Colorimetric Method for Quantitative Microdetermination Of Quaternary Ammonium Compounds losses of Quaternary Ammonium Compounds Caused by Glass Adsorption and Concentration in the Foam JORGEN FOGH, PAUL 0.H. RASMUSSEN, and KNUD SKADHAUGE Statens Seruminstitut and The Poliomyelitis institute o f the Danish National Association, Copenhagen, Denmark Work was undertalieii to find out to what degree the contradictory results generally obtained in the testing of the antibacterial effects of quaternary ammonium compounds were due to an adsorption of the compounds onto different glass surfaces. Losses of cetylpyridinium chloride, due to adsorption onto different glass surfaces, were found to vary from 0 to 707, of the initial concentration (1 to 50,000). After shaking, an appreciable increase in the concentration of the compound of the foamy phase was found, at the expense of the concentration of the liquid phase. Treatment of test tiihes with Plexiglas
H
ITHERTO inveptigation. to determine the use of quaternary ammonium compounds as antiseptics have given results that show discrepancies. The testing of the antibacterial effect in vitro has presented many difficulties. The cause of the rather contradictory results is undouhtedly the readiness with n-hich these compounds are adsorbed onto a variety of surfaces. It is therefore considered necessary to wpplement the usual bacteriological tests with an exact chemical determination of the substances under the given experimental conditions. Various methods for the quantitative determination of the content of quaternary ammonium compounds in aqueous solutions have been published-e.g., by Hartley and Runnicles (S), FlotoR- ( 5 ) , Auerbach (1,2 ) , Epton ( 4 ) , Colichman ( 3 ) )and Gain and Lawrence (6). Most of the methods described have been based on colorimetric and photometric determinations of thc combination of the quaternary compound with various stains. Such methods have been ronfinetl to the determination of relatively high concentrations. I t was found by Skadhauge and Fogh (9) that certain indicato1s in the presence of cetylpyridinium chloride produced a color not associated with this indicator at any known pH. Bromocresol purple proved to be the most suitable of the indicators examined, as the color was intenwly blue and varied distinctly with the concentration of cetylpyridinium chloride. When an anion-active substance was added, the blue color changed back to the original violet, indicating that the cetylpyridinium chloride had been withdrawn from the bromocresol purple-cetylpyridinium chloride complex. When further quantities of cetylpyridinium chloride were added, the blue color reappeared. This color, however, was rather unstable, and in some eases spontaneous decolorization occurred. Experiments seemed t o show that it might be possible, hon ever, by means of a simple technique to find an easy and exact method of microdetermination. One of the writers therefore made an elaboration of the method of analysis and some investigation\ of variouq physical and chemical phenomena concerned with the method.
(polymetacrylic acid ester) will greatly diminish the losses of quaternary ammonium compounds originating from the adsorption of the compounds. Practical applications include the possibility of controlling the actual concentrations of quaternary ammonium compounds during the antibacterial testing experiments and of determining the very small amounts which will be carried on to the different food products, when quaternary ammonium compounds are used in the dairy and other food industries, owing to the adsorption onto the different surfaces of the equipment.
In addition, other quaternary ammonium compounds 15 ere tested by the same technique, using special standard curves for each compound--e.g., Cetavlon, Imperial Chemical Pharmacy, Ltd., Manchester, and Rodalon, Ferrosan, Ltd., Copenhagen. BROMOCRESOL PURPLE.The Merck preparation was used throughout. For use, 0.2 gram of indicator was dissolved in a few milliliters of 1N sodium hydroxide and a little water. After dilution to about 200 ml. the solution was adjusted to pH 8.2 and made up nith water to 300 ml. It is important that exactly the same quantity of the indicator should be added to the test solution as to the blank, whereas the strength of the indicator does not play so great a role. A deviation of from 5 to 10% is of no great importance. If a somewhat weaker indicator solution is used, the standard curve shows a deflection at a lower concentration than that shown in Figure 2. With an indicator solution 50'0 stronger than the one mentioned, the standard curve is rectilinear to over 40y of cetylpyridinium chloride per ml. The use of such a strength, however, has the disadvantage that the test solutions will readily decolorize, a phenomenon that has been observed only seldom when using a concentration of the indicator of p H 8.2. The indicator solution employed is considered stable, as no change in the color has been observed during 4 months. BUFFER SOLUTIONS used were 0.5N disodium phosphate (pH 8.2) and 0.5Ndisodium phosphate plus 2% of an anion detergent (Teepool 410). PLEXIGLAS.For the purpoqe of fixing the standard curves, the test tubes, pipets, and cuvettes were prepared with Plexiglas (polymetacrylic acid ester) This preparation was made as follows. 4 solution of Plexiglas in chloroform (1 to 2%) was used. Plexiglas readily dissolved and the solution w a p somewhat
0.4
4
EXPERI\IERThL
Reagents. CETYLPYRIDINIUM CIILORIUE (Gcrmidin V, Bionova, Ltd., Copenhagen) was used as a standard. This comniercial product is said to contain 10Crof cetylpyridinium chloride i n water solution. This statement v a s checked in comparative examinations, using pure cetylpyridinium chloride as well as the commercial product for the prrparation of the standard curves.
I
4Q1
' 'shoo'
I
' 660' ' '
I
160
'
Figure 1. Spectrum of Bromocresol Purple Measured against a Similar Solution without Cetylpyridinium Chloride
392
V O L U M E 2 6 , NO. 2, F E B R U A R Y 1 9 5 4 viscous. The test tubes were filled with the Plexiglas solution and emptied immediately after. The upper parts of the tubes were dipped in the Plexiglas solution, and after it had dripped off for a few seconds, the tubes were inverted and left to dry for about 15 minutes. Test tubes prepared in this way can be used only once, as the Plexiglas film has a tendency to come off when the tubes are rinsed and dried after use. SPECTROPHOTOMETER. All measurements were made using a Beckman DU spectrophotometer with I-em. cuvettes. The wave length chosen for the mrasuremcnts was 620 m p , as shown in Figure 1. Method of Analysis. I)mErnIrh-.+rrioS O F C E ' I w . P Y R r D I x I u x CHLORIDE IN COLORLES~ LIQUIDS. -1quantity of 4.00 ml.of the cetylpyridinium chloride solution to be tested (containing up to 257 per ml.) is mixed with 0.100 ml. of indicator solution and 0.20 ml. 0.LY disodium phosphate. -1similar mixture, without cetylpyridiniuni chloride, is used as a hlank. The measurements are made in the Beckman spec-trophotometer xith 1-em. cuvettes a t 620 mp. 1,:tmbert-Beer's law applies to concentratioiis up to a little more than 257 per ml. In the case of higher concentrations the curve is flattened (Figure 2 ) . The experiniental error was found to be lees than +2% for concentrations between 10 and 257 per ml. of cetylpyridinium chloride.
393 rubber stopper should be large enough to be pressed against the mouth of the tube, as the Pleviglas film breaks if the stopper is inserted into the tube. The tube can also be closed by a finger with a stall. The sanie stopper or finger stall should not be used for both the actual and the blank value when the method for colored liquids is used, a? the relatively large excess of anion detergent may then interfere with the determination of the actual value. Mixing by pouring from one tube to another by inverting the tube against a finger without a stall, resulted in a 1.4 and a 3.9y0 loss of ci.tylpyridinium chloride, respectively, determined as the average of four measurements. K:E su LT s
The described procedure ~vviisdeveloped as the result of a careful investigation of the reaction conditions. The experimental findings are summarized in the follo\ving paragraphs. The influence of pH on the intensity of the color was examined by means of a number of phosphate buffer solutions ranging from pH 6.22 to 9.87 (Table I). The actual values of T:iliIe I were measured against blank values with the same pFI. The pH should be kept constantly on the basic side of the color range of the indicator. It was found practical to use a p H of 8.2, as a more basic reaction will rcadily cause the precipitation of a numhcr of substances.
Table I.
Influence of pH on Intensity of Color PH
E
6.22 6 65 7.12 7 62 8 12 8 65 9 02 9 50 9 Si
0.418 0.420 0,424 0.441 0.445 0.445 0.419 0.451 0.451
Talde 11. Influence of Ternperatwe on Intensity of Color Temp., O C. 18 22 28 35
Figure 2. Standard Curve for Determination of Cetylpyridinium Chloride Concentrations up to 257 per M1. 1 ye, of bromocresol purple solution and test tubes
Density, % 98.7-100.2 98.9-101.1 100.4-101.0 101.2-103.5
-1s shown in Table 11, :I va1,iation of the temperature within certain limits has no iniportant c~i'fr~ct 011 tlie intensity of the color. When the influenc~of diti'rrmt vations on the reaction was tested the presence of up to 1% of ralciuni, magnesium, ferrous, with Plexiglas -.- l / sprepared %. bromocresol purple solution ferric. and cupric ion, in the measuring solutions caused no appreUke of non-Plexiglak-prepared test tubes ciable deviations in the cletrrminationr of the cetylpyridinium chloride concentrations. Tap \r.:cter niay be well used as a diluent. DETERMINATIOS OF C E , ~ Y L ~ ~ Y R I I )CHLORIDE I S . I C ~ I IK COLORED In t,he preliminary experiment h of preparing a standard curve fur the spectrophotomctric nieaaurements, ordinary untreated LIQCIDS. Two identical mixtures are prepared, containing 0.100 glass tubes \$-ere employotl. IIo\r.evcr, these attempts ~ v e r enot ml. of bromocresol purple and 4.00 ml. of the cetylpyridinium successful, owing to wiriatioii~hi tlie intensities of the color. Bs chloride solution. To one mixture is added 0.20 nil. of 0.5N the quaternary nmmoniuni i ~ ~ i i ~ ~ ) u uare i i d smarkedly surface disodium phosphate (mixturt: 1). To the other mixture is added 0.20 ml. of a buffer consisting of 0 . 5 5 disodium phosphate and active substances, it w a s thought t h a t the adsorption onto glass might possibly account Eo], tlics tli.vrepancies found in the stand2% of Teepool 410 (mixture 2 ) . Mixture 1 is used for measurard mcasurings. The test tulm ~vei'every scratchrd and there ing the actual value while mixture 2 is used as a blank. It was seemed to be a relationship Iwtween the degree of scratching found that even intensely colored liquids, in which it was inipossible to observe any difference in color with the naked eye: could anti the values of cetylpyricliliiuin c.liloride found by the photobe measured easily with the technique used. This was also the nietric measurements. To clucitiate this in detail, tliiw new case with even rather opaque liquids. test tubes, macroscopically free of scratches, as well as three The indicator, buffer, and cet,ylpyridinium chloride solutions scratched tubes, were choreii for :in experiment. The should be carefully mixed after they have been run out of the Irere left for 5 minutes in a 1 to 50,000 solution of cet~ylpyridinium chloride. Then 4.00 ml. of the cetylpyridinium chloride solution pipet into the test tubes. This is best done by inverting the test were transferred by nieanS of a pipet to Ann-less test tubes contube and closing the mouth with a rubber stopper. When using test tubes prepared with Plexiglas, it is important that the taining indicator and buffvr solution. and were used for spectro-
-
----
394
ANALYTICAL CHEMISTRY
photometry. As shown in Table 111, a loss of cetylpyridinium chloride averaging 27% was found with the scratched tubetr. The pronounced adsorDtion of cetvl~vridiniumchloride into the scratched surface of the test tubeswas also illustrated in a subsequent experiment by the appearance of an intense blue color in a transverse scratch in the tube after it had been left far some time in a mixture of cetylpyridinium chloride solution, indicator, and buffer (Figure 3). In order to find out whether and to what extent 'cetylpyridinium chloride is adsorbed by apparently unscratched test tubes, a 1 to 50,000 cetylpyridinium chloride solution was transferred through a series of new, unscratched tubes. The concentration oE cetylpyridinium chloride was measured a t every transfer. I n these experiments, too, a loss of cetylpyridinium chloride was ascertained which must be considered due to an adsorption of the substance onto the tubes. The tubes, however, did not adsorb cetylpyridinium chloride to the same degree. Reproducible results could not be obtained in numerous repeated atFigure 3. Phototempts to determine the loss of cetylgraphio Dernonstration of Adpyridinium chloride after the solution sorption of Cetylhad been transferred 10 times. Losses pyridinium Chlovarying from 0 to 70% were observed ride onto Transwhen using cetylpyridinium chloride in verse Seratoh of Test T u b e a concentration of 1 t o 50,000. Similar observations have been made Note the block line by Gershenfeld and Brillhart (7), among others. Thev. however.,exolained the . loss as an adsorption to impurities in the test tube used. According to these authors, no loss of cetylpyridinium chl6ride occurs (as measured by testing the antibacterial property of the solution), if the test tubes are cleaned in the following manner: Wash in soapy water and rinse three times in tap water, then leave for 10 minutes in chromic-sulfuric acid, rinse 3 times in tap water and 4 times in distilled water, and finally dry. This method of cleaning wa8 used in two experiments parallel to the one described above, in which the solution was transferred repeatedly. Here, too, losses of oetylpyridinium chloride of 15 and 24% were ascertained. I n an experiment in which the solution was transferred 10 times, using only two test tubes, the loss of cetylpyridinium chloride was found to he somewhat lower than the average loss found when the solution was transferred to a number of tubes. This observation might perhaps indicate a saturation of the tubes with cetylpyridinium chloride. To explore this possibility, the fallowing experiments were carried out. A number of test tubes were left for about 18 hours in 1to 50,000 and 1to 1000 solutions of cetylpyridinium chloride, respectively The tubes were then carefully rinsed in distilled water and dried. When the concentration of cetylpyridinium chloride was determined after transfer experiments, the loss of cetylpyridinium chloride was found to he 9 and 1S%, respectively. Apparently then, the test tubes cannot he saturated with cetylpyridinium chloride (or more correctly, perhaps, the cetylpyridinium chloride adsorbed onto the glass can he removed again by rinsing). I n transfer experiments using test tubes prepared with Plexiglas, the loss of cetylpyridinium chloride was found to be much lower than in the case of untreated glass tnbes. Five transfer experiments with 10 test tubes showed a maximal loss of 6% and an average loss of 2.1% of cetylpyridinium chloride added. The color resulting from the mixture of hetylpyridinium chloride, indicator, and buffer is of maximum intensity immediately after the mixing process, and the intensity of the color I,
Table 111. Comparison of Intensities f r o m Scratched and Unscratched Test T u b e s Tubes
E Values from Teat Tubes Withoutscrateher With scratches
1 2 3
0.341 0.345 0.346
0.2418 0.2528 0.2616
Mean
0.344
0.2521
AT. 1099 about 27%.
generally is relatively stable. A loss of 1888 than 3% was observed even after 8 hours a t room temperature. However, spontaneous decoloriestion sometimes occurred in one of a whole range oE test tubes. The decolorization occurred a t highly varying times after the mixing of oetylpyridinium chloride and indicator, from a few minutes up to several hours. This decolorization occurred very suddenly, beginning in the upper part of the liquid. The p H of such decolorized solutions showed no change. Nevertheless, keeping the pH on the hasic side of the calor range of the indicator (pH 8.2) made these decolorizations occur only very seldom. In view of the demonstration of the loss of cetylpyridinium chloride by glass adsorption, it wa.6 found to he important not to transfer the test solution more than once (from tube to cuvette) prior to the spectrophotometric analysis. No difference could be demonstrated in the concentration of cetylpyridinium chloride a t different levels of a 1 to 50,000 solution. After vigorous shaking, causing a high foam formation, analysis showed the liquid phase to contain 9.8% of eetylpyridinium chloride less than before shaking. The foam thus cantains an essentially higher concentration of cetylpyridinium ehloride tban the liquid phase does. This phenomenon was further illustrated by the following experiment. A current of air was blown through 100 ml. of a 1 to 50,000 solution of cetylpyridinium chloride in a suction flask and the foam was passed onto another flask. Four fractions were collected from the second flaslc-13, 12, 9, and 5 ml., respectively. After the foam had settled, the different fractions were analyzed. The results are given in Figure 3. The foam m ~ not s very dense with the procedure used. Undoubtedly i t will he possible to obtain considerably higher concentmtions of cetylpyridinium chlaride in the foam than those shown in Figure 4. SUMMARY
Rased on the ability of bromocresol purple to produce a blue calor with different quaternary ammonium compounds, a spectraphotometric method of analyzing cetylpyridinium chloride in concentrations ranging from 0 to 257 per ml. has been devised.
"I
30
F i g u r e 4. Analysis of Cetylpyridinium Chloride Conoentrations i n Four Fraotions of Foam
V O L U M E 26, NO. 2, F E B R U A R Y 1 9 5 4 The results of the method were found to be independent of variations in temperature within certain limits and uninfluenced by the presence in the test solution of calcium, magnesium, ferrous, ferric, and cupric ion up to concentrations of about 1%. The p H should be kept constantly on the basic side of the color range of the indicator (pH 8.2). The tendency of the quaternary ammonium compound to be adsorbed onto glass (especially if very scratched) has been demonstrated. This factor apparently has not been sufficiently considered in previous publications. By treating tubes, cuvettes, and pipets with Plexiglas, this adsorption can be diminished to a great extent, The concentration of cetylpyridinium chloride in the foamy phase was found to be appreciably higher than in the liquid phase. Standard curves for the microdetermination of other quaternary
395 ammonium compounds may well be elaborated using the technique here described. LITERATURE CITED
(1) Auerbach, M. E., IXD.ENG.CHFIM., ASAL.ED., 15, 492 (1943).
(2) Ibid., 16, 739 (1944). (:3)
(4) (5) (6) (7)
Colichman, E. L., AN.AI..CHEY..19, 430 (1947). Epton, S. R., Nature, 160, 795 (1947). Flotow, E., Pharm. Zentrulholle, 83, 181-5 (1912). Gain, J . F., and Lawrence, C. A , , Science, 106, 525 (1947). Gershenfeld, L., and Brillhart, R. E., Am. J Pharm., 122, 454
(1950). (8) Hartley, G. S., and Runnicles, G . F., Proc. R o y . SOC.(London), 168, 420 (1938). (9) Skadhauge, K., and Fogh, ,J., Acto P ( ~ t hMicrobiol. . Scand., 32, 290 (1952). R E C E I V Efor D review February 6, 1953. Accepted October 22, 1953
Determination of Nitrogen in Zirconium by Micro-Kjeldahl Steam Distillation J. F. R O D G E R S and G. J. H A R T E R Westinghouse Electric Corp., Pittsburgh 30, Pa.
A
RAPID and accurate routine method for the determination of
nitrogen in zirconium was needed for the control of processes for producing zirconium metal .4ftpr various methods and types of apparatus were tried ( 2 , S ) , the micro-Kjeldahl steam-distillation method developed by Beeghly ( 1 ) for steel was finally adopted. A few slight changes in the apparatus were made which improved the distillation process. The 500-ml. steam-genertiting flask was replaced with a 1000nil. flask, and a Glass-Col spherical heating mantle (three-necked type) was substituted for the open coil type of heater used to heat the steam flask. Associated equipment was carefully arranged for greatest efficiency. The advantages resulting from these modifications are that twice as many distillations can be made before refilling the generating flask; an even supply of steam is obtained by using the snugfitting Glass-Col heaters regulated by a Variac; heat radiation losses to the room are reduced considerably; and an appreciable increase in the t o t d number of determinations per day is realized
Reagents and apparatus are the same as in Beeghly's method except that a mixture of hydrochloric and hydrofluoric acids is used as the dissolving solution, and a Fisher electrophotometer is used for measuring the color of the nesslerized distillate. The electrophotometer housing was modified to accommodate 50-ml. Nessler tubes in place of the 23-ml tubes supplied with the instrument. Because of the action of the hydrofluoric acid on the distillation flask, it is necessary to test the usability of the flask after a number of runs have been made in them by lightly tapping the bulb of the flask on the work table. This is a very practical means for determining whether or not the flask is safe to use again. The standard-taper ground-glass neck of the flask is salvaged from the bulb when it breaks, and may be rejoined to another bulb at low cost of replacement when the services of a glass blower are available. REAGENTS
Dissolving solution, hydrochloric acid 1 to 1 Hydrofluoric acid, 48%. Ammonia-free distilled water. Nessler's reagent prepared as directed by Beeghly ( 1 ) .
It is very important to work in a room free from all nitric and ammonia fumes, as minute traces of either will affect the accuracy of the results. APPARATUS
A sketch of the distillation unit is shown in Figure 1.
Hot Water Bath. The bath is made of a '/lc-inch sheet of stainless steel, painted inside and out with a corrosion-resistant black paint. The inside dimensions are 48 X 6 X 6 inches, with a double row of 3-inch square, egg box partitions for holding 32 flasks. The heating element is a 500-watt immersion heater controlled by a Variac. CENERAT/NC FLASK PROCEDURE
Figure 1.
Diagram of Micro-Kjeldahl Unit
Approximately 1.0 gram of zirconium metal is placed in a 100-ml. distillation flask and dissolved with 25 ml. of 1 to 1 hydrochloric acid and 15 drops of hydrofluoric acid by heating in a hot water bath. Khile the sample is dissolving, steam from nitrogen-free water is paqsed through the apparatus until the distillate gives a negative test with Nessler's reagent. The nitrogen-free water is prepared by passing ordinary distilled water through a 24-inch glass column 3 inches in diameter con-
