Determination of Volatile Fatty Acids by
the Partition Method 0. L. OSBURN, H. G. WOOD, AND C. H. WERKMAN U. S. Department of Agriculture and Iowa Agricultural Experiment Station, Ames, Iowa
T
HE volatile fatty acids most frequently encountered in
to the barium hydroxide is added and the barium sulfate removed by decantation and filtration. I n this alternative procedure certain contaminating hydroxy acids will be present in the solution, and may interfere with subsequent analysis. In the procedures given below the concentration of acids should be about 0.03 M with respect to total volatile acid present.
fermentation studies are formic, acetic, propionic, and butyric. Usually but one or two of these acids are present, although three- and four-acid mixtures occur. Experience has shown that there are three conditions under which the determination of these four acids will usually be carried out: (1) I n routine studies and in plant control workthe number and identity of the acids present are always known and the analysis consists merely in determining the percentage of each acid present. (2) During physiological investigations in which the fermentations take place under varying environmental conditions, or when mixed cultures or new species are being used, doubt often exist8 as to the exact qualitative compositiofi of the fatty acid niixture. In such mixtures, the identification of the acids present by the preparation of organic derivatives or by fractional crystallization of the silver salts requires too much time, except for the final identification of the acids produced by R new species. (3) Higher fatty acids may be produced in small amounts in addition to the four acids mentioned above, and cause serious errors in the analysis of the other acids. Also, lactic, pyruvic, and possibly other hydroxy or keto acids may be present, and these are sufficiently volatile with steam to be partly carried into the distillate with the volatile acids. All such acids will be referred to here as ‘Lforeign”acids. They may have been produced by the fermentation, or added intentionally to the medium as part of the substrate. The purpose of this paper is to show the application of the partition method (4, 7) to the rapid analysis of volatile fatty acid mixtures under the above conditions. The partition method has been used in this laboratory for the past 5 years and has been modified from time to time to meet the demands of fermentation research.
Conventions Used To facilitate description of the method and to make possible the use of the nomograms (Figures 1 and 2), certain abbreviations and conventions have been adopted. The percentage of each acid present will be expressed as molar p e r c e n t a g e the per cent of the total molarity which is due to each acid present. Assuming that the entire acid distillate contains the equivalent of 10 cc. of 1.0 M acetic acid, 10 cc. of 1.0 M propionic acid, and 30 cc. of 1.0 M butyric acid, or 50 cc. of 1 M acid in all, then the molar percentage of acetic acid is 10/50 X 100 or 20, that of propionic is 20, and that of butyric is 60. If the molar percent-
ages of formic, acetic, propionic, and butyric acids are represented by F, A , P,and B, respectively, the com osition of the mixture P B = 100, in wKich A = 20, P = 20, may be written A and B = 60. Since only monobasic acids are considered, the terms “molarity” and “normality” have identical significance.
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Principles of the Partition Method The partition method is based on the distribution of the fatty acids between two immiscible solvents, such as water and ethyl ether, when dilute water solutions of the acids are shaken with acid-free ether in separatory funnels. In this paper the percentages of the acids which remain in the aqueous phase are taken as the partition constants. The four sets of partition constants shown in Table I were determined as follows:
Preparation of Fermentation Liquors for Analysis
Solutions of the pure acids in water were adjusted to approximately 0.03 M each. For each of the constants in column 1 of Table I, 60 cc. of the acid solution and 100 cc. of pure, acidfree ethyl ether were shaken vigorously in a separatory funnel for 1 minute. Two minutes were allowed for the two phases to separate, and 50 cc. of the aqueous phase were removed and titrated with 0.05 N alkali in presence of phenolphthalein, the cubic centimeters of alkali required being designated as N I . The cubic centimeters of alkali required t o titrate 50 cc. of the original acid solution were designated as Ne. The partition constant for the acid is N , / N a X 100. The other constants in columns 2 to 4 (Table I) were determined in like manner, using the volumes of acid solution and of ether shown in the column headings.
It is often desirable to separate certain neutral volatile products (alcohols, acetone, etc.) from fermentation liquors before the volatile acids are removed. If unfermented sugar is present, it is not advisable to neutralize the liquor before distilling off the alcohols. The pH of the solution is adjusted to about 4 with sulfuric acid (just blue to congo red paper) and the neutral products, together with part of the volatile acids, are distilled. The distillate is neutralized with sodium hydroxide and distilled until all of the neutral products are removed-i. e., one-half the volume is distilled. The two residues are combined, the pH is again adjusted to 4,and the liquid distilled with steam until 2 liters of distillate are collected. The volume of liquid in the distilling flask should be kept between 75 and 150 cc. If the concentration of the acids in the 2 liters of distillate is too low for the analysis, the entire distillate should be neutralized with alkali and evaporated to 150 cc. The residue is acidified, saturated with magnesium sulfate, and distilled with steam as described by Olmstead, Whitaker, and Duden (3). One liter of distillate should be collected. Alternatively, if greater concentration is desired, the original distillate may be neutralized with standard barium hydroxide and evaporated to the desired volume. Sulfuric acid exactly equivalent
TABLEI. PARTITION CONSTANTS OF ORUANIC ACIDS 60 cc. of Acid 100 cc. of Acid 150 cc, of Acid 00 CC. of Acid 100 cc. of Ether 20 CC. of Ether 20 cc. of Ether 200 CC. of E t h e r Formic 63.5 90.8 92.4 48.1 Acetic 58.2 88.8 91.8 42.2 Propionic 27.2 78.0 85.9 16.2 10.3 57.3 73.5 5.0 Butyric Valenc 7.5 39.0 55.2 4.3 Lactic 84.5 92.7 93.0 77.5
To secure thorough mixing of the two phases it is important that the total capacity of the separatory funnels be 50 to 100 cc. greater than the combined volumes of ether and acid solutions used. 270
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Bring the acid solution and the ether to be used t o
a temDerature of 25" C. PiDet 60 cc. of the acid solution into a separatory finnel of about 250-cc.
capacity, add 100 cc. of pure ethylether, andshake the mixture vigorously for 1 minute. Towels should be wrap ed around the funnels to prevent heating by the {and. If the temperature of the laboratory differs more than *3' C. from 25" C., precautions should be taken t o keep the temperature of the mixture at approximately 25" C. After the mixture is shaken, allow it t o stand for 2 minutes for the two phases to separate. Remove 50 cc. of the aqueous phase and titrate with 0.05 N sodium hydroxide (phenolphthalein), designating the cc. of alkali as N1. Titrate 50 cc. of the original mixture, the cc. of alkali required being Na. Calculate K1-i. e., NJN2 X 100. n l , This procedure applies only when the two FIGURE 1. NOMOGRAM FOR DETERMINATION OF ACIDSIN TWO-ACID MIXTURES acids present are known, If they are acetic and butyric acids, locate K , on the A-BKI line in Figure 1. If acetic and propionic acids ace present, locate KI on the A-PKI line. Read the percentage of acetic In the following discussion the partition constants of the acid on the left-hand ordinate. Subtract from 100 to get the individual acids will be referred to as f1, al, pl, and bl, for the molar percentage of the other acid. From these data calculate constants in column 1 of Table I. The subscripts denote the amount of each acid present in the original solution. For the set of constants referred to in Table I. These constants example, if the 50 cc. of acid solution titrated contained 30 cc. of 0.05 N acid and if there were 2 liters of the solution, there vary somewhat with conce'ntration of the acids in solution. would be 60 cc. of 1 N acid present. If analysis showed 70 molar The constants in Table I were determined for the acids in per cent of acetic acid and 30 per cent of butyric acid, there 0.03 M solutions and are valid only for concentrations rangwould be 42 cc. of 1 N acetic and 18 cc. of 1 N butyric acids ing from 0.02 to 0.04 M . present. If two or more acids are together in a solution, each has its ANALYSISOF THREE-ACIDMIXTURES,ONE OF WHICHIs own partition constant and acts independently of the other FORMIC ACID. The partition constants of formic and acetic acids present. If a mixture contains acetic and propionic acids are so close together (Figure 3) that accurate determinaacids in equimolar portions-i. e., if A = 50 and P = 50tions of either in the presence of the other cannot be made by the partition constant obtained by shaking 60 cc. of the the partition method. The formic acid in such mixtures may acid solution with 100 cc. of ether (column 1, Table I) is be determined by the method of Auerbach and Zeglin ( I ) , 50 X 0.582 50 X 0.272 = 42.7 Ki of Osburn, Wood, and Werkman (d), or of Weihe (6). Express the amount of formic acid found as molar per cent of The partition constants are written decimally in all calcuthe total acid as follows: lations as a matter of convenience. K1designates the partition constant for any mixture of acids when 60 cc. of acid solution and 100 cc. of ether are used to determine the constant. Likewise K z is found by using 100 cc. of acid solution apd 20 cc. of ether as shown in column 2 of Table I, etc. Since no other mixture of acetic and propionic acids could give 42.7 as the value for K,, it follows conversely that if one partition constant, such as K1, is determined for the mixture, the percentage of each acid present can be found. This may be done either graphically or by means of equations. If two acids are present in solution only one partition constant need be determined, but there must be two equations. Using a mixture of acetic and propionic acids as an example and the conventions already described, equations may be written as follows: alA p,P = K1 A P = 100
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These equations may be solved for A and P in terms of KI. Similar equations may be written for any two-acid mixture, and such equations are given below. The graphical method is more convenient. If K1is determined for a mixture of acetic and propionic acids it is only necessary to locate K1 on the A-PKI diagonal of Figure 1 and read the molar percentages of A and P on the ordinate. Diagonal lines are given for acetic-propionic and acetic-butyric acid mixtures for both K1 and Kz values. Similar charts may be made for any two-acid mixtures. The construction of Figure 1 is given below. If three acids, such as acetic, propionic, and butyric, are present it is necessary to determine two partition constants, K1 and Kz, and Figure 2 is used as described below.
Procedure DETBRMINATION O F -ACIDS IN TWO-ACID MIXTURES.The concentration of acid in the solution should be about 0.03 M .
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FIGURE 2. NOMOGRAM FOR DETERMINATION OF ACIDS IN THREE-ACID MIXTURES
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FIGURE 3. PARTITION CONSTANTS FOR VARYING RATIOS OF WATERTO ETHERUSED IN THEIR DETERMINATION Suppose the total acid solution contains 50 cc. of 1 M acid and that 0.230 gram of formic acid is found present. The cc. of 1.0 M 0 230 formic acid will be -= 5.0 cc. (1 cc. of 1 M formic acid con0.046 5 tains 0.046 gram). According t o the conventions F = - X 100 50 = 10 molar per cent. To determine the other acids find KI for the mixture as described above. Before using the nomogram (Figure 1) K1 has t o be corrected for the known percentage of formic acid and a new constant K: calculated as follows: K:
Ki - fiF 100 -F __ 100
Locate K: then on the diagonal corresponding to the two acids known to be present (aside from formic) and find the percentages of the two acids from the ordinate. Multiply each of these percentages by loo- t o get the true percentages of the two 100 acids in the solution. Example: The constant, K1,for mixture 5, Table 11, was 40.25 and F was equal t o 10.0. Hence K;
40.26
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0.635 X 10 100 10 100
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37.66
This value of K: was located on the A-PK1 line and gave 33.0 and 67.0 for A and P. These two values, each multiplied by loo- lo(i. e., gave 29.8 and 60.2 for A and P, respec100 100 P = 100. The constants given in Table tively. Thus F A I1 are the K1 values. The results shown in Table I1are representative.
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Find K1 for the mixture. Determine K I for the mixture by shaking 100 cc. of the acid solution with 20 cc. of ether and titrating 50 cc. of the aqueous phase and 50 cc. of the original solution. Calculate K z in the same manner as KI. Assume (as an exampIe) that the mixture is thought to contain acetic and propionic acids and no others. Locate K1 on the A-PK1 diagonal, move horizontally to the same ordinate on the A-PKz diagonal, and read, at the top of the chart, a corresponding value of Kz. Call this value Kzc. If only acetic and propionic acids are present, K2, the experimentalvalue, and Kzcshould agree in value to within 0.3 or 0.4 unit. Usually these two values agree within 0.2 unit. If appreciable quantities of other acids are present or if the ualitative assumptions were wrong, there would be a large %ifferenee between Kz and Kzc. Suppose, for example, that mixture 3, Table 11, had been assumed t o contain acetic and propionic acids and that the above procedure had been carried out. The KZ value found for this mixture was 83.4 and K ~ c (assuming an acetic-propionic mixture) was 8 6 . 0 4 . e., KZ- KZC = -2.6. If mixture 2, Table 11, had been thought t o contain acetic and butyric acids K2 - KZCwould have been 80.5 - 73.4 = +7.1. Making correct assumptions, K z - Kzc for mixture 3 is -0.2 and for mixture 2, K z - Kzc = -0.1. Such false assumptions are not likely to be made but serious errors sometimes occur in the determination of acetic, propionic, and butyric acids because of the presence of small amounts of lactic or pyruvic acid or of acids above butyric acid in the series. The differences (Kz - Kzc) caused by a few such “foreign” acids in two-acid mixtures are shown in the first seven mixtures of Table 111. It has been found that the algebraic sign of K z - Kzc will always be negative if the numerical values of the partition constants of the foreign acid are either greater or smaller than the constants of the two acids being determined. Referring to the curves of Figure 3, if any acid shown is taken as a “foreign” acid in the analysis of an acetic-propionic acid mixture, for example-the Kz - Kzc difference will be negative, and the curve of this foreign acid will be outside the curves of the acids being determined. For an acetic-butyric acid mixture, propionic acid would give a positive value for Kz - Kzc, since the curve lies between the curves for these two acids. Otherwise, for an acetic-valeric acid mixture, both propionic and butyric acids would give positive values. Beyond this the procedure offers no means of identifying the “foreign” acids present. If formic acid is present both K1 and Kz should be corrected as described above:
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METHODSOF CONFIRMING QUALITATIVE ASSUMPTIONS. The exact qualitative composition of an acid mixture is sometimes in doubt. A mixture may be known to contain acetic and butyric acids and the presence of another acid may be suspected, or it may not be known whether a mixture is one of acetic and butyric acids or acetic and propionic acids, etc. The procedures given above tell nothing as to the accuracy of the analysis. Confirmation is offered by the following simple procedure:
100
100
The K : and K ; values calculated are then used in the same manner as K1 and Kz given above.
DETERMINATION OF ACETIC, PROPIONIC, AND BUTYRIC ACIDS MIXTURE.Determine K1 and Kz as described above. Locate K1 on the K1 line of Figure 2 and KZon the Kz line. Lay a straight edge across these two points and read A on the A line, P on the P line, and B on the B line of the chart. If formic acid is present, determine F and calculate K: and K,1 exactly as described above. Locate K i on the K1 line and Kj on the KZ line and read values of A , P, 100 - F to and B. Multiply each of the values by 100 TABLE11. DETERMINATION OF ACIDSBY PARTITION METHOD get the percentage of each acid present. The results -Acid Present-Acid Foundshown in the last four mixtures of Table I1 are typiExpt. Formic Acetic Propionic Butyric Formica Acetic Propionic Butyric Ki K2 cal of many analyses made in this manner. Equa% % % % % % % % tions for A , P, and B in terms of K I and KZare given .. 33 57 .. 045 88 20 .. 45 below. 0 ... . 32 35 .. 02 67 74 .. 08 1 0 33.3 66.6 25.0 75.0 2 0 0 83.4 60.4 83.7 83.4 0 .. 16:3 3 0 16.6 .. It is possible to detect the presence of “foreign” 66.8 4 6 9 . 8 24.63 70.0 0 .. 3 0 . 2 30 0 Rn 40.25 83.0 29.8 6 10 60:2 10 .. 60 0 acids in mixtures of formic, acetic, propionic, 82.2 10 19:9 49.2 70.1 10 6 0 20 70 and butyric acids. The constants in columns 3 32.8 35:O 32.0 32.0 76.0 33.3 33.3 7 0 33.3 .. 30 50 0 20 8 . . 49.8 20.2 30.0 37.7 77.3 and 4 of Table I and the lines K3 and K4 on 60 44.9 82.9 49.4 31.8 9.0 30 .. 10 10 9 10 34.2 22.0 38.0 74.8 20 20.0 20 10 20 40 Figure 2 are for this purpose. If acids above 20 butyric acid in the series are thought t o be presa Actual amounts present. No determinations were made on formic acid. IN A
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TABLE111. DETECTION OF FOREIGN ACIDSIN MIXTURESOF
dividuals to duplicate partition constants when working with the same purified sample; but when Composition of Foreign different acids have been purified and used some Mixture Acid, F A P B % KI K2 Ka K4 K I- Kic K3 - K3c K4 - K4c discrepancies have been found. Obviously the ... .. 50 . . 40 .. Formic, 10 difficulty lies in the purification of the acids. ... .. .. Formic 10 . . 40 50 .... .. .. Butyri; 10 10 The many different values published for the . . 80 . . .. 30 .. Valeric,’ IO 60 Duclaux constants of these same acids have ... .. io 40 . . 40 Valeric, 10 ... .. Lactic 10 . . 70 . . 20 probably arisen in part from the same cause. 20 50 . . 30 Propidnic, -6:i +o:i 50 30 None 10 The constants given in Table I were taken as -0.2 +0.9 . . 20 30 40 Lactic, 10 average values of carefully purified acids, and the 10 . . 30 20 30 Lactic, plus 35.2 72.6 81.6 25.6 ,. - 0 : s + 0 . 8 calculations given below are based upon them. Valeric, 10 . , 40.6 20.2 30.2 Valeric. 9 . 1 33.1 72.8 82.0 22.5 . . -0.5 -0.06 It is possible that others may be able to use the methods given here without having to determine new sets of constants and standardize the apparatus, ent,-determineK1, K2,and K3by using thevolumes of acid soluas has always been necessary with the Duclaux (8) method. tion and ether shown in Table I. Locate K1 and K2 as deThe following equations give the percentages of the acids in scribed, and read K3c on the K3 line a t the point of intersecterms of the constants used. Terms for formic acid (as known tion of K 3and the straight edge. If appreciable quantities of values) are included in each equation. If formic acid is not higher acids are present, K3 - K3c will differ by more than in the mixture, the formic acid term becomes zero and has no 0.4 unit. effect on the other values. For acetic-propionic acid mixtures, Nonvolatile acids, such as lactic or pyruvic, are detected using K1 by using K4 in the same manner. The data for the last four mixtures of Table I11 show that K 3 - KBcis not significantly alA plP = K 1 - f1 F affected by the nonvolatile acids, and that K4 - K4c does not A+P=100-F show the presence of highly volatile acids (valeric, capric, caproic, etc.). from which, substituting the constants in Table I This latter procedure can be used only to detect large A = K 1 - looPI --(fl - PI) F = 3.226 K1 - 87.75 - 1.17F (1) errors in analysis. As the mixtures become more complex, (a1 - PI) (a1 -PI) the differences in the K and Kc values become less, and it is K I 1 0 0 ~ 1 I (ai more difficult to get satisfactory agreement between K3 P = jl)F = 187.73 - 3.226K 0.17 F (2) (PI - all (PI -a11 and K3c or K4 and K4c with mixtures of known purity. If the procedure is used with suitable precaution it can be of For acetic-butyric acid mixtures considerable service. a1 A bl B = K1 - jl F Construction of Nomograms A B = 100 - F (fi - bi) = Figure 1 is drawn on ordinary cross-section paper. It is from which A = Ki - 100 bi (a1 - bl) (a1 - bl) possible to secure paper such that the KI and K2 scales may 2.088 KI - 21.5 - 1.11 F (3) be read directly to 0.1 or 0.2 unit. The K1 values are on the horizontal axis and the diagonals A-PKI and A-BKI are B = K1 - looal &LZ@F = 121.5 - 2.088 K 1 O.llF (4) (bl - all (bl - all drawn a s shown. The K2 values are placed at the top merely as a matter of convenience. The K2 values-i. e., a z , f ~ , For acetic-propionic-butyric acid mixtures which may also and b2 of Table I-are used when drawing the A-BK2 and contain formic acid and for which K1 and K2 are determined, A-PK2 lines. The two systems are entirely independent of the following equations may be solved for A , P , and B: each other and the Kz scale need not have the same number of lines of the cross-section paper per unit as the K1 scale. 0.582 A 0.272 P 0.103 B = K1 - 0.635 F Figure 2 may be drawn to any convenient siae. The A 0.888 A 0.78 P 0.573 B = Kz - 0.908 F and P lines may be made 250 mm. in length and 200 mm. A P B = 100 - F apart. The B line is exactly half-way between A and P from which and has half their length. The lines for the four constants K 1 . . . K4 terminate on the diagonal from P = 0 to A = 100. A = 164.71 4.508 K I - 3.68 Kz - 1.161 F (5) Their upper ends are on the line from A = 0 to P = 0 which P = 10.432 Kz - 6.86 K1 - 529.1 0.155 F (6) passes through the point B = 100. To calibrate the K l . , . K4 B 462.62 2.35 K I - 6.75 KI 0.0135 F (7) lines, use the corresponding constants for the pure acids given The procedures given in this paper as confirmatory tests in Table I. Each line is calibrated downward from the value should be used for the most part as time-saving devices. of the constant for butyric acid to that for acetic acid. The extra time required to determine K2 and to apply the Each of the lines, K 1 , . . K4,is located laterally by the expresprocedure does not exceed 10 minutes, and experience has sion shown that if for two acid mixtures K2 - K2c is within 0.2 or 0.4 unit, it may safely be assumed that the analysis is correct both quantitatively and qualitatively and no extended search for other acids is necessary. Such assurance is a great where D is the distance between the A and P lines and a, p , satisfaction in fermentation work. Similar methods may be and 6 are the constants of Table I corresponding to the line developed for mixtures of acids other than those given here. being located. The intervals so calculated are measured The effect of any “foreign” acid on the difference K2 - Kzc from the P line. can be calculated if the partition constants, x1and x2,and the Discussion molar percentage, X,of the acid are known. If acetic and The acids used for the determination of the partition conpropionic acids are determined in the presence of the foreign acid by using Figure 1, it is obvious that false readings, stants were purified by several redistillations, taking for each A , and P,, will be obtained. If x1 and X are substituted in redistillation the fraction nearest the correct boiling point of Equations 1 and 2 above they become: the pure acid. It has always been possible for different inVOLATILE FATTY ACIDS
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(9)
The last term in each of these equations shows the manner in which A , and P, must be corrected in order to obtain A and P , the correct molar percentages of acetic and propionic acids in the mixture. Call these terms the correction factors for X. The value of KZ determined experimentally is
Kz
=
+ pzP + ZZX
UZA
The value of Kzc, determined as described above is
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+ PZ ( P - P J + ZZX
of the volume of solution are distilled and two distillation con-
(10)
I n these equations A - A , and P - P, represent the numerical values of the correction factors for X . By substituting the correction factors in Equations 8 and 9 for A - A , and Pi- P, in Equation 10 we obtain
The effect of any third acid on the analysis of any two-acid mixtures may be calculated by substituting the constants of the acids concerned in Equation 11. By similar methods the effect of foreign acids on K S - Kac and K4 K4c for aceticpropionic-butyric acid mixtures may be calculated. Using the constants in Table I
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Ks - K ~ c= (0.02554 xz - 0.6204 $1 - 0.382) X 23X Kc - K4c = (0.221 - 0.909~2- 0.07) X $. 3 s The molar percentage of a “foreign” acid is seldom known in fermentation studies. These calculations are intended t o be of use when procedures are being worked out for the analysis of various acid mixtures. The methods given here reach their maximum effectiveness only when the proper sets of partition constants are selected. In Figure 3 the partition constants have been plotted against the ratios of acid solution to ether used in determining the constants. The ratios range from 0.3 (60 cc. of solution and 200 cc. of ether) to 7.5 (150 cc. of solution and 20 cc. of ether). For the K1 system (Figure 1) the ratio is 0.6 and for the K z system 5.0. These two constants were used for the two-acid mixtures given above because calculations by means of Equation 11 showed that they gave the maximum magnitude to the difference, Kz - KZC,when adjacent acids were present as “foreign” acids-i. e., they give the confirmatory procedure its maximum sensitivity. If 60 cc. of acid and 25 cc. of ether were used to get K1, a better differentiation between acetic and butyric acids would be obtained, but no possible value of Kz could be found to give a suitable difference, K Z - KZC. Et can be seen from Figure 2 that for three-acid mixtures the spacing of the K1 and Kz lines largely determines the accuracy of the method. If constants are used which place the K 1and K zlines close together the method becomes useless. The expressions used to calculate the spacing of KI and K2 o n Figure 2 show that their relative positions are determined and (” If constants had by the ratios of (’ (PI (Pz be)’ been selected which made these ratios equal in value, the Kl and Kz limes would coincide. If simultaneous equations were used to calculate A , P , and B, the equations under these conditions would be indeterminate and no solution would be
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possible. In general, if the constants p1 and pz for propionic acid are spaced a t proportionately equal intervals between a1 - bl and a2 - bz, the equations are indeterminate, and K, and Ks will coincide on Figure 2. By using the ratios of 0.6 and 5.0 for K1 and K2, it can be seen from Figure 3 that the constants for propionic acid have been made to approach the constant of acetic acid for K1 and to approach the constant for butyric acid for K2, thereby spacing K1 and KS a t the maximum distance apart in Figure 2. In terms of simultaneous equations the determinants are made larger. Distillation methods, such as the Duclaux method (2) and the Virtanen and Pulkki modification (5) of the Duclaux method, do not offer the advantages given above for the partition method. In the Virtanen and Pulkki procedure one-fourth and one-half
Kzc - WL PZP, Hence Kz - KZC= uz ( A - A , )
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stants are calculated which corres ond to the two partition constants used here. They are, for t i e one-half volume, al = 36.6, pl = 57.7, bl = 74.0, and, for the one-fourth volume, ue = 17.2, pa = 30.8, and bz = 43.0. For two-acid mixtures Kljz is used and the results are probably as accurate as they are for the partition method. The other constant, K1/4, cannot be used successfully, however, in the detection of foreign acids. If these two sets of constants (u1, pl,, bl, and a2,pz, bz) are used to construct a nomogram such as Figure 1, for a mixture of acetic and propionic acids which contains 10 per cent of butyric acid as a “foreign” acid, the calculated difference K1/4 - K1/4c, corresponding to the authors’ K z - K ~ chas , a value of only 0.14 as compared to the authors’ value of 1.5 (mixture 3, Table 111). It has been shown above that if mixture 2 (Table 11) had been falsely assumed t o be a mixture of acetic and butyric acids, K z - Kec would have been 7.1. For the same mixture under the same conditions K1/4 - Kl/rc is only 0.6. For mixture 3 (Table 11), assuming an acetic-propionicacid mixture, K2- Kzcwould be -2.6 and Kli4 - K I / ~would c be 0.22. For confirming the analysis the partition method apparently has 10 times the sensitivity of the distillation method. For three-acid mixtures the two constants are used as they are for the partition method, except that Virtanen and Pulkki did not use the equation A P B = 100 but used A P B = cc. of 0.1 N acid. If their two sets of constants are used to construct a nomogram similar in size to Figure 2, the K I / and ~ Kl/z lines would be only 3.3 mm. apart as com ared to 26 mm. for K1 and Kz. Any error which occurred in t i e determination of K1/4 or K I / Zwould cause about eight times as much error in A , P, and B as would an error of e ual magnitude in K1 or Kz. Experience with the distillation mekods and with the partition method indicates that about the same errors are to be expected in the determination of the constants in the different procedures.
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The ethyl ether used should be free of acid. It is sometimes necessary to purify the ether by shaking it with 1 N alkali and storing it for a day or so over calcium chloride. The use of isopropyl ether (6) has been discontinued because of the difficulty of keeping it acid-free. The volumetric glassware should be standardized and all titrations carefully made. No precautions have been taken to exclude carbon dioxide during the titrations.
Summary The partition method described by Werkman (7) has been used extensively in this laboratory for 5 years and has been modified to meet the demands of fermentation research. Procedures are given for the determination of the acids in two-acid mixtures by using one partition constant. By the additional use of a second partition constant it is possible t o detect the presence of acids other than the two acids assumed to be present. Nomographic methods are used. The two partition constants may also be used to determine the acids in three-acid mixtures. Procedures are given for the detection of large errors in the analysis of three-acid mixtures by the further use of two additional partition constants. If formic acid is present in any of the acid mixtures, it is
JULY 1.5, 1936
ANALYTICAL EDITION
determined separately and treated as a known quantity. The other acids are determined in the presence of formic acid.
Literature Cited (1) Auerbach, F., andZeglin, H.,Z. physilz. Chem., 103, 161-77 (1922). (2) Duclaux, E., Ann. d. 2’Ecole h’ormale Supbrieure, 2, 270 (1865); Ann. chim. phys., 2, 289 (1874); Trait6 d e Microbiologie, 3, 384 (1900). (3) Olmstead, W. H., Whitaker, W. M., and Duden, C. W., J.Biol. Chem., 85, 109 (1929).
275
(4) Osburn, 0.L., W-ood, H. G . , and Werkman, C. H., IND. ENQ. CHEM.,Anal. Ed., 5, 247 (1933). (5) Virtanen, A. I., and Pulkki, L , Ann. Acad. Sci. Fennicae, 29A, No. 25 (1927); J. Am. Chem. SOC.,50, 3138 (1928). (6) Weihe, H. D., and Jacobs, P. B., IXD. ENQ. CHEM.,Anal Ed., 8, 44 (1936). (7) Werkman, C. H., IND. ENQ.CHEM.,Anal. E d . , 2, 302 (1930); Iowa State Coll., S. Sci., 4, 459 (1930); 5, 1, 121 (1930). RECEIVED April 3, 1936. Paper J-191 of the Iowa Agricultural Experiment Station. Project 67.
An Overhead Heater for Rapid Evaporation, Drying, and Charring LESLIE F. NIMS
AND
M. K. HORWIT”, Yale University School of Medicine, New Haven, Conn.
The heater is made from the top unit (17.5 X 35 cm., 7 X 14 inches) of a muffle furnace (multiple-unit furnace, Type 56, Hevi Duty Electric Co.), with the radiating face of the unit protected by a sheet of stainless steel (Ka2SMO of the Crucible Steel Co. or equivalent is satisfactory). The apparatus is supported on two ring stands by a suitable brass framework. The heating unit is connected directly to a 220-volt line and draws about 17.5 amperes when hot. Under these conditions the radiating Burface becomes a bright cherry red. The amount of heat received by the dishes is regulated by raising or lowering the unit. The efficiency of the radiator could be increased by thermal insulation for the top surface, but it has been found convenient to use the top surface as a high-temperature hot plate. A smaller unit (top unit of a multiple-unit furnace, Type 52), 12.5 X 25 cm. (5 X 10 inches), drawing 10.5 amperes when connected directly to 110 volts, has proved useful for analyzing samples in small crucibles.
FIGURE 1. RADIANT HEATER A S USEDFOR EVAPORATION OF FLUIDS
In Table I are given the times necessary to complete t h e procedures there listed. The radiant heater would be especially useful in those methods that require frequent evaporations, such as microdeterminations of bromine or iodine in biological materials, where as much as 24 hours may be saved on a single analysis.
Hot plate aerves only as convenient support for dishes
T
HE foaming, spattering, and creeping of fluids that often accompany some of the procedures in quantitative analyses can be obviated by the proper application of heat ( I ) , Figure 1 illustrates a simply constructed overhead heater, made from a standard commercial unit, sufficiently large to allow the simultaneous treatment of ten 50-cc. or four 200-cc. samples. With this apparatus, troublesome evaporations can be carried out expeditiously-for example, 200 cc. of urine can be taken to dryness in half an hour with no signs of ebullition or foaming. Materials such as casein, blood, and sugar, which are difficult to ash by the usual methods, can be quickly dehydrated and charred under the heater. The residues may then be safely placed in a hot muffle furnace t o complete the ashing.
TABLEI. TIMEREQUIRED Material
Operation
Time
Mi%. Water Urine Blood
Evaporate 200 cc. to dryness Evaporate 200 cc. to dryness and char Evaporate 10 cc. t o dryness char, add 1 cc. of concd. nitric acid and evaiorate to dryness Casein 10 grams plus 70 cc: of water and 4 cc. of 8 N potassium carbonate. Evaporate to dryness and char Sodium chloride 0.5 gram plus 2 co. of concd. sulfuric acid. Evaporate to dryness and heat to cessation of fuming Glucose 10 grams, thoroughly charred
30 45
45 60) 90) 40,
Literature Cited (1) Fresenius, C. R., and Cohn, A. I., “Quantitative Chemical Analysis,” pp. 86,87, and 90, New York, John Wiley & Sons, 1903. RECEIVED May 9, 1936.
