WO2013054129A1

WO2013054129A1 - Novel chromatography method using mesoporous silicon imide derivatives as the stationary phase

Info

Publication number: WO2013054129A1
Application number: PCT/GB2012/052534
Authority: WO
Inventors: Stephen Malcolm Kelly; Fei Cheng; Stuart Paul KITNEY; Vincent ROCHER
Original assignee: University Of Hull
Priority date: 2011-10-12
Filing date: 2012-10-12
Publication date: 2013-04-18
Also published as: GB201117630D0; GB201200652D0

Abstract

The invention provides a method for the chromatographic separation of chemical species, the method comprising the provision of a chromatograph comprising a mesoporous silicon imido nitride (Si₂N₂(NH)₂), silicon diimide (Si(NH)₂) mesoporous gel or silicon nitride (Si₃N₄) mesoporous ceramic stationary phase. Typically, the mesoporous gel is prepared by means of a sol-gel process which is optionally partially or completely pyrolysed to form a mesoporous silicon imido nitride or silicon nitride mesoporous ceramic. The chromatographic separation method generally comprises liquid phase column chromatography or gas chromatography and is particularly applied to the separation of acid-sensitive organic molecules which may include proteins or compounds used in the preparation of pharmaceuticals, and the removal of catalytic amounts of metal ions from reaction products. The invention also provides a chromatograph comprising the disclosed stationary phase and envisages the preparation of a chromatograph by incorporating this stationary phase in a chromatographic column.

Description

NOVEL CHROMATOGRAPHY METHOD USING MESOPOROUS SILICON IMIDE DERIVATIVES AS THE

STATIONARY PHASE

Field of the Invention

[0001] This invention relates to a novel chromatographic separation technique. More particularly, the invention is concerned with an enhanced technique for liquid phase column chromatography which employs a new material as the stationary phase.

Background to the Invention

[0002] Various chromatographic techniques are routinely used as standard procedures for the separation of chemical species and are well known in the art. One of the most important of these techniques is column chromatography, a typical example of which involves a stationary phase - generally a solid adsorbent - being placed in a vertical glass column and a mobile phase, usually a liquid, being added to the top of the column such that it flows down through the column, either by means of gravity or external pressure. Column chromatography is generally used as a purification technique wherein desired compounds may be isolated from a mixture.

[0003] In such a case, the mixture to be separated is applied to the top of a column and a liquid solvent (the eluant) is passed through the column. This technique is an example of liquid phase chromatography, wherein equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Since the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation of components will be achieved. The individual components, or elutants, are collected as the solvent exits from the bottom of the column.

[0004] A m ore soph i sti cated ch romatog ra ph ic tech n iq ue th a n si m ple col u m n chromatography is high-performance liquid chromatography (or high-pressure liquid chromatography - HPLC). Th is is a chromatographic technique that can separate a mixture of compounds and is commonly used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of a mixture.

[0005] HPLC typically utilises different types of stationary phases, a pump that moves the mobile phase(s) and analyte through the column , and a detector to provide a characteristic retention time for the analyte. Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. Thus, HPLC is a form of l i q u id chromatography that utilises a smaller column size, smaller media inside the column, and higher mobile phase pressures. It is found that pressures in HPLC columns can reach up to about 400 atmospheres, whilst Ultra High Pressure Chromatography - which is a variant of HPLC - can involve pressures as high as 1000 atmospheres.

[0006] A further chromatographic technique having widespread use is size exclusion chromatography (SEC), which is used for the purification and analysis of synthetic and biological polymers, such as proteins, polysaccharides and nucleic acids. Biologists and biochemists typically use a gel medium - usually polyacrylamide, dextran or agarose - and operate under low pressure, whilst polymer chemists usually employ either a silica or crosslinked polystyrene medium under a higher pressure.

[0007] The advantages of SEC include good separation of large molecules from small molecules with a minimal volume of eluate, and the ability to apply various solutions without interfering with the filtration process, both of which may be achieved whilst preserving the biological activity of the particles to be separated. The technique is generally combined with others that further separate molecules by means of other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With size exclusion chromatography, it is possible to achieve short and well-defined separation times and narrow bands, thereby leading to good sensitivity. In addition, there is no sample loss, because solutes do not interact with the stationary phase. However, disadvantages are associated with the technique; thus, for example, only a limited number of bands can be accommodated in view of the short time scale of the chromatogram and, in general, there has to be a 10% difference in molecular mass in order to achieve good resolution.

[0008] A requirement for SEC is that the analyte should not interact with the surface of the stationary phases. Differences in elution time are based solely on the volume into which the analyte percolates. Thus, a small molecule that can penetrate every corner of the pore system of the stationary phase effectively percolates into the entire pore volume and the interparticle volume (-80% of the column volume) and will elute later, when the corresponding volume of mobile phase has passed through the column; at the other extreme, a very large molecule that cannot penetrate the pore system percolates only into the interparticle volume (-35% of the column volume) and will elute earlier, when this volume of mobile phase has passed through the column. The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates, and this results in the separation of a solution of particles on the basis of particle size. Thus, provided that all the particles are loaded simultaneously or near- simultaneously, particles of the same size should elute together. [0009] Liquid-phase chromatographic techniques involving purification over columns currently use stationary phases based on two types of porous silica gel (Si0₂), these comprising either undecorated silica gel (direct phase), su ch as DAVI S I L^®, or functionalised silica gel with long hydrocarbon chains (reverse phase) (L.F. Giraldo, B.L. Lopez et al (2007), "Mesoporous silica applications", Macromolecular Symposia, 258, 129- 141 ; I.M. El-Hahhal and N.M El-Ashgar (2007), "A review on polysiloxane-immobilized ligand systems: Synthesis, characterization and applications", Journal of Organometallic Chemistry, 692(14), 2861 -2886). Silica gel has a high surface area (800 m²g^"1) and is intrinsically acidic (pH 4), which can lead to the degradation of acid-sensitive compounds when using it as a support material for column chromatography. Organic compounds incorporating common protective groups, such as those with acetal, thioacetal, ether or silane protective groups, are especially labile and prone to decomposition on silica gel columns. In addition, functionalised silica gel columns often require a number of extra washing steps in order to regenerate the columns after use.

[0010] Basic alumina (pH 7-10) offers an alternative to silica gel for use in liquid phase column chromatography, but it is hydroscopic, decomposes a large range of organic compounds, often requires an aqueous pre-treatment to control compound specific activity and affinity, and has a low surface area (200 m²g^"1) (J-J- Kirkland (1996), "Stability of silica- based monofunctional C-18 bonded-phase column packing for HPLC at high pH", Journal of Chromatographic Science, 34(7), 309-31 3; Y. Mao and B. M. Fung (1997), "Use of alumina with anchored polymer coating as packing material for reversed-phase high- performance liquid chromatography", Journal of Chromatography A, 790(1 -2), 9-15).

[0011] Zirconia may allow for the treatment of acid-sensitive compounds, but has important drawbacks in terms of cost, ease of use and reusability (M. Grun, A.A. Kurganov et al (1996), "comparison of an ordered mesoporous aluminosilicate, silica, alumina, titania and zirconia in normal-phase high-performance liquid chromatography", Journal of Chromatography A, 740(1 ), 1 -9).

[0012] In view of the importance of these chromatographic techniques in the separation of various chemical species, it is necessary to provide methods which can reliably and efficiently separate acid-sensitive organic molecules of the type mentioned above, and it is this requirement that the present invention seeks to address.

[0013] In WO 2006/046012, the present inventors disclosed various novel nanoporous materials, in addition to methods for their manufacture and use. Specifically, the inventors were concerned with certain silicon nitride materials, and their use as filters and catalytic materials, and described a sol-gel procedure for the preparation of materials based on silicon nitride and silicon oxynitride. The disclosed sol-gel preparation procedure allowed the size and size-distribution of the pores to be controlled and optimised and facilitated the fabrication of filters of a defined size, thickness and shape.

[0014] The preparation of nanoporous ceramics had, in fact, previously been well documented in the prior art. Thus, for example, Cheng and various collaborators have variously described the preparation of a mesoporous silicon boron nitride via a nonaqueous sol-gel route (F. Cheng, B. Toury, F. Lefebvre, and J.S. Bradley, Chem. Commun., 2003, p 242-243), the preparation of mesoporous silicon boron imide gels from single source precursors via a non-aqueous sol-gel route (F. Cheng, S.J. Archibald, S. Clark, B. Toury, S.M. Kelly and J.S. Bradley, Chem. Mater., 2003, 15, p 4651 -4657), and the application of a sol-gel technique to the preparation of 2,4,6- tris[tris(dimethylamino)silylamino]borazine (F. Cheng, B. Toury, S.J. Archibald and J.S. Bradley, J. Organometallic Chem., 2002, 657, p 71 -74). In addition, the synthesis of a carbon-free, porous silicon diimide gel from tris(dialkylamino)silazanes by means of non oxide sol-gel chemistry has been reported (R. Rovai, C. W. Lehmann and J.S. Bradley, Angew. Chem. Int. Ed., 1999, 38, No. 13/14, p 2036-2038).

[0015] In WO 2006/046012, there is provided a nanoporous, preferably mesoporous, non-oxide material which comprises a silicon imido nitride derivative, and which preferably comprises a plurality of nanoscale pores which have an average pore diameter which most preferably falls in the range of from 1 .5 to 5 nm, and which is typically prepared by means of a sol-gel procedure.

[0016] A particular advantage associated with the use of the sol-gel procedure in the preparation of silicon imido nitride derivatives is the ability to form different shapes without the necessity for slurry powder processing. A further advantage which results from the use of this technique to prepare these materials is that they can be fabricated to form nanoporous, especially mesoporous, membranes with a defined shape and size.

[0017] The sol-gel technique also allows for the preparation of a series of nanoporous, especially mesoporous, aerogels and ceramic materials having a controlled composition in addition to a defined and reproducible pore-size distribution. This facilitates improvements in the degree of selectivity of absorption. In addition, the technique facilitates the preparation of nanoporous, especially mesoporous, aerogels and ceramics having a specific shape and defined dimensions either by means of mechanical compaction in a metallic die followed by cutting, or by the use of a dipping procedure for filter formation on a microporous membrane support of suitable dimensions.

[0018] Thus, the inventors in WO 2006/046012 provided low-cost mesoporous non-oxide aerogels and ceramic materials which were easily manufactured and had a reproducible pore-size and size-distribution, thereby offering significant economic and practical advantages over the prior art. The use of these materials as filters and catalytic materials was also reported, and studies of their uses as possible filters for the filtration of gas were also discussed by F. Cheng, S.M. Kelly et al (2006), "Preparation and characterization of a supported Si₃N₄ membrane via a non-aqueous sol-gel process", Journal of Membrane Science, 280(1 -2), 530-535, whilst their use as supports for metallic catalysts was further reported by F. Cheng, S. M. Kelly et al (2006), "Preparation of mesoporous Pd/Si₃N₄ nanocomposites as heterogeneous catalysts via three different chemical routes", Chemistry of Materials, 18(25), 5996-6005.

[0019] The present inventors have now established that mesoporous silicon imido nitride derivatives of the type disclosed in WO 2006/046012, prepared by sol-gel techniques, provide suitable alternatives to silica for use as the stationary phase in various chromatographic techniques. Specifically, it has been found that silicon diimide gel (Si(NH)₂) offers an isoelectronic alternative to silica in chromatography.

Summary of the Invention

[0020] Thus, according to a first aspect of the present invention, there is provided a method for the chromatographic separation of chemical species, said method comprising the provision of a chromatograph comprising a stationary phase which comprises mesoporous silicon imido nitride (Si2N₂(NH)₂), silicon diimide (Si(NH)₂) mesoporous gel or silicon nitride (Si₃N₄) mesoporous ceramic.

[0021] Said mesoporous silicon imido nitride stationary phase comprises a silicon diimide mesoporous gel which is partially pyrolysed. Consequently, said silicon imido nitride comprises from 0.0001 to 100% silicon diimide gel and from 100 to 0.0001 % silicon nitride ceramic material, and all possible combination ratios therebetween. I n certain embodiments of the invention said stationary phase comprises 100% silicon diimide mesoporous gel. In other embodiments of the invention, said silicon diimide gel is completely pyrolysed such that said stationary phase comprises 100% silicon nitride mesoporous ceramic.

[0022] Typically, said mesoporous gel is prepared by means of a sol-gel process. A silicon nitride mesoporous gel prepared in such a manner provides a large specific surface in addition to basic properties which are associated with the presence of pendant N H_n groups. Typically, some collapse in pore structure is observed on pyrolysis of the gel, such that increasing proportions of the ceramic material contribute to smaller average pore sizes. This is largely a consequence of the collapse and filling of some pores during pyrolysis, rather than a change in the actual size of individual pores, and this effect also contributes to a decrease in specific surface (surface area per unit of mass) of the material. Some variation in the size of individual pores does also occur on pyrolysis, due to local movements of silicon and nitrogen atoms and whilst, on average, pore size is slightly increased, individual pores may show either a small increase or a small decrease in size. The net result is a broadening of the pore size distribution in the pyrolysed (ceramic) material.

[0023] Accord i n g to preferred em bod i ments of the i nvention , said method for chromatographic separation of chemical species comprises liquid phase column chromatography. Further preferred embodiments comprise high performance liquid chromatography (HPLC), gas chromatography (GC), size exclusion chromatography (SEC), thin layer chromatography (TLC) and solid phase extraction (SPE).

[0024] In certain embodiments of the invention, said silicon diimide mesoporous gel prepared by a sol-gel process, or its partially or fully pyrolysed silicon imido nitride or silicon nitride derivatives may be ground to a fine powder which typically may be poured into a column comprised of, for example, glass or metal for use as a chromatograph. In alternative embodiments of the invention, said silicon diimide, silicon imido nitride or silicon nitride may be cast directly as a monolith in a column. Following use, the column could then be regenerated and reused.

[0025] In particular embodiments of the invention, said method of chromatographic separation is applied to the separation of acid-sensitive organic molecules. Said organic molecules may, for example, include proteins, wherein acidic conditions may catalyse the breaking of internal peptide bonds, or compounds which find use in the preparation of materials such as pharmaceuticals which may, for example, include intermediates with protected functional groups in which the protection can be removed in presence of acids (e.g. aldehydes or ketones protected by diols).

[0026] In alternative embodiments of the invention, said method of chromatographic separation may be applied to the separation of catalytic amounts of metal ions from reaction products. In such embodiments, said silicon diimide, silicon imido nitride or silicon nitride materials act as ion scavengers for the removal of said metal ions. Typical examples of ions which may be removed in this way include platinum and palladium ions.

[0027] According to the invention the chemical species to be separated are comprised in a mobile phase which is passed through said stationary phase according to standard chromatographic techniques. Amongst the solvents which may be used in the mobile phase according to the method of the invention are those solvents which are less susceptible to oxidising amine groups to hydroxyl groups; particularly suitable solvents include, for example, THF, diethylether, dichloromethane and alkanes. [0028] According to a second aspect of the present invention , there is provided a chromatograph comprising a silicon diimide, silicon imido nitride or silicon nitride stationary phase. Said silicon diimide comprises a mesoporous gel which is typically prepared by means of a sol-gel process. Said silicon diimide is optionally partially pyrolysed to form a silicon imido nitride material, or totally pyrolysed to form a silicon nitride ceramic. The ceramic may be finely grou nd and the powder used to pack columns for use as a stationary phase for chromatographic separation.

[0029] A further aspect of the invention envisages the preparation of a chromatograph by incorporating a silicon diimide, silicon imido nitride or silicon nitride stationary phase in a chromatographic column.

Brief Description of the Drawings

[0030] Embodiments of the invention are further described hereinafter with reference to the accompanying drawing, wherein:

Figure 1 illustrates the results obtained from the elution of Sudan Red I in methanol on silicon diimide gel, silica gel and cellulose TLC plates.

Description of the Invention

[0031] Embodiments of the first aspect of the present invention provide a method for the chromatographic separation of chemical species, said method comprising the provision of a chromatograph comprising a silicon imido nitride (Si2N₂(NH)₂), silicon diimide (Si(NH)₂) or silicon nitride (Si₃N₄) stationary phase. Said silicon diimide comprises a mesoporous gel which is typically prepared by means of a sol-gel process. Said silicon diimide mesoporous gel is optionally partially pyrolysed to form a silicon imido nitride, or completely pyrolysed to form a silicon nitride ceramic material. Said stationary phase material provides a large specific surface in addition to basic properties which are associated with the presence of pendant NH_n groups (where n is an integer from 0 to 3, and the value of n is typically determined according to the pH of the solution in contact with the gel or ceramic).

[0032] Typically, said stationary phase material is basic (pH generally 7-10, such that solutions in contact with it reach equilibrium at a pH in the region of 9-10, as opposed to 4 in the case of silica), has a large surface area, and has residual -NH_n groups.

[0033] The stationary phase material may be ground to a fine powder, typically having a particle diameter of 0.5-300 μηι, more typically 0.5-100 μηι, more especially 0.5-50 μηι, and particularly in the range of 0.5-5.0 μηη, which is then packed into an HPLC style column . The ceramic material typically has slightly lower surface area and porosity compared to the gel , but has much better mechanical and thermal stability. Thus, increasing proportions of th e ceramic generally provide increased stability to high temperature (up to 1000°C), high pressure and mechanical stress, so that such materials are particularly suitable for HPLC applications.

[0034] The pore size of the material is generally in the range of 3.5-4 nm in the ceramic form and 3.6-3.8 nm in the gel form, which is in a similar range to the pore sizes of zeolith gels, so that the material is useful for separating proteins in the 2000-4000 Da range.

[0035] The pore size can, however, be varied by the addition of more pores using methods well known to those skilled in the art, such as the use of templates or alternative drying methods. Materials generated using such techniques typically have a pore size in the range of 1 nm-100 nm, and the size range of said materials may or may not overlap with the range of 3.5-4 nm for the materials in the ceramic form and 3.6-3.8 nm for the materials in the gel form.

[0036] The surface area of the material is generally in the region of 300-1000 m²/g for the ceramic material and 300-1200 m²/g for the gel version, which is a similar order of magnitude to silica.

[0037] In use, the interactions of the material with the solute typically involve surface nitrogen atoms and amine groups, strong electron donor interactions, as well as a good dispersive component, coming mostly from acid-base exchanges and hydrogen bonding, such that it is possible to control the retention times of solutes.

[0038] Silicon imido nitride, silicon diimide and silicon nitride materials employed for the purposes of the invention are stable in contact with water and air. Some oxidation may be observed on the surface of the materials when in contact with water and/or air, but once the materials have been oxidised at the surface, no further oxidation occurs. The basic surface can be regenerated by washing with THF saturated with NH₃.

[0039] The materials are found to be stable at pH values of up to 13, which is significantly higher than is the case with currently available column chromatographic materials. In view of the presence of surface amine groups, the materials behave like soft bases and are capable of tolerating a wider range of functional organic groups than either basic alumina or silica gel. As noted above, the materials can also be regenerated after use as a chromatographic stationary phase, for example by washing with a THF/NH₃ mixture. Their thermal stability, chemically inert nature, reusability and recyclability thereby provide significant economic and environmental benefits. [0040] Suitable preparative techniques for these materials involve a combination of a non-aqueous sol-gel process and solid state chemistry, as reported by R. Rovai, C.W. Lehmann et al (1999), "Non-ox i d e s o l-gel chemistry: Preparation from tris(dialkylamino)silazanes of a carbon-free porous, silicon diimide gel", Angewandte Chemie-lnternational Edition, 38(13-14), 2036-2038, and F. Cheng, S. Clark et al (2004), "Preparation of mesoporous silicon nitride via a non-aqueous sol-gel route", Journal of the American Ceramic Society, 87(8), 1413-1417.

[0041] Thus, for example, a solution of tris(dimethylamino)silamine in THF is first heated with a drop of trifluoromethanesulfonic acid for 16 hours. The solution is cooled down and THF saturated with ammonia (NH₃) is added in a proportion of 0.1 mL for 10 mL of initial solution. After 8 hours, gaseous ammonia is bubbled in the solution for 10 to 20 minutes and the solution is then left to gel slowly overnight. The solvent-filled gel is dried at 50°C first under a stream of nitrogen, then using a light vacuum (3 mm of mercury). The product, a silicon diimide gel, can be transferred to a glove box for processing, or heated at 1000°C for 2 hours under an ammonia stream for transformation into a silicon nitride ceramic.

[0042] The silicon imido nitride, silicon diimide and silicon nitride stationary phase materials used in the method of the present invention may be prepared from tris(dimethylamino)silamine (Si(NH)2[N(CH₃)₂]₃) according to these prior art methods, and are obtained as white solids which are typically ground and sieved into fine powders having average particle sizes in the region of 0.5-1 50 pm before being used to fill a standard chromatography glass column. Thus, the materials, as fine powders, may be poured into a glass or metal column over a cotton plug or frit by preparing and drying the gel/ceramic in a separate flask, then grinding it and filling the column.

[0043] Alternatively, the stationary phase material may be cast as a monolith directly within the column, and then dried by a flow of, for example, a supercritical diethylamine, following which drying step, the filled column is ready for use. The material may also be cast in other different shapes, such as spheres, by using appropriate moulds and drying in a similar manner.

[0044] In a particular embodiment of the invention, a silicon diimide gel was used which had a large specific surface (approximately 500 m²g^"1) and an almost monodisperse pore size (3.4 nm). These values compared favourably with the corresponding values (540 m² g^"1, and 5.5±1 .5 nm, respectively) of a standard silica sample (DAVISIL^® supplied by Sigma Aldrich) which, for present purposes, was used as a reference solid phase.

[0045] The surface energy of the said gel sample was determined by means of the advancing and receding contact angles of different liquids through a drop under air according to the method of Vanoss et al (C.J. Vanoss, R.F. Giese et al (1992), "Determination of Contact Angles and Pore Sizes of Porous-Media by Column and Thin- Layer Wicking", Journal of Adhesion Science and Technology, 6(4), 413-428. Subsequent calculations gave a result of 52.6 mJm^"2 with a strong electron donor contribution (46 mJm^"2) and smaller dispersive interactions (23.4 mJm^"2). When compared to results gathered from similar studies of silica and alumina surfaces (E. Chibowski (1992), "Solid- Surface Free-Energy Components Determination by the Thin-Layer Wicking Technique", Abstracts of Papers of the American Chemical Society, 203, 262-COLL; W. Wu, R.F. Giese et al (1996), "Change in surface properties of solids caused by grinding", Powder Technology, 89(2), 129-132), silicon diimide gel demonstrates electron-donor interactions which are of the order of those observed with silica, but are smaller than those associated with alumina, whilst the dispersive interactions are weaker than those of both silica and alumina.

[0046] The present inventors have successfully demonstrated the use of the mesoporous gels and ceramics previously described as the stationary phase in column chromatography in the efficient purification of acid-sensitive compounds.

[0047] Thus, the use of a porous silicon diimide gel as a stationary phase for the chromatographic separation of acid-sensitive compounds according to an embodiment of the invention was tested in two steps. The first step consisted of a series of batch experiments wherein 0.1 mol L^"1 solutions of 2-phenoxytetrahydropyran in tetrahydrofuran were stirred over either porous silicon diimide or silica. The formation of decomposition products from this THP-protected phenol was monitored at regular intervals over time by proton NMR spectroscopy.

[0048] It was observed that the solution in contact with silica soon became acidic (pH = 4), and NMR measurements showed that the Hp triplet was gradually replaced over 3 hours by a composite peak comprised of the H_Q broad phenolic singlet and the H_Y hemiacetal triplet. The amount of protons represented by this composite peak also increased relatively to the number of protons of the phenyl ring, in agreement with the replacement of one H_b by one H_a plus one H_g. These analyses showed that 2- phenoxytetrahydropyran was hydrolysed to form phenol and tetrahydropyran by the acidic nature of the silica gel at room temperature.

[0049] No such changes were observed with the solution in contact with the silicon diimide gel under identical conditions. The triplet at 5.35 ppm was still observed with the same relative intensity as at the start of the reaction, and this remained the case even after 24 hours of interaction. As such, no hydrolysis of the 2-phenoxytetrahydropyran had occurred and no formation of phenol and tetrahydropyran was deemed to have taken place in the reaction mixture. Furthermore, it was also observed that the mixture became basic (pH between 9 and 10), in agreement with previous characterisations of silicon diimide gel.

[0050] In the second stage of the testing, 2-phenoxytetrahydropyran was synthesised from the reaction of 3,4-dihydropyran and phenol in dichloromethane for 24 hours in the presence of p-toluene-sulphonic acid as a catalyst. Following removal of the solvent under vacuum, the product was analysed by NMR spectroscopy, which revealed the presence of traces of unreacted phenol and dihydropyran alongside 2-phenoxytetrahydropyran formed in the reaction as the major component of the crude reaction product.

[0051] The raw product was then purified using column chromatography with silicon diimide gel as the stationary phase and a 50/50 mixture of dichloromethane and hexane as the mobile phase to yield fractions containing pure 2-phenoxytetrahydropyran. No 2- hydroxytetrahydropyran could be detected, indicating that no decomposition of the 2- phenoxytetrahydropyran had occurred on the column during the purification by chromatography.

[0052] Consequently, it became evident from these studies that when used in a high-pH environment, mesoporous silicon imido nitride, silicon diimide mesoporous gel and silicon nitride mesoporous ceramic are more stable, less hydroscopic and more reproducible in their characteristics than either basic alumina or base-doped silica gel. Due to these qualities, these materials provide an alternative chromatographic stationary phase for the identification or purification of acid-labile chemical compounds such as pharmaceutical intermediates or final products. The materials shows chemical stability at extreme pH values (9-10) when compared with standard reverse-phase chromatographic techniques, sustainable operation at basic pH and excellent specific surface and capacity to interact with solutes.

[0053] The invention will now be further illustrated, though without in any way limiting the scope thereof, by reference to the following examples and associated illustration.

Examples

Example 1

[0054] This experiment demonstrates the use of mesoporous silicon diimide gel as a stationary phase for chromatographic applications involving TLC.

[0055] A spot of Sudan Red I in methanol was applied as usual close to the bottom of a silicon diimide gel plate, which was then put in a chromatography tank containing methanol as the mobile phase. The methanol eluant rose up the silicon diimide gel stationary phase at a similar rate to that observed for the reference silica gel and cellulose TLC plates using the same analyte and eluant. The TLC plate was removed from the tank and the retention factor (Rf) determined. The experiment was repeated with several silicon diimide gel TLC plates and the chromatographic effect was compared with that of the reference silica gel and cellulose TLC plates. The results are illustrated in Figure 1 , which shows the results obtained from the elution of Sudan Red I in methanol on (from left to right) silicon diimide gel (according to the invention), silica gel (reference) and cellulose (reference) TLC plates. It is evident that a clear chromatographic effect is seen for the silicon diimide gel TLC plate, comparable with that observed for the silica gel TLC plate, whereas almost no chromatographic retardation is seen for the cellulose TLC plate.

Example 2

[0056] This experiment was designed to test the stability of acid-sensitive organic compounds in the presence of silicon diimide gel. Identical solutions (1 mol L^~1) of 2- phenoxytetrahydropyran in tetrahydrofuran were stirred over identical amounts of either porous silicon diimide gel or silica gel at room temperature. THP is a typical protecting group used for alcohols and phenols in organic synthesis. The composition of these solutions was monitored at regular intervals over time by 1 H-NMR spectroscopy for the acid-catalysed formation of decomposition products from the THP-protected phenol, as shown in Scheme 1 .

Scheme 1 2-phenoxytetrahydropyran (Ηβ @ 5.17 ppm s,), phenol (Ηα @ 5.37 ppm, t) and tetrahyd ropy ran (Ηγ @ 5.22, t).

[0057] The solution in contact with silica gel became acidic (pH = 4) quite quickly. The NMR measurements showed that the Ηβ triplet was gradually replaced over 3 hours by a composite peak comprised of the Ha broad phenolic singlet and the Ηγ hemiacetal triplet. The amount of protons represented by this composite peak also increased relative to the number of protons of the phenyl ring, in agreement with the replacement of one Ηβ by one Ha plus one Ηγ. These analyses show that 2-phenoxytetrahydropyran is hydrolysed to form phenol and tetrahyd ropyran by the acidic nature of the silica gel at room temperature. No such changes were observed in the solution in contact with the silicon diimide gel under identical conditions. The triplet at 5.35ppm was still observed with the same relative intensity as at the start of the reaction and even after 24 hours of interaction. The test mixture containing silicon diimide gel gradually became basic (pH = 9-10) due to the presence of silicon diimide gel.

Example 3

[0058] This experiment involved the purification of 2-phenoxytetrahydropyran by column chromatography using silicon diimide gel as the stationary phase. The 2- phenoxytetrahydropyran was synthesised from 3,4-dihydropyran and phenol in presence of p-toluene-sulfonic acid as a catalyst in dichloromethane. After removal of the solvent under vacuum, analysis of the reaction mixture by 1 H-NMR spectroscopy revealed the presence of a mixture of unreacted phenol and dihydropyran alongside 2- phenoxytetrahydropyran, as expected for such an equilibrium-based reaction.

[0059] This raw product mixture was then eluted through a standard chromatography column packed with silicon diimide gel as the stationary phase using a 50%/50% mixture of dichloromethane and hexane as the mobile phase. Fractions containing pure 2- phenoxytetrahydropyran were collected and confirmed as such using 1 H-NMR spectroscopy. It should be noted that no traces of the original reactants or degradation products were observed in these fractions, which is confirmation of the successful separation of the THP-protected product from the starting reactants, and of the successful protection of the desired product from hydrolysis during the chromatographic separation of the desired final product.

[0060] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0061] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0062] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Claims

1 . A method for the chromatographic separation of chemical species, said method comprising the provision of a chromatograph comprising a stationary phase which comprises mesoporous silicon imido nitride (Si2N₂(NH)₂), silicon diimide (Si(NH)₂) mesoporous gel or silicon nitride (Si₃N₄) mesoporous ceramic.

2. A method as claimed in claim 1 which comprises liq uid phase col umn chromatography.

3. A method as claimed in claim 1 or 2 which comprises gas chromatography, high performance liquid chromatography, size exclusion chromatography or th i n layer chromatography.

4. A method as claimed in claim 1 , 2 or 3 wherein said silicon diimide mesoporous gel has a pore size in the range of 3.6-3.8 nm and a surface area of about 300-1200 m²/g.

5. A method as claimed in claim 1 , 2 or 3 wherein said silicon nitride mesoporous ceramic comprises a silicon diimide gel which is completely pyrolysed.

6. A method as claimed in claim 1 , 2, 3 or 5 wherein said silicon diimide mesoporous ceramic has a pore size in the range of 3.5-4 nm and a surface area of about 300-1000 m²/g.

7. A method as claimed in claim 1 , 2 or 3 wherein said mesoporous silicon imido nitride stationary phase comprises a silicon diimide mesoporous gel which is partially pyrolysed.

8. A method as claimed in claim 7 wherein said silicon imido nitride comprises from 0.0001 to 100% silicon diimide gel and from 100 to 0.0001 % silicon nitride ceramic material.

9. A method as claimed in any one of claims 1 to 7 wherein said mesoporous gel is prepared by means of a sol-gel process.

10. A method as claimed in claim 9 wherein said mesoporous gel is prepared from tris(dimethylamino)silamine (Si(NH)2[N(CH₃)₂]₃).

1 1 . A method as claimed in any one of claims 1 to 10 wherein said stationary phase material is ground to a fine powder which is pou red i nto a colu m n for use as a chromatograph.

12. A method as claimed in claim 1 1 wherein said powder has an average particle diameter in the range of 0.5-300 μηι, optionally in the range of 0.5-100 μηι, optionally in the range of 0.5-50 μηι, optionally in the range of 0.5-5 μηι.

13. A method as claimed in any one of claims 1 to 10 wherein said stationary phase material is cast directly as a monolith in a column.

14. A method as claimed in any preceding claim wherein said stationary phase material has pH 7-10 and residual -NH_n groups.

15. A method as claimed in any preceding claim when applied to the separation of acid-sensitive organic molecules.

16. A method as claimed in claim 15 wherein said organic molecules include proteins or compounds which find use in the preparation of pharmaceuticals.

17. A method as claimed in claim 16 wherein said compounds which find use in the preparation of pharmaceuticals comprise intermediates with protected functional groups in which the protection can be removed in presence of acids.

18. A method as claimed in claim 17 wherein said intermediates comprise aldehydes or ketones protected by diols.

19. A method as claimed in claim 16 wherein said proteins are in the in the 2000-4000 Da range.

20. A method as claimed in any one of claims 1 to 14 when applied to the separation of catalytic amounts of metal ions from reaction products.

21 . A method as claimed in claim 20 wherein said metal ions comprise platinum or palladium ions.

22. A method as claimed in any preceding claim wherein said stationary phase material is regenerated after use as a chromatographic stationary phase by washing with a THF/NHs mixture.

23. A chromatograph comprising a mesoporous silicon imido nitride (Si2N₂(NH)₂), silicon diimide (Si(NH)₂) mesoporous gel or silicon nitride (Si₃N₄) mesoporous ceramic stationary phase.

24. A chromatograph as claimed in claim 23 wherein said silicon diimide mesoporous gel has a pore size in the range of 3.6-3.8 nm and a surface area of about 300-1200 m²/g.

25. A chromatograph as claimed in claim 23 wherein said silicon nitride mesoporous ceramic comprises a silicon diimide gel which is completely pyrolysed.

26. A chromatograph as claimed in claim 23 or 25 wherein said silicon diimide mesoporous ceramic has a pore size in the range of 3.5-4 nm and a surface area of about 300-1000 m²/g.

27. A chromatograph as claimed in claim 23 wherein said mesoporous silicon imido nitride stationary phase comprises a silicon diimide mesoporous gel which is partially pyrolysed.

28. A chromatograph as claimed in claim 27 wherein said silicon imido nitride comprises from 0.0001 to 100% silicon diimide gel and from 100 to 0.0001 % silicon nitride ceramic material.

29. A chromatograph as claimed in any one of claims 23 to 28 wherein said mesoporous gel is prepared by means of a sol-gel process.

30. A chromatograph as claimed in claim 29 wherein said mesoporous gel is prepared from tris(dimethylamino)silamine (Si(NH)2[N(CH₃)₂]₃).

31 . A chromatograph as claimed in any one of claims 23 to 30 wherein the pore size of said silicon diimide mesoporous gel is varied by the addition of more pores, and wherein said pore size is in the range of 1 -100 nm.

32. A chromatograph as claimed in any one of claims 23 to 31 wherein said stationary phase material is ground to a fine powder which is poured into a column for use as a chromatograph.

33. A chromatograph as claimed in claim 32 wherein said powder has an average particle diameter in the range of 0.5-300 μηι, optionally in the range of 0.5-100 μηι, optionally in the range of 0.5-50 μηι, optionally in the range of 0.5-5 μηι.

34. A chromatograph as claimed in any one of claims 23 to 31 wherein said stationary phase material is cast directly as a monolith in a column.

35. A chromatograph as claimed in one of claims 23 to 34 wherein said stationary phase material has pH 7-10 and residual -NH_n groups.

36. A chromatograph as claimed in any one of claims 23 to 35 wherein the stationary phase materials can be regenerated after use by washing with a THF/NH₃ mixture.

37. The preparation of a chromatograph by incorporating a mesoporous silicon imido nitride (Si2N₂(NH)₂), silicon diimide (Si(NH)₂) mesoporous gel or silicon nitride (Si₃N₄) mesoporous ceramic material in a chromatographic column.

38. The use of a chromatograph as claimed in any one of claims 23 to 36 for the chromatographic separation of chemical species.

39. A method for the chromatographic separation of chemical species, said method comprising the provision of a chromatograph as claimed in any one of claims 23 to 36.