Steam vs. Ether in Separation of Acids from Bacteriological Media JAMES B. MCNAIR,818 S. Ardmore Ave., Los Angeles, Calif.
T
HE experiments outlined in this paper were carried out to show the great inefficiency in the estimation of organic acids separated from bacteriological media by the customary steam distillation, that this separation may be done much better by ether, and that the time and tediousness of the operation are much less when ether is used. For more than twenty years it has been customary in some laboratories to obtain the volatile acids from bacteriological media by steam distillation. The medium is acidified with sulfuric or phosphoric acid. This distillate is neutralized with sodium or barium hydroxides and concentrated by evaporation. The concentrate is subjected frequently to analysis for acids according to the method of Duclaux (9). The nonvolatile (fixed) acids are considered as remaining in the residue in the distillation flask, from which they are obtained by extraction with ether. I n order to determine the amount of acids produced by the bacteria, an aliquot of uninoculated media is subjected to the same analysis as an inoculated portion and the difference in acidity between the inoculated and uninoculated media is taken as the acidity produced by bacteria. Methods similar to the foregoing have been used by Harden (IC), Selieber ( I O ) , Fred and Peterson (12), Zoller and Clark (6), and others, in determining the acids produced by bacteria; by Jensen (16) in studying the volatile aliphatic acids of Swiss cheese; by Freudenreich and Jensen (IS) in their researches in Emmenthaler cheese; by Currie (7) in his investigation of Roquefort cheese; by Susuki, Hastings, and Hart (9Q)in their studies on the acids of Cheddar cheese; by Richmond (18) and Edelstein and Csonka (IO) in determining the acids formed by decomposition in milk; by Dox and Neidig (8) in their work on the volatile aliphatic acids of corn silage; by Woodman and Burwell (21) in the detection of formic acid as a food preservative, and by many investigators of the acids of wine. ERRORS IN OLD METHOD BY HEAT PH DECREASE IN HEATIKG MEDIA. The importance of the effect of heat on the production of acid in bacteriological media has not been sufficiently emphasized in connection with the separation of acids by steam distillation, It has been noticed by workers in this subject that under the influence of heat bacteriological media tend to become more acid in reaction. Anthony and Ekroth (2) found that '%he point a t which no further acidity is produced was not reached with meat infusion (which had been previously subjected to boiling over the open flame for one to two hours) even after prolonged autoclaving a t 15 pounds pressure for eight hours." Clark (6) found that a meat infusion containing 0.5 per cent potassium monophosphate, brought to a reaction of pH 7.19 before sterilization, changes to pH 6.70 after sterilization. McIntosh and Smart (16) confined their observations to the effect of the maximum amount of heat employed, under ordinary conditions, to sterilize the media. Broth subjected to three steamings of 20 minutes each caused a decrease in pH of 0.13; after the same number of autoclavings a t 115" C. for 20 minutes, the figure was 0.23. In the case of 1 per cent glucose broth, the reaction decreased as much as pH 1.2 during autoclaving.
It is obvious that the increase in acidity which takes place during sterilization can also continue during the hours of steam distillation. ACID INCREASE IN HEATING SUGARSWITH AMINOACIDS, Mudge (17) maintains that it is the presence of an unstable sugar molecule together with an amino acid which gives rise to part of the acidity of sterilization. He bases this conclusion on experiments with a 0.5 per cent solution of alanine, and to a 1 per cent solution of maltose or raffinose. Solutions were also used which contained 0.5 per cent of alanine and 1 per cent of the sugars mentioned. These solutions were sterilized in the autoclave for periods of 15 minutes, 30 minutes, and 1 hour. All of the solutions were neutral to phenolphthalein a t the start. The results show that alanine alone and sugar alone give no acidity; a mixture of alanine and raffinose remains neutral even after heating for an hour; but a solution containing a mixture of alanine and maltose becomes acid, presumably according to the equation: H&-CH-NH2-COOH
+ H& :O = H&-CH-NCHZ-
COOH
+ Hz0
ACIDINCREASE IN HEATING SUGARS WITH MINERAL ACIDS. It is evident, however, that this is not the only possible source of acidity. The prolonged action of mineral acids upon glucose, accelerated by heat, results in the well-known cleavage of glucose into levulinic and formic acids. To prove this, the following experiment was performed: One and one-fourth grams of c. P. dextrose were dissolved in 250 cc. distilled water; 1.6 cc. of 25 per cent H,PO( were added. This was steam-distilled. The first 500 cc. of distillate contained 0.4 cc. of 0.1 N acid; the second, 0.25 cc.; the third and fourth, 0.65 cc.; the fifth was lost; the sixth, 0.30 cc,; the seventh, 0.25 cc.; the eighth, 0.30 cc: the washings from condenser, 0.15 cc.; total, 2.30 cc. of 0.1 k acid. This distillate was evaporated on the steam bath to about 100 cc. and then treated with the mercuric chloride-formic acid reagent (for details of formic estimation, see the official method, 3 ) . As a result, 0.0232 gram of mercurous chloride was obtained, equivalent to 2.03 cc. of 0.1 N formic acid. MOLECULAR CHANGESAhfONG ORG.4h-112 ACIDS. Besides the changes in acidity caused by the possible decomposition of proteins, fats, and carbohydrates, there is the probable change in the structure of the acids themselves. Erlenmeyer ( 1 ) found that formic acid is formed by heating lactic acid with dilute sulfuric acid. To demonstrate this reaction under the usual conditions of media distillation, the following experiment was carried out: Three cubic centimeters of 25 per cent H3POa (U. S. P.) and 86.65 cc. of 0.1 N barium lactate were diluted to 250 cc. with distilled water. The reaction of the mixture was then blue t o Congo red, and brown t o thymol blue. This liquid was steam-distilled under constant volume. The first liter of distillate contained 2.4 cc. of 0.1 N acid; the second, 2.1 cc.; the third, 1.9 cc.; total, 6.4 cc. of 0.1 N acid. The distillate was evaporated on the steam bath and treated with the mercuric chlorideformic acid reagent (8). As a result, 0.0046 gram of mercurous chloride was obtained, equivalent t o 0.51 cc. of 0.1 N formic acid. As precautions were taken against carbon dioxide, the remainder of the acidity of the distillate (5.89 cc. of 0.1 N ) was caused by lactic acid.
62
January 15, 1933
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I N D U S T R I A I, A N D E N G I N E E R I N G C H E M I S T R Y
From the foregoing evidence it is clear that, during the steam distillation of bacteriological media practiced for the recovery of volatile acids and their separation from nonvolatile acids, an increased amount of acid as well as molecular change among the acids themselves is liable to occur. This is especially true if the steam distillation lasts for 10 hours, a s is sometimes necessary. FAILURE TO SECURE COMPARABLE DATA. It is likewise evident that, a t the time distillation is begun, the inoculated and the uninoculated media do not have the same amounts or varieties of the various constituents. For instance, carbon dioxide may have been formed and partly escaped, or various acids may have been formed, or the sugar may have been partially decomposed in the inoculated media. Therefore, when both the inoculated and uninoculated media are subjected to distillation, they are not comparative and the effect of heat may differ in the production of the various acids involved.
evaporation, for the salts of the weak organic acids are appreciably hydrolyzed, and an odor of the acids can readily be detected above a hot solution neutral to phenolphthalein. The spontaneous decomposition of calcium butyrate a t room temperature is shown by an odor of butyric acid from a bottle containing the salt. To determine the loss of formic acid during evaporation, two portions of approximately 0.2 gram of calcium formate were dissolved in 300 cc. distilled water; 150 cc. of each of these solutions were tested ERRORS IN OLD METHODCAUSEDBY INCOMPLETE f o r formic acid by the mercuSEPARATION OF ACIDS ric chloride reagent (3). The first portion contained 0.08231 As has been shown above, a considerable amount of lac- gram (1.765 cc. of 0.1 N); the tic acid may be carried over in the distillate during steam second portion, 0.09094 gram distillation. (1.929 cc. of 0.1 N ) of formic The following experiments show that at least 25 per cent acid. One hundred fifty cubic of the volatile acids may remain behind in the distillation centimeters of each of the flask unless the flask contents is sufficiently acidified with solutions were evaporated in 25 per cent H,PO,, or by other adequate means previous to 26.5-em. porcelain evaporation distillation. dishes on a steam bath. Three A mixture of 9.69 cc. of 0.1 N formic, 9.365 cc. of 0.1 N acetic, liters of water were gradually 10.08 cc. of 0.1 N propionic, 9.744 cc. of 0.1 N lactic, and 10.00 added to each, and the evapoCC. of 0.1 N succinic acids was diluted to 150 cc. with distilled water. The mixture therefore contained a total of 48.874 cc. ration continued almost to of 0.1 N acid, of which 19.744 cc. were nonvolatile and 29.13 cc. dryness. As a result, the first volatile. When this was subjected t o steam distillation and the portion contained 0.08056 gram volume kept constant, the first liter of distillate contained 21.78 (1.709 cc. of 0.1 N) and the CC. of 0.1 N acid; the second and third liter, 0.05 cc.; total, second p o r t i o n c o n t a i n e d 21.785 cc. of 0.1 N acid. As there were 29.13 cc. of volatile acid in the distilling flask, from which only 21.78 cc. distilled, 0.08899 gram (1.887 cc. of 0.1 7.35 CC. or 25.22 per cent of the volatile acid remained undistilled. N ) of formic acid by the mercuric chloride method. There I n a duplicate experiment in which the same acids in w e r e , t h e r e f o r e , losses of similar amounts were used, the first liter of distillate con- 0.00175 gram or 2.14 per cent, tained 20.97 cc. of 0.1 N acid; the second and third, 0.1 cc.; and 0.00195 gram or 2.16 per and the fourth, 0.1 cc. As only 20.97 cc. of 0.1 N acid out cent during evaporation. (In of 29.13 cc. of 0.1 N volatile acid distilled, 8.16 cc. or 28 per concentrations of formic acid of less than 0.5 gram per liter, cent remained undistilled. the average error is *0.0005 gram or k 0 . 2 per cent for ERROR IN OLD METHOD IN CONCENTRATINQ the mercuric chloride method.) DISTILLATE
Loss OF ACIDITY. In order to determine the amounts and varieties of the various acids present in the distillate, it is common practice to neutralize and concentrate the distillate by evaporation on a steam bath. I n this process a loss of acidity is generally experienced and has frequently been mentioned (11). A medium of meat infusion, 1.0 per cent Difco pe tone, 2
per cent dextrose, and 1 per cent potassium monophospfate was made up t o 250 cc., including 6 cc. of 25 per cent HaP04to make the liquid acid t o Congo red. This was steam-distilled until the last 500 cc. of distillate contained only 0.8 cc. of 0.1 N acid. The total distillate contained 44.60 cc. of 0.1 N acid. This was evaporated on a steam bath t o about 50 cc. The liquid after evaporation contained 39.66 cc. of 0.1 N acid or only 88.94 per cent of the original acidity of the distillate. A duplicate experiment resulted in 43.20 cc. of 0.1 N acid in the distillate, from which 40.12 cc. or 92.96 per cent remained after evaporation. This loss is greater when evaporation is continued to dryness. Part of it may be due to decomposition by heat, as observed by Browne (6). There is also a loss of acid by
EXTRACTION OF ACIDSBY ETHER
63 Wo4
,
FIGURE1. ETHERExTRACTION APPARATUS
From evidence presented above, it is desirable to find a method for the separation of acids from bacteriological media which will eliminate the effects of heat on the media. This is successfully carried out in the ethereal extraction of the nonvolatile acids from the distillation residue in the old method. Harden (14) in 1900 was able to remove completely lactic and succinic acids by continuous extraction in 10 hours. It was demonstrated as long ago as 1872 by Berthelot and Jungfleisch (4) that formic, acetic, butyric, and succinic acids map be extracted from aqueous solutions by shaking with ether. It was thought therefore that the extraction of all of the aliphatic acids met with in bacteriological media might be extracted with ether. EXTRACTION APPARATUS.An ether extraction apparatus similar to Figure 1 was used in the following experiments.
ANALYTICAL EDITION
64
A is the condenser, D the extraction tube, C the inner tube to hold the material to be extracted, E the flask for boiling the ether and also to retain the extract. In the apparatus illustrated, the inner tube is able to hold 100 cc. of media and have about 4 inches (10.2 cm.) of tube above. It is necessary t o have considerable space above the medium in the inner tube in order to allow complete separation of the medium and ether. This is to prevent the overflowing of the medium to E . The distance between the top of the funnel and the top of the medium must be great enough to allow the ether to flow out of the bottom of the funnel stem. This distance increases with an increase in the specific gravity of the medium. EXTRACTION OF FORMIC AND ACETIC ACIDS. To test the efficiency of the apparatus t o extract formic and acetic acids, 14.52 cc. of 0.1 N acetic, 9.365 cc. of 0.1 N formic, 10.8 cc. of 25 per cent HsP04 (U. S. P. sirupy) acids, and water sufficient to make 100 cc. were extracted with ether. At the end of 19.5 hours 0.0008 gram (or 0.017 cc. of 0.1 N) formic acid remained to be extracted (determined by mercuric chloride reagent, 5). EXTRACTION OF LACTIC ACID. Lactic acid (9.9 cc. of 0.1 N ) placed in the ether extractor was diluted with water t o 100 cc., and 5.5 grams of sulfuric acid (95 er cent sulfuric acid, specific gravity 1.84) were added. Two Kundred cubic centimeters of ether were placed in the lower flask. After extraction had proceeded for a certain number of hours, the electric hot plate was taken away, 100 cc. of water were added t o the ether in the lower flask, and the ether was distilied off. The a ueous residue was titrated with 0.1 N barium hydroxide in %e presence of phenolphthalein; this was allowed to stand overnight and was then filtered through No. 589 S. & S. filter paper; the precipitate was washed with water; the precipitate and filter were placed in a weighed platinum crucible and heated 16 minutes at a dull red heat. From the weight of barium sulfate in the crucible the amount of free sulfuric acid was calculated. Zinc sulfate solution (0.2 N ) was added to the filtrate, treated on a steam bath for 6.5 hours, and filtered as the preceding solution; the precipitate and filter paper were treated as above. From the weight of zinc oxide in the crucible the amount of lactic acid was calculated. The results of the extraction, found in Table I, show that 9.65 cc. out of 9.9 cc. of 0.1 N lactic acid, or 97.47 per cent, were extracted. OF EXTRACTION OF LACTIC ACID TABLEI. RJOSULTS
TIME EXTRACTED Hours 6.5 9.5 9.26 8.5 7.3
41.05
TITER 6.26 2.15 0.55 0.40 0.30
TOTALLACTICACIDAND H&Oc ACCORDINQ TO HzSOi LACTICACID Bas01 Cubic centimeters of 0.1 N acid + 6.92 7.22 0.30 2.56 0.24 2.32 0.27 0.46 0.18 0.16 0.21 0.06 0.025 0.09 0.068
- 9.65
0.848
9.686
10.54
COMPARISON OF ETHER EXTRACTION TVITH STEAM DISTILLATION Ether extractions were made of a water solution of mixtures of the following acids: 9.696 cc. of 0.1 N formic, 9.365 cc. of 0.1 N acetic, 10.08 cc. of 0.1 N propionic, 9.744 cc. of 0.1 N lactic, 10.00 cc. of 0.1 N succinic, and sufficient water to make I00 cc. In 7.5 hours 44.85 cc. of 0.1 N acid were extracted, consisting of about equal quantities of volatile and nonvolatile acids. During the next 20 hours the remainder of the acid (mostly nonvolatile) was extracted. Three more similar experiments showed 44.15 cc. of 0.1 N, 45.45 cc. of 0.1 N, and 49.05 cc. of 0.1 N , respectively, extracted in 7.5 hours. The experiments shown in Table I1 demonstrate the increase in the amount of acid formed during the steam distillation of a medium. The medium consisted of one per cent Parke-Davis peptone, one per cent dextrose, one per
Vol. 5 , No. 1
cent dibasic potassium phosphate adjusted to pH 7.3 with sodium hydroxide. Before distillation, 25 per cent HPPOJ was added until the reaction was blue to Congo red. TABLE11. INCREASE IN ACID FORMED ACIDPBR 100 cc. OF MEDIUX Total volatiIe and nonvolatile Volatile Nonvolatile Cc.O S N Cc. 0.1 N Cc. 0.1 N
MEDIUM Sterilized uninoculated: Steam Ether Difference Inoculated with 90-H-1: Bteam Ether Difference
8.40 0.57 1.83
4.96 4.80 0.15
3.45 1.77 1.68
48.22 32.07 16.15
8.80
39.42
...* ....
.... ....
These experiments show in the case of the uninoculated medium an increase of 1.83 cc. of 0.1 N, or 27.86 per cent by steam distillation; in the case of the inoculated medium, an increase of 16.15 cc. of 0.1 N or 50.36 per cent by steam distillation. TIMEREQUIRED.The customary estimation of acids in bacteriological media by steam distillation involves three steps: (1) the time occupied in distillation, (2) the time consumed in the concentration of the distillate, and (3) the time involved in the ether extraction of the residue in the distilling flask. The new method of ether extraction eliminates the time (about 10 hours) taken up by the first two steps and requires only the time for the third step; that is, both the volatile and nonvolatile acids may be extracted in the same amount of time required for the extraction by the old method of the nonvolatile acids. SUMMARY Among the evidence against steam distillation may be included (A) errors caused by heat: (1) p H decrease in heating media, ( 2 ) acid increase in heating sugars with amino acids, (3) acid increase in heating sugars with mineral acids, (4) molecular changes among the organic acids-e. g., lactic to formic, and (5) failure to secure comparable data when the same medium is heated before and after the action of bacteria; (B) errors caused by incomplete separation of acids: (1) variable amounts of lactic acid may be carried over in the distillate, and ( 2 ) as much as 28 per cent of volatile acid may remain in the distilling flask; and (C) errors in concentrating distillates: acidity may be lost during the evaporation of the distillate. The extraction of acids with ether is already successfully carried out. An apparatus is described in which formic, acetic, propionic, lactic, and succinic acids may be successfully extracted in 40 hours, and comparison of extraction by steam distillation and ether is given, showing that steam distillation may result in the formation of 27 to 50 per cent more acidity and that a large amount of time is saved by ether extraction.
LITERATURE CITED (1) Allen, “Commercial Organic Analyses,” 4th ed., Vol. VII, p. 433, Blakiston, 1913. (2) Anthony, B. van H., and Ekroth, C. V., Coll. Stud. Bur. of Laboratories, New York, Vol. 17, p. 294 (1914-15).
(3) Assoc. Official Agr. Chem., Official and Tentative Methods, 2nd ed., 1925. (4) Berthelot and Jungfleisch, Ann. chint. phys., [4] 26, 396-407 (1872). (5) Browne, C. A., Jr., J . Am. Chem. Soc., 21, 807-27 (1899). (6) Clark, W. M., $. Infectious Diseases, 17, 108 (1915). J. Agr. Research, 2, 1-14 (April, 1914). (7) Currie, J. N.,
January 15, 1933
INDUSTRIAL A N D ENGINEERING CHEMISTRY
(8) Dox, A. W., and Keidig, R. E., Agr. Expt. Sta., Iowa State Coll. Agr. Mech. Arts, Research Bull. 7 (1912). (9) Duclaux, E., “Trait6 de Microbiologie,” Vol. 3, p. 385, Masson,
Paris, 1900. (10) Edelstein, F., and Csonka, F. V., Biochem. Z., 42, 372 (1912). (11) Eyre, J. W. H., Brit.Med. J . , 1900, 11, 921. (12) Fred, E. B., and Peterson, W. H., J . Infectious Diseasss, 27, 539-49 (1920). (13) Freudenreich, E. de, and Jensen, O., “Annuaire agricole de la Suisse,” Part 4, Berne, 1906. (14) Harden, A., J . Chem. Soc., 79, 610-28 (1901).
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(15) Jensen, Landw. Jahrb. Schweiz, 18, 319-405 (1905). (16) McIntosh, J., and Smart, W. A M., Brit. J . Exptl. Path., 1, 9-30 (1920). (17) Mudge, C. S., J . Bact., 2, 403-15 (1917). (18) Richmond, H. D., and Miller, E. H., Analyst, 31, 318-35 (1906). (19) Selieber, M. G., Compb. rend., 150, 1267-70 (1901). (20) Susulri, S. K., Hastings, E. G., and Hart, E. B., J. Biol. Chem., 7, 431-58 (1910). (21) Woodman, A. G., and Burwell, A. L., Tech. Quart., 21, 1 (1908). R ~ C I D I VJune E D 2 , 1932
Automatic Titrating Devices K. HICKMAN AND C. R. SANFORD, Eastman Kodak Co., Rochester, N. Y.
T
HE paper industry has a very real need for a recording
titrator, for a device which will determine the total acidity of alum baths, wash waters, etc. The present potentiometric recorders do not entirely meet the need, which is for an indication of reserve acidity and not for the potential of hydrogen ions. Further, it is found that total acidity and potential are not simply or constantly related to one another under working conditions in a paper mill. The problem has been approached in two ways: directly, by the mechanical manipulation of solution volumes; and indirectly, by the diffusive mixing of parallel liquid streams.
The means chosen for securing the performance is illustrated in Figures 1 and 2. The first requisite, a timing unit to control the sequence of events, is provided by bulb A and appendages ( 2 ) . When the bulb, which is fed with water or the unknown (dilute solution) under approximate control from tap B, has filled to level c, the solution passes over siphon C, loading the mercury column, D, which completes
DIRECTMECHANICAL TITRATOR
-4repeating cycle has been secured which comprises the following operations: (a) A sample of standard volume is withdrawn from the unknown liquid. (b) The sample is discharged into a reaction vessel together with a smtable quantity of indicator solution. ( c ) The mixture is observed b an optical device which (d) Controls the addition of tge second component, the estimating fluid, in volume sufficient t o reach neutrality, and (e) Activates a en t o record the volume on a chart. cf) The titratel solution is discharged t o waste, the chart moved t o a new position, and (9) The cyrle repeats.
FIGURE2. VIEW OF SET-UPOF TITRATOR
a circuit through the electrode, E . The current actuates a magnetic valve, F , which allows the contents of the reaction vessel, G, to pass to waste. Soon after the emptying of G, bulb A should be full enough for its contents to siphon rapidly down tube H , unload mercury column D,and refill the reaction vessel. During the passage of this unit volume of fluid, a constant and repeatable suction impulse (negative pressure X time of flow) is exerted a t each point in the column, notably a t points I and J, from which tubes communicate with constant-level reservoirs of reagents. Through one tube a chemical color indicator (phenolphthalein, methyl red, etc.) is imbibed. The other tube is used only when pure water is fed to bulb A , and its purpose is to admit a predetermined quantity of the liquid to be titrated. Thus, OF DIRECT MECHANICAL TITRATION two general levels of solution strength can be manipulated, FIGURE1. DIAGRAM
