Journal of Chromatography A, 1331 (2014) 80–89

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Two-dimensional countercurrent chromatography × high performance liquid chromatography for preparative isolation of toad venom Ying-Kun Qiu a,∗ , Xia Yan a , Mei-Juan Fang a,b , Lin Chen a , Zhen Wu a,b,∗ , Yu-Fen Zhao a,b a b

School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen 361100, China College of Chemistry & Chemical Engineering, Xiamen University, South Si-Ming Road, Xiamen 361005, China

a r t i c l e

i n f o

Article history: Received 21 October 2013 Received in revised form 9 January 2014 Accepted 11 January 2014 Available online 17 January 2014 Keywords: 2D CCC × LC Flow programming Bufo bufo gargarizans Toad venom

a b s t r a c t In this work, a new on-line two-dimensional chromatography coupling of flow programming countercurrent chromatography and high-performance liquid chromatography (2D CCC × HPLC) was developed for preparative separation of complicated natural products. The CCC column was used as the first dimensional isolation and a preparative ODS column operated in reversed-phase (RP) mode as the second dimension. The CCC was operated at a controlled flow rate to ensure that each fraction eluted within one hour, corresponding to the isolation time of the 2nd dimensional preparative HPLC. The eluent from the 1st dimensional CCC was diluted using a makeup pump and trapped onto holding column, before been eluted and transferred to the 2nd dimensional HPLC. The performance of the holding column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure, for the solution to the issue of online trapping of low pressure eluent from a CCC column. Satisfactory trapping efficiency and tolerable CCC pressure can be achieved using a commercially available 15 mm × 30 mm i.d. ODS pre-column. The present integrated system was successfully applied in a one-step preparative separation of 12 compounds, from the crude methanol extract of venom of Bufo bufo gargarizans. Compounds 1–12 were isolated in overall yield of 1.0%, 0.8%, 2.0%, 1.3%, 2.0%, 1.5%, 1.9%, 3.6%, 6.1%, 4.8%, 3.5% and 4.1%, with HPLC purity of 99.9%, 99.7%, 90.6%, 99.9%, 77.0%, 99.9%, 90.4%, 99.9%, 52.0%, 99.9%, 99.3%, and 85.0%, respectively. All the results demonstrate that the flow programming CCC × HPLC method is an efficient and convenient way for the separation of compounds from toad venom and it can also be applied to isolate other complex multi-component natural products. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Natural products continue to play a vital role in drug discovery and development [1,2]. It is very important to rapidly, efficiently, economically and in an environment-friendly way to isolate and purify the target natural product in current drug discovery processes. Chromatographic isolation is a key technique to obtain pure compounds for structural elucidation, for pharmacological testing or development into therapeutics. In the past few years, countercurrent chromatography (CCC), a unique liquid–liquid partition chromatographic method with a support-free liquid stationary phase, has been developed for resolving the complex natural product extracts into pure components [3]. CCC eliminates irreversible absorptive loss of samples onto the solid support matrix used in

∗ Corresponding authors. Tel.: +86 592 2189868; fax: +86 592 2189868. E-mail addresses: [email protected], [email protected] (Y.-K. Qiu), [email protected] (Z. Wu). 0021-9673/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2014.01.029

conventional chromatography. CCC method has been successfully applied to the analysis and separation of natural products [4,5]. Some articles have been published with regard to separating a range of compounds by stepwise elution CCC or three-phase CCC in recent years [6,7]. Moreover, High Performance Countercurrent Chromatography (HPCCC) has become established where separations times are in minutes rather than hours and preparative HPCCC has shown very high isolation efficiency [8]. However, because of the narrow range of separation utility and that gradient elution in this technique is not as straightforward as that in high-performance liquid chromatography (HPLC), classic CCC has always been considered as a time-consuming and less efficient chromatographic tool, especially when compared with preparative HPLC [9,10]. Multidimensional (MD) chromatography is an approach capable of providing greater resolution. The most common use of MD separation is two-dimensional liquid chromatography (2D LC × LC), which has been widely used in analysis of complex samples. Recently, an integrated counter current chromatography (2D CCC × CCC) [7], which provided for higher resolution of complex

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samples and larger peak capacity than a single CCC process, had been developed. Coupling of CCC and preparative HPLC for isolation of galactolipids [11] and phthalides [12], which represented offline and heart-cutting combination of CCC and HPLC, respectively, have been reported. Applications of 2D CCC × LC [13,14] in which a macroporous resin column was packed as the 2nd dimensional chromatography, for the purpose of desalination, have also been reported. However, the reported CCC × LC system was not a strict two dimensional chromatography, because the 2nd dimensional column did not provide additional peak capacity. We report here a new 2D CCC × HPLC system for preparative separation of complex natural products, where CCC column was applied as the first dimension and preparative HPLC column as the second one, equipped with a makeup pump and a 10-port switching valve in conjunction with two reversed phase holding columns (Fig. 1). Unlike in analytical on–line 2D LC × LC system, the second-dimensional chromatography in preparative isolation system always requires a certain period of time to complete its separation period, resulting in a limited separation power [15]. Therefore, flow programming CCC technology was employed into this system to solve the limitation in operation time for multiple targeting components. The flow programming scheme aimed at controlling the flow rate of the elution from the first-dimension column while a fraction was transferred to and analyzed on the second-dimension column. This work developed a new 2D flow programming-CCC × LC protocol, and applied it in the separation of the crude methanol extract of toad venom (Bufo bufo gargarizans). In the traditional Chinese medicine, toad venom, with bufadienolides and indole alkaloids as major components, had been used for the alleviation of human sufferings and for the treatment of various diseases, including cancer, arrhythmia and heart diseases [16,17]. Although numbers of chromatographic methods have been used for the separation of components in toad venom, to the best of our knowledge, this is the first study to demonstrate the application of a 2D flow programming-CCC × LC separation system for the isolation and purification of ingredients from the toad venom. 2. Experimental 2.1. Chemicals and materials All solvents used for the preparation of crude extracts and CCC separations were of analytical grade (Jinan Reagent Factory, Jinan, China). HPLC grade solvents for HPLC were purchased from Merck, Darmstadt, Germany. The dried toad venom of B. bufo gargarizans were purchased from Luyan Pharmaceuticals (Xiamen, China) and identified by Professor Yan Qiu (Xiamen University, Xiamen, China). A voucher specimen (20120201-TV) has been deposited at School of Pharmaceutical Sciences, Xiamen University.

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of the apparatus can be regulated with a speed controller in the range between 0 and 1000 rpm. The system was also equipped with a Büchi C-601 pump, a C-615 pump controller (Büchi Labortechnik AG, Flawil, Switzerland), and a ProStar 218 photodiode array detector (Varian Inc. Corporate, Santa Clara, USA). The data were collected with a Varian Star Workstation 6.41 (Varian Inc. Corporate, Santa Clara, USA). The LC system using a Varian binary gradient LC system (Varian Inc. Corporate, Santa Clara, USA) containing two solvent deliver modules (PrepStar 218), a photodiode array detector (ProStar 335) and a fraction collector (ProStar 704). Preparative-HPLC control and data acquisition were also performed by Varian Star Workstation. An Agilent 1100 system HPLC has been used for the analysis of the crude extract and the isolated fractions. It was equipped with a G1379A degasser, a G1311A QuatPump, a G1367A Wpals, a G1315B diode assay detector (DAD), and an Agilent ChemStation for LC. 2.3. Toad venom extract preparation In the present study, methanol extract from toad venom was used as a model complex mixture to evaluate the performance of the total system. The venom of B. bufo gargarizans (100 g) was extracted with methanol twice and concentrated under reduced pressure at 40 ◦ C to resulting in a 28.0 g residue. The residue was stored in a refrigerator (5 ◦ C) for further use. 2.4. Selection of two-phase solvent systems The two-phase solvent system was selected according to the distribution ratio (KD) of each target component. The KD value was determined by HPLC analysis. A suitable amount of crude extract was dissolved in the lower phase of a two-phase solvent system. The solution was analyzed by HPLC. The peak area was recorded as A1 . Then equal volume of the upper phase was added to the solution and the two phases were mixed thoroughly. After partition equilibration, the lower phase solution was determined by HPLC again, and the peak area was recorded as A2 . The KD value was calculated according to the following equation: KD = (A1 − A2 )/A2 . 2.5. Preparation of two-phase solvent system and sample solutions n-Hexane–ethyl acetate–methanol–water (4:6:5:5, v:v:v:v) was used as the two-phase solvent system for CCC separation. It was prepared by adding the solvents to a separation funnel according to the volume ratios and mixed thoroughly. After equilibrium was established, the upper phase and the lower phase were separated and degassed by sonication for 30 min shortly before using. The sample solution for CCC separation was prepared by dissolving 100 mg of the methanol extract into 2 ml of the two-phase solvent (1:1, v/v) before use.

2.2. Instrumentation 2.6. Classical CCC separation procedure All instruments used in this study are commercially available. The CCC instrument employed in the present study was a TBE-300 high-speed counter-current chromatograph (Tauto Biotechnique, Shanghai, China) with three multilayer coil separation columns connected in series (i.d. of the tubing = 1.5 mm, total volume = 300 ml) and a 20 ml sample loop. The revolution radius was 5 cm, and the ˇ values (ˇ = r/R, where r is the rotation radius or the distance from the coil to the holder shaft, and R is the revolution radius or the distances between the holder axis and central axis of the centrifuge) of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal. The revolution speed

The CCC column was first filled with the upper phase as stationary phase, and then the apparatus was rotated at 900 rpm, and the lower phase as mobile phase was pumped through the column at a flow-rate of 2 ml min−1 from the head end of the column to the tail end at room temperature. When a hydrodynamic equilibrium was established in the column and the mobile phase started emerging in the effluent, 2 ml of the sample solution containing 100 mg of the methanol extract was injected through the injection valve. The effluent was monitored by the Varian DAD detector at 254 and 296 nm and recorded as Fig. 2(a).

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Fig. 1. Scheme for flow programming CCC × preparative HPLC system.

2.7. Interface between CCC and preparative HPLC A three-way valve (VICI, Schenkon, Switzerland) was equipped at the post-end of the CCC to collect the eluent without toad venom component, until the first CCC peak emerged. A Büchi pump was used for the addition of water as makeup fluid, which was passing through a dynamic mixer (ChuangXinTongHeng Science and Technology, Beijing, China) to dilute the elution from 1st dimensional CCC. The tandem CCC and HPLC columns were interfaced by two equivalent holding columns and an electronically controlled Valco Cheminert 2-position 10-port switching valve (model EDU10UW, VICI, Schenkon, Switzerland) that make up an integrated online column switching CCC × preparative HPLC system. In order that the dilution actually induced a good accumulation (peak concentration) on the solid-phase cartridge entrance, the trapped analytes were washed out of the holding column in the back-flush mode (Fig. 1). 2.8. Holding column and dilution parameter selection Three types of commercially available ODS pre-columns in different sizes, 15 mm × 21.2 mm i.d. (Phenomenex Security Guard C18 column, bed volume, b.v.: 5.3 ml), 15 mm × 30.0 mm i.d. (Phenomenex Security Guard C18 column, b.v.: 10.6 ml) and 50 mm × 20.0 mm i.d. (Varian Dynamax Microsorb 100-5 C18, b.v.: 15.7 ml), were used for holding capability evaluation. Each CCC eluent was pumped at a flow rate of 0.3 ml min−1 to a dynamic mixer (or a tee), in which the eluent was diluted by water into different ratios, and loaded onto the pre-column under evaluation. Samples trapped on each column were then completely eluted with methanol and weighed for recovery rate. Recovery rate comparisons for different parameters were shown as Fig. 3.

first CCC peak containing the first targeted component started to emerge, the three-way valve was switched and the effluent was firstly diluted at a certain ratio with water and directed to the first holding column (15 mm length × 30 mm i.d.). After all effluents of the first CCC peak have been eluted and adsorbed on the first holding column, the 2-position 10-port switching valve was switched and LC pumps began to deliver mobile phase solution to desorb and isolate the first targeted peak into its constituent components as the start of a step-by-step process. Meanwhile, when all the effluent of the second CCC peak is trapped on the second holding column, the CCC is kept on the same rotation while the CCC pump is set to another flow rate according to the program in Fig. 2b, in order that the second CCC peak width can fit with the demand of the first CCC peak’s HPLC separation. Once the first isolation has finished, the 2position 10-port switching valve was switched again and the CCC pumps were set to another flow rate for eluting the other components successively, as the second CCC peak trapped on the second holding column was isolating on the HPLC column. A reversed-phase preparative column (Shim–pack VP-ODS, 250 mm × 20.0 mm i.d.) was used as the 2nd dimensional stationary phase. The mobile phase was methanol (A) or acetonitrile (B), and water (C) in a linear gradient mode as follows. A:C from 5:95 to 60:40 for 30 min, and 60:40 to 100:0 during 30–50 min, and then the mobile phase was changed to B and C at a ratio of 28:72 and kept until 60 min, for the isolation of 1st dimensional CCC fraction 1 (Fr. 1). The mobile phase of Fr. 2-7 was programmed as: B:C from 28:72 to 54:46 during 0–40 min, and run back to 28:72 at 60 min. The flow-rate of the mobile phase was 8.0 ml min−1 . All effluents through the HPLC column were monitored by a DAD detector at 296 and 254 nm and manually collected into test tube. Seven preparative HPLC chromatograms were recorded and cascaded by Varian Star Workstation v6.41 as shown in Fig. 4.

2.9. 2D CCC × LC separation procedures 2.10. Qualitative and quantitative HPLC analysis The present flow programming 2D CCC × LC system configuration is shown as Fig. 1. Several time dependent system configurations have been designed according to the elution sequence of targeted components. A representative flow programming CCC × LC has been performed as following: CCC pumps delivered the desired upper or lower phase of selected two-phase solvent system for the first dimension of CCC separation. When the

Qualitative and quantitative analysis of the total extract and purified fragments isolated by the 2D CCC × HPLC were analyzed with a SGE Protocol C18 column (250 mm × 4.6 mm i.d., 5 ␮m, SGE Analytical Science, Melbourne, Australia). The mobile phase used was acetonitrile (A) and water (B) in a linear gradient mode as follows: A from 28% to 54% and B from 72% to 46% during 0–30 min.

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Fig. 2. CCC for the separation of ingredients from toad venom. (a) The classical CCC purification and (b) the flow programming CCC profile. Conditions: (i) (a, b) CCC conditions: solvent system: n-hexane–ethyl acetate–methanol–water (4:6:5:5, v/v/v/v); stationary phase: upper phase; mobile phase: lower phase; column temperature: 30 ◦ C; rotational speed: 900 r/min; initial stationary phase retention: 65%; detection wavelength: 296 nm; injection mode: injection after equilibrium; injection volume: 2 ml. (ii) (a) Classic CCC status: flow rate: 2.0 ml min−1 . Sample weight: 100, 120, 150 and 200 mg; (iii) (b) flow programming CCC status: flow rate of CCC pump (P1) was programed according to different fraction as follow: 0.2, 0.3, 0.4, 0.3, 0.4, 0.9 and 2.0 ml min−1 for Fr. 1–7, respectively. Sample weight: 100 mg.

The flow-rate of the mobile phase was 1.0 ml/min and the effluents were monitored at 296 nm by a DAD detector (Fig. 5). 2.11. Conventional preparative HPLC isolation In order to compare with conventional preparative HPLC isolation method, the crude toad venom extract was dissolved in methanol at a concentration of 20 mg ml−1 . The injected sample volume was chosen to be 1 ml and 5 ml, subjected to the same preparative HPLC column (Shim–pack VP-ODS, 250 mm × 20.0 mm i.d.) for isolation.Acetonitrile (A)/water (B) were employed as mobile phase, at gradient mode: A from 28% to 54% for 40 min, and run back to 28% at 60 min, as being applied in the isolation of fractions 2–7. The HPLC chromatograms were shown in Fig. 6. 2.12. Structural identification Identification of the compounds was carried out by electrospray ionization mass spectrometry (ESI-MS), one dimensional (1D) and two dimensional (2D) nuclear magnetic resonance spectra. Positive

ESI-MS were measured on an AB MD-SCIEX Advantage spectrometer. NMR experiments were carried out using a Bruker Avance 400 FT-NMR spectrometer with C5 D5 N (for isolated bufadienolides) or DMSO-d6 (for the new compound) as solvent and tetramethylsilane (TMS) as internal standard.

3. Results and discussion 3.1. Selection of solvent system and Classical CCC separation The selection of the two-phase solvent system is the most important step in performing CCC. The n-hexane–ethyl acetate–methanol–water system has been widely used as the two phase solvent system in CCC separation due to its wide polarity range by changing the ratio of the four solvents. In order to choose the optimal two-phase solvent system for CCC separation, the distribution ratio of the target compounds, bufadienolides, in different solvent volume ratios were determined (shown in Table 1). According to the KD values, the solvent system containing n-hexane–ethyl acetate–methanol–water (4:6:5:5, v:v:v:v) was selected. This

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Fig. 3. Evaluation of solid-phase enrichment and water-dilution interface between CCC and HPLC. The total weight of each CCC eluent fraction was firstly averaged by duplicated classic CCC separation (n = 3) for the calculation of recovery rate (n = 3). The weight percentage in total sample of each fraction was: 51.4 ± 3.7% (Fr. 1), 6.1 ± 1.3% (Fr. 2), 5.2 ± 0.6% (Fr. 3), 3.6 ± 0.8% (Fr. 4), 3.5 ± 0.7% (Fr. 5), 6.8 ± 0.3% (Fr. 6) and 21.9 ± 0.4% (Fr. 7). Then each CCC eluent was pumped at a flow rate of 0.3 ml min−1 to a dynamic mixer (or a tee), in which the eluent was diluted by water into different ratios, and loaded onto the pre-column under evaluation. (a) Column size selection: Three commercial available ODS pre-columns in different size, 15 mm × 21.2 mm i.d. (column bed volume, b.v.: 5.3 ml), 15 mm × 30.0 mm i.d. (b.v.: 10.6 ml) and 50 mm × 20.0 mm i.d. (b.v.: 15.7 ml), were evaluated. In addition, the system pressure was also recorded (*). (b) Dilution ratio evaluation for 15 mm × 30.0 mm i.d. column, in terms of recovery rate and system pressure (#). (c) Mixer comparison: The recovery rates on 15 mm × 30.0 mm i.d. column were compared, when a tee or a dynamic mixer was equipped to the post CCC column dilution interface. (d) Sample trapping capacity of 15 mm × 30.0 mm i.d. column in different CCC injection amount (100 and 200 mg). Evaluation condition: CCC injection amount, 100 mg for (a, b and c); dilution ratio, 1:4 for (a, c and d); 15 mm × 30.0 mm i.d. column for (b, c and d); dynamic mixer for (a, b and d).

system is simple, and the two phases reached equilibrium very quickly, which produced a good preliminary separation of toad venom. Considering that the CCC eluent will be captured onto a solid phase column under reversed-phase mode in the next isolation step, mobile phase with lower solvent strength should be adopted. Using the optimized solvent system, with the upper phase as stationary phase and lower one as mobile phase, the classical CCC separations have been performed as Fig. 2(a). In this case, chromatographic performance has been found to decrease with

increasing sample load on the CCC column, which was due to the loss of stationary phase. In details, only about 45% stationary phase could be retained till the isolation ending when more than 150 mg of crude toad venom extract was subjected to the CCC column, owing to the existence of polar constituents such as indole alkaloids in Fr. 1. This high stationary phase loss led to significant resolution decrease of Fr. 3, 4 and 5. However, seven major fractions could be well resolved when less than 120 mg of sample was loaded, as the initial and final stationary phase retention was about 65% and

Table 1 The distribution ratio values of target compounds in several solvent systems. Solvent system (H:E:M:W)a

4:6:4:6 4:6:5:5 4:6:6:4 4.5:5.5:4.5:5.5 4.5:5.5:5:5 5:5:3.5:6.5 5.5:4.5:3:7 6:3:2:5 6:4:3:7 a b

KD valuesb 2

3

4

6

7

8

9

10

11

12

0.32 0.13 0.05 0.12 0.08 0.15 0.21 0.07 0.10

0.21 0.07 0.03 0.07 0.04 0.07 0.07 0.03 0.04

0.56 0.23 0.08 0.21 0.12 0.25 0.26 0.12 0.16

0.77 0.26 0.09 0.27 0.15 0.39 0.47 0.21 0.30

0.68 0.22 0.08 0.23 0.12 0.35 0.43 0.19 0.27

1.48 0.43 0.13 0.49 0.25 0.82 1.00 0.44 0.66

2.22 0.64 0.19 0.75 0.37 1.31 1.63 0.72 1.09

3.56 0.93 0.27 1.24 0.57 2.55 3.36 1.53 2.68

7.97 1.81 0.49 2.73 1.15 6.44 7.21 3.46 7.33

7.48 1.83 0.53 2.74 1.18 6.46 7.17 3.73 7.69

H: n-hexane; E: ethyl acetate; M: methanol; W: water. The KD values of compounds 1 was close to zero, and that of compound 5 was unable to be determined accurately for its peak resolution was low.

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Fig. 4. Integrated CCC × reverse phase HPLC separation of methanol extract of toad venom. (i) The same CCC status as that of Fig. 2(b) was applied in the 1st dimensional flow-programing CCC isolation. (ii) The makeup pump (P2) was turned on and its flow rate was programed according to CCC fraction as follow: 1.2, 1.2, 1.2, 1.2, 1.2, 1.8 and 1.8 ml min−1 for Fr. 1–7, respectively, in order that the CCC eluent was diluted by water at ratio of 1:2–1:6. (iii) Condition of 2nd dimensional HPLC: Mobile phase system was methanol (A) or acetonitrile (B), and water (C) in a linear gradient mode. A:C from 5:95 to 60:40 for 30 min, and 60:40 to 100:0 during 30–50 min, and then the mobile phase was changed to B and C at a ratio of 28:72 and kept until 60 min, for the isolation of Fr. 1. B:C from 28:72 to 54:46 during 0–40 min, and run back to 28:72 when 60 min, for Fr. 2-7. (iv) Sample weight of isolated fractions: 1.0 mg (Fr. 1-1), 0.8 mg (Fr. 1-2), 2.0 mg (Fr. 2-1), 1.3 mg (Fr. 2-2), 2.0 mg (Fr. 3-1), 1.5 mg (Fr. 3-2), 1.9 mg (Fr. 3-3), 3.6 mg (Fr. 4-1), 6.1 mg (Fr. 5-1), 4.8 mg (Fr. 6-1), 3.5 mg (Fr. 7-1), 4.1 mg (Fr. 7-2).

60%, respectively. By optimizing the sample loading amount, high CCC chromatographic reproducibility (n > 3) was achievable, which makes the isolation reproducible and automatable.

3.2. Flow programming CCC In order to allow the target elution from first-dimension column to be transferred to and analyzed on the second-dimension column, flow programming CCC technology was employed to increase residence time in this integrated CCC × HPLC system. The flow programming scheme refers to flow reducing or pausing elution from the first-dimension column while a fraction is transferred to and analyzed on the second-dimension column. This somewhat alleviated the time constraints of the second-dimension, and provided good chances for separation of more comprehensive components. When the flow programming scheme was applied, the CCC chromatography fitted the demand of the 2nd dimensional HPLC isolation well, with increasing tR value and baseline peak width (Table 2). In addition, our results have demonstrated that the flow programming scheme CCC process is practicable and able to produce higher resolution than common CCC operation, which has also been confirmed by recent studies on similar technique of stopand-go CCC [18]. We found a slight increase of isolation resolution with decrease of CCC flow rate (Table 2), except for the peak of none-preserved fragment (Fr. 1). Separation efficiency impairment caused by axial diffusion of the peaks was not happened, which was a common problem in traditional chromatography at the time

of flow rate reduction. This mainly lies in the special operate manner of CCC, which uses two immiscible liquid phases without any solid support. The liquid stationary phase is held in place by centrifugal force while the mobile phase is pumped through it [19]. Sample keeps been partitioned between the two phases, when the CCC multilayer coil columns retain rotating. As the times of mass transfer process increase with reducing of flow rate, the effect of axial diffusion is counteracted and the theoretical plate number of the CCC column is likely to increase. 3.3. Evaluation of solid-phase enrichment and water-dilution interface between CCC and HPLC Solid-phase extraction (SPE) has been successfully used for online analytes enrichment in analytical HPLC analysis [20], preparative HPLC fractions post-collection concentration [21], and sample transition for 2D CCC × CCC [7]. Meanwhile, in order to capture the sample eluent from CCC, it is necessary to dilute the eluted solution before subjected to the holding column. In the present study, each CCC eluent fraction was used to evaluate the performance of the solid-phase enrichment and water-dilution interface. 3.3.1. Sorbent type and holding column size Since the 2nd dimensional HPLC is the main constraint on separation capacity and efficiency, the holding column should use the same sorbent type with that of the 2nd column. And in order to preserve separation efficiency, the sorbent bed of pre-column is usually 1/25–1/5 of the corresponding

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Fig. 5. HPLC analysis of crude extract and isolated fractions by flow programming CCC × HPLC. The analysis was performed on a SGE ProteCol C18 column (250 mm × 4.6 mm i.d., 5 ␮m) with a guard column (10 mm × 4.6 mm i.d., 5 ␮m). The mobile phase used was acetonitrile (A) and water (B) in a linear gradient mode as follows: A from 28% to 54% and B from 72% to 46% during 0–30 min. The flow-rate of the mobile phase was 1.0 ml min−1 and the effluents were monitored at 296 nm by a DAD detector. The column temperature was kept at 30 ◦ C. Compound purity: 99.9% (1), 99.7% (2), 90.6% (3), 99.9% (4), 77.0% (5), 99.9% (6), 90.4% (7), 99.9% (8), 52.0% (9), 99.9% (10), 99.3% (11), and 85.0% (12). (The relative contents in percentage were calculated with area normalization method.)

ratio, 15 mm × 21.2 mm i.d. (b.v.: 5.3 ml), 15 mm × 30.0 mm i.d. (b.v.: 10.6 ml), and 50 mm × 20.0 mm i.d. (b.v.: 15.7 ml), were evaluated. Fig. 3(a) shows that better recovery rate could be achieved when a larger holding column was used. But the difference between the 15 mm × 30.0 mm i.d. and the 50 mm × 20.0 mm i.d. holding

preparative column during conventional HPLC separation. Furthermore, owing to the demand of being connected to the high pressure HPLC flow path, the holding column should be pressure tolerable, which makes commercial available ODS pre-columns of preparative HPLC a good choice for this 2D CCC × HPLC system. In this work, three pre-column in different size and different diameter-height

Table 2 Chromatography comparison of classic and flow-programming CCC. Fr.

1 2 3 4 5 6 7

tR (min)

W (baseline, min)

Resolution (R)a

W1/2 (min)

Cb

Fb

C

F

C

F

C

F

68.54 74.63 87.00 98.88 109.81 133.12 186.98

68.46 93.62 164.09 232.30 286.16 351.34 404.45

9.09 9.08 12.03 10.29 13.81 22.66 44.99

27.68 47.06 50.08 61.16 48.83 30.70 42.52

5.91 4.92 6.52 5.41 7.26 8.49 18.83

20.13 35.24 27.68 37.75 23.92 10.32 16.11

– 1.13 2.16 1.99 1.73 2.96 3.94

– 0.91 2.24 2.08 1.75 3.81 4.02

2D flow programming CCC × HPLC system was developed. Initial exploration into preparative isolation, using online CCC × HPLC techniques. One-step preparative separation of 12 compounds from the crude extract of toad venom. a Resolution between the peak and that ahead of it. b C. classic CCC; F. flow-programming CCC.

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AU

7 6 5 4 3 2 1 0 AU 5

4

3

2

1

0 10

20

30

40 Minutes

Fig. 6. Conventional preparative HPLC isolation: the crude methanol extract of toad venom was dissolved in methanol at concentration of 20 mg ml−1 and subjected to preparative HPLC column (Shim–pack VP-ODS, 250 mm × 20.0 mm i.d.). Acetonitrile (A)/water (B) were employed as mobile phase, at gradient mode: A from 28% to 54% for 40 min, and run back to 28% when 60 min. The flow-rate of the mobile phase was 8.0 ml/min and the effluents were monitored at 296 and 254 nm by a DAD detector.

column was not significant, indicating that the column size of 10 ml is adequate to retain the analytes, when 100 mg of total crude extract was loaded onto the CCC system. 3.3.2. Diameter-height ratio, holding efficency and system pressure As far as the diameter-height ratio was concerned, the data in Fig. 3(a) suggested that the column’s holding efficiency was not mainly depended on column diameter and height. However, CCC is a medium-pressure preparative technique. In this study, the system pressure should be taken into account due to the use of the solid-phase holding interface, which may cause a rise in column pressure owing to the addition of water at the post-column stage. For preparative purposes, the larger diameter column employed in the solid-phase holding interface can produce lower back-pressure even with higher postcolumn dilution ratios, which will provide much better system tolerance. For this reason, the 15 mm × 30.0 mm i.d. holding column was selected for further study. 3.3.3. Sample retention ability, dilution ratio and sorbent mixing manner In this study, eluent from the 1st dimensional CCC chromatography was firstly mixed with water thoroughly at a ratio of 1:2–1:6, by a dynamic mixer. Fig. 3(b) shows that the fraction with less

retention ability (Fr. 1), owing to the existence of strong polar components, undergoes much greater sample loss when passing through the enrichment column. Fortunately, the recovery rate of more than 50% for Fr. 1 is still acceptable in preparative separation, while most of the fractions (Fr. 2–7) have been almost entirely captured onto the enrichment columns. To be pointed out, the system pressure increased when higher dilution ratio was adopted as expected, however, the pressure was still tolerable for the CCC system. To be highlighted, the dynamic mixer played an irreplaceably important role in this dilution process, because organic-solvent rich eluent from CCC could not mix well with water by common mixing device such as tee, which could result in the loss of most samples (Fig. 3(c)). 3.3.4. Sample holding capacity The sample holding capacity of the 15 mm × 30.0 mm i.d. precolumn was evaluated as shown in Fig. 3(d). Except for the fraction with less retention ability (Fr. 1), the CCC fractionalized sample (weighted range from 4.0 to 44.4 mg, which is suitable for a single preoperative HPLC isolation), could be captured by the column at a relatively high recovery rate (more than 80%). However, considering that lower sample quantity would bring better isolation resolution for both CCC and HPLC, the 1st dimensional CCC’s injection amount was reduced to 100 mg in the later study.

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3.3.5. Desorption Apart from the holding process, the analyte desorption is also an important step affecting the outcome of subsequent HPLC experiments. In the present work, considering the second HPLC separation, the trapped analytes were washed out of the solidphase holding column in the back-flush mode (Fig. 1) using the corresponding HPLC mobile phase, which was proved to achieve full desorption. 3.4. Integrated CCC × reverse phase HPLC separation of methanol extract of toad venom Fig. 4 illustrates the integrated flow programming-CCC × HPLC separation of the methanol extract of toad venom. The crude extract was firstly isolated by CCC, after which the eluent was then concentrated online into the solid-phase holding column and then transferred into HPLC for further high performance separation, through the valve-switching technique. By comparing the CCC chromatography in Fig. 4 and Fig. 2(b), no obvious differences were observed between the integrated system and the common CCC. This suggested that the solid-phase enrichment and water-dilution interface did not affect the chromatographic behavior of 1st dimensional CCC. On the other hand, based on the facts that front peak, peak tailing or peak broadening phenomena did not appear in the chromatography of the 2nd dimensional HPLC, the water-dilution interface prior to the HPLC system was considered to be capable of eliminating the solvent effect caused by the organic eluent of CCC. After about half a day’s separation, 12 fractions were directly produced according to the flow programming-CCC × HPLC elution profile, from 100 mg of crude methanol extract using the proposed column-switching CCC × HPLC protocol. HPLC analysis showed that from the 12 fractions, 10 compounds were purified with a purity of 85.0–99.9% in a single-step separation, except for Fr. 2-2 and Fr. 5-1, whose major component’s purity was 77.0% (5, de-Oacetylcinobufotalin) and 52.0% (9, cinobufotalin) (Fig. 5). 3.5. Conventional preparative HPLC isolation As shown in Fig. 6, the separation of the crude methanol extract of toad venom by using conventional preparative HPLC was not successful, although the analytical HPLC chromatography was quite good (Fig. 5). And the case was even worse when more sample solution was injected. Owing to the high lipophilic property, the solubility in water or water containing methanol was so poor that the toad venom extract had to be dissolved in organic solvents, whose solvent effects exhibited negative impact on resolution and in the end resulted in the failure of preparative isolation. However, as mentioned above, the solvent effects could be well eliminated applying the solid-phase enrichment and water-dilution interface. 3.6. Identification of isolated compounds Among the isolated compounds, expect for an unidentified indole alkaloid (compound 1, giving a quasi-molecular ion peak at m/z 433.2197 ([M + H]+ , calculated for C20 H25 N4 O7 , 433.2132) by HR-ESI-MS), 11 cardiac steroids, were determined by several spectral analyses. Chemical structures of those compounds at purity of more than 85%, were identified as deacetylbufotalin (2), gamabufotalin (3), arenobufagin (4), telocinobufagin (6), deacetylcinobufagin (7), bufotalin (8), bufalin (10), cinobufagin (11) and resibufogenin (12) by ESI-MS and 1 H, 13 C-NMR data (Supporting Information), which were consistent with the authentic samples or literature data [16,17,22]. The two impure compounds (5 and 9) were identified by HPLC-DAD-MS with authentic samples.

4. Conclusions In this study, we have developed a new flow programming 2D CCC × LC system for preparative separation of 12 compounds from the extract of toad venom. In summary, the online columnswitching CCC × HPLC system is capable of analytes concentration prior to further HPLC separation. This method provided an attractive solution to improve the separating power of the CCC technique. Moreover, the system provided simpler and more efficient separation without using other extra steps to remove organic solvents when compared with conventional off-line CCC-HPLC separation. Furthermore, this integrated separation system provided a versatile separation for multiple-target components compared with the reported on-line CCC × LC system developed recently [14], which was suitable for one-target separation. Unlike the recently reported CCC × LC system [13], in which the 2nd column was packed with a macro porous resin column for the purpose of desalination and did not provide additional peak capacity, the flow programming CCC × LC system is a two-dimensional (2D) chromatography in strict sense. However, the on-line CCC × LC system reported here is based on the classic CCC apparatus. As far as the recently developed HPCCC is concerned, several technical problems will be involved. Firstly, adequate isolation resolution can be provided by HPCCC solely, so the necessity of coupling CCC and LC will be challenged. We hold the view that, owing to the different separation mechanisms between HPCCC and LC, the integrated 2D CCC × LC system is still valuable, especially when those complicated samples were under isolation. Secondly, when HPCCC was used, where separations could only take minutes, it is difficult to couple with preparative HPLC. The HPLC isolation time should be shortened by adjusting mobile phase solvent to adapt the demand of HPCCC. On the other hand, preparative HPLC isolation is always difficult to separate highly lipophilic samples dissolved in organic solvents, whose solvent effects always result in the failure of preparative isolation. CCC technique had been widely applied in isolations of all sorts of components, including lipophilic compounds, by dissolving samples in two-phase solvent for further injection. The use of solidphase interface between the CCC and HPLC solved the problem of solvent effects by diluting the CCC eluent with water, enabling the preparative HPLC isolation of hydrophobic compounds with higher resolution. In conclusion, to the best of our knowledge, this twodimensional (2D) chromatography presented in this manuscript is the first exploration into the preparative isolation using online CCC × HPLC techniques. In conjunction with a solid-phase holding interface, the flow programming 2D CCC × HPLC system provided a significant orthogonality improvement, which is very efficient for the separation of toad venom and successfully separate 12 compounds, which is not revealed by traditional approach. The impact of this novel separation platform may even go beyond isolating comprehensive multi-component natural products, potentially benefitting the development of separation science. Acknowledgments The project was supported by the National Natural Science Foundation of China (No. 81102333 and No. 81273400), as well as Fundamental Research Funds for the Central Universities (No. 2010121108). The authors would also like to acknowledge the financial supports from Fujian Natural Science Foundation for Distinguished Young Scholars (No. 2012J06020) and Natural Science Foundation of Fujian Province of China (No. 2011J05101).

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.01.029. References [1] [2] [3] [4] [5] [6]

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Two-dimensional countercurrent chromatography × high performance liquid chromatography for preparative isolation of toad venom.

In this work, a new on-line two-dimensional chromatography coupling of flow programming counter-current chromatography and high-performance liquid chr...
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