High-efficiency liquid chromatographic separation utilizing long monolithic silica capillary columns

Long monolithic silica-C18 capillary columns of 100 microm i.d. were prepared, and the efficiency was examined using reversed-phase HPLC under a pressure of up to 47 MPa. At linear velocities of 1-2 mm/s, 100,000-500,000 theoretical plates could be generated with a single column (90-440 cm in length) using an acetonitrile-water (80/20) mobile phase with a column dead time (t0) of 5-40 min. It was possible to prepare columns with a minimum plate height of 8.5 +/- 0.5 microm and permeability of (1.45 +/- 0.09) x 10(-13) m(2). The chromatographic performance of a long octadecylsilylated monolithic silica capillary column was demonstrated by the high-efficiency separations of aromatic hydrocarbons, benzene derivatives, and a protein digest. The efficiency for a peptide was maintained for an injection of up to 0.5-2 ng. When three 100 microm i.d. columns were connected to form a 1130-1240 cm column system, 1,000,000 theoretical plates were generated for aromatic hydrocarbons with retention factors of up to 2.4 with a t0 of 150 min. The fact that very high efficiencies were obtained for the retained solutes suggests the practical utility of these long monolithic silica capillary columns.     

Performance of monolithic silica capillary columns with increased phase ratios and small-sized domains

Monolithic silica capillary columns for HPLC were prepared from tetramethoxysilane to have smaller sized domains and increased phase ratios as compared to previous materials, and their performance was evaluated. The monolithic silica columns possessed an external porosity of 0.65-0.76 and a total porosity of 0.92-0.95 and showed considerably higher performance and greater retention factors in a reversed-phase mode after chemical modification than columns previously reported. An octadecylsilylated monolithic silica column with the smallest domain size (through-pores of approximately 1.3 microm and silica skeletons of approximately 0.9 microm) showed a plate height of less than 5 microm at optimum linear velocities (u) of 2-3 mm/s in 80% acetonitrile for a solute having retention factors of approximately 1, and approximately 7 microm at u = 8 mm/s. With a permeability similar to that of a column packed with 5-microm particles, the monolithic silica columns were able to attain column efficiencies comparable to that of particulate columns packed with 2-2.5-microm particles, and showed performance in the "forbidden region" for the previous columns. The performance of the monolithic column can be compared favorably with that of a particle-packed column when 15,000-30,000 or more theoretical plates are desired at a pressure drop of 20-40 MPa or lower. The increased homogeneity of the co-continuous structures, in addition to the small-sized domains, contributed to the higher performance as compared to previous monolithic silica columns.     

Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.     

Moment analysis of mass-transfer kinetics in C18-silica monolithic columns

The moment analysis of elution peak profiles based on new moment equations provides information on the mass-transfer characteristics of C(18)-silica monolithic columns. The flow rate dependence of the HETP data was analyzed using the generalized van Deemter equation, after correction of these data by subtraction of the external mass-transfer contribution to band broadening. Kinetic parameters and diffusion coefficients related to the mass-transfer processes in monolithic columns were derived by taking advantage of the different flow velocity dependence of their contributions to band broadening. At high flow rates, axial dispersion and diffusive migration across the monolithic C(18)-silica skeleton contribute much to band broadening, suggesting that it remains important to reduce the influence of eddy diffusion and the mass-transfer resistance in the stationary phase to achieve fast separations and a high efficiency. Surface diffusion plays a predominant role for molecular migration in the monolithic stationary phase. Although the value of the surface diffusion coefficient (D(s)) depends on an estimate of the external mass-transfer coefficient, D(s) values of the order of 10(-7) cm(2) s(-1) were calculated for the first time for the C(18)-silica monolithic skeleton. The value of D(s) decreases with increasing retention of sample compounds. Analysis of a kind of time constant calculated from D(s) suggests that the "chromatographic corresponding particle size" is approximately 4 microm for the C(18)-silica monolithic stationary phase used in this study. The accuracy of the D(s) values determined was discussed.     

Comprehensive two-dimensional normal-phase (adsorption)-reversed-phase liquid chromatography

A comprehensive two-dimensional HPLC system has been developed. It is based on the use of a microbore silica column operated in normal-phase (adsorption) mode (NP) in the first dimension and a monolithic type C18 column operated in reversed-phase (RP) mode in the second dimension. The interface was a 10-port, 2-position valve equipped with two storage loops. The first column was operated at a flow rate of 20 microL/min in isocratic mode, while the monolithic column flow rate was 4 mL/min and was operated in gradient mode. The sample loops had a volume of 20 microL each, and the analysis time in the second dimension was 1 min. In this way, every fraction from the first dimension was transferred on-line to the second dimension switching the automated valve every minute. A photodiode array detector has been used after the secondary column. The use of normal- and reversed-phase mode in the two dimensions can be helpful in the separation of complex mixtures of a natural origin that contain uncharged molecules of comparable dimension, different in polarity and hydrophobicity. The use of a microbore column in the first dimension permits the injection of a small volume in the secondary column, making the transfer of incompatible solvents from the first to the second dimension possible. Since the mobile phase in the NP separation is always stronger than the mobile phase at the head of the secondary column operated in RP mode, the initial eluent strength is important in order to obtain an effective focusing of the sample. The use of a monolithic type column in the second dimension permits the performance of very fast analysis operating at higher flow rates without loss of resolution, due to a higher permeability and increased mass-transfer properties in comparison to conventional particulate columns. Due to the brief reconditioning time necessary for monolithic columns, repetitive gradients can be carried out, extending the field of application to mixtures that contain components with different polarities. The utility of the system has been demonstrated in the analysis of the oxygen heterocyclic fraction of cold-pressed lemon oil, made up of coumarins and psoralens. These components may contain hydroxyl, methoxyl, isopentenyl, isopentenyloxyl, and geranyloxyl groups and oxygen-containing modification of the terpenoid side-chain groups, such as epoxides or vicinal diol groups. The relative location of the components in the 2D plane varied in relation to their chemical structure and allowed positive peak identification. The UV spectra recorded with the photodiode array detector supplied additional information that was used for the characterization of the studied sample.     

Preparation and evaluation of a molecularly imprinted polymer derivatized silica monolithic column for capillary electrochromatography and capillary liquid chromatography

A method for preparation of molecularly imprinted polymer (MIP) derivatized onto the surface of a monolithic silica capillary column was successfully developed. The vinyl groups were first introduced onto the silica monolith by immobilization of gamma-methacryloxypropyltrimethoxysilane. Then the MIP coating was copolymerized and anchored onto the surface of the silica monolith. Acetonitrile was selected as porogen (solvent). The other preparation conditions, such as monomer concentration, temperature, and time of polymerization, were systematically studied. The obtained MIP-derivatized silica monolith using l-tetrahydropalmatine (l-THP) and (5S,11S)-(-)-Tr?ger's base (S-TB) as the imprinted template, respectively, was characterized in terms of the retention behavior of thiourea and toluene. Under the optimized CEC conditions, baseline enantioseparations of THP and TB were achieved in 4 min though the effective length of the columns was 8.5 cm. The result indicates that enough recognition sites were on the surface of silica monolith, resulting in strong recognition ability. Compared with a MIP organic monolith, the MIP-derivatized silica monolith exhibits better column efficiency and stability in CEC. Additionally, the comparison of these two kinds of monolithic columns was performed by capillary liquid chromatography. The separation on MIP-derivatized silica monolith was superior to that on the organic monolith.     

Calculating optimal modulation periods to maximize the peak capacity in two-dimensional HPLC

Theoretical calculations are presented to optimize modulation period for maximum total peak capacity in comprehensive two-dimensional HPLC (2D-HPLC) taking into account the effect of modulation on the apparent peak capacity of the first-dimension (1D) separation. Results indicate that modulation periods are most favorable when they are adjusted to approximately 2.2-4 times the standard deviation of a 1D peak in order to avoid excessively short run times at the second dimension (2D). Data are presented that effective peak capacities of several thousand in 60 min can be expected for practical 2D-HPLC conditions, utilizing 1D gradient elution followed by 2D isocratic elution, that remain at approximately 50-70% of the theoretical maximum peak capacity. This work suggests that lower modulation frequencies and longer 2D separation times than previously proposed are favorable under realistic chromatographic conditions, alleviating some practical problems associated with 2D-HPLC.     

Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface

Nanoscale packed-capillary liquid chromatography (LC) columns have been coupled with mass spectrometry (MS) using a coaxial continuous-flow fast atom bombardment interface. The combined system has been applied to the analysis of mixtures of peptides, including synthetic mixtures of bioactive peptides and tryptic digests of proteins. Nanoscale packed-capillary columns offer two principal advantages for LC/MS analysis--high chromatographic separation efficiencies and low mobile-phase flow rates. The high separation efficiencies facilitate the separation of complex mixtures, and the low mobile-phase flow rates reduce problems with coupling the LC effluent with the high-vacuum, high-voltage environment of sector MS ion sources. The columns used in this work were 50- or 75-micron i.d., 1-2 m long, packed with 10-micron C18 particles, using mobile-phase flow rates of 50-350 nL/min.     

Octadecylsilylated porous silica rods as separation media for reversed-phase liquid chromatography

Continuous porous silica rods consisting of a mesoporous (pore size, 14 or 25 nm) silica skeleton of ～1 μm size and through-pores of ～1.7 μm were prepared and derivatized to C(18) phase by on-column reaction with octadecyldimethyl-(N,N-diethylamino)silane. The C(18) silica rods gave plate heights of 10-20 μm for aromatic hydrocarbons in 80% methanol and 20-40 μm for insulin in acetonitrile-water mixtures in the presence of trifluoroacetic acid. The performance of the silica rods was much better at a high flow rate than that of conventional columns packed with 5 μm C(18) silica particles having 12 and 30 nm pores, especially for high molecular weight species.     

Protein-doped monolithic silica columns for capillary liquid chromatography prepared by the sol-gel method: applications to frontal affinity chromatography

The development of bioaffinity chromatography columns that are based on the entrapment of biomolecules within the pores of sol-gel-derived monolithic silica is reported. Monolithic nanoflow columns are formed by mixing the protein-compatible silica precursor diglycerylsilane with a buffered aqueous solution containing poly(ethylene oxide) (PEO, MW 10,000) and the protein of interest and then loading this mixture into a fused-silica capillary (150-250-microm i.d.). Spinodal decomposition of the PEO-doped sol into two distinct phases prior to the gelation of the silica results in a bimodal pore distribution that produces large macropores (>0.1 microm), to allow good flow of eluent with minimal back pressure, and mesopores (approximately 3-5-nm diameter) that retain a significant fraction of the entrapped protein. Addition of low levels of (3-aminopropyl)triethoxysilane is shown to minimize nonselective interactions of analytes with the column material, resulting in a column that is able to retain small molecules by virtue of their interaction with the entrapped biomolecules. Such columns are shown to be suitable for pressure-driven liquid chromatography and can be operated at relatively high flow rates (up to 500 microL x min(-1)) or with low back pressures (<100 psi) when used at flow rates of 5-10 microL x min(-1). The clinically relevant enzyme dihydrofolate reductase was entrapped within the bioaffinity columns and was used to screen mixtures of small molecules using frontal affinity chromatography with mass spectrometric detection. Inhibitors present in compound mixtures were retained via bioaffinity interactions, with the retention time being dependent on both the ligand concentration and the affinity of the ligand for the protein. The results suggest that such columns may find use in high-throughput screening of compound mixtures.     

Surface heterogeneity of six commercial brands of end-capped C18-bonded silica. RPLC separations

Single component adsorption isotherms of phenol and caffeine were measured on six different commercial brands of end-capped C(18)-bonded silica columns (five monomeric bonded phases: Kromasil, Waters Symmetry, Phenomenex, Hypersil, and Chromolith from Merck; one polymeric bonded phase, Vydac) with the same methanol/water solution (30/70, v/v) as the mobile phase. Adsorption data were acquired by frontal analysis (FA) for all these columns in the same way. Depending on their solubility in the mobile phase, the concentrations used ranged between 1 and 100 g/L and between 0.35 and 35 g/L for phenol and caffeine, respectively. Twenty-two adsorption data points were recorded over these ranges. In each case, the best isotherm model accounting for all sets of adsorption data is the bi-Langmuir model, all columns behaving as heterogeneous adsorbents despite the endcapping. Depending on the column, the high-energy sites accounts for between 30 and 40% and between 4 and 7% of the total saturation capacity for phenol and caffeine, respectively. Except for the polymeric phase (Vydac), the ratio of the adsorption constants on the high- and low-energy sites is constant at around 10 for both phenol and caffeine, corresponding to an average adsorption energy difference of 5 kJ/mol between these two sites. The exact nature of the high-energy sites is illustrated by the following properties: (i) they have a very low selectivity for caffeine, with alpha(caffeine/phenol) close to 0.4 for the five monomeric columns, which suggests the complete derivatization of residual silanols; (ii) the high-energy sites account for a large fraction of the surface area of these packing materials (35% for phenol, 6% for caffeine); (iii) there is a small adsorption energy difference between high and low adsorption energy sites (5 kJ/mol); and (iv) the adsorption constants increase with increasing surface coverage of the monomeric columns. Thus, the high energy sites cannot be residual free silanols of the bare silica. More likely, they are related to the local heterogeneity of the C(18)-bonded-layer structure. Caffeine is more strongly retained on the low-energy sites than phenol (the product q(s,) (1)b(1) is larger for caffeine) but the contribution of the high-energy sites (q(s,) (2)b(2)) is markedly lower for caffeine than for phenol, despite the larger value of the adsorption constant, b(2). Because of a larger molecular size, caffeine cannot penetrate as deeply as phenol inside the bonded layer. This explains the paradox of a stronger retention for phenol than for caffeine on end-capped C(18)-bonded stationary phases.     

Preparation and application of methacrylate-based cation-exchange monolithic columns for capillary ion chromatography

Polymer-based strong cation-exchange monolithic capillary columns with different capacities were constructed for ion chromatography by radical polymerization of glycidyl methacrylate (GMA) and ethylene dimethacrylate in a 250-microm-i.d. fused-silica capillary and its subsequent sulfonation based on ring opening of epoxides with 1 M Na(2)SO(3). The cation-exchange capacities can easily and reproducibly be controlled in the range of up to 300 microequiv/mL by changing the immersion time of the epoxy-containing polymer in the Na(2)SO(3) solution. The chromatographic performance of the produced monolithic capillary columns was evaluated through the separation of a model mixture of common cations such as Na(+), NH(4)(+), K(+), Mg(2+), and Ca(2+). As an example, these cations could be well separated from one another on a 15-cm-long cation-exchange monolithic column (column volume, 7.4 microL) with a capacity of 150 microequiv/mL by elution with 10 mM CuSO(4). The pressure drop of this 15-cm column was approximately 1 MPa at a normal linear velocity of 1 mm/s (a flow rate of 3 microL/min), and the numbers of theoretical plates for the cations were above 3000 plates/15 cm. This GMA-based cation-exchange monolithic column could withstand high linear velocities of at least 10 mm/s. Over a period of at least two weeks of continuous use, no significant changes in the selectivity and resolution were observed. The applicability of a flow rate gradient elution and the feasibility of direct injection determination of major cations in human saliva sample were also presented.     

An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation

A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved approximately 1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins.     

Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics

Application of C18 monolithic silica capillary columns in HPLC coupled to ion trap mass spectrometry detection was studied for probing the metabolome of the model plant Arabidopsis thaliana. It could be shown that the use of a long capillary column is an easy and effective approach to reduce ionization suppression by enhanced chromatographic resolution. Several hundred peaks could be detected using a 90-cm capillary column for LC separation and a noise reduction and automatic peak alignment software, which outperformed manual inspection or commercially available mass spectral deconvolution software.     

A correlation for the pressure drop in monolithic silica columns

To gain insight into how the pressure drop in monolithic silica columns is determined by the microscopic details of the pore structure, a series of well-validated computational fluid dynamics simulations has been performed on a simplified model structure, the so-called tetrahedral skeleton column. From these simulations, a direct correlation between the pressure drop and two main structural properties (skeleton thickness and column porosity) of the monolithic skeleton could be established. The correlation shows good agreement with the experimental pressure-drop data available from the literature on silica monoliths, especially when a correction for the flow-through pore size heterogeneity is made. The established correlation also yields a much more accurate representation of the relation between the flow resistance and the bed porosity than does the Kozeny-Carman model, making it much better suited for porosity optimization calculations.     

A LC/MS method for the determination of cyanobacteria toxins in water

The cyanobacteria toxins anatoxin-a, microcystin-LR, microcystin-RR, microcystin-YR, and nodularin were separated in less than 30 min on several 1 mm x 15 cm reversed-phase liquid chromatography (LC) columns, and their electrospray mass spectra were measured using injections of 50 ng or less with a benchtop time-of-flight (TOF) mass spectrometer. New data from this work include the following: (a) the impact of acetic acid concentrations in the methanol-water mobile phase on measured ion abundances; (b) the performance of the electrospray-TOF mass spectrometer as an LC detector; (c) the accuracy and precision of exact m/z measurements after LC separation with a routinely used mass spectrometer resolving power of 5000; and (d) recoveries of the five toxins from reagent water, river waters, and sewage treatment plant effluent samples extracted with C-18 silica particles enmeshed in thin Teflon membrane filter disks. This technique has the potential of providing a relatively simple and reasonable-cost sample preparation and LC/MS method that provides the sensitivity, selectivity, reliability, and information content needed for source and drinking water occurrence and human exposure studies.     

Improved sensitivity and characterization of high-speed ion chromatography of inorganic anions

In this work, a reversed-phase monolithic column was permanently coated with didodecyldimethylammonium bromide (DDAB) to perform ultrafast separations of iodate, chloride, nitrite, bromide, nitrate, phosphate, and sulfate in as little as 30 s. Separations were performed using 6 mM o-cyanophenol (pH 7.0) at flow rates up to 10 ml/min and suppressed conductivity detection. Detection limits in the parts-per-billion range were observed for all anions studied (e.g., ranging from 30 ppb for phosphate to 4 ppb for sulfate). The reproducibility was 0.7 and 0.4% RSD for retention time and peak area, respectively. Coated columns were stable for up to 12 h of continuous use at 5 mL/min (i.e., 1-min separations).     

Chip-based solid-phase extraction pretreatment for direct electrospray mass spectrometry analysis using an array of monolithic columns in a polymeric substrate

An array of eight porous monolithic columns, prepared in a Zeonor polymeric chip by UV-initiated polymerization of butyl methacrylate and ethylene dimethacrylate, was tested for solid-phase extraction (SPE) cleanup of biological samples prior to directly coupled electrospray mass spectrometry (ESI-MS). The chip, fabricated by hot embossing and thermal bonding, consists of eight parallel channels (10 mm long, 360 microm i.d.) connected via external fused-silica capillaries. The monomer mixture was aspirated simultaneously into the eight channels using a homemade vacuum manifold device and polymerized in parallel for 20 min under UV irradiation. The porous monolithic columns were then characterized by scanning electron microscopy and evaluated by ESI-MS applications with respect to sample capacity, recovery, reproducibility of peak area or peak height ratios, and linearity between peak height ratio and concentration using imipramine as a pharmaceutical test compound. The average sample capacity was estimated to be 0.30 microg with a relative standard deviation (RSD) of 26.5% for the eight monolithic columns on the same polymeric chip. For two chips prepared using the same monomer mixture, the difference in average sample capacity was 7.0%. The average recovery for the eight monolithic SPE columns on the same chip was 79.1% with an RSD of 7.9%. Using imipramine-d3 as an internal standard, the RSD of peak height ratios for the eight different columns was 2.0% for a standard solution containing 1 microg/mL imipramine. A linear calibration curve (R2 = 0.9995) was obtained for standard aqueous solutions of imipramine in the range from 0.025 to 10 microg/mL. To demonstrate the analytical potential of the chip-based SPE system, two different types of real-world samples including human urine sample and P450 drug metabolism incubation mixture were tested. Similar to standard aqueous solution, a linear correlation (R2 = 0.9995) was also found for human urine sample spiked with imipramine in the range of 0.025-10 microg/ mL. When aliquots of a human urine sample spiked with 1 microg/mL imipramine were loaded onto eight different monolithic columns, the RSD of peak height ratios was 3.8%. For a P450-imipramine incubation mixture, the formation of the N-demethylated metabolite (m/z 267.2) and the monohydroxylated metabolite (m/z 297.2) of imipramine was observed following chip-based monolithic SPE sample cleanup and preconcentration.     

Porous polymer monoliths functionalized through copolymerization of a C60 fullerene-containing methacrylate monomer for highly efficient separations of small molecules

Monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns, which incorporate the new monomer [6,6]-phenyl-C(61)-butyric acid 2-hydroxyethyl methacrylate ester, have been prepared and their chromatographic performance have been tested for the separation of small molecules in the reversed phase. While addition of the C60-fullerene monomer to the glycidyl methacrylate-based monolith enhanced column efficiency 18-fold, to 85,000 plates/m at a linear velocity of 0.46 mm/s and a retention factor of 2.6, when compared to the parent monolith, the use of butyl methacrylate together with the carbon nanostructured monomer afforded monolithic columns with an efficiency for benzene exceeding 110,000 plates/m at a linear velocity of 0.32 mm/s and a retention factor of 4.2. This high efficiency is unprecedented for separations using porous polymer monoliths operating in an isocratic mode. Optimization of the chromatographic parameters affords near baseline separation of 6 alkylbenzenes in 3 min with an efficiency of 64,000 plates/m. The presence of 1 wt % or more of water in the polymerization mixture has a large effect on both the formation and reproducibility of the monoliths. Other factors such as nitrogen exposure, polymerization conditions, capillary filling method, and sonication parameters were all found to be important in producing highly efficient and reproducible monoliths.     

Comparison between the Efficiencies of sub-2 μm C(18) Particles Packed in Narrow Bore Columns

The chromatographic performance of two types of core-shell particles and two fully porous particles packed in 2.1 ID × 50 mm columns was investigated. Comparisons of the performances of the EiS-150-C(18) to that of the Kinetex-1.7 μm-C(18), Acquity-BEH-1.7 μm-C(18), and Zorbax-XDB-1.8 μm-C(18) are made and discussed. The physical factors that govern the performance of these columns, such as particle size distribution and column external, total, and particle porosity of the C(18) packing materials were among the prime foci of investigation. The differences in the mass transfer behavior measured using naphtho[2,3-a]pyrene between these columns provides an indication of improved performance of the new EiS-150-C(18) column. The minimum reduced height equivalent to a theoretical plate (HETP) value for the EiS-150-C(18), h(min) = 1.95, was achieved and was comparable to that obtained from the C(18) phases of the Kinetex (h(min) = 2.53), the Acquity (h(min) = 2.26), and the Zorbax (h(min) = 2.57) columns. This study reveals the importance of the dimension of the shell thickness in controlling the performance of columns packed with shell particles in narrow bore columns.