Journal of Chromatography A, 1324 (2014) 115–120
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Catalyst assisted synthesis of initiator attached silica monolith particles via isocyanate-hydroxyl reaction for production of polystyrene bound chromatographic stationary phase of excellent separation efficiency Faiz Ali, Yune Sung Kim, Jin Wook Lee, Won Jo Cheong ∗ Department of Chemistry, Inha University, 100 Inharo, Namku, Incheon 402-751, South Korea
a r t i c l e
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Article history: Received 13 August 2013 Received in revised form 8 November 2013 Accepted 9 November 2013 Available online 19 November 2013 Keywords: Polystyrene bonded phase Initiator attached silica particles Catalyzed isocyanate-hydroxyl reaction RAFT polymerization Excellent high separation efficiency
a b s t r a c t Dibutyltin dichloride (DBTDC) was used as a catalyst to chemically bind 4-chloromehtylphenylisocynate (4-CPI) to porous monolithic silica particles via isocyanate-hydroxyl reaction, and the reaction product was reacted with sodium diethyldithiocarbamate (SDDC) to yield initiator attached silica monolith particles. Reversible addition-fragmentation transfer (RAFT) polymerization was taken place on them to result in polystyrene attached silica particles that showed excellent separation efficiency when packed in a chromatographic column (1.0 mm × 300 mm). The numbers of theoretical plates (N) of 56,500 is better than those of any commercially available HPLC or UHPLC column yet. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Modification of the surface of porous/nonporous inorganic materials by binding initiator moieties to their active sites followed by forming controlled polymer layers has been a well established strategy [1–6]. Such hybrid materials can be used as chromatographic stationary phases, and silica particles are the most versatile materials involved in surface modification for preparation of chromatographic media [7–19]. One of the trends of method development for better chromatographic performance has been the improvement of column efficiency by reducing particle size of stationary phase. It was initially in the 100 m range and has been gradually reduced up to the sub-2 m range [20–26]. There have been specially developed stationary phases such as core-shell particles, very fine porous particles, and monoliths for fast HPLC analyses and some recent review articles have been presented for their extensive introduction [26–29]. In view of separation efficiency of a column, core-shell particles have been regarded to be the best choice as stationary phase at present [26–28].
∗ Corresponding author. Tel.: +82 328607673; fax: +82 328675604. E-mail address: [email protected] (W.J. Cheong). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.11.027
The detailed mechanism and applications of RAFT polymerization have been well introduced in the literature [11,12,30–35]. The critical feature of RAFT polymerization for chromatographic phases is preparation of surface-bound initiator by attaching a halogen terminated ligand to the silica surface followed by coupling an initiator moiety (Fig. 1). According to the RAFT polymerization mechanism, the polymer chain is grown in the C S bond located between the spacer moiety and the diethyldithiocarbamate moiety (Fig. 1). However the stationary phases prepared in the past by RAFT polymerization had lower separation performance [11,12,14,15] in comparison to the conventional C18 phases. Preparation of ground silica monolith particles and their chromatographic application have been explored in our laboratory [36–38]. In this study, we report polystyrene bound partially sub-2 m silica monolith particulate stationary phase with excellent separation efficiency and selectivity in HPLC. This study reports the highest N values for a column packed with a stationary phase based on silica particles. Sodium diethyldithiocarbamate (SDDC) is bonded to silica surface as initiator entity after attaching 4-chloromehtylphenylisocynate (4-CPI) as a spacer (Fig. 1A). There have been some reports on catalytic effects of organometallic compounds for isocyanate-hydroxyl reaction in homogeneous systems [39–42]. The idea of using similar catalysts in a heterogeneous isocyanatehydroxyl reaction has led us to carry out the reaction between 4-CPI
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Fig. 1. Schematic pathways for synthesis of silica modified with chlorine-terminated ligand (A), initiator attached silica (B), and polystyrene bound silica (C), and the initiator silica structures prepared with 4-CPI (S1) and 3-chloropropyltrimethoxysilane (S2). The arrows denote the bond where the polymer chain is introduced and grown.
and silica silanol groups with some catalyst. Dibutyltin dichloride (DBTDC) was chosen for its good solubility in the reaction solvent. The morphology of polymer layer attached on the porous silica particles was found dependent upon the chemical structure of the initiator ligand (spacer group + diethyldithiocarbamate moiety), thus RAFT polymerization on the initiator attached silica (S1 in Fig. 1) prepared with 4-CPI without any catalyst yielded a polymer layer with some lumps of aggregated polystyrene [43] while RAFT polymerization on the initiator attached silica (S2 in Fig. 1) prepared with 3-chloropropyltrimethoxysilane yielded a polymer layer with little polystyrene lumps [44,45]. The formation of lumps was explained by the too rapid and uncontrolled polymerization owing to formation of too stable re-initiated radical species as in S1 compared to S2 [43–45]. Very recently, we have reported a column of excellent separation efficiency (N ∼ 50,000) packed with polystyrene bound silica monolith particles prepared by a S2 type initiator [46]. We suspect that the S1 type initiator silica may produce a nice and uniform polymer layer without lumps if the density of the initiator ligand is high enough to cause simultaneous and uniform growth of polymer chains on individual initiator ligands followed by termination upon congestion of the grown chains. Thus catalyzed isocyanate-hydroxyl reaction was adopted here for such purpose. Even better chromatographic performance has been obtained in this study than that of Ref. [46]. 2. Experimental 2.1. Materials All the chemical reagents and solvents were of analytical grade and HPLC grade. Screen frits (1.6 mm diameter, 0.08 mm thickness, and 0.5 m pore size) were purchased from Valco (Houston, TX, USA). Glass lined stainless steel tubing (30 cm, 1.0 mm ID, 1/8 in. OD) and silica capillary (50 m ID, 365 m OD) were obtained from Grace (Deerfield, IL, USA). 2.2. Synthesis of ground silica monolith particles (1) Partially sub-2 m porous silica monolith particles (1) have been synthesized by a renovated sol-gel procedure with multistep heating followed by grinding and calcination according to the
method of Ref. [46]. Polyethyleneglycol (molecular weight 10,000) 1620 mg and urea 1650 mg were dissolved in 15 mL 0.01 N acetic acid in a Teflon vial, and magnetically stirred for 10 min in ice/water. Then, 5 mL tetramethoxysilane was added, and the mixture was kept under stirring for 40 min. The solution was incubated at 40 ◦ C in an oven for 48 h then at 120 ◦ C in an autoclave for 48 h. The residual water due to sol–gel process was decanted off and the solid cake of monolith was dried at 70 ◦ C for 20 h, ground with mortar and pestle for 10 min, and calcined at 550 ◦ C for 50 h. 2.3. Synthesis of initiator bound silica monolith particles The mixture of 300 mg 4-CPI, 100 mg DBTDC, and 30 ml anhydrous toluene was stirred for 30 min in a 100 mL round bottom flask, and 820 mg 1 pre-dried at 120 ◦ C overnight was suspended in it, and heated at 80 ◦ C for 48 h. The product (2) was washed with anhydrous toluene and acetone (HPLC grade), filtered, and dried at room temperature in a vacuum desiccator overnight. SDDC 800 mg was dissolved in 30 mL anhydrous THF in a 100 mL round bottom flask, and the dried 2 (100 ml, RB flask) was suspended there under N2 -purge for 10 min, and heated at 55 ◦ C for 17 h under N2 -purge. The product (3) was washed with THF, 60/40 methanol/water, and acetone, filtered, and dried at room temperature in a vacuum desiccator overnight. 2.4. RAFT polymerization of styrene Styrene 5 mL dissolved in 25 mL anhydrous toluene was subjected to sonication for 10 min followed by N2 purge for 20 min and 500 mg 3 was dispersed in it. Radical polymerization was carried out at 110 ◦ C for 30 h under N2 . The product (4) was washed with toluene at 110 ◦ C and acetone at room temperature, filtered, and dried at 60 ◦ C overnight. 2.5. Characterization BEL-Japan (Osaka, Japan) BELSORP-Max was used to measure the BET/BJH nitrogen adsorption/desorption isotherms at 77 K, BJH pore sizes, and BET specific surface areas for the silica monolith particles and polystyrene bound silica monolith particles. The samples were out-gassed at 373 K for 10 h to obtain a residual pressure of less than 10−3 Torr. The amount of N2 adsorbed at a relative pressure of P/Po = 0.99 was used to determine the total pore volume
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Fig. 2. Microscopic view of silica monolith particles (A) and polystyrene bound silica monolith particles (X), SEM images of silica particles (B and C, wide and close views, respectively) and polystyrene-attached particles (Y and Z).
(micropore + mesopore). The SEM images of the phases were captured by HITACHI (Tokyo, Japan) S-4200 field emission scanning electron microscopy (FE-SEM). A Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer was used to obtain the carbon load data. A Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer was used to get particle size distribution.
Table 1 Comparison of elemental analysis results. Element
3 Prepared without DBTDC
3 Prepared with DBTDC
4
Carbon % Hydrogen % Sulfur % Nitrogen %
4.30 0.51 1.78 0.79
5.93 0.65 2.41 1.03
10.21 1.30 2.12 0.85
2.6. HPLC The HPLC system identical to that of Ref. [46] was used. Packing of micro columns was carried out according to the procedure of Ref. [46]. A 0.5 m commercial screen frit was placed in the 1/8 in. outlet of a reducing union (1/8 in.–1/16 in.), and a glass lined stainless steel column (1 mm, ID and 30 cm, long) was fitted to the outlet, and the tubing was connected to the packer. The slurry was prepared by suspending the 300 mg stationary phase in 3.3 mL methanol, and was fed into reservoir. A pressure sequence was applied. After packing, the inlet union was installed in the same way as the outlet union, and the column inlet connected to the injector, and the column outlet, to the capillary window detector. The stock sample solution was prepared by dissolving phenol (0.88 L), acetophenone (0.14 L), 4-methyl-2-nitroaniline (0.32 mg), benzene (2.93 L), and toluene (1.46 L) in 1 mL mobile phase and was stored at 4 ◦ C. The sample was further diluted for injection. 3. Results and discussion An increase in the density of initiator ligands by catalytic isocyanate-hydroxyl reaction in comparison of non-catalytic reaction was observed (Table 1). Although the increase is marginal (5.95% vs. 4.30% in carbon load), the effect was proven to be critical since polystyrene lumps were not observed on the surface of polystyrene bound monolith particles (4) and the separation performance of the column packed with 4 was found excellent as described below.
The microscopy and SEM images of the silica monolith particles (1) and polystyrene bound silica monolith particles (4) are shown in Fig. 2. Current study demonstrates the catalytic effect of DBTDC in the heterogeneous isocyanate-hydroxyl reaction. Comparison of carbon load (also nitrogen and sulfur load) of the initiator attached silica monolith particles between the batch prepared with DBTDC and the batch without DBTDC manifests the higher density of initiator ligand for the batch prepared with DBTDC (Table 1). Based on Figs. 2 and 3, formation of smooth and even polymer layer of suitable thickness is supported. The layer thickness was estimated to be 0.13 m as the half of the difference in d(0.5) before and after RAFT polymerization. The average pore size was also decreased from 343 to 221 A˚ (Table 2). It is believed that the thickness of the polystyrene layer of the outer surface is thicker than that of the inner pores since the estimated thickness based on pore size decrease is 0.006 m, which is much thinner than that (0.13 m) based on the increase of particle size. TEM photographs were measured on the silica monolith particles and the polystyrene modified silica monolith particles and given in the Supplementary Information (Supplement 1). TEM photographs of particles of hierarchical periodic pore structures are clear and very informative while those of irregular pore structures are with limited information. Unfortunately, our phases are rather in the latter class. The TEM images of the silica monolith particles clearly show amorphous character with irregular pores being
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Fig. 3. Particle size distribution and pore size distribution of 1 (䊉) and 4 (). The expression “d(x)” indicates the particle diameter corresponding to the integrated area ratio of x when integrated in the range of 0–d(x) diameter.
Table 2 BET/BJH analysis data for 1 and 4. Silica particles (1)
Pore size (Å)a Pore volume (cm3 /g)b Surface area (m2 /g)c a b c
Polystyrene bound silica particles (4)
Previous study [46]
Previous study [36]
Current study
Previous study [46]
Previous study [36]
Current study
343 1.06 136
212 0.83 283
343 1.06 136
252 0.84 131
146 0.53 161
221 0.80 125
BJH adsorption average pore diameter. Total pore volume at P/Po = 0.99. BET specific surface area.
projected as dim dark spots in the expanded view. The layer of polystyrene of the polymer bound silica monolith particles in the corresponding TEM images seems differentiated from the silica substrate, and some clear pore structures appear in the expanded view. We suspect that the layer of polystyrene in the inner pores enables the visualization of the pore structures. The 13 C CP-MAS (cross-polarization magic angle spinning) and CP-TOSS (cross-polarization total sideband suppression) NMR spectra of our phase were measured, and are given in the Supplementary Information (Supplement 2). Based on 13 C NMR data, the existence of phenyl ring (110–135 ppm), carbamate CO2 moiety (160–175 ppm), the congested group of methylene C in the polystyrene and methylene C attached to N (30–50 ppm), methylene C attached to S (20–25 ppm), and terminal methyl C (5–15 ppm) were confirmed. The dithiocarbamate CS2 moiety (190–200 ppm) was not shown in the CP-MAS spectrum but shown in the CP-TOSS spectrum. The column (1 mm × 300 mm) packed with 4 of this study showed excellent chromatographic separation efficiency as shown in Fig. 4A and Table 3. The optimal flow rate was found 15 L/min. The numbers of theoretical plates (N) were in the range of 52,400–59,500 (average 56,500) for the test solutes. The chromatographic resolutions (R) were also computed for the 4 pairs of adjacent analytes and their average was 5.88. Three batches of 4 were prepared and packed in different columns for checking reproducibility. The reproducibility in N was better than 3.9%, that in R, better than 4.2%, and that in retention times, better than 4.5%. The reproducibility in either N or retention times for repeated measurements with a single column was better than 2.0%. The chromatogram obtained with the column packed with the polystyrene bound phase of Ref. [46] under the same chromatographic conditions was given as Fig. 4B. Although the carbon load (10.2%) of the stationary phase of this study is close to that (10.0%) of the stationary phase of reference [46], the chromatographic performance (Fig. 4A) of the phase of this study has proven to be quite better than that (Fig. 4B) of prior study [46]. Not only N (56,500 vs. 49,400) but
Fig. 4. Comparison of chromatograms obtained with columns (1 mm × 300 mm) packed with the phase of this study (A and C), the phase of prior study [46] (B), and a 5 m commercial C18 phase (Lichrosopher RP18, D). Flow rate: 15 L/min, detection wavelength: 214 nm. Eluents A, B, and D: 60/40 (v/v) ACN/water with 0.1% TFA, C: 40/60 (v/v) ACN/water with 0.1% TFA. The elution order of analytes was phenol, acetophenone, 4-methyl-2-nitronaniline, benzene and toluene.
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Table 3 Comparison of N and R values among different phases.a,b Solute (for N)
Solute pair (for R)
This study (A), 60/40ACN/water N
1 2 3 4 5 Average
1–2 2–3 3–4 4–5
R
Prior study (B) [46] 60/40ACN/water
This study (C), 40/60ACN/water
N
N
R
C18, 5 m (D), 60/40ACN/water R
N
R
58,900 55,900 55,800 52,400 59,500
8.15 3.82 5.25 6.31
53,700 52,300 51,600 43,400 46,000
5.15 1.43 3.76 3.35
38,700 39,500 41,100 40,200 42,600
10.26 5.74 11.68 11.01
22,200 20,500 21,700 17,400 17,400
10.01 5.79 10.41 10.62
56,500
5.88
49,400
3.42
40,400
9.67
19,800
9.20
a
Indications A,B,C, and D are in agreement with those in Fig. 4. All the phases were packed in a column of 1 mm I.D. and 300 mm length. A: The phase of this study with the mobile phase of 60/40 (v/v %) acetonitrile/water with 0.1% TFA. B: The phase of prior study [46] with 60/40 acetonitrile/water with 0.1% TFA. C: The phase of this study with 40/60 (v/v %) acetonitrile/water with 0.1% TFA. D: The Lichrosopher RP18 (5 m) with 60/40 acetonitrile/water with 0.1% TFA. b Three batches of the phase of this study were prepared and packed in different columns for checking reproducibility. The reproducibility in N was better than 3.9%, that in R, better than 4.2%, and that in retention times, better than 4.5%. The reproducibility in N, R or retention times for repeated measurements with a fixed column was better than 2.0%. Solutes – 1: phenol, 2: acetophenone, 3: 4-mehtyl-2-nitroaniline, 4: benzene, 5: toluene.
also R (5.88 vs. 3.42) was found superior, and all the analytes were better separated in a wider retention time range as shown in Fig. 4A in comparison with Fig. 4B. Thus the solutes 2 and 3 were perfectly separated in Fig. 4A while they were a little overlapped in Fig. 4B. It seems that adoption of catalyzed isocyanate-hydroxyl reaction in binding initiator moieties and consequent increased density of surface initiator molecules enabled formation of polystyrene layer of more uniform chain lengths showing better chromatographic performance. The initiator attached monolith particles (with less initiator density) prepared without the catalyst were also used for production of polystyrene attached silica monolith particles. The plate numbers obtained with the column packed with such product was only ca 30,000. The van Deemter plots (Supplement 3 in Supplement Information) of this study showed very similar patters to those of precious study [46] except for further improved plate heights. A commercial C18 phase (5 m) was packed in the column of the same physical dimension to obtain the chromatogram for the same sample in the same eluent (Fig. 4D). Another chromatogram (Fig. 4C) was obtained with the column of our phase in a different eluent to get similar retention times as those of D chromatogram, then the average N value was a little degraded but still 40,400 with the average R value being 9.67 while the average N values of Fig. 4D was only 19,800 with the average R value of 9.20. It should also be noted that the retention time range of Fig. 4C was even narrower than that of Fig. 4D. Factors contributing to enhanced separation efficiency and satisfactory selectivity of our phase are: (1) reduced average particle size; (2) diversity of particle shape (oval, bent, spherical, etc.) resulting in partially monolithic architecture after packing; (3) pseudo core-shell type behavior of our phase as a result of RAFT polymerization [36], and (4) formation of homogenous and uniformly distributed thin polymer film crucial for good mass transfer kinetics. Adoption of catalytic isocyanate-hydroxyl reaction in this study is believed to play a critical role in the above (3) and (4) by providing affluent initiator ligands. A significant portion of deep pores located in the core of particles seems clogged during RAFT polymerization. The most efficient commercial columns are generally known to be the ones packed with core-shell type spherical silica particles of 1.7–2.7 m [26–28,47]. However, the maximum length of commercial columns packed with particles of 3 m or less is 15 cm at present. The optimum separation efficiency of 2.1 mm I.D. column packed with 1.7 m Kinetex particles was around 320,000 plates/m
[48]. The maximum column length was 15 cm, so the maximum N value would be ca. 48,000. In another study, a maximum N value of 37,500 (corresponding to HEPT of 4 m) was obtained with a column (4.6 mm × 150 mm) packed with 2.2 m core-shell particles [49]. We mentioned that the reduced plate height of core-shell particles of smaller I.D. was below that of our phase in the previous study [46]. However, there are no commercial columns packed with particles of 3 m or less whose length is longer than 15 cm. We also had difficulties to pack a column of 30 cm with 3 m particles, and the separation efficiency of such a column was not better than that of the column packed with 5 m particles. On the other hand, there is no problem to pack our phase in a column of 30 cm length owing to the monolithic character. Thus the separation efficiency of our column (30 cm) phase packed with partially sub 2 m particles was better than that of commercial columns packed with small core-shell particles. The column length of this study is as twice as that of the commercial columns packed with sub 3-m particles. Nevertheless, there was no problem of column packing with our phase owing to the monolith-like structures of particles although our phase includes a major portion of sub-2 m particles since such structures allowed faster and stronger solvent flux under the same packing pressure to yield compact packing. The sample loading capacity of our phase was found to be only marginally lower than that of the 5 m commercial C18 phase based on the frontal analysis (Supplement 4 in the Supplement information). It seems that our phase is of enough loading capacity although the major contribution of capacity is due to the rather thick polymer layers of shallow pores of monolith particles while the contribution of polymer layers of deep pores is only a minor. 4. Conclusion Adoption of catalytic isocyanate-hydroxyl reaction in preparation of initiator attached silica monolith particles with increased density of initiator ligands has been successful to obtain the polystyrene bound particles with excellent separation efficiency via RAFT polymerization without formation of polystyrene lumps, and the plate numbers obtained with the column packed with this phase was found better than that of any commercially available HPLC or UHPLC column and those of the columns packed with previously reported stationary phases based on silica monolith particles.
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Acknowledgments This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) (2012 R1A1A2006066) and by the Converging Research Center Program through the Ministry of Education, Science and Technology of Korea (2012K001534). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2013.11.027. References [1] A. Olivier, F. Meyer, J. Raquez, P. Damman, P. Dubois, Prog. Polym. Sci. 37 (2012) 157. [2] H. Wang, X. Dong, M. Yang, Trends Anal. Chem. 31 (2012) 96. [3] G.J.A.A. Soler-Illia, O. Azzaroni, Chem. Soc. Rev. 40 (2011) 1107. [4] S. Edmondson, S.P. Armes, Polym. Int. 58 (2009) 307. [5] M. Beija, J. Marty, M. Destarac, Prog. Polym. Sci. 36 (2011) 845. [6] G. Moad, M. Chen, M. Häussler, A. Postma, E. Rizzardo, S.H. Thang, Polym. Chem. 2 (2011) 492. [7] M.M. Rahman, M. Czaun, M. Takafuji, H. Ihara, J. Chem. Eur. 14 (2008) 1312. [8] B.R. Coad, B.M. Steels, J.N. Kizhakkedathu, D.E. Brooks, C.A. Haynes, Biotechnol. Bioeng. 97 (2007) 574. [9] A.K. Mallik, M.M. Rahman, M. Czaun, M. Takafuji, H. Ihara, J. Chromatogr. A 1187 (2008) 119. [10] P. Hemstrom, M. Szumski, K. Irgum, Anal. Chem. 78 (2006) 7098. [11] D. Derouet, C.N.H. Thuc, J. Appl. Polym. Sci. 109 (2008) 2113. [12] R.E. Fairhurst, C. Chassaing, R.F. Venn, A.G. Mayes, Biosen. Bioelectron. 20 (2004) 1098. [13] C. Sulitzky, B. Ruckert, A.J. Hall, F. Lanza, K.K. Unger, B. Sellergren, Macromolecules 35 (2002) 79. [14] F. Roohi, M.M. Titirici, New J. Chem. 32 (2008) 1409. [15] S. Su, M. Zhang, B. LI, H. Zhang, X. Dong, Talanta 76 (2008) 1141. [16] M. Czaun, M.M. Rahman, M. Takafuji, H. Ihara, Polymer 49 (2008) 5410. [17] K. Nagase, J. Kobayashi, A. Kikuchi, Y. Akiyama, M. Annaka, H. Kanazawa, T. Okano, Langmuir 24 (2008) 10981.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Catalyst assisted synthesis of initiator attached silica monolith particles via isocyanate-hydroxyl reaction for production of polystyrene bound chromatographic stationary phase of excellent separation efficiency Faiz Ali, Yune Sung Kim, Jin Wook Lee, Won Jo Cheong ∗ Department of Chemistry, Inha University, 100 Inharo, Namku, Incheon 402-751, South Korea
a r t i c l e
i n f o
Article history: Received 13 August 2013 Received in revised form 8 November 2013 Accepted 9 November 2013 Available online 19 November 2013 Keywords: Polystyrene bonded phase Initiator attached silica particles Catalyzed isocyanate-hydroxyl reaction RAFT polymerization Excellent high separation efficiency
a b s t r a c t Dibutyltin dichloride (DBTDC) was used as a catalyst to chemically bind 4-chloromehtylphenylisocynate (4-CPI) to porous monolithic silica particles via isocyanate-hydroxyl reaction, and the reaction product was reacted with sodium diethyldithiocarbamate (SDDC) to yield initiator attached silica monolith particles. Reversible addition-fragmentation transfer (RAFT) polymerization was taken place on them to result in polystyrene attached silica particles that showed excellent separation efficiency when packed in a chromatographic column (1.0 mm × 300 mm). The numbers of theoretical plates (N) of 56,500 is better than those of any commercially available HPLC or UHPLC column yet. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Modification of the surface of porous/nonporous inorganic materials by binding initiator moieties to their active sites followed by forming controlled polymer layers has been a well established strategy [1–6]. Such hybrid materials can be used as chromatographic stationary phases, and silica particles are the most versatile materials involved in surface modification for preparation of chromatographic media [7–19]. One of the trends of method development for better chromatographic performance has been the improvement of column efficiency by reducing particle size of stationary phase. It was initially in the 100 m range and has been gradually reduced up to the sub-2 m range [20–26]. There have been specially developed stationary phases such as core-shell particles, very fine porous particles, and monoliths for fast HPLC analyses and some recent review articles have been presented for their extensive introduction [26–29]. In view of separation efficiency of a column, core-shell particles have been regarded to be the best choice as stationary phase at present [26–28].
∗ Corresponding author. Tel.: +82 328607673; fax: +82 328675604. E-mail address: [email protected] (W.J. Cheong). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.11.027
The detailed mechanism and applications of RAFT polymerization have been well introduced in the literature [11,12,30–35]. The critical feature of RAFT polymerization for chromatographic phases is preparation of surface-bound initiator by attaching a halogen terminated ligand to the silica surface followed by coupling an initiator moiety (Fig. 1). According to the RAFT polymerization mechanism, the polymer chain is grown in the C S bond located between the spacer moiety and the diethyldithiocarbamate moiety (Fig. 1). However the stationary phases prepared in the past by RAFT polymerization had lower separation performance [11,12,14,15] in comparison to the conventional C18 phases. Preparation of ground silica monolith particles and their chromatographic application have been explored in our laboratory [36–38]. In this study, we report polystyrene bound partially sub-2 m silica monolith particulate stationary phase with excellent separation efficiency and selectivity in HPLC. This study reports the highest N values for a column packed with a stationary phase based on silica particles. Sodium diethyldithiocarbamate (SDDC) is bonded to silica surface as initiator entity after attaching 4-chloromehtylphenylisocynate (4-CPI) as a spacer (Fig. 1A). There have been some reports on catalytic effects of organometallic compounds for isocyanate-hydroxyl reaction in homogeneous systems [39–42]. The idea of using similar catalysts in a heterogeneous isocyanatehydroxyl reaction has led us to carry out the reaction between 4-CPI
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Fig. 1. Schematic pathways for synthesis of silica modified with chlorine-terminated ligand (A), initiator attached silica (B), and polystyrene bound silica (C), and the initiator silica structures prepared with 4-CPI (S1) and 3-chloropropyltrimethoxysilane (S2). The arrows denote the bond where the polymer chain is introduced and grown.
and silica silanol groups with some catalyst. Dibutyltin dichloride (DBTDC) was chosen for its good solubility in the reaction solvent. The morphology of polymer layer attached on the porous silica particles was found dependent upon the chemical structure of the initiator ligand (spacer group + diethyldithiocarbamate moiety), thus RAFT polymerization on the initiator attached silica (S1 in Fig. 1) prepared with 4-CPI without any catalyst yielded a polymer layer with some lumps of aggregated polystyrene [43] while RAFT polymerization on the initiator attached silica (S2 in Fig. 1) prepared with 3-chloropropyltrimethoxysilane yielded a polymer layer with little polystyrene lumps [44,45]. The formation of lumps was explained by the too rapid and uncontrolled polymerization owing to formation of too stable re-initiated radical species as in S1 compared to S2 [43–45]. Very recently, we have reported a column of excellent separation efficiency (N ∼ 50,000) packed with polystyrene bound silica monolith particles prepared by a S2 type initiator [46]. We suspect that the S1 type initiator silica may produce a nice and uniform polymer layer without lumps if the density of the initiator ligand is high enough to cause simultaneous and uniform growth of polymer chains on individual initiator ligands followed by termination upon congestion of the grown chains. Thus catalyzed isocyanate-hydroxyl reaction was adopted here for such purpose. Even better chromatographic performance has been obtained in this study than that of Ref. [46]. 2. Experimental 2.1. Materials All the chemical reagents and solvents were of analytical grade and HPLC grade. Screen frits (1.6 mm diameter, 0.08 mm thickness, and 0.5 m pore size) were purchased from Valco (Houston, TX, USA). Glass lined stainless steel tubing (30 cm, 1.0 mm ID, 1/8 in. OD) and silica capillary (50 m ID, 365 m OD) were obtained from Grace (Deerfield, IL, USA). 2.2. Synthesis of ground silica monolith particles (1) Partially sub-2 m porous silica monolith particles (1) have been synthesized by a renovated sol-gel procedure with multistep heating followed by grinding and calcination according to the
method of Ref. [46]. Polyethyleneglycol (molecular weight 10,000) 1620 mg and urea 1650 mg were dissolved in 15 mL 0.01 N acetic acid in a Teflon vial, and magnetically stirred for 10 min in ice/water. Then, 5 mL tetramethoxysilane was added, and the mixture was kept under stirring for 40 min. The solution was incubated at 40 ◦ C in an oven for 48 h then at 120 ◦ C in an autoclave for 48 h. The residual water due to sol–gel process was decanted off and the solid cake of monolith was dried at 70 ◦ C for 20 h, ground with mortar and pestle for 10 min, and calcined at 550 ◦ C for 50 h. 2.3. Synthesis of initiator bound silica monolith particles The mixture of 300 mg 4-CPI, 100 mg DBTDC, and 30 ml anhydrous toluene was stirred for 30 min in a 100 mL round bottom flask, and 820 mg 1 pre-dried at 120 ◦ C overnight was suspended in it, and heated at 80 ◦ C for 48 h. The product (2) was washed with anhydrous toluene and acetone (HPLC grade), filtered, and dried at room temperature in a vacuum desiccator overnight. SDDC 800 mg was dissolved in 30 mL anhydrous THF in a 100 mL round bottom flask, and the dried 2 (100 ml, RB flask) was suspended there under N2 -purge for 10 min, and heated at 55 ◦ C for 17 h under N2 -purge. The product (3) was washed with THF, 60/40 methanol/water, and acetone, filtered, and dried at room temperature in a vacuum desiccator overnight. 2.4. RAFT polymerization of styrene Styrene 5 mL dissolved in 25 mL anhydrous toluene was subjected to sonication for 10 min followed by N2 purge for 20 min and 500 mg 3 was dispersed in it. Radical polymerization was carried out at 110 ◦ C for 30 h under N2 . The product (4) was washed with toluene at 110 ◦ C and acetone at room temperature, filtered, and dried at 60 ◦ C overnight. 2.5. Characterization BEL-Japan (Osaka, Japan) BELSORP-Max was used to measure the BET/BJH nitrogen adsorption/desorption isotherms at 77 K, BJH pore sizes, and BET specific surface areas for the silica monolith particles and polystyrene bound silica monolith particles. The samples were out-gassed at 373 K for 10 h to obtain a residual pressure of less than 10−3 Torr. The amount of N2 adsorbed at a relative pressure of P/Po = 0.99 was used to determine the total pore volume
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Fig. 2. Microscopic view of silica monolith particles (A) and polystyrene bound silica monolith particles (X), SEM images of silica particles (B and C, wide and close views, respectively) and polystyrene-attached particles (Y and Z).
(micropore + mesopore). The SEM images of the phases were captured by HITACHI (Tokyo, Japan) S-4200 field emission scanning electron microscopy (FE-SEM). A Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer was used to obtain the carbon load data. A Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer was used to get particle size distribution.
Table 1 Comparison of elemental analysis results. Element
3 Prepared without DBTDC
3 Prepared with DBTDC
4
Carbon % Hydrogen % Sulfur % Nitrogen %
4.30 0.51 1.78 0.79
5.93 0.65 2.41 1.03
10.21 1.30 2.12 0.85
2.6. HPLC The HPLC system identical to that of Ref. [46] was used. Packing of micro columns was carried out according to the procedure of Ref. [46]. A 0.5 m commercial screen frit was placed in the 1/8 in. outlet of a reducing union (1/8 in.–1/16 in.), and a glass lined stainless steel column (1 mm, ID and 30 cm, long) was fitted to the outlet, and the tubing was connected to the packer. The slurry was prepared by suspending the 300 mg stationary phase in 3.3 mL methanol, and was fed into reservoir. A pressure sequence was applied. After packing, the inlet union was installed in the same way as the outlet union, and the column inlet connected to the injector, and the column outlet, to the capillary window detector. The stock sample solution was prepared by dissolving phenol (0.88 L), acetophenone (0.14 L), 4-methyl-2-nitroaniline (0.32 mg), benzene (2.93 L), and toluene (1.46 L) in 1 mL mobile phase and was stored at 4 ◦ C. The sample was further diluted for injection. 3. Results and discussion An increase in the density of initiator ligands by catalytic isocyanate-hydroxyl reaction in comparison of non-catalytic reaction was observed (Table 1). Although the increase is marginal (5.95% vs. 4.30% in carbon load), the effect was proven to be critical since polystyrene lumps were not observed on the surface of polystyrene bound monolith particles (4) and the separation performance of the column packed with 4 was found excellent as described below.
The microscopy and SEM images of the silica monolith particles (1) and polystyrene bound silica monolith particles (4) are shown in Fig. 2. Current study demonstrates the catalytic effect of DBTDC in the heterogeneous isocyanate-hydroxyl reaction. Comparison of carbon load (also nitrogen and sulfur load) of the initiator attached silica monolith particles between the batch prepared with DBTDC and the batch without DBTDC manifests the higher density of initiator ligand for the batch prepared with DBTDC (Table 1). Based on Figs. 2 and 3, formation of smooth and even polymer layer of suitable thickness is supported. The layer thickness was estimated to be 0.13 m as the half of the difference in d(0.5) before and after RAFT polymerization. The average pore size was also decreased from 343 to 221 A˚ (Table 2). It is believed that the thickness of the polystyrene layer of the outer surface is thicker than that of the inner pores since the estimated thickness based on pore size decrease is 0.006 m, which is much thinner than that (0.13 m) based on the increase of particle size. TEM photographs were measured on the silica monolith particles and the polystyrene modified silica monolith particles and given in the Supplementary Information (Supplement 1). TEM photographs of particles of hierarchical periodic pore structures are clear and very informative while those of irregular pore structures are with limited information. Unfortunately, our phases are rather in the latter class. The TEM images of the silica monolith particles clearly show amorphous character with irregular pores being
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Fig. 3. Particle size distribution and pore size distribution of 1 (䊉) and 4 (). The expression “d(x)” indicates the particle diameter corresponding to the integrated area ratio of x when integrated in the range of 0–d(x) diameter.
Table 2 BET/BJH analysis data for 1 and 4. Silica particles (1)
Pore size (Å)a Pore volume (cm3 /g)b Surface area (m2 /g)c a b c
Polystyrene bound silica particles (4)
Previous study [46]
Previous study [36]
Current study
Previous study [46]
Previous study [36]
Current study
343 1.06 136
212 0.83 283
343 1.06 136
252 0.84 131
146 0.53 161
221 0.80 125
BJH adsorption average pore diameter. Total pore volume at P/Po = 0.99. BET specific surface area.
projected as dim dark spots in the expanded view. The layer of polystyrene of the polymer bound silica monolith particles in the corresponding TEM images seems differentiated from the silica substrate, and some clear pore structures appear in the expanded view. We suspect that the layer of polystyrene in the inner pores enables the visualization of the pore structures. The 13 C CP-MAS (cross-polarization magic angle spinning) and CP-TOSS (cross-polarization total sideband suppression) NMR spectra of our phase were measured, and are given in the Supplementary Information (Supplement 2). Based on 13 C NMR data, the existence of phenyl ring (110–135 ppm), carbamate CO2 moiety (160–175 ppm), the congested group of methylene C in the polystyrene and methylene C attached to N (30–50 ppm), methylene C attached to S (20–25 ppm), and terminal methyl C (5–15 ppm) were confirmed. The dithiocarbamate CS2 moiety (190–200 ppm) was not shown in the CP-MAS spectrum but shown in the CP-TOSS spectrum. The column (1 mm × 300 mm) packed with 4 of this study showed excellent chromatographic separation efficiency as shown in Fig. 4A and Table 3. The optimal flow rate was found 15 L/min. The numbers of theoretical plates (N) were in the range of 52,400–59,500 (average 56,500) for the test solutes. The chromatographic resolutions (R) were also computed for the 4 pairs of adjacent analytes and their average was 5.88. Three batches of 4 were prepared and packed in different columns for checking reproducibility. The reproducibility in N was better than 3.9%, that in R, better than 4.2%, and that in retention times, better than 4.5%. The reproducibility in either N or retention times for repeated measurements with a single column was better than 2.0%. The chromatogram obtained with the column packed with the polystyrene bound phase of Ref. [46] under the same chromatographic conditions was given as Fig. 4B. Although the carbon load (10.2%) of the stationary phase of this study is close to that (10.0%) of the stationary phase of reference [46], the chromatographic performance (Fig. 4A) of the phase of this study has proven to be quite better than that (Fig. 4B) of prior study [46]. Not only N (56,500 vs. 49,400) but
Fig. 4. Comparison of chromatograms obtained with columns (1 mm × 300 mm) packed with the phase of this study (A and C), the phase of prior study [46] (B), and a 5 m commercial C18 phase (Lichrosopher RP18, D). Flow rate: 15 L/min, detection wavelength: 214 nm. Eluents A, B, and D: 60/40 (v/v) ACN/water with 0.1% TFA, C: 40/60 (v/v) ACN/water with 0.1% TFA. The elution order of analytes was phenol, acetophenone, 4-methyl-2-nitronaniline, benzene and toluene.
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Table 3 Comparison of N and R values among different phases.a,b Solute (for N)
Solute pair (for R)
This study (A), 60/40ACN/water N
1 2 3 4 5 Average
1–2 2–3 3–4 4–5
R
Prior study (B) [46] 60/40ACN/water
This study (C), 40/60ACN/water
N
N
R
C18, 5 m (D), 60/40ACN/water R
N
R
58,900 55,900 55,800 52,400 59,500
8.15 3.82 5.25 6.31
53,700 52,300 51,600 43,400 46,000
5.15 1.43 3.76 3.35
38,700 39,500 41,100 40,200 42,600
10.26 5.74 11.68 11.01
22,200 20,500 21,700 17,400 17,400
10.01 5.79 10.41 10.62
56,500
5.88
49,400
3.42
40,400
9.67
19,800
9.20
a
Indications A,B,C, and D are in agreement with those in Fig. 4. All the phases were packed in a column of 1 mm I.D. and 300 mm length. A: The phase of this study with the mobile phase of 60/40 (v/v %) acetonitrile/water with 0.1% TFA. B: The phase of prior study [46] with 60/40 acetonitrile/water with 0.1% TFA. C: The phase of this study with 40/60 (v/v %) acetonitrile/water with 0.1% TFA. D: The Lichrosopher RP18 (5 m) with 60/40 acetonitrile/water with 0.1% TFA. b Three batches of the phase of this study were prepared and packed in different columns for checking reproducibility. The reproducibility in N was better than 3.9%, that in R, better than 4.2%, and that in retention times, better than 4.5%. The reproducibility in N, R or retention times for repeated measurements with a fixed column was better than 2.0%. Solutes – 1: phenol, 2: acetophenone, 3: 4-mehtyl-2-nitroaniline, 4: benzene, 5: toluene.
also R (5.88 vs. 3.42) was found superior, and all the analytes were better separated in a wider retention time range as shown in Fig. 4A in comparison with Fig. 4B. Thus the solutes 2 and 3 were perfectly separated in Fig. 4A while they were a little overlapped in Fig. 4B. It seems that adoption of catalyzed isocyanate-hydroxyl reaction in binding initiator moieties and consequent increased density of surface initiator molecules enabled formation of polystyrene layer of more uniform chain lengths showing better chromatographic performance. The initiator attached monolith particles (with less initiator density) prepared without the catalyst were also used for production of polystyrene attached silica monolith particles. The plate numbers obtained with the column packed with such product was only ca 30,000. The van Deemter plots (Supplement 3 in Supplement Information) of this study showed very similar patters to those of precious study [46] except for further improved plate heights. A commercial C18 phase (5 m) was packed in the column of the same physical dimension to obtain the chromatogram for the same sample in the same eluent (Fig. 4D). Another chromatogram (Fig. 4C) was obtained with the column of our phase in a different eluent to get similar retention times as those of D chromatogram, then the average N value was a little degraded but still 40,400 with the average R value being 9.67 while the average N values of Fig. 4D was only 19,800 with the average R value of 9.20. It should also be noted that the retention time range of Fig. 4C was even narrower than that of Fig. 4D. Factors contributing to enhanced separation efficiency and satisfactory selectivity of our phase are: (1) reduced average particle size; (2) diversity of particle shape (oval, bent, spherical, etc.) resulting in partially monolithic architecture after packing; (3) pseudo core-shell type behavior of our phase as a result of RAFT polymerization [36], and (4) formation of homogenous and uniformly distributed thin polymer film crucial for good mass transfer kinetics. Adoption of catalytic isocyanate-hydroxyl reaction in this study is believed to play a critical role in the above (3) and (4) by providing affluent initiator ligands. A significant portion of deep pores located in the core of particles seems clogged during RAFT polymerization. The most efficient commercial columns are generally known to be the ones packed with core-shell type spherical silica particles of 1.7–2.7 m [26–28,47]. However, the maximum length of commercial columns packed with particles of 3 m or less is 15 cm at present. The optimum separation efficiency of 2.1 mm I.D. column packed with 1.7 m Kinetex particles was around 320,000 plates/m
[48]. The maximum column length was 15 cm, so the maximum N value would be ca. 48,000. In another study, a maximum N value of 37,500 (corresponding to HEPT of 4 m) was obtained with a column (4.6 mm × 150 mm) packed with 2.2 m core-shell particles [49]. We mentioned that the reduced plate height of core-shell particles of smaller I.D. was below that of our phase in the previous study [46]. However, there are no commercial columns packed with particles of 3 m or less whose length is longer than 15 cm. We also had difficulties to pack a column of 30 cm with 3 m particles, and the separation efficiency of such a column was not better than that of the column packed with 5 m particles. On the other hand, there is no problem to pack our phase in a column of 30 cm length owing to the monolithic character. Thus the separation efficiency of our column (30 cm) phase packed with partially sub 2 m particles was better than that of commercial columns packed with small core-shell particles. The column length of this study is as twice as that of the commercial columns packed with sub 3-m particles. Nevertheless, there was no problem of column packing with our phase owing to the monolith-like structures of particles although our phase includes a major portion of sub-2 m particles since such structures allowed faster and stronger solvent flux under the same packing pressure to yield compact packing. The sample loading capacity of our phase was found to be only marginally lower than that of the 5 m commercial C18 phase based on the frontal analysis (Supplement 4 in the Supplement information). It seems that our phase is of enough loading capacity although the major contribution of capacity is due to the rather thick polymer layers of shallow pores of monolith particles while the contribution of polymer layers of deep pores is only a minor. 4. Conclusion Adoption of catalytic isocyanate-hydroxyl reaction in preparation of initiator attached silica monolith particles with increased density of initiator ligands has been successful to obtain the polystyrene bound particles with excellent separation efficiency via RAFT polymerization without formation of polystyrene lumps, and the plate numbers obtained with the column packed with this phase was found better than that of any commercially available HPLC or UHPLC column and those of the columns packed with previously reported stationary phases based on silica monolith particles.
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