INDUSTRIAL AND ENGINEERING CHEMISTRY
340
recorder-controller. It has a range of 8 p H units, adjustable within the range 0 to 14 pH. The circuit has also been successfully used for some time in a semiautomatic titrating device (6).
Aclinowledgment The authors wish to express their appreciation of the encouragement of L. Lykken and D. J. Pompeo.
Literature Cited (1) Am. SOC. Testing Materials, Standards on Petroleum Products and Lubricants (Committee D-2), pp. 13-18 (September, 1941).
Vol. 15, No. 5
(2) Henney, K., "Electron Tubes in Industry", 2nd ed., p. 79, New York. McGraw-Hill Book Co.. 1937. (3) Lykken, L., and Rolfson, F. B., IND.ENG.CHEM., ANAL.ED.,13, 653-5 (1941). (4) Lykken, L., and Tuemmler. F. D., I bid., 14, 67-8 (1942) (5) Penther, C. J., Rolfson, F. B., and Lykken, L.. Ibid.. 13. 833-4 (194 1). (6) Pompeo, D. J., Penther, C. J., and Hallikainen, K. E., paper in preparation. (7) Rider, J. F., "Vacuum Tube Voltmeter", 1941 ed., pp. 119, 122, New York, J. F. Rider Publisher. (8) Tarnele, M. W., and Ryland, L. B., IND. ENQ.C H I X . ,ANAL. ED.,8, 16-19 (1936). (9) Tamele, M.W., Ryland, L. B., and Irvine, V. C., I b id., 13, 618-22 (1941).
Colored Chromatograms with Higher Fatty Acids MORRIS &I. GRAFF AND EVALD L. SKAU Southern Regional Research Laboratory, U. S. Department of Agriculture, New Orleans, La.
T
HE higher fatty acids, being colorless compounds of
similar chemical and physical properties, present difficulties in their chromatographic separation, and such separation b y several workers (5, 6, 7) has been successful only to a limited degree. Cassidy (1, 2, 3) has made a careful study of the adsorption isotherms for various fatty acids in relation to their resolvability upon columns of various adsorbents. Among the obstacles encountered in using chromatographic columns has been inability to establish the location of. the zones which define the separation of the various fatty acids in the mixture. This difficulty has been overcomeinsomecolorlesscompounds (I0,11,13),butnoneofthese methods has been shown to be applicable-in.the case of fatty acids, Martin and Synge (8) applied the principle of producing a color change on an adsorbent to the chromatographic separation of the acetyl derivatives of the higher monoamino acids but they employed two liquid phases. Trappe (18)observed that silicagel columns became transparent or translucent when wetted by certain solvents, and under these conditions it was possible to observe zones formed in the study of lipid mixtures. This paper describes a method for separating mixtures of certain higher fatty acids into their components with the aid of adsorption columns, using an adsorbent impregnated with a dye t o serve as an indicator. T o be suitable for following the adsorption of fatty acids on an adsorbent, an indicator must change color on the column when in contact with the fatty acid, i t must be insoluble in both the solvent for the fatty acid and the eluting agent and, in addition, i t must revert to its original color after elution of the fatty acid. Preliminary experiments showed t h a t phenolsulfonphthalein (phenol red) as manufactured by the Eastman Kodak Company satisfies the above conditions. Columns prepared with heavy magnesium oxide impregnated with this indicator proved satisfactory in bringing about and observing a separation. (Good results were obtained only when the heavy magnesium oxide manufactured b y the J. T. Baker Chemical Company and the phenol red manufactured by the Eastman Kodak Company were used.) The fatty acids mere recovered from the adsorbent b y dissolving the magnesium oxide in particular sections of the column (4) in concentrated hydrochloric acid and water, and subsequently extracting the fatty acid with diethyl ether. Magnesium oxide was found to be particularly adaptable t o
this procedure, in that it is readily soluble in the acidified water.
Reagents Magnesium oxide, U. S. P. Baker's heavy powder; phenolsulfonphthalein (phenol red), Eastman Kodak Company; petroleum ether, Skellysolve F having a boiling range of about 30" to 60" C. The petroleum ether was distilled before use.
Fatty Acid Samples Stearic acid, Eastman Kodak Company, m.- p. 69.8" C:; myristic acid, Eastman Kodak Company, m. p. a4.6" C.; oleic acid, Baker's U. S. P.
Preparation of Impregnated Magnesium Oxide About 0.5 gram of phenol red was dissolved in 10 to 15 ml. of 95 per cent alcohol. A few grams of magnesium oxide were added and stirred into a paste, which was mixed thoroughly I1
FIGURE1. ABSORPTIONCOLUMNS I, Oleic and stearic acids. a.
11. Myristic and stearic acids. Cotton p l y b. Perforated porceain disk. Zone Length of Zone I I1 Mm. Mm. 35 .. 12 21 126
1
2
3 4
-Color- I 1 2
Yellow pink Ori ins1 pink
34
Lig& Original pink pink
8
7
7 175
I1 Yellow pink Original pink Yellow in Originafpibk
May 15, 1943
ANALYTICAL EDITION
while the alcohol was being evaporated on an electric hot plate. The resulting pink powder was dried in an oven at 100" C. The dried powder was mixed with 100 grams of magnesium oxide and the mixture was screened through a 200-mesh sieve. The adsorbent was then ready for use.
Procedure The adsorption column was retained in a vertical cylindrical glass tube 11 to 12 mm. in diameter and 60 to 70 em. in length, constricted a t the lower end. A plug of absorbent cotton held in position by a perforated porcelain disk was tamped over the constriction in the glass tube. A mixture of dye-impregnated magnesium oxide and petroleum ether in the form of a slurry was poured rapidly into the tube and the adsorbent was allowed to settle as the solvent percolated out of the tube, forming a column 20 to 22 cm. in length, but the liquid was never allowed to fall below the surface of the adsorbent. The best columns were obtained when the adsorbent was allowed to settle under a layer of solvent for several hours. The fatty-acid mixtures were dissolved in about 50 ml. of petroleum ether and added to the column. The column was then cautiously filled with petroleum ether and connected by means of a siphon to a reservoir-flask of petroleum ether a t a higher level. When the fatty-acid mixture came in contact with the adsorbent, a bright yellow band was formed which separated into zones upon development. The chromatogram was developed by allowing petroleum ether to percolate through the adsorbent continuously until clearly defined zones were obtained. The volume of petroleum ether used and the rate of flow varied, depending on the packing of the column. After formation of clearly defined zones, the column mas permitted to run dry, forced from the tube, and then cut into sections as defined by the change in the color of the adsorbent. The cuts were made in order best to demonstrate the separation of the constituents of the mixture. The fatty acids were recovered from the adsorbent by dissolving the latter in hydrochloric acid and then extracting with diethyl ether. The ethereal extracts were washed free of indicator and acid by water in a separatory funnel and subsequently dried over anhydrous sodium sulfate and evaporated to dryness under a stream of nitrogeb. The residues were dried in a vacuum over phosphorus pentoxide to constant weight. Samples were removed for iodine number (9) and melting point determinations.
SEPARATION OF A MIXTUREOF OLEIC TABLEI. ADSORPTIVE .4ND STEARIC ACIDS (COMIIERCIAL GRADES) Section 1 2 3 4
Weight of Fraction
Iodine No.
Mg. 110.2 21.6 71.3
1.6 72.8 91.6
1.4
.*.
Melting Point
c.
65-69
.. .. .. ...
341
required 14 days. The appearance of the column and the definition of the zones are sketched in Figure 1 and the analytical data are tabulated in Table 11. The fractions recovered on evaporation of the ether solutions from the different sections to dryness, without recrystallization, gave successively lower melting points from the top t o the bottom of the column, indicating t h a t a separation had been accomplished. These fractions probably contained traces of solvent or other accumulated impurities. By recrystallization from acetone i t was possible t o show t h a t the acids recovered from section 1 and section 2 consisted mainly of stearic acid and myristic acid, respectively. The generality of the application of this method to chromatographic separation of a mixture of fatty acids is indicated by the fact that definite zones have been obtained with other mixtures of fatty acids. I n the case of palmitic and stearic acids, where the length of chain differs b y only two carbon atoms, zones mere developed, but i t was difficult to establish definite proof of a separation using melting points as criteria because of the proximity of their melting points.
TABLE 11. ADSORPTIVE SEPARATION OF A MIXTURE OF MYRISTIC AXD STEARIC ACIDS (COM1IERCIAL GRADES) Sections 1 2 3 4
Weight of Fraction
Melting Point
Me.
c.
44.7
56-57 51-52 44-46
36.5 31.5 0.8
Melting Point after Recrystallizin 1st time 2nd time
...
c.
63 54
....
c.
68 54
.. ..
Summary A method has been described for separating mixtures of higher fatty acids b y Tswett adsorption analysis, whereby separation into zones was observed on a column of heavy magnesium oxide impregnated with a suitable indicator. The fatty acids were recovered b y dissolving the magnesium oxide in particular sections of the column in acid and extracting with ether. B y this procedure i t was possible to demonstrate a separation of a n unsaturated fatty acid from a saturated fatty acid of the same number of carbon atoms and of two saturated fatty acids differing in chain length by four carbon atoms.
Acknowledgment Experimental B y means of the above procedure i t was possible to demonstrate a separation of (I) a n unsaturated fatty acid from a saturated fatty acid of the same number of carbon atoms and (11) two saturated fatty acids differing in length of chain b y 4 carbon atoms. In (I) a mixture of 100 mg. of stearic acid (C18H3S02), melting point 69.8" C., and 100 mg. of oleic acid (C18&02), iodine number 86.4, was used. The development of the chromatogram required 17 days. The appearance of the column and the definition of the zones are sketched in Figure 1. The analytical data shown in Table I indicate an almost complete separation of the fatty acids in the mixture. Practically all of the stearic acid was retained in section 1 and the oleic acid had moved down the column. T h e fractions recovered from sections 2 and 3 were pale yellow oils. I n (11) a mixture of 50 mg. of stearic acid (C18HsBOJ, m. p. 69.8" C., and 50 mg. of myristic acid (ClrH,02), m. p. 54.6' C., was used. The development of the chromatogram
The authors wish t o thank H. R. R. Wakeham for his interest and cooperation in the course of this work.
Literature Cited Cassidy, H. G., J . A m . Chem. S O C 62,3073 , (1940). Ibid., 63, 2735 (1941). Cassidy, H. G., and Wood, S. E., Ibid.,63, 2628 (1941). Duschinsky, R., and Lederer, E., Bull. SOC. chlm. bid., 17, 1634 (1935). Kaufmann, H. P.. Fette u. Seifen, 46, 268 (1939). Kondo, H., J. Pharm. SOC.Japan, Trans., 57, 218 (1937). Manunto, C., Helv. Chim. Acta, 22, 1156 (1939). Martin, A. J. P., and Synge, R . L. XI., Biochem. J., 35, 1358 (1941). Rosemund, K. W., and Kuhnhenn, W., 2. Untersuch. Nahr. u. Genussm., 46, 154 (1923). Strain, H. H., "Chromatographic -idsorption Analysis", New York, Interscience Publishers, 1942. Strain, H. H., J. Am. Chem. Soc., 57, 758 (1935). Trappe, W., Biochem. Z., 306, 316 (1940). Zechmeister, L., and Cholnoky, L., "Principles and Practice of Chromatography", tr. from 2d and enl. German ed. by A. L. Bacharach and F. A. Robinson, London, Chapman & Hall, 1941.
