Preparative Medium Pressure Liquid Chromatographic (MPLC) and SemiPreparative HPLC Separation of Furocoumarin Isomers

G. C. Zogg/Sz. Nyiredy/O. Sticher*

Department of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich, CH-8092 ZLirich, Switzerland.

Key Words
Column liquid chromatography Overpressured layer chromatography Furocoumarin isomers Preparative separation of plant extracts

Summary
Preparative medium pressure liquid chromatography and semi-preparative high performance liquid chromatography on silica were employed for the isolation of eight furocoumarin isomers (iso-bergapten, angelicin, psoralen, bergapten, pimpinellin, sphondin, xanthotoxin and isopimpinellin). The conditions were transposed from analytical on-line overpressured layer chromatography and analytical high performance liquid chromatography, respectively. The retention data of the analytical on-line overpressured layer chromatographic separation and the preparative medium pressure liquid chromatographic isolation show a linear relationship. Using 15 /~m TLC silica 60 as stationary phase a dry-packing procedure was developed for Labomatic medium pressure liquid chromatographic glass columns. The efficiency of the preparative separation techniques was demonstrated by the isolation of the investigated furocoumarins from Heraclei mantegazziani radix and from Pimpinellae radix (Balkan quality),

HPLC has been published by Erdelmeier et al. [13]. Using analytical off-line overpressured layer chromatography (OPLC) with HPTLC plates the same eight compounds were separated and detected in different concentrations in the roots of several plants from the family Umbelliferae [14]. The five main furocoumarin isomers from Heracleum sphondylium roots have been isolated by semi-preparative RP-HPLC [15] and by preparative on-line OPLC [16, 17]. To date all eight furocoumarin isomers have not been isolated by normal phase (NP) liquid chromatography (LC). In the past few years various methods of preparative liquid chromatography [18] have been employed for the separation of naturally occurring compounds with closely related structures. Medium pressure LC (MPLC) combines the advantages of different column LC techniques, such as open column LC, flash chromatography (which can be easily filled and refilled), low pressure LC and HPLC (e.g. small particle size, high resolution, short separation time). Proper column preparation [19], an essential prerequisite for good MPLC separations, can be carried out by the dry-filling method, which gives a 20% higher packing density than the slurry techniques [18]. In planar preparative liquid chromatography, fully on-line OPLC [20, 21] may be carried out either by several repetitive injections on HPTLC plates [22], on an analytical scale, or with larger samples on precoated preparative plates [ 17]. This paper describes the direct transfer of the optimized mobile phase from analytical scale on-line OPLC as a pilot method to MPLC and from analytical scale HPLC to semipreparative HPLC, using silica as stationary phase. The separations were carried out not only with the eight reference compounds but also with extracts from roots of different species of the family Umbelliferae.

I ntroduction
Furocoumarins occur in many plant species and are typical constituents of Umbeliferae [1], such as Heracleum, Pimpinella and Pastinaca. They are well known for their mutagenic and photosensitizing activities [2-4], and the latter are also used therapeutically [5, 6]. Analytical high performance liquid chromatography (HPLC) has attracted increasing attention for the separation of furocoumarin containing plant extracts [7-12]. The baseline separation of eight furocoumarins from Heracleum mantegazzianurn leaves by gradient reversed phase (RP) Chromatographia Vol. 27, No. 11/12, June 1989 0009-5893/89/6 0591-05 ~ 03.00/0 Originals

Experimental
Chemical and Reagents Solvents of HPLC quality from Merck (Darmstadt, FRG) were used for all separations.Chloroform had to be stabilized with 2-methyl-2-butene instead of ethanol since ethanol influences the separation. OPLC and MPLC separations

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9 1989 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

were carried out with a mobile phase A, of composition ethyl acetate, chloroform, diethyl ether, n-hexane (0.6%; 58,5%; 0.9%; 40% v/v) and an additional 0.04% water as modifier. Mobile phase B, used for the analytical and semipreparative HPLC, differed from A only in the absence of water. Analytical silica 60 F254 HPTLC alufoils (20 x 20 cm) for OPLC separations and TLC silica 60 F254 of 15/Jm average particle size for MPLC separations were obtained from Merck. Analytical and semi-preparative HPLC separations were carried out using Lichrosorb Si 60 of 5 ~m particle size (Merck, Darmstadt, F RG).

Hungary) at 25 bar external pressure. A teflon insert sheet with a eluent directing through for the solvent inlet and with a small hole for the solvent outlet at a distance of 18 cm, was placed between the water cushion and the chromatographic plate. The HPTLC alufoils were impregnated on all four sides with Impres II polymer suspension (Labor MIM, Budapest, Hungary) and one channel was scraped out for solvent outlet at a distance of 18 cm from the solvent inlet. Analytical HPLC separations were carried out with a Knauer cartridge column (250 x 4 mm I.D.), and semipreparative HPLC with a Knauer 250 x 16 mm I.D. column (Berlin, FRG). MPLC separations were performed with a dry-packed Labochrom 4D MPGC glass column, 735 x 26 mm I. D. (Labomatic, Sch6nenbuch, Switzerland). Detection at a wavelength of 313 nm was carried out with a Perkin Elmer LC 75 (Norwalk, CT, USA), equipped with a Merck Hitachi D 2000 Chromato-lntegrator (Merck, Darmstadt, FRG) for all separations except for MPLC where an LKB detector (Bromma, Sweden) coupled with an LKB 2210 recorder was used. A Gilson fraction collector FC-201 (Middleton, WI, USA) and an LKB 700 Ultrorac | fraction collector were used to recover the isolated compounds.

Samples
Reference samples of angelicin, psoralen and xanthotoxin (8-methoxypsoralen) were purchased from Roth (Karlsruhe, FRG). The other five compounds were isolated by preparative OPLC [17] from Heracleum sphondylium roots and identified at the Department of Pharmacy, ETH Zurich. The structures of all eight investigated furocoumarins are given in Fig. 1. Reference sample concentrations of between 0.2 and 0.6 mg/ml chloroform were used and the injection volume was between 10/~1 and 20 #1 for the analytical separations. Samples of Pimpinellae radix (Balkan quality) were obtained from Mfiggenburg (Hamburg, FRG), and samples of Heraclei mantegazziani radix were collected in Switzerland. The crude chloroform extracts from the dried roots, obtained by Soxhlet extraction were evaporated and redissolved in chloroform (500 mg/ml). RI

MPLC Column Preparation

Labomatic recommend that their Labochrom MPGC columns should be dry-filled with continuous axial tapping of the wall. With TLC silica 60 having an average particle size of 15 /Jm, this column packing process was unsatisfactory because it failed to give homogeneously and reproducibly packed columns with this small particle size. Drawing upon our experience of column preparation, we developed the following dry-filling procedure, which resulted in the best packed MPGC columns for our experiments:

R2

R1 R2 H Angelicin (2) H OCH3 Iso-berg~pten (1) OCH3 H Sphondin (6) OCH30CH3 Pimpinellin (5)
H

R2 R1 R2 H H H OCH3 OCH3 H OCH30CH3

Psofalen (3) Xanthotoxin(7) Bergapten (4) Iso-plmpinellin (8)

Fig. 1
Structures of furocoumarin isomersinvestigated.

Apparatus
Sample application was performed with a Waters injector U6K (Milford, Mass., USA) for analytical and semi-preparative HPLC and for the OPLC. Sample injection for the MPLC separations was carried out with a Rheodyne 4-way valve (Cotati, CA, USA). Solvent delivery for the analytical separations was by a Waters M 6000 chromatography pump (Milford, Mass., USA). For the preparative separations, either a Kontron LC 410 pump (Zurich, Switzerland) or a Labomatic MD80/100 medium pressure pump (Sch6nenbuch, Switzerland) was used. OPLC separations were performed with a Chrompres 25 overpressured layer chromatograph (Labor MIM, Budapest,

a) A teflon plug, inserted in the lower end of the vertical glass column was coupled to vacuum (12 mm Hg) during the whole packing process. b} Dry 15/~m silica 60 was slowly introduced through the upper end of the column until 2/3 of the column length was filled. c) After closing the upper end of the column with a second stopper, increasing nitrogen pressure (up to 10 bar) was applied to the column. d) In the next step, the pressure in the column was allowed to fall to atmosphere before opening the column and adding support up to the top of the cylindrical part. e) Depending on the column dimensions, steps c and d were repeated several times (1-5), before also filling the upper column section with stationary phase. f) After again subjecting the column to nitrogen overpressure, the packing difference was eliminated by vertical adjustment of the upper plug. g) Finally the connections for vacuum and the nitrogen pressure were interchanged and step f was repeated from the other end of the column, before conditioning with the appropriate mobile phase composition. Chromatographia Vol. 27, No. 11/12, June I989 Originals

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Because of this dry-packing process and the symmetrical column design, development may be carried out in either direction without any difference in resolution.

Results and Discussion

after 30 minutes equilibration time for OPLC and 4 hours for MPLC. A 450 mg sample of a furocoumarin mixture containing all the investigated compounds was separated by MPLC. Good separation was obtained between the eight isomers, except for iso-bergapten (1) and angelicin (2), (Fig. 2). All furocoumarins were isolated in pure form, 1 (16 mg), 2 (19 mg), 3 (27 mg), 4 (31 rag), 5 (128 mg), 6 (36 rag), 7 (34 mg), 8 (59 mg), with only small mixed fractions (36 mg). The chromatograms of the analytical on-line OPLC and the MPLC separations (Fig. 2) show very similar resolutions, though the average particle size of the silica was 6/~m for analytical OPLC and 15 /~m for preparative MPLC separations. MPLC separation of 500 mg crude extract from a sample of Pimpinellae radix was carried out under the same conditions on the same Labochrom MPGC column. The investigated extract contained all furocoumarin isomers tested (psoralen only as minor compound) and the chromatogram obtained (Fig. 3) shows a baseline separation for six compounds and reasonable resolution of substances 1 and 2. Seven furocoumarins were isolated in pure form, 1 (14 mg), 2 (17 mg), 4 (19 mg), 5 (82 mg), 6 (28 mg), 7 (4 mg), 8 (42 mg), wihtin 14 hours. A mixed fraction of 18 mg iso-

Generally, analytical HPLC serves as the pilot method for the various preparative column liquid chromatographic methods [23]. As a new approach, we have proposed a simple strategy to transfer the optimized TLC mobile phase via analytical off-line OPLC to MPLC [24, 25]. OPLC, representing a planar version of a column liquid chromatographic system, is a suitable pilot method for the separation of the components of interest from various biological matrices. Due to the lower costs of stationary phase and its easy replacement, sample purification is not so important as in HPLC. Using on-line OPLC the information provided can be improved, especially in the higher k' range, but the required level of instrumentalisation also increases.
Analytical On-line OPLC as Pilot Method for MPLC

The mobile phase composition was directly transposed from analytical, isocratic on-line OPLC [26] (Fig. 2) to preparative MPLC. Sample application was carried out

MPLC separation 41
I

Full on-line OPLC separation 5

MPLC separation

2
17 ~g

5
82 ms

14 mg

19 ~g

40 I(min)

8
7

t
5

k
I0 t(h) I 5 I I0

l(h)

Fig. 2 Isocratic preparative MPLC and analytical on-line OPLC separations of the furocoumarin isomersinvestigated. On-line OPLC separation: mobile phase A (without modifier); flow rate: 1.2 ml/rnin; counterpressure: 22 bar. MPLC separation: mobile phase A (without modifier); flow rate: 5 ml/min at a maximum pressure of 12 bar (other conditions see Experimental).

Fig. 3 Isocratic preparative MPLC separation of 500 mg crude extract from a sampleof Pimpinellaeradix. Mobile phase A (without modifier); flow rate: 5 ml/min at a maximum pressureof 12 bar (other conditions see Experimental).

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y 12

lx

.02,

R-squared:

.999

Semi-preparative HPLC separation 4 5

Analytical HPLC separation

tO

5 8

~6
3r 4 e

2.

, 2

, 4

. OPLC)

, 8

, 10

, 12 i.

6 k' (on-line

Fig. 4 Correlation between the k ~ values of analytical on-line OPLC and

Is 8
7

20 t{min)

'

preparative MPLC separations.

bergapten (1) and angelicin (2) was also obtained. Due to the efficiency of the MPLC system up to 5 grams, of various crude plant extracts, containing up to 500 mg of individual furocoumarins could be separated in a single run. A plot of the relationship between the analytical and preparative retention data of the two isocratic methods (Fig. 4) shows a strainght line. Thus it should be feasible to predict all kinds of retention data (e. g. separation time, resolution) in MPLC separation from the analytical OPLC separation. Such predictions should be based on analytical OPLC results either after elution of the first (two or three) substances in the MPLC separation or with the aid of a calibration between the two separation systems with standard homologues (e. g. the Kovats retention index system [27]).
Analytical and S e m i - P r e p a r a t i v e HPLC Separations

10
Fig. 5

20

30

40

50

t(min)

Isocratic analytical and semi-preparative HPLC separations of furocoumarin isomers investigated. Analytical HPLC" mobile phase B (containing 0.04% water as modifier); flow rate: 2 ml/min. Semipreparative HPLC: mobile phase B (containing 0.04% water as modifier); flow rate: 16 ml/min (other conditions see Experimental).

Semi-preparative HPLC separation 1 4 5

Using 5/~m silica, the optimized mobile phase [26] of the analytical HPLC system (Fig. 5) was transferred w i t h o u t any modification to semi-preparative HPLC. The column diameter was enlarged from 4 mm (analytical column) to 16 mm and the flow rate was increased from 2 to 16 ml/min at a maximum pressure of 300 bar. The isocratic semi-preparative HPLC separation was carried out with 30 mg of furocoumarin reference mixture. The chromatograms obtained are presented in Fig. 5. Practically the same baseline resolution was achieved as in the analytical experiments and, as expected, the separation time was doubled for the semi-preparative isolation because the high back pressure had only allowed an increase of flow by a factor of 8 than by a factor of 16. Semi-preparative HPLC separations were also carried out with various prepurified plant extracts. One of them (from Heraclei mantegazziani radix), in which all eight furocoumatins were detected, is depicted in Fig. 6. Except for psoralen, representing only a minor component of the 50 mg crude extract, which was prepurified (26 mg) over Si 60 Extrelut | cartridges, pure fractions between 1 and 7 mg ( 1 : 1 . 7 mg, 2 : 2 . 0 mg; 4 : 1 . 4 mg; 5 : 6 . 8 mg; 6:1.5 mg; 7 : 1 . 0 rag; 8 : 3 rag) were isolated within one hour.

10
Fig. 6

20

30

40

50

~" t(min)

Isocratic semi-preparative HPLC separation of 26 mg prepurified extract from Heracleum mantegazzianum roots. Mobile phase B (containing 0.04% water as modifier); flow rate: 17 ml/min (other conditions see Experimental, or text). Chromatographia Vol. 27, No. 11/12, June 1989 Originals

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After one or more MPLC separations or several semipreparative HPLC runs, the collected mixed fractions of isobergapten and angelicin can be rapidly separated by either technique, using a mobile phase composition of ethyl acetate, chloroform and n-hexane (20 %/20 %/60 %).

References
[1| R. D. H. Murray, J. M#ndez, & A. Brown, The Natural

Conclusions
dry-packing procedure developed for preparative MPLC glass columns permitted the use of silica with an average particle size of 15 pro. Because of the proposed filling process and the relatively low cost of normal phase material, MPLC separations may be carried out without prepurification of crude plant extracts or complex mixtures, in contrast to HPLC methods. Use of OPLC as pilot method also has the advantage that sample prepurification is not so important as in HPLC. The stationary phase used for OPLC may be changed after a few runs or after each separation, w i t h o u t incurring high costs, As a result of these advantages and the linear relationship between the retention data of OPLC and MPLC separations (these correlations are under study for various substances), OPLC is an appropriate pilot method for preparative medium pressure liquid chromatographic isolations from crude plant extracts.
The

The reported preparative, isocratic MPLC system, with mobile phase transfer from on-line OPLC, permits isolation of the eight furocoumarin isomers from crude plant extracts in one step. Though the semi-preparative HPLC separation described above was carried out with 5 /lm silica, the resolution was not much better than for the MPLC separation with 15 #m silica. Due to the smaller solvent consumption and the cheaper stationary phase, preparative MPLC isolations cost less for a given sample amount than semi-preparative HP LC. The above results demonstrate the suitability of the MPLC technique for isocratic isolation of natural compounds even with closely related structures.

Acknowledgment
The autors are gratful to Mr. F. Vreven (SES, Nieder-OIm/ Mainz, FRG) and to Labomatic (Sch~nenbuch, Switzerland) for the kind loan of a Chrompres 25 OPLC instrument and a MPLC system, respectively.

Coumarins, Occurrence, Chemistry and Biochemistry, John Wiley & Sons Ltd., Chichester, 1982. [2] B. Bridges, G. Strauss, Nature 283, 523 (1980). [3] O. Schimmer, R. Beck, U. Dietz, Planta Med. 40, 68 (1980). [4] O. Schirnmer, Planta Med. 47, 79 (1983). (51 E. Fahr, Pharm. Ztg. 127, 163 (1982). [6J K. Wolff, Dtsch. Med. Wochenschr. 104, 1542 (1979). [7] J. G. Montbaliu, M. 7". Rossel, M. G. Bogaert, J. Pharm. Sci. 70, 965 (1981). [8] F. R. Stermitz, R. D, Thomas, J. Chromatogr. 77, 431 (1973). I9] H.E. Nordby, S. Nagy, J. Chromatogr. 207, 21 (1981). [10] G. Innocenti, A. Bettero, G. Caporale, II Farmaco Ed. Sci. 37,475 (1982). [ 11 ] G.W. Ivie, R. C. Beier, D. L. Holt, J. Agric. Food Chem. 30, 413 (1982). [12] R. G. Enriquez, i14. L. Romero, L. A Esr Po JosephNatan, W. F. Reynolds, J. Chromatogr. 287,209 (1984). [13] C. A. J. Erdelmeier, B, Meier, O. Sticher, J. Chromatogr, 346,456 (1985). [ 14] G.C. Zogg, Sz. Nyiredy, O. Sticher, J. Liq. Chromatogr. 10, 3605 (1987). [15] C. A. J. Erdelmeier, Ph, O. thesis No. 7294, ETH ZiJrich, 1983. [16] Sz. Nyiredy, C. A. J. Erdelmeier, O. Sticher, Instrumental Preparative Planar Chromatography, in: "Planar Chromatography, Vol. 1", R. E. Kaiser ed., HiJthig Verlag, Heidelberg, 1986; p. 119. [ 17] G.C. Zogg, Sz. Nyiredy, O. Stichef, J Planar Chromatogr. 1, 261 (1988). [18] K. Hostettmann, M. Hostettmann, A. Marston, Preparative Pressure Liquid Chromatography, in Preparative Chromatography Techniques, Springer-Verlag, Berlin, 1986; p. 27. 119] M. Verzele, E. Geeraert, J. Chromatogr. Sci. 18, 559 (1980). [20] E. Mincsovics, E. Tyih~k, A. M. Siouffi, in Proc, Intern. Syrup. on TLC with Special Emphasis on OPLC, Szeged, 1984, E. Tyihdk ed., Labor MIM, Budapest, 1986; p. 251. [21] E. Mincsovics, E. Tyih~k, A. M. Siouffi, J. Planar Chromatogr. 1,141 (1988). [22l P. Oroszl#n, G. Verz#r-Petri, E. Mincsovics, T. Sz#kely, in Proc. Intern. Syrup. on TLC with Special Emphasis on OPLC, Szeged, 1984, E. Tyih~k ed., Labor MIM, Budapest, 1986; p. 343. [23] K. Jones, Chromatographia 25,547 (1988). [24] SZo Nyiredy, G. C. Zogg, O. Sticher, 3rd Washington Symposium on Preparative Scale Liquid Chromatography, Washington, USA, 1987; Abstr. p. 12. [25J Sz. Nyiredy, K. Dallenbach-TSIke, G. C. Zogg, O. Sticher, in preparation. [26] G. C. Zogg, Sz. Nyiredy, (3. Sticher, J. Planar Chromatogr., 1,351 (1988). {27] E. Kov#ts, Holy. Chim. Acta 41, 1915 (1958) Received: Dec. 29, 1988 Accepted; Jan. 6, 1989 8

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