Online polar two phase countercurrent chromatography × high performance liquid chromatography for preparative isolation of polar polyphenols from tea extract in a single step

Accepted Manuscript Title: Online polar two phase countercurrent chromatography×high performance liquid chromatography for preparative isolation of polar polyphenols from tea extract in a single step Author: Wei-Bin Chen Shu-Qi Li Long-Jiang Chen Mei-Juan Fang Quan-Cheng Chen Zhen Wu Yun-Long Wu Ying-Kun Qiu PII: DOI: Reference:

S1570-0232(15)30049-0 http://dx.doi.org/doi:10.1016/j.jchromb.2015.06.011 CHROMB 19481

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

16-12-2014 3-4-2015 12-6-2015

Please cite this article as: Wei-Bin Chen, Shu-Qi Li, Long-Jiang Chen, Mei-Juan Fang, Quan-Cheng Chen, Zhen Wu, Yun-Long Wu, Ying-Kun Qiu, Online polar two phase countercurrent chromatographytimeshigh performance liquid chromatography for preparative isolation of polar polyphenols from tea extract in a single step, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.06.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Online polar two phase countercurrent chromatography × high performance liquid chromatography for preparative isolation of polar polyphenols from tea extract in a single step by Wei-Bin Chen a, Shu-Qi Li b, Long-Jiang Chen a, Mei-Juan Fang a, Quan-Cheng Chen a, Zhen Wu a, Yun-Long Wu a, * , Ying-Kun Qiu a, *

a

School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, South Xiang-An Road, Xiamen, 361102, China

b

College of Chemistry and Chemical Engineering, Xiamen University, South Si-Ming Road, Xiamen, 361005, China

Submitted to Journal of Chromatography B

* Corresponding author: Tel.: +86-592-2189868; Fax: +86-592-2189868; E-mail address: [email protected] (Ying-Kun Qiu) and [email protected] (Yun-Long Highlights ?

Wu) Highlights

 2D CCC × HPLC system was developed for preparative separation of polar compounds.  10 pure compounds were one-step preparative separated from crude tea water extract.  Environmental friendly aqueous two phase CCC solvent system was used. Abstract

Herein we report an on-line two-dimensional system constructed by counter-current chromatography (CCC) coupling with preparative high-performance liquid chromatography (prep-HPLC) for the separation and purification of polar natural products. The CCC was used as the first dimensional isolation column, where an environmental friendly polar two-phase solvent system of isopropanol and 16% sodium chloride aqueous solution (1:1.2, v/v) was introduced for low toxicity and favorable resolution. In addition, by applying the stop-and-go flow technique, effluents pre-fractionated by CCC was further purified by a preparative column packed with octadecyl silane (ODS) as the second dimension. The interface between the two dimensions was comprised of a 6-port switching valve and an electronically controlled 2-position 10-port switching valve connected with two equivalent holding columns. To be highlighted here, this rationally designed interface for the purpose of smooth desalination, absorption and desorption, successfully solved the solvent compatibility problem between the two dimensional separation systems. The present integrated system was successfully applied in a one-step preparative separation and identification of 10 pure compounds from the water extracts of Tieguanyin tea (Chinese oolong tea). In short, all the results demonstrated that the on-line 2D CCC × LC method is an efficient and green approach for harvesting polar targets in a single step, which showed great promise in drug discovery.

Keywords:

Counter-current

chromatography;

Two-dimensional

liquid

chromatography;

Tieguanyin tea; Polar two-phase solvent system; Tea polyphenols

1. Introduction Pure natural components from medicinal plants are the main sources of commercial pharmaceutical products and have been driving drug discovery over the past few centuries. As most target compounds are present in complicated mixture at low concentrations, numerous effects have been devoted to develop efficient, economical and environmental-friendly isolation as well as purification methods. As a typical example, high-performance liquid chromatography (HPLC) was developed for resolving the complex natural product extracts into pure components several years and has become a conventional preparative method. Besides to HPLC, counter-current chromatography (CCC), a unique liquid–liquid partition chromatography without any solid matrix, was demonstrated to eliminate irreversible adsorption of samples on solid support in the conventional chromatographic column [1] and was suitable for large-scale isolation, which made it widely accepted in preparative separation of many kinds of natural products [2]. However, the peaks of two or more components would overlap in a CCC chromatogram if they have similar polarities, the overlapping components required further isolation, which rendered multi-dimensional chromatography with sufficient resolving power for the separation of components in many complex natural samples [3]. Multidimensional chromatography was based on the combination of different techniques involving a variety of separation mechanisms. Typically, two-dimensional liquid chromatography (2D-LC) was the most popular method in multidimensional chromatography. In addition, an integrated counter current chromatography (2D CCC × CCC) [4] has been demonstrated to show higher resolution and larger peak capacity

compared with single CCC process. Recently, coupling of CCC and preparative HPLC was successfully applied for galactolipids [5] and phthalides [6] isolation, which represented off-line and heart-cutting combination of CCC and HPLC, respectively. These successful reports have demonstrated that 2D chromatography has shown a great potential to improve separation capacity of natural products, but it remains a challenge for achieving optimized additional peak capacity and separation power. To address this issue, we recently reported a new hybrid two-dimensional chromatography by on-line coupling of CCC and LC for preparative separation of complex natural products, where CCC column was applied as the first dimension and preparative HPLC column as the second one, interfaced by a makeup pump and a 10-port switching valve in conjunction with two reversed phase holding columns [7]. This interface enabled the transfer of 1D effluents in on-line mode, which avoided sample contamination, deterioration and personal error, thus made our on-line 2D CCC × HPLC separation system an ideal mode for the analysis of complex samples. Similar to classical on-line 2D-LC, the 2D CCC × HPLC system required that the second-dimensional analysis be completed during the time needed to collect the fraction, transfer and analyze it, and restore the column to the initial conditions of the analysis. Therefore, flow programming or stop-and-go technique [8] was applied to solve the problem of operation time limitation, especially during the multiple targeting components separation process. This technique referred to reduce or to pause elution from the first-dimension CCC column while a fraction is transferred to and analyzed on the second-dimension HPLC column, which alleviated the time constraints of the second dimension. Apart from the above conditions, the selection of separation system was crucial for successful purification of target product in CCC, which was usually based on the characteristics of the target product, especially the polarity [9]. In addition to the conventional multi-component organic/aqueous (MCO/Water) solvent systems, such as hexane–ethyl acetate–methanol–water

(HEMWat), chloroform–methanol–water (ChMWat), and ethyl acetate–n-butanol–water (EBWat), a promising organic/salt polar two-phase system [10, 11] have been presented to separate several natural products such as flavones. These biphasic systems had the advantages of higher polarity compared with conventional organic/aqueous systems and lower cost compared with aqueous polymer two-phase systems, as well as the lower environmental toxicity, and its principle could be attributed to salting-out, a very common but not simple physical phenomenon extensively exploited by biopolymer science, ion-exchange chromatography and counter-current separations. Furthermore, this novel approach has been demonstrated to be favorable for the separation of polar natural products. However, the salting-out process required an extra step, desalting the salt containing effluent, which could induce the blocking of the 2nd dimensional HPLC column. Rationally designed elution program which led to complete desalination should be applied on the interface. This work applied the newly developed 2D CCC × LC protocol based on our recent publication (Fig. 1) in the separation of the total tea extract (TTE) of Tieguanyin tea (Camellia sinensis (L.) O. Kuntze), a commonly consumed beverage associated with many health benefits including the prevention of cancer and heart disease [12]. Tea polyphenols, such as flavanols and flavonols which account for 30% of leaf dry weight, were responsible for those beneficial health effects [13]. A great number of papers described HPLC basic methods for the simultaneous separation of several tea polyphenols with the aim to have short separation time, efficient conditions of analyte extraction, detection and characterization [14, 15]. Whilst different groups of tea polyphenols were isolated, a broad range of species within this class could not be preparatively separated in single step isolation by HPLC. Therefore the on-line CCC×LC separation system was developed for the simultaneous separation of the TTP extracts. As a result, 10 highly represented components in tea samples were isolated, and identified by UV, MS, as well as 1D and 2D NMR. Although numbers of chromatographic methods have been used for

the separation of tea polyphenols in tea extract, to the best of our knowledge, this is the first study to demonstrate the application of a 2D CCC × LC separation system in a single step for fast and easy isolation as well as purification of targeting ingredients from TTE extracts.

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. Tieguanyin tea was commercially available and was purchased from Anxi County of Fujian province.

2.2. Instrumentation For the first dimensional CCC system, TBE-300 high-speed CCC (Tauto Biotech. Co., Ltd, Shanghai, China), equipped with three multilayer coil separation column connected in series (i.d. of the tubing is 1.5 mm, total volume is 300 ml) and a 20 ml sample loop, was employed as the CCC instrument in this study. The revolution radius was 5 cm, and the β values of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal. A speed controller was applied to regulate the revolution speed of the apparatus in the range between 0 and 1000 rpm. In addition, a SP930D gradient pump (Young Lin Instrument Co. Ltd., Korea) and a ProStar 218 photodiode array detector (Varian Inc. Corporate, Santa Clara, USA) were equipped to construct an integrated 1D system. The data were recorded with a Varian Star Workstation 6.41 (Varian Inc. Corporate, Santa Clara, USA). For the second dimensional LC system, a Varian binary gradient LC system (Varian Inc. Corporate, Santa Clara, USA) contained two solvent deliver modules (PrepStar 218), a photodiode array detector (ProStar 335) and a fraction collector (ProStar 704), was applied. Preparative-HPLC control and data acquisition were also performed by Varian Star Workstation.

Last but not least, an Agilent 1100 system, which was equipped with a G1379A degasser, a G1311A QuatPump, a G1367A Wpals, a G1315B diode assay detector (DAD), and an Agilent ChemStation for LC, was further applied for the HPLC analysis of the total tea polyphenols extract and the isolated fractions.

2.3. Sample preparation Tieguanyin tea (2.0 kg) was extracted with water thrice and concentrated under reduced pressure at 40 ˚C. The concentrate was subjected to a Diaion HP20 macro-porous resin (Mitsubishi Chem, Tokyo, Japan) column (600 mm × 65 mm i.d.), and eluted with H2O and 95% EtOH 6000 ml respectively. 95% EtOH eluent was collected and concentrated to afford TTE for further separation.

2.4. Selection of CCC solvent system The two-phase solvent system was selected mainly according to the partition coefficient (KD) of each target component, settling time and phase ratio of a system. Several concentrations of sodium chloride solution (8%, 10%, 13%, 14%, 15%, 16%, 17%, 18%) were placed in test tubes separately, mixed with isopropanol at the rate of 1:1 or 1:1.2 (v/v, water/organic solvent). After shaking them vigorously, test tubes were standing still to record settling time and phase ratio. To get the KD values, 4 mg of the TTE extract was dissolved into equal volumes (1 mL) of aqueous phase (lower phase) and organic phase (upper phase) of the thoroughly equilibrated two phase solvent system in a 15 mL bottle. After the equilibration was established, both the solutions of upper phase and lower phase were directly analyzed by HPLC and the peak area of each component in the upper phase and lower phase were recorded as A1 and A2, respectively. The partition coefficients were then calculated by the following equation: KD=A1/A2.

2.5. Preparation of two-phase solvent system and sample solutions Hydrophilic organic/salt-containing polar two-phase system was prepared by thoroughly mixing the desired amount of isopropanol with sodium chloride solutions in a separatory funnel

at room temperature, allowing the two clear phases to form. The two phases were separated shortly before use and degassed in an ultrasonic water bath for 30 min. The sample solution for CCC separation was prepared by dissolving certain amount of the water extract into 10 mL of the two-phase solvent (1:1, v/v) before use.

2.6. Conventional CCC isolation of sample The column was first filled with the upper phase as stationary phase, and then the apparatus was rotated at 900 rpm (reversed mode), and the lower phase as mobile phase was pumped through the column at a flow-rate of 1 mL/min from the head end of the column to the tail end. Retention rate of stationary phase was measured by the excurrent stationary phase from tail end. When a hydrodynamic equilibrium was established in the column and the mobile phase started emerging in the effluent, 10 mL sample solution was injected through the injection valve. Chromatogram of 274 nm and 310 nm were recorded as Fig. 2(a).

2.7. Interface between CCC and prep-HPLC A 2-position 6-port switching valve (Valve A, Valco Cheminert EDU6UW, VICI, Schenkon, Switzerland) was equipped at the post-end of the CCC to bypass the CCC eluents without Tieguanyin tea component. A medium pressure pump (C-601, Büchi Labortechnik AG, Flawil, Switzerland) was used for the addition of water as makeup fluid, which was passed 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 a 2-position 10-port switching valve (Valve B, Valco Cheminert EDU10UW, VICI, Schenkon, Switzerland) and two equivalent holding columns. After the first CCC peak emerged, the ON-OFF status of CCC pump and the flow-path of CCC eluents were controlled by the position of Valve A as follow: CCC pump was kept turning ON and the CCC eluents were subjected to the dynamic mixer for dilution when Valve A was at position A; CCC pump was switched off and only water from makeup pump was passing through the holding column for the purpose of

desalination at position B. After desalination completed, the trapped analytes were washed out of the holding column in the back-flush mode. The sketch map was shown in Fig. 1.

Insert Fig. 1 here.

2.8. On-line 2D CCC×HPLC separation of sample A schematic diagram of novel stop-and-go 2D CCC × LC system configuration was shown as Fig. 1. Several time dependent system configurations have been designed according to the different peak width of conventional CCC chromatogram of the sample (Fig. 2 (b)). A representative stop-and-go CCC × LC was 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 first CCC peak started to emerge, the 6-port valve (Valve A) was switched to position A and the effluent was firstly diluted at a certain ratio with water and directed to the first holding column (15 mm × 30 mm i.d.). After all the effluent of the first CCC peak has been eluted and adsorbed on the first holding column, the 6-port valve was switched to position B and the salt remained in the holding column was washed out with water by makeup pump. Meanwhile, the CCC pump switched off while the CCC coil was kept rotating. After desalination completed, the 6-port valve (Valve A) and the 2-position 10-port switching valve (Valve B) were switched simultaneously. The HPLC pumps began to deliver the 2nd dimensional mobile phase to desorb and isolate the first CCC peak into its constituent components, while the CCC pump turned on to deliver the second CCC peak onto the second holding column. Once the second CCC peak was adsorbed completely, the 6-port valve was switched again for desalination, until the first CCC peak’s HPLC isolation completed. Then, both the 6-port valve and the 10-port valve were triggered back simultaneously for the next CCC fraction trapping so that two trapping columns could be used alternatively.

A reversed-phase preparative column (Cosmosil ODS- PAQ column, 250 mm × 20 mm i.d., 5 m, Nakalai Tesque Co. Ltd., Kyoto Japan) was used as the 2nd dimensional stationary phase. The mobile phase was methanol (A) and water (B) in a gradient elution mode as follows: A:B from 20:80 to 80:20 for 50 min, and 80:20 to 20:80 during 50 ~ 51 min, then kept 20:80 until 60 min. The flow-rate of the mobile phase was 8.0 mL/min. All effluents through the HPLC column were monitored by a DAD detector at 274 and 310 nm and collected into test tubes by fraction collector. After weighted, these samples were analyzed by HPLC. Eight preparative HPLC chromatograms were recorded and cascaded by Varian Star Workstation v6.41 as shown in Fig. 3.

2.9. HPLC analysis of the total extract and purified fragments Analyses of the total extract and purified fragments isolated by the 2D CCC × HPLC were performed on a Cosmosil ODS- PAQ column (250 mm × 4.6 mm i.d., 5 m, Nakalai Tesque Co. Ltd., Kyoto, Japan). The mobile phase was methanol (A) and water (B) according to the following gradient program (v/v): 0-25 min: 20-80% A, 25-27 min: 80-20% A, 27-30 min: 20% A. The flow-rate of the mobile phase was 1.0 mL/min and the effluents were monitored at 274 nm by a DAD detector (Fig. 4).

2.10. Structure identification of components 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 a Thermo Q Exactive LC-MS/MS spectrometer. NMR experiments were carried out using a Bruker Avance III 600 FT-NMR spectrometer with DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard.

3. Results and discussion 3.1.

HPLC analysis of the total tea extract The TTE of Tieguanyin tea was used as model complex mixture to evaluate the

performance of the on-line CCC × HPLC system. According to analysis HPLC chromatogram shown in Fig. 4, caffeine (1), EGCG (3) were the major component present in the water extract, and epicatechin (2), ECG (5), 4G-α-D-glucopyranosyl-rutin (7) also had relatively high contents. Other components were in low concentration or poorly separated.

3.2.

Solvent system selection for 1st dimensional CCC seperation The selection of solvent system for the target compound was the most important step in

CCC. As components such as catechins and flavonoid glycosides are strong polar compounds, which are soluble in water or other polar solvents such as methanol and ethanol, polar two-phase solvent system was selected for evaluation. In addition, the partition coefficient (KD) value of main components was carefully measured using the simple test tube method [16 ]. The organic–aqueous two-phase solvent system examined in the present study was composed of isopropanol or methanol or ethanol/ sodium chloride solution at several desired composition ratio according to the method by S. C. Li et al. [1]. Table 1 illustrated the change of KD values of main components at different volume ratios of the above solvent system. By adding sodium chloride in water above 15%, the isopropanol/sodium chloride solution mixture was separated into two immiscible polar phases, but no layered solution was observed at all concentration of methanol or ethanol/sodium chloride. As the content of sodium chloride increased, the KD value of each component was increased. Apart from an appropriate KD value, satisfactory retention of the stationary phase in the column was also necessary, because higher retention of the stationary phase would lead to better peak resolution. The amount of stationary phase retained in the column was highly correlated with the settling time of the two phases in a test tube [17]. Thus measurement of the settling time of the two-phase solvent system was used for the selection of

solvent system. System 4~6 listed in Table 1 provided satisfactory settling time less than 40 s, as well as suitable VU/L value below 2:1, which would avoid wastage of organic solvent [9]. According to the equation of VR = VC [1 + (KD – 1) Sf] (VR: retention volume, VC: total column capacity, Sf: stationary phase retention ratio), solvent systems 4 were selected for further evaluation. Because stationary phase retention ratio (Sf) of more than 50% is always required for a classic CCC separation, while the KD values of some components in system 5, 6 were too large to complete CCC isolation in an acceptable time.

Insert Table 1 here.

3.3.

Optimization of CCC conditions

Although the selection of the two-phase system was important, the flow-rate of the mobile phase, rotation mode and revolution speed also played critical roles in the separation process, especially with regard to retention of the stationary phase. As shown in Table 2, the lower phase was chosen as the mobile phase and was introduced through the head end toward the tail end of the column under reversed mode, which provided the system relatively high retention of the stationary phase. It also prevented trapping air bubbles in the flow cell of the detector by introducing the effluent from the lower end of the cell [17]. Different flow rate (1.0 and 2.0 mL/min) of the mobile phase and different revolution speed (500 and 900 rpm) of the selected system were also examined. High rotary speed could also increase the retention of the stationary phase. The experimental results also clearly showed that high flow rate was unfavorable to the retention of the stationary phase. Slow flow speed, with higher Sf value, could produce a good separation, along with more separation time. However, in this CCC × HPLC system, the extended separation time of 1st dimensional CCC was favor to meet the separation time of 2nd dimensional HPLC.

The isopropanol/sodium chloride aqueous solution system had a small density difference between its two phases, which made it difficult to retain an acceptable amount of stationary phase in hydrodynamic CCC columns. The hydrostatic CCC columns (CPC) had been reported suitable for the small density difference solvent system [18], as it allowed running the CPC columns at high rotor rotation speed without excessive pressure for the rotating seals. However, as the data shown in Table 2, the ideal CCC stationary phase retention ratio of 50% could also approach (about 46%), by using common hydrodynamic CCC at an optimal condition as following: lower phase used as mobile phase and pumped through the column under reversed mode at flow rate of 1 mL/min, with resolution speed of 900 rpm. In conclusion, the isopropanol-16% sodium chloride solution (1:1.2, v/v) was chosen as optimal solvent system under an optimal CCC condition, with a comprehensive consideration of both KD value and retention rate of stationary phase.

Insert Table 2 here.

3.4.

Classical CCC separation and stop-and-go technique Using the optimized CCC conditions, 150 mg, 500 mg and 1.0 g water extract of

Tieguanyin tea were subjected to the CCC column respectively. Fig. 2(a) showed a typical separation program. Clearly, chromatographic performance decreased with increasing sample load on the CCC column. 1.0 g of the sample resulted in a low resolution, owing to the loss of stationary phase. Thus 500 mg of sample amount was selected in consideration of sufficient amount of compounds needed to be analyzed by HPLC and identified by NMR. The chromatogram was fractionated into fractions 1–8. In the integrated 2D system, the second dimensional preparative HPLC separation time may longer than the CCC peak width. In order to allow fractions from first-dimension column to be

transferred to and analyzed on the second-dimension column, stop-and-go technique (Fig. 2(b)), instead of flow programming, was employed on the 1D chromatography to increase residence time in this integrated CCC × LC system. The flow programming scheme applied in our previous study referred to flow-rate changing of 1D CCC mobile phase while a fraction was analyzed on the 2D column, provided good chances for separation of more comprehensive components in the 2D system. To be pointed out here, the determination of flow-rate program might need repeated CCC experiments. On the other hand, due to the usage of the polar two phase solvent system in CCC, it’s necessary to execute process of desalination. It was more convenient to fulfill desalination using stop-and-go technique. In this study, the continuous CCC effluent was cut according to the CCC peak, by switching the 6-port valve. The first dimensional CCC separation was paused while the eluted CCC peak was isolated on the second dimensional preparative HPLC column. After analysis of the previous fraction finished, the CCC pump restarted and the next CCC fraction was then enriched onto the other holding column. Finally, the flow-rate of 1 mL/min was not high and the peak width of most CCC fractions was close to the separation time of the 2nd dimensional HPLC. When the stop-and-go technique was applied, both CCC and HPLC isolation could be well done.

Insert Fig. 2 here.

3.5.

Construction of the CCC‒HPLC interface and desalination project A newly developed solid-phase holding interface [7] was applied with an improvement to

desalt the 1st dimensional effluents. The interface between CCC and HPLC can not only contributed to the adsorption of components on trapping columns, but also removed the salt and residual solvent. After overall consideration of separation efficiency, pressure tolerance and sample adsorption capacity, two commercial available 15 mm × 30.0 mm i.d. ODS pre-columns

of preparative HPLC were selected as holding columns in this study. Owing to the existence of strong polar components, the fraction would undergo a great sample loss when passing through the holding column without any aqueous dilution. As shown in Table 3, the sample retention ability enhanced with the dilution radio in the range of 1:1~1:4 and 1:3 was finally chosen in consideration of the optimal system pressure. By taking the advantages of this novel design, most salts in the effluents of the components from CCC peak would be removed by water dilution during the holding column absorption process. In addition, an extra water elution of the holding column was set, by switching the 6-port valve to position B (Fig. 1), to ensure complete desalination and prevent crystallization of salt on the HPLC column.

Insert Table 3 here.

3.6.

Application of on-line CCC×LC separation Based on the above experiments, we constructed an on-line CCC×LC separation system.

The TTE was firstly isolated by CCC, after which the CCC eluent was diluted 1:3 by water pumped via make up pump and concentrated on-line into one of the solid-phase holding column. Once the enrichment completed, the 2-position 6-port valve was switched and CCC pump was stopped while the holding column was washed with water. Then, both the 6-port valve and the 10-port valve were switched. The sample trapped on the holding column was eluted to the preparative HPLC column for further second dimension separations. Through the valve-switching technique, 8 fractions were continuously prepared in turns (Fig. 3).

Insert Fig. 3 here.

After 12 hours’ separation, 10 pure compounds were obtained from 8 fractions at a purity of more than 90% in a single-step separation, weighted from 1.2 ~ 12.0 mg. Compounds 1 ~ 10 were isolated in overall yield of 0.42%, 0.52%, 1.22%, 0.24%, 0.34%, 0.42%, 2.4%, 0.46%, 1.30% and 0.52%, with HPLC purity of 97.4%, 90.7%, 90.6%, 99.9%, 99.9%, 99.9%, 99.9%, 99.9%, 94.7%, and 99.9%, respectively. By using this tandem CCC×HPLC system, components that were poor resolved in HPLC, such as compounds 8 and 9, were also obtained with high purities (Fig. 4).

Insert Fig. 4 here.

3.7.

Identification of isolated chemical components The chemical structures of isolated components were further identified by ESI-MS,

1

H-NMR, and 13C-NMR. Chemical structures of those compounds were identified as caffeine (1)

[19], epicatechin (2) [20], EGCG (3) [21], 3''-O-methyl EGCG (4) [22], ECG (5) [21], myricetin 3-O-β-D-glucopyranoside (6) [ 23 ], 4G-α-D-glucopyranosyl-rutin (7) [ 24 ], quercetin 3-O-β-D-glucopyranoside

(8)

[ 25 ],

kaempferol

3-O-[β-D-glucopyranosyl-(1→4)]

[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (9) [26 ] and kaempferol-3-O-rutinoside (10) [25] (Supporting Information), which were consistent with the authentic samples or literature data.

4. Conclusion An on-line coupling of CCC and HPLC system was constructed for one-step separation of complex samples. The system provided simpler and more efficient separation without using other extra steps to remove organic solvents and salt when compared with conventional off-line CCC-HPLC separation. In conjunction with a solid-phase holding interface, the stop-and-go 2D

CCC × HPLC system provided a significant orthogonality improvement, which was very efficient for the separation of polar compounds. Meanwhile, the incompatibility of solvent system in two dimensions can also be resolved so that one-step isolation was finally achieved. Furthermore, comparing to traditional separation system, less time consuming as well as enhanced isolation efficiency was observed. Considering that less organic solvent was used in our system, we suggested that the feature of eco-friendly chromatography would gave a promising practical potential in future. In conclusion, to the best of our knowledge, our results have demonstrated for the first time that the on-line hybrid 2D polar two phase CCC×HPLC presented in this manuscript provided a remarkable separation power on polar targeting components from complex natural products such as tea extracts in a single step, which might widely benefit the development of separation science.

Acknowledgment The project was supported by the National Natural Science Foundation of China (No. 81302652, No. 81102332, and No. 21303145). The authors would also like to acknowledge the financial supports from Natural Science Foundation of Fujian Province of China (No. 2014J01063) and the Fundamental Research Funds for the Central Universities (No. 2013121037). This research was also financially supported by Xiamen science and technology project (Grant No. 3502Z20123015). Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jchromb.2015.06.011 Appendix B [{(Appendix A)}] Supplementary data The following are Supplementary data to this article: mmc1

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Figure Captions

Fig. 1. Scheme for stop-and-go CCC × preparative HPLC system

Fig. 2. CCC for the separation of water extract from Tieguanyin tea. (a) The classical CCC purification and (b) the stop-and-go CCC profile. (i) CCC conditions: (a, b) solvent system:

isopropanol and 16% sodium chloride aqueous solution (1:1.2, v/v); stationary phase: upper phase; mobile phase: lower phase; column temperature: 30 ˚C; rotational speed: 900 r/min; initial stationary phase retention: 46 %; detection wavelength: 274 nm; injection mode: injection after equilibrium; injection volume: 10 ml; flow rate: 1.0 ml/min. (ii) Sample weight: (a) 150, 500, and 1000 mg; (b) 500 mg. (iii) (b) Valve switching and CCC stop-and-go program: The 2-position 6-port switching valve (Valve A) was at position A and the CCC pump kept turned ON when a CCC peak fraction was eluted. Once the elution of the CCC peak finished, the 6-port valve was switched to position B to stop the CCC pump and to begin with the desalination process. Then the two valves (A and B) were triggered back simultaneously after desalination. (iv) (b) Desalination time: One minute, if the CCC peak fraction width was more than 60 minutes; otherwise, desalination process was lasted until the completion of the 2nd HPLC isolation, such as Fr. 3.

Fig. 3. Integrated stop-and-go CCC × reverse phase preparative HPLC separation of water extract from Tieguanyin tea. (i). The same CCC protocol as that of Fig. 2 (b) was applied in the 1st dimensional stop-and-go CCC isolation. (ii). The makeup pump was kept turned on at flow rate of 3 ml/min after the first CCC peak emerged, in order that the CCC eluent was diluted by water at ratio of 1:3. (iii). Condition of 2nd dimensional HPLC: Mobile phase system was methanol (A) and water (B) in a linear gradient mode. A:B from 20:80 to 80:20 for 50 min, and 80:20 to 20:80 during 50 ~ 51 min, then kept 20:80 until 60 min. (iv). Sample weight of isolated fractions: 1.3 mg (Fr. 3-1), 1.6 mg (Fr. 3-2), 0.5 mg (Fr. 3-3), 0.6 mg (Fr. 3-4), 12.0 mg (Fr. 3-5), 0.8 mg (Fr. 4-1), 2.1 mg (Fr. 4-2), 0.9 mg (Fr. 4-3), 1.0 mg (Fr. 4-4), 1.0 mg (Fr. 4-5), 6.5 mg (Fr. 4-6), 1.7 mg (Fr. 4-7), 2.6 mg (Fr. 5-1), 0.5 mg (Fr. 5-2), 1.3 mg (Fr. 5-3), 2.6 mg (Fr. 5-4), 1.3 mg (Fr. 5-5), 0.7 mg (Fr. 5-6), 0.4 mg (Fr. 5-7), 0.7 mg (Fr. 5-8), 3.7 mg (Fr. 6-1), 0.6 mg (Fr. 6-2), 1.0 mg (Fr. 6-3), 0.6 mg (Fr. 6-4), 6.1 mg (Fr. 7-1), 0.5 mg (Fr. 7-2), 0.7 mg (Fr. 8-1), 1.7

mg (Fr. 8-2), 0.5 mg (Fr. 8-3). (v). Fractions with high purity were combined with each other as shown to obtain 10 pure compounds.

Fig. 4. HPLC analysis of crude extract and isolated compounds by stop-and-go CCC × HPLC. The analysis was performed on a Cosmosil ODS- PAQ column (250 mm × 4.6 mm i.d., 5 μm) with a guard column (10 mm × 4.6 mm i.d., 5 μm). The mobile phase used was methanol (A) and water (B) in a linear gradient mode as follows: 0-25 min: 20-80% A, 25-27 min: 80-20% A, 27-30 min: 20% A. The flow-rate of the mobile phase was 1.0 ml/min and the effluents were monitored at 274 nm by a DAD detector. The column temperature was kept at 30 ˚C. Compound purity: 97.4 % (1), 90.7% (2), 90.6% (3), 99.9 % (4), 99.9 % (5), 99.9 % (6), 99.9 % (7), 99.9 % (8), 94.7 % (9) and 99.9 % (10). (The relative contents in percentage were calculated with area normalization method.)

Table Error! Main Document Only. Relevant parameter for selection of CCC solvent system

Phase ratio

Settling

Partition coefficient (KU/L) of main components

(upper: lower, v/v)

time(s)

1

2

3

4

5

6

7

8

9

10

Solvent system

1

15%a, 1:1b

no delamination

-

-

-

-

-

-

-

-

-

-

-

2

15%, 1:1.2

2:1

47

1.49

1.76

2.49

2.12

3.15

1.74

1.19

1.82

1.46

1.89

3

16%, 1:1

1.5:1

85

1.83

1.92

3.84

3.40

5.55

1.90

1.26

2.10

1.59

1.96

4

16%, 1:1.2

1.8:1

37

2.37

2.47

4.94

6.33

8.74

2.54

1.31

2.78

2.10

3.87

5

17%, 1:1

1.2:1

36

2.86

2.66

3.47

4.16

9.57

2.83

1.39

2.93

1.94

3.90

6

17%, 1:1.2

1.7:1

29

2.95

2.76

10.19

10.47

12.13

2.33

1.47

2.19

2.25

3.67

b Volume ratio of salt-containing aqueous solution and isopropanol.

Table Error! Main Document Only. Optimization of 1D CCC conditions Stationary/mobile phase a

Rotation mode/speed (rpm)

Flow rate (mL/min)

Sf (%) d

L/U b

REV c/900

2

13.3

U/L

REV/900

2

26.7

U/L

FWD c/900

2

6.7

U/L

REV/500

2

4.3

U/L

REV/900

1

46

a Solvent system: isopropanol-16% sodium chloride solution (1:1.2, v/v)

b “U” and “L” stands for upper phase and lower phase respectively

c “FWD” and “REV” stands for forward rotation mode and reverse rotation mode respectively

d Sf: Stationary phase retention ratio

Table Error! Main Document Only. Recovery rate of CCC fractions 1-8 trapped on the holding columns (X ± RSD, %, n = 3)

Dilution ratio

Fr. 1

Fr. 2

Fr. 3

Fr. 4

Fr. 5

Fr. 6

Fr. 7

Fr. 8

1:1

55.4 ± 2.5

53.9 ± 3.9

63.9 ± 3.2

61.9±2.4

67.2±2.9

70.5±3.4

72.4±3.3

75.3±2.7

1:2

61.1 ± 3.8

60.5 ± 4.8

78.1 ± 5.2

76.1±3.2

80.3±3.4

81.4±3.2

83.2±2.6

86.4±3.6

1:3

65.7 ± 3.0

64.7 ± 2.6

89.7 ± 4.3

88.2±3.5

93.6±3.7

94.6±2.5

92.6±3.1

95.3±2.7

1:4

66.4 ± 3.5

63.7 ± 3.3

90.4 ± 1.9

89.1±2.9

95.8±2.1

96.5±2.1

93.4±2.4

96.7±2.4