Porous particles for liquid chromatography and processes for the preparation thereof

Abstract

Superficially porous silica particles are provided as well as a one-pot process for making the superficially porous particles, the process comprising hydrolyzing and condensing a silica precursor comprising a functional group to form superficially porous particles, the superficially porous particles comprising silica microparticles having silica nanoparticles bound to the surface of the microparticles. The nanoparticles provide a porous outer layer on the microparticles. The superficially porous particles are useful as a stationary phase in liquid chromatography and allow for fast mass transfer and separation of samples.

Claims

1. A process for making superficially porous silica particles, the process comprising, in a basic medium that comprises water and an organic solvent, in the presence of a hydrophilic polymer acting as a colloid stabilizer and in the presence of a surfactant, hydrolyzing and condensing a silica precursor comprising a functional group selected from mercapto, amino, hydroxyl and epoxy, to form superficially porous silica particles, the superficially porous silica particles comprising substantially non-porous silica microparticles having silica nanoparticles bound to the surface of the microparticles, wherein hydrolyzing and condensing the silica precursor is performed as a one-pot synthesis and comprises forming the microparticles first, followed by controlled growth of the surface-bound nanoparticles on the surface of the microparticles.

2. The process according to claim 1 wherein the functional group is a mercapto group.

3. The process according to claim 2 wherein the precursor comprises a mercapto-silane.

4. The process according to claim 3 wherein the mercapto-silane comprises 3-mercapto-propyl-trimethoxy-silane or 3-mercapto-propyl-triethoxy-silane.

5. The process according to according to claim 1 wherein the silica precursor is used as a sole silica source.

6. The process according to according to claim 1 wherein the basic medium has a starting pH in the range 9 to 11.

7. The process according to claim 1 wherein the organic solvent comprises methanol.

8. The process according to claim 1 wherein the surfactant comprises a quaternary ammonium surfactant.

9. The process according to claim 8 wherein the quaternary ammonium surfactant comprises cetyltrimethylammonium bromide (CTAB) or cetyltrimethylammonium chloride (CTAC).

10. The process according to claim 1 wherein the hydrophilic polymer comprises poly(vinyl alcohol) (PVA) or poly(vinyl pyrrolidone) (PVP).

11. The process according to claim 1 further comprising calcining the superficially porous silica particles.

12. The process according to claim 1 wherein the microparticles have an average particle size as measured by laser diffraction in the range 1 m to 20 m.

13. The process according to claim 1 wherein the nanoparticles have an average particle size measured by dynamic laser scattering (DLS) of not greater than 500 nm.

14. The process according to claim 1 wherein a pore size of the superficially porous particles calculated by non-local density functional theory (DFT) is less than 2 nm.

15. The process according to claim 1 wherein the nanoparticles bound to the surface of the microparticles form a monolayer.

16. The process according to claim 1 wherein the microparticles have an average particle size as measured by laser diffraction in the range 1 m to 10 m.

17. The process according to claim 1 wherein the hydrophilic polymer and surfactant are included in and mixed with the basic medium, followed by adding the silica precursor to the basic medium and stirring the reaction ingredients during the hydrolysis and condensation.

18. The process according to claim 17 wherein a duration of the hydrolyzing and condensing is least 30 minutes as measured from a start of the adding the silica precursor to the basic medium.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows an SEM image of spheres-on-sphere silica particles synthesized according to the present invention.

(2) FIG. 1B shows the N.sub.2 isotherm for the silica particles shown in FIG. 1A.

(3) FIG. 1C shows the pore size distribution for the silica particles shown in FIG. 1A.

(4) FIG. 2 shows SEM images of the growth of spheres-on-sphere silica particles over time (in minutes).

(5) FIG. 3 shows schematically an apparent synthesis mechanism according to the present invention.

(6) FIG. 4 shows SEM images of spheres-on-sphere silica particles synthesized according to the present invention using different NH.sub.3 concentrations.

(7) FIG. 5 shows SEM images of spheres-on-sphere silica particles according to the present invention after ultra-sonication.

(8) FIG. 6 shows a chromatogram obtained from a column packed with spheres-on-sphere particles according to the present invention using a normal phase test mixture containing 1-100 g/ml of toluene, o-nitroaniline, p-nitroaniline and m-nitroaniline in heptane:dioxane (0.7% vv) at 0.3 mL/min flow rate.

(9) FIG. 7 shows chromatograms obtained in a similar manner to that of FIG. 6 but with higher flow rates.

(10) FIG. 8 shows a chromatogram obtained from a column packed with functionalized spheres-on-sphere particles according to the present invention using a reverse phase test mixture, TM-2, containing 1-100 g/ml of benzamide, acetophenone, benzophenone and biphenyl in water:acetonitrile (50% vv).

(11) FIG. 9 shows a chromatogram obtained from a column packed with functionalized spheres-on-sphere particles according to the present invention using a reverse phase protein mixture, containing 2-10 mg/ml of Ribonuclease A, Cytochrome C, lysozyme, Trypsin and BSA in 0.1% vv TFA-water.

(12) FIG. 10A shows an additional mobile phase gradient profile (Gradient Method 1) and FIG. 10B shows another additional gradient profile (Gradient Method 2) that were each used to obtained chromatograms with the reverse phase protein mixture.

(13) FIG. 11 shows a chromatogram of the reverse phase protein mixture obtained using the Gradient Method 1.

(14) FIG. 12 shows chromatograms of the reverse phase protein mixture obtained using different flow rates and using Gradient Method 1.

(15) FIG. 13 shows chromatograms of the reverse phase protein mixture obtained using different flow rates and using Gradient Method 2.

(16) FIG. 14 shows a further optimised gradient profile (Gradient Method 3) used to obtained chromatograms with the reverse phase protein mixture.

(17) FIG. 15 shows a comparison of chromatograms obtained using Gradient Method 3 (top trace) and gradient Method 2 (bottom trace).

(18) FIG. 16 shows an SEM image of unpacked spheres-on-sphere silica material according to the present invention after performing chromatographic separations.

DETAILED DESCRIPTION OF THE INVENTION

(19) In order to further understand the invention, but without limiting the scope thereof, various examples are now described with reference to the accompanying drawings. Whilst the following are only examples of the invention, it will be apparent that many features evident from the examples could be generalized, such as the preferred order of addition of reagents for instance.

(20) Material Characterization

(21) SEM

(22) Morphologies of the silica microparticles were observed by a Hitachi-S4800 scanning electron microscope (SEM). One drop of the suspension in ethanol was deposited on a SEM stud and allowed to dry overnight. The samples were then coated with gold using a sputter-coater (EMITECH K550X) for 3 min at 30 mA for SEM imaging. To observe the particles using a STEM detector, one drop of the suspensions was deposited on a TEM grid and then observed directly after drying overnight.

(23) Particle Size

(24) The silica microparticles were measured using a Malvern (Malvern, Worcestershire, UK) Mastersizer 2000 equipped with dispersion unit. The analysis was performed using HeNe laser in conjunction with blue light source to provide superior sensitivity across a wide size range. The angular intensity of scattered light is detected by forward and backscatter detection for enhanced sizing performance. The scattering of the particles is accurately predicted by the Mie scattering model. The measurements were carried out six times to obtain the average particle size distribution at 2000 rpm dispersion. The silica nanoparticles as suspensions in water were characterized by dynamic laser scattering (DLS) using Viscotek Model 802 DLS system to obtain hydrated particle sizes. The measurements were carried out in a quartz cell at room temperature and the average particle size distribution was obtained after six measurements.

(25) pH

(26) The pH of the solution mixture was continuously measured for a period of at least 2 hours at regular intervals.

(27) Surface Area and Pore Volume

(28) The Brunauer-Emmett-Teller (BET) surface area and pore volume by N.sub.2 sorption at 77K were measured using a Micromeritics ASAP 2020 adsorption analyzer. Pore size distributions were calculated from Barrett-Joyner-Halenda (BJH) desorption data and non-local density functional theory (DFT) data. Samples were degassed for 10 h at 120 C. before analysis.

(29) HPLC

(30) For HPLC characterization of the column, an Agilent 1200 series high performance liquid chromatography system was used, comprising a vacuum degasser, quaternary pump, ALS auto-sampler, heated column compartment and UV-Vis detector. Data analysis was performed using Agilent Chemstation software, version B.02.01 (Agilent Technologies, USA).

Example 1Preparation of Spheres-on-Sphere Silica

(31) The following chemicals and reagents were used: (3-Mercaptopropyl)trimethoxysilane (MPTMS) (95%), cetyltrimethylammonium bromide (CTAB) (98%), ammonium hydroxide solution (reagent grade, 28-30% NH.sub.3 basis), hydrophilic polymers: poly(vinyl alcohol) (PVA) (Mw 10K), poly(vinyl pyrrolidone) (PVP) (Mw 8K), hydroxypropyl methylcellulose (HPMC) (Mw 10K), and methanol (analytical grade). Trifluoroacetic acid (TFA) and Chloro(dimethyl)octylsilane (C8) were purchased from Sigma-Aldrich and used as received. Distilled water was used throughout the experiment.

(32) The hydrophilic polymer (0.25 g) and CTAB (0.1 g) were dissolved in 5 g water. To this solution, 8 mL methanol was added while stirring. The purchased ammonium hydroxide solution (0.5 g) was diluted by mixing with 2 mL water and then 2 mL of the diluted ammonia solution was added into the reaction mixture. After stirring for 15 min, 0.5 mL MPTMS was added drop-wise over a 30 seconds period. The concentrations of polymer and surfactant in these examples were referred to the volume of water. The reaction was stirred for 24 hours at room temperature. The resulted silica particles were collected by centrifuging the suspensions. These collected silica particles were calcined in a furnace (Carbolite, CWF1500) to remove the organic template and other organic components such as the functional groups. The calcining condition: heat at 1 C./min in air to (550-1250) C., hold for 300 min, and then cool down to room temperature at 5 C./min.

(33) When the MPTMS was added, spheres-on-sphere particles were formed in a one-pot synthesis. Using PVA as the hydrophilic polymer, the particle size distribution of the spheres-on-sphere particles was in the range of 2.5-12.9 m and peaked at 5.67 m. The values of d.sub.0.1 and d.sub.0.9 were 3.21 m and 9.89 m respectively. The resultant SEM image of the spheres-on-sphere particles is shown in FIG. 1A. Large silica microspheres were seen to be coated with silica nanoparticles ranging 200-400 nm in size, which were orderly arranged on the surface of the microspheres. The BET surface area after calcination was 204 m.sup.2/g. The N.sub.2 isotherm showed a type I isotherm (shown in FIG. 1B) which is normally obtained for microporous silica. Due to the BJH model limitations it is unable to calculate pores less than 2 nm. Thus, for microporous materials, non-local density functional theory (DFT) method should be used. The pore size distribution calculated from the DFT data showed micropores peaks at 0.91 and 1.51 nm (FIG. 10).

Example 2Growth Study

(34) A time study was conducted in order to see how the spheres-on-sphere particles grow during the one-pot synthesis. This was done by examining samples at regular time intervals. To obtain each sample, it was centrifuged at 13000 rpm to ensure complete extraction of the silica particles from the suspension, which were then thoroughly washed with water to remove any unreacted silica precursor. The obtained samples were analysed by SEM and the images are shown in FIG. 2 (the number on each image indicates the time in minutes after the start of the reaction when the sample was extracted). It appears that there are two stages of nucleation occurring. The first stage comprises the growth of silica microparticles and lasts for a period of about 20 minutes. Then, a second stage of nucleation occurs on the surface of the formed microparticles. At 180 minutes, it appeared that the spheres-on-sphere particles have fully formed. The reaction was monitored for 24 hours but no further significant growth was seen during this time. Without the scope of the invention being bound by this theory, an illustration of the apparent synthesis is shown in FIG. 3.

Example 3pH Study

(35) The pH of the solution was about 9.9 before the start of the hydrolysis and was just below 10.5 by the time the process was complete (150 mins). In comparison with TEOS in conventional processes, the present pH pattern with time was rather different since usually a pH decrease is observed due to the formation of silicic acid. Without being bound by any theory, it appears that this behaviour may be caused by the thiol group as it deprotonates under basic conditions.

(36) The reaction conditions described above involved the use of 2 ml of the 25% wt diluted NH.sub.3 solution during the synthesis and this resulted in the formation of orderly densely arranged nanoparticles of 200-400 nm size on the surface of the microparticles. The pH effect was further investigated using more diluted NH.sub.3 solutions of 5% wt and 1% wt. In these cases, the SEM images (see FIG. 4 images C and D) showed that the nanoparticles increased in size with denser packing with reduced NH.sub.3 concentration. For comparison, the SEM of the above example using the 25% wt NH.sub.3 solution is shown in image B of FIG. 4. It appears that the silica nanoparticles are strongly fused or aggregated on the surface with the addition of 1% wt NH.sub.3. The precipitation of particles proceeds more slowly in the more dilute cases, which leads to larger particles forming. This has an effect on particle size distribution and larger particles are formed. After calcination, the samples made using the more dilute NH.sub.3 solutions also had lower surface areas (30 m.sup.2/g and 19 m.sup.2/g). The use of undiluted (i.e. 100% wt) NH.sub.3 solution yielded reduced coverage of nanoparticles as shown in image A of FIG. 4.

Example 4Particle Stability

(37) The spheres-on-sphere particles were ultra-sonicated to observe the stability of the nanoparticles on the surface. The particles were ultra-sonicated at 140 W for 8 hours in water and it was observed that some nanoparticles had come off the surface of the microspheres, but not all of them, as revealed by SEM (see FIG. 5). This indicates that the nanoparticles are strongly attached to the surface as the surface remains relatively densely covered even after such a strong sonication.

Example 5Double-Coating Layer

(38) The particles obtained using the method described above resulted in a single-coating layer of nanoparticles on the surface. However, by adding further MPTMS during the nanoparticle growth stage (i.e. after 20 minutes), at 30 minutes, without removing the pre-formed microspheres, more nanoparticles were seen to grow on the surface. With increasing MPTMS amount in solution, the spacing between the nanoparticles decreased and much denser coating was formed. This process enabled the formation of a double-coating layer when a further 0.5 ml of MPTMS was added at 30 minutes, although the double-coating layer was observed to come off with washing so was not apparently as stable as the single layer, but stability may be improved with calcination.

Example 6Controlling the Particle Formation

(39) The effect of the surfactant on the spheres-on-sphere morphology was significant. By varying the surfactant concentration the size of the nanoparticles on the surface were reduced. In contrast to the CTAB amount in the Example 1 above (2% wv based on the 5 mL added water volume), a high concentration of CTAB (10% wv) contributed to the formation of smaller nanoparticles on the surface at around 128 nm and the microspheres size remained unchanged. The nature of the surfactant counter-ion was investigated by replacing the CTAB surfactant with the same surfactant cation but with chloride counter-ion (CTAC). The use of the different surfactant counter-ion during the reaction resulted in the same spheres-on-sphere structure.

(40) Controlling the uniformity of the stationary phase particles is very important for chromatographic applications. Increasing the PVA concentration by a factor of 2 improved the size distribution of the spheres-on-sphere particles to d.sub.0.1-d.sub.0.9=2.68-6.81 m. Another hydrophilic polymer, poly(vinyl pyrrolidone) (PVP) (Mw 8K), was introduced into the reaction in place of the PVA and this also yielded spheres-on-sphere structures, as did another hydrophilic polymer, hydroxypropyl methylcellulose (HPMC) (Mw 10K). The polymer concentration of 5% wv (based on the 5 mL of added water) was kept the same for the samples prepared using the other polymers. The particle size distribution using PVP was better than with the PVA polymer. With PVP a smaller and narrower particle size distribution around 2.73 m was achieved with d.sub.0.1-d.sub.0.9=1.54-4.76 m (compared to the cases above of PVA (5% wv) d.sub.0.1-d.sub.0.9=3.21-9.89 m, PVA (10% wv) d.sub.0.1-d.sub.0.9=2.68-6.81 m). The surface nanoparticles were also smaller with the PVP at 75 nm.

(41) HPLC

Example 7Column Packing and Characterization

(42) The calcined spheres-on-sphere particles were packed into a 2.150 mm stainless steel column using a synchronic column packing method. The slurry was prepared using 0.3 g of silica particles in 15 ml methanol. The slurry was poured into a 15 ml reservoir and packed at 60K bars.

(43) The columns prepared as above were used for normal phase (NP) testing conditions. The freshly made column was washed with isopropanol (1 hours), then with heptane (1 hour). The NP test mix contained 1-100 g/ml of toluene, o-nitroaniline, p-nitroaniline and m-nitroaniline in heptane:dioxane (0.7% vv).

(44) For reverse phase (RP) chromatography the column was functionalised by flushing with chloro(dimethyl)octylsilane in toluene. The column was heated at 100 C. for 24 hours. The exact procedure was repeated twice to ensure a good coverage. Finally, the column was washed with toluene (1 hour), acetonitrile (1 hour) and acetonitrile:water (1 hour). The RP test mix, TM-2, contained 1-100 g/ml of benzamide, acetophenone, benzophenone and biphenyl in water:acetonitrile (50% vv). The RP protein mix contained 2-10 mg/ml of Ribonuclease A, Cytochrome C, lysozyme, Trypsin and BSA in 0.1% vv TFA-water.

Example 8Normal-Phase (NP) HPLC

(45) The as-prepared spheres-on-sphere particles made using the 5% NH.sub.3 solution described above were calcined at 600 C. and the calcined particles were packed into the column by the Example 7 method without further treatment other than being washed with isopropanol (1 hours) and then with heptane (1 hour).

(46) The column was tested using the NP test mix at 0.3 mL flow rate and with UV detection at 254 nm. FIG. 6 shows the resultant chromatogram. The material exhibited good efficiency of separation of small molecules with plate numbers in the range of 15000-21000 plates/m (p/m) based on m-nitroaniline peak at the optimum flow rate. The efficiency could be further enhanced if the particles were classified to produce a much tighter particle size distribution. The spheres-on-sphere particles were advantageous for rapid diffusion of these analytes with good separation characteristics. The stationary phase produced well resolved peaks even at higher flow rates of 0.6 and 1.0 mL/min as shown in FIG. 7. The analytes were separated within less than two minutes, but there was a slight drop in efficiency and plate numbers of 9000 p/m were found based on m-nitroaniline peak at 1 mL/min.

Example 9Reverse-Phase (RP) HPLC

(47) Spheres-on-sphere particles are desirable for reverse-phase separation of bio-macromolecules due to excellent mass transfer characteristics. The column containing the 600 C. calcined material was functionalized with chloro(dimethyl)octylsilane as described above and tested under reverse-phase conditions.

(48) Gradient Elution

(49) The water:acetonitrile mobile phase strength was increased over time during the RP chromatographic separation to achieve faster separation than under isocratic conditions. The analysis of the TM-2 mixture at 1 mL/min and UV detection at 254 nm was performed using a linear solvent gradient with acetonitrile increasing over the range 5-70% vv. The solvent gradient returned to initial conditions after 5 minutes. The peaks were well resolved and the total analysis time was 4 minutes as shown in FIG. 8. The gradient method could be further developed in order to achieve much faster separation time.

(50) Protein Separation

(51) Spheres-on-sphere particles morphology is suitable for the separation of bio-macromolecules. The nanoparticles packed on the surface provide inter-particle pores that permit large molecules to enter the pore structure with faster diffusion distances. A gradient elution study with the protein mix of Ribonuclease A, Cytochrome C, lysozyme, Trypsin and BSA proteins was performed at 25 C. with UV detection at 220 nm. The mobile phase used comprised 0.1% vv TFA-water and 0.1% vv TFA-acetonitrile. Since acetonitrile absorbs at 220 nm a baseline drift was visible. A number of gradient methods were tested in order to achieve the best separation of these proteins. A simple linear gradient method was first set using 5-70% vv acetonitrile 0.1% vv TFA mobile phase at 1 mL/min with a backpressure of 139 bars and a 1 l injection. Re-equilibration time between each run is important to ensure repeatable separation, thus the column was allowed to equilibrate for 10 minutes before each run. The protein separation using this simple linear gradient is illustrated in FIG. 9. The proteins, which are large macromolecules, were well separated in less than 4 minutes, with excellent peak shapes and resolution. This is due to the excellent kinetic properties of the spheres-on-sphere particles. However, the lysozome and trypsin could not be resolved under these simple linear gradient conditions. Hence, further gradient methods were developed to improve the separation of all five proteins. FIGS. 10A and 10B respectively show further gradient profiles, Gradient Method 1 and Gradient Method 2, used with the same solvent system.

(52) The gradient profiles Gradient Method 1 and Gradient Method 2 showed a good separation of the protein mixture at 1 ml/min (see FIG. 11 for Gradient Method 1). All five proteins were fully resolved within 4 minutes (Gradient Method 1) and 3 minutes (Gradient Method 2). The resolution was maintained for each peak with excellent peak shapes. However, there was some loss of resolution between lysozome and trypsin caused by the faster elution of these proteins under Gradient Method 2. Higher flow rates were also tested under these gradient conditions. Because of the excellent kinetic properties of the spheres-on-sphere particles, even faster separations of such compounds are possible at higher mobile phase velocities (e.g. flow rates such as 1.5 and 1.75 ml/min). The results of the separations at the different flow rates are shown in FIG. 12 for Gradient Method 1 and in FIG. 13 for Gradient Method 2. As shown in FIGS. 12 and 13, the separation of proteins at 1.75 ml/min was completed in about 3.5 minutes (Gradient Method 1) and 2.5 minutes (Gradient Method 2) respectively. Again, resolution was maintained with good peak shapes. The back pressure of the column increased to 189 bars at 1.5 ml/min and 241 bars at 1.75 ml/min, well within the operating range of the HPLC system.

(53) FIG. 14 shows a further optimised gradient profile that was used (Gradient Method 3). From the chromatogram using this mobile phase gradient, the BSA peak was much sharper and was slightly shifted as shown in FIG. 15 (top trace) compared to Gradient Method 2 (bottom trace). Gradient Method 3 effectively improved the separation of lysozome and trypsin peaks. Hence, depending on the gradient applied during separation, the peaks can easily be accelerated through the column or delayed in the column. In any case, the spheres-on-sphere particles demonstrated the ability to rapidly separate higher-molecular mass compounds such as proteins.

(54) After completing the chromatographic testing, the column bed had not changed and no void was observed after the column was unpacked and the morphology was observed by SEM. The SEM image in FIG. 16 shows the unpacked spheres-on-sphere silica-C8 material with the spheres-on-sphere morphology retained. This suggests that the nanoparticles are strongly attached to the surface of the microparticles.

(55) As used herein, including in the claims, unless the context indicates otherwise, singular forms of the terms herein are to be construed as including the plural form and vice versa. For instance, unless the context indicates otherwise, a singular reference, such as a or an means one or more.

(56) Throughout the description and claims of this specification, the words comprise, including, having and contain and variations of the words, for example comprising and comprises etc, mean including but not limited to, and are not intended to (and do not) exclude other components.

(57) It will be appreciated that variations to the foregoing embodiments of the invention can be made while still falling within the scope of the invention. Each feature disclosed in this specification, unless stated otherwise, may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

(58) The use of any and all examples, or exemplary language (for instance, such as, for example, e.g. and like language) provided herein, is intended merely to better illustrate the invention and does not indicate a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(59) Any steps described in this specification may be performed in any order or simultaneously unless stated or the context requires otherwise.

(60) All of the features disclosed in this specification may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. Likewise, features described in non-essential combinations may be used separately (not in combination).