Hydrodynamic chromatography of macromolecules on 2 μm non-porous spherical silica gel packings

Non-porous spherical 2μm silica gel particles have been tested as packing for hydrodynamic chromatography of macromolecules (HDC). Columns packed with these particles in 250×4.6mm i.d. columns can be operated very efficiently (e.g. a reduced plate height of about 2) when the detection cell is miniaturized. The packing is suitable for the separation of polystyrenes of Mw of 104–107 by hydrodynamic chromatography and the retention behaviour of polystyrenes agrees well with the known theoretical models. Preliminary results on the applicability of HDC for the separation of biopolymers are presented.

Role of the functional group in n-octydimethylsilanes in the synthesis of C8 reversed-phase silica packings for high-performance liquid chromatography

Abstract C8 reversed-phase packings have been sysnthesised by reaction of a 25-nm pore-size high-performance liquid chromatographic silica (10 μm, as(BET) = 297 m2 g-1) with 2,4-lutidine as base and dichloromethane and N,N-dimethylflormamide as solvents, or without solvents and with the following silanes: n-octyldimenthylchlorosilane (C8-Cl), n-octyldimethylhydroxysilane (C8-OH), n-octyldimethylmethoxysilane (C8-OCH3), n-octyldimethylethoxysilane (C8-OC2H5), n-octyldimethyl(dimethylamino) silane [C8-N(CH3)2], n-octyldimethyl(trifluoroacetoxy)silane (C8- OCOCF3), and bis-(n-octyldimethylsiloxane) (C8-O-C8). C8-Cl, C8-OH and C8-OCH3 each form a reactive intermediate with 2,4-lutidine, favouri…

Size Exclusion Chromatography of Proteins on Improved Bonded Silica Columns

Publisher Summary This chapter discusses the size exclusion chromatography (SEC) of proteins on improved bonded silica columns. Over the past few years, the use of high performance liquid chromatography (HPLC) has been extended to protein chemistry as a powerful separation and isolation technique. A spectrum of variants providing high resolution potential—ion exchange, reversed phase, hydrophobic interaction, affinity, and size exclusion—is available. Of these, SEC is the simplest with respect to method development and equipment design: proteins are separated according to their size in a short and predictable time span under isocratic conditions. The SEC columns are packed with micro-partic…

High-performance liquid chromatography of amino acids, peptides and proteins

Abstract The thermodynamic constants, associated with the interaction of three proteins with triazine dye affinity sorbents, have been derived from bath and frontal analysis experiments. In cases where mass-transfer restrictions are very high, calculation of the thermodynamic constants directly from frontal analysis experiments could not be achieved. In such cases, a portion of the adsorbate was always present in the effluent, a situation which has its effect as the split peak phenomenon. With Fractogel-based triazine dye affinity sorbents none of the test proteins applied in frontal analysis were adsorbed. A similar behaviour was observed for a Cellufine sorbent during the adsorption of hu…

Investigation of the porosity of silica and alumina with chemically bonded polystyrene

The effect of modification of porous silica and alumina with a copolymer of styrene and vinylsilane on the porosity of oxides have been investigated by means of low temperature nitrogen sorption. Only small changes of the specific surface area and of the specific pore volume were observed on modification of oxides with non-cross-linked copolymer. The specific surface area significantly increased after the cross-linking of the deposited polymeric layer while the specific pore volume remains almost unaffected. A broad hysteresis loop appears in the nitrogen sorption isotherm for the alumina modified with the cross-linked polymer. The porosity of oxides modified by chemisorption method differe…

Effect of polystyrene coating on pore, structural and chromatographic properties of silica packings

Abstract A series of mesoporous and macroporous silicas with a mean pore diameter (PD), between 6 and 200 nm were reacted with a copolymer of styrene and vinyl-methyldiethoxysilane. The mass load of coated silicas corresponded to the monolayer capacity of the polymer calculated on the basis of the molecular cross-sectional area, except for the two mesoporous silcias of PD = 6 and 7.5 nm. Depending on the pore size, changes occurred in the specific surface area, as, between the native and the coated product. A comparison of the specific pore volume, νp, of silicas before and after polymer immobilization indicated a major loss of νp for the two mesoporous silicas whereas this effect was much …

Evaluation and comparison of tailor-made stationary phases based on spherical silica-based beads for capillary electrochromatography via peptide separation analysis.

Small cyclic peptides have been employed to elucidate the performance of novel sorbents as stationary phases in capillary electrochromatography (CEC). In this paper chain length dependencies for ordinary liquid chromatographic sorbents are reported together with findings acquired on beads specifically designed to suit CEC. The latter, tailor-made, spherical, porous silica exhibits a distinguished surface modification to meet the criteria anticipated to enhance performance profiles in CEC. With well-characterised peptides resembling the analytes, probing of the CEC system in a systematic manner (predominantly via the organic modifier content of the background electrolyte (BE)) reveals insigh…

Separation of unmodified α-amino acid enantiomers by reverse phase HPLC

A novel chiral phase system is presented for the resolution of unmodified α-amino acid racemates which is composed of a reverse phase packing coated with N-alkyl-L-hydroxyproline (where alkyl is n-C7H15-, n-C10H21- and n-C16H33-) and a hydro-organic eluent containing copper(II)acetate. The factors controlling retention and enantioselectivity such as concentration of Cu(II)ion and pH of the eluent, addition of NH4Ac to the eluent, type and content of organic solvent of hydro-organic eluent and column temperature were examined. The extremely high enantioselectivity observed (α up to 16) is assumed to be caused by a three site sorbate-sorbent interaction involving bidentate coordination of two…

A model for polybutadiene coatings on porous silica

Non-wetting viscous liquids such as oligobutadiene prefer “active” sites such as pores during the process of physisorption. Thus, polybutadiene (PBD) coatings on porous silica do not result in a homogeneous polymer film but in an inhomogeneous loading where the bulk polymer is mainly sited in the pores of the silica. An increasing polymer loading leads to increasingly filled pores instead of a thicker polymer film. We cannot exclude the possibility that most of the surface is covered at least with a thin polymer film since the chromatographic behaviour is relatively good for polypeptides, which are highly susceptible to the silanol groups of silica.

The Properties Of Commercial Alumina Base Materials And Their Effect On The Manufacture Of Active Porous Alumina Supports By Means Of Extrusion

The behaviour of 60 commercial base aluminas (macrocrystalline non-porous gibbsites, microcrystalline non-porous and porous boehmites, thermally activated porous boehmites and gibbsites) was studied via paste processing and extrusion in order to produce active porous alumina pellets. The most decisive properties of the base materials with respect to extrusion were: the degree of dehydration, the mean of aggregate size, the shape of aggregate size distribution, the shape of aggregates, the size of primary crystallites and the specific surface area. The relevance of these properties to those of the final pellets, i.e. crushing strength, porosity, pore volume distribution and specific surface …

Standardization of sorption measurements and reference materials for dispersed and porous solids

Comparative study of Zorbax Bio Series GF 250 and GF 450 and TSK-Gel 3000 SW and SWXL columns in size-exclusion chromatography of proteins.

Abstract A reduction of the mean particle diameter of silica-based packings in the size-exclusion chromatography (SEC) of proteins to about 5 μm generates the expected increase in column plate number over the traditional 10 μm SEC columns, as demonstrated for the Zorbax Bio Series GF 250 and GF 450 and TSK-Gel 3000 SWXL columns. The slightly lower column efficiency of the TSK-Gel 3000 SWXL compared with the GF 250 column is compensated by the fact that the phase ratio of the 3000 SWXL column is higher by a factor of two. Hence both columns show nearly the same peak capacity of about 20–30. When the ionic strength of the eluent was changed by varying the salt concentration, the elution volum…

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography

Following previous studies of the use of non-porous monodisperse 1.5-microns n-octyl- and n-octadecyl-bonded silicas in gradient elution of proteins, this work was aimed at elucidating further the properties of this novel column material for peptide and protein separations in comparison with wide-pore silicas. First, it is demonstrated that with short columns (e.g., 35 X 8 mm I.D.) packed with these non-porous reversed-phase materials, mixtures of small peptides and mixtures of proteins can be very efficiently resolved. When the chain length of the bonded ligand was varied, the retention of a test set of proteins in gradient elution followed the ligand sequence C18 greater than C8 approxima…

Die Charakteristika der Optimierung in einzelnen HPLC‐Modi

Packings and stationary phases for biopolymer separations by HPLC

Packings and stationary phases applied to high resolution separations of proteins, enzymes, and nucleic acids must satisfy a series of distinct criteria that are different from those usually required by HPLC of low molecular weight non-biologically active analytes. These requirements have been met through substantial improvements in classical gel media together with novel developments in silica supports, and have led to a family of products with tailor-made and reproducible properties. Supports consisting of cross-linked organic gels, and inorganic materials (mostly silicas) are now available with graduated particle sizes, pore sizes, porosities and surface areas as well as non-porous beads…

ChemInform Abstract: The Synthesis of Micrometer- and Submicrometer-Size Spheres of Ordered Mesoporous Oxide MCM-41.

TEMPLATE-FREE AND ALKALI-FREE SYNTHESIZED SILICATES WITH MFI-STRUCTURE -A COMPARATIVE STUDY

ABSTRACT Zeolites and gallosilicates with an MFI-structure which were crystallized from inorganic reaction systems and from alkali-free, tetrapropylammonlumbromide (TPABr) containing systems are compared. The strength, nature and distribution of the acidic sites were determined by FTIR-spectroscopy, TPD, electron microprobing, 29 si, 27 Al and 1 H MAS NMR spectroscopy. The influences of the different trivalent elements as well as the strength, type and distribution of the acidic sites on the catalytic performance of the different MFI-silicates are discussed.

Native and modified alumina, titania and zirconia in normal and reversed-phase high-performance liquid chromatography

Chromatographic properties of silica, alumina, titania and zirconia have been investigated in normal phase mode in the separation of test mixtures of basic, neutral and acidic compounds. In contrast to silica the chromatographic behaviour revealed the basic properties of the alumina, titania and zirconia surfaces. Therefore, separation of basic compounds on these packings seems very promising. Lypophilic packings have been synthesized by modification of titania, zirconia and alumina with organosilanes and polymers and tested for the separation of basic compounds and proteins. High hydrolytic stability of the modified packings was observed during separations with strong alkali and acidic elu…

Analysis of neuropeptide Y and its metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger.

A novel restricted access cation exchanger with sulphonic acid groups at the internal surface was proven to be highly suitable in the sample clean up of peptides on-line coupled to HPLC-electrospray ionization (ESI)-MS. Neuropeptide Y (NPY) and several of its fragments in plasma were subjected to the sample clean-up procedure. The peptides were eluted by a step gradient from the restricted access column, applying 10 mM phosphate buffer pH 3.5 from 5 to 20% (v/v) of acetonitrile with 1 M NaCl and transferred to a Micra ODS II column (33x4.6 mm). The separation of the peptides and their fragments was performed by a linear gradient from 20 to 60% (v/v) acetonitrile in water with 0.1% formic ac…

Flow-through pore characteristics of monolithic silicas and their impact on column performance in high-performance liquid chromatography

Abstract In order to elucidate the role of the flow-through characteristics with regard to the column performance in high-performance liquid chromatography (HPLC) native and n -octadecyl bonded monolithic silica rods and columns, respectively of 100 mm length and 4.6 mm ID with mesopores in the range between 10 and 25 nm and macropores in the range between 0.7 and 6.0 μm were examined by mercury intrusion/extrusion, scanning electron microscopy, image analysis and permeability. The obtained data of the flow-through pore sizes and porosity values as well as surface-to-volume ratio of the stationary phase skeleton enabled to predict their influence to the chromatographic separation efficiency…

ChemInform Abstract: Nonporous Silica Microspheres in the Micron and Submicron Size Range: Manufacture, Characterization and Application

Microporous hypercrosslinked polystyrene Styrosorb as a restricted access packing in sample clean-up for high performance liquid chromatography. Part I: Evaluation of restricted access properties

Styrosorb is a beaded microporous polystyrene with particle sizes between 2 and 4 μm. In spite of hypercrosslinkage the material was seen to swell in organic solvents. The native material is functionalized with Tris-groups at the outer surface of the particles. The average pore diameter was determined as 1.45±0.3 nm from size exclusion data using polystyrene and polyethylene standards in THF. The reversed phase behavior of the restricted access phase Styrosorb was investigated by injection of two test mixtures. Mixture I contained five aromatic amines, mixture II consisted of AmperozideR and four related compounds. The optimum range of mobile phase composition was assessed so that analytes …

Reversed-phase high-performance liquid chromatography of proteins and polypeptides on polystyrene-coated silica supports

Abstract A new type of hydrolytically stable reversed-phase packing material prepared by multi-point covalent binding of polystyrene chains onto the surface of porous silica was examined in the high-performance liquid chromatography of proteins and polypeptides. Whereas wide-pore material was shown to give a rapid and efficient resolution of proteins, packings with smaller pores provided better selectivities for peptides.

Non-porous microparticulate supports in high-performance liquid chromatography (HPLC) of biopolymers — concepts, realization and prospects

Packing meterials with surface barriers, controlled distribution and topography of ligands for sample clean-up and analysis of biologically active solutes in HPLC

Various types of, so-called, restricted access packings have been developed for sample clean-up and analysis of biologically active solutes by means of HPLC. The unique feature of these packings is that they prevent the access of matrix components such as protein whilst selectively retaining the drug components and their metabolites. This results from the fact that the particles of the packings possess a surface barrier for large solutes at the external or internal surface area and exhibit a controlled gradient in the chemical surface composition. They are grouped, according to their particle structures, into internal-surface reversed phases (ISRP), shielded hydrophobic phases (SHP), semi-p…

BONDED SILICA PHASES FOR THE SEPARATION OF BIOPOLYMERS BY MEANS OF COLUMN LIQUID CHROMATOGRAPHY

Monomeric vs. polymeric bonded iminodiacetate silica supports in high-performance ligand-exchange chromatography

The synthesis of monomeric and polymeric bonded iminodiacetate-groups on silica supports is described. The polymeric support offers advantages over the monomeric, such as (i) preparation is much simpler, (ii) higher surface concentration of functional groups can be achieved without loss of efficiency and (iii) hydrolytic stability. Cu2+ loaded supports were examined for their kinetic performance, in particular for the C-parameter of the Knox equation, and for their hydrolytic stability. Calculation of the surface coverage shows that only a rather thin polymeric layer is formed; this is in agreement with the results of the kinetic performance. The retention of α-amino acids on the Cu2+ loade…

Application of a weakly basic dimethylamino-modified silica ion exchanger to the separation of oligonucleotides

Abstract LiChrosorb RP-8, RP-18 and Diol as well as a newly synthesized basic dimethylamino-modified silica ion-exchanger (DMA-silica) were applied for the separation of adenylic acid, cytidylic acid and uridylic acid oligoribonucleotides. On LiChrosorb RP-8 and RP-18, respectively, in aqueous buffered eluents (K 2 HPO 4 - H 3 PO 4 ), the retention of oligonucleotides was increased with decreasing number of nucleotide units in the solute, i.e., with increasing hydrophobic character. The elution behaviour of ologonucleotides on LiChrosorb Diol followed the same order but took place according to a size-exclusion mechanism. The retention of oligonucleotides on DMA-silica is assumed to be based…

Pore structural characteristics of mesostructured materials prepared under different conditions

This paper presents an overview of the pore structural properties of different mesostructured materials prepared at room temperature, namely MCM-41, MCM-48 and MMS. The stability towards water vapour of some samples is inferred by XRD and nitrogen adsorption after and prior to water adsorption. It is shown that MMS materials are more stable than MCM-41. A new method for the stabilisation of MCM-41 using tetraethoxysilane (TEOS) in hexane as silicification agent is proposed and it is shown to be effective in stabilising the pore structure and to increase significantly the surface hydrophobicity.

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography

Abstract The reversed-phase chromatography of proteins by gradient elution with acidic, low-ionic-strength aqueous—organic eluents is often associated with losses of the biological activity of the protein. In this study, the enzymatic activities of catalase, horseradish peroxidase and pepsin were examined under static and dynamic column conditions on non-porous, monodisperse 1.5-μm reversed-phase silicas with various n -alkyl ligands. Catalase readily lost its enzymatic activity under the influence of the acidic aqueous—organic eluents in the absence of the reversed-phase packing, whereas peroxidase was partially deactivated as a result of combined mobile phase and stationary phase effects …

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography

Abstract The linear solvent strength model of Snyder was applied to describe fast protein separations on 2.1-μm non-porous, silica-based strong anion exchangers. It was demonstrated on short columns packed with these anion exchangers that (i) a substantially higher resolution of proteins and nucleotides was obtained at gradient times of less than 5 min than on porous anion exchangers; (ii) the low external surface area of the non-porous anion exchanger is not a critical parameter in analytical separations and (iii) μg-amounts of enzymes of high purity and full biological activity were isolated.

Hydrolytically stable chemically bonded silica supports with metal complexating ligands: Synthesis, characterization and use in high-performance ligand-exchange chromatography (HPLEC)

A silica of 14 nm pore size was reacted according to two procedures with the following silanes: γ-aminopropyltriethoxysilane (1), N-aminoethyl-N′-aminopropyltrimethoxysilane (2), N-aminoethyl-N′-aminoethyl-N″-aminopropyltrimethoxysilane (3), N-(3-triethoxysilanepropyl)-N, N-diacetic dimethylester (4), N-(3-trisodiumsilanolatepropyl)-N, N-diacetic acid disodium salt (5) and N-(3-trisodiumsilanolatepropyl)-ethylenediamine-N, N′, N′-triacetic acid trisodium salt (6). The reaction of silanes 1–4 with the silica was carried out under anhydrous conditions (procedure A). Silanes 1–6 were subjected to surface modification under essentially hydrous conditions applying a two-step procedure (procedure…

Packing technology, column bed structure and chromatographic performance of 1-2-μm non-porous silicas in high-performance liquid chromatography

This work is aimed at further elucidating the aggregation behaviour of micron- and submicron-size non-porous silicas and the column performance of 1–2-μm C18 silicas in reversed-phase high-performance liquid chromatography of low-molecular weight compounds. It is demonstrated that highly ordered, dense, porous aggregates of such silica beads were obtained by gravity settling and centrifugation. The slurry techniques applied at constant flow-rate and a pressure up to 50 MPa provided less-ordered aggregates, but generated an acceptable performance of columns when 1–2-μm C18 silica beads were employed. To operate columns of 53 mm × 4.6 mm I.D., the maximum flow-rate needs to be ca. 2.5 ml/min …

Protein mapping by two-dimensional high performance liquid chromatography.

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We …

Control of catalytic properties of ZSM-5 made by fast and template- free synthesis.

Abstract The fast template-free synthesis of ZSM-5 was accomplished in both static and stirred autoclaves employing sodiumwaterglass.silica hydrogel and pyrogenic silicas as silica sources. Highly pure crystalline ZSM-5 was obtained in the sodiumwaterglass reaction system in the stirred autoclaves without seeds within 3 hours. Addition of 2% (w/w) of seeds reduced the reaction time to 2 hours. The catalytic activity and shape selectivity of the H-ZSM-5, tested by the disproportionation of ethylbenzene, was found to be largely controlled by the synthesis parameters and by the posttreatments. The homogeneous aluminum distribution across the crystals achieved by this synthetis route resulted i…

The application of porous silica layers in open tubular columns for liquid chromatography

Two methods to realize a porous retentive silica layer on the inner wall of 10–25 µm fused silica capillaries for OTLC, etching and precipitation of silica from solution, have been investigated. Etching of the fused silica capillaries with 1M KOH, creates an activated surface, but the capacity of the silica layer is too small to serve as retentive layer in OTLC. Better prospects are offered by the precipitation of silica from a solution of polyethoxysiloxane, dynamically coated on the inner wall of the fused silica capillary. It appears to be possible to deposite a porous silica layer up to 0.8 µm thick (in a 25 µm capillary) by this method, which seems to be suitable for liquid-solid an dy…

Packings of an unidimensional regular pore structure as model packings in size-exclusion and inverse size-exclusion chromatography

Using porous aluminas and aluminosilicates with a regular and well-defined pore structure an attempt was made to correlate the SEC data of polystyrenes with established theoretical models. The pore structure of the two types of packings was extensively characterized by means of transmission and scanning electron microscopy, nitrogen sorption and mercury porosimetry. The SEC distribution coefficients measured experimentally were correlated with the distribution coefficients calculated from the theoretical models for flexible polymers. A good correlation was observed for the packing of the simplest pore morphology, but remarkable deviations appeared for the materials with a more complex pore …

Sorption Studies on Large ZSM-5 Crystals: The Influence of Aluminium Content, The Type of Exchangeable Cations and the Temperature on Nitrogen Hysteresis Effects

Abstract In comparison to polycrystalline powder large ZSM-5 crystals are best suited for high resolution sorption experiments. The nitrogen isotherms on HZSM-5 at 77 K reveal a pronounced hysteresis loop. The shape and position of the hysteresis relative to the pressure axis has been found to be affected by the aluminium content of the zeolite, the type of framework cation and the sorption temperature. The occurence of the hysteresis and its changes are assigned to a transition from a liquidlike to a solid-like adsorbed phase of nitrogen. The micropore volume v mp derived from the argon and nitrogen isotherm (s. Fig. 2b, first plateau) and calculated according to DUBININ-RADUSHKEVICH was c…

Influence of synthesis conditions on the morphology of Dodecasil 1H

Abstract The synthesis of Dodecasil 1H was studied in the system SiO2/1-adamantylamin/NH3/water with the objective of controlling the particle size and the morphology of the crystals. Since nucleation occurs in a very early stage of the synthesis, the heating rate to the final temperature and/or holding points during heat up have a strong influence on both parameters. Crystals smaller than 150 μm could only be obtained by the use of seed crystals, which are completely consumed in the synthesis with subsequent new nucleation. Using seed crystals, the expensive template 1-adamantylamine can be substituted with cheaper reagents such as sodium dodecyl sulfate or trimethylethylammonium bromide a…

ChemInform Abstract: A Novel Pathway for Synthesis of Submicrometer-Size Solid Core/Mesoporous Shell Silica Spheres.

Preparative separation of proteins and enzymes in the mean molecular-weight range of 10,000–100,000 LiChrosorb diol® packing by high-performance size-exclusion chromatography

Abstract LiChrossorb Diol® packing has been to be well-suited for the separation of proteins and enzymes according to a size-exclusion mechanism in a mean molecular-weight (MW) range between 10,000 and 100,000. Loadability of a small bore column of 6 mm I.D. (A) and a large-bore column of 23.5 mm I.D. (B), both of 250 mm in length, were examined. Defining a 20% decrease of the number of theoretical plates as loadability limit the volume load at constant mass of chymotrypsinogen as representative test solute was ≈ 100 μl for column (A) and 1500 μl for column B at 0.78 · 10-5 g/g of packing for column A and 0.78 · 10-6 g/g of packing for column B, respectively. Mass load to constant injection…

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…

Development of standard operation procedures for the manufacture of n-octadecyl bonded silicas as packing material in certified reference columns for reversed-phase liquid chromatography.

The development of standard operation procedures for the manufacture of a n-octadecyl bonded spherical silica packing from partially condensed tetraethoxysilane as silica source is described. The synthesis comprises five intermediate products and six synthesis steps which were examined according to their reproducibility and robustness. The results led to the optimisation of the manufacturing process for a n-octadecyl bonded silica. Correlations were drawn between the dynamic viscosity of the poly(ethoxy)siloxane (PES), the synthesis parameters, the resulting pore structural properties and particle size distribution of the silicas. Validated procedures were developed to manufacture spherical…

Influence of ligand density on the properties of metal-chelate affinity supports.

A new procedure has been developed to immobilize iminodiacetic acid (IDA) onto the surface of silica supports, such as LiChrospher Si-1000 and 1.5-microns nonporous silica, for use in high-performance immobilized metal affinity chromatography (HPIMAC) of proteins. This IDA immobilization method has been achieved through the synthesis of a new silylation reagent, 1-(iminodiacetic acid di-tert-butylester)-3-glycidoxy-propyltrimethoxysilane (IDA-silane). Various modified silicas of different ligand densities have been prepared by using mixtures between 1 and 100% of the IDA-silane diluted with the corresponding 3-glycidoxy-propyltrimethoxysilane (GLYMO-silane). Frontal analysis was used with t…

Retention behaviour of paracelsin peptides on reversed-phase silicas with varying n-alkyl chain length and ligand density.

As part of further investigations on the characterization of the ligand-induced conformational stabilization of peptides, two series of n-alkyldimethylsilyl bonded silicas have been prepared. In series A the n-alkyl chain length, n, of the bonded phase was varied between 1 and 20 carbon atoms at a constant ligand density. In series B the ligand density, alpha exp, was gradually changed from 0 to 4.1 mumol/m2 on a C1, C4, C6, C8 and C18 bonded phase. The retention behaviour of four peptides of the paracelsin family were examined under isocratic conditions, using a ternary mobile phase of water-methanol-acetonitrile (22:39:39, v/v/v). Plots of k' versus n showed pronounced maxima between n = …

Nonporous Silica Microspheres in the Micron and Submicron Size Range

Uniform nonporous silica microspheres in the micrometer and submicrometer size range are formed by a process which involves reacting alkoxysilanes with water in a short chain alcohol using dissolved ammonia as a morphological catalyst.

The CEC behaviour of several synthetic peptides related to the activin ��A-��D subunits

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-…

Role of solvent and base in the silanization reaction of silicas for reversed-phase high-performance liquid chromatography

Reversed-phase packings were synthesized by reaction of n-alkyldimethylchloro- and trifluoroacetoxysilanes with silicas of 15 and 25 nm pore size, employing tetrahydrofuran, N,N-dimethylformamide, benzene, diethyl ether, dichloromethane and acetonitrile as solvents and triethylamine, pyridine, triazole, quinuclidine, 2,4- and 2,6-lutidine and imidazole as bases. With dichloromethane as solvent and 2,6- lutidine as base, n-alkyldimethyltrifluoroacetoxysilanes did not yield n-alkyl ligand densities higher than those obtained with n-alkyldimethylchlorosilanes under identical conditions. With a given base and given n-alkyldimethylchlorosilane the solvent type was found to exert a noticeable inf…

Super-high-speed liquid chromatography of proteins and peptides on non-porous Micra NPS-RP packings

Abstract The new generation of non-porous silica RP packings commercially available from Micra Scientific was tested for separations of peptides and proteins by means of the gradient HPLC. Extremely high-speed separations were achieved using conventional chromatographic equipment: six proteins could be completely separated within six seconds. Tryptic digest peptides could be resolved in more then 40 components within 2–3 min. The effect of the experimental parameters such as temperature, flow rate etc. was investigated.

Synthesis And Sorptive Properties Of Dodecasil 1 H With An Acessible Pore System

Abstract The synthesis of Dodecasil 1H was studied in the system SiO2/1-adamantylamine/NH3/water with the objective of controlling the particle size and the morphology of the crystals. Since nucleation occurs in a very early stage of the synthesis, the heating rate to the final temperature have a strong influence on both parameters. Using seed crystals and such a heating programme, the expensive template 1-adamantylamine can be reduced to so small amounts, that a nearly template free pore system is achieved. The emptiness of the pore system was demonstrated from adsorption measurement using He and H2.

Stabilization of MCM-41 by Pyrolytic Carbon Deposition

A new method of surface modification which is effective in stabilizing silica grades of MCM-41 in the presence of water vapor is presented. It is shown by means of XRD, low-temperature nitrogen ads...

Comparative study of the performance of spherical and angular silica and alumina supports in the 1-10-μm size range

Summary Columns packed with four different sets of well characterized packings, spherical silica, angular silica, spherical alumina and angular alumina, varying in mean particle diameter ( d p ) between 1 and 10 μm, were compared. On these columns, the chromatographic permeability ( K 10 ), the column resistance parameter (Φ) and the reduced plate height-velocity dependences were determined under identical and comparable conditions. Discrepancies were found for K 10 and Φ depending on the type of packing and the particle shape. The courses of the h versus v plot, however, were nearly identical for packings with d p > 4 μm, showing a minimum at v ≅ 5 and h = 1.5–2.5 for solutes of k ′ ≅ 0.2 …

Capillary Hysteresis in Nanopores: Theoretical and Experimental Studies of Nitrogen Adsorption on MCM-41

Capillary hysteresis in cylindrical nanopores has been studied using MCM-41 as the prime example of a mesoporous material. These materials, due to their regular pore structure, can be considered to be candidates for reference adsorbents for standardizing adsorption measurements and methods for characterization of porous solids. They provide a unique opportunity for verification of theoretical models employed for predicting phase equilibrium in confined geometry. Three samples with monodisperse pore channels have been synthesized and examined using X-ray diffraction (XRD). Nitrogen adsorption isotherms were modeled using nonlocal density functional theory (NLDFT) in a wide range of pore size…

Critical Appraisal of the Pore Structure of MCM-41

Purely siliceous and aluminosilicate types of MCM-41 were synthesized and characterized by means of X-ray diffraction (XRD), transmission electron microscopy (TEM), nitrogen sorption (NS) and size exclusion chromatography (SEC).

On-line high-pressure extraction-high-performance liquid chromatography

Abstract A coupling unit is described that allows on-line operation of a high-pressure extraction apparatus with a high-performance liquid chromatograph. The unit consists of two high-pressure sample-injection valves, connected in series. The first valve operates as a switching valve to the loop and controls the release of pressure along a packed microbore column. Two short, packed microbore columns are positioned between the first and second valve, sampling the extracted compounds over a desired period and simultaneously operating as sample loop for the second valve; the latter is the injector for the high-performance liquid chromatographic column. The unit was tested by continuous extract…

Progress in the standardisation of particle and surface characterisation

In 1999, we published a comprehensive survey of standards and certified reference materials for dispersed and porous solids. The present paper summarises and reports on new standards and proposals.

Novel general expressions that describe the behavior of the height equivalent of a theoretical plate in chromatographic systems involving electrically-driven and pressure-driven flows

Novel general expressions are constructed and presented that describe the behavior of the height equivalent of a theoretical plate (plate height), H, as a function of the linear velocity, Vx, along the axis, x, of the column and the kinetic parameters that characterize the mass transfer and adsorption mechanisms in chromatographic columns. Open tube capillaries as well as columns packed with either non-porous or porous particles are studied. The porous particles could have unimodal or bimodal pore-size distributions and intraparticle convective fluid flow and pore diffusion are considered. The expressions for the plate height, H, presented in this work could be applicable to high-performanc…

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography

Abstract In the separation of proteins and peptides by the various modes of high-performance liquid chromatography, the nature of the substrates requires the use of microparticulate silica packings with bonded ligands of appropriate design. Agglomeration of monodisperse silica hydrosols of defined size distribution into beaded particles provides a useful method of controlling the pore size, the size distribution, the particle porosity and surface area of these packings. The particle porosity is shown to be a major factor governing the packing density and packing stability of the column. For size-exclusion chromatography of proteins, parent silicas of pore size 10 and 100 nm with a narrow po…

Chapter 8 Native and Bonded Silicas in Aqueous Sec

Publisher Summary This chapter deals with the properties of packings and columns in correlation to chromatographic separations in size exclusion chromatography (SEC) and provides guidelines for optimum support design. The chapter provides a survey of the current state-of-the-art with respect to the surface modification of silica for SEC packings in aqueous eluents. The properties of commercial packings and columns are listed and compared. An advice is also discussed on the operation and maintenance of columns with a view to achieving high resolution and long life-time. In SEC, solute elution is based on equilibrium, entropy­-controlled size exclusion process. According to their size, the so…

Affinity chromatography with triazine dyes immobilized onto activated non-porous monodisperse silicas

Abstract Non-porous monodisperse silicas with a particle diameter of 2.1 μm were modified with different silanes for immobilization of various triazine dyes including Procion Red HE3B, Procion Red MX5B, and Cibacron Blue F3GA. Lactate dehydrogenase and malate dehydrogenase from different species and aldehyde reductase from rat brain were purified by affinity elution using the substrate of the enzyme and NADH. With Cibacron F3GA the selectivity for NADH-dependent enzymes was higher than with the two Procion dyes. The utility of these immobilized triazine dye systems on non-porous silica supports for the rapid separation of Cohn Fraction III plasma proteins, including plasminogen, is also des…

SELF SIMILARITY IN SWELLING SYSTEMS: FRACTAL PROPERTIES OF PEAT

Sphagnum peat gives an example of a swelling system with a self-similar structure in sufficiently wide range of scales. The surface fractal dimension, dfs, has been calculated by means of thermodynamic method on the basis of water adsorption and capillary equilibrium measurements. This method makes possible the exploration of the self-similarity in the scale range over at least 4 decimal orders of magnitude from 1 nm to 10 μm. In a sample explored, two ranges of fractality have been observed: dfs ≈ 2.55 in the range 1.5–80 nm and dfs ≈ 2.42 in the range 0.25–9 µm.

Chromatographic peak profile of ionogenic analytes upon elution with unbuffered eluents

Experimental proof of the model was carried out for the separation of benzoic acid on a LiChrospher RP-18 column. The observed chromatographic peak profile coincides with the peak profile predicted by the model. The retention times of the dissociated and non-dissociated species determined by using an appropriate fitting procedure were 2.2 and 4.3 min, respectively. Based on these values, a theoretical peak profile was calculated, demonstrating a close agreement with the experimentally observed peak profile. An unexpectedly large difference was found for the retention times of the dissociated and non-dissociated species calculated by a fitting procedure and those experimentally measured on t…

The Relevance of Kneading and Extrusion Parameters in the Manufacture of Active Porous Aluminas from Pseudoboehmites

In order to study the effect-on crushing strength, attrition resistance, specific pore volume, and pore volume distribution of extrudates-of kneading and extrusion parameters in the manufacture of active porous aluminas, a commercial pseudoboehmite (Pural SB from Condea Chemie, Brunsbuttel, FRG) was employed. A minimum kneading torque was required to prepare an extrudable paste, predetermined by extrusion conditions. The torque had an upper limit with respect to certain extrudate properties. The amount of water and the concentration of nitric acid as binder solutions were found to have a decisive bearing on the mechanical properties and the distribution of the pore volume. Crushing strength…

Comparative study on the column performance of microparticulate 5-μm C18-bonded and monolithic C18-bonded reversed-phase columns in high-performance liquid chromatography

In this paper we report on the results of a comparative study on the performance of Purospher RP 18e, 5 μm, columns and prototypes of monolithic columns named SilicaROD from Merck, Darmstadt, Germany. The studies were performed on HPLC equipment with minimum extra column contribution. The plate height linear velocity dependency of the Purospher RP 18e column showed a minimum of H of about 10–15 μm at a linear velocity of 1 mm/s. The H versus u curves of the monolithic columns followed the same course. Yet, the curves remained flat up to a linear velocity of about 7 mm/s, where the Purospher RP 18e column could not be operated anymore due to the extremely high back-pressure. In conclusion th…

Optimisation of fast protein separations on non-porous silica-based strong anion exchangers

The adsorbed coating technology using various vinylpyrrolidone-vinylimidazole copolymer compositions was carried out on 1.7 μm non-porous monodisperse silica. It was shown that the retention properties and the loading capacity for bovine serum albumin (BSA) increases with the amount of vinylimidazole in the copolymer composition. The retention behavior of various proteins as a function of the salt composition in the eluent has been applied to find the optimal conditions for the synthesis of the anion-exchange stationary phase. The suitability of these supports for the fast separation of biological molecules is demonstrated. The best resolution and the highest speed in protein analysis was o…

Evaluation of the Stability of Pure Silica MCM-41 toward Water Vapor

Water vapor adsorption/desorption isotherms at 298 K and XRD measurements and nitrogen isotherms at 77 K before and after exposure to water vapor were determined on pure silica MCM-41 samples; samples had different pore widths and were prepared by different synthesis methods, including hydrothermal and room-temperature procedures. It was found that prolonged exposure to water vapor provoked structural alterations in all of the MCM-41 materials studied, the most significant effects being a loss of pore shape uniformity and a large decrease in pore size and, in consequence, pore volume. Analysis of the results suggests that these alterations are due to expansion of the pore walls as well as, …

Novel synthesis of spherical MCM-48

Abstract A novel synthesis route was developed for the cubic member of the M41S family, MCM-48, with a three-dimensional pore system allowing the formation of submicrometre- to micrometre-sized beads with a narrow pore-size distribution. The synthesis is based on the modified Stober method applying tetraethoxysilane, ethanol, water, ammonia and n -hexadecyltrimethylammonium bromide as template. The specific surface area, the specific pore volume and the average pore diameter were varied in the following ranges: 900–1600 m 2  g −1 , 0.5–0.9 cm 3  g −1 and 2–3 nm. Aluminium-, chromium-, gallium-, niobium- and vanadium-MCM-48 were also synthesized following this procedure.

Separation of selected peptides by capillary electroendoosmotic chromatography using 3 μm reversed-phase bonded silica and mixed-mode phases

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentrat…

Theoretical and Experimental Studies of Capillary Hysteresis in MCM-41

Mesoporous materials of MCM-41 type are considered to be reference model adsorbents due to their regular pore structure. Nitrogen adsorption isotherms on MCM-41 are modeled using the Non-Local Density Functional Theory (NLDFT). The thermal dependence of the thermodynamic hysteresis predicted by the NLDFT is confirmed by experimental measurements. The nitrogen hysteresis on MCM-41 at temperatures below 77.4 K or in pores greater than 4 nm is associated with the metastability of the adsorption branch of the isotherm. In the hysteresis regime, the desorption branch is likely to be thermodynamically stable and is recommended for calculating pore size distributions.

Ion-exchange high-performance liquid chromatography of nucleotides and polypeptides on new types of ion-exchange sorbents, based on polystyrene-coated silicas.

A novel type of ion exchanger was prepared by multipoint covalent binding of polystyrene chains onto the surface of porous silica followed by polymer-analogous modification of the bonded layer. Both anion and cation exchangers were synthesized and examined in the separation of nucleotides and proteins. Rapid and efficient separation of basic polypeptides on strong anion exchangers and that of acidic polypeptides on strong cation exchangers could be achieved. For the separation of complete mixtures of polypeptides the application of zwitter-ionic ion exchangers can be recommended.

Impact of acidic/hydrothermal treatment on pore structural and chromatographic properties of porous silicas

Abstract A series of commercial silicas and a laboratory made product were subjected to acidic/hydrothermal treatment with solutions of HCI, HNO3, H2SO4 and HF. The concentration of acid and the temperature and duration of treatment were varied. The specific surface area, content of metal impurities, types and concentration of surface hydroxyl and siloxane groups and crystallinity were determined for native and treated silicas. Only changes in the structural order of the surface could be assessed by means of 19Si cross-polarization magic angle spinning NMR spectrometry and electron diffraction. The native and acid-treated silicas were surface modified to n-octyl derivatives and tested under…

Rational design, tailored synthesis and characterisation of ordered mesoporous silicas in the micron and submicron size range

Characterization of MCM-48 Materials

Mesoporous molecular sieves of MCM-48 type were prepared by conventional hydrothermal and novel room temperature syntheses. Scanning electron microscopy (SEM) studies have shown that nonagglomerate...

High Resolution Sorption Studies of Argon and Nitrogen on Large Crystals of Aluminophosphate AlPO4-5 AND ZEOLITE ZSM-5

High resolution adsorption (HRADS) with argon and nitrogen at 77 K in the pressure range of 10−6 < p/p° < 0.5 were performed on large crystals of zeolite ZSM-5 (180 μm) and aluminophosphate AlPO4-5 (150 μm) using a novel volumetric device. Multi-step isotherms of both adsorptives on ZSM-5 could be observed for the first time. The adsorption followed by low temperature microcalorimetry resulted in distinct exothermic signals at the steps in the adsorption isotherms. Based on the results of atom-atom potential energy calculations (AAP) as well as independent model building it was shown that 24 ‘kinetic’ adsorbate molecules can be filled into a ZSM-5 unit cell. Experimental results are reasona…

Assessment and optimization of system parameters in size exclusion separation of proteins on diol-modified silica columns

Abstract On diol-modified silica columns the retention of proteins is governed by a size exclusion effect, but superimposed on this are some secondary effects, i.e. , ionic and diol-ligand interactions which can be controlled and adjusted reproducibly by varying the eluent composition. The eluent composition also affects the column efficiency and peak shape. Both dependences can be employed to obtain a better resolution of proteins than can be expected from size exclusion alone.

Intracrystalline Diffusion of Benzene in Ga-Silicate

Abstract The sorption kinetics of benzene in large Ga-MFI crystals was investigated under constant volume- variable pressure conditions. A complete analysis of the uptake curves has been performed using solution of a nonlinear Volterra equation which describes the interaction of uptake process with the apparatus. Within the time scale of uptake measurements (10 3 -10 4 s) the uptake curves were found to be consistent with the solution of the second Fick's law. Corrected diffusion coefficients were found to be essentially independent of loading within the loading range investigated and in contrast to the system benzene-HNaZSM-5 [1,2] their temperature dependence is much stronger.

Factorial Design in the Ammonia Based Synthesis of Large Uniform MFI, MEL, DOH, and DOH/DDR Type Zeolite Crystals

This study reports the synthesis of large and uniform crystals of high-silica zeolite materials. Statistic factorial design was applied in order to determine significant parameters for the rapid growth of alkali-free crystals out of ammonia-based synthesis systems. For silicalite-I, high ammonia contents at low water concentrations yielded crystals up to 300 μm in size. The number of crystal nuclei in type MFI synthesis systems was found to be significantly reduced by increasing ammonia and aluminium contents. MEL-type zeolites could be prepared only with the smaller size of 15 μm. Low water concentrations improved the product yield. Aminoadamantane as a guest molecule nucleated highly unif…

The Synthesis of Spherical Mesoporous Molecular Sieves MCM-48 with Heteroatoms Incorporated into the Silica Framework

Characterization of Diesel Soot by Sedimentation Field Flow Fractionation

Surface acidity studies on active aluminas by means of the amine titration method and heterogeneous isotopic exchange using HTO

Abstract The maximum sorption capacity of n-butylamine (A + n-but ), the exchange capacity of HTO (A HTO ) and the acid strength distribution (ASD) - titration of n-butylamine using arylmethanols as indicators - were measured for a series of commercial active aluminas. A + n-but was formally regarded as total acidity and was found to be larger than A HTO , the latter representing a measure of the Broensted acidity. On the other hand, the cumulative acidity derived from ASD gave values smaller or equal to A HTO . Changes in A HTO and A + n-but within a series of aluminas manufactured at different calcination temperatures gave the expected results which were also supported by infrared spectro…

Study on the efficiency of assembled packed microbore columns in HPLC

Stainless steel columns (internally mirror-finished, 125 or 250nm in length, of bore 1.0 or 1.6mm) were slurry-packed with 5µm and 4µm reversed phase silicas (Hypersil ODS, LiChrosorb RP-8 and RP-18 and Superspher RP-8. The HPLC equipment consisted of a pump LC5A (Shimadzu) or a pump 2150 (LKB Instruments), a rheodyne valve 7413 with loops of 0.5, 1.0 and 5.0mm3 and a Jasco-Uvidec 100-II UV detector, variously with one of three specially constructed cells of 0.2, 0.4 and 1.3mm3 volume. Columns were assembled using two types of coupling device employing a stainless steel capillary of 0.12mm bore. The effect of sample volume, design of coupling device in assembled column, detector cell volume…

Comments on the use of alumina in HPLC

Structure and properties ofn-alkyldimethylsilyl bonded silica reversed-phase packings

Summary The effects of the modifier functionality and the chain length ofn-alkylchlorosilanes on the surface structure of packings and on their retention behaviour in reversed-phase chromatography were investigated. Comparative retention studies on three silica packings that had been treated to maximum conversion withn-octyltrichlorosilane (I),n-octylmethyldichlorosilane (II) andn-octyldimethylmonochlorosilane (III) showed that the most pronounced reversed-phase character is obtained by using III as the modifying reagent. A series ofn-alkyldimethylsilyl bonded silica packings were prepared with widely differingn-alkyl chain lengths. Although the packings are very hydrophobic, the maximum su…

Porous zirconia and titania as packing materials for high-performance liquid chromatography

Abstract Porous amorphous zirconia (ZrO 2 ) and titania (TiO 2 ) packings were synthesized as rigid microparticulate beads by means of a sol—gel process. Sufficient rigidity and desired mesoporosity of the ZrO 2 and TiO 2 particles were achieved only by a gel hardening process, followed by heat treatment. The mean pore diameter, p d , the specific surface area, a s , and the specific pore volume, ν p , were controlled by the heat treatment. Typical values were p d = 8 nm, a s = 80 m 2 /g and ν p = 0.23 ml/g. ZrO 2 - and TiO 2 -based revesed-phase packings were prepared by subjecting the native materials to a specific activation process and reaction with octadecyltrimethoxysilane. Native ZrO…

Berechnung der Fraktaldimension einiger poröser Feststoffe aus der Stickstoff-Adsorptionsisotherme

Hysteresis in nitrogen sorption and mercury porosimetry on mesoporous model adsorbents made of aggregated monodisperse silica spheres

Abstract An elucidation of the hysteresis phenomena in nitrogen sorption (NS) and mercury porosimetry (MP) on porous aggregates of non-porous silica spheres of particle diameter 90–1000 nm is presented. First, it is demonstrated that highly ordered and dense aggregates of a porosity of 26–30% are formed by gravity settling and centrifugation. A hexagonal and cubic close-packed structure within the layers and a vertical stacking between the layers were observed. Minor deteriorations were seen between the ordered domains. Second, the pore size distribution (PSD) curves calculated from the adsorption branch of the nitrogen isotherms by means of the Cohan equation and from the desorption branch…

Separation of plasma membrane proteins of cultured human fibroblasts by affinity chromatography on bonded microparticulate silicas.

Abstract Adsorbents for high-performance affinity chromatography were prepared by bonding proteins and reactive Procion triazine dyes to 3-isothiocyanatopropyl- and 3-aminopropylsilicas. The materials prepared were used successfully in the separation of hydrophobic plasma membrane proteins of cultured human fibroblasts. The data obtained show that the reaction of 3-isothiocyanatopropyltriethoxysilane (ITCPS) with the surface hydroxyl groups of silica yields a new and convenient route to preparing an “activated carrier” that is capable of coupling with potential affinity ligands containing amino functional groups. The reaction and bonding procedures of 3-isothiocyanatopropyltriethoxysilane a…

Sorption Kinetics of Xenon on MFI-Type Zeolite Molecular Sieves

Kinetic uptake data for xenon adsorbed onto large and uniform silicalite-I crystals are presented over a temperature range of 121 K to 296 K. — Adsorption isotherms and corrected diffusion coefficients derived from the uptake curves are given. The heat of adsorption and activation energy of diffusion were estimated from the plots of reciprocal temperature against the logarithm of equilibrium pressure and corrected diffusion coefficients, respectively. — While the corrected diffusion coefficient is independent of coverage for the higher temperatures, it decreases significantly at the lower temperatures investigated (<170 K) when the limiting adsorption capacity has been reached. — Comparing …

Critical assessment of particle size analysis of porous silica microbead high-performance liquid chromatographic packings by photosedimentation

Abstract Particle size analysis of commercial spherical silica packings of graduated pore size by means of photosedimentation (PS) is critically examined and compared to results obtained by microscopy. To avoid particle fractionation, the suspension prepared for PS measurements should be subjected to gentle ultrasonic treatment. For computing the particle size distribution, the effective density of solvent-filled porous particles has to be inserted into the Stokes equation. Reproducibility of d p , estimated by PS is found to be largely dependent upon the accuracy of the specific pore volume determination. Satisfactory agreement of data between PS and microscopy is obtained for all products…

Selective extraction of small proteins from biological samples using a novel restricted access column with cation exchange properties

The determination of proteins utilising a polymer-based restricted access suppor material with ion exchange properties (IERAM) is outlined. Solid phase extraction coupled on-line with a mincrobore reversed phase HPLC system for the quantitation of small marker proteins is demonstrated. The cation-exchange restricted access packings were characterised with respect to their adsorption and desorption kinetics. The IERAM material was also investigated by capacity, selectivity, and biocompatibility determinations when applied to the quantification of small molecular weight proteins such as cytochrome C, Lysozyme, Ribonuclease A, Myoglobin, Insulin, human serum albumin, and a Tryptic inhibitor.

Analysis of organic pollutants in water at trace levels using fully automated solid-phase extraction coupled to high-performance liquid chromatography

A method has been developed for the determination of trace levels of 32 pesticides, 19 explosives and 16 polycyclic aromatic hydrocarbons (PAH) in water in three individual steps. Solid-phase enrichment (SPE) is coupled to high-performance liquid chromatography (HPLC) with a fully automated system. The organic pollutants are enriched on reusable cartridges packed with adsorbent materials: pesticides and explosives on a mixed bed of divinylbenzene-ethylvinylbenzene copolymers (LiChrolut EN®) and perfluorinated polyethylene (PolyF®), and polycyclic aromatic hydrocarbons on C18-modified silica (Zorbax® ODS1). Thermally assisted desorption (TAD) has been shown to increase the recovery of analyt…

The Use of 15N NMR for the Understanding of Nitrogen Physisorption.

Abstract Nitrogen adsorption at liquid nitrogen temperature, 77K, is universally employed to determine pore dimensions. The nature of nitrogen physisorbed within the micropores of ZSM-5 zeolite was studied by in situ15N NMR as it depends on the partial pressure of the nitrogen and the temperature above and below 77K. An increase in the volume of adsorption of nitrogen at 77K occurs at a relative pressure of ~0.18 for this zeolite. The nature of the nitrogen was studied below and above this increase in nitrogen adsorption. We find that two states (phases) of nitrogen are evident. We conclude that two phases can be present but that a solid phase of nitrogen is not evident even as the temperat…

Hydrodynamic chromatography of macromolecules on small spherical non-porous silica particles

Abstract Non-porous silica spheres with sizes in the range 1.4–2.7 μm were applied as packings for the hydrodynamic chromatography (HDC) of macromolecules. Highly efficient columns, with a reduced plate height below 2, were packed with these small particles. Up to molecular weights of 10 6 the elution behaviour of polystyrenes agreed very well with existing theoretical models. However, for larger polystyrenes the flow-rate exerted an influence on the relative peak positions. The applicability of HDC to rapid separations of soluble macromolecules and inorganic colloids was demonstrated.

Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogeneous peptides in multidimensional liquid chromatography

Abstract In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm ( n -octadecyl derivative). Additional characterisations were performed on restricted access materials with nitr…

Selective protein removal and desalting using microchip CE.

Abstract This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip™). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP–CZE could not be used for the rem…

Impact of the post-treatment conditions of parent silica on the silanization of n-octadecyl bonded silica packings in reversed-phase high-performance liquid chromatography

Native mesoporous silica beads were subjected to a sequence of post-treatment procedure including hydrochloric acid treatment, calcination and subsequent rehydroxylation. The post-treated silica beads were converted into RP-18 silica by silanization with monochloro- and dimethoxy-n-octadecylsilanes, respectively. The influence of post-treatments and silanization conditions on the physico-chemical characteristics and on the chromatographic behaviour of the RP-silicas was studied. Also the changes of the pore structural parameters and the silanol group densities during the post-treatment and silanization were assessed.

Novel porous carbon packings in reversed-phase high-performance liquid chromatography

Abstract Porous carbon packings of particle size 5–20 μm were prepared by calcination of purified active carbons and cokes having narrow size ranges. Highly efficient columns packed with 10-μm particles were obtained by means of the high-viscosity slurry technique, and exhibited about 3000 theoretical plates at 100 mm length and optimal eluent flow-rate. The loading of columns, θ0.1, was determined to be 25–50 μg of solute per gram of packing. Eluotropic series of solvents were found to be similar to those on pyrocarbon-modified carbons and silicas. The log k′ values for homologous compounds increased linearly with the hydrocarbonaceous surface area at constant eluent composition. Studies w…

Is multidimensional high performance liquid chromatography (HPLC) an alternative in protein analysis to 2D gel electrophoresis?

The interactive modes of High Performance Liquid Chromatography (HPLC) of proteins provide a platform for the construction of a multidimensional HPLC system coupled to mass spectrometry. We present a system composed of both anion and cation exchanger columns, in the first dimension, and n-octadecyl bonded 1.5 μm nonporous silica columns in the second dimension. Both columns are operated under gradient conditions. A system suitability test with standard proteins showed that the total analysis can be performed within about 20 minutes. The fractions taken from the ion exchanger column are directly analyzed within one minute on the reversed phase column at a high flow rate. Two reversed phase c…

Separation of polycyclic aromatic hydrocarbons under isocratic conditions by a column switching technique

A simple and efficient method of separating a 20-component PAH-mixture (RSM 1647 standard mixture +benzene, toluene, perylene and coronene) by RP-HPLC is described. Separation was by using two Superspher-100 RP-18 cartridges thermostatted at different temperatures under isocratic conditions with water-acetonitrile eluent. The analysis time with complete resolution of all components can be reduced to 15 min.

Anwendungsmöglichkeiten der HPLC bei der Trennung, Isolierung, Identifizierung und quantitativen Bestimmung von Substanzen aus komplexen Stoffgemischen—am Beispiel von Carbonsäuren in Natriumaluminatlaugen aus dem Bayerprozeß der Aluminiumoxidgewinnung

Es wird die Anwendung der Hochdruckflussigchromatographie (HPLC) in Bezug auf die Identifizierung und quantitative Bestimmung von Carbonsauren in Aluminatlaugen aus dem Bayerprozes beschrieben. Die Vorgehensweise besteht in folgenden Teilschritten: Optimierung von HPLC Phasensystemen zur Trennung von synthetischen Gemischen von Carbonsauren, Aufarbeitung der Aluminatlauge, semi-praparative Isolierung von Substanzen, Identifizierung auf flussigchromatographischem Wege und durch Massenspektrometrie, quantitative Bestimmung durch Peakhohenauswertung mit Hilfe externer Standards.

Applications of mathematical modelling to the simulation of binary perfusion chromatography

Abstract A mathematical model of binary (competitive adsorption involving two components) perfusion chromatography is used to simulate and study the behavior of a binary adsorption system. The dynamic behavior of column systems (frontal analysis) is examined for different particles sizes, column fluid superficial velocities, Vf, and intraparticle fluid velocities, νp. Column systems with perfusive (νp > 0) and purely diffusive (νp = 0) adsorbent particles are studied. The results obtained from the binary system studied in this work suggest that the times at which breakthrough begins for components 1 and 2, and the dynamic (unsteady state) relative separation between components 1 and 2 obtai…

High-resolution sorption studies of argon and nitrogen on large crystals of microporous zeolite ZSM-5

High resolution adsorption (HRADS) with argon and nitrogen at 77 K were performed on large crystals of zeolite ZSM-5 using a novel volumetric device. Multi-step isotherms for both adsorptives could be observed for the first time. The micropore filling was followed by low temperature microcalorimetry. Exothermic heats of adsorption were found to be correlated with steps in the adsorption isotherms. Based on results from atom-atom potential energy calculations (AAP) as well as from independent model building it is shown that 24 kinetic adsorbate molecules can be situated in a ZSM-5 unit cell. Localized adsorption is presented as possible filling mechanisms. Experimental results are reasonably…

Performance of porous silica layers in open-tubular columns for liquid chromatography

Abstract Progress has been made in the preparation of porous silica layers in fused-silica capillaries for open-tubular liquid chromatography. The porous silica layer is prepared by (i) static coating of the silica precursor, polyethoxysiloxane (PES), followed by (ii) converting the PES film into a porous silica layer with ammonia solution. The porous silica layer can be easily modified by silane reagents commonly used in packed column high-performance liquid chromatography. The performance of the silica layer with the different phase systems was tested with polyaromatic hydrocarbons and derivatized amino acids as samples.

Non-porous polybutadiene-coated silicas as stationary phases in reversed-phase chromatography

Abstract Non-porous silica of mean particle diameter 1.7 μm (Monospher) was coated with polybutadiene (PBD) following a published procedure. The silicas were prepared with graduated polymer loads up to 8% (w/w). Examination of the PBD-coated packings by means of electron spectroscopy for chemical analysis, scanning electron microscopy, diffuse reflection infrared fourier transform spectroscopy and differential thermal gravimetry indicated that the optimum polymer load was between 1 and 3% (w/w) with regard to a dense coverage corresponding to an average layer thickness of about 4 nm. No silanophilic interactions could be monitored using 1–3% (w/w) coated silicas under reversed-phase conditi…

Separation studies of amino acids, proteins and enzymes on bonded 1,2-dihydroxy-, 1,2-hydroxylamino-and amino silica packings

1,2-dihydroxy-3-propoxypropyl (HPPS), 1-amino-2-hydroxy-3-propoxypropyl (AHPS) and 1-aminoethyl-3-aminopropyl (AEAPS) silica were synthesized by means of both a surface modification procedure (I) and a bulk modification procedure (II). Method (I) gave a surface concentration, α, of functional groups of 2–3 μmole/m2, whereas method (II) gave values up to 5 μmole/m2. Retention times, peak asymmetries and plate heights of thiamine and ascorbic acid eluted with aqueous buffer solutions ranging from pH 5.3 to 9.2 gave only a±5% variation over periods of 12 hours and more. The recoveries of selected enzymes and proteines examined under static and dynamic conditions were between 60% and 100% depen…

Formation of hierarchically ordered silicas prepared by spray drying of nanosized spheres

We report on our strategy to synthesise defined arrangements of macro- and mesopores in one single material with spherical morphology using the spray drying technique. As starting materials, nano-sized silica spheres and colloidal suspensions were chosen. The intra-particle as well as inter-particle pore size was independently controlled which allowed to obtain materials with varied bimodal pore networks. The spherical agglomerates were characterised regarding their pore structural parameters and are potential adsorbents in liquid chromatographic separations.

Evaluation of advanced silica packings for the separation of biopolymers by high-perforamnce liquid chromatography

Non-porous monodisperse 1.5-μm silicas were allowed to react with (A) and (B) N-acetylaminopropyltriethoxysilane to generate bonded phases useful in high-performance hydrophobic-interaction chromatography (HIC). Differences in the selectivity were observed between he amide and the ether phase. Peak capacities between 10 and 30 were achieved for several proteins with the amide and ether phase packed into columns of 36 × 8 mm I.D. and elution of the proteins under chromatographic conditions in which the gradient volume, VG, was held constant by varying the gradient time between 20 and 2.5 min and the flow-rate between 0.5 and 4.0 ml/min. The S values derived from the dependences of log k′ on …

Evaluation and comparison of the pore structure and related properties of particulate and monolithic silicas for liquid phase separation processes

Two types of silicas, highly porous beads of 10 μm average particle diameter and particle porosity between 48 to 86 % and monolithic silicas with constant mesopore diameter of 12 nm, constant total porosity of 80 % at variable macropore diameter between 2 and 6 μm were subjected to pore structural characterisation and tests in high performance liquid chromatography (HPLC). The column permeability KF of the columns packed with highly porous beads was twice as much as for the monolithic silica columns. The differences in theoretical plate height - linear velocity curves could be explained on the basis of the pore connectivity values nT of the materials which were computed from the nitrogen so…

Sorption of argon and nitrogen on network types of zeolites and aluminophosphates

Abstract Synthetic zeolites and aluminophosphates comprising 10- and 12- membered ring openings, unidimensional and network type of pore systems (MFI, MEL, ERI, LTA, AEL, AFI and FAU) were used as model adsorbents to examine the impact of micropore structure on the sorption properties. Argon and nitrogen were employed as adsorptives. Adsorption measurements were carried out on gravimetric and volumetric sorption devices and also monitored by microcalorimetry. From the low coverage regime of the isotherm Henry's constants and isosteric heats of adsorption were derived. Both quantities allowed the discrimination between 10- and 12- membered ring systems. Unidimensional 10- and 12- membered ri…

Textural characterization of native and n-alky-bonded silica monoliths by mercury intrusion/extrusion, inverse size exclusion chromatography and nitrogen adsorption

Native and n-alkyl-bonded (n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25 nm and macropores in the range between 1.8 and 6.0 microm were examined by mercury intrusion/extrusion, inverse size exclusion chromatography (ISEC) and nitrogen sorption. Our results reveal very good agreement for the mesopore size distribution obtained from nitrogen adsorption (in combination with an advanced NLDFT analysis) and ISEC. Our studies highlight the importance of mercury porosimetry for the assessment of the macropore size distribution and show that mercury porosimetry is the only method which allows obtaining a combined and comprehensive structural characterization of m…

Relationship between intrinsic pore-wall corrugation and adsorption hysteresis of N-2, O-2, and Ar on regular mesopores

Gravimetric characterisation of the surface properties of a porous drug carrier

The gas adsorption method is the most common means to characterise the topology of solid surfaces with regard to its use as an adsorbent. Adsorption isotherms are determined advanta-geously using a vacuum microbalance: Thermogravimetric techniques allow the observation of sample degassing and its optimization. The dry mass is determined in situ, the mass of gas adsorbed is measured directly and different gases can be used without calibration. From the isotherm the pore size distributions, specific surface area, fractal dimension and density can be derived. Commercially available gravimetric sorption apparata and vacuum balances as well as software for data evaluation are reviewed in tables.…

Effect of Size of Solvent Molecule on the Adsorption of p- and o-Xylene on ZSM-5 Type Zeolites and Mechanism of Adsorption

Summary The adsorption isotherms of p- and o-xylene have been studied on MFI-type zeolites of different alumina to silica ratios from solvents of different molecular sizes. The adsorption is mainly controlled by the shape and size of the pore opening relative to the size and shape of the sorbate and the solvent molecules. Aluminum containing zeolites formed colored π-complexes with p-xylene.

Solute retention in reversed-phase chromatography as a function of stationary phase properties: Effect of n-alkyl chain length and ligand density

Two series of bonded phases were synthesized employing LiChrospher Si 100, 10 μm and n-alkyldimethylmonochlorosilanes as silanizing reagents. In series A the n-alkyl chain length, n, of the bonded phase was varied between 1 and 20 at a constant ligand density of 3.5±0.2μmol·m−2. In series B the ligand density, d, was gradually changed from 0 to 4.1μmol·m−2 on the C1, C4, C6, C8 and C18 bonded phases, respectively.

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 th…

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. …

Adsorption of proteins on porous and non-porous poly(ethyleneimine) and tentacle-type anion exchangers

Abstract Adsorption isotherms of proteins [bovine serum albumin (BSA), soybean trypsin inhibitor and alcohol dehydrogenase] on anion exchangers were measured by on-line and off-line methods. The poly(ethyleneimine) (PEI) type and the tentacle-type materials exhibited principally different modes of adsorption. On thin layers of PEI, bonded to non-porous silica, BSA adsorption data corresponded to a monolayer of molecules, with 80% adsorbed side-on, with a high affinity constant for binding, and 20% adsorbed more weakly. With porous material, the amount of BSA bound per unit surface with high affinity was smaller. With tentacle-type anion exchangers, adsorption exceeded a monolayer by far, an…