Quantitative Hydrogenation of Unsaturated Fatty Acid Derivatives Hydrogenation of Less Than Milligram Amounts in the Warburg Apparatus JAMES F. BIEAD AND DAVID R. HOWTON, Medical School, University of California, Los Angeles, Calif. ECENT publication (7') of a method for the determination R of unsaturation by microhydrogenation prompts a report of a procedure developed some time ago (5) to serve a similar purpose. Having in hand a promising method (6) of isolating very small, pure samples of unsaturated fatty acid esters derived from natural sources, the authors were faced with the problem of determining the extent to which these samples were unsaturated. As it did to Ogg and Cooper ( 7 ) , microhydrogenation appeared to be much more attractive than microhalogenation because of its less capricious dependence upon structural factors. Some years ago, Kuhn and Moller ( 8 ) published details of a method hased on simiIar reasoning. Although a number of microhydrogenation apparatus have been described ( d ) , these were considered unsuitable because they involved more or less complicated construction or were incapable of measuring accurately the small hydrogen uptake anticaipated in the experiments. The commercially available Barcroft-Warburg apparatus, with no more complicated modification than is customary for studies involving gases other than air, serves admirably for the microhydrogenation of less-than-milligram amounts of material containing as little unsaturation per molecule as that found in methyl oleate. Coupled with a colorimetric determination ( 1 ) of carbomethoxy content, this procedure suffices to distinguish very small amounts of such esters as methyl oleate, linoleate, :md linolenate from one another. PROCEDURE
Standard single-side-armed 15-ml. Warburg flasks are charged as follows: Approximately 5 mg. of catalyst (10% palladium black on charcoal) and 2.5 ml. of redistilled 96% ethyl alcohol are placed in the main compartment of the flasks, while 0.5 nil. of an ethyl alcohol solution of the unsaturated compound (1 to 109 moles per ml.) is added to the side arm (which should be sufficiently large to prevent spilling into the body of the flask during the equilibration-period shaking). The charged flasks are connected to the Warburg manometers and the air in them is replaced by hydrogen, by means the apparatus described by Burris ( B ) , except that screw clamps are used to prevent rising of the nianometer fluid from its reservoir bulb during evacuation. l'ahle I.
Hydrogen Uptake of Unsaturated Acid L)eri\ atives Aniount p moles
Substrate
MI:
Maleicantiydride
0.23
hlethyl oleate
0.54 1.08
Methyllinolvate
0.35
1.19
hlethyllinolenate
0.19 0.47
0.64 1.60
H l d r o g e n Uptake, p Moles Observed
Calcd
~ ~ Bonds
Found
'2.87
2.50 2.47 2.42
+5,5 f4.2 +2.1
1.06 LO4 1.02
1 81
1.81 3.62
1.85 1 67 3.48
4-2.2
3.62
-7.7 -3.9
1.02 0.92 0.96
9.38
2.29 2.47
-3.8 +3.8
1.93 2.08
1.92 4.80
1.86 5.02
+4.6
-3.1
2.91 3.14
2.37
Without disconnecting the assembly from the hydrogen reservoir, the manometer stopcocks are closed by a quarter turn, and the shaking is started. When the absorption of hydrogen by the catalyst and the solutions has stopped (this requires about, 30 minutes and is followed manometrically), the manometer stopcocks are reopened to the system, and the manometer fluid is readjusted to the zero point (usually 250 mm.). The stopcocks are again closed, the manometers are disconnected from each other, and the system is equilibrated for 5 minutes. The contents of the side arms are then tipped into the main compartmenta, and 'the shaking is continued for 5 minutes. At this time a preliminary reading is taken and, by appropriate tipping of the manometers, the side arms are rinsed with some of the reduced solution; the shaking is then continued for an additional &minute period. The final readings are corrected against a blank manometer, identically charged and manipulated, hut containing no unsaturated material, and the corrected hydrogen uptake is converted to micromoles by the usual procedures. Using this technique, the hydrogen uptake of less-than-milligram amounts of several unsaturated acid derivatives has been mevsured and is reported in Table I. The maleic anhydride was freshly sublimed; the methyl esters were purchased from the Horniel Foundation and were reported to have the following iodine numbers (Wijs): oleate, 85.43 (theory 85.6); linoleate, 172.4 (theory 172.4); linolenate, 258.6 (theory 260.4). ERRORS AND PRECAUTIONS
A survey of the literature failed tQ reveal data on the solubility of hydrogen in 96% ethyl alcohol, which are required in the evaluation of the Warburg flask constant. However, because this factor affects only about 1% of the flask constant, using data on the ~olubilityof hydrogen in absolute ethyl alcohol at 30" C. (4) probably introduces no great error. Absolute ethyl alcohol is to be avoided as a solvent in these microhydrogenations because of its usual benzene content. The reading of the manometers, especially where small differeiica of level are involved, is probably the greatest single source of low precision. To minimize the change in readings whic*hoccurs when the shaking of the flasks in the bath is iiiterruptcd, the method of reading is standardized in any convenient w t y :ind accomplished as quickly as possible. The temperature of the bath should also be standardized, and should be as close to room temperature as proper functioning bf the bath thermost:tt \sill permit. The lubricant used on the ground surfaces of the apparatus sliorild he irisolublc in 96% ethyl alcohol and must not itself ~ b , ~ undcr the conditions of the determinations. take ~u p hydrogen Tlic silirone-type high vacuum grease manufactured by the Dow Corning Company was found to be s'atisfactory from these standpoints; use of anothcr common lubricant of a different type led to continued erratic hydrogen absorption. Following preliminary examination of various hydrogenation catalysts, Adams' platinum oxide was discarded because the small amounts required were not adequately dispersed in the solution by the rather moderate shaking typical of the Warburg apparatus. .\ catalyst which proved to he eminently satisfactory both from the point of view of easy suspension in the solution and for its
1204
V O L U M E 22, N O , 9, S E P T E M B E R 1 9 5 0 activity is the 10% pnlladium on charcoal manufactured by the American Platinum Works. The volume of the solution of substrat,eis important in that i t must be large enough for adequate transfer from the side arm to the niniii coinpart,ment and amall enough for adequate equilibration with hydrogen before bhe substrate and catalyst solutions are mised. In this connection, it is wise not to fill the side arm too full, lest premature mixing occur during evacuation of the system or during the equilibration shaking period. For the type of flask used in this laboratory (American Instrument Company, Xo, 5-201 without center d l ) , 0.5 ml. of substrate solution rr-as entirely satisfactory. A cursory examination. of Table I discloses the fact thst the inetors discussed above may lead to very tLppreciahle errors in terms of per cent; however, the precision is adequate for the determination of the number of carbon-bearbon double bonds I I I less-than-milligram amounts of fatty acid derivatives.
1205 LITERATURE CITED (1) Bauer, F. C., Jr.. (19491~ .
.~...
and Himeh, E. F.. Aich. Biocha., 20, 242
(2) Johns. I. B., and Seifede, E. J., 1x0. ENO.Cnsx.. ANIT.. ED 13. 841 (1941).
(3) Kuhn, R.. and Moiler. E. F., Awew. Chem., 47, 145 (1934). (4) Muted. E. B., and Moon, C. H.. Trans. Faraday Soc., 32. 76 (1936).
( 5 ) Mead, J. F.. and Howton. D.
R.. University of California at Lo Angeles, Atomio Energy Project, Repl. U.C.L.A.20 (1949). (6) Mead. J. F..Howton, D. R.,and Kryder. G.D., work in progrear (7) Ogg. C. L.. and Cooper. F. J.. ANAL CEEM.,21, 1400 (1949). ( 8 ) Umbreit. W. W., Burris, R. H., and Stauffer, J. F.. "Mano metric Techniques and Related Methods for the Study E Tissue Metabohsm." P. 44, Minneapolis. Minn., Burgee Publishing Co., 1948. Racarvso Deeeinber 12, 1949. Based on work Derformed under Contrae:t No, AT64-1-GEN-12 the Atomic Cornmisslo"and th /e University of California at Los Angeles.
Elastomeric Test Films from Acidic latices T. B. BLEVINS, W. S. WRIGHT, AND FRED LEONARD .lrmy Prosthetics Research Laboratory, Forest Glen, Md.
HE necessity for obt.aining homogeneous "flaw-free" Tsamples for testing the stress-strain properties of elastomeric films is well recognized; Tensile strength, elongat.ion, modulus, and tear rPsistance are all affected by the presence of flaws such as air bubbles, surface rraeks, ridges, edge nicks, and t,hickness heterogeneitiesin the filmstesterl. Previous authors ( 5 )considering the problem have recommended carefully pouring the latex 011 flat g l u s plates and using controlled humidity and preeipitating atmospheres to prwent the formation of an impervious SUP fare film which would prevent further escape of water from the underlayers within the latex film. Use of this method in this 1al)oratory has in many cases yielded flawed and irregular films d acidic copolymer latices of ethyl acrylate and acrylonitrile, won after the alkalinity had been adjusted to p H 10.5 with sodium hydroxide. It became necessary therefore to devise a method for casting, from such acidic type latices, films which rvould be essentially flaw-free. Thick plaster oi Paris molds are generally used t o cast commercial articles from latex. The mechanism of film formation against such a plaster mold is usually considered as being due to absorption of water through the minute plaster of Paris pores, the size of which prevents pusnge of the solid polymer particles. The polymer film is deposited as B more or less porous mass
through which diffusion of water may occur, and drying m;t: take place simultaneously from both surfaces-.g., through th plaster mold and from the side exposed to the atmosphere. Thi has a distinct advantage over casting against glass, where dryin! may take place through one mrface only. For these reasons, i was decided to attempt t o apply this technique to casting fls films for test purposes. In the method used, thick plaster molds with an interior cavity the size and shape of a 250-ml. high-form beaker were prepared. This shape of mild was used for several reasons. Its cyliddrical shape allows the film to distribute evenly any stresses that develop in drying. In addition, under these condibions of casting, the film, during formation, isrnain+ined in contact with rm queous medium that prevents the formation of an impervious surface skin, which would cause entrapment of water in the subsurface layers. Experience has shown thst such a skin is likely to d e velop wrinkles and other flaws as the film dries. Films depwited on plaster molds were, built up rapidly and wet films 0.060 to 0.090inch thick could b r deposited in 1 to 2 hours. ~~
MOWS
The molds w r e prepared from Coe Dental Laboratories Hydrocal dental stone and distilled water. To 2100 grams of stone. 700 ml. of distilled water were slowlv added and the resulting slurry was kneaded between the ling& until d l lumps had
solution of Geen soap.anh allowed*todry,
