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Vol. 77
two exponential terms whose decay constants are given by
with a-dextrin. Long chain fatty acids, alcohols and esters fall into this category. Their chemical characteristics are not pronounced so that it is difficult to find indicators of a sensitivity adequate for use in paper chromatograms. Although various sugIn certain favorable cases these transients could gestions have been made to solve these diffic~lties,~ also be employed in deducing velocity constants. paper chromatography does not hold its due place For example, if k L 1 were very large compared to among analytical tools in the lipid field. (kz klEo), one decay constant would tend to a-Dextrin can be used successfully on chromatozero, and the other to - k - l , while if k2 were very grams of fatty alcohols, acids, methyl or ethyl esklEo), one decay con- ters and monoglycerides. Diglycerides and triglyclarge compared to ( k - l stant would tend to - klEo, and the other to - kz. erides do not respond to this indicator method, lx-ith modern, high speed computing devices i t is since they do not react readily with a-dextrin. quite feasible to find by trial the unique set of val- However, they can be detected by splitting them ues for the parameters needed to reproduce a given into their components after chromatographic sepexperimental concentration time curve for any com- aration. The necessary hydrolysis is carried out ponent of the reaction system. This is easily done in situ enzymatically by means of commercial panwith an analog computer where the programming creatin. I n this way it is possible to make visible is direct and simple. However, the usefulness of the separated di- and triglycerides on the paper. the procedure may be limited in situations where Two other indicators were found to be applicable there are wide differences in order of magnitude be- to certain types of lipids. Iodine vapors have been tween the various parameters, e.g., Eo and So. used for detecting a variety of substances4 but not U. S. NAVAL MEDICAL RESEARCH INSTITUTE for higher fatty acids. I n the lipid series we found BETHESDA,MARYLAND i t specific for spotting unsaturated components. For instance, oleic acid and its derivatives appear as brown spots on chromatograms when exposed to Indicators for the Paper Chromatography of Lipids‘ iodine vapors. The limitation of the method is BY HELMUT K. MANGOLD, BEVERLY G. LAMPAND HERMANNgiven by the fact that symmetrical oleodipahitin SCHLENK cannot be located unambiguously in actual chromatograms. A more specificreagent is lead tetraaceRECEIVED JUNE 24, 1955 tate which is applicable to the chromatography of The inclusion reaction has received considerable monoglycerides. The reagent hydrolyzes in air to attention in recent years. One of the basic princi- form brown lead dioxide ; with a-monoglycerides it ples resulting from research in this field is that undergoes the normal glycol splitting reaction and shape rather than chemical character of a molecule yields colorless lead compounds.j Consequently, determines whether or not i t can be included by a monoglycerides appear as white spots on a brown certain host. The complexing reaction can be background in chromatograms sprayed with lead carried out in minute amounts such as are used in tetraacetate solution. For the detection of di- and practical paper chromatography. This is easily triglycerides i t can be used after splitting them by demonstrated by spraying a filter paper bearing a the above-mentioned enzymatic hydrolysis. spot of octadecane with a-cyclodextrin and subseComplexing is the most universal method and is quently exposing i t t o iodine vapors. The area certainly applicable to many other compounds becovered with hydrocarbon remains white, while the sides the lipids. remainder of the paper turns bluish purple. This Table I shows R f values obtained by chromatooccurs because of different reactivities of the free graphing model mixtures and individual lipids for and of the complexed a-dextrin. Free a-dextrin identification, Different solvent systems were reacts with iodine to form a deeply colored inclusion used in ascending technique with silicone impregcomplex very similar to that resulting from starch nated paper, i.e., a reversed phase chromatography. with iodine. I n fact, the two reactions are closely Whatman No. I paper was modified according to related2 and the same phenomenon can be observed procedures reported in the literature. We found with starch on a spotted paper. Comparative examination proved a-dextrin to be the preferable impregnation with silicone6 to be the most versatile indicator in actual Chromatograms. When the a- preparation for our purpose. Since silicone is not dextrin is already occupied by a guest molecule, in washed out by the developing solvents as hydrocarthe above case the octadecane, it is no longer avail- bons are from impregnated papers, the same paper able for the iodine reaction which produces color. can be used for reversed phase chromatography in Alccordingly,the spot of hydrocarbon stays white. the second dimension. Uniform coating is easily This holds for other compounds having low chemi( 3 ) J. Asselineau, Bull. SOL. chim. France, 884 (1952) (review); cal reactivity besides hydrocarbons ; for example, H. P. Kaufmann and W. H. Kitsch, Fette und Seifen, 56, 154 (1954); esters, ethers or other types of molecules that react H. P. Kaufmann and H. G. Kohlmeyer, ibid.. 67, 231 (1955); y .
+
+
(1) Supported by research grants from the U. S. Atomic Energy Commission, the Sational Institutes of Health (P.H.S. 4226, of the Public Health Service). and by the Hormel Foundation. Presented a t the 127th meeting of the American Chemical Society, Cincinnati, Ohio, April, 1955. Hormel Institute Publication S o . 133. (2) F. Cramer, “Einschlussverbindungen,” Springer, Berlin, 1954, p. 71 ; K . E. Rundle, J. F. Forster and R. R. Baldwin, THISJ O U R N A L , 66, 2116 (1044); R. E. Rundle, ibid., 89, 1769 (l0?7).
Inouye, M . Noda and 0. Hirayama, J . A m . Oil Chemists’ Soc., 32, 132 (1955); B. D. Ashley and U. Westphal, Arch. Biochcm. Biophrs., 66, 1 (1955); J. W. Dieckert and R. Reiser, Science, 120, 678 (1954). (4) G. B. Marini-Bettolo-Marconi and S . Guarino, Expeuienlia, 6, 309 (1950); G. B. Marini-Bettolo, Estraffod a i Rendiconti dell’ I S f i f Z l o Superiove d i S a n i f a , XVII, 471 (1954). (5) J. G. Buchanan, C. A. Dekker and A. G. Long, J . C h m . SOC., 3162 (1950). (6) T. H. Kritchevsky and A. Tiselius, Science, 114, 299 (1921).
NOTES
Nov. 20, 1955
by dividing the distance from starting line to center of spot TABLE I by the distance from starting line to solvent front. f Ratios Rr VALUES'OF LIPIDS IN DIFFERENTSOLVENT SYSTEMS~ are vol./vol.
Lauric Myristic Palmitic Stearic Unsatd. fatty acids"" Oleic
Laurate Myristate Palmitate Unsatd. methyl estersa" Oleate Linoleate Linolenate
Monostearin
0.74 .65 .50 .36
1 0.9
Unsatd. or-monoglycerides"*c'd Mono-olein Monolinolein Monolinolenin
j
.32
.45 .72 ,65
Satd. 1,3-diglycerides* Dipalmitin Unsatd. 1,3-diglycerides" Diolein Satd. triglycerides' Trilaurin Trimyristin Tripalmitin Tristearin Unsatd. triglyceridesC Triolein Trilinolein Trilinolenin sym.-Diacid triglyceridesb Oleodipalmitin Oleodistearin Myristodistearin Glycerold
technique. ( b j Pancreatin.-Diand triglycerides are accessible to procedure a when split enzymatically into free acids and monoglycerides. The chromatograms were sprayed with a solution of 1% pancreatin (Takamine Labs., Clifton, S. J . ) in water and incubated for about 2 1 hours at 37" in a moisture chamber. After air-drying they mere subjected to the above a-dextrin-iodine technique. About 200 y of a saturated triglyceride can be detected. (c) Iodine.-Unsaturated alcohol.,, acids, esters aud gll-cerides can be detected b>- exposure t o iodine vapors. The spots are deep yellow or brown on a white background. Less than 20 y of unsaturated material can easily be detected in the chromatogram. ( d ) Lead Tetraacetate .-The air-dried paper strips were sprayed with a solution of 1yo lead tetraacetate in absolute benzene. TlThite spots on a brown background of lead dioxide are characteristic for monoglycerides or free glycerol. The sensitivity is 50 t o 100 y of monoglyceride in actual chromatograms. UNIVERSITY OF MIXNESOTA THEHORMEL IXSTITUTE AUSTIS, MINNESOTA .. - -.... - . .-
(I
Vol. 77
NOTES
1) J . C . ICnncn, F?. T. Clockner and R. P. Cox, Oil n ~ i Sl o o p , 22, ,r,7 (19.45). ( 1 2 ) K. Frciiclcnl,crp, 1;. €'lankenhorn and 11. Knauher. A n u . , 668, I ( l ! l & i ] ; 1). I'rencti, M. L. T.rvinr, J . 11. l ' ~ u i i r ant1 I?. h'oriierg, 'I'rris J O I . K N \ I . , 71, 3.73 (1!1 k ! l ) .
Preparation of p-Methylglutamic Acid BY D . C. MORRISOS RECEIVED APRIL22, 1955
p-hIethylglutamic acid was desired as a iiictabolite antagonist, and its synthesis was, therefore, undertaken. This compound has been prepared by Smrt and Sorm,2by a Schmidt reaction, and the aand y-methyl isomers were also obtained. The latter two amino acids had previously been synthesized by Gal, -1vakian and Martin, and by Fillman and Albertson, r e s p e ~ t i v e l y . ~ , ~ It seemed logical to suppose that the P-methyl isomer might be available by an extension of the glutamic acid synthesis of Snyder, Shekelton and Lewis,5 involving the condensation of ethyl acetamidomalonate with methyl acrylate. The reaction between ethyl acetamidomalonate and ethyl crotonate was studied, and the intermediate adtlition product was isolated as a solid of m.p. 73.j74.5'. Contrary to the results obtained in the case of the y-methyl isomer, loss of a carbethoxy group was not observed. The intermediate was hydrolyzed and decarboxylated by heating with hydrochloric acid, furnishing a solution from which dl-p-methylglutamic acid could be obtained -1second dl-form of the amino acid may be present in the mother liquors but it was not observed. The P-methylglutarnic acid gave an N-benzoyl derivative which crystallized from water as a monohydrate. In the corresponding synthesis of glutamic acid from methyl acrylate, previous workers did not isolate the addition product, but hydrolyzed the reaction mixture directly. This preparation was repeated, using ethyl acrylate, and the intermediate isolated and found to be S-acetyl-a-carbethoxyglutamic acid diethyl ester, a solid of m.p. 81-82.,5". Experimental
N-Acetyl-a-carbethoxy-p-methylglutamic Acid Diethyl Ester.--& solution of 21.7 g. (0.1 mole) of ethyl acetamidumalonate in 150 ml. of absolute ethanol was treated with a solution of 400 mg. of sodium in 10 ml. of absolute ethanol. This misture was treated slowly with 18.7 ml. (0.15 mole) of ethyl crotonate, and then refluxed 9 hours and left overiiight. T o the solution n-as added 5 ml. of acetic acid and 20 ml. of water, and most of the solvent removed by vacuum distillation. The residue \vas steam distilled under water-pump vacuum for one hour, leaving a light brown oil in a colorless solution. After ice-cooling for one hour, crystals formed, and soon all of the oil had solidified. The crystals were filtered, washed with water, and dried and weighed 17.72 g . The filtrates, on concentrating and seeding, gave a second crop of 2.29 g. The total yield was 20.01 g. (60.5y0) though more was still present in the filtrates. This compound was recrystallized several times from water, and then had m.p. 73.5-74.5'. A4mz/. Calcd. for CISH2iX07:C, 54.38; H, 7.55. Found: ( I ) : C, 54.03; H,7.02.Found(I1): C, 54.25; H, 7.03. 8-Methylglutamic Acid.-For hydroil-sis, 5 g. of the ester was refluxed overnight with 50 i d . of concentrated hydro(1) T h e work described in this paper was aided by a grant to P r t ~ f . D. M. Greenberg from the R'ational Cancer Institute, United States Public Health Service. (2) J. S m r t and F. Sorm, Coll. Czech. Chem. Commsnr.,18, 131 (1953); C. A . , 48, 3903 (1954). ( 3 ) A. 13. Gal, S A\.akian and (;, J. hlariin, THISJ O I I R N A I . , 76, ,4181 (19.X). ( 4 ) J. I,. Fillman and R' l c . Albertson, ihd , 1 4 , 4
