A Simple Method to Prepare On‐Capillary Frits for Micro‐Column Separation

Sintering stainless steel powders was initially used to prepare the inlet frit in fused silica capillaries. The use of such inlet frits and outlet frits sintered by the stationary phase itself in the capillary to retain C₁₈ particles was demonstrated to withstand the long exposure, up to a high pressure of 60 MPa, for packing and the prepare column was stable and robust enough to do the continuous chromatographic operations. Characterization of the inlet and outlet frits by scanning electron micrography showed the fused metal particles formed a porous network in the capillary inlet and the homogenous separation beddings were obtained by slurry packing.     

Fritless capillary electrochromatography

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.     

磁场辅助毛细管电色谱

磁场辅助毛细管电色谱是液相色谱研究领域中出现的新技术．它利用外加磁场的引力将置于毛细管内的具有磁响应性的硅胶微球或四氧化三铁微球固定在管内任意位置．磁场固定微球聚集体既可用作填充柱,直接用于电色谱分离;也可用作柱筛,用于填装由商品色谱填料组成的色谱柱．这一技术的优势在于制备简便易行,柱管可以再生使用,适合于微流控芯片上柱筛或柱床的制作．本文简要评述磁场辅助毛细管电色谱的进展,包括磁性色谱填料的制备,磁场固定柱床电色谱,磁性柱筛电色谱及毛细管柱内柱结构参数的测定等方面．     

Capillary electrochromatography without external pressure assistance. Use of packed columns with a monolithic inlet frit

A fused silica capillary column was packed with RP(18) silica stationary phase entrapping the particles between two frits obtained by two different procedures. The inlet frit consisted of a short organic polymer made via a thermopolymerization process while the outlet frit was prepared by sintering the octadecylsilica (ODS) material. The packed column was employed in capillary electrochromatography (CEC) experiments for the separation of three selected test compounds. Retention time and separation efficiency were evaluated. Results were compared with those ones obtained with a packed capillary containing the same stationary phase entrapped between two sinterized frits. The novel packed column exhibited comparable separation efficiency and resolution with the traditional one. However, it allowed experiments without pressure support during the runs with no bubble formation.     

New method for sintering silica frits for capillary microcolumns

One of the main steps in the manufacture of robust and efficient packed capillary microcolumns for electro- and capillary chromatography is the generation of porous devices to retain the packed beds. Frits based on sintered silica particles have been found to give the best results in terms of mechanical resistance and efficiency. The conventional procedure to produce these kinds of frits consists in a radial heating of the packed material with either a flame or an electrical resistance, but the frits thus obtained have many drawbacks as a result of the procedure rather than the silica per se as the base material. In the present work we investigated a new approach to produce silica-based retaining devices involving the frontal exposure of a short silica-particle bed packed at the end of a capillary tube. The capillary is radially insulated and frontally exposed to the heat of a muffle oven, generating a transfer of heat that is not radial but rather throughout the capillary axis. This procedure resulted in substantial advantages: an improved radial homogeneity, a protection of the external polyimide, and a generation of extremely short (400–600 μm) frits that were highly permeable and avoided bubble formation.     

Novel fabrication of on-column capillary inlet frits through flame induced sintering of stainless steel particles

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5-1 mm and capillaries with inner diameter about 50-100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C₁₈ particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.     

Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.     

Disposable microcolumns with welded metal frits

This study reports the preparation of disposable microcolumns with welded metal frits for the first time. First, the bottom of glass‐lined stainless‐steel tubing of 30 cm length, 1.6 mm od, and 0.5 mm id was welded with a stainless‐steel screen frit of 1.6 mm diameter. A micro‐welding machine was used for this. Next, the column was connected to a slurry packer and packed with porous silica particles. Then, the inlet of the column was carefully welded with another frit. The column was tested for separation of a test mix composed of phenol, 2‐nitrophenol, acetophenone, aceanilide, and benzamide. Another column of the same physical dimension was also prepared with frits that were not welded to the column. The chromatographic performances of the two groups of columns (welded frits versus non‐welded frits) were examined. The columns of welded frits showed ca. 18% better separation efficiency (number of theoretical plates) than those of non‐welded frits.     

The Development of Reversed‐Phase Capillary Electrochromatography for the Separation of Steroids

This paper describes the preparation and optimization of packed capillary columns for reversed‐phase separation of steroids with CEC. The fabrication of on‐column frits is considered to be the most important step for obtaining a reproducible packed column for CEC separation. Porous silicate frits were generated in a fused‐silica capillary by heating the silica gel/sodium hydroxide solutions electrically. The optimized conditions involve silica gel (10.8%), sodium hydroxide (5.8%), and heating time (5 sec) with heating voltage (5V) for obtaining a 100‐μ end‐frit that can withstand pressure over 6000 psi. A HPLC pump was utilized to pack the 5‐μm ODS particle slurry into the capillary column. The ODS packed capillaries were then utilized for the separation of four anabolic cholesterols with a capillary electrophoresis system without pressurization of the column. The reproducibility of the packed columns was evaluated by measuring the relative standard deviations of four steroids. The relative standard deviations of migration time for column‐to‐column, day‐to‐day, and run‐to‐run are less than 7%, 2%, and 1% for four steroids, respectively.     

Application of an in-situ thermo-polymerized porous polymer: creation of an on-column frit for a packed capillary HPLC column.

A 3-mm length of a porous monolithic polymer was prepared in a 0.32-mm inner-diameter fused-silica capillary by an in-situ thermo-polymerization method and used as an on-column frit for a packed capillary HPLC column. The on-column frit can resist high pressure up to 400 bar. A 5-microm packing material was packed in the capillary with the on-column frit by a slurry method. At pressure driving mode, separation of samples was performed using the capillary HPLC column. The in-situ frit preparation method has the advantages of easy preparation, easy control of the location of the frit and a mild preparing reaction condition.     

Comparison of different types of outlet frits in slurry‐packed capillary columns

Fused‐silica capillary columns for high‐performance liquid chromatography with 320 and 250 μm inner diameter were prepared by slurry packing with 5 and 3 μm Nucleosil C₁₈ stationary phase. Different types of mechanical and monolithic outlet frits were used and their influence on the resulting column performance was evaluated. Columns with quartz wool exhibited symmetrical peaks and low theoretical plate height, and the preparation time was short. The performance of monolithic frits varied based on type of monolith, length of the frit, and silanization procedure. The best frit performed similarly to the quartz wool ones, but the preparation took several hours. Their main advantage lies in the possibility of on‐column detection, because the detection window can be burnt immediately behind the frit.     

Polyether ether ketone encased monolith frits made of polyether ether ketone tubing with a 0.25 mm opening resulting in an improved separation performance in liquid chromatography

Tiny polyether ether ketone encased monolith frits have been prepared by modified catalytic sulfonation of the inner surface of polyether ether tubing (1.6 mm od, 0.25 mm id) followed by modified formation of organic monolith and cutting of the tubing into slices. The frit was placed below the central hole of the column outlet union and supported by a combination of a silica capillary (0.365 mm od, 0.05 mm id) and a polyether ether ketone sleeve (1.6 mm od, 0.38 mm id) tightened with a nut and a ferrule when the column was packed to prevent sinking of the frit element into the union hole (0.25 mm opening) otherwise. The column packed this way with the frits investigated in this study has shown better separation performance owing to the reduced frit volume in comparison to the column packed with a commercial stainless‐steel screen frit. This study establishes the strategy of disposable microcolumns in which cheap disposable frits are used whenever the column is re‐packed to yield columns of even better chromatographic performance than the columns with commercial frits.     

Particulate capillary precolumns with double-end polymer monolithic frits for on-line peptide trapping and preconcentration

In this work,a novel kind of particulate capillary precolumns with double-end polymer monolithic frits has been developed.Firstly,the polymer monolithic frit at one end was prepared via photo-initiated polymerization of a mixture of lauryl methacrylate and ethyleneglycol dimethacrylate with 1-propanol and 1,4-butanediol as porogens and 2,2-dimethoxy-2-phenylacetophenone as a photo-initiator in UV transparent coating capillary(100 μm i.d.).Subsequently,C18 particles(5 μm,100 A) were packed into the capillary,and sealed with the polymer monolithic frit at another end.To prevent the reaction of monomers and C18 particles,the packed C18 particles were masked during UV exposure.The loading capacity of such a precolumn was determined to be about 9 μg by frontal analysis with a synthetic peptide APGDR1 YVHPF as a model sample.Furthermore,two parallel precolumns were incorporated into a two-dimensional nano-liquid chromatography(2D nano-LC) system with dual capillary trap columns for peptide trapping and concentration.Compared to 2D nano-LC system with a single trap column,such two dimensional separations could be operated simultaneously to improve the analysis throughput.All these results demonstrated that such capillary precolumns with double frits would be promising for high-throughput proteome analysis.     

Single step on-column frit making for capillary high-performance liquid chromatography using sol-gel technology

One step frit-making in packing fused-silica capillary column of high-performance liquid chromatography was developed using sol-gel technology. Frit fabrication procedure was quite simple without sintering. On-column frit was formed through gelling of sol solution with packing materials, silica gel, and jointing the particles together with capillary wall through bonded and immobilized networks. Solvent types and proportions in sol solution were selected. And the sol solution compositions as well as amount of silica gel particles were also optimized to achieve maximum strength. Such an on-column frit of 250 microm in diameter is capable of resistant packing pressure up to 500 bars in ultra-sonic bath action. Chemical resistance to solvents and extreme pHs were also tested. Scanning electron micrograms of the frit profile showed that the evolving sol-gel network joined particles to each other and onto the column wall. Routine runs in reversed-phase mode, the frits of several columns proved to be effective enough to resist pressures without collapse.     

Towards the column bed stabilization of columns in capillary electroendosmotic chromatography. Immobilization of microparticulate silica columns to a continuous bed

This article discusses a novel method generating a continuous bed inside the CEC column. The column bed composed of microparticulate reversed-phase silica is completely immobilized by a hydrothermal treatment using water for the immobilization process. This process eliminates the manufacture of frits of both ends of the column and all problems associated with their preparation. Fundamental studies on operational parameters will be presented such as the dependence of the immobilization on the column temperature, the type of stationary phase and the column back pressure. The immobilized CEC columns show the same high column efficiency as packed columns with frits.     

Fritting techniques in chromatography

It is surprising that there has been no devoted review article for frits and relevant studies so far despite the long history of packed columns and the use of frits in them. This review was activated for such a reason. Both separate frits and in situ permanent frits have been covered since the appearance of primitive frits. The in situ fritting methods such as the formation of organic monoliths, sol–gel technology, sintering, fritless techniques such as tapered tip and capillary restrictors, and miscellaneous fritting techniques including magnetically trapped frits and single particle frits are introduced and discussed. In addition, frit‐related studies and patents are also introduced. Finally, some conclusive comments on the choice of fritting technique in different situations and future perspectives are given.     

Preparation of fritless capillary using avidin immobilized magnetic particles for electrochromatographic chiral separation

In capillary electrochromatography (CEC), magnetic particles (MPs) were packed in a fused silica capillary by using the magnetic field to be retained without frits. For a chiral CEC separation, avidin was immobilized onto the surface of the MPs (AVI-MPs) as a stationary phase by using the physical adsorption technique. The injected AVI-MPs into the capillary were stably captured with the magnet (surface magnetic flux density, 250 mT) under the separation voltage of 10 kV (190 V/cm). By employing the fritless AVI-MPs packed capillary, the chiral separation of ketoprofen was successfully attained with the packing length of only 5 cm. Effects of the modification condition of avidin, pH of background solution, and the packing length on the enantioseparation were also investigated. Under the optimal condition, furthermore, the repeatability for the retention time of ketoprofen was better than 1.5% in the relative standard deviation and the capillary-to-capillary reproducibility was also acceptable in the prepared fritless capillaries.     

Molecularly imprinted magnetic nanoparticles as tunable stationary phase located in microfluidic channel for enantioseparation

A microfluidic device integrated with molecularly imprinted magnetic nanoparticles as stationary phase was designed for rapid enantioseparation by capillary electrochromatography. The nanoparticles were synthesized by the co-polymerization of methacrylic acid and ethylene glycol dimethacrylate on 3-(methacryloyloxy)propyltrimethoxysilane-functionalized magnetic nanoparticles (25-nm diameter) in the presence of template molecule, and characterized with infrared spectroscopy, thermal gravimetric analysis, and transmission electron microscope. The imprinted nanoparticles (200-nm diameter) could be localized as stationary phase in the microchannel of microfluidic device with the tunable packing length by the help of an external magnetic field. Using S-ofloxacin as the template molecule, the preparation of imprinted nanoparticles, the composition and pH of mobile phase, and the separation voltage were optimized to obtain baseline separation of ofloxacin enantiomers within 195 s. The analytical performance could be conveniently improved by varying the packing length of nanoparticles zone, showing an advantage over the conventional packed capillary electrochromatography. The linear ranges for amperometric detection of the enantiomers using carbon fiber microdisk electrode at +1.0 V (vs. Ag/AgCl) were from 1.0 to 500 μM and 5.0 to 500 μM with the detection limits of 0.4 and 2.0 μM, respectively. The magnetically tunable microfluidic device could be expanded to localize more than one kind of template-imprinted magnetic nanoparticles for realizing simultaneous analysis of different kinds of chiral compounds.     

Use of a fritless dual tapered column and a low flow interface for capillary electrochromatography-mass spectrometry

To avoid problems associated with the use of sintered frits to retain packing material, tapered columns were investigated for use with capillary electrochromatography-mass spectrometry (CEC-MS) analysis. Taking the advantage that negatively charged stationary phase particles have a net velocity directed towards the buffer reservoir (inlet) over a wide range in pH, a fritless CEC column with a single taper tip was prepared for CEC-MS analysis. During CEC-MS analysis, the tapered end was immersed in the buffer reservoir and the unmodified end was pointed toward the ionization source. For better sensitivity, this single tapered CEC column was coupled to ESI/MS using a low flow sheath liquid interface. With this setup, occasional blockage of the ESI sprayer by stationary phase particles was observed. In addition, significant dead volume was observed because the unmodified tip could not be inserted into the very end of the sprayer of the low flow sheath liquid interface. To circumvent these problems, a dual tapered CEC column was prepared. This fritless dual tapered column CEC-MS approach alleviated the problems of frit, sprayer blockage and extensive dead volume.     

Radial heterogeneity of some analytical columns used in high-performance liquid chromatography

An on-column electrochemical microdetector was used to determine accurately the radial distribution of the mobile phase velocity and of the column efficiency at the exit of three common analytical columns, namely a 100 mm × 4.6 mm C18 bonded silica-based monolithic column, a 150 mm × 4.6 mm column packed with 2.7 μm porous shell particles of C18 bonded silica (HALO), and a 150 mm × 4.6 mm column packed with 3 μm fully porous C18 bonded silica particles (LUNA). The results obtained demonstrate that all three columns are not radially homogeneous. In all three cases, the efficiency was found to be lower in the wall region of the column than in its core region (the central core with a radius of 1/3 the column inner radius). The decrease in local efficiency from the core to the wall regions was lower in the case of the monolith (ca. 25%) than in that of the two particle-packed columns (ca. 35–50%). The mobile phase velocity was found to be ca. 1.5% higher in the wall than in the core region of the monolithic column while, in contrast, it was ca. 2.5–4.0% lower in the wall region for the two particle-packed columns.