Monolithic stationary phases for liquid chromatography and capillary electrochromatography

A monolithic stationary phase is the continuous unitary porous structure prepared by in situ polymerization or consolidation inside the column tubing and, if necessary, the surface is functionalized to convert it into a sorbent with the desired chromatographic binding properties [J. Chromatogr. A 855 (1999) 273]. Monolithic stationary phases have attracted considerable attention in liquid chromatography and capillary electrochromatography in recent years due to their simple preparation procedure, unique properties and excellent performance, especially for separation of biopolymers. This review summarizes the preparation, characterization and applications of the monolithic stationary phases. In addition, the disadvantages and limitations of the monolithic stationary phases are also briefly discussed.     

Capillary electrochromatography using fibers as stationary phases.

Fiber-packed capillary columns have been evaluated in chromatographic performance in capillary electrochromatography (CEC). The change of electroosmotic flow (EOF) velocity and selectivity using different kinds of fiber materials was examined. Although the EOF velocity among the different fiber packed columns was almost the same, retention of parabens was larger on the Kevlar-packed column than on the Zylon-packed one, and was larger on the as-span-type fiber-packed column than on the high-modulus-type packed one. Using 200 microm ID x 5 cm Kevlar packed column combined with a 100 microm ID x 20 cm precolumn capillary and a 530 microm ID x 45 cm postcolumn capillary, the separation of three parabens within 30 s was achieved. Other compounds were also separated in a few minutes by the fiber-packed CEC method.     

Chromatographic evaluation and comparison of three beta-cyclodextrin-based stationary phases by capillary liquid chromatography and pressure-assisted capillary electrochromatography

Enantiomer separations were performed on three beta-cyclodextrin-based chiral stationary phases (CSP) containing the pernaphthylcarbamoylated beta-cyclodextrin (CSP 1), peracetylated beta-cyclodextrin (CSP 2) and permethylated beta-cyclodextrin (CSP 3) as chiral selectors by capillary liquid chromatography and pressure-assisted capillary electrochromatography in this study. Triethylammonium acetate/MeOH or phosphate buffer/MeOH was used as the mobile phase. The experimental factors affecting chiral separations have been examined for each CSP, including pH of the buffers, methanol content and applied voltage. Under optimal separation conditions, a number of racemic compounds were resolved into their enantiomers on three cyclodextrin-based CSP. A comparative study on the performance of three CSP revealed the presence of carbonyl functional groups as well as aromatic rings in the cyclodextrin derivatives, enhanced the interaction between the analytes and CSP, and thus improved enantioselectivity of the CSP.     

Polar stationary phases for capillary electrochromatography

This review article summarizes the variety of polar stationary phases that have been employed for capillary electrochromatographic separations. Compared with reversed-phase stationary phases, the polar alternatives provide a completely different retention selectivity towards polar and charged analytes. Different types of polar stationary phases are reviewed, including the possible retention mechanisms. Electrochromatographic separations of polar solutes, peptides, and basic pharmaceuticals on polar stationary phases are presented.     

Sol-gel stationary phases for capillary electrochromatography

A review is presented on the current state of the art and future trends in the development of sol-gel stationary phases for capillary electrochromatography (CEC). The design and synthesis of stationary phases with prescribed chromatographic and surface charge properties represent challenging tasks in contemporary CEC research. Further developments in CEC as a high-efficiency liquid-phase separation technique will greatly depend on new breakthroughs in the area of stationary phase development. The requirements imposed on CEC stationary phase performance are significantly more demanding compared with those for HPLC. The design of CEC stationary phase must take into consideration the structural characteristics that will provide not only the selective solute/stationary phase interactions leading to chromatographic separations but also the surface charge properties that determine the magnitude and direction of the electroosmotic flow responsible for the mobile phase movement through the CEC column. Therefore, the stationary phase technology in CEC presents a more complex problem than in conventional chromatographic techniques. Different approaches to stationary phase development have been reported in contemporary CEC literature. The sol-gel approach represents a promising direction in this important research. It is applicable to the preparation of CEC stationary phases in different formats: surface coatings, micro/submicro particles, and monolithic beds. Besides, in the sol-gel approach, appropriate sol-gel precursors and other building blocks can be selected to create a stationary phase with desired structural and surface properties. One remarkable advantage of the sol-gel approach is the mild thermal conditions under which the stationary phase synthesis can be carried out (typically at room temperature). It also provides an effective pathway to integrating the advantageous properties of organic and inorganic material systems, and thereby enhancing and fine-tuning chromatographic selectivity of the created hybrid organic-inorganic stationary phases. This review focuses on recent developments in the design, synthesis, characterization, properties, and applications of sol-gel stationary phases in CEC.     

Histidine-functionalized silica and its copper complex as stationary phases for capillary electrochromatography

A histidine-functionalized silica was prepared by covalent bonding of the functional groups to silane-treated silica gel. Conversion of functional groups was confirmed by infrared (IR) spectra, elemental analysis, and potentiometry. The functionality of the silica gel is 0.293 mmol g(-1). The coordination behavior of the histidine-functionalized silica was investigated by metal capacity and electron paramagnetic resonance (EPR). EPR measurements at different copper loadings were made. The results showed that the copper histidine complex might be distorted tetragonal. Both histidine-functionalized silica and its copper complex were employed as stationary phases for packed capillary electrochromatography (CEC). Electrical current was found helpful for evaluating the properties of frit construction and the stationary phase packing. Test samples include neutral compounds, inorganic anions and organic anions. Factors influencing the separation behavior have been studied. With copper-histidine functionalized silica under the condition of citrate buffer (10 mM, pH 4.0) and applied voltage of -20 kV, the separation of benzoic acid, D- and L-mandelic acid, phthalic acid and salicylic acid could be achieved within 12 min. The column efficiency for these acids was more than 1.2 x 10(5) plates m(-1), except salicylic acid.     

Enantiomer separations by capillary electrochromatography using chiral stationary phases

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.     

Comparision of succinate- and phthalate-functionalized etched silica hydride phases for open-tubular capillary electrochromatography

Two open-tubular (OT) capillary electrochromatographic (CEC) columns were prepared by chemically bonding ionizable mono-(2-(methacryloyloxy)ethyl) succinate (MES) and phthalate-functionalized (MEP) ligands onto silica hydride-based phases through surface etching, silanization, and hydrosilation reactions, starting with a bare fused-silica tube. An analysis of the effect of performance of electrophoretic flow (EOF) on the changes in pH values, ionic strength, and the amount of acetonitrile modifiers helped to reveal that some silanol groups remained in the surface composite of the modified capillaries and to prove that MEP capillaries actually exerted greater EOF than MES ones. To explore the potential utilization of these two columns in various fields, three categories of samples, which spanned a wide range of polarities, were prepared and analyzed through many systematic trials of optimizing CEC conditions. For the separation of a mixture of nucleosides and thymine, guanine and adenine with purine uncleobases, which exhibit greater aromaticity than pyrimidine nucleobases, performed a higher retention in the MEP capillary through a π–π interaction than in the MES capillary. While four steroids were used as test samples, their migration order revealed that the MES stationary phase is hydrophilic in comparison with the MEP. An addition of methanol modifier (30%, v/v) into 10 mM borate buffer (pH 9.55 for MEP; pH 10.0 for MES) was necessary to accomplish a baseline separation of nine flavonoids in the MEP and MES capillaries. Studies on the elution order of these solutes revealed the presence of chromatographic activity in addition to electrophoretic migration. Especially in the MEP capillary, hydrophobic characteristics and π–π interactions with aromatic solutes were found and further improved to resolve an enantiomeric pair, catechin and epicatechin. Overall, the hydride-based stationary phases with ionizable ligands were successfully applied to the OT-CEC separations, and these results confidently propose an ideal route to the synthesis of a novel OT-CEC column.     

Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography

The physical adsorption method proposed previously has been successfully applied to a monolithic silica column. By virtue of the physical adsorption, a chiral stationary phase of avidin was prepared onto the silica monolith. The phase ratio of resulting stationary phase was evaluated with frontal analysis. The method proved to be comparable in phase ratio to the chemical bonding methods used in high-performance liquid chromatography (HPLC). Enantiomer separations were carried out in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) modes. Due to its larger phase ratio, the resulting column showed more powerful separation capability as compared to open-tubular CEC (OTCEC). Twelve chiral compounds were baseline-resolved. The resulting column showed high separation efficiency, with average theoretical plate numbers of 66 000/m for CLC and 122 000/m for CEC. Good reproducibility was observed, with RSD value less than 1.3% for retention time, retention factor and separation factor, and less than 6.6% for plate counts and resolution (n = 40). Fast separations were achieved with a short column. The test enantiomers were baseline-resolved within 4 min under CLC and CEC modes. In addition, field-enhanced sample injection (FESI) was coupled to CLC as well as CEC to improve the detection sensitivity.     

Capillary electrochromatography in anion-exchange and normal-phase mode using monolithic stationary phases

Hydrophilic macroporous weak and strong anion-exchange stationary phases have been prepared in a monolithic format within untreated fused-silica capillaries by the simple thermally or UV-initiated polymerization of 2-dimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of a binary porogenic mixture of dodecanol and cyclohexanol. The tertiary amino functionalities were then alkylated in situ to afford strong anion-exchangers. These new monolithic stationary phases with optimized porous properties were used for the CEC separation of various organic anions. Thus, a mixture of 2-substituted propionic acid drugs (profens) was separated in 13 min and high column efficiencies of up to 231,000 plates/m were achieved. The separation of substituted benzoic acids indicates that the selectivity results primarily from the anion-exchange interactions, while electrophoretic migration contributes only slightly. In addition, these hydrophilic anion-exchangers are also able to separate weakly acidic, neutral and basic compounds such as phenols, xanthines and aromatic amines in normal-phase electrochromatographic mode.     

Polymeric monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions of a number of groups working in the rapidly growing area of monolithic columns for capillary electrochromatography (CEC), with a focus on those prepared from synthetic polymers. Monoliths have quickly become a well-established stationary phase format in the field of CEC. The simplicity of their in situ preparation method as well as the good control over their porous properties and surface chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials. A wide variety of approaches as well as materials used for the preparation of the monolithic stationary phases are detailed. Their excellent chromatographic performance is demonstrated by numerous separations of different analytes.     

Polymer-brush stationary phases for open-tubular capillary electrochromatography

Synthesis of poly(2-hydroxyethyl methacrylate) (PHEMA) brushes from the inside of silica capillaries by surface-initiated atom transfer radical polymerization (ATRP) yields unique stationary phases for open-tubular capillary electrochromatography (OT-CEC). Although PHEMA brushes have only a small effect on the separation of a set of phenols and anilines, derivatization of PHEMA with ethylenediamine (en) allows baseline resolution of several anilines that co-elute from bare silica capillaries. Derivatization of PHEMA with octanoyl chloride (C8-PHEMA films) affords even better resolution in the separation of a series of phenols and anilines. Increasing the thickness of C8-PHEMA coatings by a factor of 2 enhances resolution for several solute pairs, presumably because of an increase in the effective stationary phase to mobile phase volume ratio. Thus, this work demonstrates that thick polymer brushes provide a tunable stationary phase with a much larger phase ratio than is available from monolayer wall coatings. Through appropriate choice of derivatizing reagents, these polymer brushes should allow separation of a wide range of neutral molecules as well as compounds with similar electrophoretic mobilities.     

Monolithic enantiomer-selective stationary phases for capillary electrochromatography

Monolithic materials have become a well-established format for stationary phases in the field of capillary electrochromatography. Four types of monoliths, namely particle-fixed, silica-based, polymer-based, and molecularly imprinted monoliths, have been utilized as enantiomer-selective stationary phases in CEC. This review summarizes recent developments in the area of monolithic enantiomer-selective stationary phases for CEC. The preparative procedure and the characterization of these columns are highlighted. In addition, the disadvantages and limitations of different monolithic enantiomer-selective stationary phases in CEC are briefly discussed.     

Monolithic stationary phases for fast ion chromatography and capillary electrochromatography of inorganic ions

The focus of this review is on current developments in monolithic stationary phases for the fast analysis of inorganic ions and other small molecules in ion chromatography (IC) and capillary electrochromatography (CEC), concentrating in particular on the properties of organic (polymer) monolithic materials in comparison to inorganic (silica-based) monoliths. The applicability of these materials for fast IC is discussed in the context of recent publications, including the range of synthesis and modification procedures described. While commercial monolithic silica columns already show promising results on current IC instrumentation, polymer-based monolithic stationary phases are currently predominantly used in the capillary format on modified micro-IC systems. However, they are beginning to find application in IC particularly under high pH conditions, with the potential to replace their particle-packed counterparts.     

Enantioseparations in non-aqueous capillary electrochromatography using polysaccharide type chiral stationary phases

Enantioseparations of chiral compounds with different structures were studied in non-aqueous capillary electrochromatography (NAQ CEC). Three different polysaccharide derivatives, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD), amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) and cellulose tris(4-methylbenzoate) (Chiralcel OJ) were used as chiral stationary phases (CSPs). Methanolic or ethanolic ammonium acetate solutions served as a mobile phase. The effect of the type of the CSP, the loading of the chiral selector on wide-pore aminopropyl derivatized silica gel and operational parameters such as apparent pH, applied voltage, etc. on the EOF and chromatographic characteristics (alpha, N, Rs) were studied. NAQ CEC represents a valuable alternative and an extension to chiral separations by HPLC with common-size columns as well as to capillary LC and CEC in aqueous buffers.     

Capillary electrochromatography with physically and dynamically absorbed stationary phases

Adsorption is always considered a troublesome effect in capillary electrophoresis (CE) and capillary electrochromatography (CEC). However, the adsorption effect can also be exploited to prepare or optimize the stationary phase in CEC. Compared with the chemical synthesis of new stationary phase materials for CEC, this method is simpler and more convenient. This review is focused on CEC with physically and dynamically adsorbed stationary phases. Separation of some acidic, basic and neutral solutes as well as enantiomers in CEC with dynamically adsorbed stationary phases are presented. The theory for the migration of charged solutes and the stationary phases currently used in CEC are also briefly reviewed.     

Succinyl methacrylate polymerized in porous-layered phases for open-tubular capillary electrochromatography: Comparison with silica hydride monolayered phases

A polymer phase, which was constructed with butyl methacrylate (BMA), an ionizable monomer (mono-(2-(methacryloyloxy)ethyl) succinate (MES)), and a crosslinking agent (ethylene dimethacrylate), was first formed in a porous-layered open-tubular (PLOT) capillary. The PLOT capillary was characterized with SEM and electrophoretic flow as the pH level, ionic strength and addition of organic modifiers in the running buffers changed. In addition, a bare capillary and a silica hydride based capillary (SiH-MES), which bore a monolayered MES phase on it, were used to compared with the BMA-MES capillary. Besides optimizing the capillary electrochromatographic (CEC) conditions for each group of analytes, which were a mixture of nucleosides and thymine, flavonoids, and phenolic acids,comparison of the separation selectivity among analytes between the BMA-MES and SiH-MES capillaries was done according to the velocity and retention factors obtained from the CEC data. Overall, the polymeric phase formed in the PLOT mode was capable of preventing blockage of the columns and was superior to the monolayered phase bonding with the same ionizable ligands for application in CEC as well as to the bare silica phase in CE.     

The analysis of pharmaceutical bases on a silica stationary phase by capillary electrochromatography using aqueous mobile phases

Summary The capillary electrochromatographic (CEC) separation of a range of pharmaceutical bases was investigated on a commercially available silica stationary phase using aqueous mobile phases. The effects of mobile phase composition, buffer pH, applied voltage, and buffer anion on the retention behaviour of these bases were studied. Promising chromatography was obtained at pH 7.8 but was later found to be irreproducible. However, successful and reproducible chromatography of the bases was achieved at pH 2.3. We have previously demonstrated that the addition of mobile phase additives such as TEA-phosphate at low pH values has resulted in excellent CEC analysis of bases on reversed-phase packing materials. The same approach was applied to the analysis of bases on the silica phase in order to improve peak shape. Excellent chromatography was obtained for the analysis of strong pharmaceutical bases such as benzylamine, nortriptyline and diphenhydramine. The experimental investigations have shown that the CEC separation of a range of pharmaceutical bases can routinely be achieved with excellent peak shapes and peak efficiencies as high as 320,000 plates m⁻¹.