Research Article Preparative isolation and purification of six volatile compounds from essential oil of Curcuma wenyujin using high-performance centrifugal partition chromatography Six volatile compounds, curdione (1), curcumol (2), germacrone (3), curzerene (4), 1,8- cineole (5) and b-elemene (6), were successfully isolated from the essential oil of Curcuma wenyujin by high-performance centrifugal partition chromatography using a nonaqueous two-phase solvent system consisting of petroleum ether-acetonitrile-acetone (4:3:1 v/v/v). A total of 8 mg of curdione (1), 4 mg of curcumol (2), 10 mg of germacrone (3), 18 mg of curzerene (4), 9 mg of 1,8-cineole (5) and 17 mg of b-elemene (6) were isolated from the essential oil (300 mg) in 500 min. Their structures were determined by comparison of their retention times and MS data with those of the authentic samples as well as NMR spectroscopic analysis. Keywords: Curcuma w enyujin / High-performance centrifugal partition chromato- graphy / Preparative separation / Volatile components DOI 10.1002/jssc.200900453 1 Introduction Essential oils (also called volatile oils) generally extracted by distillation from plants are hydrophobic liquids containing volatile aromatic compounds. The essential oils are widely used in perfumes, c osmetics as well as in food and drink as flavor additives. Some essential oils show multiple pharmaco- logical activities and have been considered as th e major active fractions of herbal medicines [1–3]. Preparative separation of the v olatile components is very important not only f or the quality control of the crude herbsandtheproductscontaining the esse ntial oil but also for the bi o-evaluat ion. However, preparative separation of these pu re vola tile components is a challenge because of their structural similarity, strongly hydrophobic properties a nd poor stability. Column chromato- graphy over silica gel is broadly used to separate the volatile components, which is time-consuming and a rduous work. I n most cases, the major components in essential oils cannot b e retained on silica gel even using hexane or heptane as mobile phase. Poor solubility in aqueous solvents prevents them from being separated in large s cale over the reverse stationary phase. GC is the conventional method for analysis of the essential components usually c oupled with a mass s pectrometer. Preparative GC (Prep-GC) has been successfully used in the separation of the volatile components nowadays [4–6]. However, t hermolabile volatile c omponents could decompose during separation by Prep-GC. Samples injected into Prep-GC mustbefullyvaporizedontocolumntoensureradial distribution of the sample across the column. The compounds of interest are eluted out at very dilute concentrations fro m the column;therefore,itisverydifficulttoextractorcondense those compounds from the gas stream. Efficient parking of large GC columns is also difficult. A ll these e co nomic and technical difficulties limit wide use of Prep-GC in chemical separation([4],Scott,R.P.W.,Chrom-Ed Book Series- GC Chromatography, http://www.library4science.com). Counter-current chromatography (CCC) including high- speed CCC (HSCCC) a nd high-performance centrifugal partition chromatography (HPCPC) is a liquid–liquid partition chromatographic t echnique based on partition of compounds between two immiscible liquid phases. T his method p rovides an advantage over the conventional column chromatography by eliminating the use of a solid support, which may result in irreversible adsorption [7, 8]. HSCCC and HPCPC have increasingly been used to isolate and purify a multitude of natural p roducts [ 7–10]. But only a few researches have been reported in the separation of one or two volatile components from essential oils w ith this technique [11–15]. Eighty-four herbal drugs including Rhizoma Curcumae (Ezhu) are among 472 Chinese herbal materials recorded in China Pharmacopoeia (2005). Their therapeutic effects have been attributed to essential oils [16, 17]. The rhizomes of Yuan-Ye Dang Xiao-Cen Li Qing-Wen Zhang Shao-Ping Li Yi-Tao Wang Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR, P. R. China Received June 29, 2009 Revised January 20, 2010 Accepted February 23, 2010 Abbreviations: CCC, counter-current chromatography; HPCPC, high-performance centrifugal partition chromato- graphy; HSCCC, high-speed CCC; Prep-GC, preparative GC Correspondence: Dr. Qing-Wen Zhang, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR, P. R. China E-mail: qwzhang@umac.mo Fax: 1853-2884-1358 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2010, 33, 1658–1664 1658 three species of Curcuma including C. wenyujin, C. phaeo- caulis and C. kwangsiensis are used as Ezhu, which displays wide and diverse medicinal value [16]. The essential oil of C. wenyujin is considered as an effective part of Rhizoma Curcumae, which is reported to possess anti-tumor [18, 19] and anti-viral activities [20, 21]. Several components including b-elemene, curcumol, germacrone and curdione have been reported to be the biologically active ingredients in the essential oil [22, 23]. Germacrone and curdione were isolated and purified using two-phase solvent system composed of petroleum ether/ethanol/diethyl ether/water (5:4:0.5:1 v/v/v/v) by HSCCC by Yan et al. [15]. The ther- mally labile compounds in ezhu oil, germacrone, curdione, furanodienone and furanodiene, were found to degrade to b-elemenone, curcumol, curzerenone and curzerene, respectively, under heat while zurzerene was transformed into callitrin and callitrisin in aqueous media in previous studies [24–26]. Therefore, it is preferable to use a non- aqueous solvent system at low temperature to separate those unstable volatile components. An HPCPC method using a nonaqueous two-phase solvent system was successfully developed for the separation of six components including curdione (1), curcumol (2), germacrone (3), curzerene (4), 1,8-cineole (5) and b-elemene (6) (Fig. 1) from the essential oil of C. wenyujin. GC-MS analysis showed that the six isolated compounds except curcumol (2) were the major components in the essential oil of C. wenyujin. 2 Materials and methods 2.1 Apparatus The separation was performed on an SIC CPC240 HPCPC (Ever Seiko Corporation, Tokyo, Japan) at room tempera- ture. Total rotor volume of this model is 240 mL and total number of cells is 3136 in which cell length is 15 mm and the distance to the cell center is 82.5 mm. Descending and ascending modes can be interconverted by a four-way switching valve. The rotation speed is up to 2000 rpm (increase and decrease by 100 steps). The two-phase solvent system was pumped with a Syknm S 1021 pump using a dual piston solvent delivery system (Syknm, Germany), equipped with HPCPC to pump. Maximum sample injection volume was 5 mL and the flow rate of the pump was set between 0 and 30 mL/min. Maximum pressure of the HPCPC equipment is 6.0 MPa. An ultra-violet multiple wavelength detector of Agilent Technologies 1200 series (Agilent Technologies, USA) performed the preliminary analysis and detection for all the effluent from CPC240 constantly. The chromatogram was recorded with Agilent ChemStation. Fractions were collected by a CF-1 fraction collector (Spectrum, USA). GC-MS analysis was performed with an Agilent 6890 gas chromatography instrument coupled to an Agilent 5973 mass spectrometer and an Agilent ChemStation software (Agilent Technologies). 2.2 Reagents and materials All organic solvents used for HPCPC were of analytical or chromatographic grade. Petroleum ether (b.p. 60–901C) of analytical grade was purchased from Uni-Chem (Guang- zhou, China). Acetonitrile and acetone of chromatographic grade were purchased from Merck (Darmstadt, Germany). The essential oil of Curcuma wenyujin was purchased from Zhejiang Ruian Pharmaceutical (Ruian, China). The refer- ence compounds of curdione (1), curcumol (2), germacrone (3), curzerene (4) and b-elemene (6) were previously isolated from the above commercial oil [25]. 2.3 Preparation of two-phase solvent system and sample solutions The two-phase solvent systems were selected mainly according to the partition coefficient K of the target compounds. The K values were determined by GC-MS analysis as follows: One drop (about 5 mg) of the essential oil was added to the mixture of equal volume of the upper phase and the lower phase of the two-phase solvent system. The solutions were then mixed thoroughly by shaking. The upper phase and the lower phase were analyzed by GC-MS, respectively, after equilibration was established. O O O OH O O O 21 43 5 6 Figure 1. Chemical structures of curdione (1), curcumol (2), germacrone (3), curzerene (4), 1,8-cineole (5), b-elemene (6). J. Sep. Sci. 2010, 33, 1658–1664 Sample Preparation 1659 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com A two-phase solvent system composed of petroleum ether/acetonitrile/acetone (4:3:1 v/v/v) was selected for the HPCPC separation after a partition experiment of the essential oil in a series of solvent systems (Table 1) by GC- MS analysis. The solvent mixture was equilibrated comple- tely in a separatory funnel at room temperature. Both of the upper and the lower phases were degassed for 30 min by ultrasonication before use. Aliquots of 300 mg of the essential oil were diluted by 2.4 mL of the mixed solution of lower phase and upper phase (1:1 v/v) of the optimized solvent system used for HPCPC separation. 2.4 HPCPC separation procedure The upper phase of the two-phase solvent system was performed as the stationary phase and the lower phase as the mobile phase in the present HPCPC separation. The upper stationary phase was first pumped into HPCPC at a flow-rate of 5.0 mL/min and the apparatus was run at 300 rpm under ascending separation mode. After about 50 min when the column was fully filled with stationary phase, the lower mobile phase was pumped into HPCPC at a flow rate of 1.5 mL/min and the rotation speed was set at 1700 rpm under descending separation mode. The sample was injected through the sample loop after hydrodynamic equilibrium was reached, as indicated by a clear mobile phase eluting at the outlet. The HPCPC separation was running for 600 min and effluent from the outlet of HPCPC was continuously monitored by an ultra-violet multiple wavelength detector at UV 210 nm. Per 3 mL fractions of effluent were collected and analyzed by GC-MS. The fractions were pooled according to GC-MS results and evaporated at 401C under reduced pressure to avoid thermal degrading. At the end of the separation, all the upper stationary phase was collected to calculate the retention of the stationary phase. 2.5 GC-MS analysis GC-MS was performed on a capillary column (30 m  0.25 mm, id) coated with 0.25 mm film 5% phenyl methyl siloxane was used for the separation. The carrier gas was high-purity helium and its flow rate was at 1.0 mL/min. The optimized GC-MS conditions were as follows: the column temperature was set at 601C for injection, then programmed at 51C/min to 1201C and held for 13 min, then at 251C/min to 1451C and held for 20 min, finally, at 301C/min to 2801C. Split injection (2 mL) was conducted with a split ratio of 10:1. The mass spectrometer was set in electron-impact (EI) mode in which the scan range was between 40 and 550 amu, the ionization energy was 70 eV and the scan rate was 0.34 s per scan. The inlet, ionization source temperatures were 250 and 2801C [27]. 3 Results and discussions The selection of the two-phase solvent system is the first and critical step for an HPCPC or an HSCCC separation. Successful separation by HSCCC or HPCPC depends on the suitable selection of a two-phase solvent system, which should have good solubility and stability for target compounds, an ideal partition coefficient (K), short settling time (o30 s) and satisfactory stationary phase retention (>50%) [9, 28]. Partition coefficient is a very important parameter for solvent system selection. A compound with a small K value may come out with the solvent front and result in a loss of peak resolution while a large K value produces excessive running time and band broadening. Ideally, the partition coefficient K has to be close to 1, usually in a range of 0.5–2 [8, 9]. However, all partition coefficients are difficult to maintain in the range of 0.5–2 when separating more than four compounds in a one-step HSCCC or HPCPC run. In practice, a K value between 0.2 and 5 can be used without the excessive elution time associated with band broadening [7, 29]. In this study, K 5 A up (peak area of compound in upper phase)/A low (peak area of compound in lower phase). Previous study indicated some components degraded in aqueous solution [26]. Hence, the non-aqueous solvent system was given priority in the selection of solvent system. Eight kinds of two-phase nonaqueous solvent systems were studied, such as petroleum ether/acetonitrile/acetone (2:1:1, 5:3:2, 4:3:1 and 7:6:1 v/v/v) and n-heptane/acetonitrile/ethyl acetate (7:6:1, 4:3:1, 5:3:2 and 2:1:1 v/v/v). With the GC-MS results and the calculation of K 5 A up /A low , the different K values of curdione (1), curcumol (2), germacrone (3), Table 1. The partition coefficient values of curdione (K 1 ), curcumol (K 2 ), germacrone (K 3 ), curzerene (K 4 ), 1,8- cineole (K 5 ) and b-elemene (K 6 ) in eight kinds of solvent systems No. Solvent systems (v/v) K 1 K 2 K 3 K 4 K 5 K 6 1 Petroleum ether/acetonitrile/ acetone (2:1:1) 0.36 0.58 0.92 2.25 2.01 6.03 2 Petroleum ether/acetonitrile/ acetone (5:3:2) 0.39 0.60 0.77 1.49 1.66 3.27 3 Petroleum ether/acetonitrile/ acetone (4:3:1) 0.33 0.56 0.76 1.74 1.94 4.53 4 Petroleum ether/acetonitrile/ acetone (7:6:1) 0.17 0.32 0.50 1.02 1.15 2.22 5 n-Heptane/acetonitrile/ethyl acetate (7:6:1) 0.28 0.54 0.72 2.08 2.17 6.49 6 n-Heptane/acetonitrile/ethyl acetate (4:3:1) 0.34 0.63 0.75 1.90 1.94 5.22 7 n-Heptane/acetonitrile/ethyl acetate (5:3:2) 0.43 0.72 0.80 1.49 1.56 2.99 8 n-Heptane/acetonitrile/ethyl acetate (2:1:1) 0.58 0.82 0.81 1.25 1.26 1.94 J. Sep. Sci. 2010, 33, 1658–16641660 Y Y. Dang et al. & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com curzerene (4), 1,8-cineole (5) and b-elemene (6) were obtained for those eight kinds of solvent systems (Table 1). The K values of nearly all target compounds in petroleum ether/acetonitrile/acetone systems could be enlarged by increasing the volume ration of acetone. In the petroleum ether/acetonitrile/ethyl acetate systems, increasing the volume ration of ethyl acetate result in the increasing of the K values of curdione (1), curcumol (2) and germacrone (3), and the decreasing of the K values of curzerene (4), 1,8- cineole (5) and b-elemene (6). The K values of all target compounds in n-heptane/acetonitrile/ethyl acetate (2:1:1 v/ v/v) system lay well in a range of 0.5–2.0. Theoretically, two compounds with a similar K value could not be separated from each other by CCC. Since K 2 and K 4 were close to K 3 and K 5 , respectively, curcumol (2) and germacrone (3), and curzerene (4) and 1,8-cineole (5) were hardly separated from each other with this solvent system (n-heptane/acetonitrile/ ethyl acetate 2:1:1 v/v/v). Other factors beside the range of K value should also be considered for the selection of the solvent system. Resolution Rs is a critical parameter to measure the quality of a separation and is expressed as the Knox equation composing three variables including the efficiency N, the selectivity a, and the capacity k 0 : Rs ¼ðNÞ 1=2 ½ða À 1Þ=a½k 0 2 =ð1 þ k 0 2 Þ=4or Rs ¼ðNÞ 1=2 ða À 1Þ½k 0 1 =ð1 þ k 0 1 Þ=4 ð1Þ Since K is more practically used than k 0 , the above equation will be given as [7]: Rs ¼ 2V s ðK 2 À K 1 Þ=ðW b1 1W b2 Þð2Þ Herein, V s is the volume of the stationary phase; K 1 and K 2 are the partition coefficients for the two adjacent peaks, respectively (where K 2 >K 1 ); W b1 and W b2 are the peak width for the two target components. It is obvious that the resolution increases with the volume V s of the stationary phase, with the selectivity (K 2 /K 1 ) or the difference between K 2 and K 1 , and the reduction in band width. Within the eight tested solvents systems, the differences between K 2 and K 3 , and between K 4 and K 5 in solvent systems (petroleum ether/acetonitrile/acetone 2:1:1 and 4:3:1 v/v/v) are 0.34 and 0.24, and 0.20 and 0.20, respec- tively, which are larger than those in the other six systems (Table 1). These two solvent systems (petroleum ether/ acetonitrile/acetone 4:3:1 and 2:1:1 v/v/v) were selected for further study. The settling times for solvent systems of petroleum ether/acetonitrile/acetone (4:3:1 and 2:1:1 v/v/v) were 7 and 14 s, respectively, which indicated that the solvent system of petroleum ether/acetonitrile/acetone (4:3:1 v/v/v) may yield a larger retention of the stationary phase than the solvent system of petroleum ether/acetonitrile/acetone (2:1:1 v/v/v). The influences of flow rate and revolution speed were also investigated. A lower flow rate usually gives a higher retention level of the stationary phase, improves the peak resolution and also requires a longer separation time. Using a lower revolution speed reduces the volume of the stationary phase retained in the column leading to lower peak resolution. On the other hand, higher revolution speeds may produce excessive sample band broadening by violent pulsation of the column because of elevated pressure [9]. When revolution speed was 1200 rpm and flow rate of the mobile phase was 2.5 mL/min, more than 50% of the stationary phase (upper layer of petroleum ether/acetoni- trile/acetone 4:3:1 v/v/v) was extruded when the mobile phase (lower layer of petroleum ether/acetonitrile/acetone 4:3:1 v/v/v) was pumped into HPCPC. When the revolution speed was increased to 1400 rpm and the flow rate of the mobile phase was decreased to 2.0 mL/min, the outflow volume of the stationary phase was about 70 mL before sample injection, which means that the retention of stationary phase was nearly 70% (total rotor volume of this model is 240 mL). The stationary phase could not be held well in the rotor and it came out with the effluent all the time during the whole process of separation. Then, the revolution speed was increased to 1600 rpm and the flow rate of the mobile phase was reduced to 1.5 mL/min, yielding nearly 50 mL of the stationary phase eluted after hydrodynamic equilibrium was attained, and the main- tenance of the stationary phase was also improved. The purity of the target components from this separation could reach 80%. At last, when revolution speed was increased to 1700 rpm and the flow rate of the mobile phase was main- tained at 1.5 mL/min, stationary phase retention was over 85% and rarely any stationary phase eluted after the sample injection. The purity of each target component increased to 85% or above in this last experiment. For the solvent system Absorbance (mAU) min0 50 100 150 200 250 300 350 400 450 mAU 0 500 1000 1500 2000 Time (Min) 1 2 3 6 5 4 Figure 2. HPCPC chromatogram of the essential oil of C. wenyujin. Experimental conditions: solvent system: petroleum ether/ acetonitrile/acetone (4:3:1 v/v/v); mobile phase: lower phase, revolution speed: 1700 rpm; flow rate: 1.5 mL/min; wavelength: 210 nm. Fractions 1–6 correspond to curdione, curcumol, germacrone, curzerene, 1,8-cineole and b-elemene, respectively. J. Sep. Sci. 2010, 33, 1658–1664 Sample Preparation 1661 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com of petroleum ether/acetonitrile/acetone (2:1:1 v/v/v), the retention of stationary phase was less than 50% when carrying out the same procedures for optimizing the flow rate and revolution speed as for solvent system of petroleum ether/acetonitrile/acetone (4:3:1 v/v/v). Thus, petroleum ether/acetonitrile/acetone (4:3:1 v/v/v) was selected as the solvent system for the HPCPC separation and other conditions were optimized as: revolution speed was 1700 rpm while flow rate of mobile phase was 1.5 mL/min. The HPCPC separation with the optimum condition described above yielded a total of 8 mg of curdione (1), 4 mg of curcumol (2), 10 mg of germacrone (3), 18 mg of curzerene (4), 9 mg of 1,8-cineole (5) and 17 mg of b-elemene (6) from the essential oil (300 mg) in 2.4 mL of the selected optimum solvent system (Fig. 2). The amount of each isolated compound from one run was enough for structural analysis by MS and NMR. Repeatability test was chosen to evaluate the system suitability of the developed assay. The HPCPC separation 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.000 200000 1000000 1800000 2600000 Time > Abundance A B C D E F G 1 5 6 4 3 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.000 600000 1400000 2200000 3000000 Time > Abundance 1 2 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 0 80000 16000 240000 320000 Time > Abundance 2 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 0 80000 160000 240000 Time > Abundance 3 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.000 200000 600000 1000000 1400000 1800000 Time > Abundance 4 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.000 40000 80000 120000 160000 Time > Abundance 5 5.00 10.00 15.00 20.0 25.00 30.00 35.00 40.00 45.00 0 50000 150000 250000 350000 450000 Abundance 6 Figure 3. GC-MS chromato- grams of (A) the essential oil of Curcuma wenyujin and (B) Fraction 1, (C) Fraction 2, (D) Fraction 3, (E) Fraction 4, (F) Fraction 5 and (G) Fraction 6 from HPCPC separation. 1, curdione; 2, curcumol; 3, germacrone; 4, curzerene; 5, 1,8-cineole; 6, b-elemene. J. Sep. Sci. 2010, 33, 1658–1664 1662 Y Y. Dang et al. & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com was repeated three times and the purity of the isolated components determined by GC-MS (Fig. 3, Table 2). Variation of purity was expressed by the RSDs, which were less than 2.1% (Table 2). The structures of compounds 1–4 and 6 were identified as curdione (1), curcumol (2), germacrone (3), curzerene (4) and b-elemene (6), respectively, in comparison of their retention times and MS data (Table 3) with those of the authentic samples isolated from this oil previously [25]. Compound 5 was identified as 1,8-cineole (5) by spectro- scopic analysis including MS (Table 3) and NMR as follows: The 1 H NMR spectrum of compound 5 showed signals at d: 1.01 (3H, s, H 3 -7), 1.21 (6H, s, H 3 -9 and H 3 -10 ), 1.36 (1H, m, H-4), 1.45 (2H, m, H a -3 and H a -5), 1.47 (2H, m, H a -2 and H a -6 ), 1.61 (2H, m, H b -2 and H b -6 ) and 2.00 (2H, m, H b -3 and H b -5 ). The 13 C NMR spectrum of compound 5 displayed signals at d: 22.8 (C-3 and C-5), 27.5 (C-7), 28.8 (C-9 and C-10), 31.5 (C-2 and C-6), 32.9 (C-4), 69.7 (C-1) and 73.5 (C-8). The NMR data above were in agreement with those of 1,8-cineole [30]. 4 Concluding remarks After only a one-step separation by HPCPC using a non-aqueous two-phase solvent system, six components, namely, curdione (1), curcumol (2), germacrone (3), curzerene (4), 1,8-cineole (5) and b-elemene (6), were successfully separated from the essential oil of C. wenyujin, which are pure enough for structural elucidation, qualitative analysis as a chemical reference. The isolated components (2, 3, 5 and 6) with purity above 90% might be suitable for biological research. The result suggested that HPCPC might be an effective and efficient method for the isolation and purification of bioactive compounds from volatile oil. The method provides an alternative approach to separate the strong hydrophobic components with similar structures. The authors would like to thank X. H. Gao and F. Q. Yang for their technical assistance in GC-MS in our laboratory. The research was supported by grants from Macao Science and Technology Development Fund (013/2008/A1) and Research committee of University of Macau (RG075/06-07S/WYT/ ICMS). The authors have declared no conflict of interest. 5 References [1] Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., Food Chem. Toxicol. 2008, 46, 446–475. [2] Chan, S. S. K., Yan, R., Li, G., in: Li, S. P., Wang, Y. T. (Eds.), Pharmacological Activity- Based Quality Control of Chinese Herbs, Nova Science-Publishing, New York 2008, pp. 273–289. [3] Zhang, Y., Cao, H., Yang, M. M., Xiao, P., in: Li, S. P., Wang, Y. T. (Eds.), Pharmacological Activity-Based Quality Control of Chinese Herbs, Nova Science- Publishing, New York 2008, pp. 311–324. [4] Jusforgues, P., in: Wilson, I. D. (Ed.), Encyclopedia of Separation Science, Elsevier-Publishing, Amsterdam 2000, pp. 529–536. [5] Adio, A. M., Konig, W. A., Phytochemistry 2005, 66, 599–609. Table 2. Purity of six components separated by HPCPC Purity Curdione (1) Curcumol (2) Germacrone (3) Curzerene (4) 1,8-Cineole (5) b-Elemene (6) First run (%) 85.0 a) 91.7 94.0 88.5 94.4 97.8 Second run (%) 86.9 92.8 94.5 89.7 93.2 96.7 Third run (%) 88.6 93.4 92.8 86.8 95.8 96.2 Average (%) 86.8 92.6 93.8 88.3 94.5 96.9 RSD (%) 2.1 0.9 0.9 1.6 1.4 0.8 a) Purity (%) 5 100  (peak area of target compound)/(total peak area of all peaks). Table 3. The retention times (RT) and MS data of six isolated components Peak Compound RT (min) MS data a) 1 Curdione 35.60 236(M 1 ,8), 180(96), 167(75), 109(80), 95(34), 82(36), 69(100), 68(64), 67(61), 55(73), 41(67) 2 Curcumol 29.48 236(M 1 ,26), 135(28), 121(100), 119(29), 107(41), 93(40), 91(29), 69(33), 55(44), 43(28), 41(46) 3 Germacrone 33.82 218(M 1 ,8), 136(49), 135(71), 121(24), 107(100), 105(20), 91(33), 79(20), 67(52), 53(22), 41(28) 4 Curzerene 23.59 216(M 1 ,13), 148(30), 133(10), 109(10), 108(100), 105(12), 93(11), 91(16), 79(17), 77(15) 5 1,8-Cineole 6.16 154(M 1 ,36), 111(47), 108(50), 93(36), 84(41), 81(61), 71(48), 69(38), 41(36), 43(100) 6 b-Elemene 16.98 204(M 1 ,2), 147(46), 121(46), 107(67), 93(100), 91(50), 81(93), 79(66), 68(67), 67(75), 41(51) a) m/z , relative intensity shown in parenthesis. J. Sep. Sci. 2010, 33, 1658–1664 Sample Preparation 1663 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com [6] Cool, L. 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