Magnetically immobilized frits for the preparation of packed columns used in capillary electrochromatography

Fabrication of porous frits to retain stationary phases is a critical issue in column preparation for capillary electrochromatography (CEC). In this work, porous frits were prepared by applying an external magnetic field to magnetically responsive particles placed inside a fused-silica capillary. Three batches of uniform magnetite spheres with particle diameters of 0.3, 0.4, and 0.6 μm and saturation magnetization values of 73.03, 74.41, and 77.83 emu/g, respectively, were used as frit particles and octadecyl- and phenyl-bonded silica gels were packed successfully into frit-containing capillaries. The performance of the resulting magnetically immobilized frits and packed columns was evaluated. The electroosmotic mobilities in capillaries containing outlet frit only were found to be reduced by 2–4% whereas the plate heights of an unretained marker increased by 30–50% as compared to those in open capillaries. These variations are believed to be associated with the inhomogeneities of the packed structure of the frits. The magnetically immobilized frits showed adequate mechanical strength to withstand the flow drag force, allowing separation in capillaries packed with 5-μm stationary phases up to 10–15 cm, thus rendering column efficiency and reproducibility comparable with those obtained with sintered frits. Taken together, retaining frits made of uniform magnetite particles serves as a viable alternative to sintered frits for column preparation, which offers several distinct advantages such as ease of preparation, improved durability as compared to sintered frits where the removal of the polyimide coating makes the packed column susceptible to breakage, and use of large-bore capillaries for semipreparative separations.     

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.     

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.     

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.     

Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis.

Reversed-phase nonporous silica (RP-NPS) of 1.5 microm dp is employed to demonstrate rapid and efficient separations in packed capillary electrochromatography (CEC). Two methods for packing capillaries and two techniques to manufacture frits used to hold the packing in place are evaluated for their effect upon separation performance using polyaromatic hydrocarbons (PAHs) and polar neutral pharmaceutical compounds. Attention is given to conditioning of the packed capillaries for high efficiency separations without necessity for sodium dodecyl sulfate (SDS). Separation conditions for the nonporous materials were modified from those previously determined on porous reversed-phase silica. Feasibility for method development and validation of a parent pharmaceutical compound and related impurities in the range of 0.1-120% of a 5 mg/mL concentration was assessed and reported. An approach to improving detection sensitivity through use of large-bore capillaries is briefly discussed.     

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.     

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.     

Development of a fritless packed column for capillary electrochromatography-mass spectrometry

A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.     

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．     

Silicate entrapped columns--new columns designed for capillary electrochromatography

Designed especially for capillary electrochromatography (CEC), silicate-entrapped columns are made by trapping particles of chromatographic packing material in a network of silica. Once entrapped, the capillary no longer requires frits. This renders a more homogeneous and stable packed bed. Accidental breakage of the fragile frits is not an issue with these robust columns. Columns packed with reverse-phase material subjected to silicate entrapment demonstrated faster separations of retained analytes and increased efficiencies compared with nonentrapped columns. The method was also used to prepare chiral CEC columns by entrapping a molecular imprinted polymeric (MIP) packing having minimal surface charge density, thus being unable alone to support sufficient electroosmotic flow for CEC.     

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.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.     

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.     

Photoinduced Polymerization for Entrapping of Octadecylsilane Microsphere for Capillary Liquid Chromatography

A short length of a sol‐gel monolith was initially prepared as the temporary frit in a 100 μm inner diameter fused‐silica capillary by an in situ photopolymerization. The packed 4 μm octadecylsilane particles were then immobilized within the identical sol‐gel solution through the same photopolymerization process. The prepared fritless capillary column was examined for the chromatographic performance by the self‐developed capillary liquid chromatography system. Baseline separation of the model analytes was achieved including thiourea, benzene, toluene and ethylbenzene with the lowest theoretical plate height about 66 μm for the retained component. A scanning electron micrograph was used to characterize the temporary frit and entrapped microspheres. The inorganic polymer matrix in the microsphere‐packed column functioned to link microspheres at specific sphere‐sphere and sphere‐capillary contact points. Furthermore, the stability and porosity of the fritless column were systematically investigated by a simple flow method.     

A simple procedure for the preparation of fritless columns by entrapping conventional high performance liquid chromatography sorbents

A rapid and direct method for immobilizing conventional high performance liquid chromatography (HPLC) packing material inside fritless capillaries has been developed. Due to the simple composition of the entrapment matrix (tetraethoxysilane, alkyltriethoxysilane, ethanol and water), straightforward manufacturing procedure and modest equipment requirement, the method can readily be transferred to any laboratory and easily automated. The entrapment procedure has minimal influence on the structure and chromatographic properties of the original reverse-phase sorbent. Various immobilization solutions have been tested, and a comparison between columns entrapped with different immobilization mixtures and conventional packed capillaries is presented. High efficiency separations were obtained using tert-butyl-triethoxysilane entrapped columns in both capillary electrochromatography (reduced plate heights of 1.1-1.4 were measured) and microliquid chromatography (reduced plate heights of 2.2-2.6 were observed) formats. Elimination of frits, stabilization of the packed bed and on-the-fly customization of column length render mechanically robust columns that are remarkably stable over time, from which manufacturing imperfections can be removed easily.     

Photopolymerized sol-gel frits for packed columns in capillary electrochromatography

Porous sol-gel frits are fabricated in a capillary column by filling it with a solution of 3-(trimethoxysilyl)propyl methacrylate, hydrochloric acid, water, toluene (porogen), and a photoinitiator (Irgacure 1800) and exposing it to UV light at 365 nm for 5 min. The separation column (30 cm x 75 microm I.D.) contains between the inlet and outlet frits a 15-cm packed segment filled with 5-microm silica particles modified with the chiral compound (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine. A detection window (1 mm long) is placed immediately after the outlet frit. To demonstrate the performance of this chiral separation column, mixtures of 16 different amino acids (three of which are not naturally occurring) derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole were separated by capillary chromatography. The enantiomeric separation of the column results in a resolution ranging from 1.21 to 8.29, and a plate height ranging from 8.7 to 39 microm.     

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.     

Direct observation of frits and dynamic air bubble formation in capillary electrochromatography using confocal fluorescence microscopy

Confocal fluorescence microscopy has been used to study the capillary electrochromatography (CEC) frits and dynamic air bubble formation under real chromatographic conditions. Confocal fluorescence microscopy provides a nondestructive way to view the three-dimensional structure of the frits with high spatial resolution. Frits prepared with four different procedures were studied: (1) sintering bare silica beads with sodium silicate; (2) sintering bare silica beads wetted with water; (3) sintering C18 beads wetted with water; and (4) sintering C18 beads wetted with water and then surfaced-recovered with C18. Frits prepared with sintering silicate-wetted beads have a high degree of heterogeneity, while the other three types of frits have similar, more homogeneous packing structures. Confocal fluorescence microscopy also provides sufficient temporal resolution for in situ observation of the dynamic processes in air bubble formation. In this study, air bubble formation is imaged during the reorganization process of the packing bed and is shown to occur close to the border between the packing bed and the outlet frit. Confocal fluorescence microscopy opens a new avenue in studying dynamic processes in situ in CEC separations.     

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.     

Preparation of the capillary-based microchips for solid phase extraction by using the monolithic frits prepared by UV-initiated polymerization.

A microfluidic solid phase extraction (SPE) array for sample enrichment was prepared by a simple method, a hot embossing technique. Five fused-silica capillaries (250 microm i.d., 380 microm o.d.) were partly embedded parallel in a polymethyl methacrylate (PMMA) microchip to serve as the extraction channels. Within each of the channels, a 2-mm-long monolithic porous polymer was prepared by in-situ photoinitiated polymerization. This then acted as the frit for packing of the extraction materials (octadecylsilica beads, ODS). By defining the light-exposure window on the channels, one can easily control the length and location of the polymer frits and the ODS beads can be packed at the desired location. With this method, solid phase extraction channels for microfluidic use can be easily prepared without complex fabrication of microstructures. Several SPE channels can be conveniently made in one microchip since the frits can be prepared in different channels through one polymerization; packing of the different channels can also be performed simultaneously. With the use of dilute ephedrine solutions, the sample loading capacity, linearity, and reproducibility were characterized. Coupled with the fast capillary electrophoresis separation, this microchip SPE array was applied for the detection of ephedrines in human urine.