, vita­mins, and alkaloids.

The procedure involves the titration of the salt in question with perchloric acid in dioxane as the titrant and glacial acetic acid as the solvent. Mercuric acetate is used to bind the halogen anions if the salt is one of an inorganic halide acid. Dr. Pifer said that depending upon the sharp­ness of the potential peak, precision of zfcO.2% can be obtained for some com­pounds.

Dr. Fritz, who delivered two papers during the afternoon, as well as participat­ing in the round table, described a method of titrating sulfa drugs using 0.1 M sodium methoxide in benzene-methanol as the titrant.

Other members of the panel discussion were E. F. Hillenbrand, Jr., Carbide & Carbon, who talked on applications, and P. C. Markunas, Commercial Solvents, who discussed accuracy and precision of the method.

• Chemical Analysis by High Frequency Methods

A subject of high interest, but still too new for symposium treatment was made the subject of a round-table discussion by the Division of Analytical Chemistry— namely, chemical analysis by high fre­quency methods. Chairman W. J. Blaedel, University of Wisconsin, in introducing the

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