Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Capillary action liquid chromatography

Capillary action LC (caLC) is introduced as a technique using capillary action as the driving force to perform LC in capillary columns packed with HPLC type microparticulate materials. A dry packing method with centrifugal force was developed to prepare capillary columns in parallel (10 columns per 3 min) to support their disposable use in caLC. Using a digital microscope for real‐time imaging and recording separations of components in a dye mixture, caLC was found to have flow characteristics similar to TLC. Based on the investigation of microparticulate HPLC silica gels of different size (1.5–10 μm) and a typical TLC grade irregular medium, Merck 60G silica, the van Deemter curves suggested molecular diffusion as the major contribution to band broadening in caLC. With Waters Xbridge 2.6 μm silica, plate heights down to 8.8 μm were obtained, comparable to those achievable in HPLC. Assisted by an image‐processing method, the visual caLC separation was converted to a classical chromatogram for further data analysis and such a facility confirmed the observation of highly efficient bands.     

Monolithic Silica Columns for HPLC,Micro‐HPLC,and CEC

Two types of monolithic silica columns derivatized to form an ODS phase, one prepared in a fused silica capillary (SR‐FS) and the other prepared in a mold and clad with an engineering plastic (poly‐ether‐ether‐ketone) (SR‐PEEK), were evaluated. The column efficiency and pressure drop were compared with those of a column packed with 5‐μm ODS‐silica particles and of an ODS‐silica monolith prepared in a mold and wrapped with PTFE tubing (SR‐PTFE). SR‐FS gave a lower pressure drop than a column packed with 5‐μm particles by a factor of 20, and a plate height of 20 μm at a linear velocity below 1 mm/s. SR‐PEEK showed higher flow‐resistance than the other monolithic silica columns, but they still showed a minimum plate height of 8–10 μm and a lower pressure drop than popular commercial columns packed with 5‐μm particles. The evaluation of SR‐FS columns in a CEC mode showed much higher efficiency than in a pressure‐driven mode.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Performance limits of monolithic and packed capillary columns in high-performance liquid chromatography and capillary electrochromatography

A method is proposed for the comprehensive characterization and comparison of columns in the high-performance liquid chromatographic (HPLC) and capillary electrochromatographic (CEC) modes. Using this approach, column parameters such as the number of plates, the eddy-diffusion and mass-transfer contributions to peak broadening, the permeability, and the analysis time are incorporated in a single graph and a comparison in terms of efficiency and speed is obtained. The chromatographic performance of silica-based and polymer-based monolithic capillary columns is discussed and a comparison is made with the performance of packed columns. Also, the potential of ultra-high-pressure liquid chromatography is discussed in this context. In the HPLC mode, the best results were obtained with silica monoliths; in the CEC mode, the low-density methacrylate-ester-based monoliths showed the best performance.     

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.     

毛细管电色谱柱制备技术的进展

介绍了毛细管电色谱开管柱、填充柱和整体柱的各种制备技术及其优势与不足,特别是对于近期发展的毛细管电色谱整体柱的制备方法及其应用进行了系统综述。引用文献100篇。     

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.     

Electrochromatography in packed tubes using 1.5 to 50 μm silica gels and ODS bonded silica gels

Summary Electrochromatography (that is HPLC where the eluent is driven along the column by electro-osmosis using fields of up to 100 kV m⁻¹) promises plate efficiencies for HPLC which are comparable to those attained in capillary gas chromatography, but this requires that narrow-bore columns can be successfully packed with submicron particles. This paper demonstrates that we have now moved a considerable distance towards this goal. We show (1) that, following theory, there is no evidence of any reduction in electroosmotic velocity in columns packed with particles down to 1.5 μm diameter, (2) that reduced plate heights as low as unity are attainable for unretained solutes using both slurrypacked and drawn-packed columns 30 to 200 μm bore and up to 1 m long when packed with conventional 3 and 5 μm silica gels or with 1.5 μm impermeable silica spheres, (3) that columns driven electrically show higher plate efficiencies than identical columns driven by pressure, and (4) that 100,000 plate HPLC separations can be achieved in relatively short times of 30 minutes using in situ derivatised drawn packed capillaries containing 3 and 5 μm ODS-silica gels.     

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

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

手性有机聚合物毛细管电色谱整体柱研究进展

毛细管电色谱具有高分离效率、多种保留机制和高选择性的优点。近年来,利用毛细管电色谱进行对映异构体的手性拆分受到了广泛关注。相对于传统的填充柱和开管柱,整体柱在手性拆分方面具有显著优势。与手性硅基整体柱相似,手性有机聚合物整体柱由于具有大孔,可产生较高的流速而压降较小。该文综述了近十年手性有机聚合物整体柱制备方法的研究进展,将手性有机聚合物整体柱的制备方法分为"原位聚合法"和"手性修饰法"两种,虽然前者制备简单并广泛应用于早期研究,但聚合混合液成分的微小改变即可引起最终聚合物的形态变化,并且大部分带丙烯基的手性选择剂较难从市场购买。因此,手性修饰法因作为手性选择剂基质的整体柱制备且优化只需进行一次的优势而受到普遍关注。亲核取代、杂环开环和点击化学是常用的修饰手段。该文总结了这两种制备方法的应用,同时对未来的研究方向提出参考性意见。     

Monolithic columns in high-performance liquid chromatography

Monolithic media have been used for various niche applications in gas or liquid chromatography for a long time. Only recently did they acquire a major importance in high-performance column liquid chromatography (HPLC). The advent of monolithic silica standard- and narrow-bore columns and of several families of polymer-based monolithic columns has considerably changed the HPLC field, particularly in the area of narrow-bore columns. The origin of the concept, the differences between their characteristics and those of traditional packed columns, their advantages and drawbacks, the methods of preparation of monoliths of different forms, and the current status of the field are reviewed. The actual and potential performance of monolithic columns are compared with those of packed columns. Monolithic columns have considerable advantages, which makes them most useful in many applications of liquid chromatography. They are extremely permeable and offer a high efficiency that decreases slowly with increasing flow velocity.     

Permeability, porosity, and structure of monolithic capillary columns in gas chromatography

The permeability of monolithic silica gel capillary columns with respect to the helium carrier gas was studied using gas chromatography. The results obtained by gas chromatography and liquid chromatography were found to be in close agreement. The permeability of monolithic capillary columns was compared to that of hollow capillary columns and columns packed with finely dispersed sorbents. It was demonstrated that the permeability of the monolithic capillary columns studied is almost three orders of magnitude lower than that of hollow capillary columns of the same diameter but two orders of magnitude higher than that of columns packed with micron-scale particles. The interstitial fraction of the monolithic columns was found to be very high, 0.95.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.     

Preparation of high efficiency and highly retentive monolithic silica capillary columns for reversed-phase chromatography by chemical modification by polymerization of octadecyl methacrylate

High efficiency and highly retentive monolithic silica capillary columns were obtained by polymerization of octadecyl methacrylate using alpha,alpha'-azobis-isobutyronitrile (AIBN) as a free radical initiator. Hybrid type monolithic silica columns (25 cm total length x 200 microm I.D.) prepared from a mixture of tetramethoxysilane and methyltrimethoxysilane were used as a support. The effects of the monomer and the radical initiator concentrations in the reaction mixture were examined. The performance of the columns was tested in terms of column efficiency and retention behavior by using alkylbenzenes and a few other compounds as solutes and compared with that of hybrid monolithic silica columns modified with octadecylsilyl-(N,N-diethylamino)silane (ODS-DEA). Highly retentive monolithic silica columns were obtained by polymerization at high monomer concentrations. Although a decrease in column efficiency was observed with the increase in the monomer concentration in a feed mixture, an improvement in efficiency was achieved (a plate height value lower than 10 microm) by increasing an initiator concentration without significant variations in column retention properties. Results obtained by polymerization using other monomers are also presented to demonstrate the applicability of the preparation method.     

Monolithic silica-based capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography

A silica-based monolithic stationary phase prepared by the sol-gel process in a 100 microm I.D. fused-silica (FS) capillary has been modified chemically with 3-mercaptopropyl trimethoxysilane followed by immobilization of a strong cation-exchange (SCX) type chiral selector, (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutane phosphonic acid, by radical addition reaction onto the reactive sulfhydryl surface. After a fine-tuning of the mobile phase composition, the enantioselective capillary column was evaluated for the separation of various chiral basic drugs by enantioselective non-aqueous capillary electrochromatography (CEC), in comparison to capillary column analogs packed with 3.5 microm silica particles having attached the same selector. The performance of the monolithic silica column was further compared to corresponding polymethacrylate-based organic polymer monoliths. The study indicated that strong counter-ions such as 2-aminobutanol or N,N,N',N'-tetramethylethylenediamine are needed, although they reduce the electroosmotic flow velocity and separation factors in comparison to less efficient counter-ions, in order to allow the elution of the oppositely charged solutes in the ion-exchange retention mode within reasonable run time and as sharp zones. In contrast, weak counter-ions such as N,N-diisopropylethylamine (Huenig base) provided stronger electroosmotic flow and much better separation factors, but relatively poor peak efficiencies. Overall, with the chemically functionalized monolithic silica column the high quality separations of packed column analogs could be approximated, with regards to both separation factors and peak performances. On the other hand, the monolithic capillary column certainly outperformed the packed column in terms of system robustness under capillary electrochromatography conditions and showed excellent column longevity. The enantioselective strong cation-exchange-type monolithic silica column performed also well in comparison to the organic polymer monolith.     

Towards the ultimate minimum particle diameter of silica packings in capillary electrochromatography

Porous silica beads with an average particle diameter between 0.2 and 3 microm have been applied as packing material in capillary electrochromatography (CEC). The experiments were directed to investigate whether it is really feasible and as promising as expected to use such small particles. In CEC, plate heights of H approximately/= 1-2 d(p) can be achieved which is smaller than the plate heights usually attained in high-performance liquid chromatography. Using a capillary packed with 0.5 microm silica beads we achieved a plate height of H = 3 d(p) indicating the presence of dispersive effects like Joule heating. Calculations demonstrate that at a field strength of about 800 V cm(-1) one third of the plate height can be lost by Joule heating effects if the heat is not removed by a cooling system. Additionally, the H(u) curve is still descending at the maximum electroosmotic flow (EOF) velocity we generated with the modified capillary electrophoresis instrument. To fully exploit the potential of submicron size silicas higher field strengths, i.e., higher EOF velocities, must be attained. To study the influence of the kind of packing on the EOF porous as well as nonporous silicas have been applied. The experiments clearly indicate that the EOF of porous and nonporous silicas is the same. Since the EOF is more or less exclusively generated by the packing material the zeta potential of n-octyl bonded 0.5 microm silica has been determined. The dependence of the zeta potential on the pH is identical to the dependence of the EOF on the pH in a packed capillary. The point of zero charge of the silica is at pH 2-3.     

Comparison of conventional microparticulate and a monolithic reversed-phase column for high-efficiency fast liquid chromatography of basic compounds

The effect of mobile phase flow on column efficiency for a neutral compound together with weak and strong bases was compared for conventional microparticulate (3/3.5 microm and 5 microm) silica RP columns and a monolithic silica RP. For benzene, the minimum plate height (Hmin) at optimum flow-rate (mu(opt)) for weak bases was similar for the 5 microm and the monolith phases. However, the monolith generated much flatter Van Deemter curves, such that at high flow-rate (5 ml min(-1)) the plate height was nearly 3.5 times lower on the monolith. For weak bases analysed in unbuffered mobile phases, and stronger bases with acid phosphate buffer, increased tailing was obtained on the monolith compared with the conventional phases. Nevertheless, Van Deemter plots on the monolith still showed some advantages over particulate phases, even when asymmetry factor was included in the calculation of the plate height. However, at pH 7 considerable tailing of strong bases was found using the monolith; it is not clear whether this results from unique features of the monolith structure, or whether it is due merely to usual problems of silica activity. Van Deemter plots for conventional phases may be improved considerably by operating the column at elevated temperatures. At pH 3, these improvements are influenced to a considerable extent by increases in Dm, as shown by measurements of Dm using the Taylor-Aris procedure. However, at pH 7.0, improvements are much too substantial to be explainable wholly on this basis.     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

Electrically assisted capillary liquid chromatography using a silica monolithic column

A silica monolithic capillary column was linked to an open capillary of the same internal diameter via a Teflon sleeve to form a duplex column to investigate the combination of chromatography and electrophoresis in the mode of electrically assisted capillary liquid chromatography (eCLC). Using a commercial CE instrument with an 8.5 cm long, 100 μm i.d. reversed phase silica monolithic section and a window 1.5 cm beyond the end of this in a 21.5 cm open section, a minimum plate height of 9 μm was obtained in capillary liquid chromatography (CLC) mode at a low driving pressure of 50 psi. In eCLC mode, high speed and high resolution separations of acidic and basic compounds were achieved with selectivity tuning based on the flexible combination of pressure (0–100 psi) and voltage. Taking advantage of the excellent permeability of silica monolithic columns, use of a step flow gradient enabled elution of compounds with different charge state.