High-performance Liquid Chromatography Packing Research Articles

Monodisperse core–shell silica particles as a high-performance liquid chromatography packing material: Facile in situ silica sol-gel synthesis

In recent years, core–shell silica particles (CSSPs) have been increasingly used for highly efficient separation at fast flow rates and relatively low back pressures in high-performance liquid chromatography (HPLC). However, material synthesis techniques for producing CSSPs economically in batch processes remain elusive. In this report, a practical and straightforward method for the preparation of CSSPs is presented. By refluxing freshly prepared nonporous silica particles in ammonia–water solution in the presence of poly(diallyldimethylammonium chloride) at 70–100 °C, CSSPs with shell thicknesses of up to 300 nm and pore sizes from 8 to 25 nm were easily prepared. The effects of the synthetic conditions on the shell thickness, surface area, and pore size were investigated in detail, and the method reproducibility was evaluated in scale-up experiments. A mechanism of CSSP formation is also proposed. The CSSPs were characterized via scanning electron microscopy, transmission electron microscopy, laser particle size (dynamic light scattering) analysis, and nitrogen adsorption and desorption experiments. The synthesized 3.4-μm CSSPs were functionalized with dimethyloctadecylchlorosilane and used as an HPLC packing material, exhibiting excellent separation performance for both small molecules and large biomolecules. In summary, we report the simplest method developed thus far for the preparation of monodisperse core–shell silica particles suitable for HPLC column packing.

Ultrasonic-Assisted Sol–Gel Synthesis of Core–Shell Silica Particles for High-Performance Liquid Chromatography

Core–shell particles (CSPs), also known as superficially porous particles or fused-core particles, consisting of a solid core and a porous shell, have emerged as an advanced technology in high-performance liquid chromatography (HPLC). We report here an ultrasonic-assisted sol–gel method for the fabrication of CSPs with uniform mesoporous silica shell using tetraethoxysilane as the silica source and dodecylamine as a catalyst, template and porogen agent in a methanol–water solution. Instead of stirring, sonication was adopted to guarantee the particles monodispersity and uniformity. The shell thickness between 300 and 700 nm could be obtained by adjusting the sonication time and the volume ratio of methanol–water. Pore sizes from 3.4 to 8.5 nm were tuned by hydrothermal treatment. The C18-bonded 2.7 μm CSPs with a shell thickness of ~ 500 nm showed some advantages over a commercial 1.7 μm BEH column, and successfully applied for the analyses of a Chinese medicine and peptides. The CSPs could be a promising candidate as HPLC packing materials.

Synthesis of Large-Pore Silica Microspheres Using Dodecylamine as a Catalyst, Template and Porogen Agent

This paper reports a strategy combing a modified Stober method and a post-hydrothermal treatment for the fabricating of silica microspheres with large pores. Dodecylamine (DDA), used as a catalyst, template and porogen agent, plays an important role in controlling both the particle morphology and the pore expansion. By adjusting the concentration of DDA (20, 40 and 80 mmol/L) and the temperature of hydrothermal treatment (80–140 °C), the pore size ranging from 3.5 to 16 nm could be obtained. The synthesized sub-2 µm silica spheres were modified with dimethyloctadecylchlorosilane (C18) and successfully applied for the separation of aromatic compounds in high-performance liquid chromatography (HPLC). The silica may be a good candidate as HPLC packing materials.

Preparation and Chromatographic Features of Fibrous Core–Shell HPLC Packing Material

In this study, fibrous core–shell silica particles were successfully synthesized via a one-step oil–water biphase stratification coating strategy. The core–shell silica particles were composed of 3-µm non-pore silica cores and thin shells (50–100 nm), which have radial-like direct channels and a large pore size (19.89 nm). The fibrous core–shell silica particles were further modified by n-octadecyltrichlorosilane and used as stationary-phase media in high-performance liquid chromatography (HPLC). The chromatographic properties of the particles were systematically studied in small-molecule and protein separation processes. The results showed that the back pressure was as low as 8.5 MPa under the 1.0-mL min−1 flow velocity. Furthermore, fibrous core–shell silica particles with an 80-nm shell were used for separating seven small molecules within 10 min and six proteins within 6 min. This work demonstrates that the fibrous core–shell silica particles could be used as an HPLC stationary phase with good performance and low back pressure, and that they have great potential for application to HPLC separation in the future.

Preparation and enantioseparation performance of β-cyclodextrin-silica hybrid chiral stationary phases

A simple preparation method for β-cyclodextrin-silica hybrid chiral stationary phases was developed. Firstly, the β-cyclodextrin-silica derivative was synthesized by the reaction of 3-aminopropyltriethoxysilane and monochlorotriazinyl β-cyclodextrin under weak base condition. Spherical β-cyclodextrin-silica hybrid materials with β-cyclodextrin in the surface of pores by covalent bonding were prepared using 1,2-bis(triethoxysilyl) ethane and the β-cyclodextrin-silica derivative under the alkaline condition by one-step polymerization reaction. The β-Cyclodextrin-silica hybrid chiral stationary phases could be directly used as high performance liquid chromatographic packings after the template removal. The hybrid materials prepared in this paper possessed regular spherical morphology, good monodispersion, high specific surface area, good mechanical property, high chemical stability and simple preparation process. It combined the chiral recognition performance of β-cyclodextrin and the outstanding performance of organic-inorganic hybrid material. The effect of the composition, ratio and pH of mobile phase on chiral separation was investigated, and the best chiral separation conditions had been optimized. The baseline chiral separations for five chiral compounds were obtained under the optimal conditions. The results of enantioseparation showed that the hybrid chiral stationary phases had favorable chiral recognition ability. Compared with the traditional preparation process of chiral stationary phases, a new thought for new type of chiral stationary phase is provided by the present method in this paper.

Comparison of Concentration Pulse and Tracer Pulse Chromatography: Experimental Determination of Eluent Uptake by Bridged-Ethylene Hybrid Ultra High Performance Liquid Chromatography Packings

Abstract Excess volume isotherms of acetonitrile and methanol sorbed on a C 18 BEH UHPLC packing were determined over a range of pressure, temperature, flow rate and eluent composition. The isotherm measurements were carried out by two independent experimental methods, viz. , concentration pulse and tracer pulse chromatographies. Isotherms were measured with both experimental techniques at 30, 45 and 60 °C. The excess isotherms increased with decreasing temperature although the variations were relatively small. Direct comparison of the two experimental techniques showed that the measured void volumes were identical within experimental error. The measured excess volumes by both techniques were comparable with the concentration pulse experiments producing slightly higher excess volume data with highly aqueous eluents. Both experimental techniques show some variations of the retention volumes with sample volume, sample composition, flow rate and column inlet pressure. The results confirmed the validity of both concentration and tracer pulse chromatographies for the determination of column void volumes and the excess volume of eluent taken up by UHPLC packings.

Preparation and Characterization of SiO2/SiO2 Core–Shell Microspheres as RP-HPLC Stationary Phase

Novel SiO2/SiO2 core–shell monodisperse silica spheres for high-performance liquid chromatography packing materials are prepared by the layer-by-layer self-assembly technique. The core–shell silica spheres consist of micrometer-sized porous silica spheres as the core and a thin mesoporous silica shell formed from multilayer nanometer-sized silica particles. In addition, a reversed-phase packing by bonding octadecyltrichlorosilane on SiO2/SiO2 particles through alkyl-modified method is prepared and characterized. The results show that the carbon content of the new reversed-phase stationary phase increases by approximately 45% compared with the uncoated octadecyl-bonded SiO2 stationary phase. Eight kinds of tested aromatic compounds are well-separated on the packing and the peaks are symmetrical, which demonstrates that the packing acts as an excellent reversed-phase chromatographic stationary phase.

Application of liquid phase deposited titania nanoparticles on silica spheres to phosphopeptide enrichment and high performance liquid chromatography packings

A novel core–shell composite (SiO 2– nLPD), consisting of micrometer-sized silica spheres as a core and nanometer titania particles as a surface coating, was prepared by liquid phase deposition (LPD). Here, we show the resulting core–shell composite to have better efficient and selective enrichment for mono- and multi-phosphopeptides than commercially available TiO 2 spheres without any enhancer. The material exhibited favorable characteristics for HPLC, which include narrow pore size distribution, high surface area and pore volume. We also show that the core–shell composite can efficiently separate adenosine phosphate compounds due to the Lewis acid–base interaction between titania and phosphate group when used as HPLC packings. After coating the silica sphere with titania by LPD, the silanol of silica spheres will be shielded and that the stationary phase, C 18 bonded SiO 2–3LPD, could be used under extreme pH condition.

Reversed‐phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar‐embedded and polar endcapped columns

We desired to evaluate the chromatographic selectivity for peptides of silica-based RP high-performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal either contains a free α-amino group (AmC series) or is N(α)-acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n-alkyl (Gly<Ala<Val<Ile in order of increasing hydrophobicity), alkyl versus aromatic side-chains (Phe, Tyr) and a side-chain also with polar character (Tyr). Peptide mixtures were run on all columns at pH 2 (linear gradients of ACN or methanol in aqueous mobile phases). Our initial results clearly demonstrate the useful complementarity of different RP high-performance liquid chromatography packing materials, with peptide mixtures showing profound selectivity differences between n-alkyl versus polar-embedded and polar endcapped C(18) stationary phases. In addition, changing the polarity of the RP matrix in combination with changing the polarity of the mobile phase (ACN versus methanol) is shown to be synergistic to maximize selectivity of peptide separations.

Titanium-Coated Silica Spheres Prepared by Self-Assembly Technique for Use as HPLC Packing

Titania-coated silica spheres were prepared by a layer-by-layer self-assembly technique for use as a high-performance liquid chromatography packing. This packing has a high surface area of 202.1 m(2)/g, a large pore volume of 0.36 cm(3)/g, and a pore diameter of 7.0 nm. Furthermore, the packing particles exhibit narrow pore size distribution and good pore structure. The chromatographic behavior of the packing was studied under both normal and reversed-phase conditions. Low column pressures were observed. Aromatic isomeric compounds were well separated on the TiO(2)/SiO(2) column under normal phase conditions; pyridine and aniline derivatives were separated on the octadecyl-bonded TiO(2)/SiO(2) (ODT) column, respectively, under reversed-phase conditions, and highly symmetrical peaks were obtained for 15 basic compounds. The chemical stability of the stationary phase was tested with sodium phosphate solution (10 mmol/L, pH = 10) at 25 degrees C and potassium phosphate solution (50 mmol/L, pH = 10) at 50 degrees C.

Synthesis of penetrable macroporous silica spheres for high-performance liquid chromatography

Silica microspheres have been synthesized by phase separation and sol–gel transition coupled with emulsion method. The as-obtained material is characterized by scanning electron microscopy, nitrogen sorption, elemental analysis and particle size distribution measurements. The results demonstrated that the material featured with hierarchically porous structure, possessing both mesopores and penetrable macropores. The mesopores provide large surface area while the macropores traverse the silica particles, which may facilitate fast mass transfer as well as guarantee low backpressure when such materials are used for packed high-performance liquid chromatography (HPLC) column. Therefore, their preliminary applications as HPLC packings in fast separation and low-pressure separation have been attempted in the present study. Benzene, benzaldehyde and benzyl alcohol were separated within two minutes on the silica column at a flow rate of 7 mL min −1. Vitamin E mixtures can also be baseline separated at a high flow rate of 8 mL min −1. In addition, thirteen aromatic hydrocarbons were well separated on the octadecyl-bonded silica (ODS) column. In comparison with a commercial Kromasil ODS column, the pressure of the proposed column is much lower (<1/2) under the same chromatographic conditions, while comparable separation efficiency can be achieved.

Options for Rapid Analysis of Peptides and Proteins, Using Wide-Pore, Superficially Porous, High-Performance Liquid Chromatography Particles with Unique Bonded-Phase Ligands

The large size and complexity of many proteins constrains the reversed-phase high-performance liquid chromatography packings that are useful for their separation. Wide-pore, superficially porous, silica-based packings with solid 4.5-microm cores and a 0.25-microm porous outer layer (Poroshell) demonstrate a variety of characteristics that are beneficial for the separation of proteins. A shorter diffusion distance allows separations of large molecules at high linear velocities. This benefit over totally porous particles is clearly shown using separations of a peptide-protein standard. The structure and reduced surface area (4.5 m2/g) of these superficially porous particles simplifies interactions with its surface, resulting in improved peak shapes and resolution. Specialized bonding chemistries for low- and high-pH operation may be used to change band-spacing and achieve atypical separations. These rapid analysis options are demonstrated using protein standards and very high molecular weight glycosylated proteins including intact monoclonal antibodies, IgM, alpha2-macroglobulin, and glycophorin. In liquid chromatography-mass spectrometry analysis of a myoglobin peptide digest, bidentate-C18-bonded superficially porous packings achieve complete runs in 4 min and demonstrate an elution pattern that is unique from that of material bonded with sterically protected C18 ligands.

Column packings for high-performance liquid chromatography and positron annihilation lifetime spectroscopy

Positron annihilation measurements as a function of temperature and the length of bonded alkyl groups have been carried out on silica gel samples. Silica gel samples were bare and bonded with alkyl group from C1 up to C18. The diameters of pores were deduced from the lifetime of trapped ortho-positronium ( o-Ps), and it was found that o-Ps lifetime provides reasonable information on the pore sizes for both bare and alkyl bonded silica gels.

Packing for high-performance liquid chromatography based on microspheric particles of (urea-formaldehyde resin)-silica gel composite

Preparation of uniform microspheric particles of the (urea-formaldehyde resin)-silica gel composite was studied. The suitability of this composite modified with C18 groups as packing for reversed-phase high-performance liquid chromatography was evaluated. The sorbent was modified using either octadecyldimethylchlorosilane or polymers containing C18 groups, and the columns packed with the resulting sorbent were studied. The experimental results showed that the polymeric modification allows efficient shielding of the surface silanol groups.

Atypical silica-based column packings for high-performance liquid chromatography

Column packings widely used for high-performance liquid chromatography (HPLC) mostly are based on porous silica microspheres with certain pore sizes and pore size distributions. Such materials have the most desirable compromise of properties that provide for effective and reproducible separations over a wide range of operating conditions. To provide desired separation characteristics, several manufacturers specially synthesize the silica particles for these packings. While such column packing materials have general utility for a wide range of needs, special silica-based particles have been synthesized with different physical conformations for special separation goals. This presentation describes some atypical types of silica-based particles with unique separation properties that enlarge the capabilities of HPLC methods.

Selectivity tuning and molecular modeling of new generation packings for RP HPLC

Retention mechanism, selectivity and physicochemical properties together with theoretical surface simulation experiments of laboratory-synthesized packing materials for high performance liquid chromatography (HPLC) containing cholesterol molecules immobilized on the high quality silica gel were studied. The chromatographic behavior of the three cholesterol-based columns (SG-CHOL, SG-MIX CHOL/AP and SG-10-CHOL) was examined with special regard to retention and selectivity and compared to reference materials: a typical octadecylsilica column (SG-C18) and an alkylamide bonded phase (SG-AP). Temperature investigations (DSC) and molecular modeling simulations didn't bring an expected confirmation of liquid crystalline properties of materials studied. The observed phase transition was attributed to conformation change within the amide bonding.

Synthesis of porous titania microspheres for HPLC packings by polymerization-induced colloid aggregation (PICA).

Porous titania microspheres with a very narrow particle size distribution (PSD) were synthesized by polymerization-induced colloid aggregation (PICA). After being sintered, the titania microspheres that are obtained have an average diameter of 3.5 microm, a surface area of 36.7 m2/g, an average pore volume of 0.30 mL/g, and an average pore diameter of 32.2 nm. The microspheres possess enough rigidity to withstand high packing pressure and are very useful as a new kind of chromatographic packing material for high performance liquid chromatography (HPLC).

N-Hydroxysuccinimide ester labeling 5′-aminoalkyl DNA oligomers: reaction conditions and purification

Difficulties were encountered in labeling 5′-aminoalkyl DNA oligomers with glycolketo electrophore N-hydroxysuccinimide esters in aqueous sodium bicarbonate (a common base for this purpose), followed by C 18-silica reversed-phase high-performance liquid chromatography (HPLC) to achieve purification. The electrophore-labeled oligomers were not separated readily either from the hydrolyzed electrophore or from the starting oligomer. This problem was overcome by conducting the reaction with triethylamine as a base, organic washing the reaction mixtures after evaporation, and separating on a C 18-poly(styrene–divinylbenzene) HPLC packing.

Capillary Electrokinetic Chromatography (CEC): An Introduction to a High-Efficiency Microanalytical Technique

Capillary electrokinetic chromatography (CEC, also called “capillary electrochromatography”) is a relatively new separation technique based on a combination of liquid chromatographic and electrophoretic separation methods. CEC offers both the efficiency of capillary electrophoresis (CE) and the selectivity and sample capacity of packed capillary high performance liquid chromatography (HPLC). These advantages are provided in part because of the favorable flow characteristics of electroosmosis, a method of pumping a liquid by applying a high potential axially to a thin, fluid-filled tube. The speed at which analytes move through the separation conduit under the influence of electroosmosis is quite uniform, regardless of the position of the analyte with respect to adjacent surfaces (within certain limits). This results in very little spreading of zones of analyte; narrow, compact bands of analytes are therefore maintained, which results in high efficiency. Because the capillaries used in CEC are packed with HPLC packing materials, the wealth of selectivities available in HPLC are also available in CEC. The high surface area of these packing materials enables CEC capillaries to accommodate relatively large amounts of sample, making detection a simpler task than it is in capillary electrophoresis (CE). This paper will briefly visit the theory and practice of CEC, and will provide examples of how CEC can be applied in sample analysis.

医薬品を対象とする高速液体クロマトグラフィー充填剤の開発と応用

High-performance liquid chromatography (HPLC) has been used for the assays of pharmaceuticals and their impurities, and drugs and their metabolites in biological fluids. However, HPLC packing materials so far developed are not necessarily suitable for the above mentioned purposes. During the past decade, we developed of new HPLC packing materials for direct serum injection analysis of drugs, and for chiral resolution of drug enantiomers. A sample preparation step such as deproteinization and/or extraction is required for HPLC assays of drugs and their metabolites in biological fluids. Direct serum injection analysis of drugs can be achieved by the use of the packing materials having properties as follows: large molecules such as proteins are eluted in the void volume without destructive accumulation, but small molecules such as drugs and their metabolites can reach the hydrophobic sites and be separated. Such a material is called restricted access stationary phase (RASP). We developed two RASPs, an improved internal-surface reversed-phase (ISRP) material and a mixed functional phase (MFP) material. The preparation methods of the ISRP and MFP materials and their applications to the assays of drugs in the serum are described. HPLC chiral stationary phases based on chiral small molecules, and macromolecules such as polysaccharides and proteins have been used for separations of drug enantiomers. Disadvantages of protein-bonded stationary phases include low capacity, lack of column ruggedness and limited understanding of the chiral recognition mechanism. We tried to overcome the disadvantages of protein-bonded stationary phases by modification of side-chain amino acid and preparation of protein-domain or -fragment column. We isolated each ovomucoid domain and examined chiral recognition ability of each domain. The ovomucoid third domain exhibited chiral recognition for only a few benzodiazepines and 2-arylpropionic acid derivatives. The chiral binding site and mechanism on the ovomucoid third domain was investigated by proton NMR measurements and molecular modeling of the ligand-protein complex. Further, new protein, ovoglycoprotein, was isolated from crude ovomucoid preparations, characterized and bound to silica gels. It was found that the chiral recognition ability of the ovomucoid reported previously came from the ovoglycoprotein, which is present in crude ovomucoid as an impurity.