1022
INDUSTRIAL AND ENGINEERING CHEMISTRY
There is evidence that the beer protein and carbohydrate components react with one another in solution. Figure 10 shows the effects of concentration on electrophoresis of whole beer protein (containing some carbohydrate). At low concentration (0.25 grams per 100 ml.) the beta peak was large, the alpha peak relatively small. At higher concentrations, the alpha peak increased rapidly in size and became relatively large compared with the beta peak. The alpha peak also increased in area relative to the beta a t low ionic strength (ionic strength = 0.02). Figure 11 shows the foaming properties of the various fractions in water (all concentrations were 0.02%). The first photograph was taken immediately after the solutions were given uniform shakings. Fractions I, IV, and V produced the most foam, fraction I11 the least. The lower photograph was taken 12 minutes later, with no further agitation of the solution. The foam on Fractions I1 and IIA persisted, while the foam on Fraction V collapsed almost completely. Fractions I and IV lost a large proportion of their originally high foam. SUMMARY
The proteins of beer have been isolated by ammonium sulfate precipitation follon~edby selective peptization of the precipitated materials. The water-soluble proteins have been separated into six fractions by alcohol precipitation from solutions at controlled temperature, pH, and ionic strength. Alcohol-soluble and alkalisoluble fractions have also peen obtained by peptization in the appropriate solvents. Progress of separation of the various constituents has been followed by means of electrophoresis and ultracentrifugation, and estimates of molecular weight have been obtained from osmotic pressure data.
Vol. 45, No. 5
Some carbohydrate material is carried along with the proteins and is concentrated in certain of the water-soluble fractions. A method for removing proteins and fractionating the residual polysaccharides has also been worked out. The compositions and properties of the protein preparations differ markedly. ACKNOWLEDGMENT
The authors wish to thank Frank Heckler, Rosalind Dean, Donald E. Heitmeier, and W. A. ITeber for assistance in various phases of the work. LITERATURE CITED
(1) Biserte, G., and Scriban, R., Brasserie, 5, 348 (1950). (2) Bishop, L. R., “Progress in Brewing Science,” Vol. 11, p. 244,
Ken. York, Elsevier Publishing Co., 1949.
( 3 ) Cohn, E. J., Strong, L. E., Hughes, W. L., Jr., Mulford, D. J.,
Ashworth, J. N., Melin, M . , and Taylor, H. L., J.Am. Chem. Sac., 68,459 (1946). (4) Fisher, R. A , , “Statistical Methods for Research Workers,” 8th ed., p. 153, Edinburgh, England, Oliver and Boyd, 1941. ( 5 ) Fuoss, R. M., and Mead, D. J , J . Phus. Chem., 47.59 (1943). (6) Hartong, B. D., Mastenbroek, G. G. A,, and Mendlik, F., “Progress in Brewing Science,” Vol. 11, p. 250, Ne%,York, Elsevier Publishing Co., 1949. (7) Lundin, H., Ibid., p. 229. (8) Quensel, O., “Untersuohungen Oeber die Gerstenglobuline,” Uppsala, Inaugural-Dissertation, 1942. (9) St.Johnston, J. H., J . I n s t . Brewing, 54, 305 (1948). (10) Sandegren, E., “Progress in Brewing Science,” Vol. 11, p. 78, New York, Elsevier Publishing Co., 1949. RECEIVED for review August 7, 1952. ACCEPTED FEBRUARY 4 , 1953. Presented before the Fermentation Subdivision of the Division of Agricultural and Food Chemistry at the 121st Meeting of the AMERICAN CHEMICAL SOCIETY,Milwaukee, Wis.
Antimicro ial Activity of uaternary Ammonium Chlori J
DERIVED FROM COMMERCIAL FATTY ACIDS R. A. RECB AND H. J. HARWQOD Research Division, Armour and Co., Chicago, I l l .
I
NCREASING utilization of higher aliphatic quaternary
ammonium salts as germicides and antiseptics has emphasized the need for a careful scrutiny of the economics of their manufacture and use. Two important factors must be considered with respect t o individuaI compounds-cost of manufacture and biological activity. Considering commercial fatty acids as the primary raw material, two major processes are applied to the production of quaternary ammonium salts. These processes are represented by the following series of reactions, in which palmitic acid is shown as the starting material. Process I (opposite) is .P four-step process and is especially useful for the introduction of substituent groups, such RS benzyl, which differ from those contained in the tertiary amine. Process I1 is a three-step process and of the two is more economical. I t can, however, be applied only to the manufacture of quaternary ammonium salts in which three of the substituent groups are methyl. A survey of the literature in the field ( 5 )reveals a large amount of work relating to the germicidal activity of quaternary- ammonium salts. A preponderance of this work pertains to compounds which contain aromatic nuclei, usually the benzyl group. The
inference might be drawn that the benzyl group is essential to high germicidal activity. Recent work in this laboratory has indioated that proximity of the phenyl group to the quaternary nitrogen actually results in decreased bactericidal activity ( 2 ) .
L
1 CH,
1
INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
May 1953
I n view of the lower costs involved in the manufacture of trimethylalkylammonium chlorides by Process 11i t became necessary t o compare the economics and the biological activities of several compounds produced by this process (in which commercial fatty acids were the primary starting material) among themselves and with the widely used benzyl-type salt derived from coconut fatty acids. The primary objective of the present work was to establish which quaternary ammonium salt, on the basis of antimicrobial activity, physical properties, and commercial availability of raw materials, was most suitable for commercial development. Factors considered were degree of unsaturation and alkyl-chain mixtures, Trimethylalkyl quaternary ammonium salts containing the mixed alkyl groups derived from coconut oil, tallow, and soybean oil were evaluated, as well as the benzyl-type salt derived from coconut oil. The trimethylalkyl quaternary salts prepared from commercial palmitic and stearic acids were also employed in a portion of the work. Preparative procedures reported are not necessarily those employed in commercial-scale production. PRFPARATION O F QUATERNARY AMMONIUM SALTS
QUATERNARYAMMONIUMSALT MIXTURES OF VARYING UNSATURATION.Samples of Arquad 18 and Arquad S (products of Armour and Co., Chemical Division) consisting of 33% solutions of trimethylalkylammonium chlorides in isopropyl alcohol were concentrated by warming under reduced pressure. The resulting quaternary ammonium salts possessed iodine values of 2.5 ( I ) and 55.8 (VII), respectively (see Table I). Mixtures of intermediate iodine value were prepared (I1 t o VI). Sample VIII, iodine value 79.0, was prepared from Armeen S (soya amine, iodine value 91) as follows. A mixture of 13.5 grams of Armeen S, 26.6 grams of methyl chloride, 16 grams of sodium bicarbonate, and 100 ml. of isopropyl alcohol was heated a t 125" C. in a rocking autoclave for 3 hours. After cooling and venting, the contents of the autoclave were filtered and the solvent was removed under reduced pressure. The quaternary ammonium salt was obtained as a viscous sirup. The yield was quantitative.
TABLEI. IODINE VALUESOF QUATERNARY AMMONIUM SALTS Iodine Value
Mixture
E,
Arquad 18 (commercial)
2.2
QUATERNARY AMMONIUM SALTS DERIVEDFROM PALMITIC, STEARIC,TALLOW, AND COCONUT FATTY ACIDS. Commercial nitriles (Arneels 16, 18, T, and C ) were hydrogenated t o the corresponding primary amines using Raney nickel catalyst. After distillation, each primary amine was converted to the corresponding dimethyl derivative by means of formic acid and formaldehyde (6). Treatment of the tertiary amine with methyl chloride in an autoclave a t 125' C. yielded the trimethylalkylammonium salt. DIMETHYLBENZYL QUATERNARY AMMONIUM SALT DERIVED FROM COCONUT FATTY ACIDS. Dimethyl coco amine prepared as described above was heated with benzyl chloride (10% excess) for 2 hours a t 120 C. Volatile impurities were removed under reduced pressure. ANALYSISOF QUATERNARY AMMONIUM SALTS. All salts were analyzed through titration of the dichlorofluorescein complex with standard sodium dodecyl sulfate solution. A purity of not less than 98% was indicated for all preparations. O
1023
DETERMINATION OF MICROBICIDAL ACTIVITY
This phase of the present investigation had two objectives: to establish the influence of unsaturation in the higher aliphatic chain upon the microbicidal activity of quaternary ammonium salts, and t o compare the activity of salts derived from commercially available fatty acids and fatty acid mixtures. DETERMINATION OF BACTERICIDAL ACTIVITY. The procedure followed in determining the bactericidal activity of the mixtures of varying unsaturation listed in Table I was a modification of the Weber and Black method (7). Initially, the attempt was made to define toxicity curves for each preparation by plotting numbers of surviving bacteria (Micrococcus pyogenes var. aureus) after exposure t o a 1 t o 120,000 concentration of the test material for several different lengths of time. Owing t o the high degree of variability in the results, it was not possible t o reproduce the configuration of the curves satisfactorily. It was, therefore, decided t o simplify the procedure by making counts a t only two time intervals but t o run enough replicates BO that significance could be attained. One series of replicate tests in which M . pyogenes was again employed proved to be insufficient alone for this purpose. A second series of tests was then made with Escherichia coli as the test organism. The results reported here are based mainly on these two sets of replicate data. BACTERIOLOGICAL PROCEDURE. A 24-hour growth of the test organism on an agar slant was washed off with sterile water. The resulting bacterial suspension was poured into a 99-ml. dilution blank and thoroughly shaken, and 5 mi. of this suspension was then pipetted into a sterile medication tube and warmed to 37' C. in a water bath. Five milliliters of quaternary ammonium salt solution (previously diluted to 1 t o 60,000 in the case of M . pyogenes and to 1 to 20,000, 1 to 25,000, or 1 t o 30,000 in the case of E. coli) was warmed to 37 C. and then rapidly pipetted into the medication tube and mixed with the bacterial suspension. After the selected intervals of time had elapsed ( 1 and 2 minutes in the case of M . pyogenes, 30 seconds and 1 minute or 1 and 2 minutes in the case of E. coli), 1.0 ml. of the quaternary-bacteria mixture was removed from the medication tube and combined with 9.0 ml. of a O.k% solution of naphuride sodium t o neutralize the quaternary ammonium salt. From this solution 0.1-ml. and 1.0-ml. quantities were plated onto glucose-tryptone extractc agar, then incubated for 2 days a t 37" C. The numbers of organisms introduced into the medication tubes were estimated by plating appropriate dilutions of the untreated bacterial suspensions. I n the series of tests comparing the activity of the following compounds three species of bacteria were employed, M . pyogenes, E. coli, and Bacillus subtilis, and a yeast, Torula cremoris. A slightly modifkd procedure was used. O
Trimethylhexadecylammonium chloride Trimethyloctadecylammonium chloride Trimethyl coco ammonium chloride Trimethyl tallow ammonium chloride Trimethyl soya ammonium chloride (sample VII) Dimethylbenzyl coco ammonium chloride
T o 5 ml. of an aqueous suspension of the above-named organisms, an equal volume of a dilution of one of the seven quaternary ammonium compounds was added. Both the bacterial suspensions previously prepared from 24-hour agar slants ( B . subtilis was grown for 5 days) and the quaternary solutions were brought t o a temperature of 20" C. before mixing. The total period of contact of culture with germicide was 30 seconds, after which 1 ml. of the bacterial suspension-quaternary mixture was withdrawn, placed in a 99-ml. dilution blank containing 0.1% naphuride sodium, and thoroughly shaken. Then 1.0-ml. and 0.1-ml. portions were plated from this dilution, using glucose-tryptoneagar containing 0.1 % naphuride sodium. A series of 9 or 10 replicate determinations was made on each organism with each test sample. Two or three of the determina
INDUSTRIAL AND ENGINEERING CHEMISTRY
1024
tions during each run were made with fresh bacterial suspension. Only one determination was made on the B. subtilis spores, as no germicidal action was observed even a t the high quaternary salt concentration of o.5yo, EFFECTOF DEGREEOF UNSATURATION. The results were recorded in terms of the number of surviving bacteria per 0.1 ml. of medication-tube mixture. The control counts estimate the initial concentrations of bacteria in the medication tubes after the addition of the 5 ml. of Arquad solution but prior t o any germicidal action. Upon examination of the data it became evident that there was an excessive number of very small and very large counts (in other words, that the distribution of counts does not even approximately follow a normal curve), that there was wide variation in results obtained from runs repeated under
,.",
Vol. 45, No. 5
apparent that in spite of significant difference among them, the five preparations all fell within a rather narrow range of effectiveness when compared with the wide spread between the initial level of bacterial counts and the zero base line. EFFECT OF VARIATION IN ALKYLGROUPS. The killing action of the series of six quaternary ammonium salts listed above was determined in terms of the number of surviving bacteria per I ml. of medication-tube mixture after 30 seconds (diluted 100 times in a 99-ml. dilution blank containing 0.1% naphuride sodium), In this phase of the experiment as in the previous one, there v a s a large amount of variation among the bacterial counts, with an excessive number of very small and very large counts. Using the method of analysis by ranks, as previously employed, it was found that the six quaternaries differed significantly in their ability t o kill bacteria. The relative bactericidal properties of the six salts with the three types of microorganisms vary. Table I1 ranks the salts in order of effectiveness for each organism. Within the groups no significant variation was noted.
TABLE 11. BACTERICIDAL EFBECTIVESESS E. coli 1.
M . puooenes
Hexadecyl Benzyl coco soya Tallow Octadecyl
1.
Tallow Octadecyl
2.
Soya Hexadecyl
T. crenoris (Yeast) 1 . Soya
2. cocoa 3. Benzyl cocoa cocoa Uncountable number of survivors.
2.
Tallow Hexadecyl Octadecyl
3.
Benzyl coco coco
No preparation is most effective against all three microorganisms. Each preparation has a somewhat different range of specificity.
-0.2
40 60 8C Iodine Value Figure 1. Relation of Degree of Saturation to Germicidal Activity
0
20
tCONTROL
1.19 x10'-0-0-0-0-0
Average ranks based o n number of surviving bacteria. Numerals refer to mixtures listed i n Table I
2.97 X IO6
No. of
supposedly the same conditions, and that the picture was complicated by the occurrence of cases in which the number of surviving bacteria was too large to count. Under these circumstances it is appropriate to treat data by the method of analysis of ranks (4). The results are plotted in Figure 1, average rank against iodine value of the preparation. The differences in average rank are highly significant when subjected to exact statistical tests. There is a general tendency for preparations of higher iodine value (lower degree of saturation) to be less effective in killing action. Preparations VI, VII, and VI11 were especially ineffective, and the conclusion is strengthened by the occurrence of many uncountable plates seeded from mixtures in which they were present Bactericidal activity does not bear a fiimple straight-line relationship to iodine value. Preparation 11, having an iodine value of 7.7, was a more effective bactericide than straight Arquad 18, which had a lower iodine value of 2.5, I n fact, No. I1 was significantly and consistently better than any other preparation tested. This statement rests on a consideration of all the available evidence, derived from tests with both organisms and a t all time intervals. 4 more general picture of the relative effectiveness of the five preparations having the lowest iodine values is presented in Figure 2. The bacterial counts were transformed by means of a coded, essentially logarithmic, scale which served to normalize the variability of the data satisfactorily. From the figure it is
x 107
4.75
7.41
1.~5~105-
surviving
4.63
bacierio per 0.1
ml.
x 104
-
1.16
medication mixture
-
x 105-
lo'-
4.49
x IO
1.10 X I 0
2.45 0
V
111
7 . 2 4 X 10'
1 0
IV
,
,
,
I
,
,
,
20 30 Iodine Value Figure 2. Mean Bacterial Counts after Treatment for 1 Minute
IO
Numeral8 refer to mixtures listed in Table I
Against the organisms M . pyogenes and T. cremoris the four leading quaternary salts are soya, tallow, octadecyl, and hexadecyl, whereas benzyl coco and coco are definitely inferior. Against E. coli, with the exception of coco none is significantly inferior. However, the killing dilutions used on X.pyogenes and T. crernoris were 1t o 80,000 and 1 to 100,000, respectively, whereas on E.coli the dilution was 1to 5000, 16 and 20 times as concentrated.
May 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
1025
OF TESTORGANISMS TABLE 111. POTENCY
Test Organisms-Inhibition, Compounda
a
16. 18.
C.
Aerobacter aerogenes
Bacterium ammonaagenes
B. subtilis
Torula cremoT(s
Micrograms/ML of Medium
Penicillium notaturn
T.
Trirnethylhexadecylammonium chloride. Trimethyloctadecylammonium chloride. Trimethyl coco ammonium chloride.
Mycobacterium smeomatis
Vibrio percolans
Micrococcus pyogenes
Pseudomonas aeruoanosa
Escherichia coli
Trimethyl tallow ammonium chloride.
BZC. Dimethylbenzyl coco arnmoniuin chloride.
DETERMINATION OF ANTIMICROBIAL ACTIVITY
A serial dilution broth screening assay was employed t o evaluate the antimicrobial activity of selected quaternary salts for a broad spectrum of organisms. This procedure evaluates the growth-inhibiting properties of the compounds. Independent experiments (to be published elsewhere) have indicated t h a t the antimicrobial activity revealed by this test is predominantly the result of killing action. ASSAYMEDIUM. The assay medium is nutrient broth of the following per cent composition:
INOCULATION AND INCUBATION. One tenth milliliter of the selected prepared inoculum is aseptically pipetted into each tube of the assay series. The racks containing the assay tubes are agitated t o ensure distribution of the inoculum, and then incubated a t 37 C. for 18hours; tubes inoculated with Mycobacterium smegmatis are incubated 72 hours. OBSERVATION.The ten tubes of an assay series are observed for visual evidence of growth. Usually two adjacent subseries are readily differentiated; the turbid tubes contain concentrations of test material permitting growth of the organisms and the clear tubes contain sufficient active material t o inhibit growth of
0.3 0.5 99.2
Beef extract (Difco) Peptone (Difco) Water
The final p H is adjusted t o 7.0 before the nutrient broth is dispensed into a series of ten shell vials alphabetized from A t o J. The A tube receives 4.9 ml. of medium. Tubes B t o J receive 2.5 ml. of medium. T h e vials are covered with aluminum caps and autoclaved for 15 minutes at 120" C. PREPARATION OF SAMPLE. The weighed samples are dissolved in sufficient 95% ethyl alcohol t o obtain a concentration of 25.0 mg. per ml. Alcohol-insoluble, water-soluble samples are prepared at the same concentration, and sterilized b y Seitz filtration. ADDITIONAND DILUTION OF SAMPLE. One tenth milliliter of the prepared sample solution is aseptically pipetted into the A tube of t h e series containing 4.9 ml. of medium. The tube is thoroughly agitated and 2.5 ml. of the contenb of the A tube is transferred t o the B tube with a sterile syringe fitted with a n 18gage 1.5-inch needle. The B tube is then mixed b y shaking, the syringe is rinsed b y twice drawing up and expelling the contents of the tube, and then 2.5 ml. is transferred to the next tube. The same syringe is used t o prepare the remaining dilutions. The excess 2.5 ml. in the J tube is discarded. This dilution scheme gives a geometric progression in multiples of one half. Tubes A t o J contain the following amounts of test material in micrograms per milliliter of assay medium: *
Tube Concentration
A 500
B 250
C 125
D 62.5
E 31.2
F
G
15.6
7.8
H
I
3.9 2.0
J 1.0
This broad range employs only 10 dilutions extending from 500 t o 1.0 micrograms per ml. of assay medium. TESTORGANISMS.The following organisms were selected as representative of a spectrum which grows readily in nutrient broth: Aerobacter aerogenes, Bacillus subtilis, Escherichia coli, Micrococcus pyogenes var. aureus, Mycobacterium smegmatis, Bacterium ammoniagenes, Pseudomonas aeruginosa, Torula cremoris, Penicillium notaturn, and Vibrio percolans. These organisms are maintained in stock on nutrient-agar slants and transferred at &week intervals. PREPARATION OF INOCULUM. Subcultures of the selected organisms are incubated in nutrient broth at 37" C. for 24 hours (except tubes inoculated with Mycobacterium smegmatis, which are incubated 72 hours). Dilutions of the culture are made in 0.9% saline t o obtain a suspension giving SOYo transmittance compared to a saline blank, a t 540 mfi in a Coleman Junior spectrophotometer Model 11, using a P C 4 filter.
I
0
Figure
3.
I
I
I
I
I
I
I
I
I
10 20 30 40 50 Wt. % Quaternary Ammonium Chloride Solubilities of Quaternary Ammonium Chlorides i n Water Pure 18. 18.
Pure trimethyloctadecyl Commercial trimethyloctadccyl
T. Trimethyl tallow
Pure 16. Pure trimethylhexadecyl 16. Commercial trimethylhexadecy 1 C. Trimethyl coco BzC. Dimethylbenzyl coco Pure 12. Pure trimethyldodecyl
the organism. Activity is expressed in terms of the lowest concentration (micrograms per milliliter) of test substance completely inhibiting visible growth. RESULTS.The five compounds tested and the organisms employed are listed in Table 111. Using the method of analysis by ranks, the order of activity of the five compounds is hexadecyl > tallow > benzyl coco > octadecyl > coco. The differences in potencies of the first four compounds in the series are not great. The coco salt is significantly inferior.
1026
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
PHYSICAL PROPERTIES O F QUATERNARY A&fMONIUM SALTS
Solubility and physical state are important to the consideration of the commercial development of quaternary ammonium salts. Of the compounds studied in the present investigation, those containing unsaturation in either the higher aliphatic chain or the benzyl group are viscous sirups. Trimethyl coco ammonium chloride is a hygroscopic solid. The other salts (trimethglhexadecyl, -octadecyl, and tallow ammonium chlorides) are obtained as crystalline solids. Aqueous-solution properties were determined with particular reference to precipitation and gel formation. DETERXIXATION OF SOLUBILITIES.-4sample of the salt was weighed into a test tube equipped with a calibrated thermometer and a stirrer, and successive dilutions were obtained by the addition of known amounts of water. At each concentration the physical behavior was observed visually as the mixtures were heated and cooled, and the temperatures a t which visible changes of state occurred were recorded. Solution temperatures were reproducible within f 0 . 1 " C. The boundaries of the liquidcrystalline phases were less accurately defined, but are correct within about 2" C. Aqueous-solubility characteristics of the quaternary ammonium salts are shown graphically in Figure 3. The solubilities of pure trimethyldodecyl- (1), trimethylhexadecyl-, and trimethyloctadecylamrnonium chlorides have been included for purposes of comparison. The shaded region for the tallow derivatives represents the range of temperatures within which the solubilities of three different lots of commercial material occurred. All the quaternary salts investigated present similar aqueous systems, of which the behavior of the octadecyl derivative is characteristic. Above line A B and t o the left of BD the system exists as clear, isotropic solution. Below A B the system exists as a mixture of crystalline quaternary salt and solution. T o the right of B D the quaternary salt exists in a liquid-crystalline phase. I n the coco and dimethylbenzyl coco aqueous systems the quaternary salts do not exist as crystalline solids, and the nearly horizontal portions of their diagrams represent the temperatures a t which water freezes out of the solutions. I n all the systems in which the quaternary salt occurs as a crystalline solid above 0" C., there is a break in the solubility curve a t very low concentration. This break represents the Krafft point, a t concentrations above which colloidal behavior occurs ( 3 ) . The solution temperatures for the various commercial quatcrnary salts (as represented by line AB in the octadecyl system) are as follows: octadecyl, 30-31 C.; tallow, 15-22' C.; hexadecyl, 6-7" C The following are the concentrations (percentage by weight) above which the quaternary salts exist in liquid-crystalline phases: octadecyl, 31.5%; tallow, 33 to 33 5 7 0 ; hesadecyl,
Vol. 45, No. 5
35%; coco, 42%; dimethylbenzyl coco, 47.5%. With the exception of the octadecyl derivative, all the salts investigated are soluble in water a t room temperature over relatively wide concentration ranges. OVER-ALL COKCLUSIONS
The bactericidal activity of mixtures of higher aliphatic quaternary ammonium salts containing varying proportions of the saturated and unsaturated Cia chain does not vary markedly with degree of unsaturation. There is, however, a significant tendency toward higher activity in the mixtures of lower degree of unsaturation. I n the series of compounds studied, no individual can be classified as most active against all organisms. I n general, the hexadecyl and the tallow derivatives are superior. On the basis of commercial availability of the raw material, and the antimicrobial activity and physical properties (solubility and crystallinity) of the product, i t is concluded that among the compounds studied the trimethylalkylammonium chloride derived from tallow fatty acids is the most suitable salt for commercial development. ACKNOWLEDGMENT
This report comprises a joint contribution of several groups from this laboratory. The quaternary ammonium salts were prepared by W. IT'. ROESand J. s. Stalioraitis. Determination of microbicidal activities was by D. R. Noel. Statistical analysis of his data was performed by F. C. Bock and Irwin Katzman. Determination of antimicrobial activities was by C. H. h'lunson, Marguerita Volini, H. L. Gordon, and R. A. West, Jr. Studies of the physical properties of the salts were by C. W. Hoerr. The generous cooperation of these investigators is gratefully acknowledged. LITERATURE CITED
(1) Bioome, F. K., Hoerr, C. W., and Harwood, H. J., J . Am. Chem. SOC.,73, 3350 (1951). (2) Cella, J. 9., Eggenberger, D. N., Noel, D. R., Harriman, L. A., and Harwood, H. J., I b i d . , 74, 2061 (1952). (3) Eggenberger, D. iY., and Harwood, H. J., Ibid., 73,3353 (1951). (4) Friedman, M., J . A m . Stat. Assoc., 32, 675 (1937).
(5) Lawrence, C. A , "Surface-Active Quaternary Ammonium
Germicides." New York, Academic Press, 1950.
(6) Ralston, A. W,, Eggenberger, D. S . , Harwood, H. J., and Du Brow, P. L., J . A m . Chem. SOC., 69, 2095 (1947). (7) Weber, G. R , and Black, L. A,, A m . J . P u b . Health, 38, 1405 (1948). RECEIVED for review October 24, 1952. ACCEPTED J a n u a r y 15, 1953. Presented before the Division of Industrial a n d Engineering Chemistry, Symposium on r a t t y Acids and Derivatives, a t the 122nd Meeting of the AMERICAN CHEMICAL SocIsru, Atlantic City, S . J.
