- Email: [email protected]
Surface modification of polytetrafluoroethylene column for two-stationary phase separations by counter-current chromatography
Accepted Manuscript Title: Surface modification of PTFE column for two-stationary phase separations by counter-current chromatography Author: Kai-jun Quan Xin-yi Huang Xiao-ting Li Gao-hong Wang Yan-juan Liu Wen-da Duan Duo-long Di PII: DOI: Reference:
S0021-9673(15)01452-1 http://dx.doi.org/doi:10.1016/j.chroma.2015.10.009 CHROMA 356923
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
1-6-2015 29-9-2015 6-10-2015
Please cite this article as:http://dx.doi.org/10.1016/j.chroma.2015.10.009 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.
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Highlights •The concept of auxiliary stationary phase (ASP) for CCC was presented. •A universal approach for in-situ synthesizing ASP on CCC column was proposed •A novel CCC column with ASP in its own right was prepared for the first time. •Chromatographic behavior of model analytes on novel CCC column was investigated. •The retention ratio of stationary phase was increased than original column after coating with ASP.
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Surface modification of PTFE column for two-stationary phase separations by counter-current chromatography
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province,
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Kai-jun Quana,b, Xin-yi Huang a ,Xiao-ting Lia,b, Gao-hong Wanga,b, Yan-juan Liua,b, Wen-da Duana,b, Duolong Dia*[email protected]
Chinese Academy of Sciences, Lanzhou Institute of Chemical Physics, No. 18.Tianshui Middle Road, Lanzhou 730000, China. Email: [email protected]; Fax: +86 931 8277088; Tel: +86 931 4968248
University of the Chinese Academy of Sciences, Beijing 100049, China
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Laboratory Director, Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, 730000, China. Tel.: 86-931-4968248, Fax: 86-931-8277088
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Abstract To improve the separation capability of CCC, a novel solid-liquid two stationary phases CCC (ASP-CCC) column was prepared employing graphene oxide（ GO） conjugated poly-dopamine (PD) coating (GO/PD) as auxiliary stationary phase (ASP). The results of Scanning electron microscopy (SEM), contact angle and Xray photoelectron spectroscopy (XPS) indicated that nanostructured GO and PD were successfully grafted on the inner wall of the PTFE column. Three alkaloid compounds were selected as the target analytes to evaluate the performance of the novel column. Because of the intermolecular force (hydrogen bond, electrostatic interaction and π-π interaction) between the ASP and model compounds, three analytes were well separated with this novel ASP-CCC column. Additionally, the novel column exhibited higher stationary phase retention ratio, about 8%, than original column without changing the chromatographic condition. Furthermore, the eluotropic sequence of analytes on novel column was in accordance with that in the original column. This suggested that the novel column is a CCC column with auxiliary stationary phase (ASP) in its own right, and the present separation mode is the combination of partition chromatography and adsorption chromatography. Keywords 1/1 Page 1 of 18
CCC; poly-dopamine; auxiliary stationary phase; adsorption; graphene oxide; alkaloid
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Introduction Counter-current chromatography (CCC), which based on the liquid-liquid partition mechanism, is a unique separation and purification technique. The irreversible absorption and degeneration of the sample on the support can be avoided because there is no use of solid support in the chromatographic column for CCC [1,2]. Therefore, CCC is especially suitable for the separation and purification of natural products [3]. It has become one of the most potential research fields of CCC for the separation and purification of natural products over the past two decades. However, because of its relatively low theoretical plates and single separation mechanism, it is very difficult to obtain satisfying results of separating two analytes with similar structures by CCC even under the optimal chromatography conditions [4]. This has largely hampered the further practical application of CCC in the separation field. In recent years, much work has been done to improve the separation efficiency of CCC by the researchers in different countries， and the research mainly focus on three aspects: One is to improve instruments [5]; another is to combine with other techniques and operation modes [6-8]; and the other is to add an additive into the solvent system [9-10]. All of above were good jobs; however, it is still necessary to develop a new way to better improve the separation performance of CCC.
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PTFE tube, with the great properties such as high strength and good flexibility, is an ideal chromatographic column substrate. However, due to high chemical resistance, it is difficult to immobilize absorbent onto inner of PTFE micro-tube. Fortunately, a universal PD-based surface modification method has been developed by Lee and coworker inspired by mussel’s unique binding capability to wet rocks [11-12]. They found that the PD permanent coating can be formed on a wide array of material surfaces including chemical resistance PTFE surfaces. The PD coating can serve as a good platform for the formation of multifunctional materials such as organic/inorganic ad-layer of proteins [13-14], metal ions [15], hydroxyapatites [16], or gold nanoparticles [17]. Moreover, Lee and co-works had developed a general strategy for one-step multipurpose surface functionalization by immersion of substrates in an one-pot mixture of a molecule and a catecholamine, which had breached the limit to molecules presenting either amine or thiol groups [18]. Thus, PD has shown many potentially exciting applications in these research fields. Recently, with dopamine serving as surface functionalization agents, Chen’s group adopted a novel non-covalent and covalent combined (layer-by-layer) LBL strategy for successful multilayer assemblies of Graphene in PTFE microtubes for micro-extraction [19]. So it`s of great potential to improve the separation efficiency by immobilizing sorbents onto the inner of PTFE column as an auxiliary stationary phase. We hope to seek out a new way to extend the research field of CCC.
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Encouraged by the aforementioned work, here, for the first time, we adopted PD as an intermediate layer and GO as the functionalization agent to in-situ prepare a novel ASP-CCC. The coating layer, expected as the second stationary phase, can make up for the deficiencies of the CCC and be the important assistance to the CCC separation due to the large surface area and abundant functional groups of GO. To immobilize GO, the inner wall of PTFE tube was modified by the PD method. In the process of coating, CuSO4•5H2O was used as auxiliary oxidant helping with finishing PD self-oxidation polymerization and FeCl3 solution as stabilizing agent improving the stability of PD coating in order to avoid the detachment of the coating when the column was revolved with high speed [20-21]. Scanning electron microscopy (SEM), contact angle and X-ray photoelectron spectroscopy (XPS) were used to characterize the properties of the coated surface of 2/2 Page 2 of 18
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PTFE. Data show that the modified surfaces exhibited much better wettability, so modification can make CCC have higher stationary phase retention about 8% than pre-modified. To explore preceding hypothesis, three alkaloid molecules (Isocorydine, Sinomenine hydrochloride and Isocorydione, the chemical structure show in Fig.1) were adopted as the model analytes to compare the chromatographic behaviour of the column with and without modified because alkaloids are not only a kind of compounds with biological activity compound but also charged molecules, which may ease interact with GO by the electrostatic interactions. The results proved that the ASP could produce different adsorption forces towards the three molecules, which could combine with intrinsic distributional effects of CCC and achieve two-dimensional separation. It can be speculated that many of molecule that participates in strong interactions with GO might be effective to apply to the present technique. The presented strategy using PD coating as a versatile platform for facile conjugation of diverse functional group may offer new processing strategies to prepare novel CCC column, but more important, we had found a new way to extend the research field of CCC.
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2. Materials and methods 2.1. Reagents and chemicals Analytical reagent grade n-Hexane used for HSCCC separation was purchased from Chongqing Chemical Reagent Co., Ltd. (Shanghai, China). Ethyl acetate and methanol used in HSCCC were of analytical grade and purchased from Yantai Shuangshuang Chemical Co., Ltd. (Shandong, China). Ultrapure water, used for preparation of all the samples and solutions, was obtained from a Spring-R10 water purification system (Research Scientific Instrument Co., Ltd, Xiamen, China). Dopamine hydrochloride was purchased from Sigma–Aldrich (MO. USA). N-[3-(Trimethoxylsilyl) -propyl]aniline were purchased from Alfa Aesar (Tianjin, China).Graphite powder (natural, microcrystal grade, 2-15 µm, 99.9995%) was purchased from Alfa Aesar. PTFE- tubes (3 mm o.d., 2 mm i.d.) were obtained from Haohai Chemical (Wuhan, China).Iron (III) chloride hexahydrate (FeCl3•6H2O, 97%.Sigma–Aldrich) 2.2. Apparatus In this study ,a preparative HSCCC apparatus TBE-300B (TautoBiotech, Shanghai, China) was used, which was equipped with three multilayer coil separation columns (the existing PTFE-tube was replaced with the same diameter of new PTFE-tube(3 mm o.d., 2 mm i.d.) and was installed according to the original way, a total column volume of 300 mL, the new PTFE-tube was called original column in the following) connected in series (inner diameter of the coiled PTFE tube = 2 mm, revolution radius of the multilayer coil = 50 mm, β value = 0.5–0.8), an plunger pump system (TBP 5002, TautoBiotech, Shanghai, China) with a TBD2000UV detector(TautoBiotech, Shanghai, China), an injection valve with a 25 mL sample loop and a water circulator(HX-1050, Boyikang Lab Instrument, Beijing, China), an air generator GA-2000A(Beijing Zhongxing Huili Science and Technology Development Co., Ltd. China.) The HPLC analysis was performed in an Agilent 1260 Series (Agilent Technologies, USA) LC system equipped with a G1311C quaternary pump, a G1315D diode array detector and a G1328C manual injector. The system was controlled by Agilent Chemstation software (Agilent Technologies, USA)
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2.3. In-situ preparation of multilayer PD-modified CCC column. A PTFE tube of 100 m, which is assembled in the CCC system as the column, washed sufficiently by continuously pumping methanol and acetone and dried under a stream of nitrogen gas before use. For PD modification, dopamine basic solution (2.0 mg•mL-1, pH 8.5 in Tris-HCl buffer, 20 mM CuSO4•5H2O as the assistant oxidant was vigorously agitated with a vortex mixer to facilitate the oxidation of dopamine [20]. 3/3 Page 3 of 18
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When the solution became slightly brown, 300 mL of solution was introduced into the PTFE tube by the peristaltic pump at a rate of 10.0 mL•min-1 for 30 min. Then the two ends of the PTFE tube were sealed and held for 24 h. In order to make the coating homogeneous, the column was rotated during incubation 24h period at low speeds with 50-60 rpm, which could avoid the PD was deposited onto one side of the inner wall of the PTFE-tube. The residual solution was pushed out by air pump and the PTFE tube was washed with deionized water for 30 min , then 300 mL ethanol solution of FeCl3 (10 ｍM) was pumped into PTFE tube and incubated for 1 h, after extrusion the residual solution and drying under a stream of nitrogen gas, PD-modified PTFE tube (PD@PTFE) was obtained. The process described above was repeated five times while the last two times using oxygen who dissolved in the solution instead of CuSO4•5H2O as the oxidant to obtain multilayer PD-modified PTFE tube, labeling it as PD5@PTFE.
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2.4. Immobilization of GO onto PD 5@PTFE Graphene oxide was prepared from graphite powder according to the improved Hummers method [22] and for graphene oxide modification, GO aqueous solution (1.0 mg•mL-1) was introduced into PD5@PTFE, the two ends were sealed, and the tube was put in an air bath and heated at 60°C for 24 h. After the tube was washed with water and dried under a stream of nitrogen gas, GO@PD5@PTFE, the novel CCC column was obtained. The schematic is shown in Fig. 2.
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2.5. Characterization of the novel PTFE column Quanta 200 scanning electron microscopy (SEM; FEI, USA) was utilized to characterize the morphology of the PTFE, PD5@PTFE and GO@PD5@PTFE. The chemical composition of the coated PTFE samples was confirmed by X-ray photoelectron spectra (XPS) on an ESCALAB 250 spectrometer equipped with a monochromatic Al X-ray source (1486.6 eV).The equilibrium contact angles of the PD5@PTFE and GO@PD5@PTFE surface slices were measured by a video-based contact angle measuring device (DSA100, KRUSS, Germany) in the sessile drop method. At least five points were obtained with the deviation range within ±2°, and the averaged value was used to evaluate their hydrophobicity.
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2.6. Sample solution Preparation. See Supporting Information
2.7. Solvent System Selection for CCC Separation The work is focused on the influence of the ASP layer to separation behavior of model analytes in BET300B system when using the novel column. For proof-the-principle experiments, the most common systems of n-Hexane: Ethyl acetate: Methanol: Water (1:1.3:1:1.3) was chosen for separation of the model analytes. 2.8. CCC Separation Procedure. At the beginning of the separation of process, the column was filled with the stationary phase (the upper phase of n-Hexane: Ethyl acetate: Methanol: Water). Then, the mobile phase (the lower phase of the system) was pumped into the coil at 2 mL•min -1 from the head toward the tail with a rotational speed of 850 rpm at 20°C. When hydrodynamic equilibrium was established, the sample was injected into the column via a sample loop. The effluent was monitored by an online UV detector at 280 nm. Stationary phase retention rate was measured while the separation procedure was performed. 2.9. Compared the separation behavior of model analytes 4/4 Page 4 of 18
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Three alkaloids (Isocorydine, Sinomenine hydrochloride and Isocorydione) were used as model analyses to investigate the mechanism of GO@PD5@PTFE as the column of BET-300B system. As section2.8, separate the mixed solution of model molecules using original and novel column, respectively. Then the separation behavior of model analytes between original and novel column was compared. 2.10. HPLC analyses and identification of HSCCC peak fractions The mixed solution of model analytes and fractions separated by HSCCC were analyzed by HPLC with ZORBAX SB-C18 analytical column with a gradient of phosphoric acid 2% (pH 6.32, adjusted with triethylamine)(A); and acetonitrile (B). The gradient conditions are as follows: 20% (B) at 0–3 min, 20–50% (B) at 3–4 min, 50% (B) at 4–12 min; flow rate, 1 ml/min; detection wavelength: 280 nm; injection volume: 10 µL .
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3. Result and Discussion 3.1. Surface characterization of GO/PD -coated PTFE surface For ease of characterization of ASP layer in the CCC column before and after its modification, PTFE chips of the same thickness were used instead of the PTFE tube. To characterize the morphological structures of PTFE, PD5@PTFE and GO@PD5@PTFE surface, SEM measurement was carried out (Fig.3). As showed in Fig. 3d, it is clear that the original PTFE has a relatively smooth surface. After coating five layers of PD on the surface of PTFE (Fig. 3e), some small thin wrinkles are observed, which is formed from polymerization of dopamine and gathering of multilayer PD [23]. After decoration of GO onto the surface of PD5@PTFE (Fig. A.3f), an increased thickness of wrinkled can be observed, indicating the successful formation of PD5/GO layer over the surface of PTFE.
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Chemical composition of the ASP was investigated by XPS. Peaks of C 1s and F 1s are observed in PTFE (Fig. 4 A), PD5@PTFE (Fig. 4 C) and GO@PD5@PTFE (Fig.4 E). However, contents of F in PD5@PTFE and GO@PD5@PTFE are found to decrease significantly compared with that of PTFE because of coating with PD and GO. After coating with PD, the N 1s peak was observed, which was attributed to the amino group from PD (Fig. S1 B, C).The Peak fitting of the C 1s bands of PTFE (Fig. 4 B) yields a major functional groups: C−F bonds from (-CF2-CF2-) n and a small quantity of C-C and C-Si may cause by contaminating. Peak fitting of C 1s bands of PD5@PTFE (Fig. 4 D), in addition to C−F, yields several functional groups including, C−C, C−O and C−N, which was from the bond of PD. Peak fitting of the C 1s bands of GO@PD5@PTFE (Fig. 4 F) also yields several functional groups including, C−C, C-O and C−N as well as C=O, which was from the bond of GO-PD [18].
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The hydrophobicity of PTFE, PD5@PTFE and GO@PD5@PTFE surfaces were evaluated by contact angle measurements. As showed in Fig. 3, the contact angle of the original PTFE was 116°(Fig. 3a). Due to the strong hydrophobicity of PD, the contact angle had declined to about 76°(Fig. 3a) when coated five layers of PD on the surface of PTFE. Correspondingly, after decoration of GO onto the surface of PD5@PTFE, the contact angle was increased to 86°(Fig. 3c), which because of the hydrophobicity of GO, again showing that PD and GO were successfully coated onto the PTFE surface. Fig. 3 3.2. Stability of GO@PD5@PTFE column. The stability of GO@ PD5 coating layer on the PTFE surface of the CCC column was investigated by running 8 consecutive separation tests using operating conditions as described in 2.8. Results show that there is a small part of the ASP layer that was observed to drop during the first three cycles, and then, bare coating 5/5 Page 5 of 18
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1 For CCC, the retention of the stationary phase Table is an important factor that can largely influence the resolution of the analytes. In general, the higher retention of the stationary phase, the higher resolution, which is as (1) [24]
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peeling is found any more. And on the current technical level, 7 - 9 times the separation can be reproducibly achieved by using the coated tubing(as shown in Fig. 7). In addition, in accordance with document report [18], the GO-PD layer has good endurance to organic solvents and was stable even when treated with 1 M HCl and 0.01 M NaOH. Fig. 4 3.3. The retention of stationary phase of the novel column The development of CCC separation largely depends on screening an appropriate solvent system, in which the retention of the stationary phase is a critical measure indicator. As is known to all, PTFE has extremely low coefficient of kinetic friction, so it has almost no resistance to fluid that flow through the PTFE tube. Therefore, PTFE is very suitable to be used as base material of countercurrent chromatography column because it could hardly produce interaction force between the inner wall of PTFE column and solvent system. However, the very weak interaction force between the inner wall of PTFE column and solvent system could impede the retention of stationary phase for further increasing. After coating with GO@PD, the surface of PTFE gets rougher (Figure 3 e, f) and the interaction force between the inner wall of PTFE column and solvent system have enhanced, followed by the maximum column pressure increase from 0.09 to 0.19 MPa . In addition, the contact angle in solvent system has a sharp decrease than original PTFE (See Supporting Information.). Delightedly our work indicated that the retention of stationary phase in novel column was higher about 8% than pre-modified column (Table 1) while the other condition without changed.
(1)
Where RS is the resolution, Sf is the retention of the stationary phase, KD1 is the partition coefficient; N and α are the theoretical plate number and the selective factor. At the same time, Sf can largely influence the retention volume (VR) of solutes, which is as (2)
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(2)
Where VC is the column volume of CCC, KD is the partition coefficient, VS is the volume of stationary phase. In this work, the influence of ASP to VC is negligible. KD is a constant. Thus,
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(3) Where subscript 1and 2 represent that on the original column and the novel column, respectively. Here, VC=300 mL while ∆Sf=0.08., 6/6 Page 6 of 18
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To prove whether the ASP is effective, the hypotheses was raised firstly that those changes in chromatographic behaviour due exclusively to the increase of the stationary phase retention and the coating on the inner surface of the column was simply to increase its friction with a liquid stationary phase, then the ∆VR(S) of analytes, which caused by the rise of Sf, could be obtained from the formulae (3). Since the KD of analytes is less than one (see support information), so the ∆VR value is negative (see Table 2), which means that the VR of analytes decreases with rise of the Sf. However, the calculation result is not in agreement with analytes had increased with 30.4, 40.7, 68.2 mL on the novel the experimental result. The actual VR of three Table 2 column while the calculation result should be decreases with 23.8, 23.5 and 13.2 mL, respectively. That is, the assumption might be wrong , which means that the ASP is effective. Therefore, it can be speculated that the ASP has some influence on the analytes, which has finally brought about the increase of VR (sign as VR(A)).
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3.4. Comparison of the separation behaviour of model analytes In order to prove the effectiveness of the coating, we compared the separation behavior of model analytes between original and novel column, the result is as shown in Fig.5: compared with the original column, significant changes of the separation behavior of model analytes was observed on the novel column, which mainly showed in the following aspects: firstly, the resolution of the analytes was improved on the novel column, especially to peak 2 and peak3 ; secondly, retention time of all analytes were lengthened, peak 3 in particular; thirdly, peak broadening of analyte 2 and 3 were observed on the novel column. Besides, the eluotropic sequence of solutes on novel column was in accordance with that in the original column but each fraction on novel column had a higher purity (as Fig. 6).Taken together, these results further suggested that ASP improved resolution by introducing intermolecular forces.
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The ASP, which consists of GO and PD, contained rich functional groups, which could form different intermolecular forces with the analytes. Hydrogen bonds can easily be generated between ketone, oxhydryl group of model analytes and oxhydryl, carboxyl groups of APS. Furthermore, electrostatic interaction may be generated because alkaloids can be protonated to form the cationic; meanwhile ASP were negatively charged due to the slight dissociation of some carboxyl groups contained in GO and hydroxyl groups contained in PD. Owing to the existence of several aromatic rings in both ASP and analytes, π-π stacking was equally participating in the adsorption process. Resulting from the difference in the structure of analytes, the intermolecular forces are also discrepant, which is conducive to the separation of analytes from each other. In addition, almost all of the Sinomenine hydrochloride and Isocorydine were distributed in the mobile phase because of their low KD（ see table 2） . That could lead them to come into contact and interact with ASP with relative less opportunity than Isocorydione that distributed in both stationary phase and mobile phase. Thanks to the synergetic effect of these factors, the analytes obtained better separation on the novel column.
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Although there are plenty of works concerning preparing for the new type of chromatographic column, no research had focused on the CCC column. This work, for the first time, established a universal approach to improve the separation efficiency of CCC though immobilize sorbents onto the inner wall of CCC column as an ASP. What's all the more remarkable is that the original coating could be degraded and removed by concentrated alkali solutions (PH>14) when it needs to be changed by other modifiers. However, there is still some shortage in our study, such as absorption loss and the rise of the retention time, all this need a 7/7 Page 7 of 18
better resolution to provide compensation. And one important future direction of the work is easier to be prepared and more systematically to be designed， which could achieve better stability and smaller adsorption losses
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4. Conclusion A novel CCC column with auxiliary stationary phase (ASP) in its own right was prepared. Scanning electron microscopy (SEM), contact angle and X-ray photoelectron spectroscopy (XPS) were used to characterize the properties of the coated surface of PTFE. Data show that the modified surfaces exhibited much better wettability, so modification can make HSCCCCCC have higher stationary phase retention than premodified. The chromatographic behavior of model analytes (Isocorydine, Sinomenine hydrochloride and Isocorydione) on the novel CCC column in TBE-300B was investigated combining experimental result and theoretical arithmetic method. The result showed that the ASP improved resolution by introducing intermolecular forces and improving the stationary phase retention ratio. And then followed by existing distributional effects of CCC， a better resolution than conventional CCC was obtained. It is important to say that the proposed approach can be applied to any type of CCC column installed in any type of CCC instrument, so the essence of this manuscript is to present a universal approach for CCC development rather than only to prepare a novel column. This approach might become one of the approaches for CCC scientists to improve their current instruments
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Acknowledgements The authors would like to thank the National Scientific Foundation of China (NSFC no.21175142) and Open Fund of Key Laboratory of Chemistry of Northwestern Plant Resources of The Chinese Academy of Science (no. CNPR-2011kfkt-02). References [1]
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catalytic reduction of 4-nitrophenol, Nanotechnology 26 (2015) 105601-105609. doi: 10.1088/0957-4484/26/10/105601 S.M. Kang, N.S. Hwang, J. Yeom, S.Y. Park, P.B. Messersmith, I.S. Choi, R. Langer, D.G. Anderson, H. Lee, One-Step Multipurpose
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77 (2014) 1336-1339. doi: 10.1016/j.porgcoat.2014.04.011 [22]
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graphene oxide, ACS nano 4 (2010) 4806-4814. doi: 10.1021/nn1006368 [23]
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of berberine in plasma and urine samples, Talanta 103 (2013) 103-109. doi: 10.1016/j.talanta.2012.10.014 [24]
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chromatographic separation technology and its application, Chemical Industry Press.,Beijing, 2005,pp.9-11. [25]
Y. Ito, Golden rules and pitfalls in selecting optimum conditions for high-speedcounter-current chromatography, J. Chromatogr. A
.1065 (2005) 145–168. doi: 10.1016/j.chroma.2004.12.044
Fig. 1 The chemical structure of model analytes Fig. 2 (A) Scheme of simultaneous polymerization of dopamine[13]. (B) Scheme of the mechanism of on-line prepared of Multilayer PD-modified PTFE Tube and GO immobilized on the PTFE-tube through the PD layer
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Fig. 3 Contact angle and scanning electron micrographs of PTFE tube: (a, d) Original PTFE; (b, e) PD5@PTFE; (c,f) GO@PD5@PTFE. Fig. 4 XPS spectra of (A) Original-PTFE, (C)PD5@PTFE and (E) GO@PD5@PTFE; peak fitting of C1s XPS spectra of (B) Original-PTFE, (D) PD5@PTFE and (F) GO@PD5@PTFE. Fig. 5 Comparison of the separation behavior of model analytes in TBE-300B between original and novel column: 1.Sinomenine hydrochloride; 2.Isocorydine; 3. Isocorydione. Experimental conditions: Apparatus: TBE-300B, 300 mL column volume with
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three coils of 2 mm id tubing. Solvent system: n-hexane–ethyl acetate/methanol/water 5 : 6.5 : 5 : 6.5 v/v.. sample: 1.0 mg of each Isocorydine, Sinomenine hydrochloride and Isocorydione in 1.0 mL of methanol; elution mode: the upper organic phase was the mobile phase ;revolution speed: 850 rpm; flow rate: 2.0 mL min-1;UV detection at 280 nm; Sf::58.3% (the Original
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column),66.2% (the novel column).
Fig. 6 HPLC chromatograms of mixed solution of model analytes and HSCCC peak fractions. Conditions: column, ZORBAX SB-C18 column (150 mm×4.6 mm i.d., 5 µm); the mobile phase consisted of eluent A, phosphoric acid 2% (pH 6.32, adjusted with triethylamine); and eluent B,
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acetonitrile. Gradient: 20% (B) at 0–3 min, 20–50% (B) at 3–4 min, 50% (B) at 4–10 min; flow rate, 1 ml·min-1; detection wavelength: 280 nm; injection volume: 10 µL ;(A) mixed solution of model analytes; (B) combined fractions, peak 1; (C) combined fractions, peak 2; (D) combined fractions, peak 3.
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Fig. 7 HSCCC chromatogram of 13 consecutive runs for separating three model analytes on GO@PD5@PTFE column. Experimental conditions: Apparatus: TBE-300B, 300 mL column volume with three coils of 2 mm id tubing. Solvent system: n-hexane–ethyl acetate/methanol/water 5 : 6.5 : 5 : 6.5 v/v. sample: 1.0 mg of each Isocorydine, Sinomenine hydrochloride and Isocorydione in 1.0 mL of methanol; elution mode: the
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upper organic phase was the mobile phase ;revolution speed: 850 rpm; flow rate: 2.0 mL min-1;UV detection at 280 nm.
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Table 1 Compared the stationary phase retention ratio and column pressure between original and novel column a Column Sf (×100%) flow rate pressure Column no. Solvent system ( V0- Ve) / RSD(×100%) mL·min-1 (-max.) MP V0 n2 1 0.07 57.7 Hexane−EtOAc 2 0.09 58.3 Original 2 −MeOH−Water 2 1.29 CCC 3 58.3 0.09 (1:1.3:1:1.3) column 2 4 0.07 57.0 2 5 0.09 59.0 2 1 0.19 66.2 2 2 0.21 66.9 Novel 2 0.7 CCC 3 0.19 65.5 column 2 4 0.19 66.2 2 5 0.23 66.2
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Note : The stationary phase retention ratio(Sf) was calculated as [25] Conditions: the same as shown in experimental section 2.7.
Table2 The properties of analytes and the ∆VR between original and novel column.
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Note: KD means the partition coefficient of analyte in the given solvent system. ∆VR(S) means the ∆VR value calculated by the formula: ∆VR12=VC·∆Sf12 (1-KD) 417 KD ∆VR12（（ S）） ∆VR12(E) ∆VR12(A)418 molecular compound 419 formula CS/CM mL mL mL 420 421 Sinomenine C19H24ClNO4 0.009 23.8 -30.4 -54.2 422 hydrochloride 423 424 Isocorydine C20H23NO4 0.020 23.5 -40.7 -64.2 425 426 427 428 Isocorydione C18H17NO5 0.452 13.2 -68.2 -81.4 429 430
∆VR(E) means the ∆VR value is the reality difference obtained from experiment result ∆VR(A) means the ∆VR value caused by the influence of the ASP.
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S0021-9673(15)01452-1 http://dx.doi.org/doi:10.1016/j.chroma.2015.10.009 CHROMA 356923
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
1-6-2015 29-9-2015 6-10-2015
Please cite this article as:
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Highlights •The concept of auxiliary stationary phase (ASP) for CCC was presented. •A universal approach for in-situ synthesizing ASP on CCC column was proposed •A novel CCC column with ASP in its own right was prepared for the first time. •Chromatographic behavior of model analytes on novel CCC column was investigated. •The retention ratio of stationary phase was increased than original column after coating with ASP.
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Surface modification of PTFE column for two-stationary phase separations by counter-current chromatography
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province,
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Kai-jun Quana,b, Xin-yi Huang a ,Xiao-ting Lia,b, Gao-hong Wanga,b, Yan-juan Liua,b, Wen-da Duana,b, Duolong Dia*[email protected]
Chinese Academy of Sciences, Lanzhou Institute of Chemical Physics, No. 18.Tianshui Middle Road, Lanzhou 730000, China. Email: [email protected]; Fax: +86 931 8277088; Tel: +86 931 4968248
University of the Chinese Academy of Sciences, Beijing 100049, China
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Laboratory Director, Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, 730000, China. Tel.: 86-931-4968248, Fax: 86-931-8277088
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Abstract To improve the separation capability of CCC, a novel solid-liquid two stationary phases CCC (ASP-CCC) column was prepared employing graphene oxide（ GO） conjugated poly-dopamine (PD) coating (GO/PD) as auxiliary stationary phase (ASP). The results of Scanning electron microscopy (SEM), contact angle and Xray photoelectron spectroscopy (XPS) indicated that nanostructured GO and PD were successfully grafted on the inner wall of the PTFE column. Three alkaloid compounds were selected as the target analytes to evaluate the performance of the novel column. Because of the intermolecular force (hydrogen bond, electrostatic interaction and π-π interaction) between the ASP and model compounds, three analytes were well separated with this novel ASP-CCC column. Additionally, the novel column exhibited higher stationary phase retention ratio, about 8%, than original column without changing the chromatographic condition. Furthermore, the eluotropic sequence of analytes on novel column was in accordance with that in the original column. This suggested that the novel column is a CCC column with auxiliary stationary phase (ASP) in its own right, and the present separation mode is the combination of partition chromatography and adsorption chromatography. Keywords 1/1 Page 1 of 18
CCC; poly-dopamine; auxiliary stationary phase; adsorption; graphene oxide; alkaloid
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Introduction Counter-current chromatography (CCC), which based on the liquid-liquid partition mechanism, is a unique separation and purification technique. The irreversible absorption and degeneration of the sample on the support can be avoided because there is no use of solid support in the chromatographic column for CCC [1,2]. Therefore, CCC is especially suitable for the separation and purification of natural products [3]. It has become one of the most potential research fields of CCC for the separation and purification of natural products over the past two decades. However, because of its relatively low theoretical plates and single separation mechanism, it is very difficult to obtain satisfying results of separating two analytes with similar structures by CCC even under the optimal chromatography conditions [4]. This has largely hampered the further practical application of CCC in the separation field. In recent years, much work has been done to improve the separation efficiency of CCC by the researchers in different countries， and the research mainly focus on three aspects: One is to improve instruments [5]; another is to combine with other techniques and operation modes [6-8]; and the other is to add an additive into the solvent system [9-10]. All of above were good jobs; however, it is still necessary to develop a new way to better improve the separation performance of CCC.
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PTFE tube, with the great properties such as high strength and good flexibility, is an ideal chromatographic column substrate. However, due to high chemical resistance, it is difficult to immobilize absorbent onto inner of PTFE micro-tube. Fortunately, a universal PD-based surface modification method has been developed by Lee and coworker inspired by mussel’s unique binding capability to wet rocks [11-12]. They found that the PD permanent coating can be formed on a wide array of material surfaces including chemical resistance PTFE surfaces. The PD coating can serve as a good platform for the formation of multifunctional materials such as organic/inorganic ad-layer of proteins [13-14], metal ions [15], hydroxyapatites [16], or gold nanoparticles [17]. Moreover, Lee and co-works had developed a general strategy for one-step multipurpose surface functionalization by immersion of substrates in an one-pot mixture of a molecule and a catecholamine, which had breached the limit to molecules presenting either amine or thiol groups [18]. Thus, PD has shown many potentially exciting applications in these research fields. Recently, with dopamine serving as surface functionalization agents, Chen’s group adopted a novel non-covalent and covalent combined (layer-by-layer) LBL strategy for successful multilayer assemblies of Graphene in PTFE microtubes for micro-extraction [19]. So it`s of great potential to improve the separation efficiency by immobilizing sorbents onto the inner of PTFE column as an auxiliary stationary phase. We hope to seek out a new way to extend the research field of CCC.
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Encouraged by the aforementioned work, here, for the first time, we adopted PD as an intermediate layer and GO as the functionalization agent to in-situ prepare a novel ASP-CCC. The coating layer, expected as the second stationary phase, can make up for the deficiencies of the CCC and be the important assistance to the CCC separation due to the large surface area and abundant functional groups of GO. To immobilize GO, the inner wall of PTFE tube was modified by the PD method. In the process of coating, CuSO4•5H2O was used as auxiliary oxidant helping with finishing PD self-oxidation polymerization and FeCl3 solution as stabilizing agent improving the stability of PD coating in order to avoid the detachment of the coating when the column was revolved with high speed [20-21]. Scanning electron microscopy (SEM), contact angle and X-ray photoelectron spectroscopy (XPS) were used to characterize the properties of the coated surface of 2/2 Page 2 of 18
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PTFE. Data show that the modified surfaces exhibited much better wettability, so modification can make CCC have higher stationary phase retention about 8% than pre-modified. To explore preceding hypothesis, three alkaloid molecules (Isocorydine, Sinomenine hydrochloride and Isocorydione, the chemical structure show in Fig.1) were adopted as the model analytes to compare the chromatographic behaviour of the column with and without modified because alkaloids are not only a kind of compounds with biological activity compound but also charged molecules, which may ease interact with GO by the electrostatic interactions. The results proved that the ASP could produce different adsorption forces towards the three molecules, which could combine with intrinsic distributional effects of CCC and achieve two-dimensional separation. It can be speculated that many of molecule that participates in strong interactions with GO might be effective to apply to the present technique. The presented strategy using PD coating as a versatile platform for facile conjugation of diverse functional group may offer new processing strategies to prepare novel CCC column, but more important, we had found a new way to extend the research field of CCC.
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2. Materials and methods 2.1. Reagents and chemicals Analytical reagent grade n-Hexane used for HSCCC separation was purchased from Chongqing Chemical Reagent Co., Ltd. (Shanghai, China). Ethyl acetate and methanol used in HSCCC were of analytical grade and purchased from Yantai Shuangshuang Chemical Co., Ltd. (Shandong, China). Ultrapure water, used for preparation of all the samples and solutions, was obtained from a Spring-R10 water purification system (Research Scientific Instrument Co., Ltd, Xiamen, China). Dopamine hydrochloride was purchased from Sigma–Aldrich (MO. USA). N-[3-(Trimethoxylsilyl) -propyl]aniline were purchased from Alfa Aesar (Tianjin, China).Graphite powder (natural, microcrystal grade, 2-15 µm, 99.9995%) was purchased from Alfa Aesar. PTFE- tubes (3 mm o.d., 2 mm i.d.) were obtained from Haohai Chemical (Wuhan, China).Iron (III) chloride hexahydrate (FeCl3•6H2O, 97%.Sigma–Aldrich) 2.2. Apparatus In this study ,a preparative HSCCC apparatus TBE-300B (TautoBiotech, Shanghai, China) was used, which was equipped with three multilayer coil separation columns (the existing PTFE-tube was replaced with the same diameter of new PTFE-tube(3 mm o.d., 2 mm i.d.) and was installed according to the original way, a total column volume of 300 mL, the new PTFE-tube was called original column in the following) connected in series (inner diameter of the coiled PTFE tube = 2 mm, revolution radius of the multilayer coil = 50 mm, β value = 0.5–0.8), an plunger pump system (TBP 5002, TautoBiotech, Shanghai, China) with a TBD2000UV detector(TautoBiotech, Shanghai, China), an injection valve with a 25 mL sample loop and a water circulator(HX-1050, Boyikang Lab Instrument, Beijing, China), an air generator GA-2000A(Beijing Zhongxing Huili Science and Technology Development Co., Ltd. China.) The HPLC analysis was performed in an Agilent 1260 Series (Agilent Technologies, USA) LC system equipped with a G1311C quaternary pump, a G1315D diode array detector and a G1328C manual injector. The system was controlled by Agilent Chemstation software (Agilent Technologies, USA)
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2.3. In-situ preparation of multilayer PD-modified CCC column. A PTFE tube of 100 m, which is assembled in the CCC system as the column, washed sufficiently by continuously pumping methanol and acetone and dried under a stream of nitrogen gas before use. For PD modification, dopamine basic solution (2.0 mg•mL-1, pH 8.5 in Tris-HCl buffer, 20 mM CuSO4•5H2O as the assistant oxidant was vigorously agitated with a vortex mixer to facilitate the oxidation of dopamine [20]. 3/3 Page 3 of 18
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When the solution became slightly brown, 300 mL of solution was introduced into the PTFE tube by the peristaltic pump at a rate of 10.0 mL•min-1 for 30 min. Then the two ends of the PTFE tube were sealed and held for 24 h. In order to make the coating homogeneous, the column was rotated during incubation 24h period at low speeds with 50-60 rpm, which could avoid the PD was deposited onto one side of the inner wall of the PTFE-tube. The residual solution was pushed out by air pump and the PTFE tube was washed with deionized water for 30 min , then 300 mL ethanol solution of FeCl3 (10 ｍM) was pumped into PTFE tube and incubated for 1 h, after extrusion the residual solution and drying under a stream of nitrogen gas, PD-modified PTFE tube (PD@PTFE) was obtained. The process described above was repeated five times while the last two times using oxygen who dissolved in the solution instead of CuSO4•5H2O as the oxidant to obtain multilayer PD-modified PTFE tube, labeling it as PD5@PTFE.
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2.4. Immobilization of GO onto PD 5@PTFE Graphene oxide was prepared from graphite powder according to the improved Hummers method [22] and for graphene oxide modification, GO aqueous solution (1.0 mg•mL-1) was introduced into PD5@PTFE, the two ends were sealed, and the tube was put in an air bath and heated at 60°C for 24 h. After the tube was washed with water and dried under a stream of nitrogen gas, GO@PD5@PTFE, the novel CCC column was obtained. The schematic is shown in Fig. 2.
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2.5. Characterization of the novel PTFE column Quanta 200 scanning electron microscopy (SEM; FEI, USA) was utilized to characterize the morphology of the PTFE, PD5@PTFE and GO@PD5@PTFE. The chemical composition of the coated PTFE samples was confirmed by X-ray photoelectron spectra (XPS) on an ESCALAB 250 spectrometer equipped with a monochromatic Al X-ray source (1486.6 eV).The equilibrium contact angles of the PD5@PTFE and GO@PD5@PTFE surface slices were measured by a video-based contact angle measuring device (DSA100, KRUSS, Germany) in the sessile drop method. At least five points were obtained with the deviation range within ±2°, and the averaged value was used to evaluate their hydrophobicity.
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2.6. Sample solution Preparation. See Supporting Information
2.7. Solvent System Selection for CCC Separation The work is focused on the influence of the ASP layer to separation behavior of model analytes in BET300B system when using the novel column. For proof-the-principle experiments, the most common systems of n-Hexane: Ethyl acetate: Methanol: Water (1:1.3:1:1.3) was chosen for separation of the model analytes. 2.8. CCC Separation Procedure. At the beginning of the separation of process, the column was filled with the stationary phase (the upper phase of n-Hexane: Ethyl acetate: Methanol: Water). Then, the mobile phase (the lower phase of the system) was pumped into the coil at 2 mL•min -1 from the head toward the tail with a rotational speed of 850 rpm at 20°C. When hydrodynamic equilibrium was established, the sample was injected into the column via a sample loop. The effluent was monitored by an online UV detector at 280 nm. Stationary phase retention rate was measured while the separation procedure was performed. 2.9. Compared the separation behavior of model analytes 4/4 Page 4 of 18
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Three alkaloids (Isocorydine, Sinomenine hydrochloride and Isocorydione) were used as model analyses to investigate the mechanism of GO@PD5@PTFE as the column of BET-300B system. As section2.8, separate the mixed solution of model molecules using original and novel column, respectively. Then the separation behavior of model analytes between original and novel column was compared. 2.10. HPLC analyses and identification of HSCCC peak fractions The mixed solution of model analytes and fractions separated by HSCCC were analyzed by HPLC with ZORBAX SB-C18 analytical column with a gradient of phosphoric acid 2% (pH 6.32, adjusted with triethylamine)(A); and acetonitrile (B). The gradient conditions are as follows: 20% (B) at 0–3 min, 20–50% (B) at 3–4 min, 50% (B) at 4–12 min; flow rate, 1 ml/min; detection wavelength: 280 nm; injection volume: 10 µL .
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3. Result and Discussion 3.1. Surface characterization of GO/PD -coated PTFE surface For ease of characterization of ASP layer in the CCC column before and after its modification, PTFE chips of the same thickness were used instead of the PTFE tube. To characterize the morphological structures of PTFE, PD5@PTFE and GO@PD5@PTFE surface, SEM measurement was carried out (Fig.3). As showed in Fig. 3d, it is clear that the original PTFE has a relatively smooth surface. After coating five layers of PD on the surface of PTFE (Fig. 3e), some small thin wrinkles are observed, which is formed from polymerization of dopamine and gathering of multilayer PD [23]. After decoration of GO onto the surface of PD5@PTFE (Fig. A.3f), an increased thickness of wrinkled can be observed, indicating the successful formation of PD5/GO layer over the surface of PTFE.
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Chemical composition of the ASP was investigated by XPS. Peaks of C 1s and F 1s are observed in PTFE (Fig. 4 A), PD5@PTFE (Fig. 4 C) and GO@PD5@PTFE (Fig.4 E). However, contents of F in PD5@PTFE and GO@PD5@PTFE are found to decrease significantly compared with that of PTFE because of coating with PD and GO. After coating with PD, the N 1s peak was observed, which was attributed to the amino group from PD (Fig. S1 B, C).The Peak fitting of the C 1s bands of PTFE (Fig. 4 B) yields a major functional groups: C−F bonds from (-CF2-CF2-) n and a small quantity of C-C and C-Si may cause by contaminating. Peak fitting of C 1s bands of PD5@PTFE (Fig. 4 D), in addition to C−F, yields several functional groups including, C−C, C−O and C−N, which was from the bond of PD. Peak fitting of the C 1s bands of GO@PD5@PTFE (Fig. 4 F) also yields several functional groups including, C−C, C-O and C−N as well as C=O, which was from the bond of GO-PD [18].
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The hydrophobicity of PTFE, PD5@PTFE and GO@PD5@PTFE surfaces were evaluated by contact angle measurements. As showed in Fig. 3, the contact angle of the original PTFE was 116°(Fig. 3a). Due to the strong hydrophobicity of PD, the contact angle had declined to about 76°(Fig. 3a) when coated five layers of PD on the surface of PTFE. Correspondingly, after decoration of GO onto the surface of PD5@PTFE, the contact angle was increased to 86°(Fig. 3c), which because of the hydrophobicity of GO, again showing that PD and GO were successfully coated onto the PTFE surface. Fig. 3 3.2. Stability of GO@PD5@PTFE column. The stability of GO@ PD5 coating layer on the PTFE surface of the CCC column was investigated by running 8 consecutive separation tests using operating conditions as described in 2.8. Results show that there is a small part of the ASP layer that was observed to drop during the first three cycles, and then, bare coating 5/5 Page 5 of 18
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1 For CCC, the retention of the stationary phase Table is an important factor that can largely influence the resolution of the analytes. In general, the higher retention of the stationary phase, the higher resolution, which is as (1) [24]
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peeling is found any more. And on the current technical level, 7 - 9 times the separation can be reproducibly achieved by using the coated tubing(as shown in Fig. 7). In addition, in accordance with document report [18], the GO-PD layer has good endurance to organic solvents and was stable even when treated with 1 M HCl and 0.01 M NaOH. Fig. 4 3.3. The retention of stationary phase of the novel column The development of CCC separation largely depends on screening an appropriate solvent system, in which the retention of the stationary phase is a critical measure indicator. As is known to all, PTFE has extremely low coefficient of kinetic friction, so it has almost no resistance to fluid that flow through the PTFE tube. Therefore, PTFE is very suitable to be used as base material of countercurrent chromatography column because it could hardly produce interaction force between the inner wall of PTFE column and solvent system. However, the very weak interaction force between the inner wall of PTFE column and solvent system could impede the retention of stationary phase for further increasing. After coating with GO@PD, the surface of PTFE gets rougher (Figure 3 e, f) and the interaction force between the inner wall of PTFE column and solvent system have enhanced, followed by the maximum column pressure increase from 0.09 to 0.19 MPa . In addition, the contact angle in solvent system has a sharp decrease than original PTFE (See Supporting Information.). Delightedly our work indicated that the retention of stationary phase in novel column was higher about 8% than pre-modified column (Table 1) while the other condition without changed.
(1)
Where RS is the resolution, Sf is the retention of the stationary phase, KD1 is the partition coefficient; N and α are the theoretical plate number and the selective factor. At the same time, Sf can largely influence the retention volume (VR) of solutes, which is as (2)
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(2)
Where VC is the column volume of CCC, KD is the partition coefficient, VS is the volume of stationary phase. In this work, the influence of ASP to VC is negligible. KD is a constant. Thus,
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(3) Where subscript 1and 2 represent that on the original column and the novel column, respectively. Here, VC=300 mL while ∆Sf=0.08., 6/6 Page 6 of 18
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To prove whether the ASP is effective, the hypotheses was raised firstly that those changes in chromatographic behaviour due exclusively to the increase of the stationary phase retention and the coating on the inner surface of the column was simply to increase its friction with a liquid stationary phase, then the ∆VR(S) of analytes, which caused by the rise of Sf, could be obtained from the formulae (3). Since the KD of analytes is less than one (see support information), so the ∆VR value is negative (see Table 2), which means that the VR of analytes decreases with rise of the Sf. However, the calculation result is not in agreement with analytes had increased with 30.4, 40.7, 68.2 mL on the novel the experimental result. The actual VR of three Table 2 column while the calculation result should be decreases with 23.8, 23.5 and 13.2 mL, respectively. That is, the assumption might be wrong , which means that the ASP is effective. Therefore, it can be speculated that the ASP has some influence on the analytes, which has finally brought about the increase of VR (sign as VR(A)).
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3.4. Comparison of the separation behaviour of model analytes In order to prove the effectiveness of the coating, we compared the separation behavior of model analytes between original and novel column, the result is as shown in Fig.5: compared with the original column, significant changes of the separation behavior of model analytes was observed on the novel column, which mainly showed in the following aspects: firstly, the resolution of the analytes was improved on the novel column, especially to peak 2 and peak3 ; secondly, retention time of all analytes were lengthened, peak 3 in particular; thirdly, peak broadening of analyte 2 and 3 were observed on the novel column. Besides, the eluotropic sequence of solutes on novel column was in accordance with that in the original column but each fraction on novel column had a higher purity (as Fig. 6).Taken together, these results further suggested that ASP improved resolution by introducing intermolecular forces.
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The ASP, which consists of GO and PD, contained rich functional groups, which could form different intermolecular forces with the analytes. Hydrogen bonds can easily be generated between ketone, oxhydryl group of model analytes and oxhydryl, carboxyl groups of APS. Furthermore, electrostatic interaction may be generated because alkaloids can be protonated to form the cationic; meanwhile ASP were negatively charged due to the slight dissociation of some carboxyl groups contained in GO and hydroxyl groups contained in PD. Owing to the existence of several aromatic rings in both ASP and analytes, π-π stacking was equally participating in the adsorption process. Resulting from the difference in the structure of analytes, the intermolecular forces are also discrepant, which is conducive to the separation of analytes from each other. In addition, almost all of the Sinomenine hydrochloride and Isocorydine were distributed in the mobile phase because of their low KD（ see table 2） . That could lead them to come into contact and interact with ASP with relative less opportunity than Isocorydione that distributed in both stationary phase and mobile phase. Thanks to the synergetic effect of these factors, the analytes obtained better separation on the novel column.
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Although there are plenty of works concerning preparing for the new type of chromatographic column, no research had focused on the CCC column. This work, for the first time, established a universal approach to improve the separation efficiency of CCC though immobilize sorbents onto the inner wall of CCC column as an ASP. What's all the more remarkable is that the original coating could be degraded and removed by concentrated alkali solutions (PH>14) when it needs to be changed by other modifiers. However, there is still some shortage in our study, such as absorption loss and the rise of the retention time, all this need a 7/7 Page 7 of 18
better resolution to provide compensation. And one important future direction of the work is easier to be prepared and more systematically to be designed， which could achieve better stability and smaller adsorption losses
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4. Conclusion A novel CCC column with auxiliary stationary phase (ASP) in its own right was prepared. Scanning electron microscopy (SEM), contact angle and X-ray photoelectron spectroscopy (XPS) were used to characterize the properties of the coated surface of PTFE. Data show that the modified surfaces exhibited much better wettability, so modification can make HSCCCCCC have higher stationary phase retention than premodified. The chromatographic behavior of model analytes (Isocorydine, Sinomenine hydrochloride and Isocorydione) on the novel CCC column in TBE-300B was investigated combining experimental result and theoretical arithmetic method. The result showed that the ASP improved resolution by introducing intermolecular forces and improving the stationary phase retention ratio. And then followed by existing distributional effects of CCC， a better resolution than conventional CCC was obtained. It is important to say that the proposed approach can be applied to any type of CCC column installed in any type of CCC instrument, so the essence of this manuscript is to present a universal approach for CCC development rather than only to prepare a novel column. This approach might become one of the approaches for CCC scientists to improve their current instruments
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Acknowledgements The authors would like to thank the National Scientific Foundation of China (NSFC no.21175142) and Open Fund of Key Laboratory of Chemistry of Northwestern Plant Resources of The Chinese Academy of Science (no. CNPR-2011kfkt-02). References [1]
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Fig. 1 The chemical structure of model analytes Fig. 2 (A) Scheme of simultaneous polymerization of dopamine[13]. (B) Scheme of the mechanism of on-line prepared of Multilayer PD-modified PTFE Tube and GO immobilized on the PTFE-tube through the PD layer
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Fig. 3 Contact angle and scanning electron micrographs of PTFE tube: (a, d) Original PTFE; (b, e) PD5@PTFE; (c,f) GO@PD5@PTFE. Fig. 4 XPS spectra of (A) Original-PTFE, (C)PD5@PTFE and (E) GO@PD5@PTFE; peak fitting of C1s XPS spectra of (B) Original-PTFE, (D) PD5@PTFE and (F) GO@PD5@PTFE. Fig. 5 Comparison of the separation behavior of model analytes in TBE-300B between original and novel column: 1.Sinomenine hydrochloride; 2.Isocorydine; 3. Isocorydione. Experimental conditions: Apparatus: TBE-300B, 300 mL column volume with
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three coils of 2 mm id tubing. Solvent system: n-hexane–ethyl acetate/methanol/water 5 : 6.5 : 5 : 6.5 v/v.. sample: 1.0 mg of each Isocorydine, Sinomenine hydrochloride and Isocorydione in 1.0 mL of methanol; elution mode: the upper organic phase was the mobile phase ;revolution speed: 850 rpm; flow rate: 2.0 mL min-1;UV detection at 280 nm; Sf::58.3% (the Original
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column),66.2% (the novel column).
Fig. 6 HPLC chromatograms of mixed solution of model analytes and HSCCC peak fractions. Conditions: column, ZORBAX SB-C18 column (150 mm×4.6 mm i.d., 5 µm); the mobile phase consisted of eluent A, phosphoric acid 2% (pH 6.32, adjusted with triethylamine); and eluent B,
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acetonitrile. Gradient: 20% (B) at 0–3 min, 20–50% (B) at 3–4 min, 50% (B) at 4–10 min; flow rate, 1 ml·min-1; detection wavelength: 280 nm; injection volume: 10 µL ;(A) mixed solution of model analytes; (B) combined fractions, peak 1; (C) combined fractions, peak 2; (D) combined fractions, peak 3.
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Fig. 7 HSCCC chromatogram of 13 consecutive runs for separating three model analytes on GO@PD5@PTFE column. Experimental conditions: Apparatus: TBE-300B, 300 mL column volume with three coils of 2 mm id tubing. Solvent system: n-hexane–ethyl acetate/methanol/water 5 : 6.5 : 5 : 6.5 v/v. sample: 1.0 mg of each Isocorydine, Sinomenine hydrochloride and Isocorydione in 1.0 mL of methanol; elution mode: the
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upper organic phase was the mobile phase ;revolution speed: 850 rpm; flow rate: 2.0 mL min-1;UV detection at 280 nm.
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Table 1 Compared the stationary phase retention ratio and column pressure between original and novel column a Column Sf (×100%) flow rate pressure Column no. Solvent system ( V0- Ve) / RSD(×100%) mL·min-1 (-max.) MP V0 n2 1 0.07 57.7 Hexane−EtOAc 2 0.09 58.3 Original 2 −MeOH−Water 2 1.29 CCC 3 58.3 0.09 (1:1.3:1:1.3) column 2 4 0.07 57.0 2 5 0.09 59.0 2 1 0.19 66.2 2 2 0.21 66.9 Novel 2 0.7 CCC 3 0.19 65.5 column 2 4 0.19 66.2 2 5 0.23 66.2
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411 412 413 414
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Note : The stationary phase retention ratio(Sf) was calculated as [25] Conditions: the same as shown in experimental section 2.7.
Table2 The properties of analytes and the ∆VR between original and novel column.
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Note: KD means the partition coefficient of analyte in the given solvent system. ∆VR(S) means the ∆VR value calculated by the formula: ∆VR12=VC·∆Sf12 (1-KD) 417 KD ∆VR12（（ S）） ∆VR12(E) ∆VR12(A)418 molecular compound 419 formula CS/CM mL mL mL 420 421 Sinomenine C19H24ClNO4 0.009 23.8 -30.4 -54.2 422 hydrochloride 423 424 Isocorydine C20H23NO4 0.020 23.5 -40.7 -64.2 425 426 427 428 Isocorydione C18H17NO5 0.452 13.2 -68.2 -81.4 429 430
∆VR(E) means the ∆VR value is the reality difference obtained from experiment result ∆VR(A) means the ∆VR value caused by the influence of the ASP.
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