V O L U M E 23, NO. 7, J U L Y 1 9 5 1 Table I.
1033 The separation of quinoline and aniline encountered in the Skraup synthesis can be accomplished by this method.
Experimental Results M.P.
Amine Mono-n-propyl Mono-n-butyl Monoisoamyl Diethyl Di-n-propyl Diisobutyl Di-sec-butyl Di-n-butyl Diisoamyl Triethyl Tri-n-butyl Tribenmyl Pyridine a-Picoline ?-Picoline 2,4-Lutidine 2-Vin ylp yridine Quinoline Quinaldine 8-Quinolinol @-Diethylaminoethanol p-Anisidine p-Phenetidine Triethanolamine Di-o-tolylguanidine Mixture of diisobutyl and di-sec-butyl Mixture of triethyl a n d tri-nbutyl Mixture of pyridine a n d apicoline Mixture of mono-n-butyl a n d monoisoamyl After 4 recrystallizationq.
(Corr.) of @-Resorcylate 123 133 138 128 97 144 146 123 146 120 121 141 145 141 125 143 113 128 145 150 D1 _.
143
*-.
1x7
Oil 107-l15n
Formula of Salt CioHiaOiN CiiHiiOlN CizHigOiN CiiHirOiN CiaHziOiN CirHzaOiN CiaHzs04N CisHasOiN Cir HzsOiN CiaHziOiN CigHaaOiK CzaHwOiN CixHiiOiN CiaHiaOiN CisHiaOiN Ci4HisOdN CuHuOiN CiaHiaOiPi CiiHiaO4N CieHiaOaN Cia HioOaN Ci4HiaOsN CisHiiOaN
..... .,.,,
138-142
.. ...
103-108
.....
130-132
.... .
132-133
, ,, , ,
8 e & 6.57 6.17 5.82 6.17 5.49 4.95 4.95 4.95 4.49 5.49 4.12 3.17 6.01 5.67 5.67 5.36 5.40 4.95 4.72 4.68 5.16 4.88 4.65
.. ..
.. .. ..
%N
Found 6.60 6.10 5.83 6.28 5.39 4.93 4.93 4.91 4.30 5.46 4.24 3.19 5.78 5.78 5.54 5.41 5.49 5.11 4.88 4.53 5.18 4.91 4.56
..
.. .. .. ..
certainty whether or not a given amine would form a salt, but the correlation between base strength and salt formation seems to be applicable in most cases. SEPARATION OF MIXTURES OF AMINES
A mixture of 0.84 gram of quinoline and 0.66 gram of aniline was added to 2.00 grams of p-resorcylic acid in 15 ml. of ether. After standing overnight the quinoline p-resorcylate was removed by filtration, washed with a small amount of ether (the salt is slightly soluble in ether), and dried in air; 1.4 grams of quinoline 8-resorcylate were isolated, representing 78% recovery of quinoline. Aniline was recovered by evaporation of the ether from the filtrate after removal of the excess @-resorcylic acid. The acid was removed by washing with successive portions of sodium carbonate solution and drying over solid sodium hydroxide; 0.35 gram of aniline was recovered, representing 53% of the original amount. A mixture of 1.00 gram of pyridine and 1.00 gram of dimethylaniline was separated by treatment with 3.0 grams of @-resorcylic acid in 25 ml. of ether in a manner similar to that described above; 2.51 grams of pyridine 8-resorcylate were isolated, representing 85% recovery of pyridine. Recovery of dimethylaniline was practically quantitative. A mixture of 1.20 grams of tri-n-butylamine and 0.93 gram of 2-naphthylamine was separated by treatment with 2.0 grams of p-resorcylic acid in 15 ml. of ether as above; 1.5 grams of trin-butylamine 8-resorcylate were isolated, representing 68% recovery of the amine, and 0.85 gram (92%) of the 2-naphthylamine was recovered. The free amines can be regenerated from the 8-resorcylates simply by shaking the salt with sodium carbonate solution or sodium hydroxide solution. It seems probable that these salts might find use in isolation of amines from reaction mixtures as well as in separation of mixtures of amines. LITERATURE ClTED
Ranon, E. S. G., J . Bid. Chem., 1 2 1 , 3 1 3 (1937). (2) Felsing, W. A., and Biggs, B. S., J . Am. Chem. SOC.,55, 3624 (1)
(1933). (3) (4)
Hall, N. F., and Sprinkle. M. R., Ibid., 54, 3469 (1932). Marcali, Kalman, and Rieman, Wm., ANAL. CHEM.,18, 708 (1946).
The fact that 8-resorcylic acid does not form salts with most aromatic amines suggested that it might be used in the separation of mixtures of various amines.
(5) “Organic Syntheses,” Coll. Vol. 11, New York, p. 557, John
Wley & Sons, 1943. RECEIVED July 15, 1950.
Paper Chromatography of Volatile Acids E. P. KENNEDY’ AND H. A. B4RKER Division of Plant Biochemistry, University of California, Berkeley, Ca1;f. URING an investigation of the fatty acid metabolism of the bacterium Clostridzum kluyveri, it became necessary to identify and separate small amounts (1 to 2 micromoles) of volatile fatty acids ranging in chain length from 2 to 6 carbon atoms. The valuable method of Elsden ( 2 ) , based on chromatography of the fatty acid mixture on silica, could not be used because it requires considerably larger amounts of material. Therefore a paper chromatographic method was developed that would permit the separation and identification of fatty acids in micromole quantities. The separation of nonvolatile organic acids by chromatography on paper has been described by Lugg and Overell ( 6 ) ,but in this method the substances to be separated are applied to the paper as the free acids, a technique that obviously cannot be used FT ith volatile acids. The conversion of volatile acids to their nonvolatile hydroxamic acid derivatives by the method of Fink and Fink ( 8 ) is objectionable because it involves several additional steps. In the method described in this report, the volatile acids are applied to the paper as ammonium salts, and the chromatograms are developed in solvents containing free ammonia, Present address, Biochemical Research General Hospital, Boston, hlass. 1
Laboratory, XIassachusett-
so that the acids are present a t all times as the completely ionized ammonium salts. The location of the spots after development is accomplished by spraying the dried paper with bromophenol blue indicator, made acid nith a little citric acid. Since this method was v\orked out, two papers by Brown and Hall ( 1 ) and Hiscov and Berridge ( 4 ) have appeared, which describe the paper chromatography of volatile acids in ammoniabutanol mixtures. The method described below, while similar in principle, differ8 in important details and in the authors’ hands appears to offer some advantages. I n particular, the recommended indicator gives a more even background and sharply defined spots, th.e use of ammonium salts eliminates the intense and frequently confusing alkaline spots due to other cations, and the pretreatment of the paper with ovalic acid eliminates “ghost” spots. EXPERIMEYTAL
Whatman S o . 1 filter paper n a s used throughout the study
It was found necessary to wash the paper before use in order to avoid troublesome streaking and ghost spots. Good results were obtained nhen the paper nas thoroughly washed before use with 1% oxalic acid, then with copious amounts of distilled water, and finall\ dried at room temperature.
1034
ANALYTICAL CHEMISTRY
Unidimensional chromatograms, made by the "ascending method," were used in most of these experiments. The chroniatograms n-ere developed a t room temperature (20-23" C.) for 6 to 8 hours. Thoroughly washed, dried papers 45 cm. in length and 25 em. in widt.h were used, and the ammonium salts of the acids were applied to a starting line 2.5 cm. from the bottom of the paper, usually in 0.01 ml. of aqueous solution, containing 0.5 t o 1.5 micromoles of each acid. If the fatty acids were added t o the paper as the sodium salts, as in the method of Brown and Hall ( I ) , the rates of migration of the acids with ethyl alcohol-ammonia as the developing solvent were considerably sloyer, and regions of excess alkalinity usually marked the t'racks of the acids. If a solution of the sodium salts was first mised with an equimolar amount of ammonium sulfate and made alkaline with a little free ammonia, and this mixture was applied t o the paper, the rates of migration were then approximately the same as those of the pure ammonium salts alone. .liter application of the salts of the acids, the paper was rolled into a cylinder and pinned together, and the chromatogram was developed. -1 number of developing solvents were used successfully. These included aqueous ethyl alcohol-ammonia solutions, aqueous acetone-ammonia, and aqueous butanol-ammonia. With the first two solvents, which are miscible with water, the Rj values could be altered considerably by varying the percentage of water. In general, the addition of a larger portion of water to the mixture caused the organic acids to travel more rapidly on the chromatogram. This flexibility of such watermiscible solvents may prove useful in separating the ammonium salts of dibasic acids which do not move appreciably in the aqueous ammonia-butanol mixture described by Brown and Hall. .\ simple solvent mixture that proved useful in this work was composed of 100 ml. of 95% ethyl alcohol, to which was added 1 nil. of concentrated ammonium hydroxide. Rr values of a number of acids in this system are shown in Table I. .Ift,er development of the chromatogram, the papers were dried in an oven a t 100" C. for 5 minutes. The spots were then located bj- spraying with a solution of 50 mg. of bromophenol blue in 100 nil. of water, made acid with 200 mg. of citric acid. Because of the buffer capacity of the acid anions, the location of the spots is shown hy the intense blue (alkaline) color of the indicator in
Table I.
Rj Values of Acids in A m m o n i a c a l Ethyl alcohol Solution .Icid Formic Acetic Propionic n-Butyric n-Valeric n-Caproic n-Heptanoic n-Octanoic Vinylacetic n-@-Ketohexanoic
R: 0 0 0 0 0
31 33
44 54 60 0 68 n 72 0 76 0 46 0 53
these regions, while the background is orange-yellow. This method of locating the spots was found to be more satisfactory than the use of bromocresol green made alkaline with a little sodium hydroxide ( I , 4 ) . In experiments in which radioactive fatty acids were to be separated, and it \vas desired to make radioautographs of the chromatograms, the apers were not sprayed with indicator, but instead were sprayez before drying with a 0.5 Jf solution of potassium monohydrogen phosphate, which fixes the acids on the paper as the potassium salts. Radioautographs of the papers may then be made by conventional techniques. LITERATURE CITED
(1) Brown, F., and Hall. L. P., .Vatwe. 166, 66 (1950).
(2) Elsden. 5.R., Biochem. J . , 40,252 (1946). (3) Fink, K., and Fink, R. AI., Proc. S O C .E.@. Bid. M e d . , 7 0 , 654 (1949). (4) Hiscox, E. R., and Berridge, S . ,J., .Vatwe. 166, 522 (1950). (5) Lugg, J. W. H., and Overell, B. T., d i ~ s t r n l i a nJ. Sei. Research, (A) 1, 98 (1948). RECEIVEDSeptember 5 , 1950. Investigation supported in part by research grants from the American Cancer Society and the Division of Research Grants and Fellowships of t h e National Inititutes of Health, United States Public Health Service.
Modified Photometric Determination of Copper in Ferrous Alloys KENDALL W. N-INCE Kaiser Steel Gorp., Fontana, Calif.
HE photometric determination of copper in ferrous alloys, T a b l e I.
according to the method of Dunleavy, \J-iherley, and Harley
(I), may be modified to obviate the use of a pH meter. Such a modification simplifies the method as an implement for the control analyst. The folloning procedure will produce a solution from which optimum extraction of the copper coniplei with a-benzoinouime is o1,t:iined
D e t e r m i n a t i o n of Steel and Cast Iron
160
20d 21c 55b
Cast iron 4g
1 12.1 12.3 la.1 12.3
12.0
2
True value,
Kiett-Summerson, %
PH B.S. Steel
2
3 0.062
%
1 0.061 0 164 0.056 0 038
0.061 0.161 0.054 0.039
0.164
0,164
11.3
3 12.0 12.1 12.3 12.3
0,040
0.040
12.0
11.4
0 236
0.252
0.238
0.240
11.4 12.4 12.3
0.060
0,050
0.054
REAGESTS
Boric Acid. Prepare 1 liter of saturated solution. Let the solution stand overnight a t a temperature of 23' C. and theii filter through a rapid pa er at 23" C. The boric acid may bc standardized with 0.2 $sodium hydroxide solution. Measure out 10 ml., add mannitol, and titrate with 0.2 N sodium hydroxide to a phenolphthalein end poiit. A titration of 42.65 nil. should be obtained. Potassium-Sodium Hydroxide Mixture. Five liters of solution contain 280.0 grams of potassium hydroxide and 395.0 grams of sodium hydroxide. Pipet 10 ml. into a 250-ml. volumetric flask, make up to the mark with distilled water, and mix well. Pipet 25 ml. into a titrating flask. Add 20 ml. (buret) of 0.2 N sulfuric acid and boil t o remove traces of carbon dioxide. Cool, add phenolphthalein, and titrate. with 0.2 N sodium hydroxide. Adjust the strength of the initial caustic solution until the 25-ml. aliquot is equivalent to 13.86 ml. of 0.2 N sulfuric acid. Sulfuric-Tartaric Acid Mixture. Dissolve 800 grams of tartaric acid in 1500 ml. of water, add 935 ml. of 2 N sulfuric acid, and dilute to 2500 ml. Pipet 10 ml. into a 250-ml. volu-
metric flask. Make up to the mark. Mix well and pipet a 25ml. aliquot into a titrating flask. Heat to boiling, add phenolphthalein, and titrate with 0.2 N sodium hydroxide. The 25ml. aliquot (1 ml. of acid mixture) is equivalent to 24.55 of 0.2 N sodium hydroxide. The above reagents appear to be relatively stable. S o appreciable alteration has been noted in solutions used over a 6-month period. PROCEDURE FOR DETERiMINATlON O F COPPER
In Steel. Weigh a 0.500-gram sample of steel into a 400-ml. beaker and add 15 ml. (buret) of boric acid solution and 7 nil. of concentrated nitric acid. Cover with a borosilicate glass cover glass and evaporate to dryness. Remove the cover glass and invert it upon the hot plate (270" (3.1. Bake the sample and cover for 3 minutes. Samples baked for 7 minutes yield equally
