Rod Column Research Articles

Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry.

An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.

Rapid separation of microcystins and nodularin using a monolithic silica C 18 column

A monolithic C 18-bonded silica rod column (Merck Chromolith) was compared to particle-based C 18 and amide C 16 sorbents in the HPLC separation of eight microcystins and nodularin-R. Two gradient mobile phases of aqueous trifluoroacetic acid modified with acetonitrile or methanol, different flow-rates and different gradient lengths were tested. The performance of the Chromolith column measured as the resolution of some microcystin pairs, the selectivity, efficiency (peak width) and peak asymmetry equalled, or exceeded, the performance of traditional particle-based columns. The Chromolith column allowed a shortening of the total analysis time to 4.3 min with a flow-rate 4 ml min −1.

State of the Art in Miniaturized Separation Techniques

Miniaturized separation techniques have become very attractive recently because they offer a number of advantages over classic ones, for example, reduced chemicals consumption, separation improvement, and better sensitivity. It is also very important that they require minute samples, which is very often of primary importance in the environmental or biomedical sciences. In general, miniaturized systems that are being developed currently are divided into column and chip ones. For best performance of the separation microsystem, it must be properly designed in the terms of volumes and shapes of the parts it consists of. Theoretical considerations present this problem. As column systems are better known and understood the microcolumns are more frequently developed. Current works are focused on the preparation of unified columns that can be used in various techniques — for example, in micro-HPLC and CEC. Because of problems occurring in packed capillary column preparation and utilization, monolithic (rod) columns are developed to overcome the problems of inhomogeneity of packing or bubble formation under electroosmotic flow conditions. The requirements of minute sample analysis have been resolved by the construction of chip devices originating from total analysis system (TAS). Such systems are primarily designed for zone electrophoresis separations; however, micellar electrokinetic chromatography, gel chromatography, electrochromatography, or liquid chromatography can be performed using a chip device. In this article the most important trends in miniaturization of the separation systems are discussed, including theory, system preparation, and performance as well as exemplary applications.

Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate–co-ethylene dimethacrylate)

Continuous rod of macroporous poly(glycidyl methacrylate–co-ethylene dimethacrylate) was prepared by a free radical polymerization within the confines of a stainless-steel column. The epoxide groups of the rod were modified by a reaction with iminodiacetic acid (IDA) that affords the active site to form metal IDA chelates used for immobilized metal affinity chromatography (IMAC). The efficiency of coupling of IDA to the epoxide-contained matrix was studied as a function of reaction time and temperature. High-performance separation of proteins, based on immobilized different metals on the column, were described. The influence of pH on the adsorption capacity of bovine serum albumin on the Cu 2+–IDA continuous rod column was investigated in the range from 5.0 to 9.0. Purification of lysozyme from egg white and human serum albumin (HSA) on the commercially available HSA solution were performed on the naked IDA and Cu 2+–IDA continuous rod columns, respectively; and the purity of the obtained fractions was detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

Direct analysis of pharmaceutical compounds in human plasma with chromatographic resolution using an alkyl-bonded silica rod column.

Monolithic columns have been successfully used with steep gradient and high flow rates for the direct analysis of a candidate pharmaceutical compound in human plasma. The monolithic columns showed excellent robustness with nearly 300 20-microL injections of plasma (diluted 1:1 with water) being made onto one column without significant deterioration in performance. The system gave excellent sensitivity with a limit of quantification of 5 ng/mL being achieved. Unlike previous methods of direct analysis the monolithic columns showed excellent resolution even after nearly 300 plasma injections. The column performance was measured before and after the analysis of the plasma samples.

A New Monolithic-Type HPLC Column For Fast Separations

Summary The application of a new silica-based, monolithic-type HPLC-column for fast separations is presented. The column is prepared according to a new sol-gel process, which is based on the hydrolysis and polycondensation of alkoxysilanes in the presence of water soluble polymers. The method leads to “rods” made of a single piece of porous silica with a defined pore structure, i. e. macro- and mesopores. The main feature of silica rod columns is a higher total porosity, about 15% higher than of conventional particulate HPLC columns. The resulting column pressure drop is therefore much lower, allowing operation at higher flow rates including flow gradients. Consequently, HPLC analysis can be performed much faster, as it is demonstrated by various applications.

Performance of an octadecylsilylated continuous porous silica column in polypeptide separations

A continuous porous silica rod column prepared by an alkoxy-derived sol-gel method in the presence of a water-soluble organic polymer was tested in the reversed-phase acetonitrile–water linear gradient elution of polypeptides with molecular masses of up to 80 000. The silica rod having the through-pore size of 1.1 μm and silica skeleton size of 0.7 μm with mesopore size of 26 nm was used. The gradient time and the linear velocity of the mobile phase were varied and the resolution was examined in terms of peak capacity. The resolution with the silica rod was less affected by the gradient steepness and the mobile phase velocity. The results indicate that the silica rod column could reduce the separation time by a factor of 3 or more compared to the conventional column packed with 5 μm silica particles.

Designing monolithic double-pore silica for high-speed liquid chromatography

A novel type of silica-based chromatographic column without particle-packed structure has been recently developed. A monolithic silica gel having continuous gel skeletons and through-pores together with open mesopores is the key material of this `rod column'. The principle and procedure of designing the built-in pore structure is explained.

Effect of domain size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography

We prepared continuous porous silica columns having double pore structures with micrometer-size co-continuous through-pores and silica skeletons which have mesopores. The through-pore volumes and the ratio of through-pore diameters to silica skeleton diameters were kept constant, and the domain sizes were varied between 2.3 and 5.9 μm. We examined the effect of the domain size on the performance of silica rod columns in reversed-phase liquid chromatography after octadecylsilylation. The rod column having a smaller domain size showed lower plate height, though not so low as expected based on their skeleton size, and a smaller effect of linear velocity of mobile phase on the plate height for alkylbenzenes. The tendency was more pronounced for a larger molecule such as insulin. It was found that flow resistance parameters and separation impedance of the rod columns were smaller than those of conventional packed columns by a factor of 5 or more and showed little dependence on the domain size. The silica monoliths with double pore structure can provide a higher efficiency at a lower back pressure than conventional particle-packed columns.

STRUCTURE DESIGN OF DOUBLE-PORE SILICA AND ITS APPLICATION TO HPLC

Utilizing the concurrence of polymerization-induced phase separation and sol-gel transition in the hydrolytic polycondensation of alkoxysilanes, a well-defined macroporous structure is formed in a monolithic wet gel. By exchanging the fluid phase of the wet gel with an appropriate external solution, the nanometer-range structure of the wet gel can be reorganized into structures with larger median pore size essentially without affecting the macroporous framework. The double-pore structure thus prepared is characterized by open pores distributed in discrete size ranges of micrometers and nanometers. A new type of chromatographic column (silica rod) has been developed using monolithic double-pore silica instead of packed spherical gel particles. Typical silica rod columns had significantly reduced pressure drops and improved analytical efficiencies which do not deteriorate even at higher sample flow rates, both arising from a greater macropore volume than particle packed columns.

Double pore silica gel monolith applied to liquid chromatography

Silica gels retaining double pore structure in the size ranges of micrometer and nanometer have been applied to the rod-shaped monolithic column for liquid chromatography. The macropore structure was designed by controlling the phase separation process induced by the hydrolysis and polycondensation of alkoxysilane, whereas the mesopore structure was tailored by the solvent exchange treatments on wet gels. The size exclusion chromatograms on polystyrene standards exhibited almost similar features for octadecyl-modified rod and conventional packed beads columns. The dependence of plate height on the velocity of mobile phase determined for amylbenzene was by far weaker in the rod column than in the packed beads column, suggesting that additional geometrical factors should be considered in describing the separation mechanism in the rod column.

Experimental Verification and Analysis of Neutron Streaming Effect through Void Holes for Control Rod Insertion in HTTR

Control rod columns of the High Temperature Engineering Test Reactor (HTTR) core have each three cylindrical void holes for insertion of the control rods. The reactivity worths of the simulated void holes were measured in the Very High Temperature Reactor Critical Assembly (VHTRC) by the pulsed neutron method with the purpose of evaluating the neutron streaming effect on the reactivity worths of the void holes. The reactivity worths were determined using the revised King-Simmons' formula. The measured results are compared with the calculated ones based on the nuclear data of ENDFIB-IV, using the Benoist's anisotropic neutron diffusion coefficients. It is concluded that the reactivity worths are enlarged up to about twice as large as that obtained using the conventional isotropic neutron diffusion coefficients, and it is also found that the neutron streaming effect that is defined as the relative fraction components exceeding the reactivity worths obtained by the conventional isotropic neutron diffusion coefficients are likely overestimated about 11 and 32% with the void holes being in the core and reflector regions, respectively. Then, the reactivity decrease due to the neutron streaming effect is able to be evaluated around 1%Δk for the initial critical core of the HTTR.

Molded continuous poly(styrene- co-divinylbenzene) rod as a separation medium for the very fast separation of polymers Comparison of the chromatographic properties of the monolithic rod with columns packed with porous and non-porous beads in high-performance liquid chromatography of polystyrenes

Gradient elution separations of polystyrene standards in a monolithic molded 50×8 mm I.D. poly(styrene- co-divinylbenzene) rod column and in 50×8 mm I.D. and 30×4.1 mm I.D. columns packed with porous and non-porous poly(styrene- co-divinylbenzene) beads has been carried out. All of these separation media differ in shape and porosity. Excellent separations of eight polystyrene standards were achieved with both the molded monolithic rod and porous beads at moderate flow-rates. However, the monolithic medium proved to be superior for high-speed separations using very steep gradients at a flow-rate of 20 ml/min. Three polystyrene standards were separated in the rod column within 4 s. The separation in the column packed with non-porous beads was poor at flow-rates of 2–8 ml/min, while higher flow-rates led to an unacceptably high back pressure.

Molded separation media: An inexpensive, efficient, and versatile alternative to packed columns for the fast HPLC separation of peptides, proteins, and synthetic oligomers and polymers

AbstractA simple molding process carried out within the confines of a chromatographic column has been used for the preparation of macroporous poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) and poly(styrene‐co‐divinylbenzene) rods. The novel monolithic separation media that are obtained are useful for the HPLC separation of biological and synthetic polymers. The presence of large pores with a diameter of about 1 μm makes the molded rod columns easily permeable to eluents. Therefore, the back pressure of these columns is modest even at high flow rates. In contrast to the conventional HPLC columns packed with beads, all of the mobile phase flows through the continuous monolithic medium. As a result of this total convection, the efficiency of the molded media is almost independent of the flow rate. This improves significantly the separation ability of the rod columns and very fast separations of macromolecules such as peptides, proteins, and synthetic polymers have been demonstrated.

Molded monolithic rod of macroporous poly(styrene-co-divinylbenzene) as a separation medium for HPLC of synthetic polymers: on-column precipitation--redissolution chromatography as an alternative to size exclusion chromatography of styrene oligomers and polymers.

A process for the separation of styrene oligomers and polymers by size and composition using a novel separation medium has been demonstrated. The process involves precipitation of the macromolecules on the molded macroporous rod columns, followed by progressive elution utilizing a simple gradient of the mobile phase. Molded macroporous rod columns are ideally suited for this technique because convection through the large pores of the rod enhances the mass transport of large analyte molecules and accelerates the separation process. Styrene oligomers and polymers are separated in a 50-mm x 8-mm-i.d. column using a solvent gradient composed of a poor solvent such as water, methanol, or acetonitrile and increasing amounts of a good solvent, tetrahydrofuran. Excellent separations are obtained, demonstrating that precipitation-redissolution can be a suitable alternate to size exclusion chromatography (SEC) of some polymers. Compared to SEC, the gradient elution separation can be achieved at higher flow rates in a much shorter time. Precipitation-redissolution with gradient elution can also be used for the separation of copolymers, for which the process is controlled not only by molecular weight but also by the composition of the copolymers.

Modified poly(glycidyl metharylate-co-ethylene dimethacrylate) continuous rod columns for preparative-scale ion-exchange chromatography of proteins

A continuous rod of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) has been prepared by a free radical polymerization within the confines of a 300 × 8 mm I.D. chromatographic column. The epoxide groups of the rod have been modified by a reaction with diethylamine that affords ionizable functionalities required for the ion-exchange chromatographic mode. The properties of this rod column have been characterized and the column has been used successfully for the chromatographic separation of proteins. The column exhibits a dynamic capacity that exceeds 300 mg at a flow velocity of 200 cm/min. An excellent selectivity allows the separation of up to 300 mg of a protein mixture in a single run.

Hydrophilization of Porous Polystyrene-Based Continuous Rod Column

Reactive porous rods of poly[(chloromethyl)styrene-co-divinylbenzene] have been prepared for the first time by copolymerization of the monomers in the presence of a porogenic diluent. A two-step modification process involving reaction with ethylenediamine followed by reaction with gamma-gluconolactone leads to a porous medium that has highly hydrophilic surface functionalities. Preliminary chromatographic evaluation with both small molecules such as alkylbenzenes in reversed-phase mode and large proteins in hydrophobic adsorption and ion-exchange modes demonstrates that the surface of the modified rods possesses a hydrophilicity that is comparable to that of the best hydrophilic HPLC packings.

Dynamical stability of an elastic column and the phenomenon of bifurcation

The article studies the stability of rectilinear equilibrium shapes of a non-linear elastic thin rod (column or Timoshenko's beam), the ends of which are pressed. Stability is studied by means of the Lyapunov direct method with respect to certain integral characteristics of the type of norms in Sobolev spaces. To obtain equations of motion, a model suggested in [16] is used. Furta [6] solved the problem of stability for all values of the parameter except bifurcational ones. When values of the system's parameter become bifurcational, the study of stability is more complicated already in a finite-dimensional case. To solve a problem like that, one often has to use a procedure of solving the singularities described in [1], for example. In this paper a change of variables is made which, in fact, is the first step of the procedure mentioned. To prove instability, we use a Chetaev function which can be considered as an infinite-dimensional analogue of functions suggested in [14, 9]. The article also investigates a linear problem on the stability of adjacent shapes of equilibrium when the parameter has supercritical values (post-buckling).

Macroporous polymeric stationary-phase rod as continuous separation medium for reversed-phase chromatography.

A macroporous poly(styrene-co-divinylbenzene) rod has been prepared by a free-radical polymerization of a mixture containing monomers, initiator, and porogenic solvent in the confines of a chromatographic column and used for the first time in the very fast reversed-phase HPLC of proteins. Characterization of the pore structure of the continuous rod by mercury intrusion porosimetry revealed a large volume of pores with a diameter of about 1 micron to pores below 100 nm. Size exclusion chromatography and scanning electron microscopy confirmed the unusual pore size distribution. The presence of large pores make the rod easily permeable to eluents, and therefore, the back pressure of the rod column is modest even at high flow rates. The efficiency of the polymerized column is almost independent of the flow rate. The slope of the line showing capacity factor vs composition of the mobile phase was determined for several proteins, and a gradient for the separation of their mixtures was developed. Excellent separation was achieved even at a high flow rate of 25 mL/min as documented by the resolution data. Tripling the length of the column did not improve the column resolution in protein separation.

Investigation of adhesive strength of metallized-polymer coatings of pump rods

The deep-pump rod method of recovering petroleum is the most widely used in domestic industry. Operating experience with deep-pump units has demonstrated that one of the weakest subassemblies affecting the remaining service life of the units and the net cost of recovered petroleum is the column of pump rods. The basic cause of the breakdown of pump rods is their corrosion-mechanical failure, which is dictated by the combined effect of loads (static and dynamic) and the aggressive components of the production being recovered (water, hydrogen sulfide, carbon dioxide, chlorides).