Process scale high-performance liquid chromatography Part II: Dissolution of silica matrices

Summary Possible contamination of purified product by the silica matrix is of considerable importance when considering process scale high-performance liquid chromatography (HPLC) as a separation methodology. The stability of silica supports in normal phase, reversed phase and affinity chromatography modes has been examined with organic solvents and water. Both inorganic and organic components were found, contributed by the support, and the chromatographic and distillation equipment. In all cases, based on assumed values of production, they were found to contribute less than 0.01% of impurities. In critical cases crystallisation or dialysis may be considered as a last stage of purification, with re-chromatography to maximise yields. This work was conducted in the Chemical Technology Unit, University of Manchester Institute of Science and Technology, P. O. Box 88, Manchester M60 1QD, UK.     

Process scale high performance liquid chromatography. Part III: Optimization procedure and economics of silica matrices

Summary An analytical procedure to optimise the solvent system for a given high-performance liquid chromatographic (HPLC) separation is described; it also distinguishes any preference between normal and reversed phase systems. These results can be applied directly to process scale HPLC. If intermediate pure samples are required a feasibility study and a proving trial continue the assessment. Support lifetime, cycle time, yield and purity are assessed practically and from this information processing costs can be calculated. Typical costs are given based upon reasonable assumptions. This work was conducted in the Chemical Technology Unit, University of Manchester Institute of Science and Technology, P. O. Box 88, Manchester M60 1QD, U.K.     

Process scale high performance liquid chromatography part IV—Examples of reversed phase separations

Summary The flexibility of process scale high performance liquid chromatography is demonstrated by three examples of reversed-phase separations (a) a standard purification (b) isolation of trace compounds, and (c) recovery from crystallisation mother liquors. It was shown that a material, thought to be acceptably pure, contained a toxic impurity, and a remaining impurity, a previously unseen component of potential therapeutic interest. Favourable cost data were indicated for example (3). This work was conducted in the Chemical Technology Unit, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M6O 1OD, UK.     

中孔分子筛SBA-15作为液相色谱固定相以及在巯基化合物分离中的应用

采用共表面活性剂法合成了孔道结构高度有序、孔径较大且粒径均匀的SBA－15颗粒，并在此基础上研究了其作为毛细管液相色谱柱的填料基体的特性。在SBA－15材料上进行C18官能团化前后，分别对4种巯基化合物即半胱氨酸、谷胱甘肽、多巴胺和6－巯基嘌呤进行了分离分析。     

Glucose, cellobiose, lactose and raffinose used as chiral stationary phases in HPLC

This paper presents the enantioseparation using glucose, cellobiose, lactose and raffinose as chiral selector bonded to silica gel via an arm in HPLC. Surprisingly, they also possess high enantioseparation selectivity, may be used in normal-phase and reversed- phase mode, and there is a big chiral discriminating complementary. This work indicates that oligosaccharides could soon become very attractive as a new class of chiral stationary phase for HPLC.     

硅胶基质高效液相色谱填料研究进展

高效液相色谱(HPLC)不仅是一种有效的分析分离手段,也是一种重要的高效制备分离技术。色谱柱是HPLC系统的核心,不同性能的填料是HPLC广泛应用的基础。硅胶是开发最早、研究最为深入、应用最为广泛的HPLC固定相基质,其制备方法主要有喷雾干燥法、溶胶-凝胶法、聚合诱导胶体凝聚法及模板法等。近年来,亚2μm小粒径硅胶、核-壳型硅胶、双孔径硅胶、介孔性硅胶、有机杂化硅胶等新型硅胶应用于HPLC并取得了色谱分离技术的飞速发展,例如基于亚2μm填料的超高压液相色谱技术、基于核-壳型填料的快速分离技术、基于杂化硅胶填料的高温液相色谱技术等。硅胶经表面化学键合、聚合物包覆等有机改性可制得先进的大分子限进填料、温敏性填料、手性填料等,大大扩展了HPLC的应用范围。本文对液相色谱用硅胶的制备方法、改性与修饰方法以及硅胶基质固定相的评价方法加以系统综述,概述了新型硅胶在HPLC中的应用进展,并对硅胶基质填料的发展方向与应用前景进行了展望。     

Monolithic Silica for Fast HPLC: Current Success and Promising Future

As an approach for fast HPLC, monolithic silica has proven to be highly effective. It is especially successful for routinely obtaining fast isocratic HPLC analyses of small drug molecules. The low cost of monolithic compared with other approaches, such as UPLC, makes it more convenient for everyday application. It is also the more developed and widely applied technique compared with superficially porous particles. It offers the possibility for gaining high plate numbers through column coupling, but not at the expense of run time if a proper flow program is subsequently applied. Good precision and batch reproducibility are now achieved with commercially available monolithic silica columns. The application of monolithic silica columns is already well developed in various fields. It invades the field of bio-analysis and proteomics. Hundreds of analytical methods have already been successfully transferred to or developed on monolithic silica columns. An updated strategy based on Snyder??s method for rapid method development using monolithic column has been provided. However, more is still to be expected from monolithic silica in term of chemistry variation, application, and instrument compatibility. The future of monolithic silica is promising when considering the high demand for fast chromatographic analysis.     

核壳型二氧化硅色谱填料的研究进展

复杂样品的高效快速分离分析是分离科学家所面临的挑战。近年来,核壳型二氧化硅色谱填料以其高效、快速和低背压的特点被广泛用于小分子、大分子和复杂样品的快速分离分析。该文系统综述了二氧化硅核壳色谱固定相快速分离的机理,制备方法及其在小分子、多肽和生物大分子快速分离分析方面的应用,同时对核壳型色谱固定相的发展进行了展望。     

Preparation and application of rifamycin-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles as chiral stationary phase for high-performance liquid chromatography

Rifamycin-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (RCD-HPS), a new type of substituted β-cyclodextrin-bonded chiral stationary phase (CSP) for high-performance liquid chromatography (HPLC), have been synthesized by the treatment of bromoacetate-substituted-(3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (BACD-HPS) with rifamycin SV in anhydrous acetonitrile. The stationary phase is characterized by means of elemental analysis and Fourier-transform infrared spectroscopy. This new CSP has a chiral selector with two recognition sites: rifamycin and β-cyclodextrin (β-CD). The chromatographic behavior of RCD-HPS was studied with several disubstituted benzenes and some chiral drug compounds under reversed-phase HPLC mobile phase conditions. The results show that RCD-HPS has excellent selectivity for the separation of aromatic positional isomers and enantiomers of chiral compounds due to the cooperative functioning of rifamycin and β-CD.     

HPLC of basic drugs using non‐aqueous ionic eluents: evaluation of a 3 μm strong cation‐exchange material

HPLC columns packed with 3 μm particle size HPLC Technology Techsphere SCX (propylsulfonic acid‐modified) silica offer considerable advantages over 5 μm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 μm column was not only more efficient for a given column length compared with 5 μm materials, but also elution times were less, a phenomenon observed in reversed‐phase systems. The high efficiencies and excellent peak shapes obtained with the 3 μm SCX‐modified packing together with the relatively low back‐pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required. Copyright © 2009 John Wiley & Sons, Ltd.     

New stationary phase for anion-exchange chromatography

This work describes the preparation of an anion-exchange phase based on silica, using a two-step modification process. First, 10 microm Davisil silica particles were silanized with chloropropyltrimethoxysilane to yield chloropropyl silica. The modified silica was then reacted with pyridine to produce positively charged propylpyridinium groups on the surface, the anion-exchange sites. The phase was characterized by thermogravimetric analysis and infrared and solid state 13C and 29Si NMR spectroscopies. HPLC separations of common inorganic anions, including chloride, nitrite, bromide and nitrate, were performed using 150 x 3.9 HPLC columns packed with the phase, using a phthalate buffer solution as mobile phase with non-suppressed conductivity detection. Efficiency and resolution were calculated and the results show that the new phase has significant promise for the analysis of these anions in environmental samples.     

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.     

新型离子交换硅胶键合相的制备及评价

二甲基氯硅烷与硅胶表面反应,形成牢固的ＳｉＨ键之后,连接上活泼的中间体——烯丙基缩甘油醚作为柔软的分子臂,最后接上二乙基氨基,由此制得了新型的离子交换硅胶键合相。经漫反射红外光谱、元素分析和高效液相色谱法对键合相进行了鉴定和评价。结果表明：键合反应按预定路线进行,键合相具有较好的色谱性能。此种方法可有效地运用于无孔硅胶填料的制备。     

New Stationary Phase Prepared by Immobilization of a Copper-Amine Complex on Silica and Its Use for High Performance Liquid Chromatography

Chromatographic silica (10 μm) was chemically modified with the silylating agent: [3-(2-aminoethyl)aminopropyl]trimethoxysilane (AEAPTS). The reaction product was characterized by elemental analysis and infrared and ¹³C and ²⁹Si NMR spectra. The chemically modified silica was treated with Cu(II) in methanol medium. This cation was strongly adsorbed through complexation by the pendant ethylenediamine groups attached to the silica surface. The complex formed on the silica surface was shown to be stable in both aqueous and non-aqueous media. The aim of Cu(II) immobilization is to use this new material as a stationary phase in High Performance Liquid Chromatography (HPLC). Separations of synthetic mixtures of aromatic amines and of polyaromatic hydrocarbons were undertaken using 150×3.9 mm HPLC columns packed with the modified silica, with and without copper ions, to follow the influence of the cation on the chromatographic separation and to verify the efficiency of the new stationary phase for HPLC.     

Preparation of On-Column Frits in Packed Fused Silica Capillaries by Sol-Gel Technology

Existing methods for preparing frits in packed fused silica capillaries as used for electrochromatography and micro HPLC are not applicable to all silica based packing materials and involve a high thermal stress for both the stationary phase and the fused silica tubing including the polyimide coating. A new procedure for the production of such on-column frits under mild conditions by a sol-gel type reaction of polydimethoxysiloxane (PDMOS) is described in this paper. Reaction conditions were established for optimum mechanical stability and high permeability of the frits. Frits produced in this manner showed no noticeable effect on the overall efficiency.     

(3,5-二甲基苯基氨基甲酸酯)衍生的纤维素手性固定相

纤维素三（3,5-二甲基苯基氨基甲酸酯）是液相色谱中使用最广泛的手性柱。该文详细地研究了不同程度衍生的纤维素（3,5-二甲基苯基氨基甲酸酯）以及不同硅胶（粗制硅胶、氨丙基粗制硅胶、精制硅胶、氨丙基精制硅胶、大孔硅胶、氨丙基大孔硅胶）作为支撑体对该柱手性分离能力的影响。自制了13根手性色谱柱,分别考察了其对16种外消旋体的拆分,分离结果显示：三取代纤维素柱 > 二取代纤维素柱 > 纤维素柱;精制硅胶和大孔硅胶优于粗制硅胶,大孔硅胶的柱压更低;硅胶的氨丙基化对手性选择性有一定的影响;这些手性柱之间具有一定的互补性,尤其是纤维素柱。该文有助于人们更深刻地理解和更好地把握高效液相色谱手性柱的制备。     

HPLC determination of sulfamethazine in milk using surface-imprinted silica synthesized with iniferter technique

A new method for the synthesis of sulfamethazine-imprinted polymer on the surface of silica via quasi-living radical polymerization and the application of the resulting polymer in determination of the SMZ in milk is developed. In the synthesis, initiator-transfer agent-terminator (iniferter) was immobilized on the silica surface using chemical reagents with good availability. The imprinting polymerization was initialized by the silica-supported iniferter under the UV radiation. The molecularly imprinted polymer (MIP) layer grafted on the silica surface was constructed by using sulfamethazine (SMZ) as the template, methacrylic acid (MAA) as the functional monomer and ethylene dimethacrylate (EDMA) as cross-linker. The resulting MIP-silica has good selectivity for SMZ and high column efficiency in the HPLC analysis. The result demonstrated that the SMZ-imprinted polymer was grafted on the silica surface successfully. Under the optimized HPLC condition, the MIP-silica has been used for the determination of SMZ in milk. The method was linear over the concentration range of 0.1-50 microgmL(-1) with correlation coefficient R>0.999. The detection limit for SMZ was 25 ngmL(-1). The recoveries were above 78% at the spiked concentration of 0.024, 0.24 and 0.48 microgmL(-1).     

锆基色谱填料的制备方法和物理化学性质

氧化锆基质色谱填料适合于碱性物质,特别是生物大分子的分离,因而具有良好的应用前景。它的制备方法对其物理化学性质有很大的影响,从而影响填料的色谱性能。该文对氧化锆微球的制备方法及其物理化学性质进行了综述。分析表明,目前氧化锆基质色谱填料的制备方法存在着难以克服的缺点,要制备理想的锆基色谱填料需要新的思路。     

Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized high-resolution chromatographic and electrokinetic separations

Classical silica technology has reached its limit with respect to an ultimate minimum particle size of about 2 microm in diameter. Here, a novel process is presented which allows one to synthesize porous silica beads and control their particle diameter in situ, within the range of 0.2-2.0 microm. As a result, no sizing is required and losses of silica are avoided. Furthermore, the process enables one to control in situ the pore structural parameters and the surface chemistry of the silica beads. Even though surface funtionalized silicas made according to this process can principally be applied in fast HPLC the column pressure drop will be high even for short columns. In addition, the column efficiency, expressed in terms of the theoretical plate height is about H-2d(p) in the best case and limited by the A and C term of the Van Deemter equation. In other words the gain in total plate number when using 1-2 microm silica beads in short columns is minimal as compared to longer columns packed with 5 microm particles. Capillary electrochromatography (CEC) as a hybrid method enables the application of micron size as well as submicron size particles. This consequently enhances column efficiency by a factor of 5-10 when compared to HPLC. The use of short CEC columns packed with submicron size silicas provides the basis for fast and efficient miniaturized systems. The most significant feature of CEC as compared to HPLC is that the former allows one to resolve polar and ionic analytes in a single run. An alternative method for miniaturization is capillary electrophoresis (CE) which generates extremely high efficiencies combined with fast analysis. Its application, however, is limited to ionic substances.     

Electrokinetic chromatography for drug analysis. Separation and determination of cefpiramide in human plasma

Electrokinetic chromatography using a fused silica capillary and sodium dodecyl sulfate (SDS) solution has been applied to the separation and determination of cefpiramide (CPM) in human plasma with the use of antipyrine (AP) as an internal standard. A plasma sample was introduced into the capillary by siphoning. The calibration plot for CPM in plasma sample showed good linearity in the concentration range over 10 to 300 micrograms/ml. This method has advantages over usual high performance liquid chromatography (HPLC) in that it needs only a very small volume (less than 10 nl) of plasma without pretreatment, and an extremely high separation efficiency (10 times or much higher plate number than usual HPLC) is obtained. The addition of SDS to the supporting electrolyte solution enabled (1) rapid release of protein-bound drug which allowed the total concentration to be determined, (2) reproducible results to be obtained by suppressing adsorption of protein onto the fused silica capillary and (3) rapid separation of drug from proteins by selective retardation of protein peaks.