July 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY LITERATURE CITED
(1) Alyea, H. N., and Backstrom, H. L. J., J . Am. Chem. Soc., 51, 90 (1929). (2) Bailey, H. S., and Ebert, H., Cotton Oil Press, 7, 35 (1923). (3) Bruere. P.. and Fourmont. A,. Ann. fals.. 25. 91 (19323. (4) Greenbank, G. R., and Holm, G. E.; IND.ENO.CHEM.,Anal. Ed., 2, 9 (1930). (5) Ibid., 17, 625 (1925). (6) Grettie, D. P., and Newton, R. C., Ibzd., 8, 291 (1931). .
I
247
(7) Issoglio, G., Ann. chim. applicata, 6 , 1 (1916). (8) Lea, C. H., Soap, 7, 83 (1931). (9) Newton, R. C., Oil Soap, 9, 247 (1932). (10) Pool, W. O., Oil Fat. Ind., 8, 331 (1931). (11) Richardson, A. S., Eckey, E. W., and Andrews, J. T. R., Ibid., 8, 409 (1931). (12) Royce, H. D., Soap, 7, 25 (1931). (13) Wheeler, D. H., M. M. A., Bull. 121 (Jan., 1932).
RECEIVED February 6, 1933.
Determination of Formic, Acetic, and Propionic Acids in a Mixture 0. L. OSBURN,H. G. WOOD,AND C. H. WERKMAN Ames Field Station, Bureau of Chemistry and Soils, in Coijperation with Bacteriology Section, Iowa Agricultural Experiment Station, Ames, Iowa
P
REVIOUS publications (6, 10-13) have reviewed the literature and discussed the need for rapid and accurate methods for the quantitative determination of the volatile fatty acids in fermenting liquors. I n these papers the application of the partition method to two acid mixtures was shown, together with theoretical discussions of the possibilities of the partition method in general. The method presented in this paper is intended to deal specifically with the problems presented under the special conditions involved in the fermentative production of propionic acid. In the procedure described, the partition method is combined with a modified mercuric oxide oxidation for the separate determination of formic acid.
PREPARATION OF FERMENTATION LIQUORS To prepare a fermentation liquor for the quantitative determination of the volatile acids, a quantity containing about 60 cc. of 1 N volatile acid is placed in a distilling flask, made neutral to phenolphthalein, and distilled until the residue in the flask reaches a volume of about 150 cc. The residue is then made just acid to Congo red paper with 1to 1 sulfuric acid, boiled under a reflux condenser to remove carbon dioxide, and subjected to steam distillation. A constant volume of liquid in the distilling flask should be maintained. Two liters of distillate should be collected. From Dyer’s work (3)it is evident that under these conditions about 95 per cent of the formic and substantially all of the acetic and propionic acids will be removed from the fermentation liquors. Prolonged steam distillation should be avoided, because under these circumstances volatile acid may be produced from unfermented carbohydrate. Lactic acid also distills over to an appreciable extent, and for this reason the fermentation liquor should not be saturated with salt before steam distilling. Under the conditions imposed by Olmsted, Whitaker, and Duden (6) lactic acid in 600 cc. of distillate has been found equivalent to 22 per cent of that present in the original liquor. Under the conditions of ordinary steam distillation some substances, such as lactic acid, distill over and may interfere with the analysis. A much more accurate analysis can be obtained if the first steam distillate is neutralized, evaporated to 150 cc., made just acid to Congo red, and redistilled according to Olmsted ( 5 ) . Under these conditions the volatile acids will all come over in 1liter of distillate. This distillate is then adjusted by dilution t o an acid concentration of approximately 0.03 N , and this 0.03 N acid solution is used for the determination of the volatile acids.
DETERMINATION OF FORMIC ACID Results obtained in this laboratory show that strong oxidizing agents, such as chromic acid or permanganate, cannot be used for the oxidation of formic acid in the presence of propionic acid, since some of the propionic acid is oxidized to acetic acid. For example, a solution of 25 cc. of 0.1 N formic acid, 25 cc. of 0.1 N acetic acid, and 50 cc. of 0.1 N propionic acid was oxidized with a dilute permanganate solution for 30 minutes at boiling temperature. The formic acid was completely oxi-
dized by this treatment. (The solution should remain acid, since ropionic acid is oxidized t o oxalic acid in alkaline solution, 4,) l f ter acidifying and distilling the residual solution subsequent t o oxidation, the distillate should have contained 25 cc. of 0.1 N acetic acid and 50 cc. of 0.1 N propionic acid. Analysis showed, however, 52.5 cc. of 0.1 N acetic and 22.5 cc. of 0.1 N ropionic acids-that is, about half of the propionic acid had gee, converted to acetic acid. The use of dichromate (9) for the oxidation of formic acid
in the presence of propionic is to be avoided for the same reason. The most convenient method of determining formic acid is the mercuric chloride method of Auerbach and Zeglin ( I ) , but serious errors are likely to be encountered by its indiscriminate use with such complex mixtures as fermentation liquors. It has been found by Weihe and by Wood in this laboratory, working independently with widely different substances, that the mercuric chloride method gives considerably higher results than are obtained by oxidation of the formic acid with mercuric oxide (unpublished data). These high results are apparently due to substances other than formic acid which reduce mercuric chloride, such as acetylmethylcarbinol. I n consequence, it seemed advisable to use mercuric oxide for the oxidation of formic acid and to determine the formic acid by the carbon dioxide evolved, inasmuch as it is highly improbable that carbon dioxide would be evolved by action of mercuric oxide from substances other than formic acid. If circumstances permit, any convenient method of determining formic acid, other than those involving oxidation with strong oxidizing agents, may be substituted.
PROCEDURE FOR DETERMINATION OF FORMIC ACID The formic acid is oxidized with mercuric oxide. The carbon dioxide is absorbed in Bowen potash bulbs and weighed (7), or, if a mixture is being analyzed from which volatile substances may be carried over into the potash I
ANALYTICAL EDITION
248
bulbs, the carbon dioxide is absorbed in a Truog tower (8) and determined volumetrically. Two hundred and fifty cubic centimeters of the acid solution, pre ared for analysis as described above, are placed in a 500-cc. balkon flask and 5 grams of mercuric oxide added. The flask is connected through a reflux condenser to a Bowen potash bulb, or, if the volumetric method is used, to a Truog tower as de'00
b 3.5
59
39
'Od
16&8
acid may also be present. If mixed cultures of organisms are being used, fatty acids other than formic, acetic, and propionic may also be present. The present method is designed t o determine formic, acetic, and propionic acids quantitatively, when no other acids are present. A provisional method for the detection, but not for the identification, of foreign acids is included. The procedure is based on the determination of formic acid in an aliquot as given above. Another aliquot of the acid mixture is then partitioned between ethyl ether and water. By means of the data obtained, it is possible either t o calculate the percentages of the other acids or to read them from the nomogram given in Figure 1. PARTITION METHOD.The partition constants used in this procedure are obtained as follows: A 300-cc. portion of the approximately 0.03 N acid solution prepared as described above, is brought to a temperature of 25" C. Fifty cubic centimeters of the solution are titrated with 0.05 N alkali in the presence of phenolphthalein. Let M ; represent the number of cubic Centimeters of 0.05 N alkali required. Another portion of 55 cc. of the acid solution and 25 cc. of pure ethyl ether is pipetted into a 225-cc. separatory funnel and the mixture is shaken vigorously for 1.5 minutes. The temperature should be maintained a t 25" C. After allowing 3 or 4 minutes for the phases to separate, 50 cc. of the aqueous phase are withdrawn into a 50-cc. volumetric flask and titrated with 0.05 N alkali. Let M t represent the number of cubic centimeters of alkali required. The partition constant is obtained from the equation K = M2/M1 X 100. It is evident that this constant is actually the percentage of the acid which remains in the aqueous phase. This constant is not to be confused with the partition coefficient used in physical chemistry.
48
15,
272
FIGURE 1. NOMOGRAM FOR DETERMINATION OF ACIDS scribed by Heck ( 3 ) . A current of carbon dioxide-free air is started through the apparatus and the acid mercuric oxide mixture in the oxidation flask is gently boiled for 20 minutes. Twenty cubic centimeters of 50 per cent phosphoric acid are then added to the reaction flask and boiling and aeration are continued for 15 minutes. If the volumetric method is used the alkali is carefully washed down from the absorption tower into the flask (S), an excess of barium chloride is added, and the excess of alkali is titrated with 0.5 N hydrochloric acid, according to the procedure of Heck ( 3 ) . Phenolphthalein is used as an indicator. Under these conditions the formic acid has been found to be completely oxidized, while the other acids present remain unaltered.
Constants for each acid were established in this manner. Solutions of the pure acids were made up t o approximately 0.03 N and 55 cc. of each solution partitioned with 25 cc. of ether as described. The constants found are: formic acid, 84.5; acetic acid, 82.1; and propionic acid, 58.8. If these three acids are present in a mixture in which the normality lies between 0.02 N and 0.04 N , the partition constant obtained for that mixture will be the sum of the partial constants of each acid, For example, assume a mixture consisting of 10 per cent of formic acid, 30 per cent of acetic acid, and 60 per cent of propionic acid. The value of K , if accurately determined, would be (84.5 X 0.10)
PrqLac- PyFormic Acetic pionic Butyric tic ruvic % % % % % % 0 0 0 0 0 100 0 0 0 0 0 100 n 0 0 0 0 100 0 0 0 0 0 100 0 0 50 0 25 25 20 40 0 20 20 0 0 22.2 55.5 11.1 11.1 0 0 0 50 0 50 0 50 0 0 0 50 0 0 50 0 50 0 0 0 50 50 0 0
...
..
..
..
THE PRESENCE
+ (82.1 X 0.30) + (58.8 X 0.60)
or the equation may be written (0.846X 10)
O F FORMIC ACIDIN TABLE I. DETERMINATION OF OTHERSUBSTANCES ACIDSPRESENT . coz
Vol. 5, No. 4
+ (0.821X 30) + (0.582x 60)
-
Kzr = 68.36
Klb
3
68.36
It must be noted that by the percentage of each acid is meant normality percentage, not percentage by weight. For example, would contain 10 cc. of 0.03 N acetic acid, and 60 cc. of 0.03 N propionic acid per 100 cc. of acid mixture. Normality percentage is used throughout thir discussion. The Percentage of each acid present will be designated as F , A , and P for formic, acetic, and propionic acid, respectively. For solutions containing only these three acids, F , A , and P must add up to 100. With these conventions, the general equations can be written:
RECOVERY CALCU- COa OF FORMIC the solution just mentioned formic acid, 30 cc. of 0.03 N LATBD FOUND ACID
% 0.0431 0,0431 0.0431 0.0431 0.0440 0.0440 0.0440 0.0440 0.0440 0.0 0.0 0.0
0.0436 0.0440 0.0423 0.0430 0.0444 0.0442 0,0437 0.0447 0.0432 0.0010 0.0015
101.1 102.0 98.3 99.7 101.0 100.5 99.4 101.6 198.3
..
0.0010
Representative analyses are shown in Table I. Formic acid is determined in the presence of various other acids as indicated, using the gravimetric method.
DETERMINATION OF MIXTURES OF ACIDS In many studies on the production of propionic acid by microorganisms, the mixtures of acids will contain acetic and propionic, with varyidg amounts of formic acid. LB;ctic
+
+
0.845F 0.8216 0.588P = K F + A+ P=100
Since F is determined separately, it is not one of the unknown values, and the equations may be written:
+
0.821A 0 6 S S P = K - 0.S45F A + P = 100F
Solving A and P in terms of K and F we have: A = 4.291K P = 352.36
- 262.36 +- 1.103F 0.103F - 4.291K
Once K and F are determined, the percentages of acetic and propionic acids are obtained by substitution in Equations 1 and 2, respectively.
INDUSTRIAL AND ENGINEERING CHEMISTRY
July 15, 1933
DETERMINATION OF FORMIC, ACETIC,AND PROPIONIC ACIDS IN A MIXTURE Formic acid in 250 cc. of the steam distillate is determined as described above. If the volume of distillate is limited and if the procedure is thought advisable, the formic acid may be determined by means of mercuric chloride according to the method described by Auerbach and Zeglin (1). This determination may be made on a smaller volume of the solution. The factor for converting mercuric chloride to formic acid is 0.09745. The formic acid is expressed in terms of normality percentage as indicated above. Fifty cubic centimeters of the acid solution are titrated very carefully with 0.05 N sodium hydroxide and phenolphthalein as indicator. Fifty-five cubic centimeters of the solution are then placed in a separatory funnel and shaken with 25 cc. of ethyl ether as described. After the two phases have separated, 50 cc. of the aqueous phase are titrated and the value of K is calculated. Example: 50 cc. of the original solution required 30 cc. of 0.05 N alkali. After partition, 50 cc. of the aqueous phase required 19.5 cc. of the alkali. K =
X IC0
=
65.0
After F and K are known, A and P are calculated by substitution in Equations 1 and 2 given above, or read directly from the nomogram. I n laboratories where a large number of determinations are to be made, it is convenient to read the values of A and P from a nomogram, such as that shown in Figure 1.
249
present. If, however, the experimental and calculated values of Kloodo not agree (within 0.4 unit), then other acids are present. Table I11 shows the effect which the presence of other acids has on the value of Kloo, as well as the errors which will occur in the determination of formic, acetic, and propionic acids when other acids are present. It can be seen that either lactic acid, butyric acid, or valeric acid will displace the determined value of K1oo in the same direction. TABLE111. DETECTION OF ACIDS OTHER THAN FORMIC, ACETIC, AND PROPIONIC PRESENT 1
--ACIDS
% 20 40 10 5 20 15 15 10 10 10
20 40 10 5 20 15 15 10 10 10
40 40 35 80 35 38 30 60 60 30
40.7 39.6 35.3 79.8 28 53.3 21.2 67.7 55.4 31
. . .. . . . .
40 20 55 15 40 38 50 25 25 50
39.5 20.0 55.0 15.2 52 32.7 63.8 22.3 34.6 59
. . ..
None None None None Butyric Lactic Valeric Lactic Butyric Lactic Butyric 5
73.44 78.35 69.59 78.71 70.51 74.82 67.64 77.15 74.34 68.64
. ..
47.00 54.24 41.93 53.88 44.77 50.23 41.12 52.71 48.95 42.20
..,
47.15 54.25 41.75 54.00 43.2 48.9 39.3 52.00 48.20 40.60
., .
-0.15 -0.01 $0.18 -0.12 $1.57 $1.33 $1.82 $0.71 $0.75 $1.70
...,
I n using the nomogram for the detection of foreign acids,
A and P are read on the A and P lines as usual for F and Kz5. The straight edge is now placed on F and on the same values of A and P except that the values are now located on the A' and P' lines. K1co is now read a t the intersection of
O the straight edge with K ~ Oline. O This value of K ~ Ocorresponds to the calculated value of K1co as given just above. If no foreign acids are present, this value as read on the Kloo line will be the same as the value of K,OO determined experimentally. Representative analyses of mixtures of acids are given An example will make the meaning clear. In the fifth mixture in Table 11. given in Table 111, F = 20 and K ~ =L 70.51. As read from the chart, A '= 28.0 and P = 52.0. The straight edge is then TABLE 11. DETERMINATION OF FORMIC, ACETIC,AND PROPIONIC laced across F = 20 on the F line, A = 28 on the A' line, and ACIDSIN MIXTURES 52 on the P' line. The value of K I Oas~ read on the Kloo line is 44.77. --ACIDS TAKEN-ACIDS FOUNDAssume a solution of such composition that F = 10 and K = 75. Lay a straight edge on the chart so that it passes through 10 on the F line and through 75 on the line K25. A and P are read directly on the lines labeled A and P. For the mixture assumed A = 58.2 and B = 31.8.
F=
Formic
Acetic
Prouionic
Formic
Acetic
ProDionic
Kzs
Since lactic acid with a constant of 84.5 and valeric acid with a constant of 7.9 bring about a deviation of the constants in the same direction, it is manifestly impossible to have any combination of acids present as impurities which would escape detection by balancing each other in their individual effect on the final constant. The only foreign acid which DETECTIONOF OTHER ACIDS IN MIXTURE might escape detection, if present in an amount over 5 For this purpose, another set of constants was established per cent, would be an acid with both constants (K25 and K ~ o o ) as before, except that 60 cc. of acid were partitioned with very close to those of one of the three acids included in the 100 cc. of ethyl ether. Fifty cubic centimeters of the aqueous quantitative method. If desired, A and P could be calculated in terms of F and phase were removed and titrated as before. These partition constants for the individual acids, which will be designated Kz5. The acids could be determined by using both constants. If the values for A and P came out the same in each case, , F = 63.5, A = 58.5, and P = 27.2. as K I ~ oare: In addition to these constants, the values of lactic, butyric, only the three acids would be present. If the percentages and valeric acids were also determined for Kl0o and for Kzj. of A and P came out different, some other acids would be For lactic acid, KIOO= 84.5; for butyric acid, 10.3; and present. for valeric acid, 7.9. The K25 values for these acids are: CONSTRUCTION OF XOMOGRAY lactic, 90.0; butyric, 31.1; and valeric, 17.0. The value of The construction of the nomogram offers no great difficulty. K100for the mixture of acids can be calculated in the same manner as for K2j. On a common base line the perpendiculars F and K26 are erected at any convenient distance apart [lo inches (25 om.) 0.635 0.585 A 0.272 P Kioo (3) serves very well]. F is 25 cm. in length, and K25 has a length in centimeters corresponding to the difference between the conThis equation can be used for the detection of foreign stants for formic and propionic acids: 84.5 - 58.8 = 25.7 cm. K25is calibrated in centimeters from K25 = 58.8 t o 84.5 as shown. acids in the following manner: F is calibrated from 0 to 50 in the opposite direction. A line K26 and F are determined, and A and P calculated or read is drawn from Kla = 84.5 to meet F extended t o F = 100. F from the chart. Using these values from F , A , and P, KIOO is may actually be extended, or the point of intersection of the calculated from Equation 3. Klao is then determined experi- diagonal with the base line may be calculated geometrically. mentally. If the calculated and experimental values of KIOO Another diagonal is drawn from F = 0 to K25 = 58.8. The agree, then no acids except formic, acetic, and propionic are two diagonals intersect at the point A = 0. The line A is %
%
%
%
%
%
20 40 10 5 0 0
40 40 35 80 30 70
40 20 55 15 70 30
20 40.4 9.7 5.9 0.0 0.0
40.7 39.6 35.3 78.8 30.0 69.9
39.5 20.0 55.0 15.2 70.0 30.1
+
+
73.44 78.35 69.59 78.71 65.75 75.10
ANALYTICAL EDITION
250
Vol. 5, No. 4
drawn parallel to F and Kza from the point A = 0 to the point of intersection of the line from F = 0 to K ~ = L 82.1 (the K2.6 value for pure acetic acid). This A line is calibrated upward from 0 to 100. The point P = 0 is located at the oint of intersection of the lines, F = 0 t o K26 = 82.1, and i t . 5 = 84.5 to F = 100. The line P is drawn parallel to KU to intersect the diagonal, F = 0 t o K ~ = s 58.8. The line is calibrated downward from 0 to 100. The A', P', and KIOO lines are located in the same manner. Some convenient unit of division is selected for the Kloo line. The length of line is then measured off from Klo0 = 27.2 to 63.5 = 27.34 units. The line is then located so that the upper end just meets the diagonal extended through P = 0 and Kta = 84.5. The extremities of the A' and P' Iines are then located in the same manner as those of A and P.
used instead of 4/45. The titrations must be done with extreme accuracy. I n the hands of a careless worker, errors may easily arise which would indicate the presence of 5 or 10 per cent of some foreign acid. The pipets and burets should be carefully standardized. All water used should be free from carbon dioxide. The method is rapid and reasonably accurate if properly executed. I n using the qualitative scheme devised here, it is advisable to ignore differences in K ~ OofO less than 0.4 units, which would indicate the presence of approximately 3 per cent of lactic or butyric acids, or 1 or 2 per cent of valeric acid. If the Kloo values are appreciably different from each other, it would be well to check the determinations before spending much time trying to identify any contaminating acids which DISCUSSION may be present. The scheme should be used with judgment. The method of analysis given is based on an approximately Unfortunately no clue will be given as to the nature of any 0.03 N acid solution as a matter of convenience. At least contaminating acids which may be encountered, If signifi2000 cc. of steam distillate must be collected in order to drive cant quantities of such acids are indicated, it is advisable all of the volatile acids over. Usually such a quantity of to take 500 cc. of the solution, distill off 250 cc., and make liquor can be used as to give 2 liters of 0.03 N acid distillate, a new set of determinations on the residue. If the conand the analyses can be made without further treatment of taminating acids are more volatile than propionic acid, the the distillate. two Kloo values will be closer together than they were before, since the impurities distill out faster than do the three acids TABLE IV. DETERMINATION OF ORGANIC ACIDS AT DIFFERENT under consideration. If such an acid as lactic is the conCONCENTRATIONS taminant, the Kloo values will be more divergent than they -ACIDS TAEHINACIDSFOUNDwere before. The ultimate identification, of course, would NORMALITY Formic Acetio ProDionio Formica Acetic Proponic involve the preparation of a derivative. % % % % % % I
25.0 25.0 24.0 0.lb 25.0 50.0 33.3 33.3 33.3 32.0 0.06 33.3 33.3 33.3 33.3 0.06 33.3 33.3 33.3 33.3 34.5 0.02 33.3 33.3 20.0 33.3 21.3 0.015 33.3 66.6 20.0 13.3 33.3 20.8 0.0150 66.6 20.0 50.0 30.0 20.0 31.4 0.020 50.0 30.0 20.0 29.5 0.020 20.0 33.3 33.3 34.5 33.3 0* 020 33.3 Actual amount of formio aoid present, n o determination made. b One-half volume each of ether and acid taken. c Double volume each of ether and acid taken.
51.0 35.0 33.3 32.2 65.2 66.0 48.6 60.5 32.2
@
The effect of concentration of the acids on the determinations is shown in Table IV. Apparently the method holds reasonably well for the ranges of concentration given, but is recommended only for 0.03 N acid, especially for the provisional detection of contaminating acids. More accurate results could be obtained with 0.1 N acids if the method were adapted to that concentration, because larger titration figures could be used-for example, there would be less experimental error involved if figures such as 40/50 could be
LITERATURE CITED (1) Auerbach, F., and Zedin, H., 2. physik. Chem., 103, 161-77 (1922). (2) Dyer, D. C., J . Biol. Chem., 28, 445-73 (1917). (3) Heck, A. F., Soil Sci., 28, 225-32 (1929). (4) MoNair, J. Am. Chem. SOC.,54, 3249-50 (1932). ( 5 ) Olmsted, W. H., Whitaker, W. M., and Duden, C. W,, J. Biol. Chem., 85, 109 (1929). CHEM.,Anal. (6) Osburn, 0. L., and Werkman, C. H., IND.ENCI. Ed., 3, 264 (1931). (7) Ibid., 4, 421 (1932). (8) Truog, E., IND.ENG. CHEM.,7, 1045 (1915). (9) Tsiropinas, F., Ibid., 9, 1110-11 (1917). (10) Werkman, C. H., Ibid., Anal. Ed., 2, 302 (1930). (11) Werkman, C. H., Iowa State College J . Sci., 4, 459-64 (1930). (12) Ibid., 5, 1-3 (1931). (13) Ibid., 5, 122-5 (1931).
RECEIVEDFebruary 3, 1933. Journal Paper B96 of the Iowa Agrioultural Experiment Station, Ames, Iowa. Project 67.
Gas Bubbler for Very Small Pressure Drops C. C. FURNAS, Department of Chemical Engineering, Yale University, New Haven, Conn.
I
T IS frequently necessary to have a gas absorber which will operate with a small pressure drop in the gas stream and yet have a small gas volume in the system. Such is often the case where thermal conductivity cells are used for gas analysis. I n such cases, in order to prevent time lag in the analysis it is essential to keep the volume of gas in the apparatus down to a minimum. This rules out the use of solid absorbent towers. If, in addition to the small gas
FIGURE 1. INCLINED ABSORPTION TUBE
volume requirement, it is necessary to have only a small pressure drop through the apparatus, it is frequently difficult to get efficient absorption of one of the gaseous constituents. By using a large glass cylinder nearly filled with liquid, placed on a slight incline, it is possible to obtain sufficient contact between gas and liquid for efficient absorption and the static head to be overcome can be kept very small. The apparatus shown in Figure 1has been found very satisfactory when filled with potassium hydroxide for absorbing carbon dioxide from gases containing 1 to 90 per cent with a gas velocity up to 50 cc. per minute. It has also proved satisfactory when filled with concentrated sulfuric acid for absorbing water from gases saturated at 25" C. RECEIV~D March 27, 1933.
