Mesoporous polybutadiene-modified zirconia for high-temperature packed capillary liquid chromatography: column preparation and temperature programming stability

In the present study, three different methods for packing of 3 microm PBD-ZrO2 particles in 0.5 mm i.d. glass-lined stainless steel columns have been examined. The two first methods were based on a traditional downstream high-pressure technique using tetrachloromethane (Method I) or aqueous Triton X-100 (Method II) as slurry solvents, while Method III was an upstream high-pressure flocculating method with stirring, using isopropanol both as the slurry and packing solvent. Method I was found to be superior in terms of efficiency, producing 0.5 mm i.d. x 10 cm columns with almost 90,000 plates m(-1) for toluene (R.S.D. = 8.7%, n = 3), using a slurry concentration of 600 mg ml(-1), ACN-water (50:50 (v/v)) as the packing solvent and a packing pressure of 650 bars. For Method I, the slurry concentration, column i.d., column length and initial packing pressure were found to have a significant effect on column efficiency. Finally, the long-term temperature stability of the prepared columns was investigated. In isothermal mode, using ACN-20 mM phosphate buffer, pH 7 (50:50 (v/v)) as the mobile phase, the columns were found to be stable for at least 3,000 void volumes at 100 degrees C. At this temperature, the solute efficiencies changed about 5-18% and the retention factors changed about 6-8%. In temperature programming mode (not exceeding 100 degrees C), on the other hand, a rapid decrease in both column efficiency and retention factors was observed. However, when the columns were packed as initially described, ramped up and down from 50 to 100 degrees C for 48 h and refilled, fairly stable columns with acceptable efficiencies were obtained. Although not fully regaining their initial efficiency after refilling, the solute efficiencies changed about 19-28% (32-37%) and the retention factors changed about 4-5% (13-17%) after running 3,000 (25,000) void volumes or 500 (3,900) temperature programs.     

Packing columns for capillary electrochromatography

Considering the current interest in capillary electrochromatography (CEC), performed in packed columns, we present the different methods used to pack capillary columns for use in CEC. General considerations on column packing are given and the column fabrication process is discussed in sufficient detail to allow instruction to those who are not experienced in the field. Five different packing methods are discussed to deliver packing material into the capillary column from a practical view point: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using centripetal forces, and packing by gravity. Entrapment of particulate material by sintering and sol-gel technology is also mentioned. Although slurry pressure packing procedures are most common, higher separation efficiencies are obtained using other packing approaches. Electrokinetic packing seems to be the simplest technique to deliver the packing material into the capillary columns. Nevertheless, as with the other packing techniques, skill and experience are required to complete all the steps involved in the fabrication of packed columns for CEC.     

Transfer, amplification, and inversion of helical chirality mediated by concerted interactions of C3-supramolecular dendrimers

The synthesis, structural, and retrostructural analysis of two libraries containing 16 first and second generation C(3)-symmetric self-assembling dendrimers based on dendrons connected at their apex via trisesters and trisamides of 1,3,5-benzenetricarboxylic acid is reported. A combination of X-ray diffraction and CD/UV analysis methods demonstrated that their C(3)-symmetry modulates different degrees of packing on the periphery of supramolecular structures that are responsible for the formation of chiral helical supramolecular columns and spheres self-organizable in a diversity of three-dimensional (3D) columnar, tetragonal, and cubic lattices. Two of these periodic arrays, a 3D columnar hexagonal superlattice and a 3D columnar simple orthorhombic chiral lattice with P222(1) symmetry, are unprecedented for supramolecular dendrimers. A thermal-reversible inversion of chirality was discovered in helical supramolecular columns. This inversion is induced, on heating, by the change in symmetry from a 3D columnar simple orthorhombic chiral lattice to a 3D columnar hexagonal array and, on cooling, by the change in symmetry from a 2D hexagonal to a 2D centered rectangular lattice, both exhibiting intracolumnar order. A first-order transition from coupled columns with long helical pitch, to weakly or uncorrelated columns with short helical pitch that generates a molecular rotator, was also discovered. The torsion angles of the molecular rotator are proportional to the change in temperature, and this effect is amplified in the case of the C(3)-symmetric trisamide supramolecular dendrimers forming H-bonds along their column. The structural changes reported here can be used to design complex functions based on helical supramolecular dendrimers with different degree of packing on their periphery.     

Ultrahigh-pressure liquid chromatography using a 1-mm id column packed with 1.5-microm porous particles

The evolution of chromatography has led to the reduction in the size of the packing materials used to fabricate HPLC columns. The increase in the backpressure required has led to this technique being referred to as ultrahigh-pressure liquid chromatography (UHPLC) when the column backpressure exceeds 10000 psi (approximately 700 bar). Until recently, columns packed with sub-2-microm materials have generally fitted into two classes; either short (less than 5 cm) columns designed for use on traditional HPLC systems at pressures less than 5000 psi (350 bar), or capillary columns (inner diameters less than 100 microm). By using packing materials with diameters <2 microm to fabricate UHPLC columns, there is an increase in efficiency and a decrease in the analysis time that are directly proportional to the size of the packing material. In order to realize and exploit the increase in efficiency, however, the columns must maintain lengths typically associated with analytical columns (15-25 cm). We have packed 1 mm diameter, 150 mm in length columns with 1.5 microm packing material, and evaluated their performance in UHPLC. The pressure required to achieve optimum linear velocities in plots of plate height versus linear velocity was in the vicinity of 1104 bar (16000 psi). The 1.5 microm particle-packed column was compared with the more traditional 150 mm long analytical columns packed with 3 microm materials. This column showed an efficiency that was approximately twice that observed with the 3 microm packed column and a concomitant reduction in the analysis time, theoretically predicted.     

A drying step in the protocol to pack capillary columns by centripetal forces for capillary electrochromatography.

Capillary columns have been packed for capillary electrochromatography (CEC) using centripetal forces. The packed columns were maintained under wet conditions or they were dried with nitrogen gas prior to forming the retaining frits. Upon fabrication of the retaining frits, the dried columns were resolvated with the mobile phase. The performance of the columns was evaluated to determine the effect of the drying step during the packing procedure. The columns submitted to the drying step showed improved separation efficiencies and stronger retention characteristics than those kept under wet conditions. The drying step allows the silica-based packing material to be better accommodated inside the capillary column. Upon solvation, the packing material "swells," resulting in a greater packing density, which allows for a stronger retention and improved separation efficiencies. The drying step led to a 13% increase in retention on columns packed with isopropanol. An increase of 15-20% in theoretical plates for the most retained compounds was also observed in such columns.     

Axial development and radial non-uniformity of flow in packed columns

Flow inhomogeneity and axial development in low-pressure chromatographic columns have been studied by magnetic resonance imaging velocimetry. The columns studied included (a) an 11.7-mm I.D. column packed with either 50 microm diameter porous polyacrylamide, or 99 or 780 microm diameter impermeable polystyrene beads, and (b) a 5-mm I.D. column commercially packed with 10 microm polymeric beads. The packing methods included gravity settling, slurry packing, ultrasonication, and dry packing with vibration. The magnetic resonance method used averaged apparent fluid velocity over both column cross-sections and fluid displacements greater than one particle diameter and hence permits assessment of macroscopic flow non-uniformities. The results confirm that now non-uniformities induced by the conical distributor of the 11.7-mm I.D. column or the presence of voids at the column entrance relax on a length scale of the column radius. All of the 11.7-mm I.D. columns examined exhibit near wall channeling within a few particle diameters of the wall. The origins of this behavior are demonstrated by imaging of the radial dependence of the local porosity for a column packed with 780 microm beads. Columns packed with the 99-microm beads exhibit reduced flow in a region extending from ten to three-to-five particle diameters from the wall. This velocity reduction is consistent with a reduced porosity of 0.35 in this region as compared to approximately 0.43 in the bulk of the column. Ultrasonicated and dry-packed columns exhibit enhanced flow in a region located between approximately eight and 20 particle diameters from the wall. This enhancement maybe caused by packing density inhomogeneity and/or particle size segregation caused by vibration during the packing process. No significant non-uniformities on length scales of 20 microm or greater were observed in the commercially packed column packed with 10 microm particles.     

Characterisation of RPLC columns packed with porous sub-2 microm particles

Eight commercially available sub-2 microm octadecyl silane columns (C18 columns) have been characterised by the Tanaka protocol. The columns can be grouped into two groups that display large differences in selectivity and peak shape due to differences in hydrophobicity, degree of surface coverage and silanol activity. Measurements of particle size distributions were made using automated microscopy and electrical sensing zone measurements. Only a weak correlation could be found between efficiency and particle size. Large differences in column backpressure were observed. These differences are not related to particle size distribution. A more likely explanation is differences in packing density. In order to take full advantage of 100-150 mm columns packed with sub-2 microm particles, it is often necessary to employ not only an elevated pressure but also an elevated temperature. A comparison between columns packed with sub-2, 3 and 5 microm versions of the same packing indicates potential method transferability problems for several of the columns due to selectivity differences. Currently, the best alternative for fast high-resolution LC is the use of sub-2 microm particles in combination with elevated pressure and temperature. However, as shown in this study additional efforts are needed to improve transferability as well as column performance.     

Characterization of the acidity of residual silanol groups in microparticulate and monolithic reversed-phase columns

The residual silanol acidity and activity of several microparticulate and monolithic C18 columns has been measured from the retention of LiNO3 in the columns with a methanol/buffer (1 mM in Na+) (60:40 v/v) mobile phase buffered to different pH values. For Luna C18 (2) and LiChrospher RP-18 columns, at least two different types of silanols with different acidity for each packing, were observed. Purospher RP-18e and Chromolith RP-18e packings present evidence of some active silanols only at pH values close to their basic pH stability limit or higher. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MSC18 and Underivatized XTerra. A modification of an equation previously proposed has been applied to all columns studied and the results obtained have been used to classify the columns according to their silanol acidity and activity. The method allows the prediction of the extent of the silanol activity of the columns studied at a particular mobile phase pH.     

不锈钢宽口径填充毛细管液相色谱柱的制备及评价

设计了一种零死体积的柱尾连接方式和一种能使匀浆填料均匀进入色谱柱管内的锥形储料池。详述了以反相 ＯＤＳ为固定相,制备高效稳定的不锈钢宽口径（０．８ ｍｍＩ．Ｄ．）填充毛细管液相色谱柱的方法。系统地考察了填装方向、加压溶剂、匀浆溶剂、匀浆浓度和填装压力等不同因素对柱效的影响。在４０ＭＰａ压强和优化条件下,可重复制得柱效达理论塔板数为７．５×１０４／ｍ。对所测试的样品,色谱峰不对称因子为１．０３～１．１０。     

New materials and packing techniques for micro-HPLC packed capillary columns

Summary Plastic tubing of polyetheretherketone (PEEK) is used for the preparation of HPLC packed capillary columns. The polymer is rigid but not fragile, has great resistance to chemical and physical agents and comes in standard 1/16″ O.D. These features can extend the column lifetime and facilitate the overall employment of HPLC capillary columns. Serial connection of short columns is also possible with no increase in dead volume. A new, rapid and effective packing method has been developed to be compatible with the characteristics of the material. Several C8 and C18 columns have been prepared and tested and some applications are shown.     

Use of ultrasound to monitor the packing of large-scale columns,the monitoring of media compression and the passage of molecules,such as monoclonal antibodies,through the column bed during chromatography

The novel use of ultrasound as a detector in pilot- and production-scale chromatography is described. The difficulties in packing production scale chromatography columns using an integral packing valve are reviewed. Results are presented from the packing of 400- and 600-mm diameter columns with various medias. From these results it is proposed that when packing large columns using a packing valve, for a given medium and column size, there is an ideal rate and pattern (or control "corridor") by which the bed builds in order to give optimum performance. Ultrasound was shown to be able to monitor the building of such a column bed as the medium was pumped into the column. It was found that the ultrasound detector was sensitive to bed compression, mobile phase composition and components such as acetone, albumin, casein and monoclonal antibodies while on the chromatography bed. This enabled the visualisation, by ultrasound, of these components as they were chromatographed through the column.     

Toward rapid preparation of capillary columns for electrochromatography use

CEC is a high performance electrodriving liquid phase separation technique. It does not need complex and sophisticated high pressure instrumentation for nanoflow driving. This is attractive for parallel multicolumn analysis. To this end, high throughput methods for column preparation are needed to support the use of multiple columns. In this study, we directly used CEC mobile phase solution as the packing solvent, and realized rapid preparation of capillary columns based on a single particle fritting technology. The method presented high preparation throughput compared with other reported methods based on various fritting technologies. The single particle fritting approach promoted column preparation throughput to 1 column/h, including all the fritting, packing and conditioning steps. The rapidly prepared columns showed consistently high efficiency of up to 150 000 plates per meter, and usefulness in reversed phase CEC of neutral, charged and biomolecules. With standard peptides as the sample, excellent long term reproducibility (better than 0.8%RSD, ten days, for retention times) was observed.     

气液色谱中串联柱、多相柱与均相柱的保留体积的热力学讨论

本文测定了一些溶质在一些二元固定液串联柱、多相柱与均相柱的比保留体积,应用三元体系的Flory-Huggins公式以及两种不同溶剂的Flory-Huggins型相互作用参数数据,揭示了溶质在这些柱中比保留体积异同的热力学原因.     

Preparation and use of packed capillary columns in chromatographic and related techniques

In this paper general considerations related to the various approaches and parameters influencing the preparation of highly efficient and stable capillary columns for use in liquid chromatography and related techniques are presented and the column production process is discussed in some detail. The different packing methods available for delivering a packing material into a capillary column are discussed from a practical viewpoint. Packing with a gas (dry packing), packing with a liquid solvent or a mixture of solvents (slurry packing), packing with supercritical carbon dioxide, electrokinetic packing, and sol-gel packing technologies are introduced and discussed throughout the paper. Practical recommendations for obtaining highly efficient (high plate numbers) and stable capillary packed columns are also addressed and discussed.     

The art and science of forming packed analytical high-performance liquid chromatography columns

Columns of packed particles still are the most popular devices for high-performance liquid chromatography (HPLC) separations because of their great utility, excellent performance and wide variety. However, the forming of packed beds for efficient, stable columns traditionally has been an art where the basics of how to form optimum beds generally was not well understood. The recent development of monolith rods was introduced in part to overcome the difficulty of producing stable beds of packing particles. However, these materials are less versatile than packed particle columns. Technology developments in recent years have produced a better understanding among those skilled in the practice of how to form optimized packed beds, and this has led to widely available, high-quality commercial columns. This presentation discusses the developments that led to the present state of column packing technology. Important steps in the packing of efficient, stable beds are described. The key step of selecting the best solvent for the slurry packing method is emphasized. Factors affecting the mechanical stability of packed columns also are discussed. The early art of packing columns now has evolved into a more scientific approach that allows the packing of good columns with a minimum of effort and time.     

Practical comparison of LC columns packed with different superficially porous particles for the separation of small molecules and medium size natural products

Commercial C(18) columns packed with superficially porous particles of different sizes and shell thicknesses (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes. Significant efficiency loss was observed on 2.1-mm id columns even with a low dispersion ultra-high pressure liquid chromatography system. The Kinetex 4.6-mm id column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes. The Kinetex 2.1-mm id column packed with 1.7-μm particles did not deliver the expected performance and the possible reasons besides extra column effect have been proved to be frictional heating effect and poor column packing quality. Different column retentivities and selectivities have been observed on the four C(18) columns of different brands for the natural product separation. Column batch-to-batch variability that has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column.     

Boron macrocycles having a calix-like shape. Synthesis, characterization, X-ray analysis, and inclusion properties

The syntheses, structure, and inclusion properties of trinuclear boron compounds having a calix-like shape are described. The compounds have been obtained via self-assembly reactions between salicylaldehyde derivatives and 3-aminophenylboronic acid, whereby the formation of three N --> B coordination bonds favored the oligomerization. The products have high melting points (>370 degrees C), are stable to moisture, and have good solubility in organic solvents; the latter property is useful for host-guest recognition experiments. The structural analysis by X-ray diffraction revealed that diverse conformations are possible because of the presence of two different units of aromatic rims. A cone-cone (double-cone) conformation is observed for three of these compounds, while the remaining one has a cone-partial cone conformation. An analysis of the molecular packing showed that the molecules are stacked in columns in two different orientations in relation to the organization of the macrocycles when referred to the N-B bonds. The inclusion properties toward primary amines and ammonium chlorides were analyzed by titration experiments and monitored by UV spectroscopy, whereby association constants on the order of 10(2)-10(3) M(-1) were determined.     

The use of microparticulate guard columns in reverse-phase high-performance liquid chromatography

Summary The use of mciroparticulate guard columns in conjunction with high efficiency reverse-phase columns has been evaluated in terms of system efficiency and peak symmetry using three standard test mixtures. The effects of linking tube dimensions, particle size of guard column packing and the use of low or zero dead volume couplings have been investigated. Both valve and stopped-flow injection techniques have been used. Recommendations are given for the most efficient use of guard columns.     

Fast supercritical fluid chromatography hydrocarbon group-type separations of diesel fuels using packed and monolithic columns

Two approaches for decreasing diesel hydrocarbon group-type separation times by normal phase supercritical fluid chromatography (SFC) are compared. Short (10-15 cm) columns with small 3 microm diameter packing are compared with monolithic Chromolith bare silica columns under high carbon dioxide flow rates approaching 5 ml min(-1). Elution times are reduced up to 13-fold on a 10 cm Chromolith column and 7-fold on the short packed columns compared with conventional length columns run at typical flow rates. Short packed columns, with their higher surface area and retention characteristics, offer higher resolutions compared with Chromolith columns. Diesel samples are separated into saturates, mono-, di-, tri-, and polyaromatics in as little as 2 min on a 10 cm packed silica column. Diesel group-type results on a 15 cm titania-silica coupled column compare favorably with results from longer columns.     

Comparison of Retention Indices of Some Monosubstituted Benzenes Calculated by Different Mathematical Methods in RP-LC

In reversed phase liquid chromatography, the retention indices of benzene and nine mono substituted benzenes with different functionality based on the alkan-2-ones and alkyl aryl ketones retention index standards have been determined by the application of two new mathematical adaptation methods, viz. a multiparametric least-squares regression iterative method based on the determination of the adjusted retention times and a local cubic interpolation method directly using the total retention times. The two methods were applied to two types of columns. The first group includes four octadecyl-C₁₈ columns with different packing materials obtained from different manufacturers, while the second comprises an octyl-C₈ column. The retention indices have been extensively studied using either methanol–water or acetonitrile–water mobile phase systems. The influences of the concentration of the organic modifier in the mobile phase (methanol or acetonitrile), the column temperature, and the column packing material on retention indices of the set of the ten monoaromatics studied were also investigated. The calculated multiparametric retention indices values, those obtained by the local cubic interpolation and Kováts’ methods are compared. Good agreement was observed between the retention indices calculated by the three methods.