V O L U M E 2 8 , NO. 12, D E C E M B E R 1 9 5 6 Table I.
Recoveries of Biphenyl Added to Blood, Urine, and Feces (5.0 ml. of blood and urine, and 5 grams of feces) Biphenyl, y
hdded
Recovered In Blood
10 20 25 40
9.2 18.5 22.7 40.5
20 25 40 40 50 75
I n Urine 17.2 30.4 41.4 40.3 44.0 77.4
Added 2 4 5 7
50 00 00 50 5 00 10.0
Biphenyl, M g . Recovered 2.30
% recovery
Av.
92 93 91 101 94
47,.
86 121 103 101 88 103 100
yo recovery 90
1975
I n preliminary work i t was found that during the evaporation of chloroform solutions of biphenyl considerable and variable amounts of biphenyl mere lost. The addition of acetic acid prior to evaporation prevented this loss. To solutions containing 5 and 10 7 of biphenyl in 10 ml. of chloroform, 0.50 ml. of acetic acid was added. The solutions were evaporated under gentle air streams again until no odor of chloroform remained. The resulting acetic acid solutions were diluted to 10.0 ml. with iso-octane and their absorbances compared a t 248 mp with that of pure biphenyl in iso-octane containing the same concentrations of acetic acid. The absorbances indicated that no biphenyl had been lost in the evaporation steps despite the marked volatility of biphenyl. The procedure used for purification of extracts of biological tissues is similar to that described by Dickey and Green (1). The preliminary wash with sodium hydroxide is intended to remove phenols that may have formed from metabolism of biphenyl. I n order to determine the efficiency of this wash, solutions containing 1 mg. each of 0- and p-phenylphenol and 4,4'dihydroxybiphenyl were carried through the procedure. No color was formed with any of these phenols. Control samples of blood, urine, and feces carried through the procedure gave only small blank readings. Although these were negligible, corrections were made. LITERATURE CITED
beginning a t the appropriate steps. The absorbances per micromole were 0.875 for the nitro derivative and 0.872 for the amino derivative. The corresponding absorbance found when biphenyl was carried through the procedure was 0.705. These results indicate that approximately 20% is lost either during or hcfore nitration.
(1) Dickey, E. E., Green, J. W., Fourdrinier Kraft Board Institute, Appleton, Wis., Project 1108-7-4 (1955). ( 2 ) Kirchner, J. G., Mller, J. 31., Rice, R. G., J . Agr. Food Chem. 2, 1031 (1954). (3) Newhall, W.F., Elvin, E. J., Knodel, L. R . , ;IXAL. CHExr. 26, 1234 (1954). (4) Tomkins, It. G., Isherwood, F. A . , Analyst 70, 330 (1945). (5) W e s t , H. D., Meharry Medical College, Nashville. Tenn., per-
sonal communication. RECEIVEDfor review April 2 5 , 1956.
Accepted August 9 , 1956.
Use of 2,4=Dinitrophenylhydrazones of p-Phenylphenacyl Esters as Second Derivatives in Identification of Organic Acids HAWKINS NG, A. DINSMOOR WEBB, and RICHARD E. KEPNER Departments of Chemistry and of Viticulture and Enology, University of California, Davis, Calif.
The 2,4-dinitrophenylhydrazones of the p-phenylphenacyl esters of 18 fatty acids were prepared. In several cases a greater difference was observed between the melting points of the hydrazones than between the melting points of the correspondingesters. The double derivatives of the straight-chain saturated acids from acetic through octadecanoic were found separable on silicic acid-nitromethane chromatographic columns. The relative rates of travel with respect to that of the hexanoate derivative were determined and should be of assistance in the identification of small amounts of unknown organic acids.
T
HE identification of small amounts of organic acids present in the volatile aroma materials from grapes and wines has heen of interest in this laboratory for some time. The p-phenylphenacyl derivatives have proved valuable for this purpose when only a single acid or a mixture of acids of low molecular weight was present in a fraction. The chromatographic method for qeparation of mixtures of p-phenylphenacyl esters on silicic acid rolumns described by Kirchner, Prater, and Haagen-Smit (3) has permitted the separation and identification of these esters
when there is a considerable difference in molecular weight of the acid portion of the esters. Column chromatographic separations of p-phenylazophenacyl esters, as developed in this laboratory ( Z ) , have provided slightly better separations of the higher molecular weight acid derivatives. When only milligram quantities of p-phenylphenacyl ester are available, purification by either recrystallization or by chromatography is sometimes not effective enough to permit positive identification. The value of being able to prepare a second derivative from the small amount of pphenylphenacyl ester available in such cases is obvious. This paper reports the melting points and the chromatographic behavior of a number of the 2,4-dinitrophenylhydrazones of the pphenylphenacyl esters of the saturated fatty acids from acetic through stearic acids. MATERIALS
2,4-Dinitrophenylhydrazine Reagent. The reagent solution for preparation of the 2,4-dinitrophenylhydrazones of the p phenylphenacyl esters was made by dissolving 5.9 grams of 2,4dinitrophenylhydrazine in 130 ml. of reagent grade concentrated hvdrochlxic acid s3lution and then dilnting it with 870 ml. of 9570 ethyl alcohol. Aliquot8 of this stock solution \?ere filtered shortly before use. p-Phenylphenacyl Esters. Esters were prepared from- knon n
ANALYTICAL CHEMISTRY
1976 acids by the method of Shriner and Fuson ( 6 ) and were urified by the chromatographic method of Kirchner, Prater, and &aagenSmit (S). Chromatographic Adsorbent. The chromatographic columns were packed with an adsorbent prepared from analytical reagent grade silicic acid (Mallinckrodt No. 2847) activated by drying for 3 days a t 105" C. and then thoroughly mixed with nitromethane (Eastman No. 189). Eight milliliters of nitromethane were mixed with 10 grams of the dried silicic acid. This is essentially the adsorbent developed by Kramer and van Duin ( 4 ) . Developing Solvent. The developing solvent for the chromatographic separation of the derivatives was prepared by saturating redistilled Skellysolve B (boiling range 60' to 70" C.) with nitromethane. The Skellysolve B was shaken in a separatory funnel with 0.01 its volume of nitromethane for 5 minutes. After separation, the lower nitromethane layer was discarded. APPARATUS
Chromatographic Columns. The chromatographic columns used were the same as those described by Ikeda, Webb, and Kepner (2). PROCEDURE
Preparation of p-Phenylphenacyl-2,4-dinitrophenylhydrazones of Acids. The double derivatives of the acids were prepared by adding slowly twice the theoretical amount of the filtered 2,4dinitrophenylhydrazine solution to a warm solution of the pphenylphenacyl ester in 95y0ethyl alcohol with stirring. -4fter standing several hours a t room temperature, the crystalline derivative was filtered from the solution, washed with small quantities of water, and dried in air. Mixtures of varying proportions of ethyl alcohol, ethyl acetate, and water were used for recrystallization. Chromatography of p-Phenylphenacyl-2,4-dinitrophenylhydrazones of Acids. The chromatographic columns were packed by transferring a slurry prepared from approximately 15 grams of the adsorbent and 30 ml. of the development solvent into the tubes through a funnel. The adsorbent waa compacted in the column by applying a pressure of 10 pounds per square inch of nitrogen a t the top of the column. The pressure was released when there was about 1 cm. of solvent left above the adsorbent in the column. From approximately 10 y to 2.5 mg. of the double derivative or mixture of the double derivatives dissolved in the minimum amount of the developing solvent was carefully pipetted into the top of the column. The solution of derivatives was then forced into the adsorbent layer by the application of pressure from a cylinder of nitrogen a t the top of the column. One or more small washings of the flask and pipet with the developing solvent were transferred into the column by the above sequence of operations, after which the reservoir of developing solvent was attached at the top and the column development was started. It was found important always to maintain a layer of solvent above the adsorbent in the column. Measurement of R Values. The facts that the chromatographic columns are packed with adsorbent in the form of a slurry and that the derivatives move very slowly with respect to the solvent front prohibit the determination of R, values as such. The relative rates of travel of the various derivatives investigated in this work were measured with respect to the rate of travel of p-phenylphenacylhexanoate-2,4-dinitrophenylhydrazoneon the chromatographic column. Three sets of measurements were usually taken; in each case the distance from the top of the adsorbent layer to the point of greatest color density in each of the derivative zones on the column was measured. The average of the ratios of distance moved by a second derivative divided by distance moved by the hexanoate derivative was recorded as the R value for the particular compound. RESULTS AND DISCUSSION
The 2,4-dinitrophenylhydrazones of 18 different saturated fatty acid p-phenylphenacyl esters were prepared by the procedure outlined above, with yields of from 93 to 100%. Repeated crystallizations from ethyl alcohol or mixtures of ethyl alcohol and ethyl acetate were usually required to secure material of constant melting point. The melting points of the p-phenylphenacyl esters and their 2,4dinitrophenylhydrazonesand the results of combustion analyses for the double derivatives prepared are listed in Table I.
Table I.
ilcid
Acetic , Propionic Butyric Isobutyric Valeric Isovaleric Hexanoic Isohexanoic Heutanoic Ocianoic Nonanoic Decanoic Undecanoic Dodecanoic Tridecanoic Tetradecanoic Hexadec-
Melting Points and Combustion Analyses of Acid Derivatives Combustion Analysis of ilelting points, o c, 2,4-DNHb 2,4-DNH of p-Phenylp-phenyl- of p-phenylphenacyl Esters phenacyl phenacyl %C % H ester ester Calcd. Found Calcd. Found 110.5-110.8 103.5-104.0 82.0-82.2 89.2-90.0 68.2-69.0 79.0-79,5 70.2-71 .O 70.9-71 . 9 64.5-65.0 68.2-69.3 20.5-71.5 I 6,0-i6.5 80.0-80.7 84,4-84.8 87,0-87,5
183.5-184.0 139.0 142.5-143.5 155,4-156 4 149.5-150.0 149.0-150.0 136.2-137.2 145.0-146.0 121.0-122.0 113.3-114.0 104.0-105.0 99.2-100 0 101.0-102.0 103.0-103.7 101.0-102.0
60 61 62 62 63 63 63 63 64 64 65 65 66 66 67
82 60 33 33 01 01 66 66 27 85 40 92 41 88 33
60 61 62 62 63 62 63 63 64 64 65 66 66 67 67
94 82 52 10 01 92 77 70 02 93 64 11 65 06 38
4.18 4.50 4.80 480 5 08 5 08 5 34 5 34 5 59 5 83 606 6 27 6 47 6 67 6 85
4.06 4.64 4.72 494 5 34 4 99 5 37 5 12 5 80 5 91 604 6 27 6 61 6 52 7 05
90.3-91,O
101.0-101 6 67 75
67 65 7 02
7 25
anoic
93.0-94.0
101.0-102.0 68 55
68 78
7 35
7' 23
anoic
96.3-97.0
101.5-102 2 69 27
69 42
7 65 7 57
Octadec0
b
.XI1 iiieltiriy point:: corrected.
L',4-Dir.itro~,hen).Ih?.drazonr.
Examination of the melting points listed in Table I shows that in several cases where the p-phenj-lphenacyl esters of acids have similar melting points, the 2,4-dinitrophenylhydrazones prepared from them show considerable differences in melting point. The Cs,iso-Ce, Cs,and COesters have an inclusive melting range of 4" C., for instance, while the corresponding double derivatives are distributed over a range of 42" C. with the closest interval being equal to 8" C. Examination of Table I also s h o w , however, that for the double derivatives of the acids of molecular weight greater than that of undecanoic acid which were investigated, the melting points were remarkably constant a t 101' to 102' C. Measurement of the melting points of both the p phenylphenacyl derivative and the p-phenylphenacyl-2,4-dinitrophenylhydrazone derivative of the acids, therefore, will be of great assistance in establishing the identity of an unknown acid in some cases, but of only limited assistance in others. The value of the melting points of the 2,4-dinitrophenylhydrazones of the p-phenylphenacyl esters as a means of identifying unknown acids is somewhat lessened because of difficulty in attaining a constant melting point by recrystallization of these derivatives. This difficulty is, however, probably not due to the possible presence of geometrical isomers, since only one zone is obtained on chromatographing each of these derivatives under conditions which have been shown to separate the syn and anti forms of certain other 2,4-dinitrophenylhydrazones ( 1 ) . It i p thought more likely that in this case the difficulty in securing constant melting points is due to polymorphism in these derivatives. This hypothesis is supported by the fact that differently
Table 11. Relative Rate of Travel of p-Phenylphenacyl-2,4Dinitrophenylhydrazones with Respect to Hexanoate Derivative R Value Acid R Value Acid 0.23 Octanoic 1.8 Acetic 0.38 Nonanoic 2.4 Propionic 0.53 Decanoic 3.3 Butyric 0.56 Undecanoic 4.0 Isobutyric 0.7'4 Dodecanoic 5.3 Valeric 0.75 Tridecanoic 6.5 Isovalerk 1.000 Tetradecanoic 8.8 Hexanoic 1. o Hexadecanoic 12 Isohexanoic 1.4 Octadecanoic 15 Heptanoic
V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6
1977
Table 111. Chromatographic Separations of p-Phenylphenacyl Ester-2,4-Dinitrophenylhydrazones (S = separated, 0 = not separated)
Acetic Propionic Butyric Isobutyric Valeric Isovaleric Hexanoic Isohexanoic Heptanoic Octanoic Nonanoic Decanoic Undecanoic Dodecanoic Tridecanoic Tetradecanoic Hexadecanoic Octadecanoic
S
.. s
8
s s ..os s 8 ..s s ..os s s . s ..os s s s s s s .. s
E
s s
.. s
. s
.. s
. . s. . s s
.. s
.. s..
colored and differently formed crystals may be obtained from the same preparation of a derivative simply by changing the solvent used for crystallization. In general, no depression of melting point was obeerved with a mixture of the two crystalline forms of a derivative of a single acid-that is, the melting range was the same as that observed when the lower melting form was melted by itself. The melting points listed in Table I are those of the higher melting, more stable crystal modifications. The greatest value of the 2,4-dinitrophenylhydrazones of the p-phenylphenacyl esters lies in the fact that extremely small amounts of the orange colored derivatives may be chromatographed to give measurable R values which may be helpful in the identification of a particular compound. Table I1 lists the R
values for the 18 derivatives investigated. All the derivatives chromatographed have significantly different R values, with the exception of the three cases of the normal and is0 acid derivatives of the same molecular weight. I t is possible that with a column of greater length the isobutyrate derivative could be separated from the normal butyrate derivative, but the two hexanoate derivatives moved with identical speeds, as nearly as could be determined. The values recorded for relative travel rates are reproducible when the chromatographic columns are carefully prepared. I t was found that a column could be used repeatedly for separations but that variations in the R values occurred as the column aged. Therefore, when a R value TTas desired to assist in the identification of an unknown acid, a freshly prepared chromatographic column was always used. In Table I11 are recorded the results of experimental chromatographic separations of various combinations of the double derivatives. In every chromatogram three different derivatives were run a t the same time, one of which was always the hexanoate or reference derivative. Examination of Table 111 shows that with the exception of the normal and is0 acid derivatives of the same molecular weight, the separations on the chromatographic column were excellent. In the case of the structural isomer8 no separation was evident. LITERATURE CITED
H. van, Rec. trav. chim. 73, 78 (1954). (2) Ikeda, R. M., Webb, A. D., Kepner. R. E., (1) Duin,
. ~ Z S ~ LCHEW. . 26,
1228 (1954).
(3) Kirchner, J. G., Prater, h. S . , Haagen-Smit, A. J., IND. Exc. CHEZI.,A s . 4 ~ ED. . 18, 31 (1946). (4) Kramer, P. J. G., Duin, H. van, Rec. trai.c h i m . 73, 63 (1954). (5) S h i n e r , R. L., Fuson. R. C., "Systematic Identification of Organic Compounds," 3rd ed., Wiley, S e w York, 1948. RECEIVED for review June 11, 1956.
Accepted August 11, 1956.
Division
of .4nalytical Chemistry, 130th Meeting, ACS, Atlantic City, N. J., September 1956. Taken from master's thesis presented to the Graduate Division. University of California, June 1956, by Hawkins Kg.
Determination of Primary Aliphatic Amines by an Acidimetric Salicylaldehyde Reaction JAMES B. JOHNSON and GREGORY L. FUNK Development Department, Carbide and Carbon Chemicals Co., Division o f Union Carbide and Carbon Corp., South Charleston, W. V a . An acidimetric method for the determination of primary amines uses an indicator end point. A measured excess of salicylaldehyde reacts with a primary amine and the excess reagent is titrated as an acid with sodium methylate in a pyridine medium. Aliphatic secondary and tertiary amines do not interfere. The interference of ammonia, alcoholamines, aniline, heterocyclic amines, and the ethyleneamines is discussed. Data are presented for the determination of 12 primary aliphatic amines. The method is rapid, easy to perform, and applicable over a wide rangeof concentrations.
T
HE methods available for the determination of primary
amines have been revien-ed by Critchfield and Johnson ( 2 ) . The reaction of salicylaldehyde with primary amines to form the corresponding imines was used by Wagner, Brown, and Peters (6) as a basis of a method for the analysis of amine mixtures. In their procedure the unreacted secondary and tertiary amines are titrated in a nonaqueous medium. Primary amine content may be calculated as the difference between a total base and
secondary plus tertiary amine determination. As this method does not employ an indicator end point, it is necessary to determine the entire potentiometric titration curve for each titration. This paper is a presentation of an unpublished method originally cited by Hillenbrand (6) and more recently by Critchfield and Johnson (8)for the direct determination of primary amines by the salicylaldehyde reaction employing an indicator end point. Moss, Elliott, and Hall (4), Fritz and Keen (S), and others have shown that many phenols may be quantitatively titrated as acids in basic solvents. The method described herein is based upon the fact that in pyridine medium, salicylaldehyde is acidic and can be titrated with sodium methylate. The reaction products of a primary aliphatic amine and salicylaldehyde are imine and water, XThich are neutral in pyridine medium. The amount of salicylaldehyde consumed, determined by titrating the exceas reagent vith sodium methylate, is a measure of the primary amine. REAGESTS
Methanol, anhydrous, Carbide and Carbon Chemicals Co. Pyridine, redistilled.
