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Polar-copolymerized approach based on horizontal polymerization on silica surface for preparation of polar-modified stationary phases
Journal of Chromatography A, 1217 (2010) 4555–4560
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Polar-copolymerized approach based on horizontal polymerization on silica surface for preparation of polar-modified stationary phases Zhimou Guo a , Chaoran Wang a , Tu Liang b , Xinmiao Liang a,b,∗ a b
Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 17 January 2010 Received in revised form 21 April 2010 Accepted 23 April 2010 Available online 29 April 2010 Keywords: Reversed-phase liquid chromatography Polar-modified stationary phases Polar-copolymerized Horizontal polymerization
a b s t r a c t A new approach for preparation of polar-modified reversed-phase liquid chromatography stationary phases was developed by using horizontal polymerization technique on silica surface, which was defined as “polar-copolymerized” approach. Based on this new approach, a representative polar-copolymerized stationary phase composed of mixed n-octadecyl and chloropropyl (C18–C3Cl) was synthesized. The resulting stationary phase named C18HCE was characterized with elemental analysis and solid phase 13 C and 29 Si NMR, which proved the chemistry of polar-copolymerized stationary phases. Chromatographic evaluation and application of the C18HCE were also investigated. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited 100% aqueous mobile phase compatibility, low silanol activity. In addition, the application results demonstrated that the C18HCE had superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phases. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Reversed-phase liquid chromatography (RP-HPLC) is the most widely used chromatography technique for analysis of pharmaceutical and biological samples due to the high separation efficiency, broad analytes coverage, MS compatibility. Although silica based conventional C18 stationary phases are the most popular and useful HPLC stationary phases, there are several distinct drawbacks in some cases, such as weak retention and poor selectivity for polar compounds, de-wetting in high aqueous mobile phase conditions, peak tailing of basic compounds [1–4]. Besides the development in silica synthesis and surface bonding technology for preparation of conventional RP-HPLC stationary phases [5,6], polar-modified C18 stationary phases including the polar-endcapped and polarembedded stationary phases were intensively studied and used to resolve the above problems [1–3,7–14]. In addition, Schurig and coworkers developed a polar headed approach for preparation of the polar-modified stationary phases [15]. However, there are also some drawbacks in these approaches. The polar-endcapped method can resolve the de-wetting problem, but the effect on the reducing silanol activity is limited at pH 2.5 [1,2]. Polar-embedded
∗ Corresponding author at: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Tel.: +86 411 84379519; fax: +86 411 84379539. E-mail address: [email protected] (X. Liang). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.04.068
approaches were developed to shield the residual active silanol and improve the polar selectivity [12,16]. However, some of the polarembedded stationary phases usually have lower stability than the conventional alkyl stationary phases [2,15,17]. In addition, the synthetic techniques for preparation of polar-embedded stationary phases are usually complicated. And they bring some other problems, such as residual active polar groups in the two-step methods, reduce the hydrophobicity of the alkyl functionalities [1,4,12,16]. Furthermore, the amount and position of polar groups incorporated in these two polar-modified approaches are restricted. The polar groups in the polar-endcapped stationary phases are arranged at the position of residual silanol and the amount is limited. The polar groups in the polar-embedded stationary phases are inserted in the alkyl groups and the amount is identical to that of alkyl groups. Herein, we proposed a new approach for preparation of polarmodified RP-HPLC stationary phases based on the horizontal polymerization technique which was developed by Wirth and coworkers [18–25]. Horizontal polymerization technique has been developed for preparation of C18 stationary phases, in which mixed self-assembled monolayers of n-octadecyl and methyl chains (C18–C1) or n-octadecyl and propyl chains (C18–C3) form into a dense, two-dimensionally cross-linked network on silica surface to reduce the activity of silanols and enhance the stability of the bonded phases [23,24,26]. To the best of our knowledge, the horizontal polymerization has not been used for synthesis of polar-modified stationary phases. In our new approach, the short alkyl chains are replaced by the polar groups. The mixture of non-
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Fig. 1. Representative surface chemistry of the polar-copolymerized, polar-embedded (amide) and polar-endcapped RPLC stationary phases.
polar silane (e.g. n-octadecyl trichlorosilane) and polar silane (e.g. 3-chloropropyl trichlorosilane) are copolymerized on the silica surface to afford the “polar/non-polar copolymerized stationary phase”, which was very different from the other two approaches (Fig. 1). This new approach is defined as “polar-copolymerized” approach. The polar-copolymerized approach is facile and robust, in which the types and ratios of the polar and non-polar silanes can be adjusted for preparation of well designed stationary phases. In this paper, we present n-octadecyl trichlorosilane and 3-chloropropyl trichlorosilane mixed stationary phase (C18–C3Cl) as an example of polar-copolymerized stationary phases. The preparation and characterization of the polar-polymerized stationary phase will be described. And its chromatographic properties and application in the separation of alkaloids will also be investigated in the present work. 2. Experimental 2.1. Chemicals and materials Spherical silica (5 m particle size; 10 nm pore size; 330 m2 g−1 surface area) was purchased from Fuji Silysia Chemical (Aichi, Japan). Octadecyltrichlorosilane, methyltrichlorosilane and 3chloropropyltrichlorosilane were obtained from ABCR (Karlsruhe, Germany). Compounds used as test probes were obtained from different commercial sources. Acetonitrile of HPLC grade was purchased from Fisher (Fair Lawn, NJ, USA). Water was purified by a Milli-Q water purification system (Billerica, MA, USA). The test probes were dissolved in methanol to about 0.2 mg mL−1 concentration. All other reagents were analytical grade reagents and used without purification. The actual sample of alkaloids from natural products was extracted from the water-extracts of Corydalis yanhusuo Wang. The preparation procedures were according to the reports [27,28]. The sample concentration is about 10 mg mL−1 . Commercial columns for comparison in the separation of alkaloids were purchased from different commercial sources. The columns are listed as follows: Zorbax Eclipse 5 m XDB C18 (80 Å pore size, Agilent, Hewlett-Packard, Palo Alto, CA), Atlantis 5 m T3 C18 (100 Å pore size, Waters, Milford, MA, USA), XBridge 3.5 m C18 (135 Å pore size, Waters, Milford, MA, USA), XTerra 5 m C18
(125 Å pore size, Waters, Milford, MA, USA), Inspire 5 m C18 (100 Å pore size, Dikma, Tianjing, China), Spursil EP 5 m C18 (100 Å pore size, Dikma, Tianjing, China), TSKgel 3 m ODS-100 V (100 Å pore size, Tosoh, Tokyo, Japan). The last two columns (Spursil EP C18 and TSKgel ODS-100 V) are polar-modified RPLC columns. All the columns are 150 mm × 4.6 mm format except for the XTerra 5 m MS C18 (100 mm × 4.6 mm). 2.2. Synthesis of the stationary phases and column packing The synthetic procedures were according to the method of Wirth et al with minor modification [25,26], which were described in brief as follows: silica gel was cleaned by boiling in 2N hydrochloric acid for 12 h, washed with water to neutral and dried at 120 ◦ C overnight. Water was adsorbed to the silica surface by flowing 50% water humidified nitrogen through the silica at least 24 h till the weight increase was to 5–6% of the silica. After humidification, 10 g of silica was placed in a flask under a blanket of nitrogen, along with a stirring bar and 30 mL of anhydrous toluene. A solution of 3.2 mL octadecyl trichlorosilane, 2.4 mL of 3-chloropropyl trichlorosilane (molar ratio of the two silanes is 1:2) and 20 mL of toluene was added to the pretreated silica gel. The reaction was allowed to continue for 24 h. The silanized silica was filtered and washed with dichloromethane, methanol, water and methanol with 20 mL of each solvent successively. The silanized silica was dried at 80 ◦ C overnight. And then the silanized silica was endcapped using a conventional liquid phase reaction by refluxing nearly 10 g of the silanized silica with an mixture of trimethylchlorosilane (3.8 mL), hexamethyldisilazane (7.0 mL) and pyridine (2.0 mL) in 100 mL of dry toluene for 48 h. The endcapped silica was filtered and washed with dichloromethane, methanol, water and methanol. Finally, the materials were dried at 80 ◦ C overnight to obtain the resulting stationary phase (C18HCE, in which H stands for horizontal, C is for chloropropyl and E represents endcapping process). A horizontal polymerized stationary phase with mixed selfassembled monolayers of n-octadecyl and methyl (C18/C1) was synthesized for comparison with C18HCE. The synthesis procedures of C18/C1 are nearly the same as that of C18HCE including the same endcapping procedure. The only difference is the amount of silanes used in the silanization: a solution of 9.5 mL of octadecyl
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Fig. 2. XPS spectra of the C18HCE stationary phase.
Fig. 3.
29
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Si CP/MAS-NMR spectra of the C18HCE stationary phase.
3. Results and discussion trichlorosilane, 0.5 mL of methyl trichlorosilane (molar ratio of the two silanes is 6:1) and 20 mL of toluene was added to the pretreated silica gel (10 g) according to Wirth’s reports [25,26]. The C18HCE (2.8 g) and C18/C1 (2.6 g) was slurry-packed into stainless steel column (150 mm × 4.6 mm), respectively with 20 mL of toluene/acetone (1/1, v/v) as slurry solvent and acetone (80 mL) as propulsion solvent under a pressure of 45 MPa.
2.3. Instrumentation and conditions Solid state 13 C and 29 Si CP/MAS-NMR characterization was performed on a Bruker (USA) DSX 300 NMR Spectrometer (300 MHz, 7.0 T). Elemental analysis was measured on a Vario EL III elemental analysis system (Elementar, Germany) and Kratos XSAM 800 spectrometer (Kratos Analytical, Manchester, UK). Chromatographic evaluation were performed on Alliance HPLC system consisted of a Waters 2695 HPLC pump and a Waters 2996 DAD (Waters, Milford, MA, USA). The mobile phases were consisting of acetonitrile (ACN), methanol and water without or with 20 mM K2 HPO4 /KH2 PO4 (pH 7.0) or 20 mM KH2 PO4 /H3 PO4 (pH 2.5), 0.1% formic acid additives as noted in the figure captions. The column temperature was held constantly at 30 ◦ C and the flow rate was 1 mL min−1 .
Fig. 4.
13
The surface chemistry of the new polar-copolymerized stationary phase was shown in Fig. 1. Alkyl (C18 ligands) and polar functionalities (3-chloropropyl ligands) were copolymerized on the silica surface. In this type of polar-modified alkyl stationary phases, the alkyl ligands provide the hydrophobicity and the presence of copolymerized polar groups could improve the wetting properties and polar selectivity. The synthesis of the new phases is very simple. In this approach, it is just needed to mix the non-polar and polar silanes in the reaction mixture, which avoid synthesis of polar-embedded alkyl silanes prior to sinalization. In addition, many kinds of non-polar ligands (such as C18, C8, phenyl) and silanes with polar groups (such as cyano, amino, sulphonic groups) could be employed to synthesis of well designed stationary phases with different separation properties. Moreover the molar ratio of non-polar ligands and polar silanes could be controlled by adjusting the feed ratio of the silanes. Organic elemental analysis (EA) results indicated that the carbon content of C18HCE stationary phase is 11.5% and the hydrogen content is 2.16%. But EA could not detect chloride element. Therefore, X-ray photoelectron spectroscopy (XPS) was employed to detect the surface elemental composition (Fig. 2). The XPS spectrum clearly showed the presence of Cl (Cl 2p: 203 eV) and C (C 1s: 285 eV) on the silica surface, which confirmed the successful bonding of chloride. The content of chlorine is about 2.45% based on the
C CP/MAS-NMR spectra of the C18HCE stationary phase.
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Fig. 5. Chromatograms of de-wetting test. Conditions: mobile phase, 10 mM NH4 Ac (pH 3); flow rate: 1.0 mL/min; column temperature: 30 ◦ C; UV detection: 260 nm. After running the first cycle (before stop flow), the flow was stopped for 30 min before the next injection and running (After stop flow).
Fig. 7. The chromatogram of accessible hydrogen bonding test. Conditions: mobile phase, ACN/water (30:70, v/v); flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 280 nm.
integral results of the XPS spectrum. But it should be noted that XPS provide semi-quantitative result. The precise surface coverage of C18HCE will be measured by other quantitative surface analysis techniques in due course. By the way, the horizontal polymerized stationary phase C18/C1 for comparison was also characterized by EA. The carbon content of C18/C1 is 19.26% and the hydrogen content is 3.56%. The 29 Si and 13 C CP/MAS-NMR spectroscopy were used to investigate the different silicon species and bonded ligands on the silica surface. According to Maciel’s NMR work [29], the NMR spectrum shown in Fig. 3 can be assigned. The peaks at −109 and −101 ppm correspond to the Q4 (siloxanes) and Q3 (free and vicinal silanols) silicon atoms. The peaks at −66 and −57 ppm correspond to the reagent silicon atoms having no terminal hydroxyl (T3 ) and one terminal hydroxyl (T2 ). And the peak at +11 ppm corresponds to the silicon atoms attached to the trimethyl groups from the endcapping process. T3 are the idea silicon atoms which are the desired, fully cross-linked reagent silicon atoms of polar-copolymerized stationary phase as shown in Fig. 1. The intensity of T3 is much larger than that of T2 and the signal of T1 silicon atoms are almost not detected, which confirm that there are few residual silanols due to the horizontal polymerization technique [19–21,23,25] and exhaustive endcapping. The results of 29 Si CP/MAS-NMR suggest the C18HCE stationary phase would provide low silanol activity.
Fig. 4 shows the 13 C CP/MAS-NMR spectrum of the C18HCE stationary phase. The peaks in the 13 C CP/MAS-NMR spectrum can be assigned according to the previous reports on the polar-modified stationary phases [2,8,12]. The resonance at 47 ppm relates to the carbon atoms attached to chloride atoms, which confirmed the presence of polar groups bonded on the surface. The peaks at 11–32 ppm are assigned to the carbon atoms on the C18 ligand and other two atoms on the C3Cl ligand. The peak M at 2 ppm corresponds to the methyl groups from the endcapping reagents. The 13 C CP/MAS-NMR spectrum proves the chemistry of the C18HCE stationary phase. The chromatographic properties of the C18HCE were investigated with series of test probes. First of all, as a type of new polar-modified alkyl stationary phase, the wetting property of the C18HCE in 100% aqueous should be investigated. In this work, wetting property of the C18HCE was investigated with a “stopflow” test in a 100% aqueous mobile phase [2]. As shown in Fig. 5a, the C18HCE column provides stable retentions before and after stopping the flow. Therefore, like polar-embedded and polar-endcapped stationary phases, the new polar-copolymerized stationary phase demonstrates excellent compatibility with 100% aqueous mobile phase. To prove the role of chloropropyl in the improvement of wetting property, “stopflow” test in the 100% aqueous mobile phase was also carried out on the horizontal polymerized alkyl stationary phase C18/C1. All the four solutes have no retention on the C18/C1
Fig. 6. The chromatogram of some compounds of the Engelhardt test mixture. Conditions: mobile phase, methanol/water (55:45, v/v); flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 260 nm.
Fig. 8. Chromatograms of silanol activity tests. Conditions: mobile phase, ACN/20 mM KH2 PO4 –H3 PO4 , pH 2.5 (30:70, v/v) (acidic condition) and ACN/20 mM K2 HPO4 –KH2 PO4 , pH 7.0 (30:70, v/v) (neutral condition); flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 280 nm.
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Fig. 9. Separations of alkaloids components extracted from a Traditional Chinese Medicines Corydalis yanhusuo Wang on C18HCE column (a and b) and ODS columns for comparison ((c) Zorbax Eclipse 5 m XDB C18; (d) Atlantis 5 m T3 C18; (e) Xbridge 3.5 m C18; (f) Xterra 5 m C18; (g) inspire 5 m C18; (h) spursil EP 5 m C18; (i) TSKgel 3 m ODS–100 V; (j) self-made 5 m C18/C1). Conditions: mobile phase (A) 0.1% formic acid water, (B) ACN; gradient for C18HCE: 0–30 min, 0% → 30% B and gradient for commercial columns: 0–30 min, 5% → 35% B; 30–40 min, 35% → 60% B; injection volume: (a, c–i) 50 L and (b) 100 L; flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 254 nm.
after stopping flow (Fig. 5b), which is the typical phenomenon of de-wetting. The opposite results in the “stopflow” test indicated that the improving wetting property of the C18HCE was brought from the copolymerized chloropropyl group. Secondly, the test probes were composed of uracil, aniline, phenol, N,N-dimethyl aniline (DMA), toluene, ethylbenzene, which are from the test mixture suggested by Engelhardt et al [30,31]. The chromatogram obtained on the C18HCE in neutral conditions (methanol/water, 55:45, v/v) is shown in Fig. 6. The retention factor and selectivity of ethylbenzene and toluene (˛E/T ) indicates the hydrophobicity of the stationary phase. The retention factor of ethylbenzene is 12 and ˛E/T is 1.76, which demonstrated the hydrophobicity of the C18HCE. The peak shape and efficiency for the weakly basic solutes (aniline and DMA) provide information about silanol activity. The tailing factors and separation efficiency of DMA is 1.01 and 11400, which indicated the silanol activity on the C18HCE stationary phase is well shielded. The weak retention and good peak shape of aniline also demonstrated the low silanol activity. The amount of accessible hydrogen bonding sites from silanol groups was tested from the selectivity between caffeine and phenol (˛C/P ) in the mobile phase of ACN/water (30:70, v/v). As shown in Fig. 7, the peak shape of caffeine is very good and ˛C/P is about 0.17, which indicated that the accessible hydrogen bonding sites on the surface of C18HCE have been reduced by this new bonding technique [1]. In addition, the silanol activity under acidic and neutral conditions were also determined by using benzylamine and phenol in ACN/20 mM phosphate buffer (pH 2.5 and pH 7.0) [1]. The residual silanols are ionized under pH 7.0 and the total ion-exchange capacity of the C18HCE stationary phase would be determined at pH 7.0. The majority of silanols are protonated at pH 2.5 and the silanophilic interaction would be tested at pH 2.5. As shown in Fig. 8, the peak shape of benzylamine and phenol under neutral condition is not tailing and the selectivity between benzylamine and phenol is about 0.20, which indicated that the silanol activity and ion-exchange capacity is not significant [1]. It should be noted that benzylamine eluted before dead time marker (uracil) at pH 2.5. The results indicated that ionic repulsion occurs between the stationary phase and benzylamine under the acidic condition, which is similar with other types of silica based stationary phases and helpful to improve the peak shape of alkaloids [1,32]. But the actual reason of
the significant positive charge was not clear at present. In summary, the results of chromatographic evaluation demonstrated the low silanol activity of C18HCE stationary phase especially under acidic conditions, which will be very beneficial in alkaloids separation. As we known, separation of alkaloids with symmetric peak shape and high loadability is a difficult work on the silica based RPLC column. In order to demonstrate the application potential, the C18HCE column was employed in the separation of alkaloids sample which was extracted from the water-extracts of Corydalis yanhusuo Wang. The main components in this sample are protoberberine alkaloids, which are quaternary alkaloids and difficult to obtain good peak shape and sample loading in the RPLC separation. Besides the horizontal polymerized stationary phase C18/C1, several commercial C18 columns including conventional alkyl bonded phases and polar-modified alkyl bonded phases (Spursil EP C18 and TSKgel ODS-100 V) on silica and organic/inorganic hybrid silica support were used for comparison. As shown in Fig. 9, the peak shape obtained on the C18HCE column was good (Fig. 9a) even with high sample loading (Fig. 9b). But on the other stationary phases (Fig. 9c–j), the peaks were obviously tailing and wider than that on the C18HCE column, which indicated that the C18HCE demonstrated superior performance in the alkaloids separation. The very different separation results obtained on C18/C1 (Fig. 9j) and C18HCE (Fig. 9a and 9b) prove the importance of polar group (chloropropyl) for the superior performance in the alkaloids separation. In addition, the retention is weaker on C18HCE column than that on the other columns at the conditions, which may be due to the low silanol activity and surface positive charge. The above results demonstrated the great power of C18HCE in the alkaloids separation especially in preparative scales. But the deeply reason of the excellent of peak shape and loadability of C18HCE in the alkaloids separation is not clear so far. 4. Conclusion This work introduced a new approach based on horizontal polymerization on silica surface for preparation of polar-modified RPLC stationary phases. This new approach (polar-copolymerized) is facile. A representative stationary phase C18HCE was synthesized with this method. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited
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typical characteristics of polar-modified RPLC stationary phases, such as 100% aqueous mobile phase compatibility, low silanol activity. Moreover, the C18HCE showed superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phase. The detailed synthetic methods, characterization, chromatographic evaluation, chemical stability test and application are undergoing in our laboratory. We believe that this polar-copolymerized approach together with the new stationary phases based on this approach will become useful in a wide range and complement the synthetic strategies and application of polar-modified stationary phases.
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Acknowledgements The authors gratefully acknowledge the financial support from the China National Funds for Distinguished Young Scientists (Grant: 20825518) and Project of Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-YW-R-214 and KSCX2-YWR-170). References [1] J. Layne, J. Chromatogr. A 957 (2002) 149. [2] X.D. Liu, A. Bordunov, M. Tracy, R. Slingsby, N. Avdalovic, C. Pohl, J. Chromatogr. A 1119 (2006) 120. [3] J.L. Rafferty, J.I. Siepmann, M.R. Schure, Anal. Chem. 80 (2008) 6214. [4] J.W. Coym, J. Sep. Sci. 31 (2008) 1712. [5] B.C. Trammell, L.J. Ma, H. Luo, D.H. Jin, M.A. Hillmyer, P.W. Carr, Anal. Chem. 74 (2002) 4634.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Polar-copolymerized approach based on horizontal polymerization on silica surface for preparation of polar-modified stationary phases Zhimou Guo a , Chaoran Wang a , Tu Liang b , Xinmiao Liang a,b,∗ a b
Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 17 January 2010 Received in revised form 21 April 2010 Accepted 23 April 2010 Available online 29 April 2010 Keywords: Reversed-phase liquid chromatography Polar-modified stationary phases Polar-copolymerized Horizontal polymerization
a b s t r a c t A new approach for preparation of polar-modified reversed-phase liquid chromatography stationary phases was developed by using horizontal polymerization technique on silica surface, which was defined as “polar-copolymerized” approach. Based on this new approach, a representative polar-copolymerized stationary phase composed of mixed n-octadecyl and chloropropyl (C18–C3Cl) was synthesized. The resulting stationary phase named C18HCE was characterized with elemental analysis and solid phase 13 C and 29 Si NMR, which proved the chemistry of polar-copolymerized stationary phases. Chromatographic evaluation and application of the C18HCE were also investigated. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited 100% aqueous mobile phase compatibility, low silanol activity. In addition, the application results demonstrated that the C18HCE had superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phases. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Reversed-phase liquid chromatography (RP-HPLC) is the most widely used chromatography technique for analysis of pharmaceutical and biological samples due to the high separation efficiency, broad analytes coverage, MS compatibility. Although silica based conventional C18 stationary phases are the most popular and useful HPLC stationary phases, there are several distinct drawbacks in some cases, such as weak retention and poor selectivity for polar compounds, de-wetting in high aqueous mobile phase conditions, peak tailing of basic compounds [1–4]. Besides the development in silica synthesis and surface bonding technology for preparation of conventional RP-HPLC stationary phases [5,6], polar-modified C18 stationary phases including the polar-endcapped and polarembedded stationary phases were intensively studied and used to resolve the above problems [1–3,7–14]. In addition, Schurig and coworkers developed a polar headed approach for preparation of the polar-modified stationary phases [15]. However, there are also some drawbacks in these approaches. The polar-endcapped method can resolve the de-wetting problem, but the effect on the reducing silanol activity is limited at pH 2.5 [1,2]. Polar-embedded
∗ Corresponding author at: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Tel.: +86 411 84379519; fax: +86 411 84379539. E-mail address: [email protected] (X. Liang). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.04.068
approaches were developed to shield the residual active silanol and improve the polar selectivity [12,16]. However, some of the polarembedded stationary phases usually have lower stability than the conventional alkyl stationary phases [2,15,17]. In addition, the synthetic techniques for preparation of polar-embedded stationary phases are usually complicated. And they bring some other problems, such as residual active polar groups in the two-step methods, reduce the hydrophobicity of the alkyl functionalities [1,4,12,16]. Furthermore, the amount and position of polar groups incorporated in these two polar-modified approaches are restricted. The polar groups in the polar-endcapped stationary phases are arranged at the position of residual silanol and the amount is limited. The polar groups in the polar-embedded stationary phases are inserted in the alkyl groups and the amount is identical to that of alkyl groups. Herein, we proposed a new approach for preparation of polarmodified RP-HPLC stationary phases based on the horizontal polymerization technique which was developed by Wirth and coworkers [18–25]. Horizontal polymerization technique has been developed for preparation of C18 stationary phases, in which mixed self-assembled monolayers of n-octadecyl and methyl chains (C18–C1) or n-octadecyl and propyl chains (C18–C3) form into a dense, two-dimensionally cross-linked network on silica surface to reduce the activity of silanols and enhance the stability of the bonded phases [23,24,26]. To the best of our knowledge, the horizontal polymerization has not been used for synthesis of polar-modified stationary phases. In our new approach, the short alkyl chains are replaced by the polar groups. The mixture of non-
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Fig. 1. Representative surface chemistry of the polar-copolymerized, polar-embedded (amide) and polar-endcapped RPLC stationary phases.
polar silane (e.g. n-octadecyl trichlorosilane) and polar silane (e.g. 3-chloropropyl trichlorosilane) are copolymerized on the silica surface to afford the “polar/non-polar copolymerized stationary phase”, which was very different from the other two approaches (Fig. 1). This new approach is defined as “polar-copolymerized” approach. The polar-copolymerized approach is facile and robust, in which the types and ratios of the polar and non-polar silanes can be adjusted for preparation of well designed stationary phases. In this paper, we present n-octadecyl trichlorosilane and 3-chloropropyl trichlorosilane mixed stationary phase (C18–C3Cl) as an example of polar-copolymerized stationary phases. The preparation and characterization of the polar-polymerized stationary phase will be described. And its chromatographic properties and application in the separation of alkaloids will also be investigated in the present work. 2. Experimental 2.1. Chemicals and materials Spherical silica (5 m particle size; 10 nm pore size; 330 m2 g−1 surface area) was purchased from Fuji Silysia Chemical (Aichi, Japan). Octadecyltrichlorosilane, methyltrichlorosilane and 3chloropropyltrichlorosilane were obtained from ABCR (Karlsruhe, Germany). Compounds used as test probes were obtained from different commercial sources. Acetonitrile of HPLC grade was purchased from Fisher (Fair Lawn, NJ, USA). Water was purified by a Milli-Q water purification system (Billerica, MA, USA). The test probes were dissolved in methanol to about 0.2 mg mL−1 concentration. All other reagents were analytical grade reagents and used without purification. The actual sample of alkaloids from natural products was extracted from the water-extracts of Corydalis yanhusuo Wang. The preparation procedures were according to the reports [27,28]. The sample concentration is about 10 mg mL−1 . Commercial columns for comparison in the separation of alkaloids were purchased from different commercial sources. The columns are listed as follows: Zorbax Eclipse 5 m XDB C18 (80 Å pore size, Agilent, Hewlett-Packard, Palo Alto, CA), Atlantis 5 m T3 C18 (100 Å pore size, Waters, Milford, MA, USA), XBridge 3.5 m C18 (135 Å pore size, Waters, Milford, MA, USA), XTerra 5 m C18
(125 Å pore size, Waters, Milford, MA, USA), Inspire 5 m C18 (100 Å pore size, Dikma, Tianjing, China), Spursil EP 5 m C18 (100 Å pore size, Dikma, Tianjing, China), TSKgel 3 m ODS-100 V (100 Å pore size, Tosoh, Tokyo, Japan). The last two columns (Spursil EP C18 and TSKgel ODS-100 V) are polar-modified RPLC columns. All the columns are 150 mm × 4.6 mm format except for the XTerra 5 m MS C18 (100 mm × 4.6 mm). 2.2. Synthesis of the stationary phases and column packing The synthetic procedures were according to the method of Wirth et al with minor modification [25,26], which were described in brief as follows: silica gel was cleaned by boiling in 2N hydrochloric acid for 12 h, washed with water to neutral and dried at 120 ◦ C overnight. Water was adsorbed to the silica surface by flowing 50% water humidified nitrogen through the silica at least 24 h till the weight increase was to 5–6% of the silica. After humidification, 10 g of silica was placed in a flask under a blanket of nitrogen, along with a stirring bar and 30 mL of anhydrous toluene. A solution of 3.2 mL octadecyl trichlorosilane, 2.4 mL of 3-chloropropyl trichlorosilane (molar ratio of the two silanes is 1:2) and 20 mL of toluene was added to the pretreated silica gel. The reaction was allowed to continue for 24 h. The silanized silica was filtered and washed with dichloromethane, methanol, water and methanol with 20 mL of each solvent successively. The silanized silica was dried at 80 ◦ C overnight. And then the silanized silica was endcapped using a conventional liquid phase reaction by refluxing nearly 10 g of the silanized silica with an mixture of trimethylchlorosilane (3.8 mL), hexamethyldisilazane (7.0 mL) and pyridine (2.0 mL) in 100 mL of dry toluene for 48 h. The endcapped silica was filtered and washed with dichloromethane, methanol, water and methanol. Finally, the materials were dried at 80 ◦ C overnight to obtain the resulting stationary phase (C18HCE, in which H stands for horizontal, C is for chloropropyl and E represents endcapping process). A horizontal polymerized stationary phase with mixed selfassembled monolayers of n-octadecyl and methyl (C18/C1) was synthesized for comparison with C18HCE. The synthesis procedures of C18/C1 are nearly the same as that of C18HCE including the same endcapping procedure. The only difference is the amount of silanes used in the silanization: a solution of 9.5 mL of octadecyl
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Fig. 2. XPS spectra of the C18HCE stationary phase.
Fig. 3.
29
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Si CP/MAS-NMR spectra of the C18HCE stationary phase.
3. Results and discussion trichlorosilane, 0.5 mL of methyl trichlorosilane (molar ratio of the two silanes is 6:1) and 20 mL of toluene was added to the pretreated silica gel (10 g) according to Wirth’s reports [25,26]. The C18HCE (2.8 g) and C18/C1 (2.6 g) was slurry-packed into stainless steel column (150 mm × 4.6 mm), respectively with 20 mL of toluene/acetone (1/1, v/v) as slurry solvent and acetone (80 mL) as propulsion solvent under a pressure of 45 MPa.
2.3. Instrumentation and conditions Solid state 13 C and 29 Si CP/MAS-NMR characterization was performed on a Bruker (USA) DSX 300 NMR Spectrometer (300 MHz, 7.0 T). Elemental analysis was measured on a Vario EL III elemental analysis system (Elementar, Germany) and Kratos XSAM 800 spectrometer (Kratos Analytical, Manchester, UK). Chromatographic evaluation were performed on Alliance HPLC system consisted of a Waters 2695 HPLC pump and a Waters 2996 DAD (Waters, Milford, MA, USA). The mobile phases were consisting of acetonitrile (ACN), methanol and water without or with 20 mM K2 HPO4 /KH2 PO4 (pH 7.0) or 20 mM KH2 PO4 /H3 PO4 (pH 2.5), 0.1% formic acid additives as noted in the figure captions. The column temperature was held constantly at 30 ◦ C and the flow rate was 1 mL min−1 .
Fig. 4.
13
The surface chemistry of the new polar-copolymerized stationary phase was shown in Fig. 1. Alkyl (C18 ligands) and polar functionalities (3-chloropropyl ligands) were copolymerized on the silica surface. In this type of polar-modified alkyl stationary phases, the alkyl ligands provide the hydrophobicity and the presence of copolymerized polar groups could improve the wetting properties and polar selectivity. The synthesis of the new phases is very simple. In this approach, it is just needed to mix the non-polar and polar silanes in the reaction mixture, which avoid synthesis of polar-embedded alkyl silanes prior to sinalization. In addition, many kinds of non-polar ligands (such as C18, C8, phenyl) and silanes with polar groups (such as cyano, amino, sulphonic groups) could be employed to synthesis of well designed stationary phases with different separation properties. Moreover the molar ratio of non-polar ligands and polar silanes could be controlled by adjusting the feed ratio of the silanes. Organic elemental analysis (EA) results indicated that the carbon content of C18HCE stationary phase is 11.5% and the hydrogen content is 2.16%. But EA could not detect chloride element. Therefore, X-ray photoelectron spectroscopy (XPS) was employed to detect the surface elemental composition (Fig. 2). The XPS spectrum clearly showed the presence of Cl (Cl 2p: 203 eV) and C (C 1s: 285 eV) on the silica surface, which confirmed the successful bonding of chloride. The content of chlorine is about 2.45% based on the
C CP/MAS-NMR spectra of the C18HCE stationary phase.
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Fig. 5. Chromatograms of de-wetting test. Conditions: mobile phase, 10 mM NH4 Ac (pH 3); flow rate: 1.0 mL/min; column temperature: 30 ◦ C; UV detection: 260 nm. After running the first cycle (before stop flow), the flow was stopped for 30 min before the next injection and running (After stop flow).
Fig. 7. The chromatogram of accessible hydrogen bonding test. Conditions: mobile phase, ACN/water (30:70, v/v); flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 280 nm.
integral results of the XPS spectrum. But it should be noted that XPS provide semi-quantitative result. The precise surface coverage of C18HCE will be measured by other quantitative surface analysis techniques in due course. By the way, the horizontal polymerized stationary phase C18/C1 for comparison was also characterized by EA. The carbon content of C18/C1 is 19.26% and the hydrogen content is 3.56%. The 29 Si and 13 C CP/MAS-NMR spectroscopy were used to investigate the different silicon species and bonded ligands on the silica surface. According to Maciel’s NMR work [29], the NMR spectrum shown in Fig. 3 can be assigned. The peaks at −109 and −101 ppm correspond to the Q4 (siloxanes) and Q3 (free and vicinal silanols) silicon atoms. The peaks at −66 and −57 ppm correspond to the reagent silicon atoms having no terminal hydroxyl (T3 ) and one terminal hydroxyl (T2 ). And the peak at +11 ppm corresponds to the silicon atoms attached to the trimethyl groups from the endcapping process. T3 are the idea silicon atoms which are the desired, fully cross-linked reagent silicon atoms of polar-copolymerized stationary phase as shown in Fig. 1. The intensity of T3 is much larger than that of T2 and the signal of T1 silicon atoms are almost not detected, which confirm that there are few residual silanols due to the horizontal polymerization technique [19–21,23,25] and exhaustive endcapping. The results of 29 Si CP/MAS-NMR suggest the C18HCE stationary phase would provide low silanol activity.
Fig. 4 shows the 13 C CP/MAS-NMR spectrum of the C18HCE stationary phase. The peaks in the 13 C CP/MAS-NMR spectrum can be assigned according to the previous reports on the polar-modified stationary phases [2,8,12]. The resonance at 47 ppm relates to the carbon atoms attached to chloride atoms, which confirmed the presence of polar groups bonded on the surface. The peaks at 11–32 ppm are assigned to the carbon atoms on the C18 ligand and other two atoms on the C3Cl ligand. The peak M at 2 ppm corresponds to the methyl groups from the endcapping reagents. The 13 C CP/MAS-NMR spectrum proves the chemistry of the C18HCE stationary phase. The chromatographic properties of the C18HCE were investigated with series of test probes. First of all, as a type of new polar-modified alkyl stationary phase, the wetting property of the C18HCE in 100% aqueous should be investigated. In this work, wetting property of the C18HCE was investigated with a “stopflow” test in a 100% aqueous mobile phase [2]. As shown in Fig. 5a, the C18HCE column provides stable retentions before and after stopping the flow. Therefore, like polar-embedded and polar-endcapped stationary phases, the new polar-copolymerized stationary phase demonstrates excellent compatibility with 100% aqueous mobile phase. To prove the role of chloropropyl in the improvement of wetting property, “stopflow” test in the 100% aqueous mobile phase was also carried out on the horizontal polymerized alkyl stationary phase C18/C1. All the four solutes have no retention on the C18/C1
Fig. 6. The chromatogram of some compounds of the Engelhardt test mixture. Conditions: mobile phase, methanol/water (55:45, v/v); flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 260 nm.
Fig. 8. Chromatograms of silanol activity tests. Conditions: mobile phase, ACN/20 mM KH2 PO4 –H3 PO4 , pH 2.5 (30:70, v/v) (acidic condition) and ACN/20 mM K2 HPO4 –KH2 PO4 , pH 7.0 (30:70, v/v) (neutral condition); flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 280 nm.
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Fig. 9. Separations of alkaloids components extracted from a Traditional Chinese Medicines Corydalis yanhusuo Wang on C18HCE column (a and b) and ODS columns for comparison ((c) Zorbax Eclipse 5 m XDB C18; (d) Atlantis 5 m T3 C18; (e) Xbridge 3.5 m C18; (f) Xterra 5 m C18; (g) inspire 5 m C18; (h) spursil EP 5 m C18; (i) TSKgel 3 m ODS–100 V; (j) self-made 5 m C18/C1). Conditions: mobile phase (A) 0.1% formic acid water, (B) ACN; gradient for C18HCE: 0–30 min, 0% → 30% B and gradient for commercial columns: 0–30 min, 5% → 35% B; 30–40 min, 35% → 60% B; injection volume: (a, c–i) 50 L and (b) 100 L; flow rate: 1.0 mL min−1 ; column temperature: 30 ◦ C; UV detection: 254 nm.
after stopping flow (Fig. 5b), which is the typical phenomenon of de-wetting. The opposite results in the “stopflow” test indicated that the improving wetting property of the C18HCE was brought from the copolymerized chloropropyl group. Secondly, the test probes were composed of uracil, aniline, phenol, N,N-dimethyl aniline (DMA), toluene, ethylbenzene, which are from the test mixture suggested by Engelhardt et al [30,31]. The chromatogram obtained on the C18HCE in neutral conditions (methanol/water, 55:45, v/v) is shown in Fig. 6. The retention factor and selectivity of ethylbenzene and toluene (˛E/T ) indicates the hydrophobicity of the stationary phase. The retention factor of ethylbenzene is 12 and ˛E/T is 1.76, which demonstrated the hydrophobicity of the C18HCE. The peak shape and efficiency for the weakly basic solutes (aniline and DMA) provide information about silanol activity. The tailing factors and separation efficiency of DMA is 1.01 and 11400, which indicated the silanol activity on the C18HCE stationary phase is well shielded. The weak retention and good peak shape of aniline also demonstrated the low silanol activity. The amount of accessible hydrogen bonding sites from silanol groups was tested from the selectivity between caffeine and phenol (˛C/P ) in the mobile phase of ACN/water (30:70, v/v). As shown in Fig. 7, the peak shape of caffeine is very good and ˛C/P is about 0.17, which indicated that the accessible hydrogen bonding sites on the surface of C18HCE have been reduced by this new bonding technique [1]. In addition, the silanol activity under acidic and neutral conditions were also determined by using benzylamine and phenol in ACN/20 mM phosphate buffer (pH 2.5 and pH 7.0) [1]. The residual silanols are ionized under pH 7.0 and the total ion-exchange capacity of the C18HCE stationary phase would be determined at pH 7.0. The majority of silanols are protonated at pH 2.5 and the silanophilic interaction would be tested at pH 2.5. As shown in Fig. 8, the peak shape of benzylamine and phenol under neutral condition is not tailing and the selectivity between benzylamine and phenol is about 0.20, which indicated that the silanol activity and ion-exchange capacity is not significant [1]. It should be noted that benzylamine eluted before dead time marker (uracil) at pH 2.5. The results indicated that ionic repulsion occurs between the stationary phase and benzylamine under the acidic condition, which is similar with other types of silica based stationary phases and helpful to improve the peak shape of alkaloids [1,32]. But the actual reason of
the significant positive charge was not clear at present. In summary, the results of chromatographic evaluation demonstrated the low silanol activity of C18HCE stationary phase especially under acidic conditions, which will be very beneficial in alkaloids separation. As we known, separation of alkaloids with symmetric peak shape and high loadability is a difficult work on the silica based RPLC column. In order to demonstrate the application potential, the C18HCE column was employed in the separation of alkaloids sample which was extracted from the water-extracts of Corydalis yanhusuo Wang. The main components in this sample are protoberberine alkaloids, which are quaternary alkaloids and difficult to obtain good peak shape and sample loading in the RPLC separation. Besides the horizontal polymerized stationary phase C18/C1, several commercial C18 columns including conventional alkyl bonded phases and polar-modified alkyl bonded phases (Spursil EP C18 and TSKgel ODS-100 V) on silica and organic/inorganic hybrid silica support were used for comparison. As shown in Fig. 9, the peak shape obtained on the C18HCE column was good (Fig. 9a) even with high sample loading (Fig. 9b). But on the other stationary phases (Fig. 9c–j), the peaks were obviously tailing and wider than that on the C18HCE column, which indicated that the C18HCE demonstrated superior performance in the alkaloids separation. The very different separation results obtained on C18/C1 (Fig. 9j) and C18HCE (Fig. 9a and 9b) prove the importance of polar group (chloropropyl) for the superior performance in the alkaloids separation. In addition, the retention is weaker on C18HCE column than that on the other columns at the conditions, which may be due to the low silanol activity and surface positive charge. The above results demonstrated the great power of C18HCE in the alkaloids separation especially in preparative scales. But the deeply reason of the excellent of peak shape and loadability of C18HCE in the alkaloids separation is not clear so far. 4. Conclusion This work introduced a new approach based on horizontal polymerization on silica surface for preparation of polar-modified RPLC stationary phases. This new approach (polar-copolymerized) is facile. A representative stationary phase C18HCE was synthesized with this method. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited
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typical characteristics of polar-modified RPLC stationary phases, such as 100% aqueous mobile phase compatibility, low silanol activity. Moreover, the C18HCE showed superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phase. The detailed synthetic methods, characterization, chromatographic evaluation, chemical stability test and application are undergoing in our laboratory. We believe that this polar-copolymerized approach together with the new stationary phases based on this approach will become useful in a wide range and complement the synthetic strategies and application of polar-modified stationary phases.
[6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
Acknowledgements The authors gratefully acknowledge the financial support from the China National Funds for Distinguished Young Scientists (Grant: 20825518) and Project of Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-YW-R-214 and KSCX2-YWR-170). References [1] J. Layne, J. Chromatogr. A 957 (2002) 149. [2] X.D. Liu, A. Bordunov, M. Tracy, R. Slingsby, N. Avdalovic, C. Pohl, J. Chromatogr. A 1119 (2006) 120. [3] J.L. Rafferty, J.I. Siepmann, M.R. Schure, Anal. Chem. 80 (2008) 6214. [4] J.W. Coym, J. Sep. Sci. 31 (2008) 1712. [5] B.C. Trammell, L.J. Ma, H. Luo, D.H. Jin, M.A. Hillmyer, P.W. Carr, Anal. Chem. 74 (2002) 4634.
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