ANALYTICAL CHEMISTRY
66 Table 11. Analysis of Olefin-ParaffinMixtures by HJdroxy Mercurial Method
.
Mixture E rbylene-ethane
(Absorption time 10 minutes) Volume of Olefin Samole. Theoretical. Determined. Difference, c u . Mm. 5% % % 54.53 0.00 0.02 +o 02 65.56 9.99 10.50 t0.51 51.44 25.36 25.33 -0.03 91.56 30.59 30.36 -0.23 55.37 49.67 49.75 + O . 08 84.58 57,35 57.11 -0.24 0,04 88.70 66.25 66.21 53.13 74.54 74.52 81.42 84.86 84.57 ,\lean +o, 18 50.12 10.79 10.68 -0.11 48.12 20.94 20.90 -0.04 48.91 30.47 30.49 +0.02 50.05 40,03 39.96 -0.07 51.90 50.24 50.22 -0.02 50.11 60.59 60.61 t0.02 50.04 69.16 69.28 48.83 79.49 79,58 90 16 90.12 -0.04 49.85 lTean *O.06
-
1::;;
Propylene-propane
$::Ai
Butene-%butane
7 18 .. 95 65 7 80.06 81.09 83.47 84.06 81.51
102..06 04 27.24 39.02 47.75 58.97 72,93 84 86
80,60
103.. 25 60 27.02 39.44 47,74 58,40 72.94 84.62 Nean
-0.22
?E;7::;:; -0.24 t0.32
Bttempts to use this absorbent with propylene-propane mixtures were unsuccessful, owing to the absorption of propane. The results \yere considerably better when "beads" previously saturated with propane were used, but this procedure was not considfor a routine method'
ered
HYDROXY MERCURIAL METHOD
Beads made from mercuric acetate and water gave good results with propylene-propane mixtures, but absorption was extremely slow. The rate of absorption could be increased considerably by the addition of benzoyl peroxide, but. other gaseous products resulted from the reaction. There n-as a volume increase even in pure propane. Since nitrates have been used as catalysts for reactions of this type ( 5 ) ,a series of absorbents x a s tried using mercuric nitrate for this purpose. The data in Table I1 were obtained with an absorbent made from 3 cc. of pou-dered mercuric acetate, 1.5 cc. of water, and approximately I gram of mercuric nitrate (Merck). Water vapor was removed with fused potassium hydroxide beads. The test for complete absorption was made as before. There is relatively little difference between the two methods in the case of ethylene-ethane mixtures, but the greater versatility of the hydroxy mercurial method recommends its use in most instances. LITERATURE CITED (1)
Birks, A . M., and Wright, G . F., J . Am. Chem. SOC., 62, 2412 (1940).
made from 3 cc. of powdered mercuric acetate (General Chemical Co.) and 2 cc. of a 1% solution of boron trifluoride ethyl etherate in ethylene glycol (Eastman Kodak Co.), and was then applied to the standard platinum loop absorbent holder. Table I gives typical results obtained with this absorbent on ethylene-ethane mixtures' A test for absorption Was made in each a fresh bead being used for this purpose.
IND.ENG.CHEM.,ANAL.ED., (2) Blacet, F. E . , and Leighton. P. .I., 3,266 (1931).
(3) Blacet, F. E.,MacDonald, G. D., and Leighton, P. A . , Ibid.,5 , 272 (1933). (4) ward, E. c.,Ibid.,10, 169 (1938). (5) Wright, G. F., J . Am. Chem.SOC.,57, 1993 (1935). PREBEXTED a t the Annual Convention of the Chemical Institute of Canada. Toronto, June 1946.
Determination of Carbon Monoxide A Microgravimetric Method JASON M. SALSBURY', JAMES W. COLE, AND JOHN H. YOE Cobb Chemical Laboratory, University of Virginia, Charlottesville, Va. A microgravimetric method, accurate to about 2940, is described for low concentrations (0.002 to 0.1%) of carbon monoxide in air. The gas is drawn over Hopcalite at 195' C. and the carbon dioxide thus formed is absorbed in microabsorption tubes containing Ascarite, the volume being measured with a flowmeter and stop watch. From the weight of carbon dioxide absorbed in the tubes the percentage of carbon monoxide is calculated.
7 ARIOUS methods for the
determination of lo^ concentrations of carbon monoxide in air have been reported (1-4). Many of them are based on oxidation, but none involves direct weighing of the oxidation product, carbon dioxide. During the course of a problem involving a search for new carbon monoxide detectors and the devising of instruments for carbon monoxide determination, a microgravimetric method was developed for analyzing low concentrations of this gas. The method is based on the oxidation of carbon monoxide by air in the presence of Hopcalite and subsequent absorption of the carbon dioxide in tubes filled with Ascarite. From 0.002 to O.lyocarbon monoxide in air may thus be determined with a n accuracy of about 27,.
APPARATUS
v
A diagrammatic sketch of the apparatus is shown in Figure 1.
1
Purification Train. For Air. Outside air is drawn successively through calcium chloride, charcoal, and soda-lime mixture (Chemical Warfare Service mixture), purified silica gel (dry), a moisture indicator (cobalt chloride on silica gel), Hopcalite (room temperature), purified silica gel (dry), calcium chloride, soda-lime, Ascarite, and finally calcium chloride. The purified air then enters the catalyst chamber. For Carbon Monoxide in Air. The air containing carbon monoxide is purified by successive passage through calcium chloride, charcoal, and soda-lime mixture (CWS mixture), soda-lime, Ascarite, calcium chloride, purified silica gel (dry), and the cobalt chloride moisture indicator. The gas then enters the catalyst chamber. Microabsorption Tubes. Four microabsorption tubes are used
Present address, American Cyanamid Co., Stamford, Conn.
V O L U M E 1 9 , NO. 1, J A N U A R Y 1 9 4 7
Figure 1.
67
Diagrammatic Shetch of Apparatus
1 1 -
with a piece of wet flannel. (This cools the tubes during a measurement and allows the air in them to expand after they are disconnected and taken t o the balance room, thus preventing room air from entering.) The gas to be analyzed is drawn through the purifying train, the Hopcalite, the microabsorption tubes, and finally through a flowmeter. The rate of flow may be any constant value between 25 and 100 ml. per m i n u t e . T h e s y s t e m is flushed immediately with a measured volume of purified air (see tables\, the tubes are weighed, and from their gain in weight the per cent of carbon monoxide is calculated.
RESULTS
essentially similar to, but slightly longer (190 mm.) than, the type recommended by Pregl ( E ) for the absorption of carbon dioxide and water in the analysis of organic compounds. Pregl's directions for filling and handling the tubes are followed in general. The first tube is packed very tightly \T- i t h Dehydrite, saturated with carbon dioxide, and flushed with uiified air a t 100 ml. per minute. The second tube contains Behydrite for one third of its length and 12- t o 20-mesh -1scarite for two thirds. The third tube contains successively Dehydrite ( l / 5 of length), 12-20 Ascarite ( 3 / ~ ) , and Dehydrite ( i / 5 j , This is mainly a safety tube. The fourth or control tube contains equal lengths of Dehydrite and 12-20 Ascarite. Small wads of cotton are placed at the ends of each tube and between the absorbing sections. The tubes should be refilled every two weeks. The ground joints of each tube are sealed with Kronig's cement and the tubes are connected in the train with "aged rubber tubing". I n weighing, the tubes are wiped and allowed to stand 10 minutes in the microbalancc instead of 5 as recommended by Pregl. The need for two ahsorption tubes for carbon dioside is evident from the results viith 10 liters of air containing O.OOScc carbon monoside: Weights of .Lbsorption Tubes Weight after absorption of C O I , mg. Weight belore absorption of CO,, nig. Gajn in weight, mg. Gain in weight of control, ma. Gain in weight due t o COX,mg.
Tube2 9.961 8.586 1.375 0.007 1.365
Tube 3 6.025 5.998 0.027 0.007 0.020
Tube 1 6.650 6.643 0.007
PROCEDURE
Outline of Method. Air containing carbon monoxide is purified, dried, and then passed over Hopcalite a t 19.5" C. to oxidize the carbon monoxide to carbon dioxide. Following the Hopcalite are microabsorption tubes containing Dehydrite and Ascarite; next is a flowmeter connected to a suction source. The volume of gas or air passed through the tubes is calculated from the flowmeter readings. Conditioning of Hopcalite. The Hopcalite is heated in a bath of boiling decahydronaphthalene (technical, boiling point 195 O C.) and purified air is drawn through for a t least 30 minutes; then 1 liter of the gas t o be analyzed is drawn through, followed by 500 ml. or more of purified air. The Hopcalite is then ready for use. This conditioning of the catalyst before the actual analysis is essential. Table I1 shows that a very low value for the expected carbon monoxide concentration is obtained when the Hopcalite is not conditioned. So long as either air or the gas to be analyzed continues to pass through the Hopcalite, no further conditioning is necessary; however, if it stands more than 5 minutes without air or gas flowing through it, conditioning appears t o be necessary to eliminate a low value for the next analysis. Typical Run. When the Hopcalite is ready for use, the second, third, and fourth absorption tubes are weighed on a microbalance; then all four tubes are connected as described above and attached to the exit end of the catalyst chamber. Each tube is covered
Microanalyses were made on two samples of carbon monoxide in air obtained from the Xational Bureau of Standards (cylinder -1,0.0025% and cylinder B, 0.00S02% carbon monoxide by volume). The authors results with these samples are recorded in Table I. I n each series the analyses n'ere made consecutively. These samples from the Sational Bureau of Standards were used to evaluate the method. I n Table I the carbon monoxide content found by the microgravimetric method was within 1% of the bureau's value on the sample of 0.0025% and Rithin 2% on the sample of O.OOSO~Ocarbon monoxide. Xext a sample of carbon monoxide in air containing about 0.1% carbon monoxide n a s prepared. Microanalyses of this mixture gave the values in column 4 of Table 11.
Table I.
Rlicroanalyes of N.B.S. Samples of Carbon JIonoxide in Air
Volume of Gas LiteTs
I'olurne of Air Liters
20.00 20.00
1.00 1.00
Series I 12 00 10.10 Series I1 10.00 10.00 10.00
co
R a r e of Concentration Comments Flow Found 3fL.i min, % Cylinder A (0,0025% CO) 100 0.00242 Hopcalite conditioned 100 0.0026a Hopcalite conditioned At'. 0,00252 + 0.00010 Cylinder B (O.OOROz% C O )
1 00
1.00 1.00
1.00 1.00
100 100
0 0079s 0.0078r
Hopcalite conditioned
100 100 100
0 . O07gs 0.0078, 0 00738 0.00791
Hopcalite conditioned
AV.
S o t included in average 0.0000s
=t
Table 11. Microanalyses of 0.1% Carbon 3Ionouide in Air Volume of Gas -111.
a
Tolurne of Air Ml.
1000 1000 1000
500 500 500
1000 1000 2000
500 1000
500
CO Rate of Concentration Flow Found Comments Ml lmin. % Series I 25 O.Ofi8ao Hopcalite not conditioned 25 0.093s 25 0 091s Series I1 25 0.099r Hopcalite conditioned 25 50
X o t included in average value.
0.09SO
0,1022
68
ANALYTICAL CHEMISTRY 4CKYOWLEDGMENT
Table 111. \Iicroanalyses of 0.01% Carbon ?Ionoxide in i i r T olunie i > f G:iq
Latrr,
1 olume of Air Lzters
CO Rate of Concentration Fluu Found Coninients 111 /man c70 Series I Hopcalite conditioned 100 0 01334
10 00 10 00 lei.70 I) 50
1 50
1.00 1 00
100 100
15 00
1.00
100
13 80 10 00
1.00 1.20
100 100
I ~
on ..
100 ~~.
4r.
The authors wish to thank Charles H. Lindsley for his intei c-: in this work and for helpful suggestions and criticisms. Thankc .ire also due the National Bureau of Standards for supplying -tandard samples of carbon monoxide-air mixtures
0 012%
o.Oi3L
0 01400 Series I1 0 01341 Hopcaliie cunditioiied Series I11 0 01380 Hopcalite conditioned 0 01346 0 01350 * 0.00024
LITERATURE CITED (1) Berger and Schienk, U. S. Bur. Mines, Tech. Paper 583 (19381. ( 2 ) Brown, Felger, and Hon-ard, IND. ENG CHEM.,ANAL. E D , 17 (.3)
287 (1945). Jacobs, “Analytical Chemistry of Industrial Poisons, Hazai d. and Solvents”, p. 320, NeK York, Interscience Publishei
3.
1941
Polis, Berger, and Schrenk, U. 9. Bur. Mines, R e p t . InvestigatiorLa 3785 (1944). (.5) Pregl, “Quantitative Organic Microanalysis”, 3rd English ed., 11. 42, Philadelphia, P. Blakiston’s Son & Co., 1937. (4)
,.1he
carbon monoxide-:iir mixture \viis thcln tlilutc~el n-itli c,iiough carbon monoxide-fret, air to yield a n estimiitcd 0.013rc; (.arbon monoxide in air. llicroanalyses of thib s:implc) g;ivct :in :iverage value of 0.0135cC; carbon monoxitlc :I? i- c1ion.n 11y the, t1at:i recorded in Tahlc 111.
H a s E v 011 work done f o r the Office of Scientific Reaeurcli and Derelopiiieiit w i t h the Rector and Visitors n i the Cni\.Fr. under Contract Su OE\Iar-13!~ sity of Yirginia.
Estimation of Types of Penicillin in Broths and Finished Products A Microbiological Method KIYOSHI HIGUCHI
AND
W. H. PETERSON, Depnrtment of Biochemistry, University of Wisconsin, .Wadison, W i s .
A method for the estimation of tlie relatiTe amounts of three penicillins in a mixture by means of a microbiological differential assay is described. Staph. aureus 209-P, Bac. brevis, and Organism E are used i n the procedure. issays on various known mixtures and reco~eriesof penicillins added to broths were carried out. Data on several assays of commercial products are presented. The possible presence of unknown penirillins in penicillin preparations is di5cussed.
T
HE term penicillin today denotes a number of closely related
compounds, a t least four of which are well recognized: G (benzylpenicillin), X (p-hydroxybenzylpenicillin), F ( Az-pentenylpenicillin), and K (n-heptylpenicillin) (d). These compounds differ not only chemically, but also in antibiotic propertiw (1, 4,5, 7 , 9). Since present methods of penicillin production usually yield mixtures, much effort is being devoted to devising methods for estimating the relative amounts of each penicillin in a given product. Partition chromatography has bevn successfully used in the quantitative fractionation of penicillin mixtures (6). Snother procedure is that of counter-current distribution (5). These methods seem particularly advantageous in the detection of unknown penicillins because they are essentially separation procedures. These physical methods have bevn rcportedly used on quantities of 25 mg. and more, which limits their application. Schmidt et al. (8)showed that penicillins F and X elicited diffrrent responses from Bac. subtilis NRRL B-558 than from tlie standard assay organism, Staphylococcus aureus KRRL B-313. By determining ratios among these responses, a n indication of the composition of a mixture of penicillins could be obtained. The present method is a n extension of the principle of ratios to three penicillins and three test microorganisms. EXPERIMENTAL
This paper describes a microbiological differential assay niethod with which an estimation of the relative amounts of three
different penicillins in a mixture is possible. Theoretically, . i c method can be extended to mixtures of more than three penicillins, but the consequent increase in errors makes its practical application difficult. Crystalline penicillins G, X, F, and K were used as standards. The three organisms used in the assay mere selected after a preliminary survey of about sixty gram-positive bacteria. The rrsponses of the organisms to the different penicillins in a liquid medium were studied (Table I), and on the basis of their differvntial responses and growth characteristics Staph. aureus 209-I’, Hac. brevis, and an unidentified spore-forming lactic, called F:, were chosen for the assay purposes. Comparisons on a weight basis of the effects of the various penicillins on the assay organisms are given in Table 11. The inhibition values are calculatcd from the data in Table I and column 3 in Table 11. For examplr, 0.025 unit per ml. of penicillin G is required to inhibit Staph. aureus, but since one unit equals 0.6 microgram then 0.025 X 0.6 = 0.015 microgram is the weight of G required for inhibition. Likewise, 0.06 X 1.11 = 0.067 microgram per ml. of Xisrequired to inhibit the growth of Bac. brevis. This latter value represents the largest quantity of any of the penicillins required t o inhibit any of the three assay bacteria. The penicillin required in thc least amount for any of the three organisms is K; only 0.011 microgram per ml. is needed to stop the growth of Staph. aureiis. However, in order to compare the results obtained in this paper with previous results, all data are expressed in terms of the standard unit, which is the activity equal to 0.60 microgram of
