72 JOURNAL OF THE AMERICAN PHARMACEUTICAL ASSOCIATIOX Vol. XLVI, No. 1

The radioactivity for the samples varied from 161 to 2101 counts per minute. The cyanocobalamin tracer reagent radioactivity varied from 394 to 4013 counts per minute. The background radioactivity varied from 11 to 171 counts per minute. At least two different types of counting assemblies were used involving Geiger Miiller and Scintillation detectors. Each collaborator selected the magnitude of the radioactivity to give optimum counting accuracy for his assembly. For instance, the collaborator who reported a high background count of 171 counts per minute used counts on the sample and standard generally in excess of 1,000 counts per minute.

Six different lots of MBl Resin (manufactured by Rohm and Haas Co.) were used by the nine collabo- rators.

Each collaborator used a different lot of alumina with two commercial sources being represented.

With Samples No. 1 and No. 2 the sample weights varied from 0.157 to 0.428 Gm. With Samples No. 3 and No. 4 the sample volume varied from 4 to 10 ml .

The weight of the residue of the sample used for the radioactivity counts was reported by one col- laborator to vary from 0.1 to 0 2 mg., and to require no sclf-absorption correction.

COMMENTS BY COLLABORATORS

No collaborator reported any serious difficulties in using the method. Some collaborators reported some mechanical difficulties such as emulsification with Samples No. 3 and No. 4 which required longer time for separation or a second centrifuging. Some collaborators found it necessary to recycle the solu- tions from Samples No. 3 and No. 4. Comments were also received on the wording of the method, and changes were made to improve clarity of ex- pression.

After preliminary results had been received, several collaborators suggested that a standdrdiza-

tion test for tracer purity be included in the method. One collaborator suggested that a tracer with a correction factor in excess of l l O ~ o would not be suitable for use. He stated his correction factor changed by 3.3% over a period of two weeks which he thought was due to decomposition of the tracer solution as prepared in the method. Another col- laborator using the same lot of tracer stored the prepared tracer solution in a refrigerator for three and one-half months, but could not demonstrate any mCdSUrdbk changes in the correction factor. A second lot of tracer solution showed no change after a nine-month period of storage in a refrigerator. With information available from this study and with the standardization of tracer reagent incorpo- rated in the method to serve as a measure of purity, it is anticipated that the radioactive cyanocobalamin solutions to be distributed will require little or no correction.

REFERENCES

(1) “Third Supplement to the Pharmacopeia of the United States,” Fourteenth Revision, Mack Publishing Company, Easton, Pa., 1952.

(2) Boxer, G. E., and Rickards, J. C.. Arch. Biochem., 30, 372(1951).

(3) Rudkin, G. O., Jr., and Taylor, R. L., Anal. Chem., 24, 1155(1952).

(4) Fisher, R. A., J. Agr. Food Chem., 1, 951(1953). ( 5 ) Chaiet, L., Miller, T., and Boley, A. E., ;b id . , 2 ,

711411454) . _ _ \ _ ” __,_ (6) Ford J. E. Brit. J . Nutrition 7 299(1953). (7) Brow’n F. B. Cain J. C. Gakt’ D. E., Smith, E.L..

(8) Krieger, C. H., J . Assoc. O l c . Agri. Chemists, 38, 71 1 (1 95.5)

and Parker, L: F. J.,’Bioch;m. J.,’59, Si(1955).

- - - \ - - - - , -

(9) Mader, W. J., and Johf, R. G., THIS JOURNAL, 44, 577(1955).

(10) Neuss, J. D., Merck & Co., Inc., private communica- tion.

(11) Bacher, F. A., Boley, A. E., and Shonk, C. E., Anal. Chem., 26 , 1146(1954).

(12) VanMelle P. J. THIS JOURNAL 45, 26(1956). (13) “First Suipleme‘nt t o the Pharmkopeia of the United

States, Fifteenth Revision,” Mack Publishing Company, Easton, Pa., 1956

(14) “Seco:? Interim Revision Announcement, National Formulary X

(15) Rosenblum. C., Merck & Co.. Inc.. private com- THIS JOURNAL, 45, No. 10, p. vii, 1956.

munication.

Notes

A Note on a New Method of Preparation of p-Aminocinnamic Acid* By JAMES J. CARROLL? and W. LEWIS NOBLES

HE PRIMARY METHODS reported in the literature dealing with the preparation of certain vinylogs of T (1-5) for the synthesis of p-aminocinnamic acid folk acid, a relatively large quantity of this acid was involve the reduction of the corresponding nitro desired. Since the methods described in the derivative or its esters. literature, based on the nitration of cinnamic acid,

involve isomer formation resulting in a subse- In pursuing a program

* Received October 1, 1956, from the University of Missis. quent loss Of large amounts Of starting a’ sippi School of Pharmacy, University, Miss.

wich, N. Y.

the undesired isomer, o-nitrocinnamic acid, a dif-

Also, as Bender (3) pointed out, some of the re- Aided by a grant from the American Cancer Society. ferent synthetic approach was felt desirable. t Present address: Norwich Pharmacal Company, Nor-

January 1957 SCIENTIFIC EDITION 73

duction procedures in the methods reported are quite troublesome.

Other substituted cinnamic acids have been pre- pared by a modification of the Doebner reaction (6), utilizing malonic acid in the presence of pyri- dine(7).

Applying this procedure in the present case, p-acetamidobenzaldehyde was condensed with ma- lonic acid, as described below, to produce a 92% yield of p-acetamidocinnamic acid. This was sub- sequently hydrolyzed to afford an 85% yield (based on starting aldehyde) of p-aminocinnamic acid.

EXPERIMENTAL

p-AcetamidocinnamicAcid.-The method of Blake, et al. (7), was utilized with some modification. In a 1-liter, three-neck flask equipped with a stirrer, thermometer, and a reflux condenser, are mixed 81.6 Gm. (0.5 mole) of p-acetamidobenzaldehyde, 106.1 Gm. (1 mole) of malonic acid and 400 ml. of pyridine (reagent grade). Piperidine ( 5 ml.) was then added. The temperature of the reaction mix- ture was raised to 85” over a two hour period and held at that temperature for an additional five hours, or until the evolution of carbon dioxide ceased. The clear, light yellow solution was cooled to 25‘, and cautiously acidified with concentrated hydrochloric acid. During this time, the tempera- ture was maintained a t 40” or slightly below. The yellow product was removed from solution by filtration and washed with glacial acetic acid, fol-

lowed by several washings with water. The pale yellow solid, m.p. 260-262’ (reported (8) 259-260’) was obtained in a yield of 94.1 Gm. (920/,).

p-Aminocinnamic Acid.-In a 3-liter, three-neck flask, fitted with a stirrer and thermometer, were placed 1500 ml. of a 15% aqueous sodium hydroxide solution and 94.1 Gm. (0.46 mole) of p-acetami- docinnamic acid. The mixture was slowly heated to a temperature of 85” during a period of one hour and then this temperature was maintained for three additional hours, by which time solution had been effected. The clear, yellow solution was cooled to 25’ and adjusted to a pH of 5 with glacial acetic acid. The resulting yellow solid was filtered off and air dried. The product thus obtained, 67.7 Gm. (92%), melted with decomposition a t 173- 175’ (reported 175-176’ (8)). The product was recrystallized from water and dilute ethanol. This treatment did not, however, produce a change in the melting point.

REFERENCES (1) Tiemann, F., and Opperman, J., Bcr., (2) Gabriel, S., ibid., 15 2299(1882) (3) Bender G . ibid. 14 ’ 2360(1881).’ (4) Miller, ‘W. b., anh Kiikelin, F., ibid., (5) Wollring, F . , ibid., 47, 114(1914). (6) Doehner, O., Ann. . 242, 265(1887). (7) Koo, J., Fish,,,M. S., Walker, G. N.

“Oraanic Syntheses, Vol. 31. Tohn Wiley

13. 2056(1880).

18, 3234(1885).

, and Blake, J., and Sons, New

Yo&, 1951; p. 35. eilbron I. Editor “Dictionary of Organic Com-

p&%~”Vol . i, O’xford Uhiversity Press, New York, 1953, p. 79.

A Note on the Determination of Ionic Copper Present in Commercially Available Copper Chlorophyllins”

By BERNARD M. BLANK and KENNETH MORGAREIDGE

N A RECENT NOTE to the editor, Wuggatzer and I Christian (1) in describing a polarographic pro- cedure for the determination of ionic copper present in commercially available copper chlorophyllins, made reference to unrevealed chemical procedures employed by manufacturers of water-soluble copper chlorophyllins. This communication will present the chemical method currently employed for the determination of ionic copper (called “free copper” by the trade) in these water-soluble copper chloro- phyllins. This procedure was adopted in 1953 and has been in use in these laboratories since that date.

The readiness with which magnesium is replaced by copper and other metallic ions in the chelate position of chlorophyll derivatives is well known. Furthermore, the fact that copper is removed with difficulty from this position has been, in the past, the basis for the conclusion that this metal, as it oc- curs in the sodium and/or potassium copper chloro- phyllin, is relatively unreactive. This is undoubt-

edly true in the case of the pure compound, Since, however, commercially available “coppered” chloro- phyllins are mixtures consisting of several derivatives and degradation products, it became of interest to determine the stability of the copper complex in commercial preparations under standardized con- ditions.

EXPERIMENTAL

The empirically devised method is as follows: An accurately weighed quantity (about 1.0 Gm.) is transferred to a beaker and 75 ml. of distilled water is added. Agitation by a motor-driven glass stirrer is maintained until solution is complete (about 3 minutes). From a buret, 1.0 N HCl is added slowly with continuous stirring until a pH of 3.0 is reached (as determined by pH meter). As the pH approaches 3, proceed with caution. Do not overshoot and back titrate with alkali. Suf- ficient time is allowed for electrolyte equilibrium to be established between additions of acid. Vigor- ous agitation is maintained during the addition of * Received August 21, 1956, from Food Research Labora-

tories, Inc., Long Island City, New York.