Fractionation of Geometric Isomers of Methyl Linolenate by Argentation Countercurrent Distribution C. R. SCHOLFIELD, R.
0.BUTTERFIELD,
and H. J. DUTTON
6 7 604
Northern Regional Research laboratory, Peoria, 111.
b
Geometric isomers of methyl linolenate were fractionated by countercurrent distribution between 0.2N A g N 0 3 in 90% methanol and hexane. Infrared analyses, capillary gas chromatography, and oxidative cleavage of monoenes produced by partial reduction with hydrazine showed that these trienoic esters were partially separated into the following six classes: ( 1 ) all-trans; (2) trans,cis,trans; (3) trans,trans,cis and cis,trans,trans; (4) cis,cis,trans and trans,cis,cis; (5) cis,frans,cis; and (6) all-cis. Positions of the various isomers of isomerized methyl linolenate on capillary gas chromatograms with Apiezon L and 100% cyanoethyl silicone columns are included.
300
c
F
H
v
Transfer Number Figure 1 , Argentation countercurrent distribution of isomerized methyl linolenate
T
of mixtures of monoenoic and of dienoic fatty acid esters by countercurrrent distribution (CCD) with 0.2N silver nitrate in 90% methanol and hexane (argentation CCD) has been described previously (8, 16). Nichols (IS) showed that it should be possible to separate cis and trans monoenoic esters because cis double bonds complex with AgN03 to a greater extent than do trans. With dienoic esters, fractionation by this CCD method depends not only on geometric configuration, but also upon relative position of double bonds along the carbon chain. If the double bonds have the same geometric configuration, dienes with double bonds separated by four methylene groups complex with AgNOs to a greater extent than those with double bonds separated by one methylene (15). In this paper, argentation CCD of mixtures of geometric isomers of methyl linolenate is described. With this complex mixture complete separation of all eight possible geometric isomers was not attained. Fractions rich in certain isomers were recovered, and the order of elution of isomers from the CCD apparatus is that expected from the relative position and configuration of the double bonds. Also, some of the peaks on capillary gas chromatograms of isomerized methyl HE FRACTIONATION
1694
ANALYTICAL CHEMISTRY
Dashed line represents triene portion of fractions estimated by gas chromatography
linolenate are identified by comparison with capillary chromatograms of CCD fractions of known composition. These identifications agree with, and extend, those reported by Litchfield (10, 11) for linolenate isomers. EXPERIMENTAL
Linolenic acid concentrate (85% linolenic, 11% linoleic, 4% oleic) from a Podbielniak centrifugal extractor (2) was used as starting material. A 100-gram portion of this concentrate was heated with 2 grams of selenium for 9 hours a t approximately 210' C. The acids were esterified with methanol and sulfuric acid catalyst and distilled to yield 36.6 grams of product. Infrared analysis indicated 111.2% trans esters compared with a methyl elaidate standard as 100%. As shown in our previous paper (15), trans bonds in 9,124inoleic isomers have only about 85% as much absorption as in monoenes. If we assume this lower absorption also occurs with trienoic esters of the linolenic type, the trans value can be corrected to 131oj,; that is, 4470 of the double bonds in the mixture are trans. The esters also contain a small amount of diene conjugation (am = 5.3). These esters (35 grams) were placed in the first four tubes of a 200-tube CCD apparatus and fractionated with 40 ml.
of 0.2N AgNOs in 90% methanol as the lower layer in each tube and with 10ml. portions of hexane as the upper layer. The single withdrawal procedure (4) was used, and the upper layer from two transfers was combined to give the curve shown in Figure 1. Dienoic ester impurities formed a band which overlapped the trienoic esters. A calculated curve for trienoic esters based on gas chromatographic data is also shown in Figure 1. Fractions were combined, as shown at the top of the figure, on the basis of infrared analyses and capillary column gas chromatograms of material from selected individual collector tubes. These fractions were distilled under vacuum from a small alembic flask. Weights of the combined fractions and analyses of the distilled material are given in Table I. The trans double bonds are calculated in the same manner as were those in the starting material. Lipoxidase conjugable esters were measured by the method of McGee (12). Diene impurities were removed from Fractions A and B by chromatography on a rubber column (7) before analysis. Capillary column gas chromatograms showed that each of the combined fractions contained two or more isomeric esters, besides small unidentified peaks believed to be conjugated esters or other impurities formed by the action of selenium.
I
I
I
I
I
40
45
50 Time, mln.
55
bo
Figure 2. Capillary gas chromatogram of isomerized methyl linolenate from a 200-foot X 0.01 -inch Apiezon L column Mt = mono-trans, Dt = di-trans
An estimate is given in Table I1 of the isomeric composition for trans content, based on the assumption that each fraction is a binary mixture of only two classes of isomers. The estimate of conjugated esters is based on absorptivities a t 233 mp. To identify some of the mono-trans and di-trans isomers, the following procedure was used: Acids were prepared from the combined fractions. These acids were partially reduced with hydrazine ( 1 7 ) ; methyl esters were formed again and passed through a rubber column to isolate the monoene fraction ( 7 ); the monoenes were separated into cis and trans fractions on a silverloaded cation exchange column (6). The position of the double bonds in the cis and trans monoenes was determined by capillary column gas chromatographic analysis and also by permanganate-periodate oxidation of the monoenes and analysis of the resulting dibasic acids (8). Configuration of the trienoic esters was deduced from the composition of the resulting monoenes. To determine the position of some of the different isomers in gas chromatograms of isomerized linolenate, capillary column gas chromatograms were run with mixtures of isomerized linolenate and some of the combined CCD fractions. Figures 2 and 3 are chromatograms of linolenate isomerized with nitrous acid according to the procedure of Harlow, Litchfield, and Reiser (6). Locations of individual isomers are marked on the curve.
we found the nitrous acid procedure used by Harlow, Litchfield, and Reiser (6) for linoleic acid to be superior because it produces a purer product with little conjugation. In later work we also used CCD with acetonitrile hexane (17) to remove dienoic and monoenoic esters in the linolenate concentrate. Results of argentation CCD were similar to those presented here for the selenium-isomerbed linolenate on which more complete analytical data were available. The gas-liquid chromatography (GLC) curves of Figures 2 and 3, however, r e p resent samples from nitrous acid isomerization. Argentation CCD of Isomerized Linolenate. The geometric isomers of methyl linolenate with eight possible components comprise a much more complex mixture than that of linoleate with four. Partly because of this complexity and partly because the range of partition coefficients for isomeric linolenates is so great that recycle operation is impractical, the separation of linolenate isomers by argentation CCD is less complete than with linoleate. Analyses of individual fractions and of combined fractions in Tables I and I1 do show that fractionation has taken place and that fractions rich in the all-trans, di-trans, mono-trans, and all-cis isomers can be recovered. If selenium-isomerized linolenate is used, conjugated impurities occur in all fractions.
Table 1.
ctc
25
30 Time, min.
35
Figure 3. Capillary gas chromatogram of isomerized methyl linolenate from a 200-foot X 0.01-inch 100% nitrile silicone column
The all-trans, di-trans, mono-trans, and all-cis ester contents of the combined fractions are estimated in Table I1 from the trans double bond analyses. To locate individual di-trans and mono-trans isomers, partial hydrazine reduction was used (16), which does not change the position or configuration of remaining double bonds, followed by analysis of the monoene fraction. Since
Analyses of Combined Fractions from Argentation Countercurrent Distribution of Isomerized Methyl Linolenate
Fractions
Wt., grams
A
3.71
F G H
6.61 1.47 2.33
trans Double bonds, %
51 35 25 13
a233
a267
4.2 5.4 6.3 7.5
0.5 0.5 0.3 0.8
Lipoxidase conjugable, %
28.6 56.5 22.5 62.4
Analysis run on triene portion of sample isolated by chromatography on a rubber column ( 7 ) . Table II.
Estimated Isomeric Composition of Combined Fractions from Argentation Countercurrent Distribution of Isomerized Methyl Linolenate
RESULTS AND DISCUSSION
Fraction
All-cis
All CCD fractionations described here were performed on linolenate isomerized with selenium. Some diene conjugation was produced during the isomerization. Also, dienoic esters formed by hydrogen transfer (9) were probably present, in addition to the dienoic impurities in the original linolenate concentrate. Later
A B C D
... ... ...
E F G H
... ...
... 18 54
Calculated percentage composition Mono-trans Di-trans All-trans
... ...
... 6 38 82 75 38
6 61 98 91 58 12
... ...
Conjugated
87 34
7 5 2 3 4 6 7 8
... ...
...
... ...
...
V O L 38, NO. 12, NOVEMBER 1966
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completion of our work, Privett and Nickel1 (14) described a similar technique using thin-layer chromatography. Although it is possible to set up equations for a quantitative estimation of the composition of each fraction, it was not considered practical for several reasons: (a) The presence of small amounts of conjugated impurities results in monoenes besides those from the 9,12,15-trienoates. (b) The values either from capillary GLC or oxidative cleavage, are not accurate enough to justify such calculations. (c) The relative rates of reduction of double bonds in different positions and of different configurations are not known to be the same. On the contrary, unpublished data a t this laboratory indicate that for cis A 15, double bonds may be reduced more rapidly than A 9 or A 12; and Asinger et al. (1) found differing reaction rates for different n-undecene isomers. I t is possible, however, to deduce the configuration of some of the parent trienes. For example, trans monoene from Fraction A is 41% A 9, 27% A 12, and 32% A 15 and cis monoene is 31% A 9, 59% A 12, and 10% A 15. The large amount of trans,trans,trans evident from the high infrared trans value is confirmed by the formation of trans monoenes with double bonds in all three positions. The high value in the cis fraction and the low value in the trans fraction for the A 12 monoene identify the principal di-trans ester as 9-transJ12-cisj15-trans. A similar composition but with less all-trans isomer is shown in Fraction B by the analyses of the monoenes: trans 51% A 9, 15% A 12,34% A 15; cis 18% A 9,74% A 12,8% A 15. Similarly, Fraction H trans monoene contains 15% A 9,74% A 12, and 11% A 15 and cis monoene contains 36% A 9, 34% A 12, and 30% A 15. These monoene analyses indicate a mixture mainly of all-cis and 9-cisJl2-transJl5cis isomers. A similar composition is suggested by monoenes from Fraction G where trans monoene is 4% A 9, 86% A 12, and 10% A 15 and cis monoene is 3970 A 9, 20% A 12, and 4170 A 15. From these results we conclude that the first di-trans isomer eluted from the CCD instrument is 9-transll2-cis,15trans and the last mono-trans isomer is 9-cis, 12-trans115-cis. Intermediate fractions yielded more complex mixtures of both cis and trans monoenes which are difficult to interpret. There appears to be little fractionation between trans,trans,cis and cis,trans,trans or between trans,cis,cis and cis,cis,trans isomers. The identification of the position of the cis,trans,cis isomer is supported by results of lipoxidase oxidation (12) which conjugates only cis,& pentadiene structures (Table I). Lipoxidase reactive esters are high in the last fractions from CCD (Fraction H) because of the 1696
ANALYTICAL CHEMISTRY
cis,cis,cis isomer; they decrease in Fraction G rich in cis,trans,cis and increase again in Fractions F and E where the other mono-trans isomers with adjacent cis bonds are present. The order of elution of isomers from the CCD apparatus is that which might be predicted from earlier work with linoleates (16). Separation by CCD depends upon differences in partition coefficients of the substances being fractionated. As no silver ion is present in the upper solvent phase, these differences depend largely upon the tendency of the substances to complex with silver ion and to remain in the lower solvent phase. With trienoic esters, because three double bonds are available for complexing, mono-, di- and trisilver complexes can be formed; and when more of these complexes are present, the equilibrium shifts toward the lower phase : Trienoate (upper phase) .............. Trienoate (lower phase)
...it I ? .
J I
Trienoate, AgN03 I.?.
$ 1
Trienoate (AgN0J2
.lT
Trienoate (&(No& In addition to the earlier knowledge that cis double bonds complex to a greater extent than trans, resulting in smaller partition coefficients for cis isomers, it has been shown (15) that with polyenoic esters the partition coefficient is smaller if double bonds are widely separated. Thus the partition coefficient for cis-9, cis-15-linoleate is less than for cis-9, cis-12 but greater than for linolenate. As expected, trans,trans,trans linolenate is the first of the 9,12,15-octadecatrienoate esters eluted. The trans,cis,trans isomer with the cis double bond, separated from other double bonds only by single methylenes, is eluted before the cis,trans,trans or trans,trans,cis. Of the mono-trans isomers the cis,trans,cis with two cis double bonds widely separated is eluted after the cis,cis,trans and trans,trans,cis. The all-cis isomer is eluted last. Capillary Gas Chromatography of Linolenate Isomers. The combined fractions in Table I and Figure 1 were prepared, as stated earlier, by combining individual adjacent CCD fractions that had similar infrared absorption and gas chromatogram curves. For purposes of making these combinations no identification of individual peaks was necessary. Litchfield et al. (10, 11) have identified the position of some of the isomers in chromatograms of isomerized linolenate. Capillary chromatograms run on our combined fractions, both alone and in combination
with isomerized linolenate, have confirmed and extended the identifications by Litchfield. Figure 2 is a chromatogram of the nitrous acid-isomerized linolenate on a 200-foot X 0.01-inch Apiezon L capillary. We confirm Litchfield’s conclusion that cis,cis,cis is in the first peak and that trans,trans,trans is in the second; also, that there are two mono-trans and two di-trans isomers in the region to the right. From chromatograms of the combined CCD fractions we can identify one of these mono-trans isomers as cis,trans,cis and one of the ditrans isomers as trans,cis,trans. Litchfield did not locate the remaining monotrans and di-trans isomers; although our results do not establish their positions beyond doubt, we believe there is a mono-trans isomer in the first peak and a di-trans isomer with trans,trans,trans in the second (Figure 2). Figure 3 is a chromatogram of the same nitrous acid-isomerized linolenate on a 200-foot x 0.01-inch column coated with General Electric experimental nitrile silicone 238-149-99 (lOOyo@-cyanoethylmethylsiloxane). Litchfield et al. (11) used the same substrate and reported a similar curve. Average equivalent chain lengths calculated from chromatograms of several fractions were trans,trans,trans 19.12, trans,cis,trans 19.51, cis,trans,cis 19.83, and cis,cis,cis 20.02. These values agree well with those they reported and, in addition, identify the position of the trans,cis,trans, and cis,trans,cis isomers. Diethylene glycol succinate on a 200foot x 0.01-inch capillary was also used, but resolution was less. The cis,trans,cis and cis,cis,cis isomers were not separated; trans,trans,trans and trans,cis,trans were slightly separated and in the same peak. ACKNOWLEDGMENT
We are indebted to C. Litchfield for the gift of the experimental 100% 0cyanoethylmethylsiloxane. LITERATURE CITED
(1) Asinger, F., Fell, B., Hadik, G., Steffan,G., Chem. Ber. 97, 1568 (1964). (2) . , Beal, R. E., Sohns, V. E., Eisenhauer.
R. A.,’ Griffin, E. L., Jr.,’ J . Am. Oil Chemists’ SOC. 38, 524 (1961). (3) Butterfield, R. O., Scholfield, C. R., Dutton, H. J., Ibid., 41, 397 (1964). (4) Craig, L. C., Post, O., ANAL.CHEM. 21, 500 (1949). (5) Emken, E. A., Scholfield, C. R Dutton, H. J., J . Am. Oil Chemist;’ SOC.41, 388 (1964).
(6) Harlow, R. D., Litchfield, C., Reiser, R., Zbid., 40, 505 (1963).
(7) Hirsch, J., Colloq. Intern. Centre Natl. Rech. Sci. (Paris), 99, 11 (1961). (8) Jones, E. P., Stolp, J. A., J. Am. Oil Chemists’ SOC. 35, 71 (1958). (9) Litchfield, C., Lord, Mrs. J. E., Isbell, A. F., Reiser, R., Ibid., 40, 553 (1963). \ - - - - I
(10) Litchfield, C., Reiser, R., Isbell,
A. F., Zbid.,
40,
302 (1963).
(11) Litchfield,
C., Reiser, R., Isbell,
A. F., Feldman, G. L., Ibid., 41, 52
(1964). (12) RlcGee, J., ANAL.CHEM.31, 298 (1959). (13) Nichols, P. L., Jr., J . Am. Chem. sot. 74, 1091 (1952). (14) Privett, 0. S., Nickell, E. C., Lipids 1, 98 (1966).
(15) Scholfield, C. R., Jones, E. P., Butterfield, R. O., Dutton, H. J.,
ANAL.CHEW35, 1588 (1963).
Agricultural and Food Chemistry, 148th Lleeting ACS, Chicago, Ill., AugustSowakowska, Janina, Selke, E., DutSeptember 1964. The Sorthern Laboratory is headquarters for the Northern ton, H. J.2 J . Am. oil C‘htmhts’ SOL 38, 208 (1961). rtilization Research and Development ( l ~ ~ ~ C $ Il.It;,, l ~ ~ ~ t o n ~ ~Division, Agricultural Research Service, U. S. Department of Agriculture. Men(1960). tion of trade or company names is for RECEIVEDfor review June 27, 1966. identification only and does not imply Accepted August 22, 1966. Division of endorsement by the Department. (16) Scholfield, C. R., Jones, E. P.,
~~~~k~7N,skzRf
Structure-Rf Correlations in the Thin Layer Chromatography of Some Basic Drugs WINSTON W. FIKE’ Cuyahogo County Coroner’s Office, Cleveland, Ohio 44 7 06
b Rf values for 140 basic drugs in five chromatographic systems have been obtained. Use of these data for the identification of the particular drugs listed is briefly discussed. Correlations of a more general nature between R, values in the five systems and the presence of particular chemical groups in these compounds are made. Steric hindrance around the group responsible for the bonding to silica in a particular system, the basicity of the compound, and the presence or absence of a pyridyl ring influence R, values to the greatest extent. A comparison has also been made of Rf values from the literature for the more common alkaloids and narcotics.
V
WORKERS have reported thin layer chromatographic studies of restricted groups of drugs-e.g., local anesthetics, analgesics, tranquilizers, etc. (5, 11, 13). Recently thin layer data for 140 drugs, many of which had not previously been reported, have appeared (16). These data were presented in a manner suitable for use in the rapid identification of a drug in biological media. I n this paper these compounds have been divided into two groups, phenothiazines and all others. This division has been made since the phenothiazines form a chemically distinctive group and give color reactions characteristic of them alone. The present emphasis is on correlations between the chemical structure of the drugs and their chromatographic properties. ARIOUS
EXPERIMENTAL
Reagents, Drug Standards, and Apparatus. These were a s reported
earlier (’7). Chromatographic Systems. Systems I, 11, and I11 consisted of silica Present address, The Wm. S. Merrell Co., Cincinnati, Ohio, 46235.
gel plates prepared with 0.1M potassium hydroxide with solvents cyclohexane-benzene-diethylamine (75: 15: 10), methanol, and acetone, respectively. Systems IV and V consisted of silica gel plates prepared using 0.1Ji sodium bisulfate with solvents methanol and 95% ethanol, respectively. Spray Reagents. A 1% solution of iodine in methanol was used as a general locating agent because it gave a positive test with all the drugs. Other sprays used were Dragendorff’s and Mandelin’s reagents prepared as noted previously ( 4 ) ; a 0.5% aqueous solution of Fast Red G, the stabilized diazonium salt of p-nitroaniline, and Forrest’s acidic ferric chloride (FPX) reagent used for phenothiazine detection (13). A 5y0 aqueous solution of sodium nitrite was commonly used as an overspray following Dragendorff’s reagent. This combination releases iodine and proved more effective in detecting small amounts of many of the drugs than the solution of iodine in methanol; it also produces a more persistent spot with some of the drugs. Chromatographic Procedure. The R f data were obtained using the same procedure given previously (7) except that silica gel G slurries were used to prepare the plates (8). RESULTS AND DISCUSSION
Average Rf values of the drugs in the five systems are reported in Tables I and 11. Each value is the average of at least three determinations on separate plates. These chromatographic systems enable one to identify a large majority of the drugs satisfactorily when used in conjunction with spray reactions. KO attempt has been made here to identify unequivocally all the drugs listed. Rather, an attempt has been made to develop data and structural correlation which should be useful as a n aid in identifying a large number of basic organic compounds. System 111 mould be the best system for a preliminary screening of the drugs
as it gives the most even distribution of R, values of those reported. The use of
ultraviolet light and the sprays listed would provide further characterization. Mandelin’s reagent and the Fast Red G spray gave distinctive colors with some of the drugs. Fast Red G gave orange or pink colors with those compounds containing either aniline or phenolic groups and a yellow color with the primary amines. The colors produced by the antihistamines with Mandelin’s reagent have been reported earlier ( 7 ) . Because some of the colors produced are not completely reproducible, they have not been reported. Relative R, values were not calculated. Mepivicaine and dyclonine would both be suitable as reference compounds since their solutions are stable and each has R, values between 0.30 and 0.70 in all five systems. Structure-R, Correlations in the Non-Phenothiazine
Drugs.
SYSTEM
I. I n this system all the very weak bases have low R,’s. -4mide groupse.g., in caffeine and iproniazidhydroxyl groups a s in 3-pyridinemethanol, and in papaverine the four methoxyl groups probably account for these low values where present. The attraction of oxygen-containing groups in steroids for silica has been noted earlier (14). It is noteworthy that neither the ester nor the keto group of themselves produce a low R,. Piperocaine with an ester group and diethylpropion with a keto group have RrJsof 0.63 and 0.76, respectively. Three compounds containing hydroxyl groups are exceptions to the above generalization. Benactyzine, pipradrol, and procyclidine have Rf values of 0.63, 0.74, and 0.78, respectively. Each has its hydroxyl group on a highly hindered tertiary carbon atom where it would be sterically prevented from hydrogen bonding to the silica. Steric hindrance of the bonding between V O L 38, NO. 12, NOVEMBER 1966
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