Germicidal Quaternary Ammonium Salts in Dilute Solution A Colorimetric Assay Method M. E. AUERBACH, Research Laboratories, Winthrop Chemical Company, Inc., Rensselaer, N. Y. A colorimetric method for the estimation of
tension of the general method has been suggested for the estimation of alkaloids (cations) with bromothymol blue as dye anion, and for the estimation of some colored sulfonic acids (anions) with quaternary ammonium cations.
some germicidal quaternary compounds in dilute solutions is, for practical purposes, specific in that inorganic alkalies and the common primary, secondary, and tertiary amines do not interfere. Therefore an ex-
T4,
HE quaternary ammonium compounds which are now
alkyl benzyl dimethylammonium chloride (referred to becoming increasingly useful as antiseptics and germicides as Z): (1, 6,6, 8,11, 12, IS) are of such potency that solutions CnHan + 1 CHI more concentrated than 1 to 500 are seldom used. Quite \/ properly, these compounds are usually standardized on the N-Cl basis of their phenol coefficient, or actual bactericidal efficiency. Several years ago, however, this laboratory was given the problem of devising a chemical assay method sufficiently simple for field use. Such a method was worked in which CnHZn+lrepresents a mixture of alkyl groups ranging out, and has proved to be of value not merely in the field, from C8H17 to C18Hs7. I n spite of the apparently indetermibut also (with suitable refinements) as a laboratory control nate constitution of this substance, the average molecular test. weight, because of rigidly standardized manufacturing conThe quaternary ammonium salts named below form colored ditions, is remarkably constant, being about 357.5. The salts with dibromothymolsulfonphthalein (bromothymol molecular weight was determined by preparing the ferriblue) and with tetrabromophenolsulfonphthalein (bromocyanide salt R3Fe(CN)8(R = quaternary ammonium cation), phenol blue). These salts are readily extracted from alkaline igniting a portion of the salt to Fe203,and calculating back aqueous solutions by a number of organic solvents, particuto the value of R. larly the chlorinated solvents. Bromophenol blue, which the the author Drefers to use. is itself insoluble in ethylene diStandard Solution chloride (for-example), either in its acid form or as thk sodium Titrate a 1o per cent solution of by the ferricyanide method salt; nor does it form salts extractable from alkaline solution From this standard prepare a working dilution containing with any of a large number of primary, secondary, and tertiary 0.1 mg. per ml. In working with other quaternary salts, a standard solution amines a t the author's disposal, or with alkaloids. It seems the compound in question might be used in place of a standard probable that all cations of the composition [ R ~ R ~ R ~ R ~ NofI + Z solution. The discussion below will apply equally to the new Or longer chained where R1, Rz, and R3 are standard, necessitating only the obvious changes resulting from a and RI is CH2, C,Hs, or longer chained aryl-alkyl, will different molecular weight. form salts with bromophenol blue which can be extracted from Method alkaline solution. It is not claimed that only quaternary Dilute the 1 to 1000 tinted tincture exactly ten times with disamines respond to this but it k true that Of 50 Or 6o tilledsalt) water. Take 2 ml. of the dilution (about 0.2 mg. of quaternonquaternary amines, all gave negative tests. These nary in a 125-ml. glass-stoppered Erlenmeyer flmk. Add 2 negative compounds include such typical amines as ethylml. of 10 per cent sodium carbonate solution and 1 ml. of 0.04 amine, dimethylamine, diethanolamine, diethylaminoethanol, per cent aqueous bromophenol blue indicator solution, dilute with phenylenediamine, and ethylenediamine. It is about 50 ml. of distilled water, add exactly 10 ml. of ethylene dichloride, stopper the flask, and shake gently for one minute. especially significant that lauryl dimethylamine, which can One gram-molecule of dye reacts with 2 gram-molecules of quaterbe considered a possible impurity in alkyl benzyl dimethyl nary salt in a manner analogous to the normal salt formation of the sulfonphthaleins(2): ammonium chloride, gives a negative test. The method consists of forming the quaternary ammonium-dye salt in carbonate solution, extracting the
(z>
color with ethylene photoelectric colorimeter dichloride, the intensity and measuring of color soinexa tracted. By means of a factor previously derived from a standard solution, the concentration of quaternary ammonium salt in the sample is readily calculated. To illustrate the method under discussion, there is described below the assay of a commercial tinted a l c o h o l - a c e t o n e s o l u t i o n c o n t a i n i n g 1 t o 1000
/ e O H
+ 2R.Cl -+
C
, e O R
'SO,
R 492
E
quaternary ammonium cation
Br
August 15, 1943
ANALYTICAL EDITION
TABLE 1.
AmAY OF
2 in Sample
AMMONIUM-DYE SALT Instrument Reading
493
gram per liter found. A tinted tincture labeled 1 to 500 assayed 1.997 grams per liter, using the same factor. Similarly, for cetyl pyridinium chloride (Id,18):
c1
MU. 0.05
0.10
0.20 0.30 0.40 0.50
38 77 152
\
230
ClcH83
300
376
To samples containing more than 0.3 mg. of 2, an extra ml. of indicator, solution was added.
That is, 357.5 mg. of 2 react with 335 mg. of bromophenol blue, or 1 mg. of 2 requires 0.94 mg. of bromo henol blue. Further, a linear relationship holds throu hout a sesirable range of concentrations, as demonstrated in f’able I. When the layers have separated, aspirate off the aqueous layer, transfer the colored ethylene dichloride layer t o a dry test tube, add 0.5 to 1 gram of dry sodium sulfate, and mix by gentle tion. Decant the clarified colored solution into a Kletb%%% mermn photocolorimeter tube and read in the instrument, using filter No. 54, or equivalent instrument and filter. In the same way run the prepared standard dilution.
Use of the Calculation Factor When colored solutions, which follow Beer’s law, are read in a photoelectric colorimeter provided with a logarithmic scale, it is true that RS x K = CS There RS = instrument reading of standard CS = concentration of standard K = a constant In the case of a 1 to 1000 standard solutio11 assayed as described above, the colored standard solution read in the instrument contained the equivalent of 0.2 mg. of original quaternary salt per 10 m1.-i. e., 0.002 per cent. The actual reading on the author’s instrument \\as 152. Then K = O Z 2 = 0,0000131, or, to put it other\\-ise, the instrument 152 reading X 0.00131 = mg. of anhydrous quaternary salt in the
2-ml. dilution taken for analysis, This factor, naturally, is valid for only one particular instrument and filter combination. Once the factor has been determined, it becomes unnecessary to prepare further standard solutions. One simply makes a convenient dilution of the unknown and takes a sample containing about 0.2 mg. of substance for test. The concentration is then calculated from the instrument reading and K , as indicated. Further, since the formation of the colored salt is a stoichiometric relationship, and since the spectral characteristics of the color have to do only with the dye anion and are independent of the particular quaternary cation in use, it is unnecessary to prepare standard solutions of new quaternary compounds, so long as the molecular weight of the new compound is known. It is only necessary to recalculate K to fit the new molecular weight. For example, when solutions of p-tert-octyl phenyl diethoxy dimethyl benzyl ammonium chloride monohydrate (11) : (cH3)2 C ~ H ~ , ~ O C H ~ C H ~ O C /IH ~ C H H ~~ O N-C~.
became available, K was recalculated as follows: KS - (for . new substance) = K I ( Z ) X 465.5 (molecular’weightof new substance) 357.5 (molecular weight of Z) KS 0.00131 X 1.302 0.0017 Using the new factor, K Z = 0.00170, an aqueous solution of the new compound labeled 1 to 1000 was assayed and 0.998
Ka = 0.00131 X
% : -- 0.00124
Using the factor Kt = 0.00124, a tinted tincture labeled 1 to 4000 assayed 0.220 gram of anhydrous cetyl pyridinium chloride per liter. Again, for 1,3-di-n-octylbenztriazolium bromide (6): C&
I
I
LAN/ 424 Ka was calculated as 0.00131 x __ = 0.00155. 357.5 Using the factor K , = 0.00155, a solution of the pure substance made up in a concentration of 1 to 1000 assayed 1.020 grams per liter.
Possible Analytical Uses of General Method Alkyl benzyl dimethyl ammonium chloride (and other quaternary salts, no doubt) forms salts with some sulfonic acid derivatives, the salts being extractable from water solution. For example, sulfanilyl azo acetyl 2R acid (Keoprontosil) is very soluble in water. On addition of the quaternary compound, the resultant salt can be extracted quantitatively with a number of water-immiscible solvents. Under the specified test conditions (more specifically, in alkaline medium) the dye salts of alkaloids are not extractable. However, these salts become extractable from acid solutions, and useful methods can be developed for the determination of minute amounts of various alkaloids in solution. I n fact, such a method has already been found useful for the determination in urine of an alkaloidlike synthetic drug, the ethyl ester of methyl phenyl piperidine carboxylic acid ( 7 ) . I n this laboratory very small amounts of strychnine have been determined in a complex solution by extracting it as the yellow bromothymol blue salt in the presence of acetate buffer pH 5.0, using toluene as solvent. A somewhat similar method was described several years ago for quinine, using eosin as the dye anion (9, 10).
Lit erature Cited Barnes, J. M., Lancet, 242, 531 (1942). Clark, W. M., “Hydrogen Ions”, 2nd ed., p. 61, Baltimore, Williams & Wilkins Co., 1927. Council on Pharmacy and Chemistry, Am. Med. Assoc., “New and Non-Official Remedies”, Supplement, p. 8, 1942. Domagk, G., Deut. med. wochschr., 61,829 (1935). Joslin, D. A., Yaw, K., and Rawlins, A. L., J . Am. P h m . Assoc., 32, 49 (1943). Kuhn, R., and Westphal, O., Ber., 73, 1109 (1940). Lehman, R. A., and ilitken, M. S., J . Lab. Clin. Med., 28, 787 (1943). Miller, B. F., and Baker, Z . , Science, 91, 624 (1940). Prudhomme, R.O.,Bull. S O C . path. ezotique, 31, 929 (1938). Prudhomme, R.0.. J . pharm. chim., 1, 8 (1940). Rawlins, A. L.,Sweet, L. -4., and Joslin, D. A., J . A m . Pharm. Assoc., 32, 16 (1943). Shelton, R . S., “Investigation of the Alkyl Pyridinium Salts”, presented a t Cincinnati Meeting of AMERICANCHEMICAL SOCIETY, 1940. Warren, M. R., et al., J . Pharm., 74,40 (1942).
