Porous Column Research Articles

Liquid Chromatography under Limiting Conditions of Desorption IV. Separation of Macromolecules According to Their Stereoregularity

Liquid chromatography under limiting conditions of adsorption (LC LCD) is a novel technique for separation of polymers that exhibit different extent of adsorption on the porous column packing of appropriate polarity. LC LCD is based on the selective ‘‘barrier effect’’ induced by a narrow zone of the adsorption promoting liquid that is introduced into column in front of separated macromolecules of different nature. The small molecules that form the barrier elute slowly because they permeate the packing pores. The partially or fully pore excluded polymer molecules elute rapidly. The barrier hinders fast elution of macromolecules that tend to be adsorbed within column packing; their elution is slowed down. At the same time, the non-adsorbed polymer species elute non-hindered. It is shown that due to the controlled barrier effect even a very small difference in the extent of adsorption between isotactic and syndiotactic macromolecules of poly(methacrylate)s can lead to the significant distinction in their retention volumes. While the direct LC LCD separation of poly(methyl methacrylate)s of different stereoregularity is often precluded by the aggregation of unlike macromolecules, the highly stereoregular poly(ethyl methacrylate)s of distinct kind can be easily discriminated irrespective of their molar mass. Simultaneously, the low molecular and oligomeric impurities can be removed and, if necessary they can be identified and quantified by independent methods. In this way, LC LCD can assist purification of stereoregular polymers and serve as the first step in the twodimensional liquid chromatography of their non-aggregated mixtures.

Development of fluorinated, monolithic columns for improved chromatographic separations of fluorous-tagged analytes

With applications that take advantage of highly selective fluorine–fluorine interactions appearing with greater frequency in the literature, the development of porous polymer monoliths from fluorous components is reported here for the purpose of chromatography of tagged analytes. With potential uses in fields as diverse as separation science and proteomics, facile fabrication of materials with fluorous specificity that can be applied in a high-throughput manner is greatly desirable. To this end, we have developed porous polymer capillary columns with varied fluorous content using a simple UV-initiated radical polymerization process and characterized them using flow-induced backpressure and scanning electron microscopy (SEM). With structural similarities assured (visually, and by backpressure variations of less than 42%), the monoliths were tested as chromatographic columns for the separation of a series of fluorous-tagged analytes under gradient conditions. It was found that columns made with fluorinated components exhibited greater selectivity for fluorous analytes than did equivalent, non-fluorinated monoliths, retaining analytes with either one or two fluorous tags for approximately 6% and 13% longer, respectively. This supports the idea of differences existing between fluorous and reverse-phase separation mechanisms, and encourages a broader range of potential applications for fluorous monoliths of this type.

Rapid Preparation of Monolithic Silica Column for Electrophoretic and Chromatographic Separation by Microwave Irradiation

A porous monolithic sol-gel column with the solution of methacryloxypropyltrimethoxysilane in toluene with an acid catalyst was quickly prepared using microwave irradiation. Three typical silica based capillary monolithic columns prepared were used to evaluate the separation of some typical model compounds in the modes of CEC, pressure assisted CEC, and low pressure driven separation. Baseline separation of the analytes including thiourea, benzene, toluene, ethyl benzene, biphenyl, and naphthalene could be obtained by the polymer with a length of 10 cm. A scanning electron micrograph of a cross section of the capillary column showed that the gel took the form of a spherical particle aggregate and adhered to the column inner wall.

Hydrophilic interaction 10 μm I.D. porous layer open tubular columns for ultratrace glycan analysis by liquid chromatography–mass spectrometry

The sensitivity of glycan analysis using nano-liquid chromatography interfaced with electrospray ionization mass spectrometry (ESI–MS) increases with the decrease of the mobile phase flow rate, accompanied by reduced ion suppression. In this study, we describe the preparation and performance of high efficiency 10 μm I.D. amine-bonded poly(vinylbenzyl chloride-divinylbenzene) hydrophilic interaction (HILIC) porous layer open tubular (PLOT) columns operated at 20 nL/min for the separation and analysis of glycan mixtures. HILIC-PLOT columns with a uniform porous polymer layer were reproducibly prepared (∼4% RSD in retention time from column-to-column) via in situ polymerization, followed by one step modification with ethylenediamine. When coupled on-line with negative ESI–MS, low detection limits (0.3 fmol) for a 3-sialyl-tetrasaccharide were achieved using a 2.5 m × 10 μm I.D. HILIC-PLOT column. A dextran ladder standard was used to evaluate the performance of the column, and high efficiency separation was achieved with detection of the dextrans up to G22 from ∼50 fmol amounts injected. As an example of the high sensitivity of the column, MS 6 characterization of glycan structures was possible from the injection of 10 fmol of a neutral and sialylated glycan. As another example of high sensitivity LC–MS analysis of 3 ng of a PNGase F digest of ovalbumin allowed 28 N-linked glycans to be confidently identified from a single analysis. High quality MS/MS spectra for each ovalbumin glycan were acquired and manually interpreted for structure analysis. The HILIC-PLOT column is a very promising approach for LC–MS analysis of glycans at the ultratrace level.

The use of partially porous particle columns for the routine, generic analysis of biological samples for pharmacokinetic studies in drug discovery by reversed-phase ultra-high performance liquid chromatography–tandem mass spectrometry

Recent years have seen the introduction of new high performance liquid chromatography (HPLC) instruments and columns that are capable of achieving high resolution, high speed liquid chromatographic separations at back pressures up to 1000 bar, so-called ultra-high performance liquid chromatography (UHPLC). Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) is gaining widespread use for this purpose, and for this approach to be successful a generically applicable, robust column is required. Here, data are presented showing the robustness of a partially porous 2.7 μm diameter particle material in this application and the accuracy and precision of an assay for a typical pharmaceutical in plasma. This stationary phase material is evaluated for performance and compared with other materials frequently used for similar analyses using a test mix currently used routinely in our laboratories to assess the performance of UHPLC–MS/MS systems. The partially porous material demonstrates similar resolving power to sub-2 μm materials under the ballistic gradient chromatography conditions employed and exhibits excellent resilience over the analysis of thousands of protein precipitated plasma extracts. It is suggested that this stationary phase material can be an invaluable tool in generic, high throughput assays for pharmaceutical bioanalysts.

Use of partially porous column as second dimension in comprehensive two‐dimensional system for analysis of polyphenolic antioxidants

In the present work, a comprehensive LC system using a microbore HPLC column in the first dimension and a partially porous column in the second dimension was developed and applied to the separation of polyphenolic components in a red wine sample. The performance of the partially porous short column (3.0 cm) was compared to that of a monolithic column, of comparable dimensions. The results obtained demonstrated the possibility to use partially porous columns to obtain fast analyses, using high flow rates, under repetitive gradient conditions and with very brief reconditioning times. A conventional HPLC system was used since the backpressure generated by the shell-packed column, even at very high flow rates, was well within the operational limits. The use of an increased column temperature (60 degrees C) allowed a further pressure-drop decrease, with no stationary phase degradation, or loss in column performance.

Methacrylate‐based monolithic column with mixed‐mode hydrophilic interaction/strong cation‐exchange stationary phase for capillary liquid chromatography and pressure‐assisted CEC

A novel porous polymethacrylate-based monolithic column by in situ copolymerization of 3-sulfopropyl methacrylate (SPMA) and pentaerythritol triacrylate in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was prepared. The monolith possessed in their structures bonded sulfonate groups and hydroxyl groups and was evaluated as a hydrophilic interaction and strong cation-exchange stationary phases in capillary liquid chromatography (cLC) and pressure-assisted CEC using small polar neutral and charged solutes. While the SPMA was introduced as multifunctional monomer, the pentaerythritol triacrylate was used to replace ethylene glycol dimethacrylate as cross-linker with much more hydrophilicity due to a hydroxyl sub-layer. The different characterization of monolithic stationary phases were specially designed and easily prepared by altering the amount of SPMA in the polymerization solution as well as the composition of the porogenic solvent for cLC and pressure-assisted CEC. The resulting monolith showed the different trends about the effect of the permeabilities on efficiency in the pressure-assisted CEC and cLC modes. A typical hydrophilic interaction chromatography mechanism was observed at higher organic solvent content (ACN%>70%) for polar neutral analytes. For polar charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Therefore, for charged analytes, selectivity can be readily manipulated by changing the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). With the optimized monolithic column, high plate counts reaching greater than 170 000 plates/m for pressure-assisted CEC and 105 000 plates/m for cLC were easily obtained, respectively.

Multidimensional LC×LC analysis of phenolic and flavone natural antioxidants with UV‐electrochemical coulometric and MS detection

A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.

Kinetic fractionation of Fe isotopes during transport through a porous quartz-sand column

Sorption and desorption processes are an important part of biological and geochemical metallic isotope cycles. Here, we address the dynamic aspects of metallic isotopic fractionation in a theoretical and experimental study of Fe sorption and desorption during the transport of aqueous Fe(III) through a quartz-sand matrix. Transport equations describing the behavior of sorbing isotopic species in a water saturated homogeneous porous medium are presented; isotopic fractionation of the system (Δ sorbed metal-soln) being defined in terms of two parameters: (i) an equilibrium fractionation factor, α e; and (ii) a kinetic sorption factor, α 1. These equations are applied in a numerical model that simulates the sorption-desorption of Fe isotopes during injection of a Fe(III) solution pulse into a quartz matrix at pH 0–2 and explores the effects of the kinetic and equilibrium parameters on the Fe-isotope evolution of porewater. The kinetic transport theory is applied to a series of experiments in which pulses of Na and Fe(III) chloride solutions were injected into a porous sand grain column. Fractionation factors of α e = 1.0003 ± 0.0001 and α 1 = 0.9997 ± 0.0004 yielded the best fit between the transport model and the Fe concentration and δ 56Fe data. The equilibrium fractionation (Δ 56Fe sorbed Fe-soln) of 0.3‰ is comparable with values deduced for adsorption of metallic cations on iron and manganese oxide surfaces and suggests that sandstone aquifers will fractionate metallic isotopes during sorption-desorption reactions. The ability of the equilibrium fractionation factor to describe a natural system, however, depends on the proximity to equilibrium, which is determined by the relative time scales of mass transfer and chemical reaction; low fluid transport rates should produce a system that is less dependent on kinetic effects. The results of this study are applicable to Fe-isotope fractionation in clastic sediments formed in highly acidic conditions; such conditions may have existed on Mars where acidic oxidizing ground and surface waters may have been responsible for clastic sedimentation and metallic element transport.

Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography–tandem mass spectrometry

In May 2007, investigators discovered that waste material from the pet food manufacturing process contaminated with melamine (MEL) and/or cyanuric acid (CYA) had been added to hog and chicken feeds. At this time, investigators also learned that adulterated wheat gluten had been used in the manufacture of aquaculture feeds. Concern that the contaminated feed had been used in aquaculture and could enter the human food supply prompted the development of a method for the determination of CYA residues in the edible tissues of fish and shrimp. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was employed as a sensitive technique for the analysis of CYA in catfish, tilapia, salmon, trout and shrimp tissue. CYA was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoring was performed on the [M−H] − m/ z 128 ion resulting in the product ions m/ z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10–100 μg kg −1 of CYA averaged 67% with a relative standard deviation (R.S.D.) of 18% ( n = 107). The average method detection limit (MDL) for catfish, tilapia and trout is 3.5 μg kg −1. An internal standard, 13C 3-labeled CYA, was used in the salmon and shrimp extractions. Average recovery of CYA from salmon was 91% (R.S.D. = 15%, n = 18) with an MDL of 7.4 μg kg −1. Average recovery of CYA from shrimp was 85% (R.S.D. = 10%, n = 13) with an MDL of 3.5 μg kg −1.

Chiral analysis of milnacipran by a nonchiral HPLC – circular dichroism: Improvement of the linearity of dichroic response by temperature control

The determination of the enantiomeric excess (e.e.) of a basic drug has been investigated in LC using a nonchiral stationary phase and a circular dichroism (CD) detector in order to avoid expensive chiral columns. The CD detector records both dichroic (Deltaepsilon) and UV (epsilon) signals at the same wavelength and calculates the anisotropy factor (g=Deltaepsilon/epsilon), which is linearly related to the e.e. The enantiomeric and chemical composition of a chiral drug can be simultaneously determined on a nonchiral HPLC support. However, the g factor from the CD signal is temperature dependent. Indeed, the temperature has an influence on the stability of the CD signal and the linear regression between g factor and the e.e. of 1R,2S-enantiomer. So, a decrease in temperature gives rise to an improvement of the above-mentioned linearity correlation. After optimization of chromatographic parameters (porous graphitic carbon-based column, methanol/ phosphate buffer as mobile phase) and selection of CD wavelength, a linear regression of g factor versus e.e. of 1R,2S-enantiomer was obtained at temperature-controlled CD detection and an LOQ of 94% was found. The enantiomeric composition of milnacipran was determined with good accuracy.

Network modeling of chromatography by stochastic phenomena of adsorption, diffusion and convection

The limitation of macroscopic models to represent structural parameters, such as topology and morphology, as well as population effects, i.e. multi-molecules movement, in the modeling of chromatography systems has implications on the understanding of the phenomenological aspects that contribute to the separation mechanisms in porous media. The representation of the porous structure of chromatographic columns by a three-dimensional cubic network of interconnected sites allows a better analysis of the structural characteristics of the porous column and its connection with the phenomena of adsorption, diffusion and convection. In the present work the application of an interconnected cubic network model associated with a stochastic modeling of the adsorption, diffusion and convection phenomena leads to the proper representation of the dynamic aspects of the breakthrough curves related to separation processes in chromatographic columns. Therefore, it is possible to study the dynamics of solute retention from the molecules distribution in the separation processes throughout the column. Among the mass transfer mechanisms investigated, the convection showed to be closely related to the separation dynamics of chromatography, with the diffusion having little effects. The adsorption influenced both the separation dynamics and solute retention.

Separation of inorganic anions on a high capacity porous polymeric monolithic column and application to direct determination of anions in seawater

A commercially available 4.6 mm id x 50 mm polymethacrylate-based monolithic strong anion exchange column (ProSwift SAX-1S) designed for the separation of proteins has been successfully used to separate small inorganic anions in the presence of a seawater sample matrix. Using a hydroxide eluent with suppressed conductivity detection the ion exchange capacity of this column declined over time; however, using KCl as the eluent, the column performance was stable with a capacity of 530 microequiv. for nitrate. The optimum conditions for the separation of iodate, bromate, nitrite, bromide and nitrate were assessed by constructing van Deemter plots using 1.00 and 0.100 M KCl. Efficiencies of up to 26 700 plates/m were recorded using 1.00 M KCl, at a flow rate of 0.20 mL/min but iodate was not baseline resolved from the void peak. By reducing the concentration of the eluent to 0.100 M, efficiencies of up to 39 900 plates/m could be obtained at 0.35 mL/min. By employing a linear gradient ranging from 0.05 to 1.00 M KCl the ions dissolved in distilled water or a salt water matrix could be baseline separated in less than 3 min at a flow rate of 2.50 mL/min.

Experimental Study of Porous Silicon Shell Pillars under Retentive Conditions

Experimental measurements of the retention capacity and the band broadening in perfectly ordered porous shell pillar array columns are presented for a wide range of retention conditions and layer thicknesses. The porous silicon shells were obtained using electrochemical anodization of the solid silicon pillars obtained using deep reactive ion etching. Using 10-microm-wide pillars, minimal reduced plate height values of the order of h(min) = 0.4-0.5 were obtained under nonretained conditions, even in cases where the outer shell made up 20% of the total diameter. Under retained conditions, minimal plate heights around h(min) = 0.9 were obtained, even at retention factors up to k' = 12. Using a model based on Giddings nonequilibrium theory, and using a newly calculated value for the stationary zone configuration factor for the case of porous shell cylinders, a plate height model describing the band broadening in porous shell pillar arrays has been established. The validity of this model is demonstrated by showing that the geometrical parameters appearing in the model and fitted using band-broadening measurements under nonretained conditions can be used to relatively accurately predict the band broadening under retained component conditions. Using this model, some speculations on the ultimate performance of porous pillar array columns could be made.

Experimental and numerical investigations of effect of column length on retardation factor determination: A case study of cesium transport in crushed granite

This study investigated breakthrough curves (BTCs) from a series of column experiments, including different column lengths and flow rates, of a conservative tracer, tritium oxide (HTO), and a radionuclide, cesium, in crushed granite using a reactive transport model. Results of the short column, with length of 2cm, showed an underestimation of the retardation factor and the corresponding HTO BTCs cannot be successfully modeled even with overestimated fluid dispersivity. Column supporting elements, including filters and rings, on both ends of packed granite were shown to be able to induce additional dispersive mixing, thus significantly affecting BTCs of short columns while those of the long column, with length of 8cm, were less affected. By increasing flow rates from 1mL/min to 5mL/min, the contribution of structural dispersive mixing to the false tilting of short column BTCs still cannot be detached. To reduce the influence of structural dispersivity on BTCs, the equivalent pore volume of column supporting materials should be much smaller than that of packed porous medium. The total length of column supporting structures should be greatly shorter than that of porous medium column.

Fast preparation of photopolymerized monolithic columns for capillary electrochromatography

Photopolymerized sol-gel(PSG) columns were prepared using methacryloxypropyltrimethoxysilane as the monomer, toluene as the porogen and hydrochloric acid as the catalyst. Four different photoinitiators such as benzoin methyl ether, Irgacure 819, Irgacure 1700 and Irgacure 1800 were comparatively used in the reaction solution in the presence and absence of sodium dodecyl sulfate. The above eight solutions were respectively irradiated at 365 nm for 5–10 min in each capillary (75 μm inside diameter) to prepare the porous monolithic sol-gel column by a one-step, in situ, process. The chromatographic behavior of the eight PSG columns were comparatively studied, all of which exhibit reversed-phase character. Using these columns, several neutral compounds, namely thiourea, benzene, toluene, ethyl benzene, biphenyl and naphthalene can be separated from mixtures with a largest column efficiency of 74 470 plate/column for thiourea. Addition of sodium dodecyl sulfate in the polymerization process has a significant influence on the morphology and migration time.

Selective and rapid liquid chromatography/negative-ion electrospray ionization mass spectrometry method for the quantification of valacyclovir and its metabolite in human plasma

A rapid, sensitive and specific method was developed for the quantification of valacyclovir and acyclovir in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. Valacyclovir, acyclovir and ganciclovir (internal standard) were separated isocratically on a reversed-phase porous graphitized carbon analytical column (2.1 mm × 125.0 mm i.d., particle size 5 μm), using a mobile phase of acetonitrile/water with 0.05% (v/v) diethylamine (50:50, v/v) at a flow rate of 0.15 mL min −1 in 4.0 min. Detection was performed by negative electrospray ionization using the selected ion monitoring mode of the deprotonated molecular ions at m/ z 323.0 for valacyclovir, 224.0 for acyclovir and 254.0 for ganciclovir. The assay had linear calibration curves over the range 0.020–0.800 μg mL −1 for valacyclovir and 0.100–20.00 μg mL −1 for acyclovir. Accuracy and precision were within the acceptance limit of 15%. The method was successfully applied to the analysis of plasma samples obtained from patients after oral administration of valacyclovir.

Sensitive determination of a glyoxal–DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.

Artifactual production of cyanide from coffee and tea drinks in the presence of nitrite

Introduction : In cases of unknown poisoning, forensic toxicologists are required to elucidate the cause of death, and one of the most noticeable poisons is cyanide. We report on the artifactual production of cyanide from coffee and tea drinks in the presence of nitrite, which is frequently adulterated to lead to the fault result in drug testing. Methods : Cyanide was measured by head-space gas chromatography using porous polymer capillary GS-Q column and a nitrogen-phosphorus detector. Samples were mixed with sodium nitrite in a glass vial under acidic conditions of 10% phosphoric acid or 0.6 M sodium phosphate buffer (pH 5.0). After incubation for 30 min at 50 °C, the resulting head-space was injected into the gas chromatograph. Hydrogen cyanide was eluted at 2.2 min, and the peak area was converted for measuring cyanide concentration in the liquid reaction mixture. From the 50 °C incubation mixture of commercial coffee drink (or tea drink) and nitrite (50 mM), several g/mL and sub-g/mL of cyanide was produced in the presence of 10% phosphoric acid and pH 5.0 buffer, respectively. Cyanide was also produced from the reaction mixture of nitrite and polyphenol compounds such as chlorogenic acid and catechins, which are included in coffee and tea drinks, but not from a monophenol mixture. The amount of cyanide produced was dependent on substrate concentrations of nitrite and polyphenol, pH, temperature and incubation time. Conclusion : Forensic toxicologists should pay attention to this type of artifactual production of cyanide.

Preparation of polymethacrylate monolithic stationary phases having bonded octadecyl ligands and sulfonate groups: Electrochromatographic characterization and application to the separation of polar solutes for pressurized capillary electrochromatography

In this report, the preparation of porous polymethacrylate-based monolithic columns by in situ copolymerization of octadecyl methacrylate (OMA), 3-sulfopropyl methacrylate (SPMA) and ethylene dimethacrylate (EDMA) in a binary porogenic solvent consisting of cyclohexanol/1,4-butanediol are proposed. These monoliths possess in their structures bonded octadecyl ligands and sulfonate groups and are evaluated in pressurized capillary electrochromatography (pCEC) system using small neutral and charged solutes. While the sulfonate groups are meant to generate the electroosmotic flow (EOF) necessary for transporting the mobile phase through the monolithic capillary; the octadecyl ligands are introduced to provide the nonpolar sites for chromatographic retention for neutral solutes. However, incorporating the sulfonate groups in the monoliths does not only support the EOF but also exhibit hydrophilic interaction as well as electrostatic interaction/repulsion with the monoliths in addition to electrophoretic migration with polar charged solutes (e.g., nucleotides). The monolithic stationary phases at different EOF velocities are easily prepared by altering the amount of SPMA in the polymerization solution as well as the composition of the porogenic solvent. Optimum EOF velocity, the highest efficiency and adequate chromatographic retention are obtained when 0.6% SPMA is added to the reaction mixture. Under these conditions, rapid separation and high plate counts reaching greater than 170,000 plates/m are readily obtained.