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Monolithic Columns Prepared from Particles
F. Svec, T.B. Tennikova and Z. Deyl (Editors) Monolithic Materials Jo'arnal of Chromatography Library, Vol. 67 9 2003 Elsevier Science B.V. All rights reserved.
Chapter 9
Monolithic Columns Prepared from Particles Qinglin TANG 1 and Milton L. LEE 2
1Analytical Research and Development, Pharmaceutical Research Institute, Bristol-Myers-Squibb Company, Deepwater, NJ 08023 Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602-5 700
CONTENTS
9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Particle-embedded monolithic columns . . . . . . . . . . . . . . . . . . Particle-sintered monolithic columns . . . . . . . . . . . . . . . . . . . Particle-loaded monolithic columns . . . . . . . . . . . . . . . . . . . . Particle-entrapped monolithic columns . . . . . . . . . . . . . . . . . . Particle-bonded monolithic columns . . . . . . . . . . . . . . . . . . . . Particle-crosslinked monolithic columns . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
197 198 200 202 204 205 209 210 210
9.1 I N T R O D U C T I O N
Columns packed with particles, both porous and nonporous, have been widely used in high performance liquid chromatography (HPLC) for decades. Particulate packing materials have many advantages for HPLC applications. If porous, they have high surface area that leads to high sample capacity. Small particles generate high efficiency. Particles in large quantity can be manufactured reproducibly under strictly controlled conditions. Even if there are imperfections in the particles, the numerous trans-channel couplings in a packed column average laterally the impact of the imper-
198
Chapter 9
fections by repeatedly mixing mobile phase streams at junctions between particles and spreading them across the column, which results in little fluctuation of column performance from batch-to-batch. Versatile particulate packing materials are commercially available for various applications. Capillary electrochromatography (CEC) is the newest development in liquid chromatography [ 1-3]. Instead of using a high pressure pump, CEC employs high voltage across the column to drive the mobile phase through the column. Most publications about CEC describe the use of capillary columns of 50-200 ~tm i.d. packed with 1.5-10 ~tm. silica based particles. The main disadvantages of packed columns are the requirement for end-frits and the instability of the packed bed. End-frits, which are often difficult to make and which can deteriorate column performance, are needed to keep the particles in place in the column. Even if a column is tightly packed, voids are often observed in the column after use for a period of time. Monolithic columns from particles are designed to inherit the versatility of welldeveloped particulate media but avoid the end-frits and instability of packed columns. The absence of end-frits reduces mobile phase flow resistance and allows easier interfacing to mass spectrometry. Also, the wall-supported monolith is expected to be stable and to suppress zone broadening caused by the so-called "wall effect". Various methods for preparing monoliths from particles have been reported. Generally, they can be classified into six types: particle-embedded, particle-sintered, particle-loaded, particle-entrapped, particle-bonded, and particle-crosslinked monoliths. The following sections describe in detail their preparation and properties. 9.2 PARTICLE-EMBEDDED MONOLITHIC COLUMNS Tsuda et al. [4] first described a particle-embedded monolith in which silica gel was packed into a heavy-walled wide-bore glass tube that was then drawn into a capillary using a glass drawing machine. Under high temperature the glass tube melted and the particles were embedded in the wall, forming a particle-embedded monolith. Knox and Grant [5] reported a detailed procedure for fabricating particleembedded capillary monoliths using particles with sizes of 3-50 ~tm. They first dried the silica gel overnight at 400~ and cooled it in a vacuum desiccator. A heavy-walled Pyrex glass tube of 1-2 mm i.d. sealed at one end with quartz wool was packed with the cooled silica gel by vibration and tapping while the tube was rotated. Approximately a 30-cm length of the tube was packed and the packing was topped with a glass wool plug. To remove water adsorbed during the packing process, the packed tube was evacuated using a vacuum pump while it was slowly passed through a furnace at 500~ The tube was cooled under vacuum, positioned in the glass-drawing machine, and drawn to the required diameter. The drawing temperature was initially at
Monolithic Columns Prepared from Particles
199
Fig. 9.1 Photograph of a particle-embedded monolithic capillary of 40 p,m I.D. packed with 5 ~tm Hypersil silica gel (Reproduced with permission from ref. [5]. Copyright 1991 Friedr Vieweg & Sohn Verlagsgesellschaft mbH).
650~ but ended at 560-600~ depending on the final diameter of the capillary. After cooling to room temperature, a particle-embedded monolithic capillary as shown in Fig. 9.1 was obtained. This particle-embedded monolithic capillary was stable under hydrodynamic flow if the capillary-to-particle diameter ratio was less than 10. It had a flow resistance factor as low as 120 compared to 500-1000 for slurry packed columns. An efficiency of 200,000 plates per meter in capillary electrochromatography (CEC) was obtained using 5-~tm Hypersil silica-embedded in a 40 ~tm i.d. monolithic capillary derivatized with octadecyldimethylsilane (ODS). The monolithic capillary was transparent due to the thinness of the wall and, thus, could be used with fluorometric detection at excitation wavelengths higher than 240 nm. There are several major problems associated with the preparation of particle-embedded monoliths. First, water must be evaporated during heating of the silica gel in the furnace of the drawing machine in order to obtain a good column. Second, a considerable lateral force is required to reorient the silica gel particles during drawing. Third, fused silica column preforms cannot be used because silica gel will fuse well below the melting point of the fused silica. Typically Pyrex glass results in fragile capillaries that are difficult to handle. Fourth, bonded-phase silica gels cannot be directly employed because the high drawing temperature can destroy the bonded phase. Any bonding of stationary phases must be carried out in situ after drawing, which can lead to irreproducibility in column-to-column performance. Fifth, particles are retained in the capillary because of the "keystone" effect from embedding the particles in the capillary inner wall. As a result, a hydrodynamically stable particle-
References pp. 210-211
200
Chapter 9
embedded monolithic capillary with a capillary-to-particle diameter ratio greater than 10 cannot be prepared. Thus, the diameter of the particle-embedded monolithic column is limited. 9.3 PARTICLE-SINTERED MONOLITHIC COLUMNS Asiaie et al. [6] described the preparation of particle-sintered monoliths in which the particles packed in a fused silica capillary were chemically sintered together and to the capillary inner wall. In this procedure, 6-1am ODS silica particles were packed into a fused silica capillary of 75 pm i.d. using a 50:50 v/v toluene-acetone slurry mixture. The packed capillary was rinsed with 0.1 mol/L NaHCO3 for 5 min, followed by washing with water and acetone. The column, after purging with N2 for at least 1 h, was heated at 120~ in a vacuum oven for 5 h to remove the physically adsorbed water, followed by 360~ in a GC oven for 10 h. NaHCO3 at 360~ wetted and partially dissolved the surface of the ODS, forming a flux of low melting vitreous phase rich in sodium silicate that remained between the particles and between particle and capillary wall because of surface tension forces, forming a sintered monolith shown in Fig. 9.2. It was noticed that the reproducibility of preparation of sintered capillaries from ODS was higher than that from silica because of the adsorbed water, since silica has greater water-binding power than ODS. Water adsorbed to ODS was nearly eliminated during thermal treatment at 120~ while water adsorbed to silica was just partially evaporated at that temperature. The residual water was released as a sudden burst of water vapor during the sintering step at 360~ causing bed rupture and concomitant gapping. Due to the harsh experimental conditions (high temperature and basic NaHCO3) in the sintering process, the stationary phase was partially destroyed and, thus, post deactivation and functionalization of the bed was necessary. To avoid post-functionalization of the sintered monolith, Adam et al. [7] used water instead of NaHCO3 and a movable heating coil to fabricate sintered monoliths. A 10% suspension of ODS in acetone was packed into fused silica tubing using a slurry packing method under a pressure of 60 MPa. The packed capillary was then filled with water. A heating coil driven by a processing motor moved along the length of the packed capillary with a constant velocity and at a constant temperature in the range of 300-400~
As the heating coil moved along the packed capillary, the high
temperature in the heated zone led to dissolution of silica to form polysilicic acids at the surface of the particles. After cooling, the solution in the interstitial voids became supersaturated with silica sol and re-deposition of silica occurred between particles and between particle and capillary wall, forming a monolith with particles retained in their original positions. The mild sintering conditions compared to those employed by Asiaie et al. maintained the integrity of the stationary phase and made post-function-
Monolithic Columns Prepared from Particles
201
B
Fig. 9.2. Scanning electron micrographs of particle-sintered monolithic columns sintered at (a) 260~ (b) 360~ without, and (c) 360~ with NaHCO3 flux (reproduced with permission from ref. [6]. Copyright 1998 Elsevier Science B.V.).
alization of the sintered monolith unnecessary. The quality of the sintered monolith was controlled by three operational parameters for a given particulate material: (i) the temperature of the heating coil, (ii) the speed by which the coil is moved along the packed column, and (iii) the number of cycles of this operation. Both too low temperature and number of cycles resulted in a monolith of low mechanical stability. Too high temperature or more cycles led to degradation of the surface of the particles and destruction of the bonded stationary phase. The best column efficiency for a column containing Hypersil-ODS1 was obtained after two cycles at a temperature of 300400~
References pp. 210-211
202
Chapter 9
Particle-sintered capillary monoliths are mechanically strong and stable due to interparticle growth. They are also flexible because the polyimide coating on the fused silica capillary is not destroyed. The pore structure of the ODS in the sintered capillary remains essentially unchanged since irreversible destruction of the silica pore structures was observed only at temperatures above 600~
An efficiency of 125,000
plates/m in CEC was obtained using a sintered 6 ~m ODS column [6]. Under optimized operating conditions, no remarkable difference in retention factor, resolution, and efficiency were observed before and after sintering. 9.4 PARTICLE-LOADED MONOLITHIC COLUMNS Dulay et al. [8] reported the preparation of particle-loaded continuous-bed columns, in which ODS was loaded in a sol-gel matrix that was subsequently covalently bonded to the capillary inner wall. In this procedure, the sol-gel solution was prepared by mixing 0.2 mL of tetraethoxylsilane (TEOS), 0.73 mL of ethanol, and 0.1 mL of 0.12 mol/L HC1. ODS particles were added at a concentration of 300 mg/mL to the sol-gel solution to create a suspension that was sonicated for several minutes and then introduced into 75 lam i.d. fused silica tubing of about 40 cm long by applying vacuum. The column was then coiled into a loop, placed on a hot plate, and heated above 100~ for about 24 h. During this period, TEOS hydrolyzed into tetrahydroxylsilane which then polycondensed into a silica sol-gel network filled with ethanol. Upon the evaporation of ethanol, the sol-gel network and the ODS particles were converted into a particle-loaded monolith shown in Fig. 9.3. As can be seen, the ODS particles were bonded evenly across the column and not merely cast along the capillary wall. Their loading into the sol-gel matrix was achieved either by physical trapping or by chemical bonding between the sol-gel matrix and the surface of the ODS through silanol groups. The sol-gel network was integrally fixed to the walls of the capillary through covalent bonding of silanol groups between the sol-gel matrix and the capillary inner wall. In contrast to a pure sol-gel monolith, the particle-loaded monolith did not display obvious shrinkage because the ODS particles helped to decrease the stress within the matrix. However, cracks around the ODS particles were noticed, possibly because of phase separation between the hydrophobic ODS surface and the hydrophilic sol-gel matrix. Parameters controlling the success of the preparation were sol-gel matrix composition, ratio of particles to sol-gel, and suspension cast method. The sol-gel matrix solution was adjusted to a pH around 2, which enabled fast hydrolysis of TEOS, but slow polycondensation of silanol groups and, thus, slow solidification of the sol-gel matrix. The volumetric ratio of TEOS to solvent (ethanol) determined the porosity of the sol-gel matrix. The loading of ODS into the sol-gel matrix enhanced the mechani-
Monolithic Columns Prepared from Particles
203
Fig. 9.3. Scanning electron micrographs of a particle-loaded monolithic column (reproduced with permission from ref. [8]. Copyright 1998 American Chemical Society).
cal strength of the monolith and provided the chromatographic selectivity. A good suspension of ODS particles in the sol-gel matrix solution is essential for obtaining a monolithic column with homogeneous distribution of ODS particles within the monolith. The optimum concentration was found to be 300 mg ODS per mL of the sol-gel solution. At lower concentration, such as less than 200 mg/mL the selectivity of the resulting column was poor. At higher concentration, e.g., 350 mg/mL, significant heterogeneity in the ODS packing density along the column led to numerous sections of the capillary column void of ODS particles. The suspension can be pushed or drawn into the tubing. The use of vacuum to fill the small bore capillary with the ODS/TEOS slurry suspension greatly improved the reproducibility of column preparation over pressurized syringe-filling methods. Particle-loaded monolithic columns can sustain moderate pressures of up to 1.4 MPa. A 3 lam ODS-loaded monolithic column generated a specific permeability of 7.0 x 10-2 ktm2 [9,10] which is approximately 8 times greater than that of a tightly packed 3 ~tm ODS column. This high specific permeability is due to the low pressure method of preparation of the column in which 3 lam ODS particles are not as tightly packed into the tubing as they would be in a conventional packed LC column. Furthermore, large cracks around the ODS particles as shown in Fig. 9.3 and the highly porous sol-gel matrix allowed liquid to flow through the column at low pressure. An efficiency of 80,000 plates/m was obtained in CEC with a 3 ~tm ODS-loaded monolith. This moderate efficiency may be attributed to three factors. First, there may be inho-
Referencespp. 210-211
204
Chapter 9
mogeneous loading of the ODS column due to the different polarities and densities of the sol-gel solution and ODS particles. Second, some of the ODS particles can be too deeply embedded in the sol-gel matrix to play a role in the separation mechanism. Finally, cracks are often formed around the ODS particles. 9.5 PARTICLE-ENTRAPPED MONOLITHIC COLUMNS Chirica and Remcho [ 11-13] published a method for preparing monolithic columns in which particles are entrapped in a monolithic structure by an entrapping matrix. Their procedure involved four steps: (i) the fused silica capillary was packed with ODS particles using a conventional solvent slurry packing method, and then dried by purging the column with nitrogen; (ii) the dried packed capillary was filled with entrapping matrix solution; (iii) the column was cured using different methods depending on the entrapping matrix; and (iv) the cured column was dried slowly at a specified temperature. Upon drying, a monolithic column with entrapped particles shown in Fig. 9.4 resulted [13]. The properties of particle-entrapped monoliths were determined by the homogeneity of the packed bed and the characteristics of the entrapment matrix, including porosity and surface chemistry. Because of the use of high pressure during column packing, the packed bed was tight and relatively uniform throughout the entire column. This ensured homogeneity throughout the column. Different entrapping matrices, such as silicate (Kasil 2130) [11], tert-butyl-triethoxylsilane [12], n-octyltriethoxysilane [12], and methacrylate-based organic polymers [13] were investigated. Using various entrapping matrices, different packing materials can be entrapped in the monolith. All of the resultant particle-entrapped columns gave similar efficiencies which were nearly equal to those obtained for packed columns using the same particles. While the silicate matrix generated a monolith with serious peak tailing, other entrapping matrices gave satisfactory peak shapes and fine-tuned selectivity. Depending on the amount of entrapping matrix, the resultant particle-entrapped monoliths can have nearly the same or higher permeabilities than those observed for packed column beds containing the same packing materials. Retention factors for analytes on particleentrapped columns using silicate entrapping matrices are significantly reduced compared to those for conventional packed columns due to a combination of masking of the stationary phase with the entrapping silicate and hydrolysis of the stationary phase during NH4OH rinse. In contrast, other entrapping matrices displayed no significant loss Of retention. The greatest disadvantage associated with particle-entrapped monoliths is that the preparation procedures are time-consuming because drying of the entrapped column must be performed slowly. Otherwise, surface tension forces will cause column bed
Monolithic Columns Prepared from Particles
205
S'
:-
:~; Nx-
Fig. 9.4. Scaning electron micrographs of a particle-entrapped monolithic column (reproduced with permission from ref. [13]. Copyright 2000 American Chemical Society).
cracking and failure. Also the use of a retentive entrapping matrix adds to the retention of solutes, which makes it difficult to predict the overall retention behavior of analytes on the column. 9.6 SOL-GEL-BONDED MONOLITHIC COLUMNS We developed a sol-gel-bonded monolith that employed a specifically designed sol-gel matrix and the use of supercritical fluid column packing and drying [14]. The preparation procedure involved four steps: (i) packing the column with a supercritical CO2-slurry, (ii) filling the packed column with a sol solution, (iii) gelling and aging of the sol-filled packed column at room temperature, and (iv) drying the gelled, packedcolumn with supercritical carbon dioxide. A CO2 slurry packing method developed in our laboratory [ 15] was used to pack the columns. In short, one end of a fused silica Referencespp. 210-211
Chapter 9
206
capillary was connected to a stainless steel vessel into which an approximate amount of packing material was placed. The other end of the capillary column was connected to a length of 25 ~tm i.d. restrictor tubing using a zero dead-volume union in which a stainless steel frit with 0.5 lam pores was placed. The stainless steel frit was used to retain the particles in the capillary, and the restrictor tubing was used to control the packing speed. The stainless steel vessel was connected to a syringe pump, and packing of the capillary column was carried out in an ultrasonic bath at room temperature by increasing the pressure from 5-35 MPa at 1 MPa/min. Finally the column was conditioned at 35 MPa for 30 min, and then depressurized overnight. The sol solution was prepared by mixing appropriate amounts of tetramethoxysilane (TMOS), ethyltrimethoxysilane (ETMOS), methanol, trifluoroacetic acid (TFA) and water in a 0.6 mL polypropylene vial, followed by the addition of formamide. For example, to obtain a 9% (precursor/solution, v/v) sol solution, the following amounts were used: 20 laL TMOS, 20 ~tL ETMOS, 200 laL methanol, 15 ~tL TFA aqueous solution at pH 2, and 200 laL formamide. The apparent pH of the solution was approximately 5, measured using short-range pH test paper. The solution was vortexed for 5 min at room temperature using a vortex mixer and introduced into a packed capillary column to a desired length (observed with the naked eye or under a microscope with 10x magnification) using a 100-~tL PEEK syringe mounted on a small syringe pump. The syringe was strong enough for filling 60 cm of capillary column packed with 5 lam ODS particles, or 45 cm of capillary column packed with 3 lam ODS particles, with the non-viscous sol solution at a speed of 1 laL/min. Both ends of the sol-filled packed capillary were sealed using a silicone septum and the column was stored at room temperature for 24 h to achieve the conversion of the sol to a gel, and for aging of the resulting wet gel. After aging, the capillary was connected to an SFC pump using a zero dead-volume union. The column was flushed with supercritical CO2 at 8 MPa inlet pressure and 40~ to replace the solvent in the column. Then, while supercritical CO2 at 8 MPa was purged through the column, the column was dried by temperature programming from 40~ and holding at 240~
to 240~
at I~
min -1,
for 1 h. Finally, the packing material at the end of the column
that was not bonded by the sol-gel was flushed out, leaving a section of unpacked capillary. A short length of the monolithic column was cut off and a gold coating of approximately 40 nm thickness was sputtered on it for observation using a scanning electron microscope. Typical scanning electron micrographs of the cross-section of a sol-gel bonded ODS monolithic column are shown in Fig. 9.5. As can be seen, the column was packed homogeneously without visible wall perturbations and the ODS particles were bonded to each other and to the capillary wall by the gel network, forming a sol-gel-bonded monolith.
Monolithic Columns Prepared from Particles
207
Fig. 9.5. Scanning electron micrographs showing cross-sectional views of the end of a sol-gel-bonded large-pore ODS monolithic column (reproduced with permission from ref. [20]. Copyright 1999 John Wiley & Sons).
The key for preparing a successful sol-gel-bonded monolith is the design of the sol-gel matrix and the drying process. Fig. 9.6 illustrates schematically the synthesis of the solgel bonded particles. At pH 2, both TMOS and ETMOS precursors quickly underwent hydrolysis in the solvent, forming tetrahydroxysilane and ethyltrihydroxysilane. After the addition of formamide, the solution pH was changed to approximately pH 5, where tetrahydroxysilane polycondensed much faster than ethyltrihydroxysilane. As a result, the sol solution was converted into a silica network endcapped with ethyl groups. The particles were bonded to each other and to the capillary inner wall through this inert sol-gel matrix, forming a wall-supported continuous-bed. In a classical sol-gel process for silica glasses, a single precursor, TMOS, is typically used [16]. However, a silica gel network fabricated from TMOS alone often shrinks and cracks during drying, and the silica gel network is active because of the exposed silanol groups on the gel surface. It was reported that a combination of alkyl derivatives of TMOS as co-precursors with TMOS resulted in a flexible gel network with a
Referencespp. 210-211
Chapter 9
208
S,(OCIt:,,)~ + tI,O
f
Me()H ,~ Si(OH),; ~ TFA, plt=2 pH=5
1
-{~i-O-~i)C:H~ (':H.~
I. C:H~Si(OCH~)~+ H~O
MeOH ,C:H~Si(OH)~ ~ ( S ']FA, pH=2 .....
t
I, -O-Sli)-
C2H~ C:H.~
i
I
-(Si-O-Si)I
o
\...._..../
~
!
o
o
-(Si O Si)I
!
C~t--1, C~H, Fig. 9.6. Synthetic scheme for sol-gel bonded particles (reproduced with permission from ref. [ 18]. Copyright 2000 Wiley-VCH Verlag GmbH).
more open pore structure [17], which could alleviate capillary stress during drying. The alkyl groups can also deactivate the gel network, thereby contributing little to the peak tailing of analytes. Even when the composition of the sol solution was well designed and controlled, extremely slow drying was required to minimize cracking of the column bed when conventional thermal drying was applied to the sol-gel matrix. This made column preparation rather time-consuming. Cracking of the column bed is believed to result from capillary forces generated during evaporation of the solvent. To avoid this, a supercritical fluid was used to dry the gel network since the surface tension of a supercritical fluid is zero [ 16]. In the fabrication of sol-gel-bonded monoliths, supercritical CO2 was pumped through the column to replace the solvent and residual reactants using a supercritical fluid chromatographic pump, and the column was then dried in supercritical CO2 under elevated temperature. It was observed that column drying with supercritical CO2 was fast and the resultant columns were crackfree. Sol-gel-bonded monolithic columns from small-pore ODS [14,18,19], large-pore ODS [18,20,21], mixed-mode ODS/SCX [18,22], and C30-silica [23,24] have been successfully prepared. Column-to-column reproducibility was found to be less than 8% for three monolithic columns containing 9% sol-gel bonded 5-~tm ODS [14]. An efficiency of 410,000 plates/m in CEC was obtained using a sol-gel-bonded 3 ~tm, 1500 A ODS column, which is nearly double the efficiency from a sol-gel-bonded
Monolithic Columns Prepared from Particles
209
3 lam, 80 A ODS column [21]. This was attributed to EOF generation in the largepores of the 3 ktm, 1500 A ODS.
9.7 PARTICLE-CROSSLINKED M O N O L I T H I C COLUMNS We have also described a particle-crosslinked monolithic column in which polymer-encapsulated packing materials packed in polymer-coated fused silica tubing was crosslinked in situ using a free radical reaction [25]. A fused silica open tubular column with 0.25 ~tm film thickness of octyl-substituted polysiloxane was packed with polybutadiene (PBD)-encapsulated zirconia packing material using a supercritical CO2 slurry packing method and placed in a GC oven and flushed with a free radical initiator, azo-tert-butane, in helium. The helium flow rate through the packed column was 0.5 cm/s. The oven temperature was elevated to 260~ 0.5~
at a rate of
and held at 260~ for 5 h. Porous packing materials could be immobilized
onto the PSB-Octyl capillary wall through free radical reactions [26] since about 50% of the vinyl groups on the PBD-encapsulated zirconia particles remained unreacted [27]. Under elevated temperature, azo-tert-butane decomposed to form free radicals and the PBD-encapsulated zirconia packing was crosslinked together and to the capillary wall through free radical initiated crosslinking reactions, forming a crosslinkedmonolithic column shown in Fig. 9.7. The experimental conditions, including the film thickness of the octylpolysiloxane coating in the open tubular column, the flow rate of the azo-tert-butane saturated helium through the column bed, and the time and temperature for the crosslinking reaction, determined the success of column preparation. When the octylpolysiloxane film thickness in the original capillary was 0.25 ktm, the crosslinked packing material stayed in the column even when 40 MPa carbon dioxide at room temperature was applied at the column inlet. For achieving a mechanically strong monolithic column, the column must be purged for half an hour with azo-tert-butane vapors in helium at a flow rate of 2 mm/s before increasing the temperature. Using a crosslinking temperature of 260~
for 5 h, stable crosslinked-monolithic 3% PBD-encapsulated zirconia
columns of 25-60 cm x 250 ~tm i.d. were obtained. The column efficiency, solute retention, and permeability of these monolithic columns were similar to those measured for columns freshly packed, but not crosslinked, with the same material under the same conditions.
References pp. 210-211
210
Chapter 9
Fig. 9.7. Scanning electron micrograph of the end of a crosslinked monolithic column (reproduced with permission from ref. [25]. Copyright 1999 John Wiley & Sons).
9.8 CONCLUSIONS
Monolithic columns prepared from particles are designed to inherit the efficiency and selectivity of versatile LC packing materials. Monolithic columns from particles are expected to be stable without the need of end-frits. Various methods used for the preparation of monolithic columns from particles have been described, each with its own merits and shortcomings. Sol-gel-bonded monolithic columns employing an inert sol-gel matrix and supercritical CO2 drying is currently the most promising method for obtaining high performance columns. 9.9 R E F E R E N C E S
1 2 3 4 5 6 7 8 9 10 11 12
V. Pretorius, B.J. Hopkins, J.D. Schieke, J. Chromatogr. 99 (1974) 23 J.W. Jorgenson, K.D. Lukacs, J. Chromatogr. 218 (1981)209 J.H. Knox, I.H. Grant, Chromatographia 24 (1987) 135 T. Tsuda, I. Tanaka, G. Nakagawa, Anal. Chem. 56 (1984) 1249. J.H. Knox, I.H. Grant, Chromatographia 32 (1991) 317. R. Asiaie, X. Huang, D. Farnan, C. Horvath, J. Chromatogr. A 806 (1998) 251. T. Adams, K.K. Unger, M.M. Dittmann, G.P. Rozing, J. Chromatogr. A 887 (2000) 327. M.T. Dulay, R.P. Kulkarni, R.N. Zare, Anal. Chem. 70 (1998) 5103. C.K. Ratnayake, C.S. Oh, M.P. Henry, J. High Resol. Chromatogr. 23 (2000) 81. C.K. Ratnayake, C.S. Oh, M.P. Henry, J. Chromatogr. A 887 (2000) 277. G. Chirica, V.T. Remcho, Electrophoresis 20 (1999) 50. G.S. Chirica, V.T. Remcho, Electrophoresis 21 (2000) 3093.
Monolithic Columns Prepared from Particles 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
211
G.S. Chirica, V.T. Remcho, Anal. Chem. 72 (2000) 3605. Q. Tang, B. Xin, M.L. Lee, J. Chromatogr. A 837 (1999) 35. A. Malik, W. Li, M.L. Lee, J. Microcol. Sep. 5 (1993) 361. C.J. Brinker, G.W. Scherer, Sol-gel Science: The Physics and Chemistry of Sol-gel Processing, Academic Press, San Diego, CA, USA, 1990. J.D. Mackenzie, Hybrid Organic-Inorganic Composites, ACS Symposium Series 585, American Chemical Society, Washington, D.C., USA, 1995, p 227-236. Q. Tang, M.L. Lee, J. High Resol. Chromatogr. 23 (2000) 73. Q. Tang, N. Wu, M.L. Lee, J. Microcol. Sep. 12 (2000) 6. Q. Tang, N. Wu, M.L. Lee, J. Microcol. Sep. 11 (1999) 550. Q. Tang, N. Wu., B. Yue, M.L. Lee, Anal. Chem., in press. Q. Tang, M.L. Lee, J. Chromatogr. A 887 (2000) 265. L. Roed, E. Lundanes, T. Greibrokk, J. Microcol. Sep. 12 (2000) 561. L. Roed, E. Lundanes, T. Greibrokk, J. Chromatogr. A 890 (2000) 347. Q. Tang, Y. Shen, N. Wu, M.L. Lee, J. Microcol. Sep. 11 (1999) 415. H. Yun, K.E. Markides, M.L. Lee, J. Microcol. Sep. 7 (1995) 153. J. Li, P.W. Carr, Anal. Chem., 68 (1996) 2875.
Chapter 9
Monolithic Columns Prepared from Particles Qinglin TANG 1 and Milton L. LEE 2
1Analytical Research and Development, Pharmaceutical Research Institute, Bristol-Myers-Squibb Company, Deepwater, NJ 08023 Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602-5 700
CONTENTS
9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Particle-embedded monolithic columns . . . . . . . . . . . . . . . . . . Particle-sintered monolithic columns . . . . . . . . . . . . . . . . . . . Particle-loaded monolithic columns . . . . . . . . . . . . . . . . . . . . Particle-entrapped monolithic columns . . . . . . . . . . . . . . . . . . Particle-bonded monolithic columns . . . . . . . . . . . . . . . . . . . . Particle-crosslinked monolithic columns . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
197 198 200 202 204 205 209 210 210
9.1 I N T R O D U C T I O N
Columns packed with particles, both porous and nonporous, have been widely used in high performance liquid chromatography (HPLC) for decades. Particulate packing materials have many advantages for HPLC applications. If porous, they have high surface area that leads to high sample capacity. Small particles generate high efficiency. Particles in large quantity can be manufactured reproducibly under strictly controlled conditions. Even if there are imperfections in the particles, the numerous trans-channel couplings in a packed column average laterally the impact of the imper-
198
Chapter 9
fections by repeatedly mixing mobile phase streams at junctions between particles and spreading them across the column, which results in little fluctuation of column performance from batch-to-batch. Versatile particulate packing materials are commercially available for various applications. Capillary electrochromatography (CEC) is the newest development in liquid chromatography [ 1-3]. Instead of using a high pressure pump, CEC employs high voltage across the column to drive the mobile phase through the column. Most publications about CEC describe the use of capillary columns of 50-200 ~tm i.d. packed with 1.5-10 ~tm. silica based particles. The main disadvantages of packed columns are the requirement for end-frits and the instability of the packed bed. End-frits, which are often difficult to make and which can deteriorate column performance, are needed to keep the particles in place in the column. Even if a column is tightly packed, voids are often observed in the column after use for a period of time. Monolithic columns from particles are designed to inherit the versatility of welldeveloped particulate media but avoid the end-frits and instability of packed columns. The absence of end-frits reduces mobile phase flow resistance and allows easier interfacing to mass spectrometry. Also, the wall-supported monolith is expected to be stable and to suppress zone broadening caused by the so-called "wall effect". Various methods for preparing monoliths from particles have been reported. Generally, they can be classified into six types: particle-embedded, particle-sintered, particle-loaded, particle-entrapped, particle-bonded, and particle-crosslinked monoliths. The following sections describe in detail their preparation and properties. 9.2 PARTICLE-EMBEDDED MONOLITHIC COLUMNS Tsuda et al. [4] first described a particle-embedded monolith in which silica gel was packed into a heavy-walled wide-bore glass tube that was then drawn into a capillary using a glass drawing machine. Under high temperature the glass tube melted and the particles were embedded in the wall, forming a particle-embedded monolith. Knox and Grant [5] reported a detailed procedure for fabricating particleembedded capillary monoliths using particles with sizes of 3-50 ~tm. They first dried the silica gel overnight at 400~ and cooled it in a vacuum desiccator. A heavy-walled Pyrex glass tube of 1-2 mm i.d. sealed at one end with quartz wool was packed with the cooled silica gel by vibration and tapping while the tube was rotated. Approximately a 30-cm length of the tube was packed and the packing was topped with a glass wool plug. To remove water adsorbed during the packing process, the packed tube was evacuated using a vacuum pump while it was slowly passed through a furnace at 500~ The tube was cooled under vacuum, positioned in the glass-drawing machine, and drawn to the required diameter. The drawing temperature was initially at
Monolithic Columns Prepared from Particles
199
Fig. 9.1 Photograph of a particle-embedded monolithic capillary of 40 p,m I.D. packed with 5 ~tm Hypersil silica gel (Reproduced with permission from ref. [5]. Copyright 1991 Friedr Vieweg & Sohn Verlagsgesellschaft mbH).
650~ but ended at 560-600~ depending on the final diameter of the capillary. After cooling to room temperature, a particle-embedded monolithic capillary as shown in Fig. 9.1 was obtained. This particle-embedded monolithic capillary was stable under hydrodynamic flow if the capillary-to-particle diameter ratio was less than 10. It had a flow resistance factor as low as 120 compared to 500-1000 for slurry packed columns. An efficiency of 200,000 plates per meter in capillary electrochromatography (CEC) was obtained using 5-~tm Hypersil silica-embedded in a 40 ~tm i.d. monolithic capillary derivatized with octadecyldimethylsilane (ODS). The monolithic capillary was transparent due to the thinness of the wall and, thus, could be used with fluorometric detection at excitation wavelengths higher than 240 nm. There are several major problems associated with the preparation of particle-embedded monoliths. First, water must be evaporated during heating of the silica gel in the furnace of the drawing machine in order to obtain a good column. Second, a considerable lateral force is required to reorient the silica gel particles during drawing. Third, fused silica column preforms cannot be used because silica gel will fuse well below the melting point of the fused silica. Typically Pyrex glass results in fragile capillaries that are difficult to handle. Fourth, bonded-phase silica gels cannot be directly employed because the high drawing temperature can destroy the bonded phase. Any bonding of stationary phases must be carried out in situ after drawing, which can lead to irreproducibility in column-to-column performance. Fifth, particles are retained in the capillary because of the "keystone" effect from embedding the particles in the capillary inner wall. As a result, a hydrodynamically stable particle-
References pp. 210-211
200
Chapter 9
embedded monolithic capillary with a capillary-to-particle diameter ratio greater than 10 cannot be prepared. Thus, the diameter of the particle-embedded monolithic column is limited. 9.3 PARTICLE-SINTERED MONOLITHIC COLUMNS Asiaie et al. [6] described the preparation of particle-sintered monoliths in which the particles packed in a fused silica capillary were chemically sintered together and to the capillary inner wall. In this procedure, 6-1am ODS silica particles were packed into a fused silica capillary of 75 pm i.d. using a 50:50 v/v toluene-acetone slurry mixture. The packed capillary was rinsed with 0.1 mol/L NaHCO3 for 5 min, followed by washing with water and acetone. The column, after purging with N2 for at least 1 h, was heated at 120~ in a vacuum oven for 5 h to remove the physically adsorbed water, followed by 360~ in a GC oven for 10 h. NaHCO3 at 360~ wetted and partially dissolved the surface of the ODS, forming a flux of low melting vitreous phase rich in sodium silicate that remained between the particles and between particle and capillary wall because of surface tension forces, forming a sintered monolith shown in Fig. 9.2. It was noticed that the reproducibility of preparation of sintered capillaries from ODS was higher than that from silica because of the adsorbed water, since silica has greater water-binding power than ODS. Water adsorbed to ODS was nearly eliminated during thermal treatment at 120~ while water adsorbed to silica was just partially evaporated at that temperature. The residual water was released as a sudden burst of water vapor during the sintering step at 360~ causing bed rupture and concomitant gapping. Due to the harsh experimental conditions (high temperature and basic NaHCO3) in the sintering process, the stationary phase was partially destroyed and, thus, post deactivation and functionalization of the bed was necessary. To avoid post-functionalization of the sintered monolith, Adam et al. [7] used water instead of NaHCO3 and a movable heating coil to fabricate sintered monoliths. A 10% suspension of ODS in acetone was packed into fused silica tubing using a slurry packing method under a pressure of 60 MPa. The packed capillary was then filled with water. A heating coil driven by a processing motor moved along the length of the packed capillary with a constant velocity and at a constant temperature in the range of 300-400~
As the heating coil moved along the packed capillary, the high
temperature in the heated zone led to dissolution of silica to form polysilicic acids at the surface of the particles. After cooling, the solution in the interstitial voids became supersaturated with silica sol and re-deposition of silica occurred between particles and between particle and capillary wall, forming a monolith with particles retained in their original positions. The mild sintering conditions compared to those employed by Asiaie et al. maintained the integrity of the stationary phase and made post-function-
Monolithic Columns Prepared from Particles
201
B
Fig. 9.2. Scanning electron micrographs of particle-sintered monolithic columns sintered at (a) 260~ (b) 360~ without, and (c) 360~ with NaHCO3 flux (reproduced with permission from ref. [6]. Copyright 1998 Elsevier Science B.V.).
alization of the sintered monolith unnecessary. The quality of the sintered monolith was controlled by three operational parameters for a given particulate material: (i) the temperature of the heating coil, (ii) the speed by which the coil is moved along the packed column, and (iii) the number of cycles of this operation. Both too low temperature and number of cycles resulted in a monolith of low mechanical stability. Too high temperature or more cycles led to degradation of the surface of the particles and destruction of the bonded stationary phase. The best column efficiency for a column containing Hypersil-ODS1 was obtained after two cycles at a temperature of 300400~
References pp. 210-211
202
Chapter 9
Particle-sintered capillary monoliths are mechanically strong and stable due to interparticle growth. They are also flexible because the polyimide coating on the fused silica capillary is not destroyed. The pore structure of the ODS in the sintered capillary remains essentially unchanged since irreversible destruction of the silica pore structures was observed only at temperatures above 600~
An efficiency of 125,000
plates/m in CEC was obtained using a sintered 6 ~m ODS column [6]. Under optimized operating conditions, no remarkable difference in retention factor, resolution, and efficiency were observed before and after sintering. 9.4 PARTICLE-LOADED MONOLITHIC COLUMNS Dulay et al. [8] reported the preparation of particle-loaded continuous-bed columns, in which ODS was loaded in a sol-gel matrix that was subsequently covalently bonded to the capillary inner wall. In this procedure, the sol-gel solution was prepared by mixing 0.2 mL of tetraethoxylsilane (TEOS), 0.73 mL of ethanol, and 0.1 mL of 0.12 mol/L HC1. ODS particles were added at a concentration of 300 mg/mL to the sol-gel solution to create a suspension that was sonicated for several minutes and then introduced into 75 lam i.d. fused silica tubing of about 40 cm long by applying vacuum. The column was then coiled into a loop, placed on a hot plate, and heated above 100~ for about 24 h. During this period, TEOS hydrolyzed into tetrahydroxylsilane which then polycondensed into a silica sol-gel network filled with ethanol. Upon the evaporation of ethanol, the sol-gel network and the ODS particles were converted into a particle-loaded monolith shown in Fig. 9.3. As can be seen, the ODS particles were bonded evenly across the column and not merely cast along the capillary wall. Their loading into the sol-gel matrix was achieved either by physical trapping or by chemical bonding between the sol-gel matrix and the surface of the ODS through silanol groups. The sol-gel network was integrally fixed to the walls of the capillary through covalent bonding of silanol groups between the sol-gel matrix and the capillary inner wall. In contrast to a pure sol-gel monolith, the particle-loaded monolith did not display obvious shrinkage because the ODS particles helped to decrease the stress within the matrix. However, cracks around the ODS particles were noticed, possibly because of phase separation between the hydrophobic ODS surface and the hydrophilic sol-gel matrix. Parameters controlling the success of the preparation were sol-gel matrix composition, ratio of particles to sol-gel, and suspension cast method. The sol-gel matrix solution was adjusted to a pH around 2, which enabled fast hydrolysis of TEOS, but slow polycondensation of silanol groups and, thus, slow solidification of the sol-gel matrix. The volumetric ratio of TEOS to solvent (ethanol) determined the porosity of the sol-gel matrix. The loading of ODS into the sol-gel matrix enhanced the mechani-
Monolithic Columns Prepared from Particles
203
Fig. 9.3. Scanning electron micrographs of a particle-loaded monolithic column (reproduced with permission from ref. [8]. Copyright 1998 American Chemical Society).
cal strength of the monolith and provided the chromatographic selectivity. A good suspension of ODS particles in the sol-gel matrix solution is essential for obtaining a monolithic column with homogeneous distribution of ODS particles within the monolith. The optimum concentration was found to be 300 mg ODS per mL of the sol-gel solution. At lower concentration, such as less than 200 mg/mL the selectivity of the resulting column was poor. At higher concentration, e.g., 350 mg/mL, significant heterogeneity in the ODS packing density along the column led to numerous sections of the capillary column void of ODS particles. The suspension can be pushed or drawn into the tubing. The use of vacuum to fill the small bore capillary with the ODS/TEOS slurry suspension greatly improved the reproducibility of column preparation over pressurized syringe-filling methods. Particle-loaded monolithic columns can sustain moderate pressures of up to 1.4 MPa. A 3 lam ODS-loaded monolithic column generated a specific permeability of 7.0 x 10-2 ktm2 [9,10] which is approximately 8 times greater than that of a tightly packed 3 ~tm ODS column. This high specific permeability is due to the low pressure method of preparation of the column in which 3 lam ODS particles are not as tightly packed into the tubing as they would be in a conventional packed LC column. Furthermore, large cracks around the ODS particles as shown in Fig. 9.3 and the highly porous sol-gel matrix allowed liquid to flow through the column at low pressure. An efficiency of 80,000 plates/m was obtained in CEC with a 3 ~tm ODS-loaded monolith. This moderate efficiency may be attributed to three factors. First, there may be inho-
Referencespp. 210-211
204
Chapter 9
mogeneous loading of the ODS column due to the different polarities and densities of the sol-gel solution and ODS particles. Second, some of the ODS particles can be too deeply embedded in the sol-gel matrix to play a role in the separation mechanism. Finally, cracks are often formed around the ODS particles. 9.5 PARTICLE-ENTRAPPED MONOLITHIC COLUMNS Chirica and Remcho [ 11-13] published a method for preparing monolithic columns in which particles are entrapped in a monolithic structure by an entrapping matrix. Their procedure involved four steps: (i) the fused silica capillary was packed with ODS particles using a conventional solvent slurry packing method, and then dried by purging the column with nitrogen; (ii) the dried packed capillary was filled with entrapping matrix solution; (iii) the column was cured using different methods depending on the entrapping matrix; and (iv) the cured column was dried slowly at a specified temperature. Upon drying, a monolithic column with entrapped particles shown in Fig. 9.4 resulted [13]. The properties of particle-entrapped monoliths were determined by the homogeneity of the packed bed and the characteristics of the entrapment matrix, including porosity and surface chemistry. Because of the use of high pressure during column packing, the packed bed was tight and relatively uniform throughout the entire column. This ensured homogeneity throughout the column. Different entrapping matrices, such as silicate (Kasil 2130) [11], tert-butyl-triethoxylsilane [12], n-octyltriethoxysilane [12], and methacrylate-based organic polymers [13] were investigated. Using various entrapping matrices, different packing materials can be entrapped in the monolith. All of the resultant particle-entrapped columns gave similar efficiencies which were nearly equal to those obtained for packed columns using the same particles. While the silicate matrix generated a monolith with serious peak tailing, other entrapping matrices gave satisfactory peak shapes and fine-tuned selectivity. Depending on the amount of entrapping matrix, the resultant particle-entrapped monoliths can have nearly the same or higher permeabilities than those observed for packed column beds containing the same packing materials. Retention factors for analytes on particleentrapped columns using silicate entrapping matrices are significantly reduced compared to those for conventional packed columns due to a combination of masking of the stationary phase with the entrapping silicate and hydrolysis of the stationary phase during NH4OH rinse. In contrast, other entrapping matrices displayed no significant loss Of retention. The greatest disadvantage associated with particle-entrapped monoliths is that the preparation procedures are time-consuming because drying of the entrapped column must be performed slowly. Otherwise, surface tension forces will cause column bed
Monolithic Columns Prepared from Particles
205
S'
:-
:~; Nx-
Fig. 9.4. Scaning electron micrographs of a particle-entrapped monolithic column (reproduced with permission from ref. [13]. Copyright 2000 American Chemical Society).
cracking and failure. Also the use of a retentive entrapping matrix adds to the retention of solutes, which makes it difficult to predict the overall retention behavior of analytes on the column. 9.6 SOL-GEL-BONDED MONOLITHIC COLUMNS We developed a sol-gel-bonded monolith that employed a specifically designed sol-gel matrix and the use of supercritical fluid column packing and drying [14]. The preparation procedure involved four steps: (i) packing the column with a supercritical CO2-slurry, (ii) filling the packed column with a sol solution, (iii) gelling and aging of the sol-filled packed column at room temperature, and (iv) drying the gelled, packedcolumn with supercritical carbon dioxide. A CO2 slurry packing method developed in our laboratory [ 15] was used to pack the columns. In short, one end of a fused silica Referencespp. 210-211
Chapter 9
206
capillary was connected to a stainless steel vessel into which an approximate amount of packing material was placed. The other end of the capillary column was connected to a length of 25 ~tm i.d. restrictor tubing using a zero dead-volume union in which a stainless steel frit with 0.5 lam pores was placed. The stainless steel frit was used to retain the particles in the capillary, and the restrictor tubing was used to control the packing speed. The stainless steel vessel was connected to a syringe pump, and packing of the capillary column was carried out in an ultrasonic bath at room temperature by increasing the pressure from 5-35 MPa at 1 MPa/min. Finally the column was conditioned at 35 MPa for 30 min, and then depressurized overnight. The sol solution was prepared by mixing appropriate amounts of tetramethoxysilane (TMOS), ethyltrimethoxysilane (ETMOS), methanol, trifluoroacetic acid (TFA) and water in a 0.6 mL polypropylene vial, followed by the addition of formamide. For example, to obtain a 9% (precursor/solution, v/v) sol solution, the following amounts were used: 20 laL TMOS, 20 ~tL ETMOS, 200 laL methanol, 15 ~tL TFA aqueous solution at pH 2, and 200 laL formamide. The apparent pH of the solution was approximately 5, measured using short-range pH test paper. The solution was vortexed for 5 min at room temperature using a vortex mixer and introduced into a packed capillary column to a desired length (observed with the naked eye or under a microscope with 10x magnification) using a 100-~tL PEEK syringe mounted on a small syringe pump. The syringe was strong enough for filling 60 cm of capillary column packed with 5 lam ODS particles, or 45 cm of capillary column packed with 3 lam ODS particles, with the non-viscous sol solution at a speed of 1 laL/min. Both ends of the sol-filled packed capillary were sealed using a silicone septum and the column was stored at room temperature for 24 h to achieve the conversion of the sol to a gel, and for aging of the resulting wet gel. After aging, the capillary was connected to an SFC pump using a zero dead-volume union. The column was flushed with supercritical CO2 at 8 MPa inlet pressure and 40~ to replace the solvent in the column. Then, while supercritical CO2 at 8 MPa was purged through the column, the column was dried by temperature programming from 40~ and holding at 240~
to 240~
at I~
min -1,
for 1 h. Finally, the packing material at the end of the column
that was not bonded by the sol-gel was flushed out, leaving a section of unpacked capillary. A short length of the monolithic column was cut off and a gold coating of approximately 40 nm thickness was sputtered on it for observation using a scanning electron microscope. Typical scanning electron micrographs of the cross-section of a sol-gel bonded ODS monolithic column are shown in Fig. 9.5. As can be seen, the column was packed homogeneously without visible wall perturbations and the ODS particles were bonded to each other and to the capillary wall by the gel network, forming a sol-gel-bonded monolith.
Monolithic Columns Prepared from Particles
207
Fig. 9.5. Scanning electron micrographs showing cross-sectional views of the end of a sol-gel-bonded large-pore ODS monolithic column (reproduced with permission from ref. [20]. Copyright 1999 John Wiley & Sons).
The key for preparing a successful sol-gel-bonded monolith is the design of the sol-gel matrix and the drying process. Fig. 9.6 illustrates schematically the synthesis of the solgel bonded particles. At pH 2, both TMOS and ETMOS precursors quickly underwent hydrolysis in the solvent, forming tetrahydroxysilane and ethyltrihydroxysilane. After the addition of formamide, the solution pH was changed to approximately pH 5, where tetrahydroxysilane polycondensed much faster than ethyltrihydroxysilane. As a result, the sol solution was converted into a silica network endcapped with ethyl groups. The particles were bonded to each other and to the capillary inner wall through this inert sol-gel matrix, forming a wall-supported continuous-bed. In a classical sol-gel process for silica glasses, a single precursor, TMOS, is typically used [16]. However, a silica gel network fabricated from TMOS alone often shrinks and cracks during drying, and the silica gel network is active because of the exposed silanol groups on the gel surface. It was reported that a combination of alkyl derivatives of TMOS as co-precursors with TMOS resulted in a flexible gel network with a
Referencespp. 210-211
Chapter 9
208
S,(OCIt:,,)~ + tI,O
f
Me()H ,~ Si(OH),; ~ TFA, plt=2 pH=5
1
-{~i-O-~i)C:H~ (':H.~
I. C:H~Si(OCH~)~+ H~O
MeOH ,C:H~Si(OH)~ ~ ( S ']FA, pH=2 .....
t
I, -O-Sli)-
C2H~ C:H.~
i
I
-(Si-O-Si)I
o
\...._..../
~
!
o
o
-(Si O Si)I
!
C~t--1, C~H, Fig. 9.6. Synthetic scheme for sol-gel bonded particles (reproduced with permission from ref. [ 18]. Copyright 2000 Wiley-VCH Verlag GmbH).
more open pore structure [17], which could alleviate capillary stress during drying. The alkyl groups can also deactivate the gel network, thereby contributing little to the peak tailing of analytes. Even when the composition of the sol solution was well designed and controlled, extremely slow drying was required to minimize cracking of the column bed when conventional thermal drying was applied to the sol-gel matrix. This made column preparation rather time-consuming. Cracking of the column bed is believed to result from capillary forces generated during evaporation of the solvent. To avoid this, a supercritical fluid was used to dry the gel network since the surface tension of a supercritical fluid is zero [ 16]. In the fabrication of sol-gel-bonded monoliths, supercritical CO2 was pumped through the column to replace the solvent and residual reactants using a supercritical fluid chromatographic pump, and the column was then dried in supercritical CO2 under elevated temperature. It was observed that column drying with supercritical CO2 was fast and the resultant columns were crackfree. Sol-gel-bonded monolithic columns from small-pore ODS [14,18,19], large-pore ODS [18,20,21], mixed-mode ODS/SCX [18,22], and C30-silica [23,24] have been successfully prepared. Column-to-column reproducibility was found to be less than 8% for three monolithic columns containing 9% sol-gel bonded 5-~tm ODS [14]. An efficiency of 410,000 plates/m in CEC was obtained using a sol-gel-bonded 3 ~tm, 1500 A ODS column, which is nearly double the efficiency from a sol-gel-bonded
Monolithic Columns Prepared from Particles
209
3 lam, 80 A ODS column [21]. This was attributed to EOF generation in the largepores of the 3 ktm, 1500 A ODS.
9.7 PARTICLE-CROSSLINKED M O N O L I T H I C COLUMNS We have also described a particle-crosslinked monolithic column in which polymer-encapsulated packing materials packed in polymer-coated fused silica tubing was crosslinked in situ using a free radical reaction [25]. A fused silica open tubular column with 0.25 ~tm film thickness of octyl-substituted polysiloxane was packed with polybutadiene (PBD)-encapsulated zirconia packing material using a supercritical CO2 slurry packing method and placed in a GC oven and flushed with a free radical initiator, azo-tert-butane, in helium. The helium flow rate through the packed column was 0.5 cm/s. The oven temperature was elevated to 260~ 0.5~
at a rate of
and held at 260~ for 5 h. Porous packing materials could be immobilized
onto the PSB-Octyl capillary wall through free radical reactions [26] since about 50% of the vinyl groups on the PBD-encapsulated zirconia particles remained unreacted [27]. Under elevated temperature, azo-tert-butane decomposed to form free radicals and the PBD-encapsulated zirconia packing was crosslinked together and to the capillary wall through free radical initiated crosslinking reactions, forming a crosslinkedmonolithic column shown in Fig. 9.7. The experimental conditions, including the film thickness of the octylpolysiloxane coating in the open tubular column, the flow rate of the azo-tert-butane saturated helium through the column bed, and the time and temperature for the crosslinking reaction, determined the success of column preparation. When the octylpolysiloxane film thickness in the original capillary was 0.25 ktm, the crosslinked packing material stayed in the column even when 40 MPa carbon dioxide at room temperature was applied at the column inlet. For achieving a mechanically strong monolithic column, the column must be purged for half an hour with azo-tert-butane vapors in helium at a flow rate of 2 mm/s before increasing the temperature. Using a crosslinking temperature of 260~
for 5 h, stable crosslinked-monolithic 3% PBD-encapsulated zirconia
columns of 25-60 cm x 250 ~tm i.d. were obtained. The column efficiency, solute retention, and permeability of these monolithic columns were similar to those measured for columns freshly packed, but not crosslinked, with the same material under the same conditions.
References pp. 210-211
210
Chapter 9
Fig. 9.7. Scanning electron micrograph of the end of a crosslinked monolithic column (reproduced with permission from ref. [25]. Copyright 1999 John Wiley & Sons).
9.8 CONCLUSIONS
Monolithic columns prepared from particles are designed to inherit the efficiency and selectivity of versatile LC packing materials. Monolithic columns from particles are expected to be stable without the need of end-frits. Various methods used for the preparation of monolithic columns from particles have been described, each with its own merits and shortcomings. Sol-gel-bonded monolithic columns employing an inert sol-gel matrix and supercritical CO2 drying is currently the most promising method for obtaining high performance columns. 9.9 R E F E R E N C E S
1 2 3 4 5 6 7 8 9 10 11 12
V. Pretorius, B.J. Hopkins, J.D. Schieke, J. Chromatogr. 99 (1974) 23 J.W. Jorgenson, K.D. Lukacs, J. Chromatogr. 218 (1981)209 J.H. Knox, I.H. Grant, Chromatographia 24 (1987) 135 T. Tsuda, I. Tanaka, G. Nakagawa, Anal. Chem. 56 (1984) 1249. J.H. Knox, I.H. Grant, Chromatographia 32 (1991) 317. R. Asiaie, X. Huang, D. Farnan, C. Horvath, J. Chromatogr. A 806 (1998) 251. T. Adams, K.K. Unger, M.M. Dittmann, G.P. Rozing, J. Chromatogr. A 887 (2000) 327. M.T. Dulay, R.P. Kulkarni, R.N. Zare, Anal. Chem. 70 (1998) 5103. C.K. Ratnayake, C.S. Oh, M.P. Henry, J. High Resol. Chromatogr. 23 (2000) 81. C.K. Ratnayake, C.S. Oh, M.P. Henry, J. Chromatogr. A 887 (2000) 277. G. Chirica, V.T. Remcho, Electrophoresis 20 (1999) 50. G.S. Chirica, V.T. Remcho, Electrophoresis 21 (2000) 3093.
Monolithic Columns Prepared from Particles 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
211
G.S. Chirica, V.T. Remcho, Anal. Chem. 72 (2000) 3605. Q. Tang, B. Xin, M.L. Lee, J. Chromatogr. A 837 (1999) 35. A. Malik, W. Li, M.L. Lee, J. Microcol. Sep. 5 (1993) 361. C.J. Brinker, G.W. Scherer, Sol-gel Science: The Physics and Chemistry of Sol-gel Processing, Academic Press, San Diego, CA, USA, 1990. J.D. Mackenzie, Hybrid Organic-Inorganic Composites, ACS Symposium Series 585, American Chemical Society, Washington, D.C., USA, 1995, p 227-236. Q. Tang, M.L. Lee, J. High Resol. Chromatogr. 23 (2000) 73. Q. Tang, N. Wu, M.L. Lee, J. Microcol. Sep. 12 (2000) 6. Q. Tang, N. Wu, M.L. Lee, J. Microcol. Sep. 11 (1999) 550. Q. Tang, N. Wu., B. Yue, M.L. Lee, Anal. Chem., in press. Q. Tang, M.L. Lee, J. Chromatogr. A 887 (2000) 265. L. Roed, E. Lundanes, T. Greibrokk, J. Microcol. Sep. 12 (2000) 561. L. Roed, E. Lundanes, T. Greibrokk, J. Chromatogr. A 890 (2000) 347. Q. Tang, Y. Shen, N. Wu, M.L. Lee, J. Microcol. Sep. 11 (1999) 415. H. Yun, K.E. Markides, M.L. Lee, J. Microcol. Sep. 7 (1995) 153. J. Li, P.W. Carr, Anal. Chem., 68 (1996) 2875.