Ground, sieved, and C18 modified monolithic silica particles for packing material of microcolumn high-performance liquid chromatography

We here report a new type of stationary phase for microcolumns. C18 modified silica monolith particles were prepared by grinding and sieving the silica monolith followed by C18 modification and end-capping, and were used as packing material. Ground silica monolith particles were not spherical but irregular with some residual monolithic network structure. The separation efficiency of the stationary phase made of sieved monolith particles (5-10 microm) was better than that of the stationary phase made of unsieved particles. The microcolumn packed with the sieved C18 ground monolith particles (5-10 microm) showed quite good separation efficiency (height equivalent to theoretical plate, HETP, as low as 15 microm) and it was even superior to the microcolumn packed with a commercial spherical 5 microm C18 stationary phase. The column pressure drop of C18 monolith particles was about two-third of that of the commercial spherical C18 phase. The preparation method of C18 stationary phase with ground and sieved silica monolith particles presumably suggests advantages of simplicity and convenience in modification and washing procedures compared to bulk silica monolith. It also showed both improved separation efficiency and low back pressure.     

Cyano bonded silica monolith—Development of an in situ modification method for analytical scale columns

This study investigates the synthesis and chromatographic behaviour of an analytical size cyanopropyl “cyano” bonded silica monolith. Surface modification was undertaken by treating a neat silica monolith with chloro(3-cyanopropyl)dimethyl silane in dry heptane over a two day period. The resulting monolith showed stability over the duration of the testing program that involved flushing the column with more than 2000 column volumes of mobile phase. Efficiency measurements before and after sylation verified that the integrity of the silica monolith itself was not affected by the modification process, the highest number of theoretical plates (N/m) using anisole was 81,650. A brief selectivity test was then undertaken to assess methylene selectivity and phenyl selectivity. Elemental analysis was used to determine the homogeneity of the carbon load throughout the monolithic bed, and was compared to two commercial C18 and one ‘self’ modified C18 silica monoliths. The development of the in situ modification is also discussed.     

金纳米粒子修饰毛细管硅胶整体柱的制备及其电色谱性能研究

通过毛细管硅胶整体柱表面修饰十八烷基硫醇金纳米粒子,制备了一种新型毛细管电色谱固定相.制备金纳米粒子修饰整体柱时,采用溶胶-凝胶法制备毛细管硅胶整体柱,并在其表面化学修饰3-巯基丙基三甲氧基硅烷;通过巯基基团固载金纳米粒子于整体柱上,再共价键合十八烷基硫醇于金纳米粒子表面.以甲苯为探针,对理论塔板高度与流动相线速度之间...     

Determination of Atenolol in Human Plasma by Pseudo Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry

Previous HPLC determination of atenolol on reversed-phase packings has often required a mobile phase containing three components: organic modifier, buffer and ion-pairing reagent or organic amine. In addition to the complexity of the eluents employed, alkyl sulphonates and organic amines in the mobile phase can reduce the life of silica-based bonded columns. A new simple, rapid and sensitive method—pseudo reversed-phase liquid chromatography/tandem mass spectrometry has been developed for the analysis of atenolol in human plasma using bare silica as the stationary phase coupled with a simple mobile phase consisted of 5% acetonitrile and 95% formate buffer. The optimization of separation is fast and easy. The assay was validated for the concentration range 1–100 ng mL^?1 with a detection limit of 1 ng mL^?1. Moreover, the silica column was durable with the mainly aqueous eluents. No obvious loss in performance was observed for 30,000 column volumes of eluent.     

Preparation of an ionic liquid-functionalized polymer monolith and its application in the separation of Chinese herb with HPLC

A porous functionalized monolithic material based on ionic liquids (ILs) was produced through in situ polymerization within the confines of a stainless steel column (50 × 4.6 mm i.d.). In the processes, 1-vinyl-3-butylimidazolium chlorine ionic liquid, 1-dodecylene, and butyl methacrylate were used as ternary monomers; ethylene dimethacrylate as the cross-linker; azobisisobutyronitrile as the initiator; and dodecanol as the porogen. The optimized monolith showed high permeability as 13.54 × 10^?14 m², and high porosity as 75.08%. Then, its chromatographic characteristic was estimated by being used as the stationary phase of high-performance liquid chromatography (HPLC) to separate the mixtures of aromatic series compounds. Finally, the monolith was used to separate gastrodin from Chinese herb gastrodia rhizome; benzene and biphenyl from the effluent water, respectively. The column efficiency of the obtained IL-based monolith was calculated by the gastrodin peak as 22,000 plates m^?1. Moreover, the repeatability of the method was studied with the RSDs calculated by retention times and peak areas of gastrodin peak as 0.79%, 1.23% (run-to-run, n = 6) and 0.86%, 1.89% (column-to-column, n = 6), respectively. The results confirmed that the produced monolith was successfully used as the stationary phase of HPLC to separate small molecules in real samples with high performance.     

Preparation of a Polymer Monolithic Column Using Ionic Liquid as Porogen and Its Application in Separations of Proteins and Small Molecules

A polymer-based monolithic column was prepared for high-performance liquid chromatography (HPLC), using ionic liquids as porogen within the confines of a stainless steel column (50 × 4.6 mm i.d.). In the process, 1-butyl-3-methylimidazolium chloride and dodecyl alcohol were used as bi-porogens, vinyl ester resin as the monomer, ethyleneglycol dimethacrylate as the crosslinker, CCl₄ as the initiator, and FeCl₂ as the catalytic agent to prepare the polymer-based monolithic column. Scanning electron microscopy, nitrogen adsorption–desorption instrument, and mercury intrusion porosimetry were used to assay the characteristics of the monolith, respectively. The optimized monolith showed uniform structure and good permeability. Then, the column was used as the stationary phase of HPLC to separate standard proteins and human plasma with gradient elution. Besides, the monolith was used to separate aromatic compounds from the mixture. The results showed that the addition of IL could effectively improve the structure of monoliths prepared by atom transfer radical polymerization technique. The results also suggested that this kind of monolith could be used as a simple, cheap and effective stationary phase for HPLC.     

Electrochromatographic performance of conventional and polar-embedded C16 silica monolithic stationary phases

A novel monolithic silica column that has a polar‐embedded amide‐secondary amine group linking with C16 functionality for RP‐CEC is described. The amide‐secondary aminealkyloxysilane was synthesized by the reaction of 3‐(2‐aminoethylamino) propyltrimethoxysilane with hexadecanoyl chloride. Then, the silylant agent was bonded to the silica monolith matrix to produce hexadecanamide‐secondary amine bonded silica (HDAIS) monolithic column. The electrochromatographic performance of HDAIS monolithic column for the separation of neutral, basic and polar solutes was studied, which was compared to that using the hexadecyl bonded silica monolithic column. The HDAIS monolithic column displayed reduced hydrophobic retention characteristics in the separation of five hydrophobic n‐alkylbenzenes and four polar phenols when compared to the hexadecyl bonded silica monolithic column. A very much reduced silanol activity of HDAIS monolithic column was observed in the separation of test basic mixture including four aromatic amines, atenolol and metoprolol with 10 mM borate buffer (pH 7.5) containing 30% v/v ACN as the mobile phase. The comparison results indicate good performance for both polar and basic mixtures on HDAIS monolithic column in RP‐CEC, and also show promising results for further applications.     

Silica‐based zwitterionic monolithic stationary phase for separation of neutral and ionized solutes using pressurized CEC

A porous zwitterionic monolith was prepared by in situ covalent attachment of lysine to a γ‐glycidoxypropyltrimethosysilane‐modified silica monolith. The prepared column was used to perform neutral and ionized solutes separations by pressurized (pCEC). Due to the zwitterionic nature of the resulting stationary phase, the monolithic column provided both electrostatic attraction and repulsion sites for electrochromatographic retention for ionized solutes. Separation of several nucleotides was investigated on the monolithic column. It was shown that the nucleotides could be separated based on hydrophilic and electrostatic interactions between the stationary phase and analyte. Besides, the separation property of the zwitterionic silica monolith was compared with the use of diamine‐bonded silica monolith as stationary phase. As expected, the lysine monolith exhibited a lower retention for the five nucleotides, which was due to the dissociation of the external carboxylic acid groups, leading to electrostatic repulsion with negatively charged solutes. Under the same experimental conditions, separation of the five nucleotides on the zwitterionic column was in less than 8 min, while that on the diamine column was in approximately 60 min.     

Preparation and Characterization of a Low-Density Imprinted Monolithic Column

This work is a first effort to prepare a low-density imprinted monolithic column for LC. The new molecularly imprinted polymer monolith was prepared with 30% (w/w) monomers in the pre-polymerization mixture while general high-density monoliths are prepared with 40% monomers. The resulting low-density (S)-naproxen-imprinted monolith produced better chiral resolution of rac-naproxen (R _s = 1.55) and column efficiencies of imprinted molecules up to 2,860 plates m^?1. Morphological characteristics of low density imprinted monolith was further studied by mercury intrusion porosimetry, scanning electron microscopy (SEM) and nitrogen sorption method. The experiments demonstrated that the strategy of low-density column may be one of a potential approach to the improvement of column efficiency of an imprinted stationary phase.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Preparation and evaluation of tandem stationary phases for μ-HPLC and capillary electrochromatography

Tandem separation beddings were prepared within the capillary tube with a photopolymerized monolith initially formed by sol-gel technology in combination with microspheric octadecylsilane material by slurry packing. The chromatographic performance of the tandem stationary phases was evaluated in detail by varying the flow rate and the composition of the mobile phase using a self-installed capillary HPLC system. For the tandem stationary phases with a forepart monolithic length of 11 cm and 1 cm the lowest theoretical plate height for the retained component was 26 µm and 34 µm, respectively. After evaluation by capillary electrochromatography, enhanced chromatographic performance was obtained using a column with 1 cm monolithic inlet frit with a theoretical plate height up to 7.20 µm. A scanning electron micrograph with different cross-sections of the column showed that a porous network formed in the center of the capillary and a homogenous slurry packing of C₁₈ was obtained at the back part.     

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx? monolithic C₁₈, 100 mm?×?4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm?×?3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm?×?4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

Study of RP HPLC Retention Behaviours in Analysis of Carotenoids

For determination of selected carotenoids, various types of columns for high-performance liquid chromatography (HPLC) with different properties have been used. The characteristics of the laboratory-used packing material containing monomeric alkyl-bonded phases (C₁₈, C₃₀) and phenyl as well as phenyl-hexyl stationary phases were studied. The retention data of the examined compounds were used to determine the hydrophobicity and silanol activity of stationary phases applied in the study. The presence of the polar and carboxyl groups in the structure of the bonded ligand strongly influences the polarity of the stationary phase. Columns were compared according to methylene selectivity using a series of benzene homologues. The measurements were done using a methanol–water mobile phase. Knowledge of the properties of the applied stationary phase provided the possibility to predict the RP HPLC retention behaviours in analysis of carotenoids including lutein, lycopene and β-carotene. The composition of the mobile phase, the addition of triethylamine and the type of stationary phase had been taken into account in designing the method of carotenoid identification. Also a monolithic column characterised by low hydrodynamic resistance, high porosity and high permeability was applied. The presented results show that the coverage density of the bonded ligands on silica gel packings and length of the linkage strongly influence the carotenoid retention behaviours. In our study, the highest retention parameters for lutein, lycopene and β-carotene were observed for C₃₀ and C₁₈ stationary phase. This effect corresponds with pore size of column packing greater than 100 Å and carbon content higher than 11 %.     

Preparation and characterization of a new HPLC C18 reversed phase containing thiocarbamate groups

A HPLC stationary phase that possesses an internal thiocarbamate functional group is described. The new C18-thiocarbamate silane was synthesized by the reaction of a trifunctional alkoxysilane with a mercaptan. The silylant agent was bonded to silica (5 μm) and the new stationary phase was then endcapped. Surface characteristics of the packing before and after chemical modification with HMDS and TMCS were determined by different physico-chemical methods, such as elemental analysis and infrared and solid-state ¹³C and ²⁹Si nuclear magnetic resonance spectroscopies. Chromatographic properties of the C18-thiocarbamate silica were evaluated under reversed phase conditions by separation of four different test mixtures that including compounds from the Engelhardt, Tanaka, and Neue test mixtures. Chromatographic evaluations of the C18-thiocarbamate phase show promising results for the separation of basic analytes.     

Investigating retention characteristics of a mixed‐mode stationary phase and the enhancement of monolith selectivity for high‐performance liquid chromatography

The synthesis and chromatographic behavior of an analytical size mixed‐mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3‐cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π‐type bonding within a single separation environment. Selectivity studies were undertaken using n‐alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π‐type selectivities afforded by the two different ligands on the mixed‐mode column.     

Custom-Made C18 Column for Simultaneous Determination of Endocrine Disrupting Substances in Water by Diode-Array and Fluorescence Detectors

A C₁₈ stationary phase was synthesized for a custom-made HPLC column. When compared to a commercial C₁₈ column, better chromatographic performances were obtained. This column was successfully applied for simultaneous determination of p,p′-DDT, o,p′-DDT, benzo(a)anthracene, benzo(b)fluoranthene, and benzo(a)pyrene in waters by high performance liquid chromatography coupled with dual detectors (diode array and fluorescence detectors) combined with solid phase extraction. Low method detection limits were obtained, i.e., p,p′-DDT: 0.5 µg L^?1, o,p′-DDT: 1 µg L^?1, benzo(a)anthracene: 2.5 ng L^?1, benzo(b)fluoranthene: 5 ng L^?1, and benzo(a)pyrene: 2.5 ng L^?1. High recoveries that ranged from 82 to 94% were obtained for all compounds.     

一种内嵌三嗪环酰胺极性基团新型固定相的制备及其在碱性化合物分离中的应用

以γ-氨丙基三乙氧基硅烷为偶联剂,三聚氯氰为反应物,采用固液表面连续反应法,依次与乙二胺、十二酰氯进行亲核取代反应,制备了一种嵌入三嗪环酰胺极性基团的新型反相色谱固定相,并采用元素分析法进行了表征。用制备的固定相装填色谱柱,以商品化C18色谱柱作为参考,对比考察了碱性化合物的分离情况。结果表明,极性三嗪环酰胺基团被成功地键合到硅胶表面,连续制备3次所得固定相的C、N、H含量的最大相对偏差均小于5%,说明制备工艺重现性良好;用制备的固定相装填的色谱柱分离5种苯胺类、4种吡啶类碱性化合物的选择性好,峰形对称。该结果为进一步推进该新型固定相的商品化提供了参考数据。     

Fast HPLC for quality control of Harpagophytum procumbens by using a monolithic silica column: method transfer from conventional particle-based silica column

The applicability of a monolithic C18-bonded silica column for the rapid HPLC separation of ingredients in medicinal plants and their phytopharmaceutical preparations has been evaluated in the author's laboratory. In this presentation, an existing method for the determination of the iridoid glycoside harpagoside in Harpagophytum procumbens (Devil's Claw) was successfully transferred from a conventional particle-based C18 silica column to a monolithic silica column. The very high porosity of the stationary phase allows chromatography with a much lower backpressure than on conventional columns. Therefore, the flow rate could be easily increased from 0.8 mL/min (particle-based column) to 5 mL/min (monolithic column) and the run-time reduced from 30 to 5 min (that is a reduction about 85% !), without losing any chromatographic resolution of the compound of interest. The amount of harpagoside was measured with the original method on a conventional particle-based silica column and on the adapted method on a monolithic silica column. The statistical mean t-test showed no significant differences of the variances and the means indicating that the fast HPLC method is an acceptable alternative. The shorter analysis time makes the method very valuable for commercial quality control of Harpagophytum extracts and its pharmaceutical preparations.     

Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ～250 Å and macropores of ～?1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ～300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene–divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.     

Faster axial band dispersion in a monolithic silica column than in a particle-packed column

The contribution of molecular diffusion to peak broadening was studied in a reversed-phase HPLC system, consisting of a monolithic silica C18 column and methanol-water mobile phase. Study on the band broadening effect of holding a solute in a column or elution at very low linear velocity of mobile phase allowed facile determination of the contribution of the molecular diffusion term. Less obstruction against molecular diffusion, or the faster axial band dispersion in a monolithic silica column than in a particle-packed column, was found both in mobile phase and in stationary phase.