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Polymeric ionic liquid modified organic-silica hybrid monolithic column for capillary electrochromatography
Journal of Chromatography A, 1246 (2012) 9–14
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Polymeric ionic liquid modified organic-silica hybrid monolithic column for capillary electrochromatography Haifeng Han a,b , Qing Wang a,b , Xia Liu a,∗ , Shengxiang Jiang a a 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 b Graduate School of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Available online 14 December 2011 Keywords: CEC Monolithic column Organic-silica hybrid Polymeric ionic liquid
a b s t r a c t A polymeric ionic liquid (PIL) modified hybrid monolithic column for CEC was synthesized on the basis of mercaptopropy-functionalized (MP-silica) hybrid monolithic column which was prepared by the in situ co-condensation of tetramethoxysilane and 3-mercaptopropyltrimethoxysilane via a sol–gel process. The PIL modified (PImC8 -silica) hybrid monolithic column, which was characterized by FT-IR and Xray Photoelectron spectroscopy (XPS), generated a strong reversed and relatively stable EOF in a wide range of pH (3.0–8.0). The PImC8 -silica hybrid monolithic column was evaluated by the separation of three aromatic hydrocarbons (AHs), four alkylbenzenes and five phenols, respectively, with phosphate buffers containing different ratios of acetonitrile as mobile phase. Reproducibilities of the column were also investigated by measuring RSDs of the migration time for AHs. RSDs of run-to-run (n = 5), dayto-day (n = 3) and column-to-column (n = 3) were in the range of 0.26–0.87%, 0.90–3.2% and 1.5–5.7%, respectively. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The capillary column is the core of CEC, which combines superiorities of the CE and HPLC. Monoliths as separation columns have been widely used in CEC due to its continuous porous bed, good permeability, high phase ratios, high performance, high speed and good selectivity [1–3]. The significant aspect of monolithic materials in general and those based on synthetic polymers is the ease of their preparation, which avoids the problems related to both frit fabrication and packing [4]. Monolithic columns are synthesized by the in situ preparation of polymer-based or silica-based monoliths with different functionalized groups [5]. Organic polymer-based, inorganic silica-based and organicsilica hybrid monolithic columns are categorized based on the nature of matrix chemistry [6,7]. Organic monolithic columns have good stability in a wide range of pH and porous properties can be easily tailored by tuning the composition of the porogenic agent, monomer and cross-linking agent in the mixed solution [8]. Inorganic silica-based columns exhibit excellent mechanical stability and good solvent resistance, prepared via a sol–gel process [9]. However, many researchers have paid attention to organic-silica hybrid monolithic columns because of the ease of preparation,
∗ Corresponding author. Tel.: +86 931 4968203; fax: +86 931 8277088. E-mail address: [email protected] (X. Liu). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.12.029
good mechanical stability and good pH stability. The organic-silica hybrid monolithic columns are synthesized through a “one pot” approach by bonding different functional groups such as octyl [10], allyl [11], vinyl [12,13], phenyl [14], amino [15], mercapto [16] and chloropropyl [3]. Ionic liquids (ILs) containing relatively large organic cations and inorganic anions possess permanent charge and low melting point. ILs have been successfully applied in the separation science due to their properties such as low melting point, good solubility of organic compounds, low volatility, stability and the highly charged nature [17,18]. IL modified monolithic columns were also synthesized via different processes and were applied in capillary liquid chromatography [19] and CEC [20,21], respectively. Recently, the polymeric forms of ionic liquids (PILs) have attracted the attention of researchers because it is a new class of polymer materials with unique mechanical and electrochemical properties as well as high thermal stabilities [22]. PILs have also been used as stationary phase coatings in solid-phase microextraction [23–25] and stationary phase of GC [26], HPLC [27] and coatings of CE [28–32]. Our group has reported PIL as additive and bonded phase in CE to separate aromatic acids, inorganic anions, organic anions and basic proteins. Mechanisms of the separation are the coelectroosmotic mode and – interactions between the analytes and imidazole ring of PIL. In order to strengthen the interactions between the stationary phase and analytes, PIL and organic-silica hybrid monolithic column are combined to prepare a novel PImC8 -silica hybrid
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monolithic column. The performance of the column was investigated by the separation of aromatic hydrocarbons, alkylbenzenes and phenols. A typical reversed phase chromatographic behavior was observed when AHs were separated on the hybrid monolithic column. The successful synthesis of PImC8 -silica hybrid monolithic column expands the PIL application in separation science. 2. Materials and methods 2.1. Apparatus All experiments were performed on an Agilent CE system (Agilent Technologies, Germany) equipped with a diode array detector at a constant temperature of 25 ◦ C. An Agilent CE Chemstation (Rev.A 09.03) was used for the data analysis and instrumental control. Fused silica capillaries (Hebei Ruifeng Instrumental Company, China) of 33.5 cm (25 cm to detector) length (id = 75 m) were used. pH values of the mobile phase were measured using a Sartourius PB-10 pH meter (Beijing, China). The morphology of the column was characterized on a JSM-5600LV scanning electron microscope (JEOL, Japan) and the columns were heated in an incubator (Binder BD, Germany). The pore diameter and volume were characterized on an Autosorb-iQ analyzer (Quantachrome Instruments U.S.). The FT-IR of the monolith was recorded on FT-IR spectrometer (Nicolet Nexus 870) with a resolution of 4 cm−1 and 64 scans in the region of 4000–400 cm−1 . The elements of the PImC8 -silica hybrid monolithic column were characterized over a Thermo Fisher Scientific K-Alpha X-ray Photoelectron spectroscopy. 2.2. Reagents (TMOS) (99%) and 3Tetramethoxysilane mercaptopropyltrimethoxysilane (MPTS) (98%) were purchased from Shanghai Bore Chemical reagent Co. (Shanghai, China). PEG (Mn = 10 000) was obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). 1-Viny-3-octylimidazolium bromide ([C8 VyIm]Br)(99%) was purchased from Shanghai Cheng Jie Chemical Co. (Shanghai, China). 2,2 -Azodiisobutyronitrile obtained from Shanghai Shanpu Chemical Co. (Shanghai, China) was recrystallized in alcohol. All other reagents used in the experiment were of analytical grade. All analytes and mobile phase were passed through 0.45 m filter prior to use. 2.3. Column preparation A bare capillary was flushed with 1 M NaOH for 1.5 h and kept for a night, H2 O for 30 min, 1 M HCl for 2 h, H2 O for another 20 min and methanol for 20 min in sequence, and dried by purging nitrogen gas at 120 ◦ C for further use. 220 mg of PEG was dissolved in 2.5 ml of 0.01 M acetic acid in a glass vial, and then 0.9 ml of TMOS and 0.3 ml of MPTS were added. The solution was stirred at 0 ◦ C for 1 h until a homogenous solution was obtained. The precondensation mixture was sonicated for 3 min at 0 ◦ C, and introduced into the pretreated capillary to an appropriate length. The capillary was then placed in an incubator at 55 ◦ C for 12 h with both ends sealed to complete the condensation reaction. The obtained mercaptopropyl modified (MP-silica) hybrid monolithic column was then rinsed with 0.01 M ammonium hydroxide and kept at 120 ◦ C for 3 h, followed by washing with water and methanol. A chloroform solution containing 50 mg/ml [C8 VyIm]Br and 5 mg/ml AIBN was pumped through the MP-silica hybrid monolithic column for 10 min. And then, the column was heated at 70 ◦ C for 12 h with both ends sealed in an incubator. The monomer [C8 VyIm]Br was polymerized on the MP-silica hybrid monolith through surface radical chain-transfer polymerization. The PImC8 silica hybrid monolithic column was flushed with methanol to
Fig. 1. Scheme of PImC8 -silica hybrid monolithic column preparation.
remove the residue. Fig. 1 depicts the procedure of column preparation. 2.4. Capillary electrochromatography NaH2 PO4 (100 mM) solution was adjusted with NaOH and H3 PO4 to different pH values and used as the phosphate buffer. Mobile phases were then prepared by mixing phosphate buffer, distilled water, acetonitrile in certain ratios. Pressure injection model was used and injection pressure was 900–940 mbar and injection time was 0.01 min. The detection wavelengths were kept at 254 nm for aromatic hydrocarbons, 214 nm for phenols and EOF marker (thiourea). The retention factor (k ) was defined as (tR − t0 )/t0 , where tR and t0 represent the retention time of the aromatic hydrocarbon and thiourea in the study, respectively. 3. Results and discussion 3.1. Characterization of MP-silica and PImC8 -silica hybrid monolithic column The morphology of the MP-silica hybrid monolithic column was characterized by SEM. As can be seen in Fig. 2, the MP-silica column has meso- and macroporous structures, leading to a lower backpressure and providing a decreased mass-transfer resistance and a large surface area. The black dots in Fig. 2(D) are mesoporosities of the column. The mesoporosities of the material in the column was also characterized by an Autosorb-iQ analyzer and the average mesoporosities was 6.079 nm. The permeability of the columns was characterized by the back pressure, which was measured by pumping methanol through the column at a flow rate of 0.01 mL/min. The backpressure of the PIL-silica hybrid column was about 2.4 MPa, the similar with our previous IL-silica hybrid column [20] (about 2.3 MPa). The presence of PImC8 on the monolith was confirmed by the FT-IR spectra. As shown in Fig. 3(A), carbon–carbon and carbon–nitrogen double bond stretching vibration around 1560 cm−1 and imidazole ring stretching vibration at 878 cm−1 confirmed the presence of PILs in the PImC8 -silica hybrid monolithic column. The PImC8 monolith was also analyzed by XPS in order to explore the element types of the stationary phase. As we can see in Fig. 4, a typical N1s and Br 3d XPS spectrum of the monolith is shown at 400 eV and 70 eV, respectively, which certified again that PImC8 was modified to the PImC8 -silica hybrid monolithic column.
H. Han et al. / J. Chromatogr. A 1246 (2012) 9–14
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Fig. 2. SEM images of MP-silica hybrid monolithic column. Magnification: (A) 1200× (B) 2000×, (C) 10 000× and (D) 50 000×.
Fig. 4. XPS of PImC8 -silica hybrid monolithic column. Fig. 3. FT-IR spectra of (A) PImC8 -silica column and (B) MP-silica column.
3.2. EOF of the MP-silica and PImC8 -silica hybrid monolithic columns EOF is the basic requirement for driving buffer solution through a capillary column in CE and CEC, so measuring EOF is one of the effective ways to evaluate properties of the monolithic columns. The effect of pH value on EOF of MP-silica and PImC8 -silica hybrid monolithic columns was investigated by changing the pH value of the mobile phase. As shown in Fig. 5, the cathodic EOF increases with increase of pH for the MP-silica column, while strong anodic EOF in a wide pH range (3.0–8.0) is observed for the PImC8 -silica hybrid monolithic column, which indicates the successful polymerization of [C8 VyIm] Br onto the MP-silica column through surface radical chain-transfer polymerization. The strong reversed EOF (4.55 × 10−4 to 3.47 × 10−4 cm2 V−1 s−1 ) in the range between pH
Fig. 5. Influence of mobile phase pH on the EOF of (A) PImC8 -silica column and (B) MP-silica column. Conditions: 10 mM NaH2 PO4 buffer at different pH values; applied voltage, ±20 kV; EOF marker, thiourea.
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Fig. 6. Electrochromatograms of three AHs and EOF marker on (A) PImC8 and (B) MP-silica hybrid monolithic column. PImC8 column conditions: 20 mM NaH2 PO4 buffer containing 45% (v/v) ACN at pH 3.0; applied voltage, −20 kV; MP column conditions: 20 mM NaH2 PO4 buffer containing 45% (v/v) ACN at pH 7.0; applied voltage, +20 kV; Peak identification: 0, thiourea (EOF marker); 1, benzene; 2, naphthalene; 3, anthracene.
3.0 and 8.0 is due to the high amount of cation ring of imidazole. More silanol groups dissociate to negatively charged anions with the pH value increased, which results in a certain decreased EOF. The EOF decreases slightly (3.02 × 10−4 to 2.62 × 10−4 cm2 V−1 s−1 ) as the concentration of ACN in the mobile phase (20 mM, pH 3.0) is increased from 30 to 50%. The situation of EOF is a combined action of the decreasing dielectric constant and viscosity and the decreasing ionic strength (raising the trend of zeta potential) as the percentage of ACN is increased [33]. Feng et al. prepared a PIL modified monolithic column with free-radical copolymerization procedure [21] and our modification method was surface radical chain-transfer polymerization. Our columns generated a strongly reversed and relatively stable EOF in a wide pH range and the EOF on our column was obviously larger than that on Feng’s column. Thus, the amount of PIL was more. 3.3. Electrochromatographic performance of the PImC8 -silica column 3.3.1. Aromatic hydrocarbons Aromatic hydrocarbons are neutral compounds and cannot be resolved in CZE mode. However, the long chain of the PIL could afford the hydrophobicity and the ring of imidazole could afford aromaticity for the PImC8 -silica hybrid monolithic column when separating AHs in CEC mode. Three AHs (benzene, naphthalene and anthracene) were used as probes to test the PImC8 -silica hybrid monolithic column. For rapid and efficient separation, lower pH and concentration of mobile phase was used in order to strengthen anodic EOF and decrease the effect of Joule heat. Fig. 6 displays the separation electrochromatograms of three AHs on the MP-silica and PImC8 -silica hybrid monolithic column. The analytes are baseline separated in 5 min on the PImC8 -silica column with 45% ACN, 20 mM NaH2 PO4 (pH 3.0), while it is about 7.5 min on the MP-silica column with the same mobile phase composition at pH 7.0. The compounds are eluted in the order of thiourea < benzene < naphthalene < anthracene on both of columns, which is corresponding to hydrophobicities and aromaticities of the analytes from low to high. It can be concluded that the resolution would be improved and the migration time would be prolonged with the decrease of ACN content. The change of the retention factors (k ) of the analytes over the ACN content in mobile phase on the PImC8 -silica column was also investigated. As shown
Fig. 7. Effect of retention factor (k ) of the three AHs against ACN concentration on PImC8 -silica column. Conditions: 20 mM NaH2 PO4 containing different ACN contents; applied voltage, −20 kV.
in Fig. 7, the k decreased with the increase of the ACN content in the mobile phase, indicating a typical reversed-phase chromatographic retention mechanism [3]. The column efficiencies of 15 000 to 52 000 N/m were achieved for aromatic hydrocarbons on the PImC8 -silica column. In our experiments, the chrysene containing four rings of benzene could not be eluted using 20 mM NaH2 PO4 (pH 3.0) with 45% ACN as the mobile phase due to its high hydrophobicity and aromaticity. The thiourea and benzene would be eluted together when the content of ACN was increased to elute the chrysene (Electrochromatogram not given). Sun et al. [34] prepared poly(1-allylimidazole) modified silica as the stationary in HPLC. However, poly(1-allylimidazole) was not a polymeric ionic liquid and the EOF would not be reversed in high pH if poly (1-allylimidazole) modified to monolithic column in CEC. Thus, rapid and efficient were the advantages of polymeric ionic liquid modified monolithic column in CEC compared to poly(1-allyimidazole) modified silica as stationary phase in HPLC. Four alkylbenzenes (benzene, toluene, ethylbenzene and isopropylbenzene) were also separated according to their hydrophobicities on the PImC8 -silica hybrid monolithic column using 20 mM NaH2 PO4 (pH 3.0) with 40% ACN as the mobile phase (Fig. 8). The sequence of separation is in
Fig. 8. Electrochromatograms of four alkylbenzenes and thiourea on the PImC8 silica hybrid monolithic column. Conditions: 20 mM NaH2 PO4 buffer containing 40% (v/v) ACN at pH 3.0; applied voltage, −20 kV. Peak identification: 0, thiourea; 1, benzene; 2, methylbenzene; 3, ethylbenzene; 4, isopropylbenzene.
H. Han et al. / J. Chromatogr. A 1246 (2012) 9–14
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Table 1 Run-to-run, day-to-day and column-to-column reproducibility of migration time for PImC8 -silica hybrid monolithic column by using thiourea (EOF marker) and three aromatic hydrocarbons as test solutes. Analyte
RSDR (%)a
RSDP (%)b
RSDD (%)c
RSDC (%)d
Thiourea Benzene Naphthalene Anthracene
0.26 0.26 0.48 0.87
4.29 3.41 6.07 6.52
0.94 1.30 1.86 3.21
3.24 1.49 3.00 5.66
a b c d
Fig. 9. Electrochromatograms of five phenols on the PImC8 -silica hybrid monolithic column. The conditions as in Fig. 8. Peak identification: 1, phenol; 2, m-nitrophenol; 3, m-methylphenol; 4, 3-tert-butylphenol; 5, resorcinol.
the order of thiourea < benzene < methylbenzene < ethylbenzene < isopropylbenzene. The analytes were completely separated within 4 min, which indicated the column was able to separate aklylbenzenes in a short time by CEC. The column efficiencies were between 25 000 and 68 000 N/m. Compared with the previous work, propylbenzene was eluted in 9 min on octyl-functional monolithic column by Yan et al. [10] and isopropylbenzene was separated in 4 min on PIL modified column by us. Strong reversed and relatively stable EOF in a wide range of pH and rapid separation was the advantage of PIL modified monolithic column. 3.3.2. Phenols Phenols with different substituent would display distinct hydrophobicities and aromaticities. As we can see in Fig. 9, five phenols (phenol, m-nitrophenol, m-methylphenol, 3-tertbutylphenol and resorcinol) were separated on the PImC8 -silica column under optimal condition. The column efficiencies for the analytes were from 36 000 to 83 000 N/m. Phenol, m-nitrophenol, m-methylphenol, 3-tert-butylphenol and resorcinol are eluted sequentially. Migration time of m-nitrophenol is longer than phenol due to the hydrophobic group (nitro) of the latter. Since the nitro group is an electro-withdrawing substituent, this decreases the -electron density of the aromatic ring of m-nitrophenol. The interaction between m-nitrophenol and the stationary phase of the column is weaker and m-nitrophenol is eluted before mmethylphenol and 3-tert-butylphenol. Due to hydroxyl, which is an electron donating substituent, the -electron density of aromatic ring of resorcinol is increased. Thus, resorcinol is eluted last. We could conclude that the hydrophobicity of the long chain and the aromaticity of the imidazole of the stationary phase participate in the separation of phenols. Inorganic and organic anions were attempted to separate on the PImC8 -silica hybrid monolithic column. However, the anions were hard to elute from the column (Electrochromatogram not given) due to the strong electrostatic interaction between the negative charges of the anions and the positive charges of the imidazole cations. 3.4. Reproducibility of the PImC8 -silica column Reproducibilities of PImC8 -silica columns were evaluated by measuring RSDs of the migration time for three AHs and EOF marker (Table 1). RSDs of the migration time for the test analytes were in the range of 0.26–0.87% for five consecutive within-column runs. Both day-to-day and column-to-column reproducibilities (three
RSDR : RSD of run-to-run, n = 5. RSDP : RSD of peak area-to-peak area for run-to-run, n = 5. RSDD : RSD of day-to-day, n = 3. RSDC: RSD of column-to-column, n = 3.
injections per day per column) for monolithic columns were also evaluated in term of RSDs of migration time of analytes, which were in the range of 0.9–3.2% and 1.5–5.7%. 4. Concluding remarks A mercaptopropy-functionalized silica hybrid monolithic column was prepared by the in situ co-condensation of TMOS and MPTS. The obtained MP-silica hybrid monolith was a homogenous monolithic matrix characterized by SEM. The monomer [C8 VyIm]Br was then polymerized on MP-silica hybrid monolith through surface radical chain-transfer polymerization and the successful incorporation of PIL to the MP-silica hybrid monolith was characterized by FT-IR and XPS. The obtained PImC8 -silica hybrid monolithic column provided a reversed EOF in a wide pH range (3.0–8.0). Aromatic hydrocarbons, alkylbenzenes and phenols were well separated on the PImC8 -silica hybrid monolithic column within a short time by using a mobile phase containing 40–45% ACN in CEC. Multiple interactions including hydrophobic effect and – interactions between analytes and functionalized groups (the ring of benzene and imidazole) existed in the CEC separation. Acknowledgements The authors thank the financial support of the National Nature Science Foundation of China (Grant no. 20875096) and the National Science & Technology Major Project of China (Nos. 2011ZX05011 and 2011ZX05010). References [1] M. Chen, M. Zheng, Y. Feng, J. Chromatogr. A 1217 (2010) 3547. [2] M. Wu, R. Wu, R. Li, H. Qin, J. Dong, Z. Zhang, H. Zou, Anal. Chem. 82 (2010) 5447. [3] M. Wu, Y. Chen, R. Wu, R. Li, H. Zou, B. Chen, S. Yao, J. Chromatogr. A 1217 (2010) 4389. [4] F. Svec, J. Sep. Sci. 28 (2005) 729. [5] Z. Zhang, R. Wu, M. Wu, H. Zou, Electrophoresis 31 (2010) 1457. [6] M. Wu, R. Wu, F. Wang, L. Ren, J. Dong, Z. Liu, H. Zou, Anal. Chem. 81 (2009) 3529. [7] M. Wu, R. Wu, Z. Zhang, H. Zou, Electrophoresis 32 (2011) 105. [8] L. Yan, Q. Zhang, J. Zhang, L. Zhang, T. Li, Y. Feng, W. Zhang, Y. Zhang, J. Chromatogr. A 1046 (2004) 255. [9] M. Pursch, L.C. Sander, J. Chromatogr. A 887 (2000) 313. [10] L. Yan, Q. Zhang, Y. Feng, W. Zhang, T. Li, L. Zhang, Y. Zhang, J. Chromatogr. A 1121 (2006) 92. [11] H. Colón, X. Zhang, J.K. Murphy, J.G. Rivera, L.A. Colón, Chem. Commun. (2005) 2826. [12] Y. Tian, L. Zhang, Z. Zeng, H. Li, Electrophoresis 29 (2008) 960. [13] R. Feng, Y. Tian, H. Chen, Z. Huang, Z. Zeng, Electrophoresis 31 (2010) 1975. [14] L. Yan, Q. Zhang, W. Zhang, Y. Feng, L. Zhang, T. Li, Y. Zhang, Electrophoresis 26 (2005) 2935. [15] J. Ma, Z. Liang, X. Qiao, Q. Deng, D. Tao, L. Zhang, Y. Zhang, Anal. Chem. 80 (2008) 2949. [16] L. Xu, H. Lee, J. Chromatogr. A 1195 (2008) 78. [17] P. Sun, D.W. Armstrong, Anal. Chim. Acta 661 (2010) 1.
14 [18] [19] [20] [21] [22] [23] [24] [25]
H. Han et al. / J. Chromatogr. A 1246 (2012) 9–14 Y. Xu, E. Wang, J. Chromatogr. A 1216 (2009) 4817. P. Zhang, J. Chen, L. Jia, J. Chromatogr. A 1218 (2011) 3459. H. Han, J. Li, X. Wang, X. Liu, S. Jiang, J. Sep. Sci. 34 (2011) 2323. R. Feng, M. Shen, M.-M. Wang, H. Chen, Z.-R. Zeng, Chinese J. Anal. Chem. 39 (2011) 827. S. Ding, H. Tang, M. Radosz, Y. Shen, J. Polym. Sci., Part A: Polym. Chem. 42 (2004) 5794. F. Zhao, Y. Meng, J.L. Anderson, J. Chromatogr. A 1208 (2008) 1. E. Wanigasekara, S. Perera, J.A. Crank, L. Sidisky, R. Shirey, A. Berthod, D.W. Armstrong, Anal. Bioanal. Chem. 396 (2009) 511. Y. Meng, V. Pino, J.L. Anderson, Anal. Chim. Acta 687 (2011) 141.
[26] Y.N. Hsieh, R.S. Horng, W.Y. Ho, P.C. Huang, C.Y. Hsu, T.J. Whang, C.H. Kuei, Chromatographia 67 (2008) 413. [27] H. Qiu, M. Takafuji, T. Sawada, X. Liu, S. Jiang, H. Ihara, Chem. Commun. 46 (2010) 8740. [28] J. Li, Q. Wang, H. Han, X. Liu, S. Jiang, Talanta 82 (2010) 56. [29] J. Li, H. Han, Q. Wang, X. Liu, S. Jiang, J. Sep. Sci. 33 (2010) 2804. [30] J. Li, H. Han, Q. Wang, X. Liu, S. Jiang, Anal. Chim. Acta 674 (2010) 243. [31] J. Li, H. Han, Q. Wang, X. Liu, S. Jiang, J. Sep. Sci. 34 (2011) 1555. [32] Y. Zhou, J. Li, H. Han, X. Liu, S. Jiang, Chem. Pap. 65 (2011) 267. [33] N.J. Benz, J.S. Fritz, J. High Resolut. Chromatogr. 18 (1995) 175. [34] M. Sun, H. Qiu, L. Wang, X. Liu, S. Jiang, J. Chromatogr. A 1216 (2009) 3904.
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Polymeric ionic liquid modified organic-silica hybrid monolithic column for capillary electrochromatography Haifeng Han a,b , Qing Wang a,b , Xia Liu a,∗ , Shengxiang Jiang a a 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 b Graduate School of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Available online 14 December 2011 Keywords: CEC Monolithic column Organic-silica hybrid Polymeric ionic liquid
a b s t r a c t A polymeric ionic liquid (PIL) modified hybrid monolithic column for CEC was synthesized on the basis of mercaptopropy-functionalized (MP-silica) hybrid monolithic column which was prepared by the in situ co-condensation of tetramethoxysilane and 3-mercaptopropyltrimethoxysilane via a sol–gel process. The PIL modified (PImC8 -silica) hybrid monolithic column, which was characterized by FT-IR and Xray Photoelectron spectroscopy (XPS), generated a strong reversed and relatively stable EOF in a wide range of pH (3.0–8.0). The PImC8 -silica hybrid monolithic column was evaluated by the separation of three aromatic hydrocarbons (AHs), four alkylbenzenes and five phenols, respectively, with phosphate buffers containing different ratios of acetonitrile as mobile phase. Reproducibilities of the column were also investigated by measuring RSDs of the migration time for AHs. RSDs of run-to-run (n = 5), dayto-day (n = 3) and column-to-column (n = 3) were in the range of 0.26–0.87%, 0.90–3.2% and 1.5–5.7%, respectively. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The capillary column is the core of CEC, which combines superiorities of the CE and HPLC. Monoliths as separation columns have been widely used in CEC due to its continuous porous bed, good permeability, high phase ratios, high performance, high speed and good selectivity [1–3]. The significant aspect of monolithic materials in general and those based on synthetic polymers is the ease of their preparation, which avoids the problems related to both frit fabrication and packing [4]. Monolithic columns are synthesized by the in situ preparation of polymer-based or silica-based monoliths with different functionalized groups [5]. Organic polymer-based, inorganic silica-based and organicsilica hybrid monolithic columns are categorized based on the nature of matrix chemistry [6,7]. Organic monolithic columns have good stability in a wide range of pH and porous properties can be easily tailored by tuning the composition of the porogenic agent, monomer and cross-linking agent in the mixed solution [8]. Inorganic silica-based columns exhibit excellent mechanical stability and good solvent resistance, prepared via a sol–gel process [9]. However, many researchers have paid attention to organic-silica hybrid monolithic columns because of the ease of preparation,
∗ Corresponding author. Tel.: +86 931 4968203; fax: +86 931 8277088. E-mail address: [email protected] (X. Liu). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.12.029
good mechanical stability and good pH stability. The organic-silica hybrid monolithic columns are synthesized through a “one pot” approach by bonding different functional groups such as octyl [10], allyl [11], vinyl [12,13], phenyl [14], amino [15], mercapto [16] and chloropropyl [3]. Ionic liquids (ILs) containing relatively large organic cations and inorganic anions possess permanent charge and low melting point. ILs have been successfully applied in the separation science due to their properties such as low melting point, good solubility of organic compounds, low volatility, stability and the highly charged nature [17,18]. IL modified monolithic columns were also synthesized via different processes and were applied in capillary liquid chromatography [19] and CEC [20,21], respectively. Recently, the polymeric forms of ionic liquids (PILs) have attracted the attention of researchers because it is a new class of polymer materials with unique mechanical and electrochemical properties as well as high thermal stabilities [22]. PILs have also been used as stationary phase coatings in solid-phase microextraction [23–25] and stationary phase of GC [26], HPLC [27] and coatings of CE [28–32]. Our group has reported PIL as additive and bonded phase in CE to separate aromatic acids, inorganic anions, organic anions and basic proteins. Mechanisms of the separation are the coelectroosmotic mode and – interactions between the analytes and imidazole ring of PIL. In order to strengthen the interactions between the stationary phase and analytes, PIL and organic-silica hybrid monolithic column are combined to prepare a novel PImC8 -silica hybrid
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monolithic column. The performance of the column was investigated by the separation of aromatic hydrocarbons, alkylbenzenes and phenols. A typical reversed phase chromatographic behavior was observed when AHs were separated on the hybrid monolithic column. The successful synthesis of PImC8 -silica hybrid monolithic column expands the PIL application in separation science. 2. Materials and methods 2.1. Apparatus All experiments were performed on an Agilent CE system (Agilent Technologies, Germany) equipped with a diode array detector at a constant temperature of 25 ◦ C. An Agilent CE Chemstation (Rev.A 09.03) was used for the data analysis and instrumental control. Fused silica capillaries (Hebei Ruifeng Instrumental Company, China) of 33.5 cm (25 cm to detector) length (id = 75 m) were used. pH values of the mobile phase were measured using a Sartourius PB-10 pH meter (Beijing, China). The morphology of the column was characterized on a JSM-5600LV scanning electron microscope (JEOL, Japan) and the columns were heated in an incubator (Binder BD, Germany). The pore diameter and volume were characterized on an Autosorb-iQ analyzer (Quantachrome Instruments U.S.). The FT-IR of the monolith was recorded on FT-IR spectrometer (Nicolet Nexus 870) with a resolution of 4 cm−1 and 64 scans in the region of 4000–400 cm−1 . The elements of the PImC8 -silica hybrid monolithic column were characterized over a Thermo Fisher Scientific K-Alpha X-ray Photoelectron spectroscopy. 2.2. Reagents (TMOS) (99%) and 3Tetramethoxysilane mercaptopropyltrimethoxysilane (MPTS) (98%) were purchased from Shanghai Bore Chemical reagent Co. (Shanghai, China). PEG (Mn = 10 000) was obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). 1-Viny-3-octylimidazolium bromide ([C8 VyIm]Br)(99%) was purchased from Shanghai Cheng Jie Chemical Co. (Shanghai, China). 2,2 -Azodiisobutyronitrile obtained from Shanghai Shanpu Chemical Co. (Shanghai, China) was recrystallized in alcohol. All other reagents used in the experiment were of analytical grade. All analytes and mobile phase were passed through 0.45 m filter prior to use. 2.3. Column preparation A bare capillary was flushed with 1 M NaOH for 1.5 h and kept for a night, H2 O for 30 min, 1 M HCl for 2 h, H2 O for another 20 min and methanol for 20 min in sequence, and dried by purging nitrogen gas at 120 ◦ C for further use. 220 mg of PEG was dissolved in 2.5 ml of 0.01 M acetic acid in a glass vial, and then 0.9 ml of TMOS and 0.3 ml of MPTS were added. The solution was stirred at 0 ◦ C for 1 h until a homogenous solution was obtained. The precondensation mixture was sonicated for 3 min at 0 ◦ C, and introduced into the pretreated capillary to an appropriate length. The capillary was then placed in an incubator at 55 ◦ C for 12 h with both ends sealed to complete the condensation reaction. The obtained mercaptopropyl modified (MP-silica) hybrid monolithic column was then rinsed with 0.01 M ammonium hydroxide and kept at 120 ◦ C for 3 h, followed by washing with water and methanol. A chloroform solution containing 50 mg/ml [C8 VyIm]Br and 5 mg/ml AIBN was pumped through the MP-silica hybrid monolithic column for 10 min. And then, the column was heated at 70 ◦ C for 12 h with both ends sealed in an incubator. The monomer [C8 VyIm]Br was polymerized on the MP-silica hybrid monolith through surface radical chain-transfer polymerization. The PImC8 silica hybrid monolithic column was flushed with methanol to
Fig. 1. Scheme of PImC8 -silica hybrid monolithic column preparation.
remove the residue. Fig. 1 depicts the procedure of column preparation. 2.4. Capillary electrochromatography NaH2 PO4 (100 mM) solution was adjusted with NaOH and H3 PO4 to different pH values and used as the phosphate buffer. Mobile phases were then prepared by mixing phosphate buffer, distilled water, acetonitrile in certain ratios. Pressure injection model was used and injection pressure was 900–940 mbar and injection time was 0.01 min. The detection wavelengths were kept at 254 nm for aromatic hydrocarbons, 214 nm for phenols and EOF marker (thiourea). The retention factor (k ) was defined as (tR − t0 )/t0 , where tR and t0 represent the retention time of the aromatic hydrocarbon and thiourea in the study, respectively. 3. Results and discussion 3.1. Characterization of MP-silica and PImC8 -silica hybrid monolithic column The morphology of the MP-silica hybrid monolithic column was characterized by SEM. As can be seen in Fig. 2, the MP-silica column has meso- and macroporous structures, leading to a lower backpressure and providing a decreased mass-transfer resistance and a large surface area. The black dots in Fig. 2(D) are mesoporosities of the column. The mesoporosities of the material in the column was also characterized by an Autosorb-iQ analyzer and the average mesoporosities was 6.079 nm. The permeability of the columns was characterized by the back pressure, which was measured by pumping methanol through the column at a flow rate of 0.01 mL/min. The backpressure of the PIL-silica hybrid column was about 2.4 MPa, the similar with our previous IL-silica hybrid column [20] (about 2.3 MPa). The presence of PImC8 on the monolith was confirmed by the FT-IR spectra. As shown in Fig. 3(A), carbon–carbon and carbon–nitrogen double bond stretching vibration around 1560 cm−1 and imidazole ring stretching vibration at 878 cm−1 confirmed the presence of PILs in the PImC8 -silica hybrid monolithic column. The PImC8 monolith was also analyzed by XPS in order to explore the element types of the stationary phase. As we can see in Fig. 4, a typical N1s and Br 3d XPS spectrum of the monolith is shown at 400 eV and 70 eV, respectively, which certified again that PImC8 was modified to the PImC8 -silica hybrid monolithic column.
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Fig. 2. SEM images of MP-silica hybrid monolithic column. Magnification: (A) 1200× (B) 2000×, (C) 10 000× and (D) 50 000×.
Fig. 4. XPS of PImC8 -silica hybrid monolithic column. Fig. 3. FT-IR spectra of (A) PImC8 -silica column and (B) MP-silica column.
3.2. EOF of the MP-silica and PImC8 -silica hybrid monolithic columns EOF is the basic requirement for driving buffer solution through a capillary column in CE and CEC, so measuring EOF is one of the effective ways to evaluate properties of the monolithic columns. The effect of pH value on EOF of MP-silica and PImC8 -silica hybrid monolithic columns was investigated by changing the pH value of the mobile phase. As shown in Fig. 5, the cathodic EOF increases with increase of pH for the MP-silica column, while strong anodic EOF in a wide pH range (3.0–8.0) is observed for the PImC8 -silica hybrid monolithic column, which indicates the successful polymerization of [C8 VyIm] Br onto the MP-silica column through surface radical chain-transfer polymerization. The strong reversed EOF (4.55 × 10−4 to 3.47 × 10−4 cm2 V−1 s−1 ) in the range between pH
Fig. 5. Influence of mobile phase pH on the EOF of (A) PImC8 -silica column and (B) MP-silica column. Conditions: 10 mM NaH2 PO4 buffer at different pH values; applied voltage, ±20 kV; EOF marker, thiourea.
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Fig. 6. Electrochromatograms of three AHs and EOF marker on (A) PImC8 and (B) MP-silica hybrid monolithic column. PImC8 column conditions: 20 mM NaH2 PO4 buffer containing 45% (v/v) ACN at pH 3.0; applied voltage, −20 kV; MP column conditions: 20 mM NaH2 PO4 buffer containing 45% (v/v) ACN at pH 7.0; applied voltage, +20 kV; Peak identification: 0, thiourea (EOF marker); 1, benzene; 2, naphthalene; 3, anthracene.
3.0 and 8.0 is due to the high amount of cation ring of imidazole. More silanol groups dissociate to negatively charged anions with the pH value increased, which results in a certain decreased EOF. The EOF decreases slightly (3.02 × 10−4 to 2.62 × 10−4 cm2 V−1 s−1 ) as the concentration of ACN in the mobile phase (20 mM, pH 3.0) is increased from 30 to 50%. The situation of EOF is a combined action of the decreasing dielectric constant and viscosity and the decreasing ionic strength (raising the trend of zeta potential) as the percentage of ACN is increased [33]. Feng et al. prepared a PIL modified monolithic column with free-radical copolymerization procedure [21] and our modification method was surface radical chain-transfer polymerization. Our columns generated a strongly reversed and relatively stable EOF in a wide pH range and the EOF on our column was obviously larger than that on Feng’s column. Thus, the amount of PIL was more. 3.3. Electrochromatographic performance of the PImC8 -silica column 3.3.1. Aromatic hydrocarbons Aromatic hydrocarbons are neutral compounds and cannot be resolved in CZE mode. However, the long chain of the PIL could afford the hydrophobicity and the ring of imidazole could afford aromaticity for the PImC8 -silica hybrid monolithic column when separating AHs in CEC mode. Three AHs (benzene, naphthalene and anthracene) were used as probes to test the PImC8 -silica hybrid monolithic column. For rapid and efficient separation, lower pH and concentration of mobile phase was used in order to strengthen anodic EOF and decrease the effect of Joule heat. Fig. 6 displays the separation electrochromatograms of three AHs on the MP-silica and PImC8 -silica hybrid monolithic column. The analytes are baseline separated in 5 min on the PImC8 -silica column with 45% ACN, 20 mM NaH2 PO4 (pH 3.0), while it is about 7.5 min on the MP-silica column with the same mobile phase composition at pH 7.0. The compounds are eluted in the order of thiourea < benzene < naphthalene < anthracene on both of columns, which is corresponding to hydrophobicities and aromaticities of the analytes from low to high. It can be concluded that the resolution would be improved and the migration time would be prolonged with the decrease of ACN content. The change of the retention factors (k ) of the analytes over the ACN content in mobile phase on the PImC8 -silica column was also investigated. As shown
Fig. 7. Effect of retention factor (k ) of the three AHs against ACN concentration on PImC8 -silica column. Conditions: 20 mM NaH2 PO4 containing different ACN contents; applied voltage, −20 kV.
in Fig. 7, the k decreased with the increase of the ACN content in the mobile phase, indicating a typical reversed-phase chromatographic retention mechanism [3]. The column efficiencies of 15 000 to 52 000 N/m were achieved for aromatic hydrocarbons on the PImC8 -silica column. In our experiments, the chrysene containing four rings of benzene could not be eluted using 20 mM NaH2 PO4 (pH 3.0) with 45% ACN as the mobile phase due to its high hydrophobicity and aromaticity. The thiourea and benzene would be eluted together when the content of ACN was increased to elute the chrysene (Electrochromatogram not given). Sun et al. [34] prepared poly(1-allylimidazole) modified silica as the stationary in HPLC. However, poly(1-allylimidazole) was not a polymeric ionic liquid and the EOF would not be reversed in high pH if poly (1-allylimidazole) modified to monolithic column in CEC. Thus, rapid and efficient were the advantages of polymeric ionic liquid modified monolithic column in CEC compared to poly(1-allyimidazole) modified silica as stationary phase in HPLC. Four alkylbenzenes (benzene, toluene, ethylbenzene and isopropylbenzene) were also separated according to their hydrophobicities on the PImC8 -silica hybrid monolithic column using 20 mM NaH2 PO4 (pH 3.0) with 40% ACN as the mobile phase (Fig. 8). The sequence of separation is in
Fig. 8. Electrochromatograms of four alkylbenzenes and thiourea on the PImC8 silica hybrid monolithic column. Conditions: 20 mM NaH2 PO4 buffer containing 40% (v/v) ACN at pH 3.0; applied voltage, −20 kV. Peak identification: 0, thiourea; 1, benzene; 2, methylbenzene; 3, ethylbenzene; 4, isopropylbenzene.
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Table 1 Run-to-run, day-to-day and column-to-column reproducibility of migration time for PImC8 -silica hybrid monolithic column by using thiourea (EOF marker) and three aromatic hydrocarbons as test solutes. Analyte
RSDR (%)a
RSDP (%)b
RSDD (%)c
RSDC (%)d
Thiourea Benzene Naphthalene Anthracene
0.26 0.26 0.48 0.87
4.29 3.41 6.07 6.52
0.94 1.30 1.86 3.21
3.24 1.49 3.00 5.66
a b c d
Fig. 9. Electrochromatograms of five phenols on the PImC8 -silica hybrid monolithic column. The conditions as in Fig. 8. Peak identification: 1, phenol; 2, m-nitrophenol; 3, m-methylphenol; 4, 3-tert-butylphenol; 5, resorcinol.
the order of thiourea < benzene < methylbenzene < ethylbenzene < isopropylbenzene. The analytes were completely separated within 4 min, which indicated the column was able to separate aklylbenzenes in a short time by CEC. The column efficiencies were between 25 000 and 68 000 N/m. Compared with the previous work, propylbenzene was eluted in 9 min on octyl-functional monolithic column by Yan et al. [10] and isopropylbenzene was separated in 4 min on PIL modified column by us. Strong reversed and relatively stable EOF in a wide range of pH and rapid separation was the advantage of PIL modified monolithic column. 3.3.2. Phenols Phenols with different substituent would display distinct hydrophobicities and aromaticities. As we can see in Fig. 9, five phenols (phenol, m-nitrophenol, m-methylphenol, 3-tertbutylphenol and resorcinol) were separated on the PImC8 -silica column under optimal condition. The column efficiencies for the analytes were from 36 000 to 83 000 N/m. Phenol, m-nitrophenol, m-methylphenol, 3-tert-butylphenol and resorcinol are eluted sequentially. Migration time of m-nitrophenol is longer than phenol due to the hydrophobic group (nitro) of the latter. Since the nitro group is an electro-withdrawing substituent, this decreases the -electron density of the aromatic ring of m-nitrophenol. The interaction between m-nitrophenol and the stationary phase of the column is weaker and m-nitrophenol is eluted before mmethylphenol and 3-tert-butylphenol. Due to hydroxyl, which is an electron donating substituent, the -electron density of aromatic ring of resorcinol is increased. Thus, resorcinol is eluted last. We could conclude that the hydrophobicity of the long chain and the aromaticity of the imidazole of the stationary phase participate in the separation of phenols. Inorganic and organic anions were attempted to separate on the PImC8 -silica hybrid monolithic column. However, the anions were hard to elute from the column (Electrochromatogram not given) due to the strong electrostatic interaction between the negative charges of the anions and the positive charges of the imidazole cations. 3.4. Reproducibility of the PImC8 -silica column Reproducibilities of PImC8 -silica columns were evaluated by measuring RSDs of the migration time for three AHs and EOF marker (Table 1). RSDs of the migration time for the test analytes were in the range of 0.26–0.87% for five consecutive within-column runs. Both day-to-day and column-to-column reproducibilities (three
RSDR : RSD of run-to-run, n = 5. RSDP : RSD of peak area-to-peak area for run-to-run, n = 5. RSDD : RSD of day-to-day, n = 3. RSDC: RSD of column-to-column, n = 3.
injections per day per column) for monolithic columns were also evaluated in term of RSDs of migration time of analytes, which were in the range of 0.9–3.2% and 1.5–5.7%. 4. Concluding remarks A mercaptopropy-functionalized silica hybrid monolithic column was prepared by the in situ co-condensation of TMOS and MPTS. The obtained MP-silica hybrid monolith was a homogenous monolithic matrix characterized by SEM. The monomer [C8 VyIm]Br was then polymerized on MP-silica hybrid monolith through surface radical chain-transfer polymerization and the successful incorporation of PIL to the MP-silica hybrid monolith was characterized by FT-IR and XPS. The obtained PImC8 -silica hybrid monolithic column provided a reversed EOF in a wide pH range (3.0–8.0). Aromatic hydrocarbons, alkylbenzenes and phenols were well separated on the PImC8 -silica hybrid monolithic column within a short time by using a mobile phase containing 40–45% ACN in CEC. Multiple interactions including hydrophobic effect and – interactions between analytes and functionalized groups (the ring of benzene and imidazole) existed in the CEC separation. Acknowledgements The authors thank the financial support of the National Nature Science Foundation of China (Grant no. 20875096) and the National Science & Technology Major Project of China (Nos. 2011ZX05011 and 2011ZX05010). References [1] M. Chen, M. Zheng, Y. Feng, J. Chromatogr. A 1217 (2010) 3547. [2] M. Wu, R. Wu, R. Li, H. Qin, J. Dong, Z. Zhang, H. Zou, Anal. Chem. 82 (2010) 5447. [3] M. Wu, Y. Chen, R. Wu, R. Li, H. Zou, B. Chen, S. Yao, J. Chromatogr. A 1217 (2010) 4389. [4] F. Svec, J. Sep. Sci. 28 (2005) 729. [5] Z. Zhang, R. Wu, M. Wu, H. Zou, Electrophoresis 31 (2010) 1457. [6] M. Wu, R. Wu, F. Wang, L. Ren, J. Dong, Z. Liu, H. Zou, Anal. Chem. 81 (2009) 3529. [7] M. Wu, R. Wu, Z. Zhang, H. Zou, Electrophoresis 32 (2011) 105. [8] L. Yan, Q. Zhang, J. Zhang, L. Zhang, T. Li, Y. Feng, W. Zhang, Y. Zhang, J. Chromatogr. A 1046 (2004) 255. [9] M. Pursch, L.C. Sander, J. Chromatogr. A 887 (2000) 313. [10] L. Yan, Q. Zhang, Y. Feng, W. Zhang, T. Li, L. Zhang, Y. Zhang, J. Chromatogr. A 1121 (2006) 92. [11] H. Colón, X. Zhang, J.K. Murphy, J.G. Rivera, L.A. Colón, Chem. Commun. (2005) 2826. [12] Y. Tian, L. Zhang, Z. Zeng, H. Li, Electrophoresis 29 (2008) 960. [13] R. Feng, Y. Tian, H. Chen, Z. Huang, Z. Zeng, Electrophoresis 31 (2010) 1975. [14] L. Yan, Q. Zhang, W. Zhang, Y. Feng, L. Zhang, T. Li, Y. Zhang, Electrophoresis 26 (2005) 2935. [15] J. Ma, Z. Liang, X. Qiao, Q. Deng, D. Tao, L. Zhang, Y. Zhang, Anal. Chem. 80 (2008) 2949. [16] L. Xu, H. Lee, J. Chromatogr. A 1195 (2008) 78. [17] P. Sun, D.W. Armstrong, Anal. Chim. Acta 661 (2010) 1.
14 [18] [19] [20] [21] [22] [23] [24] [25]
H. Han et al. / J. Chromatogr. A 1246 (2012) 9–14 Y. Xu, E. Wang, J. Chromatogr. A 1216 (2009) 4817. P. Zhang, J. Chen, L. Jia, J. Chromatogr. A 1218 (2011) 3459. H. Han, J. Li, X. Wang, X. Liu, S. Jiang, J. Sep. Sci. 34 (2011) 2323. R. Feng, M. Shen, M.-M. Wang, H. Chen, Z.-R. Zeng, Chinese J. Anal. Chem. 39 (2011) 827. S. Ding, H. Tang, M. Radosz, Y. Shen, J. Polym. Sci., Part A: Polym. Chem. 42 (2004) 5794. F. Zhao, Y. Meng, J.L. Anderson, J. Chromatogr. A 1208 (2008) 1. E. Wanigasekara, S. Perera, J.A. Crank, L. Sidisky, R. Shirey, A. Berthod, D.W. Armstrong, Anal. Bioanal. Chem. 396 (2009) 511. Y. Meng, V. Pino, J.L. Anderson, Anal. Chim. Acta 687 (2011) 141.
[26] Y.N. Hsieh, R.S. Horng, W.Y. Ho, P.C. Huang, C.Y. Hsu, T.J. Whang, C.H. Kuei, Chromatographia 67 (2008) 413. [27] H. Qiu, M. Takafuji, T. Sawada, X. Liu, S. Jiang, H. Ihara, Chem. Commun. 46 (2010) 8740. [28] J. Li, Q. Wang, H. Han, X. Liu, S. Jiang, Talanta 82 (2010) 56. [29] J. Li, H. Han, Q. Wang, X. Liu, S. Jiang, J. Sep. Sci. 33 (2010) 2804. [30] J. Li, H. Han, Q. Wang, X. Liu, S. Jiang, Anal. Chim. Acta 674 (2010) 243. [31] J. Li, H. Han, Q. Wang, X. Liu, S. Jiang, J. Sep. Sci. 34 (2011) 1555. [32] Y. Zhou, J. Li, H. Han, X. Liu, S. Jiang, Chem. Pap. 65 (2011) 267. [33] N.J. Benz, J.S. Fritz, J. High Resolut. Chromatogr. 18 (1995) 175. [34] M. Sun, H. Qiu, L. Wang, X. Liu, S. Jiang, J. Chromatogr. A 1216 (2009) 3904.