HORACE P. WARRINGTON, dr. Sterling- Winthrop Research Instifufe, Rensselaer,
b Mixed alkylbenzyldimethyl ammonium chlorides are quantitatively hydrogenated f b yield a mixture of alkyldimethylamines. The homolog distribution of these resulting amines is rapidly and accurately determined by gas chromatography. Isothermal gas chromatographic operation yields data on the important CIS and C14 homologs which are sufficient for control purposes, but programmed temperature operation using a light loaded column will give a more precise determination of all the compounds present in the mixture. HE germicidal effectiveness of the mixture of Cs to CI8 alkylbenzyldimethyl ammonium chlorides known as benzalkonium chloride Cd) is gcmer9y zftributed to preponderance of the Clz and Clr.komologs present. Usually, methods of analysis for benzalkonium chlorides are based on the salt-forming characteristic of the quaternary nitrogen, but the results of this type of method do not give any indication of the homolog distribution. The homolog distribution has been determined by a laborious fractional distillation of the alkyldimethylamine mixture from which benzalkonium chloride is prepared. Using gas chromatography, the composition of the alkyldimethylamine mixture could be determined more rapidly and accurately than by fractional distillation, A solution of benzalkonium chloride is readily debenzylated by hydrogenation ( I , 8 ) yielding toluene and the original alkyldimethylamines. Thus, after quantitative debenzylation, the homolog distribution in benzalkonium chloride can be determined by gas chromatography. ~8
N. Y.
was most conveniently accomplished using a modified Parr hydrogenation apparatus (3). Gas Chromatography Operational Conditions. When using the Vapor Fractometer, the best over-all separations were found using a PerkinElmer C (silicone oil D.C. 200) I / h inch-diameter column, 2 meters long operated a t a temperature sf 225" C. and a helium carrier gasrpressure of 20 p.s.i,g. a t a column outlet flow rate of 38 cc. per minute. This column has been used under these conditions with great regularity for the past 3 years and still gives excellent separations. The column used for programmed temperature operations is a 0.75% General Electric Co. silicone gum. 8E 30 on 100- to 140-mesh Gas Chrom P as purchased from the F %I M Scientific Corp. The, solumn temperature was linearly pmgr'ammedfrom 60Oto276" C. at- a temper4ture increase of 9' C. per minute and was held a t 276' C. until the end of the chromatogram. The injection port of the F & M apparatus was operated at 225" 6.with the flame ionization detector being held a t 210' C. Nitrogen was used as the carrier gas at a pressure of 60 p.s.i.g. and a column exit flow rate of 85 cc. per minute. Debenzylation of Benzalkonium Chloride. A 5-ml. aqueous sample containing 10% benzalkonium chloride is placed in a micro reaction vessel (S), ea. 30 mg. of lOyo palladium on Darco is added and the sampie is hydrogenated with shaking a t room temperature for 30 min. (Platinum may be used in this ease but the hydrogenation will take longer.) Since the solvent and palladium cannot be presaturated
with hydrogen, the gage only serves to show when no more hydrogen is being taken up. Upon completion of the debenzylation, 1 ml. of 0.1N HCl is added to the reaction vessel and the solution is filtered through Whatman No, 1 paper into a BO-ml. separatory funnel. The reaction vessel is rinsed with,,three 5-ml. portions of distilled water and the rinse solutions are passed through the filter paper into the separatory funnel. The filtrate is made strongly alkaline with 10% sodium hydroxide solution and extracted with 15, 10, and 5 ml. of methylene chloride. After passing the methylene chloride solutions through a very small cotton plug, they are gently evaporated on a steam balh t o a 1-to 2-ml. volume. RESULTS
Because of the wide boiling range covered by CSto Cls alkyldimethylamine mixtures, isothermal operation yields very sharp CSand GO isomer peaks and a very flat CIRisomer peak (Figure 1). At 225" C. the column is at its recommended maximum temperature and increasing the helium pressure to 25 p.s.i.g. does not appreciably sharpen the CISpeak while it does decrease the resolution between the Ce and Cl0 isomers. A solution containing 270 stearyldimethylamine will show very little deviation from the base line at the retention time for the C18 homolog when a 5-pl. sample is used, and can be measured only to a fair degree of accuracy when a 10-pl. sample is used. Figure 2 shows the resolution obtained for the alkyldimethyh,mines and some
RANGE SETTING
- *r
I
n
----2-t7--d--i---.S
f
c (4
EXPERIMENTAL
Appaatus. Isothermal gas chromatogra hic analyses were performed on a Perkn-Elmer Vapor Fractometer Model 154B connected to a Leeds & Northrtlp Model G 5-mv. recorder. Programmed temperature gas c h r o m tographic procedures were carried out. using a n F & M Model 609 gas chromatograph attached to a XlinneapolisHoneywell Model Y143, I-mv. recorder equipped with a Disc integrator. y y drogenation of benzalkonium chloride 1
e
ANALYTICAL
CHEMIST
06 b
m
0 E V
sp BO
Figure 1. amines
Cj6
~
55
50
45
40 35 SO Time In Mlnutrs
C/P
j\J&
E:
25
20-
15
ib
9
Isothermal gas chromatograph of commercial mixture of alkyldimethyl-
Conditions: column, Perkin-Elmer C, 2 rneierrj He pressure, 20 p.r.i.8.; temperature, 225' C.i rample iize, 5 p!. Each tertiary amine is identified by its number of eorbon atoms in the alkyl chain
10-
Table 1. Comparison of Gas Chromatographic Methods Used on a Solution of Known Content of Alkyldimethylamines
1.61.4-
reJ 1,P2 0
5: 1.0W t0.8-
Iso-
thermal Homolog method
I):
8
0.6-
0'4
'
C8 ClO -x-
ALKYLDIMETHYLAMINES CHLORIDES ALCOHOLS
c 1 2 c 1 4
Cl6 C18
4.7 10.8 38.0 31.7 8.0 5.7
1.2-
g 1.0-
grammed method Theory 10,6
4.5 11.0
38.9
38.2
31.1 8.3 6.7
31.3 8.5 6.6
Z
W t; 0.8-
U
8 0.6-I
0.40.2-
I
I
8 10 12 14 16 18 NUMBER OF C ATOMS IN THE CHAIN
Figure 2. Cdmparison of isothermal gas chromatographic 'separations between each alkyldimethylamine and its alcohol and chloride analog Perkin-Elmer C column
potential impurities, namely, the Cs to Cls alcohols and chlorides. Other high temperature columns prepared by Perkin-Elmer were examined for this method of analysis. Of these columns, the 2-meter K (Carbowax 1500) column shows excellent resolution between a particular alkyldimethylamine and its alcohol and chloride analogs up to CIE (Figure 3), but the retention time for t h e CISanalogs and the Cl6 alcohol is too great for practicality. Various columns
/
Z
pro-
4.5
/
/
/
YF
Weight, % Temperature
+-
/
f
I
4
0 12 14 16 16 NUMBER O F C ATOMS I N THE CHAIN
containing less than 1% of liquid substrate have also been tried at lower temperatures but sample sizes which show adequate sized Cl* peaks overload the column in the CIZand C14 isomer regions. Coupling a light loaded column to an instrument with a very sensitive flame ionization detector and the capability of linearly increasing the column temperature yields chromatograms in which the CIS alkyldimethylamine peak is as sharp as is the Cs homolog peak (Figure 4). Peaks of compounds which are retained by the column longer than stearyldimethylamine also show up readily. The 0.75% silicone gum column used shows such a large pressure drop with the increasing tempera-
Figure 3. Comparison of isothermal gas chromatographic separations between each alkyldimethylamine and its alcohol and chloride analog Perkin-Elmer K Column
Table II. Comparison of Results Found for Alkyldimethylamines vs. Debenzylated Benzalkonium Chloride
Weight % DebenOriginal zylated benzalkBenzalkalkylonium Homo- dimethyl- onium chloride chloride" log amine C8
ClO c 1 2
C14 Cl6 C18
0.18 1.41 63.8
25.4 7.94 1.36
0.20 1.35
0.17 1.25 63.1 26.0
64.4 25.4
7.86 1.49
7.38 1.36
a Calculated from the results of wt. yo of debenzylated benzalkonium chloride.
Figure 4.
Pro&?mrned temperature gas chromatograph of commercial mixture
of alkyldimethytdhn'nes Condition% colu'mn,0.75~sriliconegum SE 30 on 100- to 140-mesh Gas Chrom PI Nz pressure, 60 p.r.i.g. at flow rate of 85 cc./min.j temperature, 60" C. start, increase rate of 9" C./min., limit 276' C.j sample size, 0.5 MI sCnsltiv3y, i / I 000th Each tertifiry amink is idenlifled by its number of carbon otomr in the alkyl chain
,
ture that the maximum nitrogen tank reducer valve pressure of 60 p.s.i.g. must be used so that the flow controller is capable of maintaining a constant mass flow of carrier gas through the column. Table 1 compares the values found on a solution prepared from the pure individual alkyldiðyIamines. Values for the isothermal chromatograms were computed from the peak areas corrected for thermistor response to a weight relationship. The response of the flame ionization detector to the members of this amineseries is so close to their weight relationship that no correction factor was applied to their areas. To show that hydrogenation does not alter the amine ratios, the analytical results of the commercid sample of VOL 33, NO. 13, DECEMBER 1961
0
1899
alkyldimethylamines shown in Figure 4 were compared to the results from a chromatogram of a debenzylated sample of the bennalkonium chloride made from this a l k y l d ~ e t h y l a m ~mixture. e Results of this comparison are sh0n.n in Table I1 with the calculated amounts
of benzalkonium chloride which each aminerepresents. LITERATURE CITED
(1 Adams, Re,ed., “Organic Reactions,” VU, p. 278, Wiley, New York, 1963. (2) Schulenberg, J. W., Sterling-Winthrop Research Institute, Rensselaer, N. Y.,
private communication. (3) Southworth, B. C., ANAL. GHEX. 28, 1611 (19561. (4) U. S. Pharmacopeia, 16th Rev,, p. 82, The United States Pharmacopeia Convention, Inc., 1960. RECEIVED for review July 3, 1961. Accepted October 9, 1961.
PETER E. MANNII’ and JOSEPH E. SlNSHElMERl Colleges o f Pharmacy, University of Rho& Island, Kingsfon, Is. 9., and The Yniversify o f Michigan, Ann Arbor, Mich. Variously substituted hydroxytones were studied, to determin whether differences could be detecte in their rate of reaction with blue tetrazolium. In many cases substantial erences were found which could be related to either the alpha or beta i group with position of the h respect to the keto or to whether the hydroxyl group i s primary, secondary, or tertiary. A correlation of the electron-donating ability of substituent groups with formazan development is indicated. Results also suggest the possibility of assaying for individual ketols and mixtures of ketols. Three compounds of medicinal interest tetracycline hydrochloride, alloxantin dihydrate, and erythromycin base prove reactive toward blue tetrazolium, and their quantitative determination via a “tetrazolium reactionlu seems feasible.
-
EYER and Lindberg (8) as well as
Rosenkrantz (11) have demonstrated the usefulness of rate studies involving blue tetrazolium for the structure elucidation of Ac3-ketosteroids and catecholamines, respectively. As a consequence of their investigation of steroids, Meyer and Lindberg (8) established that increased reactivity is to be expected from those compounds which contain an a-keto1 moiety. However, no data were elicited to indicate the effects of varying substituents upon reactivity of the ketol grouping. Therefore, it seemed worthwhile to investigate the rate of reaction of model compounds with blue tetrazolium. The information gleaned from such studies is useful not only as a tool for structure elucidation, but also for the quantitative analysis of individual ketols and of ketol mixtures. 1 Present addrew, College of Pharmac University of Michigan, Ann Arbor, Mi&
1900
*
ANALYTICAL CHEMISTRY
TIME (MIN.1
Figure 1. Rate of formazan formation for 1 X 1 O-b solutions of a-hydroxyketones A. Dihydroxyacetone (reeorded as monomer) B. 3-Hydroxy-?-butanone C.
Hydroxypropanone
EXPERIMENTAL
All spectrophotometric measurements were made with a Beckman Model DU spectrophotometer. All melting points are corrected. Unless otherwise specified, reagents and test compounds may be obtained from Eastman Organic Chemicals. Reagents. Blue tetrazolium (Dajac Laboratories), A commercial sample was purified by recrystallization from 95% ethyl alcohol-anhydrous ether according to the method of Weichselbaum and Margraf (13). Purification was continued until a portion of the salt in absolute ethyl alcohol showed a constant absorbance a t 254 mh.
Absolute ethyl alcohol. Following distillation from 2,4-dinitrophenylhydrazine (10 grams per liter) the alcohol was dried by treatment m t h sodium and diethyl phthalate as described by Fieser (4). It is necessav to keep the aqueous concentration of reagents a t a minimum because water has been shown to inhibit the reaction (0) *
Tetramethylammonium
hydrodde.
A 0.03OON solution was prepared by dilution of a 10% aqueous solution with
absolute ethyl alcohol. Test Compounds. Solids were recrystallized to constant melting point. Li uids were fractionally distilled un%er nitrogen. Significant disagree-
