High-performance Liquid Chromatography Packing Research Articles

HPLC on Calixarene Bonded Silica Gels. I. Characterization and Applications of the p-tert-Butyl-Calix[4]arene Bonded Material

Molecules containing cavities of well-defined dimensions are able to include guest molecules stereoselectively. This work is concerned with the introduction and evaluation of a newly developed high-performance liquid chromatographic packing material based on calix[4]arene chemically bonded to silica gel. It is well documented that calixarenes representing cyclic condensates of para substituted phenols and formaldehyde have the ability to form intracavity inclusion complexes with ions and organic molecules in aqueous solutions. The silica bonded macrocyclic ligands form truncated cones similar to cyclodextrins. Data are presented illustrating the resolution characteristics for disubstituted aromatics, peptides, nucleosides, and nucleobases. The results indicate that the calix[4]arene silica gel behaves predominantly as a reversed-phase material. Some effects support the assumption that the cavity acting as an electron donator can influence the elution order

Composite sorbents for liquid chromatography. A size exclusion study of dextran gel incorporated into porous solid particles

The novel class of column packings for high-performance liquid chromatography was prepared. These materials are formed by silica-, polyhydroxyethylmethacrylate-, or carbon-based macroporous solid particles with pores filled by a soft dextran network. They were examined by inverse size-exclusion chromatography. The analytes, which are small enough to penetrate the dextran network, can interact selectively with the support surface. Such interactions may be either suppressed by an appropriate deactivation of the support surface prior to dextran incorporation or utilized for special applications in which retention of small molecules of analytes is combined with steric exclusion of large molecules of interference.

Synthetic Analytical Studies on Organic Reagents for Biomaterials

Synthetic analytical chemistry: Analytical with the aid of synthetic organic chemistry is proposed by the present author as a new field of analytical chemistry. Such is developing on the basis of investigations of π-electron systems such as non-benzenoid aromatics (e.g. annulenes) or conjugated polyene and polyyne systems. Various kinds of organic reagents for analyses (ultraviolet/visible and near-infrared absorption, fluorescence, chemiluminescence and new silica gel column packings for high-performance liquid chromatography) were developed and applied to the analyses of many biomaterials. In this paper, synthetic analytical is illustrated mainly by the author′s own studies.

Siliceous sorbents with immobilized carbowax 20M as column packings for liquid chromatography: II. Application in high-performance liquid chromatography

The chromatographic properties of siliceous supports with thermally immobilized Carbowax 20M as packings for high-performance liquid chromatography are described. Such materials were examined as sorbents in normal- and reversed-phase chromatographic systems and as macromolecular sieves for size-exclusion chromatography of biopolymers. Additionally, the stability of the Carbowax layer was determined.

Direct resolution of organic acid enantiomers on a novel polymer-based stationary phase

A chiral packing for high-performance liquid chromatography was obtained by copolymerization of urea with formaldehyde in the presence of an optically active amino acid derivative. Spherical particles with a mean bead size of 6 μm were evaluated as stationary phases with respect to their chemical stability and chromatographic performance. Electrostatic interactions govern the overall kinetics of the retention in the chromatographic process, so that ion pairing seems to be responsible for the chiral recognition. Increasing the column temperature improves the chromatographic pattern considerably, and the resolution and peak shape are similar to those in the usual high-performance liquid chromatographic separations. The support is easy to pack and yields good column lifetimes. Its application to the resolution of some organic acid racemates is illustrated.

Importance of intraparticle convection in the performance of chromatographic processes

Large-pore materials are used in separation engineering as high-performance liquid chromatographic packings and adsorbents; however, they find also many applications in reaction engineering as catalyst supports and ceramic membrane reactors and in biotechnology as supports for mammalian cell cultures or biomass growth. In these large-pore materials, mass transport by intraparticle forced convection should be considered in process analysis. A brief historical survey of research work concerned with intraparticle forced convection is given, showing that the concept to be retained is that of effective diffusivity augmented by convection. The behaviour of the height equivalent to a theoretical plate as a function of bed superficial velocity is explained on the basis of the above concept. Analogies between slab and spherical particle geometries are discussed.

Evaluation of the application of liquid-phase titration to the examination of the adsorption activity of silica-based high-performance liquid chromatographic packings

The theoretical basis of the chromatographic titration method was investigated and the correctness of the determination of the strong silanol fraction was confirmed. Application of liquid-phase titration to the comparison of reversed-phase high-performance liquid chromatographic packings is described and the possibility of sorbent evaluation is discussed.

Semipermeable-surface reversed-phase media for high-performance liquid chromatography

Polyoxyethylene was both adsorbed hydrophobically (through the use of non-ionic surfactants) and covalently bonded to reversed-phase high-performance liquid chromatographic packings, thereby establishing a semipermeable hydrophilic layer over the alkylsilane surface. This layer restricts proteins from adsorbing to the alkylsilane phase while permitting penetration and chromatographic separation of small molecules. Biological fluids containing low-molecular-weight analytes may be injected directly, without sample pretreatment or the use of micellar eluents. In the case of adsorbed coatings, surfactant loading was determined primarily by the surface area (over the reversed phase) occupied by the polyoxyethylene head group. Semipermeability of the hydrophilic layer was demonstrated by observing changes in retention of both small molecules and proteins with increasing eluent ionic strength. Coated column stability was evaluated with regard to cumulative eluent volume and repeated serum injections.

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.

Size-exclusion chromatography of lignocellulosics in wheat straw

The application of Fractogel TSK HW-40, a semi-rigid, hydrophilic vinyl polymer, and Vydac 214 TP, a wide-pore high-performance liquid chromatographic packing, to the fingerprinting of extracts of wheat straw, both untreated and treated with sodium hydroxide, is reported. Whereas separation on Fractogel TSK is typical size-exclusion chromatography, chromatography on the Vydac column is influenced by both adsorption effects and the molecular size of the solutes. Chromatograms showing significant differences among samples are discussed in relation to sample pretreatment and extraction, molecular weight distribution and electrochemical properties of the main peak.

Interactions of human serum albumin with a modified poly(vinyl alcohol) gel packing for high-performance liquid chromatography

The interactions of human serum albumin (HSA) with a poly(vinyl alcohol) gel packing (Asahipak GS-520) for high-performance liquid chromatography of proteins were investigated. Under certain conditions, the elution of HSA from the GS-520 column was retarded and its chromatogram was split into two peaks, indicating weak adsorption of HSA onto the gels and also the existence of two subfractions, i.e. human mercapto-albumin (HMA) and human non-mercapto-albumin (HNA). The chromatograms were confirmed to be greatly influenced by the salt composition, the pH, and the temperature of the isocratic mobile phase. It is characteristic for the adsorption of HSA onto the gels to be suppressed at a pH near its isoelectric point. The HSA-gel interaction parameters calculated using an adsorption chromatography theory demonstrate that the adsorption of HSA is caused by enthalpy-driven interactions, which are depressed by lowering the pH, in addition to hydrophobic interactions. Under the recommended chromatographic conditions for high resolution of HMA/HNA, it was found that the HSA samples possessed some subfractions besides HMA and HNA fractions.

Copolymer of Di (methacryloyloxymethyl) naphthalene and divinylbenzene as a column packing for high-performance liquid chromatography

A new porous polyaromatic ester packing was synthetized for high performance liquid chromatography. The relationship between retention and chain length of the members of homologous series of alkylbenzenes, N-alkylanilines, alkylarylethers, alkylbenzoates and alkylarylketones on this new stationary phase using different eluents was investigated. Using the alkylarylketone scale the retention indices of the homologues and test compounds were calculated. The results were compared with those obtained for poly (styrene-divinylbenzene) polymers. For both types of packing the first members of each homologous series gave non-linear behaviour. The methylene group index increments are different for the studied homologous series; thus there is no simple additivity of the retention indices. The efficiency of the porous polymeric columns is a function of the capacity factor of the solute and the organic component of the eluent.

Preparation and characteristics of silica-based packings for high-performance liquid chromatography, modified with amino and carboxyl groups

Preparation and characteristics of silica-based packings for high-performance liquid chromatography, modified with amino and carboxyl groups

Application of carbon microbeads for column packing for high-performance liquid chromatography

Carbon microbeads have been recognized as an advanced carbonaceous material with exciting possibilities just like carbon fibre. Among their potential applications are high-density, high-strength, isotropic carbon solids [1] and bulking agents for liquid chromatography [2]. The microbeads have usually been obtained by carbonizing pitch, separating them from each other with solvent or in a high-temperature centrifuge. To name a few advantages of carbon microbeads, they offer (a) high resistance to very acidic and very basic reagents, (b) low expansion and shrinkage coefficients and (c) the ability to withstand an operating temperature of 250 °C. Recently we have developed a method for obtaining carbon microbeads from n-paraffin or a mixture of n-paraffin and coal tar pitch (CTP) under mild conditions at 873 K under a nitrogen pressure of 2 MPa [3, 4]. In this letter a study is made on the application of carbon microbeads for column packing for highperformance liquid chromatography (HPLC) for the separation of aromatic and polar compounds. n-Paraffin (melting point 341 to 343 K) was used as the starting material for production of carbon microbeads. Carbonization of paraffin was performed in a 15 ml autoclave. The autoclave charged with an 0.8 g sample was pressurized with nitrogen at an initial pressure of 5 MPa and put into an image furnace for carbonization at 873 K (the yield of carbon microbeads was 18 wt %). The rate of heating was 5 K min-~. The carbon microbeads formed in the autoclave were taken out and were heat-treated further at 973, 1073 and 1173 K for I h under nitrogen gas flow. Heat-treated carbon microbeads thus obtained were tested as a Figure 1 SEM micrographs of carbon microbeads from n-paraffin used for HPLC column packing.

Influence of the porous silica gel structure on the coverage density of a chemically bonded C 18 phase for high-performance liquid chromatography

In high-performance liquid chromatography (HPLC), silica gels are the most often used supports for chemically bonded stationary phases. The influence of the physical (volume and diameter of pores) and chemical (hydroxyl group concentration) structures of various silica gel supports on blocking effectively the binding of silanol groups by monochlorooctadecylsilane was investigated. Using silica gel supports with a pore volume ⩾1.5 cm 3/g and a mean pore diameter ⩾14 mm as starting materials, reproducible packings for HPLC having a high coverage density (α RP ⩾4 μmol/m 2) were obtained. Retention behaviour is discussed in terms of a schematic representation of bonded-phase structure.

Determination of surface tension of packings for high-performance liquid chromatography

Determination of surface tension of packings for high-performance liquid chromatography

Chromatographic and solid state nuclear magnetic resonance study of the changes in reversed-phase packings for high-performance liquid chromatography at different eluent compositions

A number of long and short chain reversed-phase packings for high-performance liquid chromatography from two different silica substrates, derivatized with mono- or trifunctional silane reagents, were studied under simulated routine conditions. The changes in the properties of the packings are described in terms of loss of silanes, gain in silanol content and rearrangements of the silica-to-silane bondings. Chromatographic techniques, solid-state NMR spectroscopy and elemental analyses were used to characterize and partially to quantify these changes. As expected, long chain phases are more stable than short chain phases with the same silane-to-silica attachment. More surprisingly, the reversed phases derived from monofunctional silanes are much more stable than phases with the same silanes chain length prepared from trifunctional silanes. An explanation of the changes is offered in terms of siloxane hydrolysis and rearrangement plus concomitant polymerization of multifunctional silanes. The impact on the chromatographic behaviour is discussed.

Investigation of high-performance liquid chromatographic packings by liquid mixture adsorption

The adsorption isotherms of methanol—benzene and benzene— n~heptane mixtures on four different silica gels were recorded to determine the types of adsorption isotherms. Based on the results, qualitative statements could be made regarding the relationship between chromatographic and mixture adsorption properties.

Factor analysis and experiment design in high-performance liquid chromatography : VII. Classification of 23 reversed-phase high-performance liquid chromatographic packings and identification of factors governing selectivity

The chromatographic behaviour of 63 solutes was investigated in reversed- phase high-performance liquid chromatographic (RP-HPLC) systems with the same mobil phase and 23 different commercially available packings. The results provide a new insight into variations of selectivity with column type. The factors affecting solute selectivity in RP-HPLC systems, emerging from correspondence factor analysis (CFA), are grouped into types: hydrophobic factor and chemical and/or steric factors. The relative importance of these factors is considered. Physical and chemical properties of packings, expected to affect the solute selectivity, such as the nature of the organic ligand, carbon loading, end-capping procedure, shape of silica, monomeric or polymeric layer and compression technique, are compared. It is shown that only the three first influence the selectivity. The CFA results also permit a classification of the packings on a relative scale of “hydophobicity”. To position any ne RP-HPLC packing on this scale and to estimate its “hydrophobicity”, a test with sets of only four or five test compounds is proposed.

Performance evaluation of non-porous versus porous ion-exchange packings in the separation of proteins by high-performance liquid chromatography

The performance of a non-porous, anion-exchange packing was evaluated and compared with a number of similar porous high-performance liquid chromatography packings. The non-porous columns were found to be equally efficient for proteins spanning a wide range of molecular weights, while the porous columns exhibited decreasing efficiency as the proteins became larger. The porous materials also exhibited size exclusion effects that were not seen with the non-porous materials, which partially accounts for the loss of efficiency with large proteins. When increasingly steep gradients were employed, the loss of resolution was less with the non-porous materials.