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.     

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.     

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.     

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.     

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.     

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.     

Capillary electrochromatography with segmented capillaries for controlling electroosmotic flow

Capillaries consisting of two segments each packed with a different stationary phase were introduced for the control and manipulation of the electroosmotic flow (EOF) in capillary electrochromatography (CEC). This kind of column configuration was called segmented capillary where one segment was packed with octadecyl silica (ODS) and served as the separation segment while the other segment was packed with bare silica and functioned as the EOF accelerator segment. The average flow in the segmented capillary increased linearly with increasing fractional length of the EOF accelerator segment, and consequently the analysis time was reduced. Under a given set of conditions, the average flow can be varied over a certain range that extends from the EOF in the individual ODS capillary at the lower end to the EOF in the individual bare silica capillary at the higher end. The pore size of the bare silica in the EOF accelerator segment influenced the average flow in the segmented capillary. Because of the difference in the EOF of the individual segments, the average flow across the segmented capillary is partially degenerated from EOF to viscous flow. Furthermore, the retaining frits in CEC columns are restrictive points which slow down the average flow, thus furthering the degeneration of the flow from EOF to viscous flow. In other words, in CEC columns containing retaining frits, the flow of the mobile phase is not only based on electroosmosis but is contaminated by a viscous component.     

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.     

Fluorescence imaging of frit effects in capillary separations

The gradient of five dansylated amino acids in a capillary-based separation system commonly used in capillary liquid chromatography (LC) or capillary electrochromatography (CEC) was followed and examined in detail using a detection method based on laser-induced fluorescence imaging. The detection system consisted of an XeCl excimer laser and an image-intensified charge-coupled device (CCD) camera. Fluorescence intensity profiles along the capillary column were displayed and continuously updated on a computer screen. The detector system enabled the separation dynamics in the column to be monitored. The experiments were focused on the course of events, especially at interfaces. The processes occurring at the beginning of the reversed-phase packing material were studied as well as at the transition from stationary phase to the outlet frit and the open tubular area. Striking differences in signal intensity and separation efficiency were revealed depending on where on the column detection was performed. Furthermore, adsorption of the analytes on the frits was observed.     

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.     

A Simple Design to Realize Micro-column Separation by Conventional Analytical HPLC

The conventional analytical HPLC was successfully developed for micro‐column separation by using a simple eluate splitting system, self‐preparation of packing column and on‐capillary column detector in our laboratory. Porous inlet frit in fused silica capillary was rapidly prepared by sintering stainless steel powders under 500 meshes for about 20 s. The use of such frits or metal meshes in capillary to retain C₁₈ particles of chromatographic packing was demonstrated to be stable and specially robust with continuous packing and long chromatographic runs. Furthermore, the chromatographic behavior was detailedly evaluated by changing the flow rate and the percentage of mobile phase using the prepared capillary column. Under the optimal experimental conditions, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a high column efficiency near 70000N (plates/m) by the developed capillary‐HPLC.     

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.     

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.     

1 μm硅胶颗粒固定相的加压电色谱分离性能

制备了1 μm无孔硅胶颗粒。通过电动填充法得到总长度为45 cm(固定相填充长度为20 cm)、内径为100 μm的毛细管色谱柱。以乙腈-水体系作为流动相,详细考察了碱性化合物在该色谱柱上的加压电色谱(pCEC)分离性能,讨论了流动相比例、缓冲液浓度、pH值及操作电压等因素对分离的影响。实验结果表明,裸硅胶柱在乙腈-水体系分离碱性样品中表现出典型的反相色谱分离性能;缓冲液浓度的改变则对分离影响不大。当pH值改变时,碱性化合物的解离程度发生变化,它们与固定相之间的作用力发生变化,使得分离度发生相应的变化。分离柱效随施加电压的增加而增加,在1 kV电压下,裸硅胶柱对邻甲苯胺的柱效为35000理论塔板/m。     

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.     

Visualization of sample introduction in liquid chromatography columns. The effect of the frit diameter.

A previously developed on-column detection technique using 35 mm SLR cameras [J. Chromatogr. A 826 (1998) 1] was employed to visualize colored sample bands as they elute through frits of differing diameter. Head fittings containing a 4.0 mm frit and a 15.9 mm frit were mounted in a 17 mm I.D. glass column packed with C18 silica with an average particle size of 21 microns. A carbon tetrachloride mobile phase of matching refractive index to that of the silica provides clarity along the column diameter during band migration. The photographs of the migrating sample zones were scanned and analyzed with appropriate imaging software. The smallest diameter frit induced severely parabolic sample distributions at the column inlet compared to the larger frit. Local axial dispersion coefficient values, expressed as local reduced plate height, were calculated as well as local zone velocities at the column inlet. The results demonstrate clearly the need to match the diameter of the inlet frit to the I.D. of the column so as to avoid the initial onset of zone broadening due to the frit.     

Chromatographic behavior of poly(styrene-divinybenzene) encapsulated packing material for capillary electrochromatography

Poly(styrene-divinylbenzene) encapsulated silica (PS-DES) was synthesized and its characteristics as capillary electrochromatography stationary phase were studied. On capillary columns packed with this stationary phase, the effects of acetonitrile concentration as well as pH values on electroosmotic flow, and the effect of the latter on column efficiency were evaluated. Aromatic hydrocarbons, polar and basic medicinal compounds were successfully separated by using reversed-phase elution. It was evident that selectivity based on pi-pi electronic interaction between solute and stationary phase molecules contributed to the separation. Peaks in the chromatograms obtained were generally symmetrical. At a pH of the mobile phase as high as 11.2, the stationary phase was still stable after more than 160 h of exposure.     

Histidine-functionalized silica and its copper complex as stationary phases for capillary electrochromatography

A histidine-functionalized silica was prepared by covalent bonding of the functional groups to silane-treated silica gel. Conversion of functional groups was confirmed by infrared (IR) spectra, elemental analysis, and potentiometry. The functionality of the silica gel is 0.293 mmol g(-1). The coordination behavior of the histidine-functionalized silica was investigated by metal capacity and electron paramagnetic resonance (EPR). EPR measurements at different copper loadings were made. The results showed that the copper histidine complex might be distorted tetragonal. Both histidine-functionalized silica and its copper complex were employed as stationary phases for packed capillary electrochromatography (CEC). Electrical current was found helpful for evaluating the properties of frit construction and the stationary phase packing. Test samples include neutral compounds, inorganic anions and organic anions. Factors influencing the separation behavior have been studied. With copper-histidine functionalized silica under the condition of citrate buffer (10 mM, pH 4.0) and applied voltage of -20 kV, the separation of benzoic acid, D- and L-mandelic acid, phthalic acid and salicylic acid could be achieved within 12 min. The column efficiency for these acids was more than 1.2 x 10(5) plates m(-1), except salicylic acid.     

Preparation of various silica-based columns for capillary electrochromatography by in-column derivatization

Chemically bonded silica gels were prepared in a capillary by pumping an ethanolic solution of a silylating reagent, such as octadecyltrimethoxysilane, 3-aminopropyltrimethoxysilane and dimethyloctadecyltrimethoxysilylpropylammonium chloride into a heated capillary packed with bare silica particles. The silylation reactions were completed in a short time and thus-prepared columns showed high column efficiency and high reproducibility. Examples are shown for the separation of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of aldopentoses on a 3-aminopropylated silica column and benzoate homologues as well as PMP derivatives of the component monosaccharides of glycoproteins on an octadecylammonium column. Since the presence of frit filters hampers high efficiency separation, an attempt was made to fix the bed of modified silica gel particles to the capillary inner wall by a cross-linking technique. The results indicated that this technique is promising.     

毛细管反相电色谱法分离行为的研究

对乙睛-水-磷酸二氢销体系毛细管反相电色谱分离行为进行了研究。采用柱上紫外检测,在75μmi.d.×30cm的毛细管ODS（3μm）填充柱上获得了小于2.0的折合培板高度。同时还研究了乙睛的比例、电解质的浓度和电场强度等因素对电渗流和往效的影响。