Spherical Versus Irregular-Shaped Silica Gel Particles in HPLC

Abstract

With optimised packing procedures, spherical shaped silica gel particles produce 1.5 to 2 times more plates in HPLC than irregular shaped silica gel particles. The lowest reduced plate height obtained by us so far is for 5 μm ROSiL-C₁₈-HL-D and is h: 1.62 for k': 4.5. It is suggested to transform h into 100/h% and to name this the “Chromatographic efficiency”, or a % of the ideal 100% limit. This limit would be an h value equal to the mean particle diameter. Spherical and irregular silica gel particles of 5 and 10 μm particle diameter and with similar physical characteristics have the same permeability in HPLC columns.

Whether a correct column packing procedure is used can be shown by the constancy of plate number and column permeability in function of different packing pressures.     

气溶胶粒子通过填充柱的保留时间分布测定

采用亚微米单分散聚苯乙烯球形硬气溶胶粒子和脉冲进样技术,测定了气溶胶粒子通过无规则石英砂填充柱的保留时间分布,从保留时间分布曲线得到了气溶胶粒子在填充柱中的平均保留时间和穿透率.研究了平均保留时间和穿透率与流体流速、填充柱的长度、填料粒度和气溶胶粒子大小之间的关系.研究发现,流速越大,保留时间分布曲线越尖锐,流速越小,保留时间分布曲线越平坦;气溶胶粒子的穿透率随着柱长的增加而降低,随流速、气溶胶粒子粒径和石英砂颗粒大小的减小而减小;平均保留时间随柱长增加而增大,随流速增大而减小,随气溶胶粒子粒径减小而减小,而与石英砂颗粒大小几乎无关.     

基于L-谷氨酸的手性硅胶球的制备及其应用

无机介孔硅球因其具有足够的机械强度、热稳定性,以及适应多种流动相的优点,成为高效液相色谱(HPLC)柱填料中使用最广泛和最重要的材料.但在此研究领域中,并未见球形的全无机手性硅胶用作HPLC手性固定相.该文以无机球形介孔硅胶作为研究对象,通过堆砌硅珠法,以硅溶胶为原料,L-谷氨酸(L-Glu)为手性源,在手性环境中制造...     

Theoretical study of the effect of frit quality on chromatography using computational fluid dynamics

Summary Frits at both ends of a chromatographic column, especially for a preparative column, have significant influence on the flow distribution within the column and thus the column efficiency. However, frits have received little attention from chromatographers in the past. Here a theoretical study was conducted with the aid of CFD software FLUENT to investigate the effect of frits on the performance of homogeneous and heterogeneous chromatographic columns. A dimensionless number,FQ, was applied to characterize frit quality. This study visualized how frit quality affects the flow distribution and the concentration band, the shape of eluted pulse at the colum exit and column efficiency. Simulation results show that the development length of the flow distribution is related toFQ but has nothing to do with the packing heterogeneity. The curvature of the concentration band in a column depends onFQ and packing quality. This study shows column efficiency can be improved significantly by increasingFQ and/or frit permeability.     

Hydrodynamic impact of particle shape in slurry packed liquid chromatography columns

We report on a series of flow velocity and efficiency profiles, which were measured across the cross section of preparative chromatographic columns packed with different stationary phase materials using computed tomography. It is shown that this non-invasive technique is very useful for visualization of the inner part of a packed column and measurement of the spatial resolved column packing properties. For evaluation of the influence of the particle shape on the velocity distribution and column performance, irregular and spherical reversed phases were studied in detail. The results showed a decreasing velocity towards the column wall most certainly due to a lower permeability. This effect was much less pronounced in the case of spherical particles, indicating a more homogenous packing structure. The influence of the column packing pressure, as a possible measure for improvement of the packing homogeneity was also studied. It was shown that under the same packing conditions spherical particles always lead to a more homogeneous packing. The overall results of this work contribute to the origin of the fact that spherical material is superior to irregular one from the hydrodynamic point of view.     

Column radial homogeneity in high-performance liquid chromatography

The radial distribution of analyte molecules within an elution band in HPLC was determined by local, on-column, fluorescence detection at the column outlet. Several optical fiber assemblies were implanted in the exit frit at different points over the column cross-section and the fluorescence of a laser-dye analyte was measured. The individual elements of a diode array were used as independent detectors. The distribution of the mobile phase velocity across the column was measured for a number of standard size analytical HPLC columns of different efficiencies, operated at different mobile phase linear velocities. The dependence of the column efficiency on these profiles is discussed.     

Dry-packed capillary columns for micro HPLC

A dry-packing method has been developed which enables the preparation of packed capillary columns for micro HPLC from 250 μm i.d. tubing and 5 μm packing materials. Pressurized gases, such as hydrogen and argon, were used to transport the packing media, and ethanol or methanol were used as discharge agents. By changing the gas pressure, the packing density could be easily adjusted. It was found that, within experimental limits, the higher the packing density, the greater the column efficiency. Comparison between dry- and slurrypacked columns showed that the former had greatly improved stability; the efficiency of dry-packed columns was about the same as, or even better than, that obtained by slurry-packing.     

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 porous ultra-pure silicas with a specific surface area of 350 m2 g(-1) +/- 5% R.S.D., a specific pore volume of 1.0 ml (-1) +/- 3.7% R.S.D., an average pore diameter of 12.0 nm +/- 0.5% R.S.D. and an average particle diameter of 5 microm. Results are presented on trial batches and the final master batch which were both used as packing materials in reversed-phase liquid chromatography (RP-LC) columns. The latter columns were certified and accepted as an HPLC column as reference material (BCR-722) by the European Commission, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.     

Comparison between the loading capacities of columns packed with partially and totally porous fine particles. What is the effective surface area available for adsorption?

The adsorption isotherms of phenol, caffeine, insulin, and lysozyme were measured on two C(18)-bonded silica columns. The first one was packed with classical totally porous particles (3 microm Luna(2)-C(18)from Phenomenex, Torrance, CA, USA), the second one with shell particles (2.7 microm Halo-C(18) from Advanced Materials Technology, Wilmington, DE, USA). The measurements were made at room temperature (T=295+/-1K), using mainly frontal analysis (FA) and also elution by characteristic points (FACP) when necessary. The adsorption energy distributions (AEDs) were estimated by the iterative numerical expectation-maximization (EM) procedure and served to justify the choice of the best adsorption isotherm model for each compound. The best isotherm parameters were derived from either the best fit of the experimental data to a multi-Langmuir isotherm model (MLRA) or from the AED results (equilibrium constants and saturation capacities), when the convergence of the EM program was achieved. The experiments show than the loading capacity of the Luna column is more than twice that of the Halo column for low-molecular-weight compounds. This result was expected; it is in good agreement with the values of the accessible surface area of these two materials, which were calculated from the pore size volume distributions. The pore size volume distributions are validated by the excellent agreement between the calculated and measured exclusion volumes of polystyrene standards by inverse size exclusion chromatography (ISEC). In contrast, the loading capacity ratio of the two columns is 1.5 or less with insulin and lysozyme. This is due to a significant exclusion of these two proteins from the internal pore volumes of the two packing materials. This result raises the problem of the determination of the effective surface area of the packing material, particularly in the case of proteins. This area is about 40 and 30% of the total surface area for insulin and for lysozyme, respectively, based on the pore size volume distribution validated by the ISEC method. The ISEC experiments showed that the largest and the smallest mesopores have rather a cylindrical and a spherical shape, respectively, for both packing materials.     

A particle size distribution analysis of stressed HPLC column packing material

Summary A particle size distribution analysis has been completed on three different HPLC column packing materials including silica gel (Si60) and two bonded phases (RP8 and RP18). The stationary phases were subjected to 18 hours stress with 1 N or 3 N KOH and found to have quantitatively different distribution patterns initially, at 13 hours and finally at 18 hours although the average particle diameters for the Si60 and RP8 were the same or higher at 18 hours as initially. Thirteen hoursstress with sodium octanesulfonate, tetrabutylammonium phosphate and ammonium acetate at exaggerated conditions also resulted in distributional changes with the Si60 and RP8 decreasing in average particle diameter when exposed to ammonium acetate and tetrabutylammonium stressing respectively.     

A Simple Method to Prepare On‐Capillary Frits for Micro‐Column Separation

Sintering stainless steel powders was initially used to prepare the inlet frit in fused silica capillaries. The use of such inlet frits and outlet frits sintered by the stationary phase itself in the capillary to retain C₁₈ particles was demonstrated to withstand the long exposure, up to a high pressure of 60 MPa, for packing and the prepare column was stable and robust enough to do the continuous chromatographic operations. Characterization of the inlet and outlet frits by scanning electron micrography showed the fused metal particles formed a porous network in the capillary inlet and the homogenous separation beddings were obtained by slurry packing.     

Measurement of the eddy diffusion term in chromatographic columns. I. Application to the first generation of 4.6mm I.D. monolithic columns

The corrected heights equivalent to a theoretical plate (HETP) of three 4.6mm I.D. monolithic Onyx-C(18) columns (Onyx, Phenomenex, Torrance, CA) of different lengths (2.5, 5, and 10 cm) are reported for retained (toluene, naphthalene) and non-retained (uracil, caffeine) small molecules. The moments of the peak profiles were measured according to the accurate numerical integration method. Correction for the extra-column contributions was systematically applied. The peak parking method was used in order to measure the bulk diffusion coefficients of the sample molecules, their longitudinal diffusion terms, and the eddy diffusion term of the three monolithic columns. The experimental results demonstrate that the maximum efficiency was 60,000 plates/m for retained compounds. The column length has a large impact on the plate height of non-retained species. These observations were unambiguously explained by a large trans-column eddy diffusion term in the van Deemter HETP equation. This large trans-rod eddy diffusion term is due to the combination of a large trans-rod velocity bias (?3%), a small radial dispersion coefficient in silica monolithic columns, and a poorly designed distribution and collection of the sample streamlets at the inlet and outlet of the monolithic rod. Improving the performance of large I.D. monolithic columns will require (1) a detailed knowledge of the actual flow distribution across and along these monolithic rod and (2) the design of appropriate inlet and outlet distributors designed to minimize the nefarious impact of the radial flow heterogeneity on band broadening.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Characterisation of RPLC columns packed with porous sub-2 microm particles

Eight commercially available sub-2 microm octadecyl silane columns (C18 columns) have been characterised by the Tanaka protocol. The columns can be grouped into two groups that display large differences in selectivity and peak shape due to differences in hydrophobicity, degree of surface coverage and silanol activity. Measurements of particle size distributions were made using automated microscopy and electrical sensing zone measurements. Only a weak correlation could be found between efficiency and particle size. Large differences in column backpressure were observed. These differences are not related to particle size distribution. A more likely explanation is differences in packing density. In order to take full advantage of 100-150 mm columns packed with sub-2 microm particles, it is often necessary to employ not only an elevated pressure but also an elevated temperature. A comparison between columns packed with sub-2, 3 and 5 microm versions of the same packing indicates potential method transferability problems for several of the columns due to selectivity differences. Currently, the best alternative for fast high-resolution LC is the use of sub-2 microm particles in combination with elevated pressure and temperature. However, as shown in this study additional efforts are needed to improve transferability as well as column performance.     

Development of a fritless packed column for capillary electrochromatography-mass spectrometry

A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.     

The application of retention indices using the alkylarylketone scale to the separation of the barbiturates by HPLC. II The effect of the stationary phase

Summary The capacity factors and retention indices, based on the alkylarylketone scale, of ten barbiturates and a set of five column test compounds have been compared on a series of different octadecylsilyl bonded (ODS) silica columns under identical elution conditions of 40:60 methanol-buffer pH 8.5 at 30 °C. The retentions were highly reproducible on columns prepared from the same batch or different batches of ODS-Hypersil, but large differences in the capacity factors were found when column packings from different manufacturers were compared. Retention indices were more reproducible than capacity factors but they could not compensate for differences between the different packing materials. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Properties of monolithic silica columns for HPLC.

Monolithic silica columns and their use in high peak-capacity HPLC separations are reviewed. Monolithic silica columns can potentially provide higher overall performance than particle-packed columns based on the variable external porosity and variable through-pore size/skeleton size ratios. The high permeability of monolithic silica columns resulting from the high porosity is shown to be advantageous to generate large numbers of theoretical plates with long capillary columns. High permeability together with the high stability of the network structures of silica allows their use in high-speed separations required for a second-dimension column in two dimensional HPLC. Disadvantages of monolithic silica columns are also described.     

Fabrication of ZIF‐8@SiO2 Core–Shell Microspheres as the Stationary Phase for High‐Performance Liquid Chromatography

The unique features of high porosity, shape selectivity, and multiple active sites make metal–organic frameworks (MOFs) promising as novel stationary phases for high‐performance liquid chromatography (HPLC). However, the wide particle size distribution and irregular shape of conventional MOFs lead to lower column efficiency of such MOF‐packed columns. Herein, the fabrication of monodisperse MOF@SiO₂ core–shell microspheres as the stationary phase for HPLC to overcome the above‐mentioned problems is reported. Zeolitic imidazolate framework 8 (ZIF‐8) was used as an example of MOFs due to its permanent porosity, uniform pore size, and exceptional chemical stability. Unique carboxyl‐modified silica spheres were used as the support to grow the ZIF‐8 shell. The fabricated monodisperse ZIF‐8@SiO₂ packed columns (5 cm long × 4.6 mm i.d.) show high column efficiency (23 000 plates m^?1 for bisphenol A) for the HPLC separation of endocrine‐disrupting chemicals (bisphenol A, β‐estradiol, and p‐(tert‐octyl)phenol) and pesticides (thiamethoxam, hexaflumuron, chlorantraniliprole, and pymetrozine) within 7 min with good relative standard deviations for 11 replicate separations of the analytes (0.01–0.39, 0.65–1.7, 0.70–1.3, and 0.17–0.91 % for retention time, peak area, peak height, and half peak width, respectively). The ZIF‐8@SiO₂ microspheres combine the advantages of the good column packing properties of the uniform monodisperse silica microspheres and the separation ability of the ZIF‐8 crystals.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.     

Rapid, high-resolution high-performance liquid chromatographic analysis of antibiotics

A HPLC column devised for high separation speed combined with highly practical operating features has been found useful for separating antibiotics. Important characteristics involve compromises in packing particle size, column configuration and support-stationary phase combinations. We determined that these columns are useful for rapid, high-resolution separations with unmodified state-of-the-art HPLC equipment without the extra-column band-broadening effects typical of so-called “fast” HPLC columns. The proposed columns feature efficient sterically-protected monofunctional silane stationary phases that provide good separation reproducibility and high column stability. The combination of these unique bonded silanes and a highly purified, less-acidic silica support give superior peak shapes for antibiotic compounds. The proposed column configuration can halve separation times and double peak heights without loss in resolution, compared to widely used analytical columns. Increased mobile phase flow-rates permit even faster separations of antibiotics with only modest loss in resolution and peak heights for trace analyses in biological systems.