High-performance Liquid Chromatography Packing Research Articles

Improved silica-based column packings for high-performance liquid chromatography

Undescribed adsorption of basic compounds and the low hydrolytic stability of alkyl bonded-phase ligands can be strongly affected by the existence of highly-acidic, essentially non-hydrogen bonded (or isolated) SiOH groups on silica packings. We have previously proposed that the support for stable silica reversed-phase packings with low adsorptivity for basic compounds should contain the highest, and not the lowest, number of homogeneously distributed, associated SiOH groups. To obtain such desirable supports we fully hydroxylate calcined silica by special techniques involving the dissolution and redeposition of silici acid. This rehydroxylation process results in no substantial change in the surface area or pore size of the silica support. Subsequently, the properties of these silicas were investigated by chemical, spectroscopic and chromatographic techniques. Fully hydroxylated silicas exhibit: (1) a larger number of associated silanols; (2) higher pH values; (3) markedly lowered adsorptivity for basic compounds; (4) significantly improved hydrolytic stability of bonded-phase ligands; and (5) increased mechanical stability. Large pore-size, reversed-phase packings made from this kind of support produced much better separations of basic peptides and proteins than conventional column packings.

Separation of proteins on a polymeric fluorocarbon high-performance liquid chromatography column packing

A rigid, inert fluorocarbon resin has been evaluated as an high-performance liquid chromatography packing for the separation of proteins and peptides. Samples are separated with gradient elution of the mobile phase from aqueous buffer to acetonitrile. The selectivity and retention of the fluorocarbon resin packing for proteins are similar to that of alipathic hydrocarbon-bonded silica packings.

Role of the functional group in n-octydimethylsilanes in the synthesis of C 8 reversed-phase silica packings for high-performance liquid chromatography

C 8 reversed-phase packings have been sysnthesised by reaction of a 25-nm pore-size high-performance liquid chromatographic silica (10 μm, a s(BET) = 297 m 2 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 (C 8-Cl), n-octyldimethylhydroxysilane (C 8-OH), n-octyldimethylmethoxysilane (C 8-OCH 3), n-octyldimethylethoxysilane (C 8-OC 2H 5), n-octyldimethyl(dimethylamino) silane [C 8-N(CH 3) 2], n-octyldimethyl(trifluoroacetoxy)silane (C 8- OCOCF 3), and bis-( n-octyldimethylsiloxane) (C 8-O-C 8). C 8-Cl, C 8-OH and C 8-OCH 3 each form a reactive intermediate with 2,4-lutidine, favouring the reaction with the hydroxyl groups of the silica. In the C 8-Cl reaction with silica, an amount of water larger than half of the stoichiometric amount of C 8-Cl prresent in the starting reaction leads to a drastic decline in the ligand density of bonded C 8 groups. This is associated with the formation of C 8-O-C 8, the latter exhibiting an extremely low reactivity. The highest reactivity of the silanes studied was observed for C 8-N(CH 3) 2, since this already contains a reactive Si bond and furthermore eliminates the volatile dimethylamine, which is a co-product of silanization. The ligand density of the bonded C 8 groups in the reaction of C 8-N(CH 3) 2 with silica was affected scarcely at all by the thermal pretreatment temperature (up to 800 K) of the silica, and the hydroxyl group concentration of the silica showed an approximately linear decrease at temperatures of 500–900 K. Under comparable conditions, the reactives of the silanes followed the sequence C 8-N(CH 3) 2 > C 8-OCOCF 3 > C 8-Cl ⋍ C 8-OH ≈ C 8-OCH 3 ≈ C 8-OC 2H 5 ⋍ C 8-O-C 8, with a maximum lignad density of 4.10 μmol m -2 for C 8-N(CH 3) 2. In accordance with their reaction mechanisms, distinct patterns in the course of the ligand densities achieved by the silanes as a function of the reaction temperature were obtained.

Determination of silanol groups on packings for high-performance liquid chromatography by means of isotopic exchange

A fast, easy, and non-destructive methosd has been developed for the determination of silanol groups in liquid chromatography columns by using common high-performance liquid chromatography instrumentation. The method is based on the complete exchange of the isolanol protons with deuterium.

Comprehensive characterization of some silica-based stationary phase for high-performance liquid chromatography

Comprehensive studies have been carried out on the effect of the silica support in preparing optimized bond-phase packing for high-performance liquid chromatography. Different covalently bonded reversed-phase silica-based chromatographic materials have been investigated with respect to lingand density, residual silanol group type, and concentration. Results from diffuse-reflectance Fourier-transform infrared spectroscopy, elemental and thermogravimetric analysis, and 1H solid-state- and 29Si cross-polarization magic-angle spinning nuclear magnetic resonance techniques were combined with liquid chromatographic data to provide quantative insights regarding the properties of silica-based bonded-phase packings. We have determined that unfavorable adsorption of basic compouds and low hysdrlytic stability of alkyl bonded-phase ligands can attrilbuted to the existence of siolated, non-hydrogen-bridged, high acidic SiOH groups. Contrary to comon belief, a stable silica support with the lowest adsorptivity for basic compounds must contain the highest possible (and not the lowest) concentration of homogeneously distributed, associated or bonded SiOH groups to ensure a minium concentration of deleterious highly acidic, isolated silanols. Under these conditions, silica-based bonded-phase packings exhibit higher hydrolytic staility and a substantially lower order of adsorptivity towards basic organic compounds.

Chromatographic Characteristics of polymer-based high-performance liquid chromatography packings

Rigid macroporous polystyrene-based and polyacrylamide-based packings have been developed for reversed-phase high-performance liquid chromatography. Data on pore size distribution, total pore volume and surface area have been obtained for comparison with silica-based packings. Columns containing these polymer-based packings have good permeability with maximum working pressures of 5000 p.s.i. and 3500 p.s.i. for polystyrene-based packing differs from that for the polyacrylamide-based packing. The dependence of column efficiency on teh flow velocity of the mobile phase as a function of solute type and temperature has been studied. Plate height is reduced as a function of solute type and temperature has been studied. Plate height is reduced considerably at higher temperature. This is tentatively interpreted in terms of a reduction in solute dispersion due to improved mass transfer in the stationary phase.

Factor analysis and experiment design in high-performance liquid chromatography : V. Selectivity of chalcone configuration isomers on 23 reversed-phase packings

The selectivity of 22 pairs of configuration isomers of X,Y-substituted chalcones (XC 6H 5CH = CHCOC 6H 5Y) on 23 reversed-phase high-performance liquid chromatographic (HPLC) packings has been studied. The main factors influencing the selectivity of these isomers were analysed with principal component analysis (PCA). The chromatographic behaviour of the E-s- cis and Z-s- cis chalcones was found to be well related to the substitution pattern on the chalcone core. PCA helps to formulate a general rule for selection of stationary phases suitable for separation of the configuration isomers and enables a classification of the HPLC packings.

Direct coupling of packed fused-silica liquid chromatographic columns to a magnetic sector mass spectrometer and application to polar thermolabile compounds

Fused-silica columns of 0.22 mm I.D. packed with normal small-particle high-performance liquid chromatography packing material were studied. The shape of the column end allows direct connection to a combined electron impact—chemical ionization (EI—CI) ion source. To inject small volumes a syringe-loaded micro-sample injector is used with the time splitting technique. Mass spectra obtained from easily vaporized compounds are similar to, but display less thermal decomposition than, normal EI spectra. Mass spectra of underivatized native mono- and disaccharides give useful structural information. Results from the analysis of cardiac glucosides are also shown. The system provides high-efficiency chromatography with a universal detector, suitable for the analysis of non-volatile and labile compounds.

High-performance liquid chromatographic purification of deoxynucleoside 5′-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis

This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2′-deoxynucleoside 5′-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP,dGTP,dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N 6-etheno-dATP (εdATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pur dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. εdAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.

Analysis of Proteins with new, mildly hydrophobic high-performance liquid chromatography packing materials

A series of packings that had alkyl and aryl groups incorporated into a hydrophilic polymeric matrix were developed for hydrophobic interaction chromatography. Using an inverse salt gradient of ammonium sulfate or sodium chloride, a series of proteins were purified and the activity of trypsin was monitored using post-column detection. Retention times of proteins generally increased in the following order of ligands: hydroxypropyl, propyl, benzyl, isopropyl, phenyl and pentyl.

The evaluation of “reversed phase” high-performance liquid chromatography packing materials

For the evaluation of reversed phase packing materials a mixture of acetylacetone, I-nitronaphthalene and naphthalene is proposed. This will reveal the usual optimum kinetic chromatographic parameters (the naphthalene peak), the degree of activity or endcapping status of the column (the ratio of the I-nitronaphthalene and naphthalene retention times) and trace metal activity (the shape and intensity of the acetylacetone peak).

Pyrolysis of reversed-phase high-performance liquid chromatographic packing materials

Pyrolysis coupled with gas chromatography and or mass spectrometry, allows the identification of the bonded chain in reversed-phase high-performance liquid chromatographic stationary phases. It is possible to distinguish whether an octadecylated reversed-phase was prepared with a trifunctional(e.g., trichloro or monofunctional dimethyl, e.g., dimethyl-ethoxy) octadecylsilane from the relative heights of the heptadecene and octadecene peaks. The nature of the pyrolysis products was investigated. No carbon chains are formed with more carbon atoms than in the bonded chain. The peak area ratio of methane to that of the combined C 4 products allows one to deduce whether the reversed-phase was deactivated as not by reaction with a trimethylsilylating reagent (end-capping).

Chemically-bonded chiral column packings for high-performance liquid chromatography

N-Protected aminoacids have been chemically bonded to aminopropylated silica gel, and the resulting chiral phases have been applied to the LG resolution of racemic mixtures of polar compounds. Best results were obtained with surfacebound N-formyl-isoleucine and N-formylvaline, with which baseline resolutions of a range of aminoacid and aminoalcohol derivatives were achieved.

Endcapping of high performance liquid chromatography packings derived from silica gel

AbstractThe effect of endcapping a reversed phase chromatographic packing material derived from silica gel depends on the degree of derivatization of the phase. The effect can be dramatic for phases with low loading. Whether an octadecylated silica gel has been endcapped or not can be ascertained by chromatographing the pair naphthalene/1‐nitronaphthalene with a water/methanol eluent producing at least k' ≈ 10 for naphthalene. The ratio of the two k' values is 1.4 or higher for an endcapped material, while it is only 1.1 ‐1.2 for a non‐endcapped phase. A similar approach fails to give conclusions of similar utility for octylated silica gels.

High-performance liquid chromatographic purification of endogenous tumour cell growth inhibitory peptides

Five endogenous growth inhibitors of JB-1 ascites tumour cells have been further purified and characterized. Probably because of the high biological activity of most of the inhibitors which are low molecular weight peptides, these have been extremely difficult to handle using conventional purification methods. However, high-performance liquid chromatography (HPLC) turned out to be a very powerful and useful technique in the last purification steps. Although many types of HPLC packings were tried, only a few of them (Nucleosil 5C-18 and Nucleosil 5CN) behaved satisfactorily for the present purpose.

Comparison of styrene—divinylbenzene copolymer and C 18 silica reversed-phase packings for high-performance liquid chromatography of gibberellins

Comparison of styrene—divinylbenzene copolymer and C 18 silica reversed-phase packings for high-performance liquid chromatography of gibberellins

Hydrophobic chromatography with dynamically coated stationary phases : II. Dynamic cation-exchange separations of tyrosinyl peptides

A new class of dynamically produced ion exchanger is shown to be the product of equilibration of a silica high-performance liquid chromatographic packing material and eluents composed of aqueous-organic solutions of both a non-ionic and an anionic surfactant. This new technique, conveniently called “dynamic soap chromatography”, may be used to separate mixtures of peptides and non-ionic organic compounds with the same efficiency as may be obtained from the other modes of high-performance liquid chromatography now in common use.

Isocratic multi-column high-performance liquid chromatography as a technique for qualitative analysis and its application to the characterisation of basic drugs using an aqueous methanol solvent

A variety of high-performance liquid chromatography packing materials were prepared and their chromatographic properties compared for separating several basic drugs using a single solvent system. The three most promising packing materials (silica, a mercapto propyl modified silica and a n-propyl sulphonic acid modification) were subsequently used to provide retention volume data for a large number of drugs.

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

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 studied except for that containing large macropores.

Relationship between chromatographic performance and surface structure of alumina microspheres

Spherical alumina high-performance liquid chromatography packing material has been thermally modified at temperatures ranging from 200 to 950°. The chromatographic performance of the modified columns was tested using solutes of varying column capacity ratio ( k′ = 0–14), with n-hexane as the eluent. The parameters k′, α, and ϕ′ have been obtained and conclusions concerning the solute adsorption mechanisms at various temperatures have been postulated. The effects of pre-treatment of the alumina on the column characteristics prior to column packing are also discussed.