56
INDUSTRIAL AND ENGINEERING CHEMISTRY
accumulation of water in the boiler. To cut down the amount of heat received by D,round-bottomed flasks of about 5-cc. caacity are used for extractors whose capacity is 5 cc. or less. he ratio of L to M (Figure 1) should be about 1.7. In the smaller extractors, where the smaller opening a t H causes increased resistance, this ratio is increased to 2.0. Some examples of dimensions are given in Table I.
5
Figure 2 illustrates another type of liquid-liquid extractor for solvents lightcr than water. This form has a shorter path for vapor flow between boiler and condenser, resulting in easier circulation of extracting solvent. It has the disadvantage that sampling of extracted liquor is not feasible; with the type previously discussed spot samples can ‘be withdrawn by introducing a capillary pipet to the bottom of B , Figure 1.
Determination
Vol. 17, No. 1
Figure 3 illustrates a 25-cc. extractor for solvents heavier than water. The shape shown for the extraction chamber B, has been found satisfactory. The 15-mm. glass tubing of &ch B is made is heated over a short distance and pushed in to about half the depth of the extraction chamber with the edge of a knife to form each “step”. The bubbles of solvent descending throu h B are delayed at each step, thus allowing more time for equili%rium to be established between the solvent and the aqueous solution.
LITERATURE CITED (1) Gadamer, Ann., 237, 68 (1887). (2) Lie5, M., Marks, C., and Wright, G. F., Can. J . Research, B15, 529 (1937). (3) Morton, “Laboratory Technique in Organic Chemistry”, New York, McGraw-Hill Book Co., 1938. (4) Spencer, E. Y . ,and Wright, G. F., J . Am. Chem. SOC.,63, 1281 (1941).
OF
Carbon, Hydrogen, and Chlorine in Gaseous Compounds
Application of the Microtrain E. W. BALE, H. A. LIEBHAFSKY, AND L.
B. BRONK, Research Laboratory, General Electric Co.,
khenectady,
N.Y.
The policy of this laboratory has been to use the microtrain in the study of problems different from the routine determination of carbon and hydrogen, and to modify the train in whatever way these problems seemed to require. O n the whole, this policy has been successful, although the work has not always been carried far enough to give results of the highest precision. The following determinations have been carried out on the train modified in various ways: the ultimate analyses of gaseous hydrocarbons, of chlorinated hydrocarbons, and of hydrogen chloride; the ultimate analysis of the highly volatile liquid methylene chloride;
an estimate of completeness of combustion when only tracer of material remained unburned; an estimate of the vapor pressure of camphor, a relatively nonvolatile substance. Combined chlorine was determined b y weighing an absorption tube containing silver. Before quantitative results could be obtained, it was necessary to explore the fixing of chlorine by silver rt various temperatures. Although this exploration needs to be extended, it seems clear that the process involves reactions other than the simple oxidation of silver b y elementary chlorine. Voriour techniques found advantageous in this laboratory are described.
THE
chloride, these being the two simplest chlorinated hydrocarbons. For this work, the microtrain was modified by including a different type of gasholder, and a separate quartz tube containing silver, which permitted (for the first time to the authors’ knowledge) the direct weighing of the chlorine as silver chloride. These applications have in turn suggested others. For example, instead of a-ialysing an exhaust gas, one can determine carbon in an atmosphere saturated with the vapor of an organic compound, which makes it possible to measure vapor pressures in the difficult range near 0.1 mm. Highlyvolatile liquids, usually awkward to handle quantitatively according to the common methods, have been analyzed without difficulty by vaporizing weighed samples thereof in a closed space prior to combustion. When the present work was prepared for publication, it was discovered (not unexpectedly) that previous investigators--Kirner . ( 6 ) ,for example-had modified the microtrain in ways suggestive of those described belovv. Pregl himself (IO) used a gasholder to collect the gas from his microtrain for recirculation to ensure complete combustion. In view of this fact, the authorfi were surprised to find only one other mention of a gasholder being used with the train (If), and here neither reference nor data are included. Marion and Ledingham (8) have analyzed microsamples of gaseous hydrocarbons by injecting the sample from a gas buret into the oxygen stream through a jet at the inlet of the combustion tube. Huffman ( 5 )and Belcher and Spooner (8) have removed the silver from the combustion tube for the successful, subsequent (but indirect) determination of sulfur; the latter authors also attempted to estimate chlorine indirectly on their silver fillings. Their result8 were always low, probably because
quantitative determination of carbon and hydrogen by microcombustion is of comparatively recent origin. It is consequently not surprising that much of the effort in this field has thus far been concentrated on the most challenging problem involved-namely, the obtaining of routine, accurate results on small samples of organic solids. Necessary as this work has been, and valuable though its results are, the emphasis on routine and on accuracy has inevitably fostered a conservative attitude toward radical changes, with the result that the versatility of the microtrain has not always been widely appreciated. In the wide range of problems with which this laboratory is concerned, high precision, while welcome, is not usually an indispensable requirement. It has accordingly often been possible to modify the microtrain to suit a particuIar problem without stopping to prove that the modification in question brought with it the ultimate in precision. The microdetermination of carbon in steels and alloys has been reported ( I ) ; the simultaneous determination of carbon, hydrogen, and chlorine in gases and vapors is described below. About a year ago, it became necessary to determine the completeness of combustion when a hydrocarbon fuel was burned with a large excess of oxygen. The combustible compounds of carbon and hydrogen remaining in the exhaust gas corresponded to less than 0.1 mg. of total carbon and hydrogen per liter. By the introduction of a suitable gasholder, and by proper manipulation, the problem WBS solved on the microtrain. Subsequently it became desirable to investigate the simultaneous determination of carbon, hydrogen, and chlorine; work was therefore begun on the ultimate analysis of methyl chloride and of methylene
ANALYTICAL EDITION M E
SCALE-CM.
Figure 1.
Diagram of Train Arranged for Combustion of Highly
A . Quutx pnheater (platinum roll RUlns et Po00 C.) B. Kralrl bubble counter and ebaorpUon tube C. Sample tube for voletlle liquids D. G I . w n c l o w d Iron weisht E. Sepwatory lunnel Alkd with m.tsuIy F. G.1 holder (135-ml. volume when w e l d with 1-m. dopth of mercury) C. Quwtx combustion tub. (pktlnem-(oll Allins et P(xl0 C.) 1,s. 3,4, 5,6.7. StowoJO
d v e r chloride was lost to the combustion tube; this difficulty disappertra when the tube containing the silver is weighed, as it is in the authors' method. A P P A R A T U S AND G E N E R A L M A N I P U L A T I O N
Figure 1 shows the combustion train in its most extended form, as it is used for the analysis of volatile liquids. (Gascylinders, pressure regulator, absorption tubes, and Mariotte bottle are not shown.) Parts of the train are described below in more detail; in general, the gas to be burned is forced through by allowin mercury to flow from the separatory funnel, E , to the gasholfer, F. M is a grounded wire; if there is no grounding, tiny bubbles of mercury continually leap up from the mercury surface in F, and there is the possibility of an explosion hazard owing to the accumulation of static charge. The pressure regulator and the Mariotte bottle are used in the conventional wa to control the rate of flow of gas (8 ml. per minute in all work) tzrough the train. Cylinder oxygen is used for combustion, and 150 cc. of cylinder air for sweeping. Suitable electrical furnaces, each mounted on a movable stand to facilitate chan es in the train, are used for heatin the preheater, A , the com%ustion tube, GIand the chlorine &e, L. The interchangeable joint in the sample tube, C, and all stopcocks are lubricated with Apieaon r a s e , which, being intended for high-vacuum work, has a negligi le vapor pressure. All parts are butted together and connected with impregnated tubing. The platinum-foil fillings for the preheater and the combustion tube are made b wadding sheets 0.0013 cm. thick and 12.7 cm. square annealeland etched by the vendor, into cylinders of the desired size. The furnaces are adjusted to maintain the middle near 800" C. The silver fillin6 for the chlorine tube, L,consists of a l-cm. roll of fine silver mre a t each end, with electrolytic silver crystals between. The ends of the chlorine tube are drawn down and terminate in capillaries. Satisfactory analytical results were obtained with the hottest section of the silver filling near 600" C.; at temperatures of 400" and below the results were unsatisfactory; silver chloride begins to volatilize appreciably at temperatures hi her than 600". Stanfard tubes are used for the absorption of carbon dioxide and of water, The tare tube, standard also, contains both Ascarite and Anhydrone; it follows the other absorption tubes in the train. All tubes, including the chlorine tube, are wiped each da before the first run; for wei hings durin the reat of the day, o d y the ends of the tube are cfeeaned. In %ot, humid weather, the tubea stand beaide the balance for 30 minutes to ensure reschink equilibrium; only 5 minutes' standing is required in the mnter. The use of air in sweeping the train makes it unneceaeary to aspirate the absorption tubes.
Volatile
Liquids
H. Platlnum-loil W l e I. aur(rcepiiluv 1. Rubber atopper K. A i r jet L. Quartz tobe ( t i l v ~Rlllng at 6000 C.) M. Wlre to sround NP. Alisnment ol appw~tuae1 asen horn above
The chlorine tube requires s ecial handling. Upon bein removed from the train, it is pLced inside a brass tube, w%ich promoteacooling and protects the silver chloride from light. The silver filling may be trusted to absorb 15 mg. of chlorine before cleaning is required; the filling is cleaned in the tube b the action of 10% potassium cyanide solution. After a thorou riming, the cleaned tube is fired in hydrogen and subsequengy in oxygen.
E
ANALYSISO F PUREGASES.
The sampling of pure gases for analysis w119 a c c o m l i s h e d a s follows: The gas to e! analyzed was filled into a small bulb (Figure 2) a t a ressure somewhat above atmospKeric, and allowed to reach room temperature, whereupon the excess of gas was allowed to esca e. From the volume of the b d , as determined by calibration with mercury, the volume of the gas sample a t standard conditions was calculated from the as laws. The weight of the O ' P s , d e could then be calculated from the known density at 0" C. S C A L E - CM. (This procedure is tantamount to assuming that the gas is equally Figure 2. Sampling Vmcl imperfect a t 25" and a t 0" C., IO, 11 sto~odu which is permissible; it is not permissible to w u m e that the gas is ideal.) The samp!: was then introduced into the asholder by the following o rations: (1) The mercury was &awn from the gat+ holder, r b y evacuatin the funnel E, while admitting oxygen through stopcock 6; (27 the gashofder was evacuated through stopcock 6; (3) the long arm of the sample bulb (Figure 3) w m filled with mercury to stopcock 11, and the short arm was then attached by rubber tubing to stopcock 6 of the gasholder; (4) the pas sample was expanded into the gasholder; ( 5 ) the last rtion of as in the sample bulb was pushed into the holder by &ng the %ulb with mercury from the long arm; (6) the mholder was filled with oxygen to atmospheric pressure. i n s error due to the presence of room air in the open ends of the t u b at stopcocks 6 and 10 is negligible. During all these operations, the rates of flow of mercu and of oxygen must be carefully adjusted to avoid serious di%culties, such as drawing sulfuric acid from the remure regulator into the reheater. The dikted gas is finally forced tfkough the combustion train in the manner already described. Experiments showed that incomplete combustion resulta when too rich a mixture is forced through the triin: 1 volume of sample to 30 of oxygen gave in-
58
INDUSTRIAL A N D ENGINEERING CHEMISTRY
complete combustion in the case of methyl chloride; when the ratio was 1 to 65, combustion was complete. ANALYSISOF A HIGHLYVOLATILE LIQUID. By means of the usual technique involving a preliminary evacuation, {,he liquid was forced by atmospheric pressure into a weighed glass bulb, 5 mm. in diameter, through a capillary stem 4 cm. long. The bulb was next surrounded by two small b l o - h of aluminum, whose interior surfaces had been grooved to fit it. The assembly was then immersed in acetone cooled with solid carbon dioxide. After the sample has been frozen, the protruding stem of the bulb was cleaned by entle warming with 0 5 IO a microburner, and t f e n sealed. (An abu solutely clerrri seal was always obtained in SCALE -CH. this way; without the metal blocs, t h e r e w s occasional carbonization.) After being Figure 3. weighed, the sealed bu!b was introduced L a r g e Gasinto the sample tube, C (Figure 1). C was h o l d e r (2.3 then connected to the bubble counter, and Liters) oxygen was passed through it for 10 minutes. 0. Glrir-lo-metsl At the end of that time, stopcocks 1 and 2 real R. Metal Rttlng were closed, and the assembly of the train S. Outlet to comwas completed by connectin the outlet end bustion train of .C to the gaaholder, F. %e sample bulb 8, 9. Stopcocks was then smashed by means of the glassenclosed weight, D,actuated by an external magnet. The mixture of oxyger and gaseous sample was then allowed to expand into the evacuated gasholder, F . The procedure from this point on was essentidy identical with that followed in the analysis of a pure gas after the sample had been transferred to the gasholder. Various other schemes were tried, but that described proved the most convenient. COMPLETENESS OF COMBUSTION
The problem was to estimate traces of incompletely burned material in a hot exhaust gas containing excess oxygen, 4.5 carbon dioxide, and 5% water vapor, both percentages being by volume.
Vol. 17, No. 1
make assumptions about the composition of the unburned material. Since a thorough discussion of the matter would require too much space, suffice it to say that combustion must have been about 99.9% complete, and that this fact could not have been established by any of the conventional methods. The results on the standard propane mixture are good enough to recommend the micromethod as a simple, approximate approach that can often be substituted to advantage for more elaborate schemes. DETERMINATION OF VAPOR PRESSURE
Camphor is relatively nonvolatile, so that its determination in saturated air does not diffw greatly from t,hc foregoing experiments on the completeness of combustion. A single experiment was done to show that the combustion of gases in the microt,rain can be a convenient way of determining vapor prcssures. An ordinary absorption tube filled with synthetic camphor was attached to the bubble counter and swept with oxygen for 30 minutes. It was then connected to the combustion tube followed by the usual absorption tubes, and 4@0ml. of oxygen saturated with camphor were passed through the train at the usual rate. The net weight of carbon dioxide absorbed was 2.961 mg., which leads to a vapor pressure of 0.32 mm. at 30.5" C., the temperature of the camphor tube. This result is in good agreement with 0.39 mm., the corresponding value from the International Critical Tables. At the present time, there are serious discrepancies among the published values for the vapor pressure of cnmphor, and the method proposed here might well be used to improve the situation. Under many conditions, this method of arriving at vapor pressures will be preferable to obtaining them by simple weight loss because ample amounts of the substance can be used to ensure saturation; the weight of carbon dioxide will often exceed the corresponding weight of test substance; and difficulties in weighing the test substance are avoided.
Table 1.
Dcterminrtion of Completeness of Combustion For 1 Liter of Gsl
Correated for Blanks
Sample Ha0 cos The Sam le was transferred via a metal line directly into the Ye. Me. Iar e asho7der (Figure 3) by means of a cop r fitting, R, at0.596. 0.137 Exhaust gas 1 tacted to the holder by a Fernico seal. Enougfmercury to seal 0.086. 0.071 the vertical tube was always left in the holder; a representative 0.314. 0.089 sample was obtained by allowing thorough purging before the AI. 0.332 0 . OQQ holder was disconnected from the line. (Less than b l a n h ) 0.028 Exhaust gas 2 When the sample waa brought to the laboratory, a separatory 0.123 0.165 Propane 0,081 0.148 funnel containing the mercury to be used in forcin the exhaust Propane (theoretical) gas through the train was attached a t R. The com%ustion train Water values erratic, but improved in subsequent. iirniler determinaconsisted of the following parts connected in series to 8: bubble tioru. Completeness of combustion calculated on basis of CO, data. Calculationa based on water data would not have altered conclusions. counter, B (Figure l), which included absorbents for the water and carbon dioxide in the sample; combustion tube and inlet, I , G (Figure 1); water and carbon dioxide absorption tubes; tare tube; and Mariotte bottle, which served to measure the 500 ULTIMATE ANALYSES OF VARIOUS COMPOUNDS ml. of gaa taken for a determination, the rate of flow being maintained at 8 ml. per minute. After the sample, which contained Final results from the work on the ultimate analysie of various its own oxygen, had been burned, the gasholder was removed; compounds are reported in Table 11. With the chlorine comthe train was then connected to the preheater and swept with 150 pounds, a great deal of exploratory work had to be done before ml. of air. The following blanks were evaluated and applied: (1) the satisfactory results could be obtained. Data from this work are water and carbon dioxide obtained when 500 ml. of exhaust gas not included i s Table 11, and the work is discussed briefly in the were passed through the bubble-counter assembly with the comfollowing section. bustion furnace at room temperature; (2) the water and carbon dioxide absorbed when the air used for sweeping was passed Hydrocarbons were analyzed according to HYDXOCARBON~. through a train with the combustion furnace at room temperathe method for pure gases-that is, by use of the small sample ture, and (3) with the combustion furnace a t 900' C. bulb (Figure 2). The data in Table I1 for propane are satisfacSince the amount of unburned material in the exhaust gas was tory and require no comment. The analysis of methane also protoo small for estimation by the conventional methods, it was not ceeded smoothly, but the results for t F o different products diffeaaible to prepare a standard exhaust gas for evaluating the refered widely because only methane 2 was of good quality. In the liability of the micromethod. An acceptable substitute w a ~ case of methane 1, a commercial gas that may have been only 85% found in room air containing propane in a concentration correure, the total exceeds 100% because the actual density must sponding to 0.041 m , of carbon (or 0.148 mg. of carbon dioxide) Eave been greater than the density of pure methane, which was and 0.009 mg. of hyzrogen (or 0.082 mg. of water) per liter. This used in calculating the weight of the Sam le standard mixture waa successfully analyzed with reaults given in CHLORINE COMPOUNDS.Hydrogen choride and methyl chloTable I, where data for the exhaust gases are also recorded. ride were analyzed by the method for pure gases. Methylene chloride was analyzed by the met,hod for highly volatile liquids. The data in Table I are not in themselves sufficient for a calI n these analyses, of course, the chlorine tube, L, was included in culation of the completeness of combustion; it is necessary to the train.
ANALYTICAL EDITION
January, 1945
Table II. Ultimate Analysis of Various Gases Sample P r o p a n e (theoretical) Propane Hydrogen chloride (theoretical) Hydrogen chloride Methyl chloride (theoretical) M e t h y l chloride 1 l l e t h y l chloride 2 M e t h a n e (theoretical) Meth:ine 1 Xethane 2 Methylene chloride (theoretical) Methylene chloride
%C
% H
81.71 81 5 81 8
18,29 18.8 18.8 2.76
% CI
97:i3
;:$;99 20 .. 72
2i:79 23 6 23.9 23.6 23.8 74.87 84.7 83.7 75.1 74.8 75.7 14 14 14 6 14 5 14.4
2 95 5.99 6 00 6.07 6.01 5.98 25.13 26.1 26.1 25 9 25.4 25.8 2.37 3.4 3.5 3.1
90.2 70.22 69.6 70.2 70.4 69.9
83'49 80.8 80.3 80.9
Total
e6
100 00 100 3 100 6 100 00 95 6 93 0 93 2 100 00 99 2 100 2 100 0 99 7 100 00 110 8 109 8 101 0 100 2 101 5 100 0 98 8 98 3 98.4
Table I1 shows that, the results for methyl chloride from two sources were srrtirfactory. The results for methylene chloride are probably satisfactory; a test with sodium showed that the liquid used contained some active hydrogen, and this probably explsiris why a total of 1007, was not quite obtained. It is possible, however, that here, though to a much less extent than with hydrogen chloride, not all the chlorine was fixed by the silver. FIXING OF CHLORINE BY SILVER
The first attempts to determine chlorine in chlorinated hydrocarbons by direct A-eighing were consistently unsatisfactory, mainly because the silver tube gained too little weight. It seemed advisable, therefore, t o study the behavior of chlorine and of hydrogen chloride in t,he train. Although it is common knowledge that chlorine is completely retained by a universal combustiontube filling, so many complex reactions are possible under these conditions that this experience can scarcely be used to judge whether or not chlorine can be completely fixed in an absorption tube containing only silver. The most obvious way in which silver could fix chlorine is by the reaction Ag '/tClz = AgCl (1)
+
in which chlorine act3 as oxidizing :\gent; but the reaction Ag
+
l/r02
+ IICl = AgCl + '/*HI0
(2)
in which thc oxidizing agent is oxygen, must also be considered. The gaseous equilibrium in the Deacon process IICl
+ '/,On S '/tClz + '/,HzO
(3)
if maintained, would furnish a mechanism by which silver chloride torni:ition could occur principally by the faster of the two steps, Rcactioris 1 and 2. (In the equations, all substances except silver and silLer chloridc are gases; silver chloride melts a t 455" C.) In espcsriments with chlorine and hydrogen chloride, a standard absorption tube filled with potassium iodide crystals was inserted in the train at i w i o u s points to serve as a qualitative chlorine indicator. Chlorine \vi11 of course rapidly liberate iodine from the iodide; the corresponding reactions of chlorine compounds formed during combustion might be more complicated. No difficulty was ever encountered in handling or weighing the absorption tube containing potassium iodide; even when freshly prepared, the weight of the tube WRS constant to within 0.10 mg. Unfortunately, its capacity to absorb chlorine completely is very limited, and some of the iodine formed always volatilizes. It is, however, qualitatively satixfactory. The combustion experiments with chlorine, done by various modifications of the method for pure gases, were not qumtitative because an accurate sample of thc gas could not be forced through the train by the authors' technique. Chlorine, even when dry,
59
reacts instantly with mercury. A thin laycr oi sulfuric acid protected the mercury sufficiently, ho\vever, to make rough experiments possible. In none of theseexperiments, in which the silver tube was at different temperatures not exceeding600" C., wasmore than 75% of the chlorine fixed by the silver. Both absorption tubes generally gained tvcight; there was never any gain i n weight nor change in color of the potassium iodide tube when it was placed beyond the carbon dioxide tube. JVhen it was placed after the silver tube, however, it often gained weight without bccoming colored; and, under these conditions, the water and the carbon dioxide tubes gained weight also. The simplest explanation for this puzzling behavior would assume the formation of some chlorine oxidcs in the silver tube, the presumption being that these react with the potassium iodide to give higher-valent iodine compounds, but that they react less rapidly than does chlorine under the same conditions. In any event, the work showcd that Reaction 1 cannot be relied upon to give complete fixing of chlorine by silver. The combustion experiments with hydrogen chloride were also complicated, though t o a less extent, by reaction with mercury. Equilibrium calculations show that hydrogen chloride at room temperature tends to react with mercury, giving calomel, until the pre.qsure of hydrogcn chloridc becomes comparable with that of the hydrogen produced. Titration exporiments on the hydrogen chloride used showcd that the strengtl~of this gas was reduced from 99 to 97% during 2 hours in contact with a small pool of pure mercury; a trace of calomcl \vas found. Tile rate of cnlome1 formation under these conditions \vas not great enough to influence apprwiably the results in t hc combustion exptlrinients \\it 11 hydrogen chloride. Results of the last three experiments \\it!) Iiydrogen vllloritle are given in Table 11. These were done by the method for pure gases, except that the combustion furnace \vas heated only in the third. As Table I1 shows, virtually identical rcfiults were obtained in all three. I n every case the hydrogen value is correct, but the chlorine value is low. Since not all of the missing chlorine could hnve gone to form calomel, it follows that the fixing of chlorine is incomplete for a mixture of hydrogen chloride and oxygen, whether or not this mixture has been previously passed over hot platinum. But complete fixing of the chlorine is much more nearly appro? ched here than it was with mixtures of chlorine and oxygen; the hydrogen chloride contained, it ]vi11 be remembered, only 9970 IIC1. The conclusion would therefore seem to be that, Renction 2, and perhaps Reaction 3, can help in the capture of chlorine by silver; Reaction 1 is not the only n a y in which this can be accomplished. For methylene chloride, which has the hydrogen-chlorine ratio of hydrogen chloride, Table I1 shows that the chlorine is completely (or almost completely) retained in the silver tube. This suggests that carbon compounds may also play a role in the formation of silver chloride. This suggestion is reinforced b y the fact that Pregl (IO, p. 34) obtained correct carbon and hydrogen results for trichlorodinitrobcnzene, with its one hydrogen atom, though his use of a universal filling keeps the two cases from being strictly comparable. The results in Table I1 for methyl chloride, which contains both carbon and an excess of hydrogen over ehlorine, are quantitative. This important subject deserves further investigation. At the present time, one may conclude only that the presence of combined hydrogen (Reactions 2 and 3) appears to help in the fixing of chlorine by silver, and that carbon compounds may also play a part. It would seem worth while to use the experimental metho d of ~ this paper for obtaining a t least the analytical data necessary to complete the series CI,, HCl, CH,, CH,Cl, CHICl!, CHCl,, CCI,. DISCUSSION OF TECHNIQUE
The basic apparatus and technique used in this laboratory differ from the commonly accepted practice (9) in the following re-
60
INDUSTRIAL AND ENGINEERING CHEMISTRY
spects: The rate of gas flow during combustion and sweeping is 8 ml. per minute; electrolytic silver crystals are substituted to somc extent for silver wire (7‘); a tare absorption tube is always employed (9, p. 136); a large-capacity preheater filled with crumpled platinum foil is used (3,4) ; and platinum foil is used also in the high-temperature zone of the combustion tube. Five other fillings for the combustion tube were tried under comparable conditions. The foil, or even an empty tube with no filling ( 0 , seemed as effective as any of the other fillings in promoting complete combustion. S o particular difficulty was experienced during this work in maintaining reasonable blanks. For a total gas volume of 260 ml. (the sum of the volumes of oxygen and of air in most analyses) the water blank !vas always less than 0.100 mg.; that for carbon dioxide less than 0.040 mg. (often 0.000 to 0.010 mg.); the chlorine blank usually lay betwen -0.010 and $0.010 mg. These blanks could be maintained even though the microtrain was frequently opened, though whole sections were introduced or removed, and though the stopcocks and joints were lubricated. Furthermore, the train is located in a laboratory where many kinds of experimental work are done, and the balance is in a n adjoining room where temperature and humidity changes are much more limited. According to the authors’ experience, the items listed below also play an important role in the satisfactory operation of the microtrain: (1) No time is wasted in ‘Witch hunts” for sources of high blanks. Duplicates of all parts are kept; when the train requires
Vol. 17, No. 1
cleaning, all parts are replaced. (2) The temperature of rubber connections is never allowed to rise above that of the room. (3) Sample sizes and rates of gas flow are adjusted to avoid any condensation of water. (4) Finally, the absorption tubes are always allowed to come into equilibrium with the atmosphere of the balance room before they are weighed. LITERATURE CITED (1) Balk E. W., Liebhafsky, H. A., and Winslow, E. H., IND.ENO.
CHEM.,ANAL. ED., 15, 68 (1943). (2) Belcher, R.,and Spooner, C. E., J. Chem. Soc., 1943, Part 11. 313. (3) Bock, F., and Beaucourt, K., Mikrochernie, 6 , 133 (1928). (4) Hallett, L. T.,IND.ENO.CEEM.,ANAL.ED., 14, 974 (1942). (5) HutTman, E. W. D., paper presented at A.C.S. hfeeting, April,
1938, Dallas; see Mikrochemie, 25, 384 (1938).
(6) Kirner, W. R., IND.ENO.CHEM.,ANAL.ED., 10, 342 (1938).
(7) MacNevin, W., Zbid., 341 (1938). (8) Marion, L., and Ledingham, A. E., Ibid., 13, 269 (1941). (9) Niederl,
J. B., and Niederl, V., “Organic Quantitative Microanalysis”, 2nd ed., New York, John Wiley & Sons, 1942. (10) Pregl, F.,“Quantitative Organic Microanalysis”, 2nd English ed. by E. Fyleman, p. 78, Philadelphia, P. Blakiston’s Son and Co., 1930. (11) Scott’s Standard Methods of Chemical Analysis, N. H. Furman, ed., 5th ed., Vol. 2,p. 2480,New York, D.Van Nostrand Co., 1939. P B X S ~ N T Ebefore D the Division of Analytical and Micro Chemistry, Symposium on Practical Applications of Microchemistry. at the 108th hfeeting of the AMZRICAN CHEMICAL SOCIETY, New York, N. Y.
Use of Lactobacillus arabinosus 17-5 For Microassay of Pantothenic Acid EDWARD H. HOAG, HERBERT P. SARElT, AND VERNON H. CHELDELIN, Oregon State College, Corvallis, O f e . A rapid microbiologlcal asny method for prntothenic acid uses L. arabinosus as the test orgrnism. With the growth medium used, a m y values with good recoveries of rdded pantothenic acid may b e obtoined turbidimetrically after 14 hours‘ growth, or titrimetrically after 94 to 30 hours. The response to pantothenic rcid in the effective essay rrnge (0.01 to 0.08 microgrrm of pantothenic acid) is greater and more rapid for this orgrnlsm then for L. can;. G o o d agreement is obtained betwern prntothenic acid values of foodstuffs rs determined b y the two organisms. However, a w y valuer of yeast concentrates by the chick method i r e higher than those obkeined microbiologically.
M
ICROBIOLOGICAL m y methods for pantothenic acid using L. casei as the test drganism have received wide application. The original method of Pennington, Snell, and Williams (8) together with more recent modifications (IO, 11) has proved generally useful and satisfactory for routine determinations of pantothenic acid in a wide variety of materials. Certain difficulties are encountered with L. easei, however, even with the use of modified media and extraction procedures. The organism responds erratically in the presence of small amounts of starch, fats, and fat acids (1, a), so that i t is neceaeary to digest samples of test materials enzymatically, and to extract them with ether to remove excess fat prior to the performing of assays (10). Furthermore, even though w a y s with this organism can be performed much more rapidly than animal rssoys, a further shortening of the time required would enhance the other desirable aspects of a microbiological method. Previous studies in this laboratory have shown that Lactobocillua arabinosw 17-6 responds more rapidly to graded amounts of pantothenic acid than does L.cadci (4). This organism ia eLe0
known not to be stimulated apprebiably by starch or fat acids (6). Therefore it was decided to investigate its potentialities for u98 in pantothenic acid asays, as compared to L. msei. EXPERIMENTAL
L. arabinosus is maintained on CULTURE^ AND INOCULUM. sast agar stabs 89 described by Pennington, Snell, and Williams. $0 prepare cultures for inoculum, cells from a suitable stab culture are tmmferred into a tube containing 5 ml. of the baed medium diluted with 5 ml. of water, to whch have been added 1 microgram of pantothenic acid, 5 mg. of yeast extract (Difco) and 5 mg. of liver extract (Lederle). The inoculum is incubated at 37‘ C. for 16 to 24 hours, and 1 ml. of the cell suspension is then diluted in 15 ml. of 0.9% saline. For asaays, one drop of the resulting &lute suspension is added to each test culture. The yeast and liver extracts are added to the inoculum in order to encourage very rapid growth of the culture, as this emma in the authon’ experience to be a factor contributing toward subsequent rapid growth and high acid production in the teat cultures. A short growth period (not exceedin 24 hours) is desirable to obtain maximum viability. I n adfition, .when necessary, the grown culture may be stored satisfaotonly m
Table
1.
Composition of &sal Medium
Alkali-treated peptone (plm sodium acstate)
Glucwe
Sodium acetats Acid-hydrolysed cuein (technical) Autolyaed y e u t Noritetreated Rice bran concehtrate (Vitab), Noriktreatad Cystine hydrochloride Riboflavin Salts A and B (8) Distilled water to 1000 ml.
pH
-
6.8
B Gram 10 40 24 4
2 16 200 mg. m10 0 ml. 7 each
