Gas Liquid Chromatography of Some Naturally Occurring Couma rins STEWART A. BROWN and J. P. SHYLUK Prairie Regional laboratory, National Reseurch Council, Saskatoon, Sask., Canada
b
The gas liquid chromatography of
15 neutral and five phenolic coumarins has been studied. A column of succinate-ethylene glycol polyester on Chromosorb W had the most general application for neutral coumarins and was the only one examined which resolved pimpinellin, bergapten, and sphondin. The retention times exhibited a marked correlation with the number of ether linkages in the molecule. Osthol and psoralen, which are not separated by this column, were readily resolved on a column of phthalate-ethylene glycol polyester supported on Gas Chrom P. Phenolic coumarins were best chromatographed as their acetates on a column of silicone stopcock grease supported on Celite 545. lmperatorin and umbelliprenin, coumarins of the isoprenoid ether type, could not be recovered from the columns, presumably because of decomposition even at relatively low temperatures. The procedures have been applied with considerable success to coumarin fractions from three plant species.
R
in this laboratory on the biosynthesis of certain coumarins have been handicapped] m has most work involving the isolation of coumarins, by the lack of really satisfactory purification techniques. The problem is especially troublesome in the case of plant species, such as some umbelliferae, which elaborate complex mixtures of coumarins. The older purification methods, which leaned heavily on crystallization, have been supplemented in recent years by adsorption column chromatography (1, 2, 12). Although thjs latter tool has greatly simplified some isolation procedures, many coumarins, especially minor components of a mixture] are still difEcult t o obtain in the pure state, or even to identify with certainty. Gas liquid chromatography has been used in this laboratory to separate and purify coumarin and herniarin (7methoxycoumarin) in studies of their biosynthesis in lavender (a). The present paper describes the extension of this technique to other neutral (nonphenolic) coumarins, and to several ECENT STUDIES
1058
ANALYTICAL CHEMISTRY
phenolic coumarins in the form of their acetates. Quite satisfactory resolutions of complex coumarin fractions from Angelica archangelica L. and Heracleum sibiricum L. have been achieved. EXPERIMENTAL
Apparatus. INSTRUMENT. The gas chromatograph, of a conventional design (6), was constructed in these laboratories and was operated in conjunction with a Brown continuous recorder. A filament current of 200 ma. was used throughout. COLUUNS. All liquid phase-support ratios are weight per weight. BEG No. 1. This column consisted of succinate-ethylene glycol polyester on a support of 60-80 mesh Chromosorb W, a flux-calcined, acid-washed diatomaceous silica (Johns-Manville) ina ratio of 1 to 20. The liquid phase was dissolved in a volume of chloroform sufficient to cover the solid, and the solid support was added. After 15 minutes the mixture was poured into a shallow tray and evaporated with gentle mixing a t room temperature. When visibly dry it was heated a t 60"to 80" C. in a vacuum oven for a t least one hour. (A satisfactory preparation should require no sieving a t this stage.) The dried material was poured in small aliquots, with tapping, into a 2.4meter length of copper tubing of 5 mm. 0.d. The column was operated a t 208" C., with a helium flow rate of 40 ml. per minute (measured by the soap-bubble method a t the exit), and the injector temperature was 245" C. The recorder sensitivity setting was 5 mv. SEG No. 2. This column was prepared as above with a liquid phase to support ratio of 1 to 6. The column dimensions were 0.61 meter X 5 mm. A helium flow rate of 100 ml. per minute was used. The column temperature was 208" C., and the injector temperature, 245" C. The recorder sensitivity was 1 mv. PhEG. This column was prepared as above from phthalate-ethylene glycol polyester (4) on 60- to 80-mesh Gas Chrom PI a flux-calcined diatomaceous earth aggregate (Applied Science Laboratories, State College, Pa.) in a ratio of I to 10. The column dimensions were 1.2 meters x 5 mm., and the helium flow rate was 100 ml. per A column temperature of minute. 206" C. and an injector temperature of 245" C. were used. The recorder sensitivitywas 1 Inv.
SILICONE GREASE. The liquid phase of this column was prepared from DowCorning high vacuum stopcock grease according to Cropper and Heywood (6, 7). It was dispersed in ethyl acetate and applied to 40- to 60-mesh acidwashed diatomaceous silica (JohnsManville Celite 545) in a 1 to 6 ratio. The column dimensions were 0.61 meter X 5 mm., and the helium flow rate was 100 ml. per minute. A column temperature of 178" C. and an injector temperature of 210" C. were used. The recorder sensitivity was 1 mv. Chromatographic Procedure. The samples to be chromatographed were dissolved in acetone, and volumes of 5 to 10 ll., containing about 0.1 mg. of each compound, were injected with a microliter syringe (Hamilton Co., Inc., Whittier, Calif.). Retention times were measured from the initial emergence of the solvent to the peak of the compound in question. Samples of emerging compounds were collected by sublimation of the walls of small test tubes held over the exit tube. Acetylation of Phenolic Coumarins. The coumarin or crude phenol fraction to be acetylated was mixed with six times its weight of acetic anhydride and one-quarter its weight of anhydrous sodium acetate. The mixture was heated on a steam bath under an air condenser for one hour, and then poured into several volumes of water. I n the case of pure coumarins, the crystalline product was recrystallized from water. I n the case of the crude fractions from plants, the oily product was extracted into ether, the solvent has blown off, and the residue was dried in vacuo. RESULTS AND DISCUSSION
Neutral Coumarins. The SEG No. 2 column was the only one examined which would satisfactorily resolve the furanocoumarins, pimpinellin, bergapten, and sphondin. It does not separate osthol from psoralen, but these two coumarins are readily resolved on the PhEG column. The latter column was 1.2 meters in length, but a much shorter column would have been adequate for this purpose. The relative retention times for 13 of the 1.5 neutral coumarins investigated are listed in Table I, and a chromatogram of a synthetic mixture of 11 of these on the SEG No. 2 column is reproduced in Figure 1. Difficulties
in procuring authentic samples of the less common coumarins have prevented a more extensive survey, and conclusions on the basis of a limited number are necessarily subject to revision. There is, however, a correlation between retention times on the SEG column and the number of ether linkages in the coumarin. Coumarin itself, with no ether linkage, had the shortest retention time-1.4 minutes. It was followed by the monoethers-herniarin, angelicin, seselin, osthol, and psoralen-in the range of 4.9 to 8.1 minutes. The diethers - isobergapten, bergapten, aesculetin, dimethyl ether, sphondin, and xanthotoxin-followed in the range 12.2 to 19.3 minutes. The trietherspimpinellin and isopimpinellin-emerged a t 15.1 and 33 minutes, respectively, the former falling among the diethers. The tendency for additional ether linkages to increase the retention times is thus quite marked. The same general order of emergence by class was also observed with other columns, although some variation was noted in individual coumarins. These observations are consistent with the finding of Narasimhachad and von Rudloff (IO) that retention time increases with an increase in the number of methoxyl substituenb. In addition to the compounds listed in Table I, two isoprenoid ethers-imperatoriu and umbelliprenin-were investigated, but neither could be recovered from the columns used, even a t column temperatures as low as 100" C. At this temperature, in each case, a peak characterized by severe tailing emerged just behind the solvent peak. The materials represented by these peaks were collected and chromatopraphed on paper together with samples of the solutions which had been injected. The emergent material in each case differed from the original compound, providing a clear indication of decomposition. Imperatorin is known to be unstable to heat (8),and isomeriees to allo-imperatorin as shown :
Table 1.
SEG No. 1
1.
Coumarin
bH ah-IMPERATORIN
A similar cleavage of the ether linkage may also take place in other coumarins of this type, and a more extended study of their behavior would be of interest. The one example of osthol suggests that
No.2
PhEG
2.
Herniarin
HJCO
so
3. Angelicin
0.21
0.29
1.0
1.0
1.0
1.1
1.3
0-
Po
1.25
HJCOQO
1.65
6.5
6. Psoralen
1.65
1.75
7. Isobergapten
2.5
8. Pimpinellin
3.1
4. Seselin
1
/
5. Osthol
CH2- C H S ( C H 3 z
9. Aesculetin
dimethyl ether
3.15
10. Bergapten
3.4
11. Xanthotoxin
3.6
12. Sphondin
3.9
13. Isopimpinellin
6.7
Phenolic Coumarinsb
Silicone Grease HOa 0
2. 7-Hydroxy-S-
met hoxymumann
3. Daphnetin 4. Scopoletin
1 .(J
1.35 H
o
m OCH,
o
H
o e OH
o
I
\o \ F,,?
Column SEG
Neutral Coumarins
1. Umbelliferone
O-CH~-CH=C(CH~I I MPERATORI N
Relative Retention Times of Coumarins"
H::mO
2.25 2.25
0
5. Aesculetin a
Based on herniarin
coumanns.
2.95 = 1.0 for neutral
coumarins, and umbelliferone = 1.0 for phenolic
* Chromatographed aa the acetates. VOL 34, NO. 9, AUGUST 1962
1059
c
z
Y
Y
1,
3
Ln
VI
IC
0
30
20
RETENTION
TIME
(minutes1
Figure 1 . Chromatogram of synthetic mixture of neutral coumarins (SEG No. 2 column) 1.
2. 3. 4. 5.
6. lrobergapten 7. Pimpinellin 8. Bergapten 9. Sphondin
Coumarin Herniarin Angelicin Seselin Ortho1 psoralen
+
10.
RETENTION
ImlnUteSI
Figure 2. Chromatogram of neutral lactone fraction from Heracleum sibiricum (SEG No. 2 column) 1. 2. 3. 4.
lropimpinellin
isoprenoid residues joined to the coumarin nucleus by carbon-carbon linkages behave normally on these columns. Neutral coumarin fractions isolated from three plant species have been chromatographed on SEG columns. Lavender (Laoandula o%nulis Chaix.) contains only coumarin and herniarin. These were easily separated, on a preparative scale, on the SEG No. 1 column. Retention times were 2.5 and 11 9 minutes, respectively. Several injections allowed collection of enough material for the measurement of C14 which had been incorporated from labeled precursors (3). No fluorescent or phenolic contaminants were revealed by subsequent paper chromatography in 1% acetic acid. The gas liquid chromatogram of a lactone fraction recovered by standard methods (2, 8) from roots of He-racleum sibiricum is reproduced in Figure 2. The material represented by each peak,
TIME
Unidentified Angelicin Irobergapten Pimpinellin
except peak No. 1, was collected, and its identity was confirmed by paper chromatography, together with authentic samples, in 1% acetic acid. Excellent separation was obtained except in the case of pimpinellin (peak 4, which contained appreciable amounts of bergapten. Bergapten (peak 5 ) was only slightly contaminated with pimpinellin. (Collectors were changed a t the inflection point.) Baerheim Svendsen and Ottestad (2) reported the presence of isobergapten, pimpinellin, bergapten, sphondin, and isopimpinellin in fruits of these species. Angelicin is apparently either absent from the fruits or is not present in high enough concentration to be detected by their methods. A lactone fraction from Angelica urchangelicu roots yielded the chromatogram reproduced in Figure 3. Of the eight peaks, only three have been identified. Peak 1 is angelicin; peak 2 is osthol; and peak 5 is bergapten.
5. Bergapten 6. Sphondin 7. lropimpinellin
Peaks 3 and 8 represent pure compounds, as judged by paper chromatography. The compound from peak 3 had an R j = 0.11 in 1% acetic acid and a greenish-white fluorescence after being sprayed with 2N sodium hydroxide. The compound from peak 8 had an R f = 0.35 and a bluish-purple fluorescence. Peak 4 was predominantly a compound with Rj = 0.54 and a bright ice-green fluorescence closely resembling that of angelicin. It was slightly contaminated with material from peaks 3 and 5. Peaks 6
r
2
0
RETENTION I
I
0
10
I 20
RETENTION
TIME
I
I
30
40
(mInUteS1
Figure 3. Chromatogram of neutral lactone fraction from Angelic0 urchungelica (SEG No. 2 column; for interpretation, see text)
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ANALYTICAL CHEMISTRY
4
TIME
(minutes)
Figure 4. Chromatogram of synthetic mixture of phenolic coumarin acetates (siIicone.grease coIumn) 1.
2. 3. 4.
Umbelliferone 7-Hydroxy-8-methoxycoumarin Daphnetin rcopoletin Aesculetin
+
and i were mixtures of two and three fluorwent compounds, respectively, all in small amounts. None of the unit lentified compounds is umbelliprenin, imperatorin, or xanthotoxin, all known to o w i r in this species (9,11). Phenolic Coumarins. Phenolic couniarins are normally removed during standard isolation procedures, together with other phenols, by extraction of an ethereal solution with dilute aqueous alkali. The problem of separating them chromatographically from neutral coumarins does not arise. Fewer of them are apparently found in nature than neutral coumarins, and it is rare t o find a species in which more than two have been reported. -45these coumarins tend to vaporize only with difficulty, excessively high temperatures were found necessary to elute them from even relatively short columns in reasonable times. The peaks usually tailed badly. A much more satisfactory approach involved acetylation of the free phenolic groups with acetic anhydride and sodium acetate, a reaction which is conveniently conclucted and gives excellent yields of the esters. The relative retention times of the five phenolic coumarin acetates studied are given in Table I, and a chromatogram of a synthetic mixture is reproduced in Figure 4. The separations were satisfactory except for daphnetin and scopoletin, which emerged together as a single peak. As no species appears to be known which contains both of these (9, I I ) , their failure to separate is not a practical drawback. High vacuum sublimation, over a 100’ to 220’ C. range, of phenolic reactions from Heracleum and Angelica
yielded in each case an oily material, which was acetylated and chromatographed on the silicone grease column. The Heracleum extract showed 10 minor peaks, but paper chromatography together with available authentic compounds in no case yielded any clue to identity. Several of the compounds were fluorescent, and could have been acetates of phenolic coumarins. The Angelica mixture also contained a number of minor peaks, and a major component with a relative retention time of 3.7 (umbelliferone = 1). When chromatographed on paper and then sprayed with 2N sodium hydroxide, it fluoresced a brilliant greenish-blue, and it may also be the acetate of a phenolic coumarin. At least two phenolic coumarins of which no samples were available occur in Angelica ( I O ) . Umbelliferone, which was expected in both sublimates on the basis of earlier reports (2, l o ) , was not detected, and paper chromatography of the solution before acetylation confirmed its absence from both mixtures. The techniques described in this paper, in conjunction with existing preparative and analytical methods, promise to be of value in investigations of naturally occurring coumarins. Isolation techniques used heretofore, even those employing column chromatography, often required kilogram quantities of dried plant. The use of gas liquid chromatography, together with paper chromatography, should permit a survey of coumarin patterns on less than 100 grams of dried plant or its equivalent of fresh material. I n most cases it should be possible to obtain fractions sufficiently pure for positive identification by mixed melt-
ing point, and for radioactivity determinations in biosynthetic studies. ACKNOWLEDGMENT
’The authors are indebted to the following for providing authentic samples of various coumarins used: A. Baerheim Svendsen, Oslo; Gerhard Billek, Vienna; Asima Chatterjee, Calcutta; Giovanni Rodighiero, Padova; G. H. S. Towers, Montreal. A number of helpful suggestions from B. M. Craig and E. von Rudloff during the course of this investigation are also gratefully acknowledged. The gas chromatograph employed was constructed by T. M. Mallard of the National Research Council laboratory at Saskatoon. LITERATURE CITED
(1) Baerheim Svendsen, A., “Zur Chemie Sorwegischer Umbelliferen,” p. 24,
Johan Grundt Forlag, Oslo, 1954. (2) Baerheim Svendsen, A., Otteatad, E., Pharm. Acta Helv. 32, 457 (1957). (3) -Brown, S. A., National Research
Council, Saskatoon, Sask., Canada, unpublished data, 1962. (4)Craig, B. M., Chem. & Ind. (London) 1960,i442. (5) Craig, B. M., Murty, N. L., J . Am. Oil C’hemistsSOC.36, 549 (1959). 16) Cropper, F. R., Heywood, A., Nature 172, 1101 (1953). ( 7 ) Zbid., 174, 1063 (1954). (8) Dean, F. M., Progr. Chem. Org. Nut. Prod. 9, 225 (1952).
(9) Karrer, W., “Konstitution und Vor-
kommen
der organischer Pflanzen-
stoffe.” DD. 535. 554-5. Birkhauser Verlag, ~ t i t t g a r,’t1958. ’ (10) Xarasimhachari, N., Rudloff, E. M. von, Can. J . Chem. 40, 1123 (1962). (11) Spath, E., Ber. 70A, 83 (1937). (12) Stanley, W. L., Vannier, S. H., J . Am. Chem. Soc. 79, 3488 (1957).
RECEIVED for review April 4, 1962. Accepted May 31, 1962.
Horizontal Chromatography Accelerating Apparatus Description of Apparatus and Applications J. F. HERNDON, H. E. APPERT, J. C. TOUCHSTONE, and C. N. DAVIS The Malvern Institute, Malvern, Pa.
An apparatus is described which permits horizontal chromatography, or ion exchange separations, to be carried out on strips of media, or circular disks. The apparatus permits these techniques to be accelerated at choice by the introduction of rapid solvent flow rate, heat, and centrifugal force. This apparatus has found use in accelerating a wide variety of chromatog raphic separations.
C
has been employed to accelerate horizontal paper chromatographic separation of compounds in mixtures ( I , 6, 7, 10-14). Recently, Tata and Hemmings (21) described centrifugally accelerated paper strip chromatography using an instrument of their own design. Roberts et al. (16-20)used elevated temperatures t o accelerate horizontal chromatograms. Williams (26) has speeded separation ENTRIFUGAL FORCE
in adsorption columns by centrifugation. Izmailov and Shraiber (8) originally described thin layer, open column chromatography-more recently improved by Kirchner, Miller, and Keller (9) and Reitsema (16). Tandem chromatography described by Tuckerman, Osteryoung, and Nachod (24) has introduced a novel means for accomplishing two-dimensional chromatography with strips. VOL. 34, NO. 9, AUGUST 1962
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