Separation of acidic and basic compounds in capillary electrochromatography with polymethacrylate-based monolithic columns

Methacrylate-based monolithic columns with electroosmotic flow (EOF) or very weak EOF are prepared by in situ copolymerization in the presence of a porogen in fused-silica capillaries pretreated with a bifunctional reagent. Satisfactory separations of acidic and basic compounds on the column with EOF at either low or high pH are achieved, respectively. With sulfonic groups as dissociation functionalities, sufficient EOF mobility still remains as high as 1.74 x 10(-4) cm2 s(-1) V(-1) at low pH. Under this condition, seven acidic compounds are readily separated within 5.7 min. Moreover, at high pH, the peak shape of basic compounds is satisfactory without addition of any masking amines into running mobile phase since the secondary interaction between the basic compounds and the monolithic stationary phase are minimized at high pH. Reversed-phase mechanism for both acidic and basic compounds is observed under investigated separation conditions. In addition, possibilities of acidic and basic compound separations on a monolithic column with extremely low EOF are discussed.     

S-citalopram imprinted monolithic columns for capillary electrochromatography enantioseparations

In this study, the molecular imprinting method was used to separate enantiomeric forms of chiral antidepressant drug, R,S-citalopram (R,S-CIT) in aqueous solution by CEC system combining the advantages of capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). For that, an amino acid-based molecularly imprinted monolithic capillary column was designed and used as a stationary phase for selective separation of S-citalopram (S-CIT) for the first time. S-CIT was selectively separated from the aqueous solution containing the other enantiomeric form of R-CIT, which is the same in size and shape as the template molecule. Morphology of the molecularly imprinted (MIP S-CIT) and non-imprinted (NIP S-CIT) monolithic capillary columns was observed by scanning electron microscopy. Imprinting efficiency of MIP S-CIT monolithic capillary column used for selective S-CIT separation was verified by comparing with NIP S-CIT and calculated imprinting factor (I.F:1.81) proved the high selectivity of the MIP S-CIT for S-CIT. Cavities formed for S-CIT form enabled selective (α = 2.08) separation of the target molecule from the other enantiomeric R-CIT form. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 7.68 × 10⁻⁶ m²/Vs for R,S-CIT at pH 7.0 10 mM PB and 50% ACN ratio. The performance of both MIP S-CIT and NIP S-CIT columns was estimated by repeating the R,S-CIT separations with intra-batch and inter-batch studies for reproducibility of retention times of R,S-CITs. Estimated RSD values that are lower than 2% suggest that the monolithic columns separate R,S-CIT enantiomers without losing separation efficiency.     

Simple capillary flow porometer for characterization of capillary columns containing packed and monolithic beds

A simple capillary flow porometer (CFP) was assembled for through-pore structure characterization of monolithic capillary liquid chromatography columns in their original chromatographic forms. Determination of differential pressures and flow rates through dry and wet short capillary segments provided necessary information to determine the mean diameters and size distributions of the through-pores. The mean through-pore diameters of three capillary columns packed with 3, 5, and 7 μm spherical silica particles were determined to be 0.5, 1.0 and 1.4 μm, with distributions ranging from 0.1 to 0.7, 0.3 to 1.1 and 0.4 to 2.6 μm, respectively. Similarly, the mean through-pore diameters and size distributions of silica monoliths fabricated via phase separation by polymerization of tetramethoxysilane (TMOS) in the presence of poly(ethylene glycol) (PEG) verified that a greater number of through-pores with small diameters were prepared in columns with higher PEG content in the prepolymer mixture. The CFP system was also used to study the effects of column inner diameter and length on through-pore properties of polymeric monolithic columns. Typical monoliths based on butyl methacrylate (BMA) and poly(ethylene glycol) diacrylate (PEGDA) in capillary columns with different inner diameters (i.e., 50–250 μm) and lengths (i.e., 1.5–3.0 cm) were characterized. The results indicate that varying the inner diameter and/or the length of the column had little effect on the through-pore properties. Therefore, the through-pores are highly interconnected and their determination by CFP is independent of capillary length.     

Ionic liquids monolithic columns for protein separation in capillary electrochromatography

A series of ionic liquids (ILs) monolithic capillary columns based on 1-vinyl-3-octylimidazolium (ViOcIm⁺) were prepared by two approaches (“one-pot” approach and “anion-exchange” approach). The effects of different anions (bromide, Br⁻; tetrafluoroborate, BF₄⁻; hexafluorophosphate, PF₆⁻; and bis-trifluoromethanesulfonylimide, NTf₂⁻) on chromatography performance of all the resulting columns were investigated systematically under capillary electrochromatography (CEC) mode. The results indicated that all these columns could generate a stable reversed electroosmotic flow (EOF) over a wide pH range from 2.0 to 12.0. For the columns prepared by “one-pot” approach, the EOF decreased in the order of ViOcIm⁺Br⁻ > ViOcIm⁺BF₄⁻ > ViOcIm⁺PF₆⁻ > ViOcIm⁺NTf₂⁻ under the same CEC conditions; the ViOcIm⁺Br⁻ based column exhibited highest column efficiencies for the test small molecules; the ViOcIm⁺NTf₂⁻ based column possessed the strongest retention for aromatic hydrocarbons; and baseline separation of four standard proteins was achieved on ViOcIm⁺NTf₂⁻ based column corresponding to the highest column efficiency of 479 000 N m⁻¹ for cytochrome c (Cyt c). These results indicated that the property of ILs based columns could be tuned successfully by changing anions, which gave these columns potential to separate both small molecules and macro biomolecules.     

High-performance liquid chromatographic enantioseparations on capillary columns containing crosslinked polysaccharide phenylcarbamate derivatives attached to monolithic silica

Monolithic capillary columns containing native silica gel were covalently modified with 3,5-disubstituted phenylcarbamate derivatives of cellulose and amylose and applied for enantioseparations in capillary LC. The method previously used for covalent immobilization of polysaccharide phenylcarbamate derivatives onto the surface of microparticulate silica gel was successfully adapted for in situ modification of monolithic fused-silica capillary columns. The effects of the nature of polysaccharide and the substituents, as well as of multiple covalent immobilization of polysaccharide derivative on chromatographic performance of capillary columns were studied. The capillary columns obtained using this technique are stable in all solvents commonly used in LC and exhibit promising enantiomer resolving ability.     

A novel ionic liquid monolithic column and its separation properties in capillary electrochromatography

A novel ionic liquid (IL) monolithic capillary column was successfully prepared by thermal free radical copolymerization of IL (1-vinyl-3-octylimidazolium chloride, ViOcIm⁺Cl⁻) together with lauryl methacrylate (LMA) as the binary functional monomers and ethylene dimethacrylate (EDMA) as the cross-linker in binary porogen. The proportion of monomers, porogens and cross-linker in the polymerization mixture was optimized in detail. The resulting IL-monolithic column could not only generate a stable reversed electroosmotic flow (EOF) in a wide pH range (2.0–12.0), but also effectively eliminate the wall adsorption of the basic analytes. The obtained IL-monolithic columns were examined by scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR). These results indicated that the IL-monolithic capillary column possessed good pore properties, mechanical stability and permeability. The column performance was also evaluated by separating different kinds of compounds, such as alkylbenzenes, thiourea and its analogues, and amino acids. The lowest plate height of ∼6.8 μm was obtained, which corresponded to column efficiency (theoretical plates, N) of ∼147,000 plates m⁻¹ for thiourea. ILs, as a new type of functional monomer, present a promising option in the fabrication of the organic polymer-based monolithic columns in CEC.     

A novel monolithic column for capillary electrochromatographic separation of oligopeptides

A monolithic column was prepared using l-phenylalanine as template and a covalent approach through the formation of Schiff base with o-phthalaldehyde (OPA). OPA, allylmercaptan, l-phenylalanine, and triethylamine were stirred at first, then methacrylic acid, 2-vinylpyridine, ethyleneglycol dimethacrylate, α,α-azobisisobutyronitrile, and 1-propanol were added to the reaction mixture. The resulting material was introduced into a capillary column. Following thermal polymerization, the template was then extracted with a mixture of HCl and methanol. The column was employed for the capillary electrochromatographic separation of oligopeptides. A capillary column of 75 (50) cm × 75 μm ID with a mobile phase of phosphate buffer (pH 7.0, 40 mM)/methanol (5%, v/v), an applied voltage of +15 kV, and detection at 214 nm, could baseline separate angiotensin I, angiotensin II, [Sar¹, Thr⁸] angiotensin, oxytocin, vasopressin, tocinoic acid, β-casomorphin bovine, β-casomorphin human, and FMRF amide within 20 min. The separation behavior of the templated polymer was also compared with that of the non-templated polymer. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving hydrophobic, hydrogen bonding, electrostatic as well as the Schiff base formation with OPA in the cavity of the templated polymer.     

Sol-gel monolithic anion-exchange column for capillary electrochromatography

A single-step approach has been used to prepare a monolithic electrochromatographic column by sol-gel processing of an organofunctional silicon alkoxide precursor that contains a propyl-N,N,N-trimethylammonium group. We have found that the time of adding the porogen, poly(ethylene glycol), during the sol-gel reaction affected the separation performance. Since the surface charge of this material is switchable in sign upon manipulation of solution pH, the direction and magnitude of the electroosmotic flow (EOF) can be controlled by adjusting the pH of the running electrolyte. By controlling the direction of the EOF from cathode to anode, inorganic anions can be separated in a short time. Because of the quaternary ammonium functional group, the resulting material is anion exchangeable. Interestingly, the anion-exchange selectivity of inorganic anions on this column changes with solution pH or applied voltage. The column shows excellent run to run reproducibility (R.S.D. < 0.4%), good day to day reproducibility (R.S.D. < 4%), and reasonable column to column reproducibility (R.S.D. < 9%).     

Octyl-type monolithic columns of 530 microm i.d. for capillary liquid chromatography

A novel monolithic capillary column (530 microm i.d.) was prepared for capillary liquid chromatography (CLC) by in situ copolymerization of octyl methacrylate (MAOE) and ethylene dimethacrylate (EDMA) in the presence of a porogen solvent containing 1-propanol, 1,4-butanediol, and water with azobisisobutyronitrile as the initiator. The influences of the contents of the porogen solvent, EDMA and the various concentration ratios of 1-propanol to 1,4-butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of monolithic capillary columns were characterized by SEM and mercury intrusion porosimetry, respectively. Chromatographic evaluations of the columns were performed under CLC mode. The results showed that good permeability and stability can be obtained under optimal experimental conditions. The separation results of some acid, neutral and basic analytes demonstrated the hydrophobicity and low affinity to basic analytes of the new column. Three metal ions, i.e. Mg(II), Zn(II) and Cd(II) were also separated under ion-pair mode on the new monolithic capillary column and the results were acceptable.     

Optimization of performance of monolithic capillary column in gas chromatographic separations

Dependence of monolithic column efficiency on column pressure was analyzed using modified Van Deemter relationship with incorporated inlet and outlet column pressures as independent variables. It was demonstrated that the highest column efficiency is observed at high pressures. Inlet and outlet pressure increase has to be controlled in such a way that the relative pressure approaches 1 and the pressure drop across the column is close to zero. Experimental results obtained for open and monolithic capillary columns confirm up to 50% higher column efficiency as compared to column efficiency under standard conditions found using conventional Van Deemter plot. Pressure increase also results in a decrease in the optimal carrier gas velocity and corresponding increase in the analysis time. This drawback can be compensated via an increase in the column temperature.     

Polyhedral oligomeric silsesquioxanes as functional monomer to prepare hybrid monolithic columns for capillary electrochromatography and capillary liquid chromatography

A simple approach to fabricate hybrid monolithic column within the confines of fused-silica capillaries (75 μm i.d.) was introduced. A polyhedral oligomeric silsesquioxanes (POSS) reagent containing a methacrylate group was selected as functional monomer, and copolymerized with bisphenol A dimethacrylate (BPADMA) or ethylene dimethacrylate (EDMA) in the presence of porogenic solvents via thermally initiated free radical polymerization. After optimization of the preparation conditions, two POSS-containing hybrid monoliths were successfully prepared and exhibited good permeability and stability. By comparison of the separation efficiencies of the resulting poly(POSS-co-BPADMA) and poly(POSS-co-EDMA) monoliths in capillary electrochromatography (CEC) and capillary liquid chromatography (cLC), it was indicated the former has better column efficiencies for alkylbenzenes, phenols, anilines and PAHs in CEC and cLC than the latter. Particularly, the hybrid poly(POSS-co-BPADMA) monolith is more suitable for separation of PAHs due to π–π interaction between the analytes and aromatic rings in the surface of monolithic stationary phase.     

Hybrid organic-inorganic phenyl monolithic column for capillary electrochromatography

A novel hybrid organic-inorganic silica-based monolithic column possessing phenyl ligands for reversed-phase (RP) capillary electrochromatography (CEC) is described. The monolithic stationary phase was prepared by in situ co-condensation of tetraethoxysilane (TEOS) with phenyltriethoxysilane (PTES) via a two-step catalytic sol-gel procedure to introduce phenyl groups distributed throughout the silica matrix for chromatographic interaction. The hydrolysis and condensation reactions of precursors were chemically controlled through pH variation by adding hydrochloric acid and dodecylamine, respectively. The structural property of the monolithic column can be easily tailored through adjusting the composition of starting sol solution. The effect of PTES/TEOS ratios on the morphology of the created stationary phases was investigated. A variety of neutral and basic analytes were used to evaluate the column performance. The CEC columns exhibited typical RP chromatographic retention mechanism for neutral compounds and had improved peak shape for basic solutes.     

Capillary electrochromatography with monolithic silica columns. V. Study of the electrochromatographic behaviors of polar compounds on monolithic silica having surface bound cyano functionalities

In this report, a novel polar monolithic capillary column is described for normal phase CEC (NP-CEC) of representative polar compounds including mono- and oligosaccharides, peptides, and basic drugs. The polar monolithic column, which was described in detail in the preceding paper, consisted of silica-based monolith bonded with 1H-imidazole-4,5-dicarbonitrile (IDCN) and is denoted as 2CN-OH-Monolith. Various retention parameters for neutral polar solutes (e.g., mono- and oligosaccharides) and charged polar solutes (e.g., peptides and basic drugs) were evaluated over a wide-range of elution conditions. These retention parameters yielded quantitative assessment for the polar interactions between the model solutes and the stationary phase under investigation as well as the effect of electromigration of charged solutes on their overall migration in NP-CEC. Furthermore, this investigation demonstrated that despite the possibility of achieving isocratic separation in NP-CEC for widely differing polar species, multistep-gradient elution in NP-CEC is preferred to bring about the rapid separation of a large number of polar species in a single run.     

Characterization of polymer monolithic stationary phases for capillary HPLC

Monolithic capillary columns have been prepared in fused‐silica capillaries by radical co‐polymerization of ethylene dimethacrylate and butyl methacrylate in the presence of porogen solvent mixtures containing various concentration ratios of 1‐propanol, 1,4‐butanediol, and water with azobisisobutyronitrile as the initiator of the polymerization reaction. The through pores in organic polymer monolithic columns can be characterized by “equivalent permeability particle size”, and the mesopores with stagnant mobile phase by “equivalent dispersion particle size”. Increasing the concentration of propanol in the polymerization mixture diminishes the pore volume and size in the monolithic media and improves the column efficiency, at a cost of decreasing permeability. Organic polymer monolithic capillary columns show similar retention behaviour to packed alkyl silica columns for compounds with different polarities characterized by interaction indices, I_x, but have different methylene selectivities. Higher concentrations of propanol in the polymerization mixture increase the lipophilic character of the monolithic stationary phases. Best efficiencies and separation selectivities were found for monolithic columns prepared using 62–64% propanol in the porogen solvent mixture. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra‐column contributions.     

Capillary electrochromatography with monolithic silica columns. IV. Electrochromatographic characterization of polar bonded monolithic stationary phases having surface-bound cyano functionalities

Two polar ligands, namely 3-hydroxypropionitrile and 1H-imidazole-4,5-dicarbonitrile (IDCN) were covalently attached to epoxy-activated silica-based monolithic capillary columns via an epoxide ring-opening reaction to yield CN-OH-Monolith and 2CN-OH-Monolith, respectively. The silica monolith was prepared by a sol-gel process, and the resulting "rod-like" stationary phase was subjected to pore tailoring with an alkaline solution to convert small pore domains to mesopore domains, thus yielding a monolith with bimodal pore structure consisting of flow through pores (i.e., flow channels for mobile-phase flow) and mesopores that provide most of the adsorption capacity of the monolith toward the separated solutes. The two polar monoliths, CN-OH-Monolith and 2CN-OH-Monolith, were evaluated in normal-phase CEC with organic-rich mobile phases less polar than the stationary phase. The 2CN-OH-Monolith bearing more polar functions than the CN-OH-Monolith exhibited more retention and improved selectivity toward model polar solutes.     

Selectivity comparisons of monolithic silica capillary columns modified with poly(octadecyl methacrylate) and octadecyl moieties for halogenated compounds in reversed-phase liquid chromatography

Stationary phase selectivities for halogenated compounds in reversed-phase HPLC were compared using C18 monolithic silica capillary columns modified with poly(octadecyl methacrylate) (ODM) and octadecyl moieties (ODS). The preferential retention of halogenated benzenes on ODM was observed in methanol/water and acetonitrile/water mobile phases. In selectivity comparison of selected analytes on ODM and ODS, greater selectivities for halogenated compounds were obtained with respect to alkylbenzenes on an ODM column, while similar selectivities were observed with a homologous series of alkylbenzenes on ODM and ODS columns. These data can be explained by greater dispersive interactions by more densely packed octadecyl groups on the ODM polymer coated column together with the contribution of carbonyl groups in ODM side chains. For the positional isomeric separation of dihalogenated benzenes (ortho-, meta-, para-), the ODM column also provided better separation of these isomers for the adjacently eluted isomers that cannot be completely separated on an ODS column in the same mobile phase. These results imply that the ODM column can be used as a better alternative to the ODS column for the separation of other halogenated compounds.     

Acrylate ester-based monolithic columns for capillary electrochromatography separation of triacylglycerols in vegetable oils

A simple and reliable method for the evaluation of triacylglycerols (TAGs) in vegetable oils by capillary electrochromatography (CEC) with UV-Vis detection, using octadecyl acrylate (ODA) ester-based monolithic columns, has been developed. The percentages of the porogenic solvents in the polymerization mixture, and the mobile phase composition, were optimized. The optimum monolith was obtained at the following ratios: 40:60% (wt/wt) monomers/porogens, 60:40% (wt/wt) ODA/1,3-butanediol diacrylate and 23:77% (wt/wt) 1,4-butanediol/1-propanol (14 wt% 1,4-butanediol in the polymerization mixture). A satisfactory resolution between TAGs was achieved in less than 12 min with a 65:35 (v/v) acetonitrile/2-propanol mixture containing 5 mM ammonium acetate. The method was applied to the analysis of TAGs of vegetable oil samples. Using linear discriminant analysis of the CEC TAG profiles, the vegetable oils belonging to six different botanical origins (corn, extra virgin olive, hazelnut, peanut, soybean and sunflower) were correctly classified with an excellent resolution among all the categories.     

Acrylic-based high internal phase emulsion polymeric monolith for capillary electrochromatography

The use of high internal phase emulsion polymers (polyHIPEs) for CEC applications has remained relatively unexplored. A few reports exist in the literature for the preparation of similar structures. In this study, polyHIPEs having high porosity, and interconnected open-cell structure, were introduced and evaluated as stationary phase for CEC. The polyHIPE monolithic columns were prepared by the in situ polymerization of isodecylacrylate (IDA) and divinylbenzene (DVB) in the continuous phase of a high internal phase emulsion (HIPE). Due to its well-defined polyHIPE structure with interconnected micron size spherical voids, the columns synthesized with different initiator concentrations were successfully used for the separation of alkylbenzenes. Furthermore, the columns indicated a strong electroosmotic flow (EOF) without any additional EOF generating monomer probably due to the presence of ionizable sulfate groups coming from the water-soluble initiator used in the preparation of polyHIPE matrix. The best chromatographic performance in the separation of alkylbenzenes was achieved by using 70% ACN in the mobile phase with high column efficiency (up to 200 000 plates/m).     

Preparation and Porous Property of C14-Monolithic Column for Capillary Electrochromatography

Capillary electrochromatography (CEC) has been performed with a series of C₁₄, methlyacrylamide based monolithic columns. These monoliths with different porosities were prepared by in-situ copolymerization in fused-silica capillaries. The porous properties of monoliths were further observed using scanning electron micrographs (SEM) and measured using a mercury porosimeter. The effect of various alcohols as porogens on porous structural properties and chromatographic behaviors were also investigated. The effects of organic additive, pH value and ionic strength in mobile phase on electroosmosis flow (EOF) and separation were further discussed. Meanwhile, the baseline separation of 6 neutral compounds can be well obtained. In addition, the monolithic column demonstrates the high column efficiency and satisfactory reproducibility.Acknowledgements We gratefully acknowledge the support of the National Natural Science Foundation of China.     

Silica-based monolithic columns versus conventional particle-packed columns for liquid chromatographic analysis of tetracycline, oxytetracycline and chlortetracycline

The rise of monolithic stationary phases offers to routine and research laboratories several advantages. In spite of their recent discovery, they have rapidly become highly popular separation media for liquid chromatography. Time reduction and economic reasons like e.g. a diminished use of mobile phase are the most important ones. At the same time, it was reported that these columns offer a faster and better separation. The aim of this article was to investigate the transferability of methods originally developed on conventional particle-packed C₁₈ columns (XTerra RP18 and Zorbax RX), onto the more recent monolithic columns. Both types, conventional particle-packed and monolithic columns, were able to separate tetracycline, oxytetracycline and chlortetracycline from their respective impurities with sufficient resolution, but showed remarkably shorter analysis times and lower backpressures, improving the lifetime of the column.