- Email: [email protected]
Study of triacontyl-functionalized monolithic silica capillary column for reversed-phase capillary liquid chromatography
Journal of Chromatography A, 1216 (2009) 2597–2600
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Study of triacontyl-functionalized monolithic silica capillary column for reversed-phase capillary liquid chromatography Yashun Chen, Jie Chen, Li Jia ∗ Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China
a r t i c l e
i n f o
Article history: Received 28 September 2008 Received in revised form 9 January 2009 Accepted 16 January 2009 Available online 23 January 2009 Keywords: Triacontyl-functionalized monolithic silica capillary column Reversed-phase capillary liquid chromatography Performance
a b s t r a c t A novel stationary phase triacontyl-functionalized monolithic silica capillary column was successfully prepared for reversed-phase capillary liquid chromatography. The performance of the monolithic silica capillary column coated with triacontyl chain for the separation of alkylbenzenes, xylene isomers, polycyclic aromatic hydrocarbons, and mixture of ␣- and -carotenes was studied, which was compared to that using the monolithic silica capillary column coated with octadecyl chain. The comparison results showed that triacontyl-functionalized monolithic silica capillary column would be a promising media to be used for the separation of isomeric solutes with long chain in reversed-phase capillary liquid chromatography. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Monolithic column has rapidly become highly popular separation media in chromatography. In comparison with the traditional packed columns, monolithic column offers many improvements including ease in construction, high surface area and porosity, fast mass transfer, absence of end frits, and elimination or significant reduction of certain operation problems inherent in packed columns due to the presence of end frits. In addition, the monolithic column can achieve higher separation efficiency at a similar pressure drop resulting from their unique structures [1]. They consist of one single piece of highly porous material with bimodal pore size distribution, in which the macropores influence the permeability and the mesopores determine the surface area. A large number of monolithic materials have been described in a variety of excellent reviews [2–4]. So far, silica-based monolithic columns have been extensively studied in liquid chromatography (LC) [5–7], especially octadecyl (C18) columns in reversed-phase LC [8,9]. However, C18 columns are not ideal for the separation of isomeric solutes with long chain, such as carotenoid [10], tocopherols [11] and fatty acids [12]. The packed triacontyl (C30) column has demonstrated better performance than C18 column for the separation of these solutes [10–12]. The use of packed C30 stationary phases in LC was first reported in 1987 for the separation of polycyclic aromatic hydrocarbons
∗ Corresponding author. Tel.: +86 20 85211543; fax: +86 20 85216052. E-mail addresses: [email protected], jiali [email protected] (L. Jia). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.01.058
(PAHs) [13], and oligonucleotides [14]. Since then, the alkylbenzenes [15], aldehyde and ketone derivatives [16] and fullerenes [17] were also separated with C30 stationary phases. So far, the use of C30 stationary phases has expanded to the enrichment of PAHs [18], the separation of oligosaccharides [19], amino acids [20], derivatized regioisomeric 1, 3 diacylglycerols [21], and analogs of the sodium channel blocker tetrodotoxin [22]. Sander and co-workers investigated the effect of phase length on column selectivity by utilizing the C30 stationary phase for the separation of carotenoid and PAHs, etc. [11,23]. A comprehensive review described the application of packed C30 columns in the analysis of carotenoids, retinoids and other nutrients in complex and natural-matrix samples [24]. To the best of our knowledge, C30 functionalized monolithic silica capillary column has never been reported. Considering the advantages of monolithic column, the preparation of C30 silica-based monolithic column was developed in this paper. Its performance for the separation of some model compounds was compared with that using C18 silica-based monolithic column. 2. Experimental 2.1. Reagents and chemicals Ammonium hydroxide (NH3 ·H2 O (25%, w/w)), sodium hydroxide (NaOH) and acetic acid were purchased from Guangzhou Chemical Regent Factory (Guangzhou, China). Polyethylene glycol (PEG, Mw 10,000), o-xylene and p-xylene were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Tetramethoxysilane was purchased from Acros Organics (Geel,
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Fig. 1. SEM photographs of the cross section of monolithic silica capillary column before modification with C18 chain and C30 chain at magnification of (A) 2000× and (B) 4000×.
Belgium). Toluene, ethylbenzene, propylbenzene, butylbenzene, phenanthrene and anthracene were purchased from Alfa Aesar (Heysham, UK). Dimethyltriacontylchlrosilane and dimethyloctadecylchlorosilane were purchased from Fluorochem Company (Derbyshire, UK). Carotene (mixture of ␣- and -carotenes) and -carotene were obtained from Sigma (St Louis, MO, USA). These reagents were used without any further purification. Methanol (HPLC grade) was purchased from SK Chemicals (Ulsan, South Korea). Water used in the experiments was obtained from an Elga water purification system (ELGA, London, UK). 2.2. Instrumentation The capillary LC system was comprised of a microflow pump (MP711, GL Sciences, Tokyo, Japan), an UV detector with an optical fiber flow cell (MP701, GL Sciences, Tokyo, Japan), and a manual injection valve (Valco VICI, Switzerland) with an internal sample loop of 50 nl. Chromatographic data acquisition was performed with the software HW-2000 workstation (Shanghai, China). All separations were carried out at room temperature, which was kept at 25 ◦ C using air conditioning system. A fused-silica capillary (GL Sciences, Japan), 8 cm × 50 m i.d., was used to connect the outlet end of analytical column with the optical fiber flow cell (light path 3 mm). For column fabrication, temperature-controlling process was carried out using a gas chromatography (GC) system 7890 II (Techcompany, Shanghai, China). Fused-silica capillaries (100 m i.d. × 365 m o.d.) were purchased from Hebei Yongnian Ruipu Chromatogram Equipment Company (Hebei, China). Scanning electron microscopy (SEM) of the silica monolith was carried out on a LEO-1530VP scanning electron microscope (Oberkochen, Germany) 2.3. Pretreatment of capillary A fused-silica capillary was first rinsed with 1 M NaOH for 2 h, then was put in a thermostated water bath at 40 ◦ C for 3 h. Next the capillary was rinsed by water for 30 min, 1 M HCl for 1 h, water for 30 min, respectively. Then it was dried with nitrogen at 180 ◦ C in a GC oven for 3 h prior to use. 2.4. Preparation and chemical modification of monolithic silica columns The procedure for the preparation of a monolithic silica column was based on the method outlined by Ishizuka et al. [25] with some modifications. Briefly, 3.13 ml TMOS, 0.625 g PEG and 6.50 ml acetic acid (0.01 M) were mixed by stirring for 45 min at 0 ◦ C, then the solution was sonicated for 30 s. After the transparent sol was injected into the pretreated capillary, the capillary was heated at 40 ◦ C in a
thermostated water bath for 17 h. Then, the column was rinsed with 0.01 M ammonium hydroxide and heated at 120 ◦ C for 60 min. This process was repeated three times. Next, the column was washed with water and dried in a GC oven, and the final heat treatment of the column was performed at 300 ◦ C for 20 h to remove the organic moieties and complete the formation of silica monolith. 2.4.1. Chemical modification of monolithic silica columns with C18 chain After the silica column was washed with toluene for 1 h, a solution consisting of 10% (w/v) dimethyloctadecylchlorosilane in toluene was pushed through the silica monolith and reacted at 120 ◦ C for 10 h. This process was repeated three times. Then the column was rinsed by toluene and methanol in sequence. 2.4.2. Chemical modification of monolithic silica columns with C30 chain Dimethyltriacontylchlrosilane (0.05 g) was added to 0.5 ml of toluene. The mixture was heated and held at 70 ◦ C until dimethyltriacontylchlrosilane dissolved in toluene. The resultant homogeneous solution was pushed through the silica column, which was washed with toluene for 1 h in advance, and reacted at 120 ◦ C for 10 h. This process was repeated three times. Finally the column was rinsed with toluene and methanol in sequence. 3. Results and discussion 3.1. SEM observation The SEM photographs of the cross section of monolithic silica columns before modification with C18 chain and C30 chain are shown in Fig. 1. As can be seen from the micrographs, the large through pores are interconnected. Sufficient attachment of silica monolithic skeletons to the capillary wall can prevent the shrinkage of the whole network structure. Ammonium hydroxide was used as the mesopores tailor to yield appropriate mesopores of the monolithic columns to offer sufficient large surface area that would be useful for chromatography. 3.2. Comparison of C30 and C18 columns by separation of alkylbenzenes, xylene isomers, PAHs and carotenes Fig. 2 shows the van Deemter plots of C30 and C18 monolithic silica capillary columns obtained with toluene and butylbenzene as solutes and 80/20 (v/v) methanol/water as the mobile phase. The permeability, K, was evaluated according to Eq. (1) [26], where P, , L and stands for column back pressure, dynamic viscosity of
Y. Chen et al. / J. Chromatogr. A 1216 (2009) 2597–2600
Fig. 2. van Deemter plots of the C30 and C18 monolithic silica capillary columns obtained using toluene and butylbenzene as solutes. Experimental conditions: C30 monolithic column, 30 cm × 100 m i.d.; C18 monolithic column, 30 cm × 100 m i.d.; mobile phase, 80/20 (v/v) methanol/water; detection wavelength, 214 nm.
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Fig. 4. Chromatograms of the mixture of ␣- and -carotene using C30 and C18 monolithic silica capillary columns. Experimental conditions: C30 monolithic column, 30 cm × 100 m i.d.; C18 monolithic column, 30 cm × 100 m i.d.; mobile phase, methanol; detection wavelength, 450 nm. Peak identification: 1, ␣-carotene; 2, carotene.
eluent, column length, and linear velocity of eluent, respectively. K=
L P
(1)
The permeability of the C18 column was calculated to be 4.58 × 10−14 m2 , while the permeability of the C30 column was calculated as 1.43 × 10−13 m2 . The results indicated that the C30 column provided more permeability than the C18 column. It was supposed that the difference in the bonding density for the two columns resulted in the difference in the permeability. And the bonding density for the C30 column was less than that for the C18 column. The performance of C30 monolithic silica column was compared with that of C18 monolithic silica column by the separation of alkylbenzenes, xylene, PAHs and carotenes. At first, the separation of four alkylbenzenes using C30 and C18 monolithic silica capillary columns were investigated with 80/20 (v/v) methanol/water as the mobile phase. As shown in Fig. 3, the separation performance using C30 column was inferior to that using C18 column for the alkylbenzenes. The retention factors (k) of toluene, ethyl-
Fig. 3. Chromatograms of the mixture of four alkylbenzenes using C30 and C18 monolithic silica capillary columns. Experimental conditions: C30 monolithic column, 30 cm × 100 m i.d.; C18 monolithic column, 30 cm × 100 m i.d.; mobile phase, 80/20 (v/v) methanol/water; detection wavelength, 214 nm. Peak identification: 1, toluene; 2, ethylbenzene; 3, propylbenzene; 4, butylbenzene.
benzene, propylbenzene and butylbenzene using C18 column were 0.74, 1.08, 1.72, and 2.77, respectively, while using C30 column, k of the analytes were 0.18 (toluene), 0.24 (ethylbenzene), 0.35 (propylbenzene), and 0.49 (butylbenzene), respectively. The number of theoretical plates (N) of the C18 and C30 were calculated using butylbenzene to be 3.78 × 104 , and 2.11 × 104 , respectively. Comparing with the C18 column, the less k and less N for the C30 column resulted from the less bonding density of C30 on the silica monolith. Next, the separation of o-xylene and p-xylene isomers using C30 and C18 columns was performed with 80/20 (v/v) methanol/water as the mobile phase (figure not shown). o-Xylene and p-xylene isomers was partially separated with the C18 monolithic column. Under the same conditions, o-xylene and p-xylene isomers were not resolved at all with the C30 monolithic column. The resolution (Rs) between o-xylene and p-xylene for the C18 and C30 columns were 0.7 and 0, respectively. The k of o-xylene for the C18 and C30 columns was 0.71 and 0.27, respectively. The separation of the isomers with small molecules may be strongly influenced by the bonding density of alkyl group on the silica monolith. Then, the separation of phenanthrene and anthracene isomers using C30 and C18 columns was investigated with 80/20 (v/v) methanol/water as the mobile phase (figure not shown). The Rs between phenanthrene and anthracene for the C18 and C30 columns was 1.0 and 0.8, respectively. The k of phenanthrene and anthracene for the C18 column was 1.72 and 1.94, respectively. For the C30 column, the k of phenanthrene and anthracene was 0.81 and 0.92, respectively. Based on the resolution and k for the separation of phenanthrene and anthracene using the C18 and C30 monolithic columns, the bonding density and the length of the alkyl group on the silica monolith play similar roles for controlling the selectivity of phenanthrene and anthracene. At last, the separation of ␣- and -carotene isomers with long chain was carried out using methanol as the mobile phase. As we know, C18 bonded phases are not ideal for the isomers with long chain since the dimensions of the analytes exceed the thickness of the stationary phase [10–12]. In conventional LC, the problem has been alleviated by synthesizing the bonded phases with longer chain length to the silica gels [27,28] or by using supercritical fluid chromatography [29,30]. As shown in Fig. 4, the use of C30 column for the separation of ␣- and -carotene isomers showed higher degree of selectivity than that using C18 column. ␣- and -carotene isomers were separated baseline using C30 column, while using C18
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column, the carotene isomers just obtained partial separation. The Rs between ␣- and -carotene using C18 and C30 columns was 1.1 and 2.5, respectively. The k of ␣- and -carotene using C18 column was 17.59 and 19.15, respectively, while using C30 column, the k of ␣- and -carotene was 11.51 and 14.42, respectively. The results showed the length of the phase is the primary factor controlling the selectivity for the separation of the isomers with long chain. From the experimental results, we can conclude that the bonding density and the length of the alkyl group on the silica monolith combined to influence the separation selectivity of analytes for C18- and C30-functionalized monolithic silica capillary columns. For small molecules, the bonding density of the alkyl group on the silica monolith plays main roles, while for the isomers with long chain, the length of the alkyl group on the silica monolith is a key factor. 4. Conclusions A kind of new C30-functionalized monolithic column for reversed-phase capillary LC was prepared in a fused-silica capillary via sol–gel process. The monolithic silica capillary column bonded with C30 offers advantages over the column bonded with C18 for the separation of the mixture of ␣- and -carotenes. The C18-functionalized monolithic silica capillary column shows a good performance for the separation of some small molecules because of the higher amount of alkyl chain bonded to the silica skeleton. It can be expected that C30-functionalized monolithic silica capillary column will be a promising media for the separation of isomeric solutes with long chain in reversed-phase LC. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20575041), Ministry of Education Science Foundation of China for Returnees, and Special Foundation of Guangzhou for Public Instruments.
References [1] C. Yang, T. Ikegami, T. Hara, N. Tanaka, J. Chromatogr. A 1130 (2006) 175. [2] G. Guiochon, J. Chromatogr. A 1168 (2007) 101. [3] H.F. Zou, X.D. Huang, M.L. Ye, Q.Z. Luo, J. Chromatogr. A 954 (2002) 5. ´ [4] E. Kłodzinska, D. Moravcova, P. Jandera, B. Buszewski, J. Chromatogr. A 1109 (2006) 51. ˜ [5] O. Núnez, T. Ikegami, W. Kajiwara, K. Miyamoto, K. Horie, N. Tanaka, J. Chromatogr. A 1156 (2007) 35. [6] F. Pellati, S. Calò, S. Benvenuti, J. Chromatogr. A 1149 (2007) 56. [7] S. Nonaka, M. Tsunoda, C. Aoyama, T. Funatsu, J. Chromatogr. B 843 (2006) 170. [8] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem. 68 (1996) 3498. [9] L. Jia, N. Tanaka, S. Terabe, J. Chromatogr. A 1053 (2004) 71. [10] L.C. Sander, K.E. Sharpless, N.E. Craft, S.A. Wise, Anal. Chem. 66 (1994) 1667. [11] C.A. Rimmer, L.C. Sander, S.A. Wise, Anal. Bioanal. Chem. 382 (2005) 698. [12] K. Takayama, H.C. Jordi, F. Benson, J. Liq. Chromatogr. 3 (1980) 61. [13] L.C. Sander, S.A. Wise, Anal. Chem. 59 (1987) 2309. [14] K. Makino, H. Ozaki, H. Imaishi, T. Takeuchi, T. Fukui, Chem. Lett. 16 (1987) 1251. [15] I.Z. Atamna, G.M. Muschik, J. Liq. Chromatogr. 12 (1989) 2227. [16] W. Poetter, S. Lamotte, H. Engelhardt, U. Karst, J. Chromatogr. A 786 (1997) 47. [17] H. Ohta, K. Jinno, Y. Saito, J.C. Fetzer, W.R. Biggs, J.J. Pesek, M.T. Matyska, Y.L. Chen, Chromatographia 42 (1996) 56. [18] L.W. Lim, Y. Okouchi, T. Takeuchi, Talanta 72 (2007) 1600. [19] T. Kato, K. Abe, K. Nakamura, M. Tachibana, H. Koizumi, K. Tani, N. Kiba, Bunseki Kagaku 52 (2003) 741. [20] H. Peters, X. Hou, B. Jones, Appl. Spectrosc. 57 (2003) 1162. [21] H. Takano, Y. Itabashi, Bunseki Kagaku 51 (2000) 437. [22] Y. Shoji, M. Yotsu-Yamashita, T. Miyazawa, T. Yasumoto, Anal. Biochem. 290 (2001) 10. [23] L.C. Sander, M. Pursch, S.A. Wise, Anal. Chem. 71 (1999) 4821. [24] L.C. Sander, K.E. Sharpless, M. Pursch, J. Chromatogr. A 880 (2000) 189. [25] N. Ishizuka, H. Kobayashi, H. Minakuchi, K. Nakanishi, K. Hirao, K. Hosoya, T. Ikegami, N. Tanaka, J. Chromatogr. A 960 (2002) 85. [26] R.T. Kennedy, J.W. Jorgenson, Anal. Chem. 61 (1989) 1128. [27] C.M. Bell, L.C. Sander, J.C. Fetzer, S.A. Wise, J. Chromatogr. A 753 (1996) 37. [28] J. Wegmann, K. Albert, M. Pursch, L.C. Sander, Anal. Chem. 73 (2001) 1814. [29] E. Lesellier, C. West, A. Tchapla, J. Chromatogr. A 1018 (2003) 225. [30] E. Lesellier, K. Gurdale, A. Tchapla, J. Chromatogr. A 844 (1999) 307.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Study of triacontyl-functionalized monolithic silica capillary column for reversed-phase capillary liquid chromatography Yashun Chen, Jie Chen, Li Jia ∗ Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China
a r t i c l e
i n f o
Article history: Received 28 September 2008 Received in revised form 9 January 2009 Accepted 16 January 2009 Available online 23 January 2009 Keywords: Triacontyl-functionalized monolithic silica capillary column Reversed-phase capillary liquid chromatography Performance
a b s t r a c t A novel stationary phase triacontyl-functionalized monolithic silica capillary column was successfully prepared for reversed-phase capillary liquid chromatography. The performance of the monolithic silica capillary column coated with triacontyl chain for the separation of alkylbenzenes, xylene isomers, polycyclic aromatic hydrocarbons, and mixture of ␣- and -carotenes was studied, which was compared to that using the monolithic silica capillary column coated with octadecyl chain. The comparison results showed that triacontyl-functionalized monolithic silica capillary column would be a promising media to be used for the separation of isomeric solutes with long chain in reversed-phase capillary liquid chromatography. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Monolithic column has rapidly become highly popular separation media in chromatography. In comparison with the traditional packed columns, monolithic column offers many improvements including ease in construction, high surface area and porosity, fast mass transfer, absence of end frits, and elimination or significant reduction of certain operation problems inherent in packed columns due to the presence of end frits. In addition, the monolithic column can achieve higher separation efficiency at a similar pressure drop resulting from their unique structures [1]. They consist of one single piece of highly porous material with bimodal pore size distribution, in which the macropores influence the permeability and the mesopores determine the surface area. A large number of monolithic materials have been described in a variety of excellent reviews [2–4]. So far, silica-based monolithic columns have been extensively studied in liquid chromatography (LC) [5–7], especially octadecyl (C18) columns in reversed-phase LC [8,9]. However, C18 columns are not ideal for the separation of isomeric solutes with long chain, such as carotenoid [10], tocopherols [11] and fatty acids [12]. The packed triacontyl (C30) column has demonstrated better performance than C18 column for the separation of these solutes [10–12]. The use of packed C30 stationary phases in LC was first reported in 1987 for the separation of polycyclic aromatic hydrocarbons
∗ Corresponding author. Tel.: +86 20 85211543; fax: +86 20 85216052. E-mail addresses: [email protected], jiali [email protected] (L. Jia). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.01.058
(PAHs) [13], and oligonucleotides [14]. Since then, the alkylbenzenes [15], aldehyde and ketone derivatives [16] and fullerenes [17] were also separated with C30 stationary phases. So far, the use of C30 stationary phases has expanded to the enrichment of PAHs [18], the separation of oligosaccharides [19], amino acids [20], derivatized regioisomeric 1, 3 diacylglycerols [21], and analogs of the sodium channel blocker tetrodotoxin [22]. Sander and co-workers investigated the effect of phase length on column selectivity by utilizing the C30 stationary phase for the separation of carotenoid and PAHs, etc. [11,23]. A comprehensive review described the application of packed C30 columns in the analysis of carotenoids, retinoids and other nutrients in complex and natural-matrix samples [24]. To the best of our knowledge, C30 functionalized monolithic silica capillary column has never been reported. Considering the advantages of monolithic column, the preparation of C30 silica-based monolithic column was developed in this paper. Its performance for the separation of some model compounds was compared with that using C18 silica-based monolithic column. 2. Experimental 2.1. Reagents and chemicals Ammonium hydroxide (NH3 ·H2 O (25%, w/w)), sodium hydroxide (NaOH) and acetic acid were purchased from Guangzhou Chemical Regent Factory (Guangzhou, China). Polyethylene glycol (PEG, Mw 10,000), o-xylene and p-xylene were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Tetramethoxysilane was purchased from Acros Organics (Geel,
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Y. Chen et al. / J. Chromatogr. A 1216 (2009) 2597–2600
Fig. 1. SEM photographs of the cross section of monolithic silica capillary column before modification with C18 chain and C30 chain at magnification of (A) 2000× and (B) 4000×.
Belgium). Toluene, ethylbenzene, propylbenzene, butylbenzene, phenanthrene and anthracene were purchased from Alfa Aesar (Heysham, UK). Dimethyltriacontylchlrosilane and dimethyloctadecylchlorosilane were purchased from Fluorochem Company (Derbyshire, UK). Carotene (mixture of ␣- and -carotenes) and -carotene were obtained from Sigma (St Louis, MO, USA). These reagents were used without any further purification. Methanol (HPLC grade) was purchased from SK Chemicals (Ulsan, South Korea). Water used in the experiments was obtained from an Elga water purification system (ELGA, London, UK). 2.2. Instrumentation The capillary LC system was comprised of a microflow pump (MP711, GL Sciences, Tokyo, Japan), an UV detector with an optical fiber flow cell (MP701, GL Sciences, Tokyo, Japan), and a manual injection valve (Valco VICI, Switzerland) with an internal sample loop of 50 nl. Chromatographic data acquisition was performed with the software HW-2000 workstation (Shanghai, China). All separations were carried out at room temperature, which was kept at 25 ◦ C using air conditioning system. A fused-silica capillary (GL Sciences, Japan), 8 cm × 50 m i.d., was used to connect the outlet end of analytical column with the optical fiber flow cell (light path 3 mm). For column fabrication, temperature-controlling process was carried out using a gas chromatography (GC) system 7890 II (Techcompany, Shanghai, China). Fused-silica capillaries (100 m i.d. × 365 m o.d.) were purchased from Hebei Yongnian Ruipu Chromatogram Equipment Company (Hebei, China). Scanning electron microscopy (SEM) of the silica monolith was carried out on a LEO-1530VP scanning electron microscope (Oberkochen, Germany) 2.3. Pretreatment of capillary A fused-silica capillary was first rinsed with 1 M NaOH for 2 h, then was put in a thermostated water bath at 40 ◦ C for 3 h. Next the capillary was rinsed by water for 30 min, 1 M HCl for 1 h, water for 30 min, respectively. Then it was dried with nitrogen at 180 ◦ C in a GC oven for 3 h prior to use. 2.4. Preparation and chemical modification of monolithic silica columns The procedure for the preparation of a monolithic silica column was based on the method outlined by Ishizuka et al. [25] with some modifications. Briefly, 3.13 ml TMOS, 0.625 g PEG and 6.50 ml acetic acid (0.01 M) were mixed by stirring for 45 min at 0 ◦ C, then the solution was sonicated for 30 s. After the transparent sol was injected into the pretreated capillary, the capillary was heated at 40 ◦ C in a
thermostated water bath for 17 h. Then, the column was rinsed with 0.01 M ammonium hydroxide and heated at 120 ◦ C for 60 min. This process was repeated three times. Next, the column was washed with water and dried in a GC oven, and the final heat treatment of the column was performed at 300 ◦ C for 20 h to remove the organic moieties and complete the formation of silica monolith. 2.4.1. Chemical modification of monolithic silica columns with C18 chain After the silica column was washed with toluene for 1 h, a solution consisting of 10% (w/v) dimethyloctadecylchlorosilane in toluene was pushed through the silica monolith and reacted at 120 ◦ C for 10 h. This process was repeated three times. Then the column was rinsed by toluene and methanol in sequence. 2.4.2. Chemical modification of monolithic silica columns with C30 chain Dimethyltriacontylchlrosilane (0.05 g) was added to 0.5 ml of toluene. The mixture was heated and held at 70 ◦ C until dimethyltriacontylchlrosilane dissolved in toluene. The resultant homogeneous solution was pushed through the silica column, which was washed with toluene for 1 h in advance, and reacted at 120 ◦ C for 10 h. This process was repeated three times. Finally the column was rinsed with toluene and methanol in sequence. 3. Results and discussion 3.1. SEM observation The SEM photographs of the cross section of monolithic silica columns before modification with C18 chain and C30 chain are shown in Fig. 1. As can be seen from the micrographs, the large through pores are interconnected. Sufficient attachment of silica monolithic skeletons to the capillary wall can prevent the shrinkage of the whole network structure. Ammonium hydroxide was used as the mesopores tailor to yield appropriate mesopores of the monolithic columns to offer sufficient large surface area that would be useful for chromatography. 3.2. Comparison of C30 and C18 columns by separation of alkylbenzenes, xylene isomers, PAHs and carotenes Fig. 2 shows the van Deemter plots of C30 and C18 monolithic silica capillary columns obtained with toluene and butylbenzene as solutes and 80/20 (v/v) methanol/water as the mobile phase. The permeability, K, was evaluated according to Eq. (1) [26], where P, , L and stands for column back pressure, dynamic viscosity of
Y. Chen et al. / J. Chromatogr. A 1216 (2009) 2597–2600
Fig. 2. van Deemter plots of the C30 and C18 monolithic silica capillary columns obtained using toluene and butylbenzene as solutes. Experimental conditions: C30 monolithic column, 30 cm × 100 m i.d.; C18 monolithic column, 30 cm × 100 m i.d.; mobile phase, 80/20 (v/v) methanol/water; detection wavelength, 214 nm.
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Fig. 4. Chromatograms of the mixture of ␣- and -carotene using C30 and C18 monolithic silica capillary columns. Experimental conditions: C30 monolithic column, 30 cm × 100 m i.d.; C18 monolithic column, 30 cm × 100 m i.d.; mobile phase, methanol; detection wavelength, 450 nm. Peak identification: 1, ␣-carotene; 2, carotene.
eluent, column length, and linear velocity of eluent, respectively. K=
L P
(1)
The permeability of the C18 column was calculated to be 4.58 × 10−14 m2 , while the permeability of the C30 column was calculated as 1.43 × 10−13 m2 . The results indicated that the C30 column provided more permeability than the C18 column. It was supposed that the difference in the bonding density for the two columns resulted in the difference in the permeability. And the bonding density for the C30 column was less than that for the C18 column. The performance of C30 monolithic silica column was compared with that of C18 monolithic silica column by the separation of alkylbenzenes, xylene, PAHs and carotenes. At first, the separation of four alkylbenzenes using C30 and C18 monolithic silica capillary columns were investigated with 80/20 (v/v) methanol/water as the mobile phase. As shown in Fig. 3, the separation performance using C30 column was inferior to that using C18 column for the alkylbenzenes. The retention factors (k) of toluene, ethyl-
Fig. 3. Chromatograms of the mixture of four alkylbenzenes using C30 and C18 monolithic silica capillary columns. Experimental conditions: C30 monolithic column, 30 cm × 100 m i.d.; C18 monolithic column, 30 cm × 100 m i.d.; mobile phase, 80/20 (v/v) methanol/water; detection wavelength, 214 nm. Peak identification: 1, toluene; 2, ethylbenzene; 3, propylbenzene; 4, butylbenzene.
benzene, propylbenzene and butylbenzene using C18 column were 0.74, 1.08, 1.72, and 2.77, respectively, while using C30 column, k of the analytes were 0.18 (toluene), 0.24 (ethylbenzene), 0.35 (propylbenzene), and 0.49 (butylbenzene), respectively. The number of theoretical plates (N) of the C18 and C30 were calculated using butylbenzene to be 3.78 × 104 , and 2.11 × 104 , respectively. Comparing with the C18 column, the less k and less N for the C30 column resulted from the less bonding density of C30 on the silica monolith. Next, the separation of o-xylene and p-xylene isomers using C30 and C18 columns was performed with 80/20 (v/v) methanol/water as the mobile phase (figure not shown). o-Xylene and p-xylene isomers was partially separated with the C18 monolithic column. Under the same conditions, o-xylene and p-xylene isomers were not resolved at all with the C30 monolithic column. The resolution (Rs) between o-xylene and p-xylene for the C18 and C30 columns were 0.7 and 0, respectively. The k of o-xylene for the C18 and C30 columns was 0.71 and 0.27, respectively. The separation of the isomers with small molecules may be strongly influenced by the bonding density of alkyl group on the silica monolith. Then, the separation of phenanthrene and anthracene isomers using C30 and C18 columns was investigated with 80/20 (v/v) methanol/water as the mobile phase (figure not shown). The Rs between phenanthrene and anthracene for the C18 and C30 columns was 1.0 and 0.8, respectively. The k of phenanthrene and anthracene for the C18 column was 1.72 and 1.94, respectively. For the C30 column, the k of phenanthrene and anthracene was 0.81 and 0.92, respectively. Based on the resolution and k for the separation of phenanthrene and anthracene using the C18 and C30 monolithic columns, the bonding density and the length of the alkyl group on the silica monolith play similar roles for controlling the selectivity of phenanthrene and anthracene. At last, the separation of ␣- and -carotene isomers with long chain was carried out using methanol as the mobile phase. As we know, C18 bonded phases are not ideal for the isomers with long chain since the dimensions of the analytes exceed the thickness of the stationary phase [10–12]. In conventional LC, the problem has been alleviated by synthesizing the bonded phases with longer chain length to the silica gels [27,28] or by using supercritical fluid chromatography [29,30]. As shown in Fig. 4, the use of C30 column for the separation of ␣- and -carotene isomers showed higher degree of selectivity than that using C18 column. ␣- and -carotene isomers were separated baseline using C30 column, while using C18
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column, the carotene isomers just obtained partial separation. The Rs between ␣- and -carotene using C18 and C30 columns was 1.1 and 2.5, respectively. The k of ␣- and -carotene using C18 column was 17.59 and 19.15, respectively, while using C30 column, the k of ␣- and -carotene was 11.51 and 14.42, respectively. The results showed the length of the phase is the primary factor controlling the selectivity for the separation of the isomers with long chain. From the experimental results, we can conclude that the bonding density and the length of the alkyl group on the silica monolith combined to influence the separation selectivity of analytes for C18- and C30-functionalized monolithic silica capillary columns. For small molecules, the bonding density of the alkyl group on the silica monolith plays main roles, while for the isomers with long chain, the length of the alkyl group on the silica monolith is a key factor. 4. Conclusions A kind of new C30-functionalized monolithic column for reversed-phase capillary LC was prepared in a fused-silica capillary via sol–gel process. The monolithic silica capillary column bonded with C30 offers advantages over the column bonded with C18 for the separation of the mixture of ␣- and -carotenes. The C18-functionalized monolithic silica capillary column shows a good performance for the separation of some small molecules because of the higher amount of alkyl chain bonded to the silica skeleton. It can be expected that C30-functionalized monolithic silica capillary column will be a promising media for the separation of isomeric solutes with long chain in reversed-phase LC. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20575041), Ministry of Education Science Foundation of China for Returnees, and Special Foundation of Guangzhou for Public Instruments.
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