- Email: [email protected]
cation-exchange monolithic column with a dynamically modified cationic surfactant
Journal of Chromatography A, 1216 (2009) 7728–7731
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Capillary liquid chromatography using a hydrophilic/cation-exchange monolithic column with a dynamically modified cationic surfactant Jian Lin, Jia Lin, Xucong Lin, Zenghong Xie ∗ Department of Chemistry, Fuzhou University, Fuzhou 350002, China
a r t i c l e
i n f o
Article history: Received 23 May 2009 Received in revised form 25 August 2009 Accepted 28 August 2009 Available online 1 September 2009 Keywords: Monolithic stationary phases Dynamically modified capillary liquid chromatography Hydrophilic interaction Reversed-phase mode
a b s t r a c t A novel form of reversed-phase liquid chromatography (RPLC) by the dynamically modified hydrophilic interaction monolithic column has been described in this paper. A porous poly(SPMA-co-PETA) monolith with strong cation-exchange (SCX) was prepared and the resulting monolith showed a typical hydrophilic interaction chromatography (HILIC) mechanism at higher organic solvent content (ACN% > 50%). The good selectivity for neutral, basic and acidic polar analytes was observed in the HILIC mode. In order to increase the hydrophobic interaction, the monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX monolithic material, and the resulting hydrophobic layer was used as the stationary phase. Using the dynamically modified SCX monolithic column, neutral, basic and acidic hydrophobic analytes were well separated with the RPLC mode. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction Nowadays, a novel porous material known as monoliths is an alternative to column packed with particulate stationary phase, which has been extensively studied and gradually applied in capillary liquid chromatography (CLC) and capillary electrochromatography (CEC) [1–6]. Reversed-phase liquid chromatography (RPLC) based on nonpolar stationary phases is a powerful separation mode because of its versatility and ability to retain and resolve a wide range of hydrophobic to slightly polar analytes [7–14]. However, for highly polar analytes, these hydrophobic monoliths show limited extent of retention in the reversed-phase mode [15]. Hydrophilic interaction chromatography (HILIC) based on polar stationary phases has gained increasing attention because of effective solution to the separation of polar analytes [15–20]. But due to the low hydrophobicity, the ability of these polar monoliths to separate hydrophobic analytes was poor. Only few studies have been reported for the efficient separation of various characters of compounds [21], and it is still a somewhat troublesome task in a single polar or nonpolar stationary phase. The production of monolith having both strong hydrophobic and hydrophilic interaction is rather a difficult process, and it cannot be achieved by simple polymerization of polar and nonpolar monomers. Therefore, the further development of resoluble methods to offer effective retention for
∗ Corresponding author. Tel.: +86 591 22866131. E-mail address: [email protected] (Z. Xie).
a wide range of hydrophobic to highly polar compounds in one column is necessary. This report demonstrated a novel conversion of chromatographic mode by the dynamically modified hydrophilic interaction monolith for the separation of various compounds. Chromatography on dynamically modified monolithic columns has been widely reported for use [22–25], which was a simple and convenient technique to introduce desired chromatographic functionality onto the monolithic stationary phase to exert selectivity. In this work, a porous monolith with strong cation-exchange (SCX) was prepared. The poly(SPMA-co-PETA) monolithic column was applied to the separation of polar analytes in a HILIC mode, and then the hydrophilic interaction monolith was dynamically modified with cetyltrimethylammonium bromide (CTAB), which showed excellent separation of hydrophobic analytes based on a RPLC mode. 2. Experimental 2.1. Reagents and materials 3-Sulfopropyl methacrylate potassium salt (SPMA), pentaerythritol triacrylate (PETA), 2,2 -azobisisobutyronitrile (AIBN), 3-(trimethoxysilyl)propyl methacrylate (␥-MAPS), benzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4,5trihydroxybenzoic acid, alkyl benzenes, polycyclic aromatic hydrocarbons were purchased from Aldrich (Milwaukee, WI, USA). Uracil, uridine, guanine, guanosine, cytosine, cytidine were purchased from Sigma (St. Louis, MO, USA). HPLC-grade methanol
0021-9673/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.08.071
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and acetonitrile (ACN), thiourea, acrylamide, phenol, resorcinol, catechol, hydroquinone, pyrogallol, 3,5-dinitrobenzoic acid, 4nitrobenzoic acid, 2-nitroaniline, 4-nitroaniline, diphenylamine were obtained from Chemical Reagent Corporation (Shanghai, China). Buffer solutions were prepared using sodium phosphate monobasic or triethylamine phosphate, phosphoric acid, sodium hydroxide, triethylamine, cetyltrimethylammonium bromide (CTAB), cyclohexanol and ethylene glycol were from Chemical Reagent Plant (Shanghai, China). Fused-silica capillaries with an internal diameter of 100 m and an outer diameter of 365 m were purchased from the Yongnian Optic Fiber Plant (Hebei, China). Ultrapure water used in all of the experiments was doubly distilled and purified by a Milli-Q system (Millipore, Milford, MA, USA). 2.2. Instrumentation The experiments were performed on a Trisep 2010GV cLC system (Unimicro Technologies, Pleasanton, CA, USA) equipped with a UV/Vis detector (190–800 nm), comprising two microvolume pumps, a microfluid manipulation module (including a six-port injector), and a data acquisition module, as described in literature [26]. An HPLC pump was used to flush the monolithic columns.
Fig. 1. Relationship between retention time and acetonitrile concentration on poly(SPMA-co-PETA) monolithic column. Conditions: mobile phase, 5 mM triethylamine phosphate pH 6.00 in ACN/H2 O; pump flow: 0.02 ml/min; applied pressure 7.0 MPa.
2.3. Preparation of the monolithic column Polymerization solutions weighing 2.0 g each were prepared from monomers (SPMA and PETA) and porogenic solvents in ratios of 20:80 (w/w) monomers/solvents. AIBN (1.0%, w/w, with respect to monomers) was dissolved in 0.4 g of solution consisting of 80.0% (w/w) of SPMA and 20.0% (w/w) of PETA, respectively. The binary porogenic solvents contained 55.0% (w/w) of cyclohexanol and 45.0% (w/w) of ethylene glycol. The mixture was then sonicated to obtain a clear solution, and purged with nitrogen for 5 min, a small part of the mixed solution was removed for capillary preparation using a 100 L syringe. A pretreated 55 cm capillary was attached to the syringe inlet, and the polymerization mixture was sucked into the capillary for 35 cm length. The capillary was plugged at both ends with rubber stoppers and was submerged in a 60 ◦ C water bath for 20 h. The prepared monolithic capillary column was washed with methanol and water by an HPLC pump to flush out the residual reagents. A detection window was created at 1–2 mm after the end of the polymer bed using thermal wire stripper. Finally, the column was cut to a total length of 50 cm with an effective length of 30 cm. 3. Results and discussion 3.1. Retention properties of poly(SPMA-co-PETA) monolith in aqueous acetonitrile In order to investigate the chromatographic properties of poly(SPMA-co-PETA) monolith in aqueous ACN mobile phases, toluene, acrylamide, and thiourea were used as test compounds. The solvent was selected as the void time marker in this system. The influence of ACN content in the mobile phase on the retention factor (k ) of three test compounds is shown in Fig. 1. The hydrophilic solutes, thiourea eluted after acrylamide and toluene when the ACN concentration was increased from 40% to 95%. The retention time of thiourea leveled off initially as the ACN content in the mobile phase increased from 30% to 50% and then increased when the ACN content further increased to 95%. Acrylamide behaved similar to thiourea but with much less retention due to the lower hydrophilicity. These results suggest a typical HILIC retention mechanism at higher ACN content in the mobile phase (>50%). For the hydrophobic solute toluene, the k values were decreased with the increment
Fig. 2. Hydrophilic interaction chromatography for the separation of (a) phenols, (b) benzoic acid derivatives and (c) nucleobases. Conditions: (a) mobile phase, 5 mM triethylamine phosphate, pH 6.00, at 95% (v/v) ACN; pump flow: 0.02 ml/min; applied pressure 5.1 MPa; detection wavelength, 214 nm. (b) Mobile phase, 5 mM triethylamine phosphate, pH 6.00, at 90% (v/v) ACN; pump flow: 0.02 ml/min; applied pressure 5.3 MPa; detection wavelength, 214 nm. (c) Mobile phase, 40 mM triethylamine phosphate, pH 4.00, at 80% (v/v) ACN; pump flow: 0.02 ml/min; applied pressure 3.5 MPa; detection wavelength, 254 nm.
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of the content of ACN from 30% to 50% and then approximately constant at higher ACN content. This means a RPLC-mode separation dominantly worked on this stationary phase at lower organic solvent content (ACN% < 50%). 3.2. Hydrophilic interaction chromatography The poly(SPMA-co-PETA) surface can provide a hydrophilic and ion-exchange environment. A series of neutral, basic and acidic polar analytes were selected as model compounds. The result was shown in Fig. 2. As expected for a hydrophilic monolith, the retention of phenols and benzoic acid derivatives increased with an increase in the number of hydroxyl groups within the molecule (Fig. 2a and b). Moreover, the positional isomers of phenols can be readily separated. The results showed that the extent of hydrophilic interaction depends on the number of the hydroxyl groups and the position. A baseline separation without obvious peak tailing of the nucleobases was obtained in Fig. 2c. Upon conjugation of the bases with ribose to yield nucleosides, the neutral uridine was more retained than uracil, indicating that a ribose moiety confers on the solute molecules an increased hydrophilic character [14]. Cytosine (pKa 4.5), which is about the same polarity as uracil, eluted last. This may be attributed to strong electrostatic interaction with the sulfonic acid groups of the stationary phase at pH 4.00 buffer. The separation of all the nucleobases were primarily based on both hydrophilic interaction and electrostatic interaction mechanisms on the poly(SPMA-co-PETA) stationary phase. 3.3. Reversed-phase liquid chromatography The poly(SPMA-co-PETA) monolithic column can also provide hydrophobic interaction at lower ACN content. However, as shown in Fig. 3a, the four alkyl benzenes were not efficiently separated Fig. 4. Effect of mobile phase on retention factor of neutral solutes. Plot a: effect of CTAB concentration, ACN and 20 mM sodium monophosphate (pH 2.50) kept at 50% and 10%, but changing the ratio of 50 mM CTAB solution to water. Plot b: effect of ACN fraction, 50 mM CTAB solution and 20 mM sodium monophosphate (pH 2.50) kept at 12% and 10%, but changing the ratio of ACN to water; others as in Fig. 3b.
even at relatively low ACN content (30% ACN). This is mainly due to the low hydrophobicity of monolith. In order to increase the hydrophobic interaction of poly(SPMA-co-PETA) monolith, dynamically modified chromatography was introduced in this paper.
Fig. 3. Chromatograms for the separation of five neutral solutes in (a) poly(SPMAco-PETA) monolith and (b) dynamically modified poly(SPMA-co-PETA) monolith. Conditions. (a) Mobile phase, 5 mM triethylamine phosphate, pH 2.50, at 30% (v/v) ACN. (b) Mobile phase, ACN/50 mM CTAB/20 mM sodium monophosphate (pH 2.50)/water = 50/10/12/28. Pump flow: 0.02 ml/min; applied pressure 10.5 MPa; detection wavelength, 214 nm.
3.3.1. Characterization of the CTAB dynamically modified poly(SPMA-co-PETA) monolith The poly(SPMA-co-PETA) monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, CTAB, which was added to the mobile phase. The separation mechanism of dynamically modified poly(SPMA-co-PETA) monolith was similar to that of dynamically modified SCX packing material CEC [27]. The adsorbed CTAB molecules formed a hydrophobic layer on the SCX monolithic material surface, which acted as the stationary phase as C18 chains on the ODS packings. Neutral compounds could be separated in this system based on the partitioning between the mobile phase and hydrophobic layer. The efficiency of the columns and the reproducibility of the retention factors were evaluated with four homologous benzene compounds. Void time was measured by baseline disturbance. The highest number of theoretical plates reaching greater than 100 000 plates/m was obtained, and the relative standard deviation for retention factor was less than 1.0%. Fig. 3b shows the separation of the four neutral alkyl benzenes on cLC with dynamically modified poly(SPMA-co-PETA) monolith. The hydrophobicity of SCX monolith could be increased by dynamic
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separation of acidic, basic and neutral hydrophobic compounds using dynamically modified poly(SPMA-co-PETA) monolith with a low-pH mobile phase. Excellent separation of all eight compounds was accomplished. Under these conditions, chromatography plays a major role in the separation process of weakly acidic and neutral compounds. The basic compounds were positively charged at low-pH mobile phase, but good peak symmetry for the basic compounds was observed. The peak tailing of basic compounds can be effectively eliminated with dynamically modified poly(SPMA-coPETA) monolith because the surfactant used has strong affinity to sulfonic acid groups than the common basic compound does. 4. Conclusion A porous poly(SPMA-co-PETA) monolithic capillary column, which was prepared by one-step copolymerization of SPMA and PETA, has been successfully used as a stationary phase in HILIC mode. Typical HILIC retention was obtained at high organic solvent content (ACN% > 50%). The hydrophobicity of poly(SPMA-co-PETA) monolith was increased by dynamic adsorption of CTAB, and the separation mechanism of dynamically modified poly(SPMAco-PETA) monolith was mainly based on RPLC mode. Efficient separation of a wide range of hydrophobic to highly polar compounds was performed in different modes for one column. Fig. 5. Separation of hydrophobic solutes at low pH. Conditions: mobile phase, ACN/50 mM CTAB/20 mM sodium monophosphate (pH 2.50)/water = 60/12/10/18. Pump flow: 0.02 ml/min; applied pressure at (a) 13.0 MPa and (b) 7.0 MPa; detection wavelength, 214 nm.
adsorption of CTAB, and neutral alkyl benzenes were well separated in this system. 3.3.2. Effects of the composition of mobile phase The effect of the composition of mobile on the k values of four neutral solutes was shown in Fig. 4. The effect of CTAB on retention factors is very similar to the isotherm of Langmuir adsorption (Fig. 4a). In the range of low CTAB concentrations, k values increase with increasing CTAB concentration very quickly, while in the range of high CTAB concentrations, k values increase relatively moderately. This result means that an increase in CTAB concentration will increase the hydrophobicity of the stationary phase and, therefore, the retention of solutes. It was found that the k values decreased with increasing ACN fraction (Fig. 4b). First, an increase in the ACN fraction will increase the elution strength of the mobile phase as in RPLC, which results in a decrease in the k values of the neutral solutes. Second, the amount of CTAB adsorbed on the SCX monolithic material surface decreases with increasing the ACN content because of the elution strength of mobile phase increased, and thereby the hydrophobicity of the monolith decreases, which also results in a decrease in the k values for the neutral solutes. 3.3.3. Separation of hydrophobic analytes The typical model solutes of different hydrophobic character were used to investigate the performance of the dynamically modified monolithic stationary phases. As shown in Fig. 5a, 10 neutral nonpolar analytes were separated using 60% ACN content in the mobile phase. Alkyl benzenes eluted much faster than PAHs due to the fact that they are less hydrophobic solutes, and these behaviors were mainly based on a reversed-phase mechanism. The results show the dynamically modified poly(SPMA-co-PETA) monolith can offer enough retentivity toward nonpolar solutes due to a relatively strong hydrophobic interaction. Fig. 5b shows the simultaneous
Acknowledgments This work was supported by funding from the National HighTech R&D Program (the 863 program) (2006AA09z161), National Natural Science Foundation (20575012), Science Foundation of Fujian Province (2007J0129), and the Key Science & Technology Project of Fujian Province (2007Y0062, 2008Y0301). References [1] S.M. Fields, Anal. Chem. 68 (1996) 2709. [2] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem. 68 (1996) 3498. [3] U. Pyell, J. Chromatogr. A 892 (2000) 257. [4] H. Zou, X. Huang, M. Ye, Q. Luo, J. Chromatogr. A 954 (2002) 5. [5] F. Svec, E.C. Peters, D. Sykora, J.M.J. Fréchet, J. Chromatogr. A 887 (2000) 3. [6] Q. Tang, M.L. Lee, Trends Anal. Chem. 19 (2000) 648. [7] V.V. Tolstikov, A. Lommen, K. Nakanishi, N. Tanaka, O. Fiehn, Anal. Chem. 75 (2003) 6737. [8] J. Ou, L. Kong, C. Pan, X. Su, X. Lei, H. Zou, J. Chromatogr. A 1106 (2006) 106. [9] Y. Ueki, T. Umemura, Y. Iwashita, T. Odake, H. Haraguchi, K. Tsunoda, J. Chromatogr. A 1117 (2006) 163. [10] J. Zhang, S. Wu, J. Kim, B.L. Karger, J. Chromatogr. A 1154 (2007) 295. [11] E.F. Hilder, F. Svec, J.M.J. Frechet, J. Chromatogr. A 1053 (2004) 101. [12] O. Kornyˇsva, A. Maruˇska, P.K. Owens, M. Erickson, J. Chromatogr. A 1071 (2005) 171. [13] Y. Huo, P.J. Schoenmakers, W.T. Kok, J. Chromatogr. A 1175 (2007) 81. [14] D. Allen, Z. El Rassi, J. Chromatogr. A 1029 (2004) 239. [15] K. Horie, T. Ikegami, K. Hosoya, N. Saad, O. Fiehn, N. Tanaka, J. Chromatogr. A 1164 (2007) 198. [16] R. Freitag, J. Chromatogr. A 1033 (2004) 267. [17] B.W. Pack, D.S. Risley, J. Chromatogr. A 1073 (2005) 269. [18] P. Holdˇsvendová, J. Suchánková, M. Bunˇcek, V. Baˇckovská, P. Coufal, J. Chromatogr. A 70 (2007) 23. [19] M.A. Strege, S. Stevenson, S.M. Lawrence, Anal. Chem. 72 (2000) 4629. [20] Z. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Anal. Chem. 79 (2007) 1243. [21] K. Hosoya, N. Hira, K. Yamamoto, M. Nishimura, N. Tanaka, Anal. Chem. 78 (2006) 5729. [22] R. Wu, H. Zou, M. Ye, Z. Lei, J. Ni, Electrophoresis 23 (2001) 544. [23] M.C. Breadmore, S. Shrinivasan, K.A. Wolfe, M.E. Power, J.P. Ferrance, B. Hosticka, P.M. Norris, J.P. Landers, Electrophoresis 23 (2002) 3487. [24] J.P. Hutchinson, E.F. Hilder, M. Macka, N. Avdalovic, P.R. Haddad, J. Chromatogr. A 1109 (2006) 10. [25] Y. Tian, R. Feng, L. Liao, H. Liu, H. Chen, Z. Zeng, Electrophoresis 29 (2008) 3153. [26] Z. Jiang, R. Gao, Y. Zhou, Z. Zhang, Q. Wang, C. Yan, J. Microcol. Sep. 13 (2001) 191. [27] M. Ye, H. Zou, Z. Liu, J. Ni, Y. Zhang, Anal. Chem. 72 (2000) 618.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Capillary liquid chromatography using a hydrophilic/cation-exchange monolithic column with a dynamically modified cationic surfactant Jian Lin, Jia Lin, Xucong Lin, Zenghong Xie ∗ Department of Chemistry, Fuzhou University, Fuzhou 350002, China
a r t i c l e
i n f o
Article history: Received 23 May 2009 Received in revised form 25 August 2009 Accepted 28 August 2009 Available online 1 September 2009 Keywords: Monolithic stationary phases Dynamically modified capillary liquid chromatography Hydrophilic interaction Reversed-phase mode
a b s t r a c t A novel form of reversed-phase liquid chromatography (RPLC) by the dynamically modified hydrophilic interaction monolithic column has been described in this paper. A porous poly(SPMA-co-PETA) monolith with strong cation-exchange (SCX) was prepared and the resulting monolith showed a typical hydrophilic interaction chromatography (HILIC) mechanism at higher organic solvent content (ACN% > 50%). The good selectivity for neutral, basic and acidic polar analytes was observed in the HILIC mode. In order to increase the hydrophobic interaction, the monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX monolithic material, and the resulting hydrophobic layer was used as the stationary phase. Using the dynamically modified SCX monolithic column, neutral, basic and acidic hydrophobic analytes were well separated with the RPLC mode. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction Nowadays, a novel porous material known as monoliths is an alternative to column packed with particulate stationary phase, which has been extensively studied and gradually applied in capillary liquid chromatography (CLC) and capillary electrochromatography (CEC) [1–6]. Reversed-phase liquid chromatography (RPLC) based on nonpolar stationary phases is a powerful separation mode because of its versatility and ability to retain and resolve a wide range of hydrophobic to slightly polar analytes [7–14]. However, for highly polar analytes, these hydrophobic monoliths show limited extent of retention in the reversed-phase mode [15]. Hydrophilic interaction chromatography (HILIC) based on polar stationary phases has gained increasing attention because of effective solution to the separation of polar analytes [15–20]. But due to the low hydrophobicity, the ability of these polar monoliths to separate hydrophobic analytes was poor. Only few studies have been reported for the efficient separation of various characters of compounds [21], and it is still a somewhat troublesome task in a single polar or nonpolar stationary phase. The production of monolith having both strong hydrophobic and hydrophilic interaction is rather a difficult process, and it cannot be achieved by simple polymerization of polar and nonpolar monomers. Therefore, the further development of resoluble methods to offer effective retention for
∗ Corresponding author. Tel.: +86 591 22866131. E-mail address: [email protected] (Z. Xie).
a wide range of hydrophobic to highly polar compounds in one column is necessary. This report demonstrated a novel conversion of chromatographic mode by the dynamically modified hydrophilic interaction monolith for the separation of various compounds. Chromatography on dynamically modified monolithic columns has been widely reported for use [22–25], which was a simple and convenient technique to introduce desired chromatographic functionality onto the monolithic stationary phase to exert selectivity. In this work, a porous monolith with strong cation-exchange (SCX) was prepared. The poly(SPMA-co-PETA) monolithic column was applied to the separation of polar analytes in a HILIC mode, and then the hydrophilic interaction monolith was dynamically modified with cetyltrimethylammonium bromide (CTAB), which showed excellent separation of hydrophobic analytes based on a RPLC mode. 2. Experimental 2.1. Reagents and materials 3-Sulfopropyl methacrylate potassium salt (SPMA), pentaerythritol triacrylate (PETA), 2,2 -azobisisobutyronitrile (AIBN), 3-(trimethoxysilyl)propyl methacrylate (␥-MAPS), benzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4,5trihydroxybenzoic acid, alkyl benzenes, polycyclic aromatic hydrocarbons were purchased from Aldrich (Milwaukee, WI, USA). Uracil, uridine, guanine, guanosine, cytosine, cytidine were purchased from Sigma (St. Louis, MO, USA). HPLC-grade methanol
0021-9673/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.08.071
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and acetonitrile (ACN), thiourea, acrylamide, phenol, resorcinol, catechol, hydroquinone, pyrogallol, 3,5-dinitrobenzoic acid, 4nitrobenzoic acid, 2-nitroaniline, 4-nitroaniline, diphenylamine were obtained from Chemical Reagent Corporation (Shanghai, China). Buffer solutions were prepared using sodium phosphate monobasic or triethylamine phosphate, phosphoric acid, sodium hydroxide, triethylamine, cetyltrimethylammonium bromide (CTAB), cyclohexanol and ethylene glycol were from Chemical Reagent Plant (Shanghai, China). Fused-silica capillaries with an internal diameter of 100 m and an outer diameter of 365 m were purchased from the Yongnian Optic Fiber Plant (Hebei, China). Ultrapure water used in all of the experiments was doubly distilled and purified by a Milli-Q system (Millipore, Milford, MA, USA). 2.2. Instrumentation The experiments were performed on a Trisep 2010GV cLC system (Unimicro Technologies, Pleasanton, CA, USA) equipped with a UV/Vis detector (190–800 nm), comprising two microvolume pumps, a microfluid manipulation module (including a six-port injector), and a data acquisition module, as described in literature [26]. An HPLC pump was used to flush the monolithic columns.
Fig. 1. Relationship between retention time and acetonitrile concentration on poly(SPMA-co-PETA) monolithic column. Conditions: mobile phase, 5 mM triethylamine phosphate pH 6.00 in ACN/H2 O; pump flow: 0.02 ml/min; applied pressure 7.0 MPa.
2.3. Preparation of the monolithic column Polymerization solutions weighing 2.0 g each were prepared from monomers (SPMA and PETA) and porogenic solvents in ratios of 20:80 (w/w) monomers/solvents. AIBN (1.0%, w/w, with respect to monomers) was dissolved in 0.4 g of solution consisting of 80.0% (w/w) of SPMA and 20.0% (w/w) of PETA, respectively. The binary porogenic solvents contained 55.0% (w/w) of cyclohexanol and 45.0% (w/w) of ethylene glycol. The mixture was then sonicated to obtain a clear solution, and purged with nitrogen for 5 min, a small part of the mixed solution was removed for capillary preparation using a 100 L syringe. A pretreated 55 cm capillary was attached to the syringe inlet, and the polymerization mixture was sucked into the capillary for 35 cm length. The capillary was plugged at both ends with rubber stoppers and was submerged in a 60 ◦ C water bath for 20 h. The prepared monolithic capillary column was washed with methanol and water by an HPLC pump to flush out the residual reagents. A detection window was created at 1–2 mm after the end of the polymer bed using thermal wire stripper. Finally, the column was cut to a total length of 50 cm with an effective length of 30 cm. 3. Results and discussion 3.1. Retention properties of poly(SPMA-co-PETA) monolith in aqueous acetonitrile In order to investigate the chromatographic properties of poly(SPMA-co-PETA) monolith in aqueous ACN mobile phases, toluene, acrylamide, and thiourea were used as test compounds. The solvent was selected as the void time marker in this system. The influence of ACN content in the mobile phase on the retention factor (k ) of three test compounds is shown in Fig. 1. The hydrophilic solutes, thiourea eluted after acrylamide and toluene when the ACN concentration was increased from 40% to 95%. The retention time of thiourea leveled off initially as the ACN content in the mobile phase increased from 30% to 50% and then increased when the ACN content further increased to 95%. Acrylamide behaved similar to thiourea but with much less retention due to the lower hydrophilicity. These results suggest a typical HILIC retention mechanism at higher ACN content in the mobile phase (>50%). For the hydrophobic solute toluene, the k values were decreased with the increment
Fig. 2. Hydrophilic interaction chromatography for the separation of (a) phenols, (b) benzoic acid derivatives and (c) nucleobases. Conditions: (a) mobile phase, 5 mM triethylamine phosphate, pH 6.00, at 95% (v/v) ACN; pump flow: 0.02 ml/min; applied pressure 5.1 MPa; detection wavelength, 214 nm. (b) Mobile phase, 5 mM triethylamine phosphate, pH 6.00, at 90% (v/v) ACN; pump flow: 0.02 ml/min; applied pressure 5.3 MPa; detection wavelength, 214 nm. (c) Mobile phase, 40 mM triethylamine phosphate, pH 4.00, at 80% (v/v) ACN; pump flow: 0.02 ml/min; applied pressure 3.5 MPa; detection wavelength, 254 nm.
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of the content of ACN from 30% to 50% and then approximately constant at higher ACN content. This means a RPLC-mode separation dominantly worked on this stationary phase at lower organic solvent content (ACN% < 50%). 3.2. Hydrophilic interaction chromatography The poly(SPMA-co-PETA) surface can provide a hydrophilic and ion-exchange environment. A series of neutral, basic and acidic polar analytes were selected as model compounds. The result was shown in Fig. 2. As expected for a hydrophilic monolith, the retention of phenols and benzoic acid derivatives increased with an increase in the number of hydroxyl groups within the molecule (Fig. 2a and b). Moreover, the positional isomers of phenols can be readily separated. The results showed that the extent of hydrophilic interaction depends on the number of the hydroxyl groups and the position. A baseline separation without obvious peak tailing of the nucleobases was obtained in Fig. 2c. Upon conjugation of the bases with ribose to yield nucleosides, the neutral uridine was more retained than uracil, indicating that a ribose moiety confers on the solute molecules an increased hydrophilic character [14]. Cytosine (pKa 4.5), which is about the same polarity as uracil, eluted last. This may be attributed to strong electrostatic interaction with the sulfonic acid groups of the stationary phase at pH 4.00 buffer. The separation of all the nucleobases were primarily based on both hydrophilic interaction and electrostatic interaction mechanisms on the poly(SPMA-co-PETA) stationary phase. 3.3. Reversed-phase liquid chromatography The poly(SPMA-co-PETA) monolithic column can also provide hydrophobic interaction at lower ACN content. However, as shown in Fig. 3a, the four alkyl benzenes were not efficiently separated Fig. 4. Effect of mobile phase on retention factor of neutral solutes. Plot a: effect of CTAB concentration, ACN and 20 mM sodium monophosphate (pH 2.50) kept at 50% and 10%, but changing the ratio of 50 mM CTAB solution to water. Plot b: effect of ACN fraction, 50 mM CTAB solution and 20 mM sodium monophosphate (pH 2.50) kept at 12% and 10%, but changing the ratio of ACN to water; others as in Fig. 3b.
even at relatively low ACN content (30% ACN). This is mainly due to the low hydrophobicity of monolith. In order to increase the hydrophobic interaction of poly(SPMA-co-PETA) monolith, dynamically modified chromatography was introduced in this paper.
Fig. 3. Chromatograms for the separation of five neutral solutes in (a) poly(SPMAco-PETA) monolith and (b) dynamically modified poly(SPMA-co-PETA) monolith. Conditions. (a) Mobile phase, 5 mM triethylamine phosphate, pH 2.50, at 30% (v/v) ACN. (b) Mobile phase, ACN/50 mM CTAB/20 mM sodium monophosphate (pH 2.50)/water = 50/10/12/28. Pump flow: 0.02 ml/min; applied pressure 10.5 MPa; detection wavelength, 214 nm.
3.3.1. Characterization of the CTAB dynamically modified poly(SPMA-co-PETA) monolith The poly(SPMA-co-PETA) monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, CTAB, which was added to the mobile phase. The separation mechanism of dynamically modified poly(SPMA-co-PETA) monolith was similar to that of dynamically modified SCX packing material CEC [27]. The adsorbed CTAB molecules formed a hydrophobic layer on the SCX monolithic material surface, which acted as the stationary phase as C18 chains on the ODS packings. Neutral compounds could be separated in this system based on the partitioning between the mobile phase and hydrophobic layer. The efficiency of the columns and the reproducibility of the retention factors were evaluated with four homologous benzene compounds. Void time was measured by baseline disturbance. The highest number of theoretical plates reaching greater than 100 000 plates/m was obtained, and the relative standard deviation for retention factor was less than 1.0%. Fig. 3b shows the separation of the four neutral alkyl benzenes on cLC with dynamically modified poly(SPMA-co-PETA) monolith. The hydrophobicity of SCX monolith could be increased by dynamic
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separation of acidic, basic and neutral hydrophobic compounds using dynamically modified poly(SPMA-co-PETA) monolith with a low-pH mobile phase. Excellent separation of all eight compounds was accomplished. Under these conditions, chromatography plays a major role in the separation process of weakly acidic and neutral compounds. The basic compounds were positively charged at low-pH mobile phase, but good peak symmetry for the basic compounds was observed. The peak tailing of basic compounds can be effectively eliminated with dynamically modified poly(SPMA-coPETA) monolith because the surfactant used has strong affinity to sulfonic acid groups than the common basic compound does. 4. Conclusion A porous poly(SPMA-co-PETA) monolithic capillary column, which was prepared by one-step copolymerization of SPMA and PETA, has been successfully used as a stationary phase in HILIC mode. Typical HILIC retention was obtained at high organic solvent content (ACN% > 50%). The hydrophobicity of poly(SPMA-co-PETA) monolith was increased by dynamic adsorption of CTAB, and the separation mechanism of dynamically modified poly(SPMAco-PETA) monolith was mainly based on RPLC mode. Efficient separation of a wide range of hydrophobic to highly polar compounds was performed in different modes for one column. Fig. 5. Separation of hydrophobic solutes at low pH. Conditions: mobile phase, ACN/50 mM CTAB/20 mM sodium monophosphate (pH 2.50)/water = 60/12/10/18. Pump flow: 0.02 ml/min; applied pressure at (a) 13.0 MPa and (b) 7.0 MPa; detection wavelength, 214 nm.
adsorption of CTAB, and neutral alkyl benzenes were well separated in this system. 3.3.2. Effects of the composition of mobile phase The effect of the composition of mobile on the k values of four neutral solutes was shown in Fig. 4. The effect of CTAB on retention factors is very similar to the isotherm of Langmuir adsorption (Fig. 4a). In the range of low CTAB concentrations, k values increase with increasing CTAB concentration very quickly, while in the range of high CTAB concentrations, k values increase relatively moderately. This result means that an increase in CTAB concentration will increase the hydrophobicity of the stationary phase and, therefore, the retention of solutes. It was found that the k values decreased with increasing ACN fraction (Fig. 4b). First, an increase in the ACN fraction will increase the elution strength of the mobile phase as in RPLC, which results in a decrease in the k values of the neutral solutes. Second, the amount of CTAB adsorbed on the SCX monolithic material surface decreases with increasing the ACN content because of the elution strength of mobile phase increased, and thereby the hydrophobicity of the monolith decreases, which also results in a decrease in the k values for the neutral solutes. 3.3.3. Separation of hydrophobic analytes The typical model solutes of different hydrophobic character were used to investigate the performance of the dynamically modified monolithic stationary phases. As shown in Fig. 5a, 10 neutral nonpolar analytes were separated using 60% ACN content in the mobile phase. Alkyl benzenes eluted much faster than PAHs due to the fact that they are less hydrophobic solutes, and these behaviors were mainly based on a reversed-phase mechanism. The results show the dynamically modified poly(SPMA-co-PETA) monolith can offer enough retentivity toward nonpolar solutes due to a relatively strong hydrophobic interaction. Fig. 5b shows the simultaneous
Acknowledgments This work was supported by funding from the National HighTech R&D Program (the 863 program) (2006AA09z161), National Natural Science Foundation (20575012), Science Foundation of Fujian Province (2007J0129), and the Key Science & Technology Project of Fujian Province (2007Y0062, 2008Y0301). References [1] S.M. Fields, Anal. Chem. 68 (1996) 2709. [2] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem. 68 (1996) 3498. [3] U. Pyell, J. Chromatogr. A 892 (2000) 257. [4] H. Zou, X. Huang, M. Ye, Q. Luo, J. Chromatogr. A 954 (2002) 5. [5] F. Svec, E.C. Peters, D. Sykora, J.M.J. Fréchet, J. Chromatogr. A 887 (2000) 3. [6] Q. Tang, M.L. Lee, Trends Anal. Chem. 19 (2000) 648. [7] V.V. Tolstikov, A. Lommen, K. Nakanishi, N. Tanaka, O. Fiehn, Anal. Chem. 75 (2003) 6737. [8] J. Ou, L. Kong, C. Pan, X. Su, X. Lei, H. Zou, J. Chromatogr. A 1106 (2006) 106. [9] Y. Ueki, T. Umemura, Y. Iwashita, T. Odake, H. Haraguchi, K. Tsunoda, J. Chromatogr. A 1117 (2006) 163. [10] J. Zhang, S. Wu, J. Kim, B.L. Karger, J. Chromatogr. A 1154 (2007) 295. [11] E.F. Hilder, F. Svec, J.M.J. Frechet, J. Chromatogr. A 1053 (2004) 101. [12] O. Kornyˇsva, A. Maruˇska, P.K. Owens, M. Erickson, J. Chromatogr. A 1071 (2005) 171. [13] Y. Huo, P.J. Schoenmakers, W.T. Kok, J. Chromatogr. A 1175 (2007) 81. [14] D. Allen, Z. El Rassi, J. Chromatogr. A 1029 (2004) 239. [15] K. Horie, T. Ikegami, K. Hosoya, N. Saad, O. Fiehn, N. Tanaka, J. Chromatogr. A 1164 (2007) 198. [16] R. Freitag, J. Chromatogr. A 1033 (2004) 267. [17] B.W. Pack, D.S. Risley, J. Chromatogr. A 1073 (2005) 269. [18] P. Holdˇsvendová, J. Suchánková, M. Bunˇcek, V. Baˇckovská, P. Coufal, J. Chromatogr. A 70 (2007) 23. [19] M.A. Strege, S. Stevenson, S.M. Lawrence, Anal. Chem. 72 (2000) 4629. [20] Z. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Anal. Chem. 79 (2007) 1243. [21] K. Hosoya, N. Hira, K. Yamamoto, M. Nishimura, N. Tanaka, Anal. Chem. 78 (2006) 5729. [22] R. Wu, H. Zou, M. Ye, Z. Lei, J. Ni, Electrophoresis 23 (2001) 544. [23] M.C. Breadmore, S. Shrinivasan, K.A. Wolfe, M.E. Power, J.P. Ferrance, B. Hosticka, P.M. Norris, J.P. Landers, Electrophoresis 23 (2002) 3487. [24] J.P. Hutchinson, E.F. Hilder, M. Macka, N. Avdalovic, P.R. Haddad, J. Chromatogr. A 1109 (2006) 10. [25] Y. Tian, R. Feng, L. Liao, H. Liu, H. Chen, Z. Zeng, Electrophoresis 29 (2008) 3153. [26] Z. Jiang, R. Gao, Y. Zhou, Z. Zhang, Q. Wang, C. Yan, J. Microcol. Sep. 13 (2001) 191. [27] M. Ye, H. Zou, Z. Liu, J. Ni, Y. Zhang, Anal. Chem. 72 (2000) 618.