ANALYTICAL CHEMISTRY
484
were grown in a medium containing 1 gram of potas~iumnitrate, 2 grams of potassium hydrogen phosphate, 2 grams of magnesium sulfate, 1 gram of calcium chloride and 1 liter of water. Before experimentation, the cells were centrifuged, washed twice, and resuspended in a 0.2M bicarbonate-carbonate mixture a t 1)H 8.8. The sodium salts of the carbonates were used.
I
LIWTI~OOII
210 220
Figure
230
240
t DUX
e30
Figure 12. Influence of Formate on the Oxidation-Reduction Potential and Gas Production of E. coli Cells Suspended in 0.01M Phosphate Buffer
t LWT
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Du
om
EM
m wa
SIO
SZQ
w
11. Oxidation-Reduction Potential and Changes by C. m;;wusii in Nitrogen
Gas
used for simultaneous recording of oxidation-reduction potentials and pH. LITERATURE CITED (1)
The results in hydrogen were obtained with pyrogallol prerent. -4s shown in Figure 10, whenever the light was turned on the oxidation-reduction potential rose more than 150 mv.; when the lights Tvere turned off, the oxidation-reduction potential returned to its original level. The gas produced was also in part coiisumcd. -4gain when the light was turned on, the osidation-reduction potential rose and gas was produced. In nitrogen the results were similar so far as osidation-reduction potential change and gas exchange were concerned (Figure 11). One experiment, using Escherichia coli as the biological s) stem, was performed. Sodium formate was used as the substrate. As hydrogen was produced from the formate by the bacterial cells, the oxidation-reduction potential dropped sharply (Figure 12). ACKNOWLEDGM E S T
T h c authors !+ish to acknowledge the contribution of E. D. Haller of Beckman Instruments, Inc.. \Tho designed tlic circuit
Arnold, W., Burdette, E. W., and Davidson, J. B., Science, 114,
364-7 (1951). (2) Burk, D., Brenneman, J., Laughead, T., Riley, V., and Wight, K. $1.. “Photosynthetic Production and Utilization of Hydrogen Gas by .4lgae,” presented before the Chemical Society of Washington, College Park, Md., May 8, 1952. (3)
Cannan, R. K., Cohen, B., and Clark, W. AI,, U . S. Public Health
(4)
Clark, W. M., Cohen, Barnett, and Gibbs, H. D., P u b . Hcaltii
Reports, 1926, Suppl. No. 55. Repts., 40, 1131-203 (1925). (5) Elema, H., Kluyver, A. J., and van Dalfsen, 8. W., Biochem. Z . , 270, 317-41 (1934).
(e) Hewitt, L. F., “Oxidation-Reduction
Potentials in Bacteriology and Biorhemistry,” 6th ed., p. 36, Edinburgh, F. and S. Livingston, Ltd., 1950. (7) Kluyver, A. J., and Hoogerheide, J. C., Biochem. Z., 272, 19i215 (1934). (5) Lipmann, F.,Zbid., 265,133-44 (1933). (9) Spruit, C. P. J., Acta Botan. Need., 1, 551-79 (1953). (10) TT‘assink, E. C., Antonie van Leeuwenhoek. J . Microbiol. Serol., 12,1281-93 (1947). RECEIVED for review September 8, 1953. dccepted January 6 , 1 9 j 4 . Presented before the Division of Biological Chemistry at the 122nd Meeting SOCIETY,Atlantic City, N.J . , September 1932. of the AXERICASCHEXIICAL
Manometric Method for Study of Solid-Gas Reactions at Moderate Temperatures PHILLIP SOUTERI, J O H N R. WILLIMOTT*, and T. G. HUNTER Chemical Engineering Department, University o f Sydney, Sydney, Australia
T
HE authors have recently had occasion to study the oyygen uptake of coal particles, in connection with an investigation being carried out on the spontaneous combustion of coal from the Greta seam of ?Jew South Wales, Australia. .1 literature survey revealed that many of the methods previously used for the study of solid-gas reactions of this nature 1
Present address, Chemical Engineering Department, K.S TV University
of Technology, Sydney, Australia. 2
Present address, I.C.I.A.N.Z. Ltd., Yarrawonga, Victoria, Australia.
suffer from various disadvantages. These methods are not discussed in detail here, as several reviews of the subject are available ( 4 5 ) . Some require highly specialized apparatus; others involve tedious analyses of the gaseous phase throughout the course of the experiment; some employ a static bed of solids, with gaseous phase flowing through, leading to possible irregularities in the results due to channeling, etc.; still others have a static bed of solids in contact mith a stationary body of gas leading to an indeterminate gaseous composition within the bed.
485
V O L U M E 26, NO. 3, M A R C H 1 9 5 4 During an investigation of the problem of spontaneous combustion of coal, a study was made of the rate and extent of oxygen absorption by coal, between mom temperatures and 120" C. Warburg's manometric method as commonly employed in biochemical work was found most satisfactory, provided certain modifications of the normal technique were made in view of the higher temperatures used and. the type of reaction studied. The procedure descrihed gives reliable results. It is simple and flexible and uses standard laboratory equipment. It would apear that the method could be advantageously employed in the study of a wide range of solid-gas reactions.
The equipment used and the procedure finally developed are described below and were found to satisfy the five conditions, specified above. APPARATUS
The chief mod6catian of the standard Warburg equipment that proved necessary was the use of oil as a heating bath liquid. This, in turn, necessitated efficientstirring to improve heat transfer to the reaction flasks and greater care over temperature control: A standard 16-position Warburg-type bath illustrated in Figure 1 was used, fitted with a very efficient stirrer and three ai! immersion-type heaters. Of these, one was employed to bring. the bath to the required temperature, a second, fitted with an energy regulator was used to maintain nearly constant temperature. while the third. fitted with a three-heat switch, acted at a much lower wattage in conjunction with a mercury-xylene regulator. In this way, temperature control accurate to within =tO.O2" C. could be achieved anywhere between the range 50" t o ,"no
I*"
It was desired to develop a method for examining the rate and extent of oxygen uptake by coal which would eliminate these disadvantages, and yet would he simple to operate, use only standard laboratory equipment, give reliable results, be sufficiently flexible to allow the effect of the variables to be investigated, and permit close standardization of experimental conditions. Gaseous absorption and evolution in biochemical systems may be very conveniently studied manometricdly by such methods as that of Warburg. Details of this and several sirnilm techniques are given in a monograph by D i o n (e). These methods are generally used to measure the oxygen uptake 01 carbon dioxide evolution of biological preparations in the presence of liquid media, under conditions similar to those occurring in viva. Blaskett has reported some measurements of oxidation rates of c a d a t 32' C. using such equipment ( 1 ) . It was therefore considered that the method might be adaptable to the higher temperatures required in this work. Modifications of the standard biochemical method proved necessary for two reasons: T h e standard method involves the presence of aqueous solutions, thus keeping the oxygen saturated with water vapor, and the temperatures used in the present series of investigations were of the order of 120" C. or le88, whereas in biochemicd work 40" C. is a ma
7 ,
U.
Borosilicate elass flasks of the standard Warburg singleside arm pattern, & shown in Figure 2, attached tomanometers graduated in millimeters, were employed. Brodie's d u t i o n ( 2 ) m s iiaerl for 8.8 t,he manometric fluid. These flask -... .. convenience ~~~.~~~~ and manometerunits were sttaohed to pivoted rocker arm8 in such a way that the flasks were completely immersed in the oil ~~~
~~~~
~
~
of the units could be agitated 2multanea;sly a t a selected speed. Rates of about 90 complete oscillations per minute were ordinarily used. As a lubricant for the ground-glass joints, Gargoyle Savarex No. 2 grease proved satisfactory a t temperatures as high as 115' C. while completely immersed in oil. The oil used in the heating bath was a mineral quenching oil which had satisfactory viscosity and heat-transfer charsetcristics. PRELIMINARY WORK AT IIIGH TEMPERATURE
Preliminary work, based on the teohnique described by Dixon
(e),involved weighing a suitable amount of coal into a clean dry
flask, and adding 0.2 ml. of 20% potassium hydroxide solution into the center well to act as an abscNrbent fo; carbon dioxide. Concertina filter paper was used to ir,crease the absorbing surface. The flasks were then flushed wi th oxygen and introduced into the bath. After some trial run8 n t t e m p e r a u c s 111 ~ i i rL W L W UL' 80" C., i t was found t h a t mater had to be rigorously excluded from the system. Any rrster present, either that resulting from the oxidation of the coal or that present in the potassium hydroxide s o l u t i o n , c a u s e d erratic behavior. Not only did increases i n p r e s s u r e oocnr where decreases had been expected, but difficulty was experienced in duplicae ing successive readings. T h e former effect was due to the evaporation of water and the latter to the condensation of this a,ater in the capillary tube, producing gas lock8 which prevented the true pressure's being recorded by the manometers. I n view of the fact that ter had to he excluded, the of potassium hydroxide ution as an absorbent for ,ban dioxide was precluded. empirical approach was therefore a d o p t e d a n d Standard Warburg-Type Bath ~
Figure 1.
A N A L Y T I C A 1, C H E M I S T R Y
486
various possible combinations of the following solid absarbents for carbon dioxide and water were tried: phosphoms pentoxide; anhydrous magnesium perchlorate (Dehydrite); sodium oxide on asb e s t o s (Carbosorb); and potassium hydroxide pellets. These were used both with and withoutcoalandin a t m w pheres of purified oxygen, nitrogen, and carbon dioxide. The effect of placing the absorbents in various positions in the flasks-i.e., in the center well, in the side arm, and mixed with the coal where this was f e a s i b l e wm investigated. The use of cones of filter paper on which the absorbents could hespresd out to increase their surface area was also examined This preliminary work, carried out a t temperatures of 105" and 115' C., was aimed essentially at ascertainingunder what conditions reasonably reproducible results could be obtained and what qualitative effeots the various combinations exhibFigure 2. Standard ited. Since such a variety Warhurg Single-Sideof combinations were tried, Arm Flask Attached no attempt was made a t this to Manometer stage to treat the results uumericallv. The followinx . summarizes the findings and their interpretation: Filter Paper in contact with any of the abmrhents used led to erratic results, although when not in contact with the absorbents it affected the absorption byonly a small, nearly constant amount. Phosphorus Pentoxide proved unsuitable for removing water vapor because of the formation of a skin of phosphoric acid. Anhydrous Magnesium Perchlorate (Dehydrite) was found to be very convenient as an absorbent far water vapor, giving closely reproducible results. It could he either mixed with the coal or placed in the side arm equally effectively. It gave no measurable reaction with carbon dioxide or oxygen. Sodium Oxide on Asbestos (Carbosorb) could ,no! be mined with the coal. Separately, It absorbed carbon dioxide readdy, but gave ermtio results because the Carhosorh itself yielded water vapor under the conditions of the experiment. Potassium Hydroxide Pellets placed in the side arm absorbed carbon dioxide readily and reproducibly and was not affected by oxygen or nitrogen. At this stage, further work was confined to coal, Dehydrite, and potassium hydroxide. It was found that the coal itself absorbed carbon dioxide only very slightly. Coal, Dehydrite, potassium hydroxide, and oxygen gave absorptions which agreed closely between duplicates. Coal, Dehydrite, and oxygen also gave close agreement between duplicates and the absorption under these conditions wa6 practically identical with that occurring when potassium hydroxide was also present. Since i t had been shown that the Dehydrite itself did not measurably absorb carbon dioxide, it was therefore concluded that any carbon dioxide given off by the coal under these experimeutal conditions must have been less than the limits of accuracy of the technique being used.
This conclusion is justified by the results of earlier workers. Thus Winmill (8)found that even a t the highest temperatures he used (up to 160' C.) very little carbon dioxide was formed in the initial stages of the reaction in csmparison with the amount. of oxygen absorbed, but that relatively more appeared later in the oxidation process. This mas confirmed by Graham (S), using other coal types. Since in the present work only the initial rates of oxygen absowtion were being measured, it was decided to dispense with the use of potassium hydroxide, thereby simplifying the procedure and subsequent calculations. Potassium hydroxide need not he used unless i t is desired to carry out more prolonged experiments or to investigate the formation of carbon dioxide over a longer period. Carbon monoxide is the only other gaseous oxidation product likely to be formed to any ext,ent by the reaction. This was neglected, as it has been shown that initially carbon monoxide is produced in considerably smaller amounts than is carbon dioxide
(a). PROCEDURE
The procedure finally developed on the basis of the preliminary work is described here. It was used for determining the rates of oxygen uptake by especially prepared samples of coal. Only slight modificat,ions, however, would be needed to adapt this method to the study of a wide range of other solid-gas reactions or to the study of the evolution of gas by a solid. The clean reaction flasks were weighed, and evacuated storage ampoules containing specially prepared samples of coal were then broken. These samples had previously been sieved into narrow sine ranges from about 1.1 mm. down to about 0.04 mm. Approximately 1 gram of cod was then accurately weighed into each flask, which amount proved satisfactory for particle si5e8 of the order of 100 mesh. althoueh the amount should he increased or
The flasks were then immediately connected to their respective manometers, using Gargoyle Savarex No. 2 grease as a
then attached to t h e side arms and the ground-glass'jbints and stoppers were ground in a8 well as possible. The flasks were then immersed in the constant temperature bath. Three minutes after immersion readines were cornmeneed. Each samule was ex&ed to air for the 8ame length of time he-
~~~~~~
~~~~~
~~~~
oxygen a t the tcmieratures'used in the experiments. Since it moved convenient to immerse the flasks in two groups
pressure or bath temperature occurring during the course of the experiment. Readings obtained on the manometers, when suitably corrected by the blank8, were expressed as millimeters of manometric fluid. In order to convert these readings to absolute units and to permit a comparison of different experiments, the apparatus must be cahhrated. This may be done by filling with mercury and weighing, as described by Dixon (S), the appropriate equation for the present case being
x
=
hvo
(A)
(273 F)
where x = the amount of gas absorbed or evolvea, CUDIC millimeters a t standard temperature and pressure h = the change in manometric reading, millimeters of manometric fluid DC = the volume of gas space in the vessel, cubic millimeters, and is given by the volume of the empty
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4
487
vessel less the volume of solids used, calculated from their weight and density T = temperature, degrees absolute PO = a pressure of 1 standard atmosphere, in millimeters of manometric fluid. For convenience, Brodie’s solution may be used which has a density adjusted to give a POof 10,000 mm. a t standard atmospheric pressure of 760 mm. of mercury. This equation contains no term corresponding to the prevailing atmospheric pressure, as i t is only a difference in standard volumes that is being calculated. Changes in atmospheric pressure during the course of each experiment are automatically corrected for by the blank. ACCURACY OF RESULTS
As a check on the reproducibility of the results obtained by the method, an experiment v a s conducted investigating the effect of using different weights of coal. A temperature of 94.3” C. was used. Coal used was Bellbird Tops from the Greta seain of Sew South Wales, Amtralia, giving the following analysis: Proximate Air-Dried Basis, % RIoisture 2.4 Ash 8 7 Vol m a t t e r 49 4 Fixed carbon 39 5 100 0 Total sulfur 5 9
-
D r y , Ash-Free Basis, % 55 6 79 2
Vol. matter Carbon Hydrogen 6 5 Total sulfur 6 6 Calorific value, 15,020 B t u . per lb.
Coal size was through 6400 mesh per square centimeter retained on 10,000 mesh per square centimeter. (Approximately through 200 retained on 230 mesh per inch on the standard screen scale; screen aperture 0.060 to 0.075 mm.) Dry oxygen was used as an atmosphere and the weight of the coal n a s between 0.2 and 0.8 gram in eleven different runs. The weight of Dehydrite was approximately 0.5 gram. Table I gives the results of this experiment, and a plat of the rate of oxygen absorption in cubic millimeters per minute against the JTeight of coal used gave points falling closely along a straight line.
Table I. Weight of Coal Used, G. 0.2148 0.2218 0.2954 0.3630 0.3852 0.4417 0.4550 0.4728 0.6025
0.7306 0.8040
Oxygen Absorption by Coal R a t e of 02 Absorption Cu. mm./min. 3.38 3.88 4.84 6.28 6.21 7.20 7.05 8.22 11.02 12.23 14.16
Cu. mm./g.-niin. 15.7 17.5 16.4 17.3 16.1 16.3 15.5 17.4 18.3 16.7 17.6
The results tabulated in Table I werecalculated from the total amount of absorption occurring in 2 hours. During this period readings uere taken every 10 minutes and from a plot of these readings the initial absorption rates were determined graphically. Very close agreement betu een the figures determined graphically and those given in Table I was obtained. The straight line of best fit through the points representing the results recorded in Table I, when extrapolated to a 1.00-gram weight of coal used gives a rate of 17.00 cu. mm. per gramminute. On comparing this with the mean of the rates given in the third column of Table I, Le., 16.88 cu. mm. per gram-minute, it can be seen that the agreement is good, thus showing that it is satisfactory to express the results on the basis of unit weight of coal. Had an appreciable systematic drift from a straight line appeared, such close agreement would not have been obtained,
shoTTing that no blanketing effects, caused by the topmost coal particles preventing access of oxygen to those below, occur. I n later experiments using varying temperatures and particle sizes, it proved desirable to use an amount of coal such that the total absorption measured \?-as of the order of 500 mm. on the scale. An absorption of this order necessitates only one reset of the manometer, hence the amount of nitrogen introduced does not exceed 2% of the total volumes of the flask; on the other hand, readings taken to within j=lmm. \Till prove sufficiently accurate. The present results show that it is permissible to adjust the weizht of coal used in this way. Further, the variance of the figures quoted in the third column of Table I is 0.71, giving a probable error of 0.57, or 53.4%, and the probable error of the mean of the 11 readings is 0.17, or il.O%. While for many purposes an accuracy of +3.4% for a single reading would be quite satisfactory, considerable improvement can, of course, be achieved by carrying out experiments in duplicate, the mean of TT hich would have a probable error of &2.4’%. I t is doubtful that more than t n o replicates would be justified unless results of the highest accuracy are required. The authors have, in their work on coal, used duplicates throughout. Severtheless, with solids of a more homogeneous nature than coal and in experiment- designed to provide closer standardization of conditions than has been possible in the present work, results of a considerably higher accuracy could no doubt be achieved without excessive experimental replication. SUMMARY AND CONCLUSIONS
A method which is a modification of a technique commonly used for the examination of tissue preparations, cultures, etc., in biochemical TTork, has been developed for the study of solid-gas reactions and tried out for determining the rates of 0x2 gen absorption by coal a t moderate temperatures. Standard, interchangeable equipment is used, of a type readily available in chemical laboratories. The procedure described is simple, yet as many as 14 runs may be carried out stimultaneouslv. The present results showed that the probable error of an individual determination was of the order of 5 3 % . Finally, the method described appears to be sufficiently flexible to permit a Considerable variety of experiments t3 be performed. Thus, such variables as particle size, temperature, composition of gases, humidity, etr., may all be studied without any great change in the normal procedure. Although the solid used in this work has been coal, i t would appear that the method could be advantageously employed to study many other solid-gas reactions. ACKKOWLEDGMENT
The authors desire to thank the Joint Coal Board (Australia) which has financed this nork, and the Coal Research Section of the Commonwealth Scientific and Industrial Research Organization, which analyzed the coal used. LITERATURE CITED
(1) Blaskett, D. R., -4ustralia Council Sci. Ind. Research, Rept. 164 (1947).
( 2 ) Dixon, M., “Rlanometric hlethods,” London, Cambridge Uni-
versity Press, 1943. (3) Graham, J. I., Trans. Inst. Mining Engrs. (London),48, 521-34, (1914-15). (4) Schmidt, L. D. (H. H. Lowry, Ed.), “Chemistry of Coal Ctilisation,” Chap. 18, Kew York, John Wiley & Sons, 1945. (5) Scott, G. S.,U. 5. Bur. Mines, Bull. 455 (1944). (6) Winmill, T. F., Trans. Inst. Mining Engrs. (London), 48, 514-20 (19 14-1 5). R E C E I V E for D review July 21, 1952. Accepted November 16, 1953.
