Preparative separation of a terpenoid and alkaloids from Tripterygium wilfordii Hook. f. using high-performance counter-current chromatography

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Journal of Chromatography A, 1213 (2008) 145–153

Contents lists available at ScienceDirect

Journal of Chromatography A

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reparative separation of a terpenoid and alkaloids from Tripterygium wilfordiiook. f. using high-performance counter-current chromatographyomparison of various elution and operating strategies

aoyu Yea,b, Svetlana Ignatovac, Houding Luoa, Yanfang Lia, Aihua Penga, Lijuan Chena,∗, Ian Sutherlandc,∗∗

State key laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, ChinaCollege of Chemical Engineering, Sichuan University, Chengdu 610065, ChinaBrunel Institute for Bioengineering, Brunel University, Kingston Lane, Uxbridge, Middlesex UB8 3PH, UK

r t i c l e i n f o

rticle history:eceived 17 April 2008eceived in revised form7 September 2008ccepted 26 September 2008vailable online 17 October 2008

eywords:ripterygium wilfordii Hook. f.igh-performance counter-current

a b s t r a c t

This paper describes how high-performance counter-current chromatography (HPCCC) was used strategi-cally for the separation of Tripterygium wilfordii Hook. f. Due to the complexity of Chinese herbal medicines,the initial ethanol crude extract was fractionated into seven fractions using medium-pressure liquidchromatography (MPLC). One terpenoid (triptolide) and three alkaloids (peritassine A, wilforgine andwilforine) were further separated from one of the MPLC fractions. This fraction (1.25 g) yielded 8 mg oftriptolide and 28 mg of peritassines A after one HPCCC column pass and 30 mg of wilforgine and 120 mgof wilforine after a second column pass with respective purities of 97%, 93.6%, 95.0% and 94.4%, whichwere determined by high-performance liquid chromatography (HPLC). This was a one-step HPCCC sepa-ration, using an n-hexane–ethyl acetate–methanol–water (4:5:4:5, v/v) solvent system, where increases

hromatographylution extrusionerpenoidslkaloids

in theoretical plates have been sacrificed in favour of increasing throughput. Structures were identifiedby electrospray ionization mass spectrometry (ESI-MS), 1H nuclear magnetic resonance (1H NMR) and13C nuclear magnetic resonance (13C NMR). Comparison of three different modes of eluting compoundsretained in the liquid stationary phase: elution extrusion; dual mode and simple pump-out showed thatsimply pumping out the column contents at high flow gave better resolution and was eight times faster

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than the other two well-ufrom Tripterygium wilfordi

. Introduction

Tripterygium wilfordii Hook. f. is a Chinese herbal medicine (Leiong Teng) that grows in many parts of southern China. Most ordi-ary Chinese people called it “qi bu si”, which can be interpreted asseven steps to death” due to its extreme toxicity. Because of this,t must be initially boiled for at least 60 min before mixing withther herbs to prevent the recipient having any harmful effects1]. Tripterygium wilfordii Hook. f. contains two major groups of

hemicals [2], namely terpenoids such as triotolide, tripdiolide,nd trioterolide, as well as alkaloids including peritassines A, wil-orgine, wilforine and euonine. Triptolide was first isolated fromripterygium wilfordii Hook. f. and structurally characterized in

∗ Corresponding author. Tel.: +86 28 85164063; fax: +86 28 85164060.∗∗ Corresponding Author. Tel.: +44 1895 266920; fax: +44 1895 274608.

E-mail addresses: [email protected] (L. Chen), [email protected]. Sutherland).

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021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.09.116

d methods. Triptolide and peritassines A were isolated for the first timek. f.

© 2008 Elsevier B.V. All rights reserved.

972 [3] and has been used since for the treatment of a vari-ty of autoimmune diseases and as an immuno-suppressant inatients with organ and tissue transplantations [4,5]. Recently,riptolide was shown to have anti-cancer effects by suppressinghe growth of a broad range of human tumor cells and inducingpoptosis [6,7]. Its high bioactivity and its significant therapeuticffects make it an important candidate for developing rapid purifi-ation methodology from the crude extract of Tripterygium wilfordiiook. f. using high-performance counter-current chromatogra-hy (HPCCC) where separation times are short and recoveriesigh.

Counter-current chromatography (CCC), using a liquid station-ry phase of a two-phase solvent system, was first invented by Ito inhe late 1960s [8]. With the development of CCC technology [9–11]

nd its operational strategy [12–14], more and more research hasocused on the separation of natural products [15–17] and biology

olecules [18,19] due to the unique advantages of this technology,uch as the elimination of irreversible absorption; high recoveryf target compounds and high throughput compared with other

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raditional separation methods such as column chromatographynd thin-layer chromatography (TLC).

To date, two papers have been published on the separationf crude from Tripterygium wilfordii Hook. f. using CCC. Wei etl. used multidimensional CCC with solvent systems composed

f n-hexane–dichloromethane–methanol–water (3:22:17:8, v/v)nd chloroform–methanol–water (4:3:2, v/v) for the separationnd purification of tripdiolide [20]. The whole procedure for thewo-step separation took about 6.5 h without allowing time to con-

alct

Fig. 1. Chemical structures of the terpenoid (C1 triptolide) and the three alkaloids (C2 p

1213 (2008) 145–153

entrate the fraction from the first step. Also both of their stepssed chlorinated solvents which are not considered to be verynvironmentally friendly. Ou Yang et al. separated four alkaloidswilfortrine, wilfordine, wilforgine and wilforine) from the sameerb with a solvent system composed of light petroleum–ethyl

cetate–ethanol–water (6:4:5:8, v/v) [21]. While this was an excel-ent example of high yield and high purity of four separateompounds in one isocratic run, the separation time was still inhe order of 5 h.

eritassines A; C3 wilforgine and C4 wilforine) from Tripterygium wilfordii Hook. f.

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This paper focuses (Fig. 1) on the separation of one terpenoidtriptolide) and three alkaloids (peritassine A, wilforgine and wil-orine) from an interesting fraction from Tripterygium wilfordiiook. f. ethanol extract initially fractionated by MPLC, whichroved important as a pre-purification step despite its excessivese of time and solvent. The emphasis is on increasing throughputrom 140 to 150 mg/h [20,21] to more than 650 mg/h in a simi-ar capacity, but higher performance centrifuge. The new high-gigh-performance CCC centrifuges now available and used in thisaper will give high retention of stationary phase for a given flowate leading to higher resolution separations and a larger num-er of theoretical plates. However, in this paper we are sacrificingheoretical plates in favour of high throughput (i.e. high sampleoncentration, high sample volume and high flow rate)—importantperformance” criteria for scale up. Separation times are signifi-

antly shorter (2 h compared to 5–6.5 h). A new versatile method ofperating a two-column coil planet centrifuge is presented, whichetects eluents between the columns, harvests compounds that areesolved and passes on unresolved compounds to the second col-mn. Triptolide and peritassines A are isolated for the first time

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ig. 2. Medium-pressure liquid chromatography and fraction selection. (A) MPLC setup.riptolide standard (blue, triptolide peak tR = 5 min) and fraction D (black). MPLC conditioichloromethane–methanol (100:0, 12 l; 100:1, 16 l; 50:1, 12 l; 10:1 12 l); flow rate: 140 monditions: column: reversed-phase Sunfire C18 column (4.6 mm × 150 mm i.d., 5 �m); m5–95% A in 30 min; flow rate: 1.0 ml/min; DAD, and chromatogram was show at maximum

s referred to the web version of the article.)

1213 (2008) 145–153 147

rom Tripterygium wilfordii Hook. f. In addition three different meth-ds for eluting retained compounds, with high distribution ratios,re presented for otherwise identical separation conditions. Theesults show that marginally better resolution can be obtained, butn a significantly shorter time, if a simple pump out method is usednstead of the more sophisticated elution–extrusion [14] and dual

ode [13] methods. This is the first time such a comparison haseen made.

. Experimental

.1. Reagents

All solvents used for HPCCC separation were of analytical grade

nd for HPLC detection were HPLC grade and were all purchasedrom Fisher Chemicals (Loughborough, UK). A triotolide standardas bought from Mansite Biotech (Chengdu, China). HPLC wateras purified from a Millipore laboratory ultra pure water system

0.4 �m filter) (Watford, UK).

(B) MPLC separation chromatogram and (C) HPLC analytical chromatogram for thens: stationary phase: silica gel; mobile phase: different proportions of a mixture ofl/min; detection wavelength: 254 nm. Fractions were collected every 500 ml. HPLCobile phase (A: Methanol, B: 0.1% aqueous formic acid): A and B in gradient mode:

plot. (For interpretation of the references to color in this figure legend, the reader

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.2. Apparatus

A medium-pressure liquid chromatography (MPLC) separationystem (Büchi, Flawil, Switzerland), comprising a C-605 pump, a C-15 pump manager, a C-635 UV detector, a C-660 fraction collectornd a Sepacorerecord 1.0 workstation, was employed to initiallyurify the ethanol extract of Tripterygium wilfordii Hook. f.

HPCCC was performed on a Midi-DE centrifuge (Dynamicxtraction, Slough, UK). The apparatus has four column/coils (forimplicity, called “columns”) on two bobbins all integrated in oneachine—an analytical and preparative column on each bobbin.

he analytical columns use stainless steel tubing of 0.8 mm diam-ter with column volumes for columns 1 and 2 being 18.5 and8.0 ml, respectively. The preparative columns use a 4 mm polyflu-ro alkoxy (PFA) tubing with volumes for columns 3 and 4 being60.5 and 452.0 ml, respectively. The revolution radius or the dis-ance between the column axis and central axis of the centrifugeR) for all of these columns is 11 cm with a ˇ value varying from.52 at the internal terminal to 0.86 at the external terminal (ˇ = r/Rhere r is the distance from the column to the holder shaft). The

otational speed is adjustable from 200 to 1400 rpm. A rotationpeed of 1400 rpm was used in this study giving a high “g” value of40.8g. The HPCCC separation setup consisted of an analytical HPLCump Knauer 501 (Berlin, Germany); a preparative pump Knauer800 and a spectrophotometer Knauer 2501 with a preparative cellperating at 254 nm.

HPLC was performed on a Waters Alliance 2695 separations

odule (Empower software) connected to a Waters PDA2996 pho-

odiode array detection (DAD) system (190–800 nm) using a Sunfire18 column (150 mm × 4.6 mm i.d., 5 �m) (Waters, Milford, MA,SA). The MS analyses were performed with a Q-TOF Premier Masspectrometer (Waters Micromass, Milford, MA, USA) coupled with

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ig. 3. HPCCC operating modes. Position 1 (P1), standard equilibration mode with both chrough the detector after just one column before collecting fractions; position 3 (P3), cwitched to be in series but with the detector at the end of the second column instead of

1213 (2008) 145–153

n ESI source. Their structures were identified by a Bruker Avance00 NMR system.

.3. Crude preparation

Ethanol extract powder of Tripterygium wilfordii Hook. f.as obtained from Sanling Biotech. (Guilin, China). This extractas initially fractionated by MPLC. A 200-g amount of ethanol

xtract powder of Tripterygium wilfordii Hook. f. was dissolved in00 ml dichloromethane and subjected onto a 6.3 l glass column1000 cm × 9.8 cm i.d.) packed with 3.1 kg silica gel with an averageiameter of 50 �m. The extract was eluted with different propor-ions of mixture of dichloromethane–methanol (100:0, 12 l; 100:1,6 l; 100:2, 12 l; 100:10 12 l) with a Büchi C605 pump at a flow rate of40 ml/min and eluent was detected at 254 nm with a Büchi C-635V detector (Fig. 2A). Eluent was collected in bottles of 500 ml withn automatic fraction collector and analyzed by TLC. Then groupednto seven fractions according to TLC results: A, B, C, D, E, F fromtepwise elution (Fig. 2B) and fraction G from the column washedith pure methanol (not shown in Fig. 2B).

A triotolide standard was used to indentify that Fraction D con-ained triptolide. This was evaporated to dryness and used for thePCCC separation.

.4. Determination of distribution ratios or partition coefficients

The selected solvent system was first prepared and equilibrated.

2-ml volume of upper phase and lower phase was then dispensed

nto a test tube with a pipette. A 2-mg amount of crude extract washen weighed and added to the phase system. The test tube washaken vigorously until equilibrium had been established in bothhases. Equal volumes (1 ml) of upper and lower phases were then

oils in series; position 2 (P2), four-way valves A and B switched to flow the eluentolumns switched to be in series again (as P1) and finally position 4 (P4), columnsbetween the columns.

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ipetted into separate HPLC vials and evaporated to dryness underacuum. Finally, the residues were diluted with 1 ml methanol andnalysed by HPLC. The distribution ratio/partition coefficient (KD)f a particular compound in reversed-phase mode was calculateds the ratio of peak area in the upper (stationary) phase to the lowermobile) phase. In normal-phase mode, the KD value would be theeciprocal of these values.

.5. Preparation of solvent system and sample solution

The solvent system used consisted of hexane, ethyl acetate,ethanol and water with the volume ratio of 4:5:4:5. The solvent

ystem was prepared in a separating funnel, shaken until thor-ughly equilibrated and then left overnight. The sample could beasily dissolved in upper phase of the above phase system.

.6. HPCCC system setup

The separation strategy was developed on the Midi-DE analyti-al columns (1 and 2) and then scaled up to the Midi-DE preparativeolumns (3 and 4). Either the analytical columns or the preparativeolumns were connected together with two 4-way valves (Fig. 3).his enabled pairs of columns to be used singly or in series withnly one pump. In position 1 (used to equilibrate the phases), thewo coils are connected in series with the detector between them. Inosition 2, well-resolved compounds can be detected and collectedfter just passing through the first coil (Note: in this position coil 2s isolated with equilibrated phases with no mobile phase flowinghrough). In position 3 (which is the same as position 1) both coilsre back in series with the detector between them to detect whenhe partially resolved compounds have passed from coil 1 to coil. Once this has happened, the valves can be switched to position, which places the detector at the outlet of coil 2. If compoundsluting from column 2 are still not pure, they can be introducednto column 1 again and so on.

.7. CCC separation procedure

.7.1. Establishing hydrodynamic equilibriumEquilibration was performed at 30 ◦C in reversed-phase mode

ith the lower (aqueous) phase as mobile phase and upperorganic) phase as the stationary phase using the operating sce-ario shown in Fig. 3, position 1. Both preparative columns 3 and 4ere initially filled with stationary phase at flow rate of 200 ml/minith the rotor stationary. The rotational speed was then ramped up

o 1400 rpm and the pump was set at 25 ml/min. Equilibrium wasstablished when mobile phase started eluting from the outlet andhe displaced volume of stationary phase remained constant.

.7.2. Separation and fraction collectionThe sample volume for preparative CCC separation was 25 ml

ith concentration of 50 mg/ml (1250 mg), which was 5% of theolume of column 3 and 2.5% of the volume of columns 3 and 4ogether. Ten minutes after injection, four-way valve B was changedo start collecting fractions every minute (see Fig. 3, position 2).hen after the first peak eluted from column 3 (at approx. 24 min)our-way valve B was changed again to direct the eluent from col-mn 3 to go into column 4 (see Fig. 3, position 3). When peaksith compounds C3 and C4 entered column 4 (after approximately

0 min) four-way valves A and B were changed to let the eluent from

olumn 4 pass through the detector before entering the fractionollector (see Fig. 3, position 4).

Following the elution of the first 4 target compounds (C1,2, C3 and C4), three different approaches were applied to eluteompounds with high KD values retained in the column: (1)

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obile phase (A: methanol, B: 0.1% aqueous formic acid): A and B in gradientode (0–3.5 min, 55–62% A; 3.5–4.0 min, 62–66% A; 4.01–6.0 min, 66–70.5% A;

.01–7.0 min, 70.5–74.9% A; 7.01–8.62 min, 74.9–79% A; 8.62–16.0 min, 79–90% A);ow rate: 1.0 ml/min; DAD, and chromatograms are shown at Maximum plot.

xtrusion–elution [14]; (2) normal-phase elution or dual mode13] and (3) pumping out the column contents directly. For extru-ion elution, the stationary phase (upper organic phase in thisxperiment) was pumped into columns, from head to tail withhe same flow rate (25 ml/min) and rotation speed (1400 rpm). Forormal-phase elution, the original mobile phase (lower aqueoushase) became the stationary phase and the original stationaryhase (upper organic phase) was pumped as the mobile phase fromail to head with the same flow rate (25 ml/min) and rotationalpeed (1400 rpm). For the pumping out approach, the rotation wastopped, and the mobile phase (lower aqueous phase) was pumpednto the columns at 200 ml/min to push out its content.

.8. HPLC analysis and identification of CCC peaks

Two HPLC methods were developed. The first method was forhe MPLC fraction analysis. HPLC separation was performed on aeversed-phase Sunfire C18 column (150 mm × 4.6 mm i.d., 5 �m)hermostatted at 30 ◦C using a mixture of A (methanol) and B0.1% aqueous formic acid) as mobile phase in a gradient pro-ram with a flow rate of 1 ml/min: 0–30 min: 45–95% A. Eluentas monitored using DAD (all chromatograms shown at maximumlot). This method was shorten to 16 min for analysis of crude andapid screening of HPCCC fractions. The shorter gradient methodas 0–3.50 min, 55–62% A; 3.51–4.0 min, 62–66% A; 4.01–6.0 min,6–70.5% A; 6.01–7.0 min, 70.5–74.9% A; 7.01–8.62 min, 74.9–79%; 8.63–16.0 min, 79–90% A at the same temperature and flowate.

Identification of the HPCCC peak fractions was performed withlectrospray in a positive mode and 1H NMR, 13C NMR with tetram-thylsilane (TMS) as internal standard.

. Results and discussion

.1. Fraction selection for HPCCC separation

From Tripterygium wilfordii Hook. f, by MPLC fractionation, frac-ions A, B, C, D, E, F were eluted (Fig. 2) and fraction G obtainedrom the column wash off after nearly 7 h separation. On the basisf HPLC analysis, it was found that only fraction D contained theesired target. Therefore, fraction D was chosen for the Midi-DEPCCC separation (see Fig. 2C). Before CCC separation, a shortenPLC analysis was performed on fraction D as described in Section.8 (Fig. 4).

.2. Preparative HPCCC separation

The preparative HPCCC separation of 1.25 g of MPLC fraction DKC1: 0.71, KC2: 1.15, KC3: 2.4, KC4: 3.3) is shown in Fig. 5 whereompounds C1 and C2 are separated and eluted within 26 min after

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Fig. 5. (A) HPCCC chromatogram using the Midi-DE preparative column for the separation of crude extract from Tripterygium wilfordii Hook. f. Solvent system: n-hexane–ethylacetate–methanol–water (4:5:4:5, v/v); stationary phase: upper organic phase; mobile phase: lower aqueous phase; column volume: column 3 (460.5 ml) + column 4(452.0 ml) − total (912.5 ml); sample concentration: 50 mg/ml; sample volume: 25 ml; flow-rate: 25 ml/min; rotational speed: 1400 rpm; detection wavelength: 254 nm;temperature: 30 ◦C; retention of stationary phase: 70.6%. (B) HPLC chromatogram of C1, C2, C3 and C4 purified by HPCCC. Conditions: column: reversed-phase Sunfire C18

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olumn (4.6 mm × 150 mm i.d., 5 �m); mobile phase (A: methanol, B: 0.1% aqueou.01–6.0 min, 66–70.5% A; 6.01–7.0 min, 70.5–74.9% A; 7.01–8.62 min, 74.9–79% A;aximum plot.

assing through column 3 using valve positions P1 and 2 (Fig. 3).ompounds C3 and C4 however, as they are not resolved after pass-

ng through column 3 (27–56 min), they are then passed througho column 4 using valve position P3 and when they have enteredolumn 4 the valve is switched to P4 to switch the detector to theutlet of column 4 in time for the elution of the resolved com-ounds C3 and C4 (56–113 min). Note that pump-out of the retainedompounds of high KD value in the stationary phase was achievedn about 5 min (113–118 min) at a pump rate of 200 ml/min. Theield from the shaded fractions in Fig. 5A was 8 mg for C1, 28 mgor C2, 30 mg for C3 and 120 mg for C4 with respective purities of

7%, 93.6%, 95.0% and 94.4% and recoveries of 20.8%, 24.4%, 13.6%nd 55.8%. These recoveries are relatively low due to peak shav-ng to maintain purity. Note that the pump-out contained at leastix compounds of which three were major ones. The maximumbsorbance of compounds is between 219 and 229 nm. However,

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ic acid): A and B in gradient mode (0–3.5 min, 55–62% A; 3.5–4.0 min, 62–66% A;6.0 min, 79–90% A); flow rate: 1.0 ml/min; DAD, and chromatogram was shown at

etection of CCC separation was done at 254 nm to avoid the inter-erence of ethyl acetate present in the solvent system. All HPLChromatograms (Fig. 5B) were shown at maximum plot confirminghe purity of compounds.

.3. Comparison of methods for elution of compounds with highD values

After eluting C1–C4, there were a number of compounds withigh KD values (>4) still retained in the stationary phase. Extru-ion elution, dual mode elution and pumping out are commonly

sed methods to recovery such compounds. To compare the differ-nt elution modes that had time periods varying from 5 to 37 min,he elution chromatograms have to be plotted against volumeluted or number of column volumes. Fig. 6 shows three separatehromatograms run with the same separation protocol used in

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H. Ye et al. / J. Chromatogr. A 1213 (2008) 145–153 151

Fig. 6. Comparison of methods for elution compounds with high KD values. Isocraticelution protocol and conditions (0–3 column volumes) the same as Fig. 5. Elutionprotocols: (A) extrusion elution; (B) dual mode (normal-phase) elution; (C) simplepapm

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isofmatograms during their isocratic elution stages from 0 to 3 coilvolumes.

Table 1Comparison of resolution (Rs) and elution times for three major high KD value com-pounds (C5, C6 and C7).

Elution methods Resolution Flow rate(ml/min)

Elution time(min)

Rs,C56 Rs,C57 Rs,C67

Extrusion elution 0.63 0.27 1.10 25 36.5

ump-out mode. For elution of column contents: flow-rate 25 ml/min for A and Bnd 200 ml/min for C; rotational speed: 1400 rpm for A and B and 0 rpm for C; mobilehase lower (aqueous) phase for C and upper (organic) phase for A and B. Elutionode: head to tail for A and C and tail to head for B.

ig. 5A, but each having a different elution protocol between theertical dashed lines. Fig. 6A is an elution extrusion protocol wherehe mobile phase is switched from being the lower (aqueous) phaseo being the upper (organic) phase, but maintaining the same heado tail direction of flow. Fig. 6B is “dual mode” where normal-hase elution is used pumping in the opposite direction with thepper (organic) phase pumped from tail to head. Fig. 6C is thetandard pump-out procedure where the rotor is stopped and theow increased to 200 ml/min to quickly pump out the retainedhases. Note: In all cases constant volume fractions were collectednd analysed by HPLC to give the chromatograms illustrated in

ig. 7 where only the three major compounds (C5, C6 and C7)re shown. The results shown in Fig. 7 in chromatogram form tollustrate the elution order are compared in Table 1. There are ateast three minor components not shown. The results show thathe dual mode (normal-phase elution mode) is marginally better

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olume elution profile plotted as percentage purity again elution volume expresseds the number of column volumes. (A) Extrusion elution; (B) dual mode (normal-hase with reverse flow); (C) simple pump out.

n terms of resolution than elution extrusion. But surprisingly thetandard pump-out method is, by far, superior to both other meth-ds in terms of resolution and more importantly is eight timesaster. Note also the excellent repeatability between the three chro-

ual mode elution 0.69 0.59 1.32 25 36.5umping out 0.81 0.62 1.48 200 4.6

esolution and elution time comparison for three different elution methods forajor compounds C5, C6 and C7 with high KD values: (A) elution–extrusion; (B)

ual mode (normal-phase with reverse flow) and (C) simple pump-out.

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152 H. Ye et al. / J. Chromatogr. A 1213 (2008) 145–153

Table 2Comparison between A) classical and B) strategic elution modes for compounds C1 and C2.

Compound Elution time (min) Purity (maximum) Amount (mg) Recovery

(A) Classical elution mode (two columns in series) C1 25 96.8% 14.6 38.0%C2 37 95.2% 54.0 47.1%

(B) Strategic elution mode (elution after first column) C1 13 97.0% 8.0 20.8%19

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omparison between the classical way (A) of eluting compounds C1 and C2 with telative pure peaks after a single coil and then routing the remainder through the s

.4. Comparison of different separation strategies

The classic method of separation using two columns in seriesas used to separate 1.25 g of crude (Fig. 8A). This is compared

o the strategic method of Fig. 6C replotted as Fig. 8B for com-arison. Both chromatograms pumped out the coil contents at00 ml/min. The only difference between the two methods is that1 and C2 went through two columns in Fig. 8B instead of just one

n Fig. 8A. The results for compounds C1 and C2 are compared inable 2.

Obviously, using columns in series (classical method) providesetter resolution of C1 and C2 with small differences in purityTable 2). However, the strategic use of columns (Fig. 8B) could beeally useful. Firstly, it allows online detecting of samples after the

ig. 8. Comparison of a classic elution method with the columns in series (A) withstrategic elution method where purified compounds can be eluted early after

ne column volume; and strategic elution mode (B) where compounds C1 and C2ith acceptable purity were collected after being separated by the first column and3 and C4 overlapping in the first column being further separated by the secondolumn. Solvent system: n-hexane–ethyl acetate–methanol–water (4:5:4:5, v/v);tationary phase: upper organic phase; mobile phase: lower aqueous phase; columnolume: 912 ml; sample concentration: 50 mg/ml; sample volume: 25 ml; flow-rate:5 ml/min; rotational speed: 1400 rpm; detection wavelength: 254 nm; tempera-ure: 30 ◦C; retention of stationary phase: A, 70%; B, 70.6%. Note pump out mode isse in both cases.

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umns in series and a strategic way (B) where a single column is used to first elutecoil to further resolve them.

rst column. If target compounds are pure enough, they can be col-ected after the first column, halving elution time and eluting themn a more concentrated form and hence reducing solvent evapo-ation time compared to the classic method. Secondly, it showslearly how peaks are separating from one column to the other.n this experiment, C3 and C4 peaks were overlapped after the firstolumn, and after the second one, they were well separated. How-ver, in this example the throughputs of C1 and C2 for the strategicethod are only marginally better than the classical method, but

his may not be true for all samples.

.5. Structure identification

The chemical structures of the peaks were identified accordingo their MS, 1H- and 13C NMR data.

Fraction C1 was identified as containing triptolide by comparinghe retention time of HPLC and mass spectrometry data with theirtandard triptolide.

Fraction C2: ESI-MS (m/z): [M+H]+ 806.1968, molecular formula:38H47NO18. 1H NMR (400 MHz, CDCl3) ı: 1.08 (3H, d, J = 7.0 Hz),.36 (3H, d, J = 6.9 Hz), 1.54 (3H, s), 1.70 (3H, s), 1.81, 1.84, 2.01, 2.16,.21, 2.31 (each 3H, s), 2.34 (H, q, J = 7.0 Hz), 4.72 (H, d, J = 2.6 Hz), 5.22H, t, J = 2.8 Hz), 5.54 (H, d, J = 3.8 Hz), 7.05 (H, s), 7.37 (H, d, J = 5.4 Hz),.71 (H, d, J = 5.4 Hz), 9.0 (H, s). 13C NMR (100 MHz, CDCl3) ı: 10.11,1.32, 18.45, 20.42, 20.48, 21.00, 21.02, 21, 32, 21.63, 22.76, 33.21,5.66, 50.53, 52.07, 59.93, 68.65, 68.92, 70.12, 70.53, 70.69, 73.34,3.66, 75.80, 84.32, 94.22, 121.52, 125.19, 150.85, 152.86, 156.46,67.95, 168.17, 168.96, 169.11, 169.88, 170.02, 170.30, 173.59. Com-aring the above data with Ref. [22], the obtained product was

dentified as peritassines A.Fraction C3: ESI-MS (m/z): [M+H]+ 858.1735, molecular formula:

41H47NO19. 1H NMR (400 MHz, CDCl3) ı: 1.18 (3H, d, J = 6.9 Hz), 1.573H, s), 1.85, 1.97, 2.19, 2.20, 2.25 (each 3H, s), 2.03 (2H, m), 2.361H, d, J = 4.0 Hz), 2.95, 3.94 (2H, m), 5.03 (1H, d, J = 2.6 Hz), 5.411H, d, J = 6.7 Hz), 5.72 (1H, d, J = 3.70 Hz), 6.94 (1H, s), 6.98 (1H, d,= 1.8 Hz), 7.51 (1H, t, J = 1.8 Hz), 8.40 (1H, s), 8.76 (1H, dd, J = 1.8 Hz,.9 Hz). 13C NMR (100 MHz, CDCl3) ı: 11.78, 20.34, 20.92, 21.28,1.55, 23.42, 23.91, 24.46, 33.31, 33.40, 45.82, 51.13, 52.86, 60.67,8.53, 69.41, 70.06, 71.20, 71.73, 73.18, 74.84, 77.88, 83.84, 93.85,10.14, 119.13, 121.63, 124.59, 138.91, 143.97, 149.39, 152.83, 162.27,64.39, 167.83, 168.84, 169.03, 169.59, 169.87, 170.59, 173.62. Com-aring the above data with Ref. [23], the obtained product was

dentified as wilforgine.Fraction C4: ESI-MS (m/z): [M+H]+ 868.1685, molecular formula:

43H49NO18. 1H NMR (400 MHz, CDCl3) ı: 1.21 (3H, d, J = 7.0 Hz),.62 (3H, s), 1.85, 1.95, 2.18, 2.19, 2.25 (each 3H, s), 2.05 (1H, m),.37 (1H, d, J = 4.0 Hz), 2.95, 3.92 (2H, m), 5.05 (1H, d, J = 2.6 Hz),.48 (1H, d, J = 6.7 Hz), 5.76 (1H, d, J = 3.70 Hz), 6.92 (1H, s), 7.531H, t, J = 7.9 Hz), 8.07 (1H, d, J = 7.1 Hz), 8.10 (1H, d, J = 7.1 Hz), 8.77

1H, dd, J = 1.8 Hz, 4.7 Hz). 13C NMR (100 MHz, CDCl3) ı: 11.73, 20.35,0.62, 21.38, 21.57, 23.32, 23.94, 24.45, 33.32, 33.41, 45.72, 51.17,2.76, 60.64, 68.55, 69.51, 70.16, 71.23, 71.78, 73.12, 74.86, 77.89,3.85, 93.87, 121.73, 124.56, 128.71, 128.71, 128.91, 129.83, 129.83,33.71, 138.93, 152.73, 162.24, 164.49, 167.87, 168.81, 169.13, 169.52,

Page 9

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[[19] Y. Shibusawa, N. Takeuchi, K. Tsutsumi, S. Nakano, A. Yanagida, H. Shindo, Y. Ito,

J Chromatogr. A 1151 (2007) 158.[20] J. Wei, T.Y. Zhang, Y. Ito, J. Liq. Chromatogr. Rel. Technol. 28 (2005) 1903.

H. Ye et al. / J. Chromat

69.82, 170.69, 174.32. Comparing the above data with Ref. [23], thebtained product was identified as wilforine.

. Conclusion

HPCCC was used for separation and purification of target com-ounds containing in fraction D from the Tripterygium wilfordiiook.f ethanol extract which was initially fractionated by a 7 hPLC procedure. 8 mg triptolide, 28 mg peritassines A, 30 mg wil-

orgine and 120 mg wilforine, with respective purities of 97%,3.6%, 95.0% and 94.4%, were separated with a solvent systemf n-hexane–ethyl acetate–methanol–water (4:5:4:5, v/v). Resultsomparing three commonly used methods of eluting compoundsetained in the stationary liquid phase: extrusion elution, dualnormal-phase) mode elution and simple pump-out showed thathe latter was by far the best way to recover compounds with largeD values in reversed-phase mode. Finally, the classic way for usingolumns in series showed better results from the point of viewf recovery of high-purity C1 and C2. However, it was felt that inifferent circumstances the strategic method may well offer somedvantages.

cknowledgements

This project was supported by the National Natural Sci-nce Foundation of China (30772776) and the National 863

rojects (2007AA021065). The Brunel Graduate Centre are grate-ully acknowledged for their exchange student scholarship thatupported H.Y. working at Brunel Institute for Bioengineering formonths. S.I. and I.S. would like to acknowledge financial support

rom West Focus and HEIF3 for their supervision of this research.

[[[

1213 (2008) 145–153 153

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