Enantiomeric separations by capillary electrochromatography using a macrocyclic antibiotic chiral stationary phase.

Racemic mixtures of tryptophan and dinitrobenzoyl leucine have been successfully resolved by capillary electrochromatography (CEC) using the macrocyclic antibiotic teicoplanin, covalently bonded to a 5 microns silica support. Modification of a previously published packing procedure was required to pack reliable capillaries, capable of performing enantiomeric separations. Good levels of enantioselectivity were obtained in all cases, with optimised separations being performed in less than 6 min. Retention times, resolution and reproducibility are discussed.     

Enantiomers separation by capillary electrochromatography using polysaccharide‐based stationary phases

The separation of chiral compounds is an interesting and important topic of research because these compounds are involved in some biological processes, fundamentally in human health. Among the various application fields where enantiomers are remarkable, drug analysis has to be considered. Most of the drugs contain enantiomers and very often one of the two isomers could be pharmacologically more active or even dangerous. Therefore, the separation of these compounds is very important. Among the different analytical techniques usually employed, capillary electrochromatography has demonstrated great capability in enantiomers resolution. The great potential of this electromigration technique stands mainly in its high efficiency due to the use of an electrosmotic flow (flat flow profile) and on the high selectivity because of the use of a stationary phase. Chiral separation can be obtained utilizing several chiral stationary phases including a polysaccharide derivative. The aim of this review paper is to summarize the main features of capillary electrochromatography and polysaccharide derivatives of chiral stationary phase. It also report examples of practical applications utilizing this approach.     

Enantiomer separation by nonaqueous and aqueous capillary electrochromatography on cyclodextrin stationary phases

Native beta- and gamma-cyclodextrin bound to silica (ChiraDex-beta and ChiraDex-gamma) were packed into capillaries and used for enantiomer separation by capillary electrochromatography (CEC) under aqueous and nonaqueous conditions. Negatively charged analytes (dansyl-amino acids) were resolved into their enantiomers by nonaqueous CEC (NA-CEC). The addition of a small amount of water to the nonaqueous mobile phase enhanced the enantioselectivity but increased the elution time. The choice of the background electrolyte (BGE) determined the direction of the electroosmotic flow (EOF). With 2-(N-morpholino) ethanesulfonic acid (MES) or triethylammonium acetate (TEAA) as BGE an inverse EOF (anodic EOF) was observed while with phosphate a cathodic EOF was found. The apparent pH (pH*), the concentration of the BGE, and the nature of the mobile phase strongly influenced the elution time, the theoretical plate number and the chiral separation factor of racemic analytes.     

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.     

Enantiomeric separation of citalopram analogues by HPLC using macrocyclic glycopeptide and cyclodextrin based chiral stationary phases

The enantiomeric separation of a novel series of twenty-eight racemic mixtures of citalopram analogues was performed by high performance liquid chromatography (HPLC). Due to the effectiveness of citalopram as an antidepressant drug, the development of new compounds based on its chemical structure is interesting, and their enantiomeric separation is needed to allow further pharmacokinetic studies. Several bonded cyclodextrin (both native and derivatized) and macrocyclic glycopeptide based chiral stationary phases (CSPs) were evaluated for their ability to separate this set of compounds via HPLC. Polar ionic, polar organic, and reversed phase modes were tested. Twenty-five of the racemic mixtures were separated with resolutions and enantiomeric selectivities up to 2.9 and 1.33, respectively. A total of eighteen baseline separations were achieved, while seven compounds were partially separated. Vancomycin based columns operated in the polar ionic mode resulted in the greatest number of separations. Lastly, the chromatographic behaviors of similar compounds were compared based on their chemical structure and also on the chiral selectors used.     

Enantiomeric separations of cationic and neutral compounds by capillary electrochromatography with monolithic chiral stationary phases of beta-cyclodextrin-bonded negatively charged polyacrylamide gels

Enantiomeric separation by capillary electrochromatography with beta-cyclodextrin-bonded negatively charged polyacrylamide gels was examined. The columns used are capillaries filled with a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, to which allyl carbamoylated beta-CD (AC-beta-CD) derivatives covalently bind. The capillary wall is activated first with a bifunctional reagent to make the resulting gel bind covalently to the inner surface of the fused-silica tubing. Enantiomeric separations of 15 cationic compounds were achieved using the above-mentioned columns and mobile phases of 200 mmol l(-1) Tris-300 mmol I(-1) boric acid buffer (pH 7.0 or 9.0) or 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 7.0) containing an achiral crown ether (18-crown-6). Enantiomeric separations of two neutral compounds were also achieved using 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 9.0) as a mobile phase. High efficiencies of up to 150,000 plates m(-1) were obtained. Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 3 months with intermittent use, utilizing the mobile phase of 200 mmol I(-1) Tris-300 mmol I(-1) boric acid buffer (pH 9.0).     

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.     

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.     

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.     

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.     

Enantiomeric separations of primary amino compounds by capillary electrochromatography with monolithic chiral stationary phases of chiral crown ether-bonded negatively charged polyacrylamide gels

A novel enantiomeric separation method by capillary electrochromatography with chiral crown ether-bonded negatively charged polyacrylamide gels is presented. Two kinds of chiral crown ether derivatives, (+)-tetraallyl 18-crown-6 carboxylate and (+)-18-crown-6 tetracarboxylic acid 2-allyl ester were synthesized and allowed to covalently bind to a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, respectively. The gel was placed in fused-silica tubing, the walls of which had been activated with a bifunctional reagent to make the resulting gel bind covalently to the inner surface. Enantiomeric separations of 12 primary amino compounds were achieved using these columns and mobile phases of 200 mM triethanolamine-300 mM boric acid buffers with high efficiencies of up to 135000 plates m(-1). Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 13 months with intermittent use for 3 months followed by storage at room temperature for 10 months. The result of the optical purity test of alanine-2-naphthylamide is also described.     

Separation strategy for acidic chiral pharmaceuticals with capillary electrochromatography on polysaccharide stationary phases

The effect of five factors on the capillary electrochromatographic enantioseparation of acidic compounds was studied using an experimental design. The studied factors were pH, acetonitrile content in the mobile phase, temperature, buffer concentration, and applied voltage. These experiments allowed defining a generic separation strategy applicable on acidic compounds with chemical and structural diversity. The starting screening conditions consist of a 45 mM ammonium formate electrolyte at pH 2.9 mixed with 65% acetonitrile, an applied voltage of 15 kV, and a temperature of 25 degrees C. The screening phase occasionally can be followed by an optimization procedure. Evaluation of the proposed strategy pointed out that it allows achieving baseline resolution within a relatively short time when a beginning of separation is obtained at the starting conditions. This strategy revealed enantioselectivity for 11 compounds out of 15, of which 10 could be baseline-separated after the proposed optimization steps.     

The analysis of basic pharmaceutical compounds by capillary electrochromatography using continuous bed stationary phase

Summary Capillary electrochromatography (CEC) is classed as a hybrid technique between CE and HPLC and it combines the advantages of both these techniques. However, in some cases the disadvantages are also brought to light and some of these are difficult to resolve. For example the analysis of basic compounds using CEC. The problems of tailing peaks during HPLC analysis of basic compounds was resolved by end capping the residual silanol groups, but in CEC these are the groups that generate the electroosmotic flow. The analysis of basic compounds is crucial within the pharmaceutical industry where a high percentage of the drug actives are basic. Specially designed Continuous Beds stationary phases (CB) can mean that each application can have a specific stationary phase. In order to overcome the problem associated with the analysis of basic compounds using electrochromatography, we have designed a CB stationary phase with a positive charge, which could be operated using negative voltage. The resulting chromatography showed almost gaussian peaks for bases like nortriptyline which tail significantly using stationary phase typically used in CEC.     

Fast separation of pyrimidine derivatives by capillary electrochromatography on ion-exchange/reversed-phase mixed-mode stationary phases

This work describes the use of mixed-mode stationary phases which exhibit both strong ion-exchange (either cation-exchange, SCX, or anion-exchange, SAX) and reversed-phase chromatographic characteristics in capillary electrochromatographic separations of pyrimidine derivatives. Different packing materials, namely C6, SCX/C6 and SAX/C6, were compared and the influence of the composition of the carrier electrolyte (concentration of acetonitrile and pH) on the retention behavior of the selected solutes was investigated. A separation of all eight pyrimidine derivatives could be obtained on a 6.5 cm column packed with the SAX/C6 stationary phase in less than 3 min, with good peak shapes and efficiencies in the range 39,000 to 81,000 plates per meter.     

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.     

Separation of acidic compounds by strong anion-exchange capillary electrochromatography

Separation of the acidic compounds in the ion-exchange capillary electrochromatography (IE-CEC) with strong anion-exchange packing as the stationary phase was studied. It was observed that the electroosmotic flow (EOF) in strong anion-exchange CEC moderately changed with increase of the eluent ionic strength and decrease of the eluent pH, but the acetonitrile concentration in the eluent had almost no effect on the EOF. The EOF in strong anion-exchange CEC with eluent of low pH value was much larger than that in RP-CEC with Spherisorb-ODS as the stationary phase. The retention of acidic compounds on the strong anion-exchange packing was relatively weak due to only partial ionization of them, and both chromatographic and electrophoretic processes contributed to separation. It was observed that the retention values of acidic compounds decreased with the increase of phosphate buffer and acetonitrile concentration in the eluent as well as the decrease of the applied voltage, and even the acidic compounds could elute before the void time. These factors also made an important contribution to the separation selectivity for tested acidic compounds, which could be separated rapidly with high column efficiency of more than 220000 plates/m under the optimized separation conditions.     

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-μm diol-silica particles. The CSP mixed with 5-μm amino silica particles (3:1) was packed into 75-μm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor R_s=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12–20 min).     

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.