Colloids and Surfaces

A: Physicochemical and Engineering Aspects 187–188 (2001) 273–279

Chromatographic characterization of macroporousmonolithic silica prepared via sol-gel process

Norio Ishizuka a, Hiroyoshi Minakuchi a, Kazuki Nakanishi a,*,Kazuyuki Hirao a, Nobuo Tanaka b

a Department of Material Chemistry, Graduate School of Engineering, Kyoto Uni�ersity, Yoshida, Sakyo-ku,Kyoto 606-8501, Japan

b Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Abstract

A continuous porous silica monolith prepared by the sol-gel process including phase separation was aged in a basicsolvent making use of hydrolysis of urea to prepare extended mesopore structures for chromatographic applications.The dissolution–reprecipitation kinetics at the interfaces between wet gel skeletons and an external solvent affectedthe size and volume of pores formed within the skeletons. At above 200°C, the pore size attained the macroporedimensions (�50 nm). The results of chromatography indicate that the monolithic silica column with wide mesoporecould reduce the separation time compared to the conventional column packed with 5 �m silica particle. © 2001Elsevier Science B.V. All rights reserved.

Keywords: Silica; Monolithic column; Aging; Mesopore; HPLC

www.elsevier.nl/locate/colsurfa

1. Introduction

Silica gel particles have been extensively used asa packing material of columns for high-perfor-mance liquid chromatography, HPLC [1]. High-efficiency and high-speed separations in HPLCcan be realized by small particles. Columnspacked with 5 �m particles, however, are stillmost widely used, while 1.5–3 �m particles areused in short columns due to the high back-pres-

sure associated with such small particles. Sinceincreasingly higher column back pressure is re-quired to obtain a constant mobile phase velocity,there arises an instrumental limitation for theaccelerated separation by columns packed withsmall-sized particles. Many researchers have triedto overcome this limitation and to attain muchhigher efficiency. Such an attempt in HPLC in-cludes ultra-high pressure LC [2], capillary elec-trochromatography, CEC [3] and open tubechromatography [4]. Another possible approachto overcome the problem of high pressure associ-ated with small particles is to fabricate a columnmade of one piece of a porous solid with small-sized skeletons and relatively large through-pores

* Corresponding author. Tel.: +81-75-7535551; fax: +81-75-7533345.

E-mail address: kazuki@bisco1.kuic.kyoto-u.ac.jp (K.Nakanishi).

0927-7757/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0927 -7757 (01 )00642 -2

N. Ishizuka et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 187–188 (2001) 273–279274

which could provide both low pressure drop andhigh column efficiency. The skeletons can bemeso-porous or microporous to have double-porestructures, or even nonporous. The most impor-tant features are high mechanical stability of bedsand the freedom in the ratio of through-pore sizeto skeleton size. Several examples of such mono-lithic columns made of an organic polymer havebeen reported recently [5–7].

We previously reported the preparation andcharacterization of monolithic macroporous silicacolumns [8–12] that provided high performancein high-speed separations based on the small silicaskeletons with appropriate mesopores and lowpressure drop based on the large through-pores(macropore). The size of through-pores can besimilar to or much larger than the size of skele-tons, resulting in large (through-pore size)/(skele-ton-size) ratio as large as 3–5 in the case of silicamonoliths. In the case of a particle-packedcolumn, the ratio of the size of interstitial voids toparticle size is in a range, 0.25–0.4. Increased(through-pore size)/(skeleton-size) ratios can re-sult in the higher permeability and shorter diffu-sion path length to provide the higher efficiency[13]. Such double-pore silica gel monoliths havebeen prepared with the combination of the sol-gelreaction accompanied by the phase separationand subsequent post-gelation treatments that en-hance the Ostwald ripening of finely textured wetgel matrix. While the macroporous structure isformed through concurrent phase separation andgelation in the course of hydrolysis and polycon-densation of alkoxysilane in the presence of or-ganic additives, the mesopore structure is tailoredby solvent exchange and aging [14]. The gelsexhibit well-defined micrometer-range pores withcontrollable volume fraction and median size to-gether. Nakanishi et al. reported the preparationof a macroporous silica with appropriate meso-pores tailored by aging in a basic solvent makinguse of hydrolysis of urea in a closed condition instead of solvent-exchange using aqueous ammo-nium hydroxide solution [15]. Incorporation ofurea in the hydrolysis and polycondensation pro-cess leads to the formation of a gel structurewhich exhibits higher reactivity in the dissolu-tion–reprecipitation process against the basic ex-

ternal solvents. A wide variation of pore sizewithin the silica gel skeleton can be attained as aresult of the dissolution–reprecipitaion reactionof amorphous silica [16]. The exact control ofmesopore size is of much interest for the separa-tion of solutes with a wide-range of molecularsize. The urea-containing alkoxy-derived silicasystem was applied to preparation of the continu-ous porous silica monoliths. It makes the fabrica-tion process simple in the case of preparing a gelwithin a confined space such as a fused silicacapillary since the mesopores are tailored just byheating the whole gelation mold without takingthe wet gel out. We previously reported the prepa-ration of monolithic silica with mesopores in a100 �m fused silica capillary and its evaluation inmicro-HPLC and capillary electro-chromatogra-phy (CEC) [17,18]. The mesopores formed withinthe gel exhibit appropriate size and surface areasin usual chromatography.

This paper describes the pore characteristicswithin the silica gel skeleton under weakly basicaging conditions including hydrothermal ones andthe use of an octadecylsilylated silica monolith inreversed-phase elution of polypeptides.

2. Experimental

Forty-five milliliter of Tetramethoxysilane(TMOS, Shin-Etsu Chemical, Tokyo, Japan) wasadded to 100 ml of 0.01 M aqueous acetic acid inthe presence of 9.0 g of urea and 11.5 g ofpoly(ethylene oxide) (PEO, Aldrich, Milwaukee,WI, USA) with an average molecular mass of10 000. The mixture was cooled in an ice bath andstirred vigorously for 30 min to conduct hydroly-sis and polycondensation. The resultant transpar-ent solution was poured into a polycarbonatemold and allowed to gel at 40°C. Gelation tookplace within 1.5 h, and the wet gel was subse-quently aged for 24 h. The aged wet gel wastransferred to an autoclave and immersed in anappropriate amount of aqueous external solvent.The autoclave was heated up to 120, 200, 250 or300°C for various times as shown in Table 1. Thishydrothermal aging made the pH value of theexternal solvent increase up to ca. 10, which al-

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Table 1Aging conditions and resultant pore characteristics

Temperature (°C)Monolith no. Aging time (h) Meso pore size (nm) Total pore volume (cm3 g−1)

3A 8120 4.47B 200 50 40 4.55

100 49200 4.57C15 61D 4.6025040 102250 4.68E10 413 4.28F 300

lows partial dissolution–reprecipitation of theamorphous silica network. After drying at 40°Cfor 3 days, the monolithic gel was heat-treated at600°C for 2 h. The monolithic silica gel of 7.0 mmdiameter was cut to 83 mm in length, encased inheat-shrinking PTFE tubing to be used in a Z-module (Waters, Milford, MA, USA), then oc-tadecylsilylated by on-column reaction, aspreviously described [8–12].

A scanning electron microscopy system (SEM,S-510, Hitachi, Japan) was employed for the ob-servation of the macroporous morphology. Char-acterization of the pore structure of the silica gelwas performed by mercury porosimetry (PORE-SIZER-9320, Micrometrics, USA). Chromatogra-phy was carried out using a conventional HPLCsystem (Shimadzu, Kyoto, Japan). Conventionalcolumns packed with 5 �m silica C18 particle wereobtained from commercial sources; CapcellpakC18 UG (poresize 12 nm; Shiseido, Tokyo, Japan),Capcellpak C18 SG (poresize 30 nm; Shiseido,Tokyo, Japan).

3. Results and discussion

Fig. 1 shows the co-continuous morphology ofsilica gel skeletons and macropores. The macrop-orous structure is formed by spinodal decomposi-tion which leads to the formation of continuousmicrometer range domains. Since the resultantpore size reflects the domain structure frozen-inby the gel formation on the way of coarsening, itmainly depends on the parameters affecting theonset of phase separation relative to the occur-rence of sol-gel transition. It is known that thismacroporous structure is essentially completed al-

ready in the wet state after gelation [19]. Thecontinuous macropores formed in the monolithicsilica are termed through-pores as compared tothe interstitial voids in the particle-packedcolumns.

The median mesopore size increased with anincrease of pH of external solution and/or soakingtemperature during the post-gelation reactionwhich occurs at the interface between the gelskeleton and the external solvent, suggesting thatthe dissolution and reprecipitation of oligomericunits of silica is the main mechanism of themesopore evolution process. Fig. 2 and Fig. 3show the evolution of mesopore distribution withaging temperature and time in the resultant gels.The results show narrow size distributions around1 �m except F in Fig. 3. Such sharp distributionsrepresent homogeneities of the macroporous gelsand cause high efficiencies in HPLC as shownlater. Meso pore size and total pore volume are

Fig. 1. SEM photograph of typical macroporous morphologyof gel sample.

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Fig. 2. Cumulative pore size distributions of heat-treated gels;aging temperature and duration: (A) 120°C, 3 h (solid line);(B) 200°C, 50 h (broken line); (C) 200°C, 100 h (dotted line).

Fig. 4. SEM photographs of gel sample; aging temperatureand duration: (B) 200°C, 50 h; (E) 250°C, 40 h; (F) 300°C, 10h.

summarized in Table 1. At 120°C, the medianmesopore size reaches ca. 7 nm after 3 h of aging.At temperatures above 200°C, the structure growsbeyond the mesopore range within a few days.The larger mesopores are formed accompanied bythe broadening of the distribution width as theaging time and temperature increases. The medianpore size exceeds 500 nm, although the pore vol-ume becomes considerably lower than the cases atlower temperature. With prolonged aging at hightemperature such as 300°C, the appearance of gelskeletons becomes coarse as shown in Fig. 4. The

gels treated under such severe conditions becomevery fragile. Therefore, it becomes difficult toshape it into the practical column.

Fig. 5 shows the molecular weight–elution vol-ume curves measured by size exclusion chro-

Fig. 5. Molecular weight–elution volume curves obtained bysize exclusion chromatography. Solutes: polystyrene standards.Mobile phase: tetrahydrofuran. Temperature: 30°C. �, mono-lithic silica column (B); �, previous monolithic column with14 nm mesopore [9]; �, column packed with 5 �m particles[9].

Fig. 3. Cumulative pore size distributions of heat-treated gels;aging temperature and duration: (D) 250°C, 15 h (solid line);(E) 250°C, 40 h (broken line); (F) 300°C, 10 h (dotted line).

N. Ishizuka et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 187–188 (2001) 273–279 277

Fig. 6. Plots of column back pressure against linear velocity ofmobile phase. Mobile phase: 80% methanol. The pressureswere normalized to the column length of 83 mm. �, mono-lithic column (E); �, previous monolithic column (through-pore diameter: 1.3 �m, mesopore diameter: 16 nm) [10]; �,Capcellpak C18 UG (mesopore diameter 12 nm) [10].

porosimetry, however, produced a much lowerpressure drop than that produced by conventionalcolumns packed with 5 �m particles and the previ-ous monolithic column with through-pores of 1.3�m, where column length was normalized. Thisresult could be caused by the very high porosityand resultant high permeability of the presentsilica monolith as seen in Fig. 5.

Fig. 7 compares the dependence of theoreticalplate height for the elution of insulin on themobile phase velocity (van Deemter plot) with 5�m particle-packed and monolithic columns(B, D) having smaller and larger mesopores. Thedependency of plate height on eluent linear veloc-ity is extremely small with monolithic columns.The results clearly indicate the advantage of usingthe silica-based monolithic columns over usingconventional particle-packed columns to achievehigh-speed separations. The monolithic silicacolumn with small skeletons (as seen in SEMphotograph) and large mesopores, provided goodresults. This is presumably due to the short diffu-sion path length associated with the small-sizedskeletons of the silica monoliths.

A practical example of the separation at in-creased mobile phase linear velocity in monolithiccolumn (D) is shown in Fig. 8. Applying a higher

matography using polystyrene standards as so-lutes in tetrahydrofuran with the monolithiccolumns. The results for the previous monolithicsilica column and a packed column are also plot-ted [9]. In the previous study, the monolithic silicacolumn with 14 nm mesopore was shown to pos-sess an external porosity of up to 65% of thecolumn volume (86% total porosity). This is muchhigher than 39% external porosity (79% totalporosity) found with a packed column. The exter-nal porosity was derived from the elution time ofa polystyrene standard of 8 420 000 molecularweight or greater and the total porosity from theelution time of benzene. The present monolithicsilica column (B) showed even higher porosity,69% external porosity and 94% total porosity.Since the exclusion limit molecular weight is alsohigher compared to other columns, the separationof samples with high molecular weight becomespossible.

Fig. 6 shows the column back pressure againstlinear velocity of the mobile phase. The size ofinterstitial void spaces between spherical particlesis commonly 25–40% of the size of particles [13].Therefore, the size of through-pore in the presentmonolithic silica should be smaller than that ofinterstitial openings in columns packed with 5 �mparticles. The monolithic silica with through-pores of ca.1.0 �m, as measured by mercury

Fig. 7. Van Deemter plots for C18 monolithic silica columnsand silica C18 packed columns with insulin as a solute. Mobilephase: acetonitrile–water (30:70) for monolithic columns,(32:68) for Capcellpak C18 SG and UG in the presence of 0.1%trifluoroacetic acid (TFA). Temperature: 30°C. �, monolithiccolumn (D); �, monolithic column (B); �, Capcellpak C18

UG (mesopore diameter 30 nm) [9]; �, Capcellpak C18 SG (12nm) [9].

N. Ishizuka et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 187–188 (2001) 273–279278

Fig. 8. Elution of polypeptides [(1) cytochrome c ; (2)lysozyme; (3) �-lactoglobulin; (4) ovalbumin] on monolithiccolumn (D). Linear gradient from 15 to 75% acetonitrile in thepresence of 0.1% TFA. Temperature: 30°C. Linear velocity: (a)2.0 mm s−1; (b) 4.6 mm s−1. Gradient time: (a) 10 min; (b)1.5 min.

micrometer macropores formed in the continuousmicrometer-sized silica skeleton will also havepossibilities in chromatographic applications, es-pecially for the separation of molecules with veryhigh molecular mass and low diffusion coefficient.

4. Conclusion

Monolithic silica columns prepared by the sol-gel method can provide better performance, espe-cially at high linear velocities, than conventionalcolumns packed with 5 �m particles. Thinner gelskeleton and higher through-pore volume in thewell-defined structure both contribute to enhancethe analytical performance of the monolithiccolumns under higher mobile phase velocity. Inthe preparation of silica monolith with widemesopores, post-gelation treatment in basic condi-tions and temperatures up to 300°C gives rise towell-defined meso- to macropores within themicrometer-sized silica gel skeletons. The mono-lithic silica column with larger mesopores showedhigher separation efficiency than that with smallermesopores. This kind of highly permeable andwell-defined porous structure will provide highercolumn efficiencies for high-molecular-mass so-lutes with low diffusion coefficient.

Acknowledgements

The authors thank Professor K. Yanagisawa,Institute for Hydrothermal Chemistry, Faculty ofScience, Kochi University, for his kind suggestionand support of the experimental works.

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