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

Column packings widely used for high-performance liquid chromatography (HPLC) mostly are based on porous silica microspheres with certain pore sizes and pore size distributions. Such materials have the most desirable compromise of properties that provide for effective and reproducible separations over a wide range of operating conditions. To provide desired separation characteristics, several manufacturers specially synthesize the silica particles for these packings. While such column packing materials have general utility for a wide range of needs, special silica-based particles have been synthesized with different physical conformations for special separation goals. This presentation describes some atypical types of silica-based particles with unique separation properties that enlarge the capabilities of HPLC methods.     

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

Summary 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.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Endcapping of high performance liquid chromatography packings derived from silica gel

The 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 chromatography column packings with deliberately broadened particle size distribution: relation between column performance and packing structure

The effect of the addition of 25%, 50% and 75% (weight percent, wt%) of larger particles (resp. 3 and 5 μm) to a commercial batch of 1.9 μm particles has been investigated as an academic exercise to study the effects of particle size distribution on the kinetic performance of packed bed columns in a magnified way. Comparing the performance of the different mixtures in a kinetic plot, it could be irrefutably shown that the addition of larger particles to a commercial batch of small particles cannot be expected to lead to an improved kinetic performance. Whereas the addition of 25 wt% of larger particles still only has a minor negative effect, a significantly deteriorated performance is obtained when 50 or 75 wt% of larger particles are added. In this case, separation impedance number increases up to 200% were observed. Studying the packing structure through computational packing simulations, together with the experimental determination of the external porosity, helped in understanding the obtained results. This showed that small particles tend to settle in the flow-through pores surrounding the larger particles, leading to very high packing densities (external porosities as low as 32% were observed) and also negatively influencing the column permeability as well as the band broadening (because of the broadened flow-through pore size range).     

Siliceous materials with the surface polymer layer composed of dextran-polyimine mixture as column packings for liquid chromatography

Summary Forming a polymer layer on the surface of siliceous materials is one of the methods for protecting the silica skeleton from dissolution in alkaline mobile phases as well as eliminating the negative influence of silanol groups on separated molecules e.g. proteins. Polysaccharides, especially their derivatives bearing amine groups, can play the role of the surface layer. This paper discusses the possibilities of preparing such a layer by cross-linking a dextran-polyimine mixture (rather than the traditionally used DEAE-dextran) deposited on the surface of the solid material. The results presented prove the utility of synthesized materials as supports for affinity ligands in high performance affinity chromatography or as supports for complexed metal ions in ligand-exchange chromatography. The properties of the sorbents with a polymer layer can be changed both by the composition of the cross-linked mixture and by chemical modification.     

Chiral bonded packings for liquid chromatography: Preparation and evaluation of N-acylated aminoacid packings based on aminopropylated silica gel

Summary N-Protected single amino acid residues have been chemically bound to aminopropylated silica gel to give chiral HPLC column packings. Surface coverages have been determined by both elemental and aminoacid analyses. In addition, the nature of the surface-bound groups has been studied by reflectance fourier transform infrared (FTIR) spectroscopy. The resolution of a range of racemic mixtures has been achieved using the column packings.     

Column packings for high-performance liquid chromatography and positron annihilation lifetime spectroscopy

Positron annihilation measurements as a function of temperature and the length of bonded alkyl groups have been carried out on silica gel samples. Silica gel samples were bare and bonded with alkyl group from C1 up to C18. The diameters of pores were deduced from the lifetime of trapped ortho-positronium (o-Ps), and it was found that o-Ps lifetime provides reasonable information on the pore sizes for both bare and alkyl bonded silica gels.     

Microbore columns in quantitative column liquid chromatography

Summary Some quantitative implications of the use of microbore columns in column liquid chromatography are investigated. Although for several pumps the flow rate stability at 50 l min^–1 is slightly worse than that of pumps operating at 1 ml min^–1, the quantitative performance with respect to repeatability and reproducibility of response factors equals that of conventional liquid chromatography. Thermostatting is strongly recommended for microbore column operation.     

High-temperature liquid chromatography of steroids on a bonded hybrid column

A hybrid stationary phase, XTerra MS C18, has been evaluated for the high-temperature reversed-phase liquid chromatography of selected hydrophobic steroids. The effects on the retention and efficiency at temperatures up to 130°C and eluent compositions from methanol–water mixtures to superheated water were studied. The thermodynamic data of the separations were determined. It was shown that increasing the temperature enabled the percentage of methanol to be reduced. High mobile-phase flow rates could be used, but for these non-polar analytes, the retention times with superheated water as the eluent were still high.     

Relationship between the particle size distribution of commercial fully porous and superficially porous high-performance liquid chromatography column packings and their chromatographic performance

The separation efficiency and kinetics of several commercial HPLC particle types (both fully porous and superficially porous) have been investigated using a pharmaceutical weakly basic N-containing compound as a test molecule. A strong trend between the particle size distribution (PSD) of the particles and the typically employed “goodness of packing”-parameters was observed. The relative standard deviation of the PSD of the tested particles ranged between 0.05 and 0.2, and in this range, a near linear relationship between the A-term constant, the h_min-value and the minimal separation impedance was found. The experimental findings hence confirm the recent observations regarding the relationship between the narrow PSD of the recently commercialized porous-shell particles and their superior efficiency and kinetic performance. The outcome also suggests that the performance of the current generation of fully porous particle columns could be significantly improved if the PSD of these particles could be reduced.     

Control of column temperature in reversed-phase liquid chromatography

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Quantitative analysis of tylosin by column liquid chromatography

A column liquid chromatographic method suitable for the quality control of tylosin A is described. The determination can be carried out on different C₈ or C₁₈ columns, using a mobile phase containing acetonitrile, 0.2 M tetrabutylammonium hydrogensulphate, 0.2 M phosphoric acid and water. The flow-rate is 1 ml/min and detection is performed at 280 nm. The method shows good selectivity towards the major components tylosin A, B, C and D and demycinosyltylosin. Minor degradation products, mainly observed in solutions, are also separated. The compositions of several standards are compared and results for a number of commercial samples are presented.     

Low-level flow counting of liquid chromatography column eluates

Summary The principal parameters which determine the operation of a high-resolution, high-sensitivity radioactive flow monitor are described: a) Sample preparation to ensure adequate recovery of radiolabelled sample, metabolites and internal standard. b) The instrument background count rate, when no sample or radiolabel is present in the flow cell, is a function of shielding and a reduction in noise obtained with a coincidence time below one microsecond. c) The minimum detectable amount of label depends upon the machine background, HPLC eluent and scintillator flow, whether or not packed flow cells are used, flow cell geometry, and the scintillator used. d) Three types of flow cell have been designed to cover the majority of HPLC and isotope applications. e) The performance of solid and liquid scintillators. It is shown that an instrument has been designed taking account of these parameters. The resulting design satisfies present high sensitivity counting requirements and maintains the resolution of current HPLC procedures when detection is by simultaneous flow radioassay and by optical methods.     

Refractive index detection in microbore column liquid chromatography

Summary An existing commercial refractive index detector was modified for use with microbore column LC systems. The detector utilizes the Fresnel method. The effect of band dispersion and dilution at the detector side is of extreme importance in connection with the miniaturized LC system. In the modified model the original heat-exchanger tube was removed and a stainless steel capillary was used for heat-exchanging. Gaskets having different cell volumes were also examined with respect to band broadening and sensitivity. The detection limit was 10ng for di-n-pentyl phthalate. The examples include the detection of phthalates, alcohols, n-paraffins, and kerosine.