Separation of enantiomers by nanoliquid chromatography and capillary electrochromatography using a bonded cellulose trisphenylcarbamate stationary phase

A cellulose trisphenylcarbamate-bonded chiral stationary phase was applied to nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) with nonaqueous and aqueous solutions as the mobile phases. Several chiral compounds were successfully resolved on the prepared phase by nano-LC. The applicability of nonaqueous CEC on a cellulose derivative stationary phase was investigated with the organic solvents methanol, hexane, 2-propanol, and tetrahydrofuran (THF) containing acetic acid, as well as triethylamine as the mobile phases. Enantiomers of warfarin and praziquantel were baseline-resolved with plate numbers of 82,300 and 38,800 plates/m, respectively, for the first eluting enantiomer. The influence of applied voltage, concentration of nonpolar solvent, apparent pH, and buffer concentration in the mobile phase on the electroosmotic flow (EOF) and the mobility of the enantiomers was evaluated. Enantioseparations of trans-stilbene oxide and praziquantel were also achieved in aqueous CEC with plate numbers of 111,100 and 107,400 plates/m, respectively, for the first eluting enantiomer. A comparison between nonaqueous CEC and aqueous CEC based on a cellulose trisphenylcarbamate stationary phase was discussed. Pressure-assisted CEC was examined for the chiral separation of praziquantel and faster analysis with high enantioselectivity was acquired with the proper pressurization of the inlet vial.     

Enantioseparations by capillary electro-chromatography: differences exhibited by normal- and reversed-phase versions of polysaccharide stationary phases

The influence of using normal-phase and reversed-phase versions of four commercial polysaccharide stationary phases on chiral separations was investigated with capillary electrochromatography (CEC). Both versions of the stationary phases, Chiralcel OD, OJ, and Chiralpak AD, AS were tested for the separation of two basic, two acidic, a bifunctional, and a neutral compound. Different background electrolytes were used, two at low pH for the acid, bifunctional and neutral substances, and three at high pH for the basic, bifunctional and neutral ones. This setup allowed evaluating differences between both stationary-phase versions and between mobile-phase compositions on a chiral separation. Duplicate CEC columns of each stationary phase were in-house prepared and tested, giving information about the intercolumn reproducibility. In general, reversed-phase versions of the current commercial polysaccharide stationary phases are found to be best for reversed-phase CEC, even though at high pH no significant differences were seen between both versions. Most differences were observed at low pH. For acidic compounds, it was seen that an ammonium formate electrolyte performed best, which is also an excellent electrolyte if coupling with mass spectrometry is desired. For basic, bifunctional and neutral compounds, no significant differences between the three tested electrolytes were observed at high pH. Here, a phosphate buffer is preferred as electrolyte because of its buffering capacities. However, if coupling to mass spectrometry is wanted, the more volatile ammonium bicarbonate electrolyte can be used as an alternative.     

Polyacrylamide-based monolithic capillary column with coating of cellulose tris(3,5-dimethylphenyl-carbamate) for enantiomer separation in capillary electrochromatography

A hydrophilic chiral capillary monolithic column for enantiomer separation in CEC was prepared by coating cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) on porous hydrophilic poly(acrylamide-co-N,N'-methylene-bisacrylamide) (poly(AA-co-MBA)) monolithic matrix with confine of a fused-silica capillary. The coating conditions were optimized to obtain a stable and reproducible chiral stationary phase for CEC. The effect of organic modifier of ACN in aqueous mobile phase for the enantiomer separation by CEC was investigated, and the significant influence of ACN on the enantioresolution and electrochromatographic retention was observed. Twelve pairs of enantiomers including acidic, neutral, and basic analytes were tested and nine pairs of them were baseline-enantioresolved with acidic and basic aqueous mobile phases. A good within-column repeatability in retention time (RSD = 2.4%) and resolution (RSD = 3.2%) was obtained by consecutive injections of a neutral compound, benzoin, on a prepared chiral monolithic column, while the between-column repeatability in retention time (RSD = 6.4%) and resolution (RSD = 9.6%) was observed by column-to-column examination. The prepared monolithic stationary phase showed good stability in either acidic or basic mobile phase.     

Strategy for the chiral separation of non-acidic pharmaceuticals using capillary electrochromatography

In completion of an earlier defined generic chiral screening approach, a generic separation strategy for basic, bifunctional, and neutral compounds was proposed and evaluated. This strategy adds to a previously defined strategy for acidic compounds. The screening experiment of the actual strategy used a mobile phase of 5 mM phosphate buffer pH 11.5/ACN (30/70 v/v), a temperature of 25 degrees C, and a voltage of 15 kV. The selected chiral stationary phases were Chiralpak AD-RH, Chiralcel OD-RH, Chiralcel OJ-RH, and Chiralpak AS-RH, all based on polysaccharide selectors. It was seen that 31 out of 48 test compounds were partially or baseline-resolved under screening conditions. After execution of the optimization steps of the strategy, this number increased to 41, with a total of 21 baseline-separated compounds. Combined with the results obtained from the acidic test set examined in the earlier defined strategy, of all tested compounds 82.5% showed enantioselectivity and 49.2% could be baseline-separated.     

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous–organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.     

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.     

A Silica Monolithic Column with Chemically Bonded l-Pipecolic Acid as Chiral Stationary Phase for Enantiomeric Separation of Dansyl Amino Acids by CEC–MS

A silica monolithic column chemically modified with l-pipecolic acid as chiral stationary phase has been developed for chiral separation of dansyl amino acids by capillary electrochromatography–mass spectrometry (CEC–MS). The monolithic column was prepared by a sol–gel process and subsequent chemical modification by l-pipecolic acid as chiral selector with 3-glycidoxypropyltrimethoxysilane as spacer. Interestingly, it was found that the l-pipecolic acid-modified monolithic column can hold copper(II) ions tightly after loading Cu(II) ions during column preparation and conditioning and allows CEC separation to be conducted based on chiral ligand exchange with MS detection by a mobile phase without copper ions. It has been demonstrated that the chiral monolithic column operates well for enantioseparation of several dansyl amino acids by CEC–MS.

     

Fast separations of chiral β-blockers on a cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia monolithic column by capillary electrochromatography

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) is an excellent chiral selector for enantioseparation of a wide variety of chiral compounds. The monolithic chiral columns are becoming popular in liquid chromatography and capillary electrochromatography. In this work, we present the fast separation of chiral β-blockers on a CDMPC-modified zirconia monolithic column by capillary electrochromatography (CEC). The porous zirconia monolithic capillary column was prepared by using the sol-gel technology and then zirconia surface modified with CDMPC. The enantioseparations were performed in reversed-phase (RP) eluents of a phosphate solution (pH 4.4) modified with acetonitrile or alcohol. The enantioseparations of a set of eight chiral β-blockers were achieved in less than one minute. Influences of the applied voltage, column temperature, concentration of acetonitrile and the type of alcohol as the organic modifier in the mobile phase, and sample injection time on enantioseparation were investigated. CEC separations at the applied voltage of 10 kV and 15 °C in the ACN-modified mobile phase provided the best resolutions for the analytes studied. Run-to-run and day-to-day repeatabilities of the column in the RP-CEC separation were less than 1 and 2%, respectively.     

Capillary Electrochromatography as a Method Development Tool for the Liquid Chromatographic Separation of DUP 654 and Related Substances

Capillary Electrochromatography (CEC) offers a rapid, economical, and efficient means for resolving nonionic compounds in the reversed phase mode on octadecylsilane (ODS) columns. A CEC optimization on a Hypersil ODS capillary column was employed to identify a suitable mobile phase for the pressure-driven (reversed phase ODS) separation of the anti-inflammatory 2-phenylmethyl-1-naphthol (DUP 654), and its related substances. The proportions of mobile phase modifiers methanol, acetonitrile, and water as well as pH were employed as variables in a stacked mixture design. Comparable response surface profiles were obtained for the CEC separations at pH 4 and pH 8. However, subtle differences were evident in the quality of separations obtained in the liquid chromatographic (LC) mode when using a specially-prepared column packed with exactly the same stationary phase as used in the CEC experiments. A mapping of the response surface for separations on a commercially available Hypersil ODS LC column revealed obvious differences. The differences indicate that the transfer of ODS based separation methods between CEC and LC involves more than simply transferring the conditions from one mode to the other.     

Capillary electrochromatography of basic compounds using octadecyl-silica stationary phases with an amine-containing mobile phase

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6x10(4) plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1x10(5) plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8x10(5) plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1x10(5)-3x10(5) plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning.     

Capillary electrochromatographic separation of enantiomers under aqueous mobile phases on a covalently bonded cellulose derivative chiral stationary phase

A chemically bonded cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (CSP) was prepared by a radical polymerization reaction. The prepared CSP was packed into fused-silica capillaries with inner diameter of 75 microm to perform enantiomer separations in CEC. The electrochromatographic behavior of the CSP was investigated. On the prepared CSP, high EOF could be generated under acidic mobile phases, which represented an advantage for the separation of acidic enantiomers. Several neutral, acidic, and basic enantiomers were resolved on the prepared CSP under aqueous mobile phases. The column efficiencies were between 20,000 and 100,000 plates/m, which were much higher than those of HPLC. In addition, it was observed that the separation of some enantiomers benefited from the adoption of THF as mobile phase modifier.     

Recent applications of capillary electrochromatography

A review is presented of the most important recent applications of capillary electrochromatography (CEC) for the analysis of acidic, basic, and neutral compounds, of biomolecules, environmental substances, natural products, pharmaceuticals, and chiral compounds. Packed-column CEC (packed-CEC), open-tubular (OT-CEC), as well as pressure-assisted CEC (pseudo-CEC) are hereby considered. Papers published between July 1999 and April 2001 were taken into account. Applications before July 1999 have been reviewed in Electrophoresis 1999, 20, 3027-3065.     

Enantioselective reversed-phase and non-aqueous capillary electrochromatography using a teicoplanin chiral stationary phase

Enantiomeric separation of chiral pharmaceuticals is carried out in aqueous and non-aqueous packed capillary electrochromatography (CEC) using a teicoplanin chiral stationary phase (CSP). Capillaries were slurry packed with 5 microm 100-A porous silica particles modified with teicoplanin and initially evaluated using a non-aqueous polar organic mode system suitability test for the separation of metoprolol enantiomers (Rs = 2.3 and 53000 plates m(-1)). A number of pharmaceutical drugs were subsequently screened with enantioselectivity obtained for 25 racemic solutes including examples of neutral, acidic and basic molecules such as coumachlor (Rs = 3.0 and 86000 plates m(-1)) and alprenolol (Rs = 3.3 and 135000 plates m(-1)) in reversed-phase and polar organic mode, respectively. A statistical experimental design was used to investigate the effects of non-aqueous polar organic mobile phase parameters on the CEC electroosmotic flow, resolution and peak efficiency for two model solutes. Results primarily indicated that higher efficiency and resolution values could be attained at higher methanol contents which is similar to findings obtained on this phase in liquid chromatography.     

Enantioseparations in nonaqueous capillary liquid chromatography and capillary electrochromatography using cellulose tris(3,5-dimethylphenylcarbamate) as chiral stationary phase

The potential of the widely used chiral stationary phase for high-performance liquid chromatography (HPLC) enantioseparations, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC, sold under the trade name Chiralcel OD) was evaluated under the conditions of nonaqueous capillary electrochromatography (CEC). The effect of the particle size of the silica gel, the loading of CDMPC on the silica gel and nature of the organic solvent, as well as electrolyte salts on the separation characteristics were investigated. This study illustrates the applicability of CDMPC for obtaining highly efficient enantioseparations under the conditions of nonaqueous CEC. Comparative study of enantioseparations in capillary liquid chromatography (CLC) and CEC indicated the significant advantages of CEC such as higher plate number at the similar linear flow rates of the mobile phase as well as better tolerance of higher linear flow rates.     

Enantioselectivity of monolithic silica stationary phases immobilized with different concentrations cellulose tris (3,5-dimethylphenylcarbamate), analyzed with different mobile phases in capillary electrochromatography

The 3,5-dimethylphenylcarbamate derivatives of cellulose bearing 3-(triethoxysilyl)propyl residues were immobilized in a capillary format onto a monolithic silica support by intermolecular polycondensation of the triethoxysilyl groups. The resulting columns were used for chiral separations using capillary electrochromatography. The effects of the synthesizing solvent, the selector coating procedure, the chiral selector concentration onto the silica monolith and the mobile phase pH value, on the separation of enantiomers were studied. The column-to-column reproducibility and stability also were evaluated. A test set of 14 chiral substances, including acidic, neutral, bifunctional and basic compounds, was used to investigate the effects of the factors mentioned above. Twelve pairs of enantiomers showed enantioselectivity at some of the different conditions tested. The column-to-column repeatability was satisfactory, and the prepared columns were stable under the adopted analysis conditions.     

Preparation of a neutral porous monolith and its evaluation in pressurized capillary electrochromatography with neutral and charged solutes

A neutral, nonpolar monolithic capillary column was evaluated as a hydrophobic stationary phase in pressurized CEC system for neutral, acidic and basic solutes. The monolith was prepared by in situ copolymerization of octadecyl methacrylate and ethylene dimethacrylate in a binary porogenic solvent consisting of cyclohexanol/1,4‐butanediol. EOF in this hydrophobic monolithic column was poor; even the pH value of the mobile phase was high. Because of the absence of fixed charges, the monolithic capillary column was free of electrostatic interactions with charged solutes. Separations of neutral solutes were based on the hydrophobic mechanism with the pressure as the driving force. The acidic and basic solutes were separated under pressurized CEC mode with the pressure and electrophoretic mobility as the driving force. The separation selectivity of charged solutes were based on their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase, and no obvious peak tailing for basic analytes was observed. Effects of the mobile phase compositions on the retention of acidic compounds were also investigated. Under optimized conditions, high plate counts reaching 82 000 plates/m for neutral compounds, 134 000 plates/m for acid compounds and 150 000 plates/m for basic compounds were readily obtained.     

Chemically L-prolinamide-modified monolithic silica column for enantiomeric separation of dansyl amino acids and hydroxy acids by capillary electrochromatography and mu-high performance liquid chromatography

A silica-based chiral monolithic column prepared by sol-gel process and chemical modification of chiral selector was used for enantioseparation of dansyl amino acids and hydroxy acids by capillary electrochromatography (CEC) and mu-high-performance liquid chromatography (mu-HPLC). L-Prolinamide was modified as a chiral selector. The chiral stationary phase (CSP), the chiral complex of Cu(II) with L-prolinamide, provides an anodic electroosmotic flow (EOF) in CEC. The EOF was found to be dependent on applied electric field strength, the pH, and the composition of mobile phases. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pore. D-Enantiomers migrated before L-enantiomers except for dansyl-(Dns)-DL-Ser. The separation efficiencies of up to 17600 (D) and 13,200 plates m(-1) (L) were achieved for the separation of DL-indole-3-lactic acid.     

硅胶电色谱分离机理的研究

对硅胶电色谱柱的性能进行了考察,发现在水／有机溶剂流动相条件下,几乎不存在气泡问题,流动相的组成在有机溶剂浓度、电解质浓度、ＰＨ值等方面可以在较大范围变化,选用５种典型样品,对硅胶电色谱的分离机理进行了系统研究,发现有反相分离机理、正相吸附机理、离子交换机理以及电泳机理参与作用。同时考察了有机溶剂浓度、电解质浓度、ＰＨ等对分离的影响。此外,还首次提出了一种全新的电色谱模式－动态改性硅胶电色谱。     

Evaluation of polymeric methacrylate-based monoliths in capillary electrochromatography for their potential to separate pharmaceutical compounds

Polymeric methacrylate-based monoliths are evaluated in capillary electrochromatography (CEC) and pressurized capillary electrochromatography (p-CEC) for their potential in pharmaceutical analysis. Using a given polymerization mixture as a basis for the monolith synthesis, different mobile phase pH at constant organic modifier concentrations are tested in both CEC and p-CEC. The test set consists of basic, acidic, amphoteric, and neutral compounds, which are mainly pharmaceuticals. Because of the mainly hydrophobic character of the stationary phase, the interactions are largest when the compounds appear in an uncharged state, but some ion-exchange phenomena with negatively charged compounds can also be observed. In CEC, acidic substances are most retained at low pH. For amphoteric and neutral compounds, no preference regarding analyzing pH can be derived from these experiments. For basics, a high pH is chosen, but a reduced solvent strength is needed to enhance the retention of these compounds. The retention mechanism in p-CEC can also be assigned to both hydrophobic and ionic interactions. For acidic, amphoteric, and neutral compounds, acceptable retention is seen. For the basic compounds, the retention with a mobile phase containing 50% organic modifier is low, as in CEC. However, when the organic modifier content in the mobile phase is decreased, retention increases and the selectivity of the stationary phase is more pronounced. This mode of operation presents a possibility for separating some test mixtures, thus some potential for pharmaceutical analysis is seen. More efforts are needed to obtain higher efficiencies and better peak shapes, which might be solved by a further optimization of both the stationary phase synthesis and the mobile phase composition.     

Chiral metal-organic cages used as stationary phase for enantioseparations in capillary electrochromatography

Since some metal-organic cages (MOCs) have been synthesized in past several years, the applications of MOCs such as drug delivery, molecular recognition, separation, catalysis, and gas storage, etc. have been witnessed with a significant increase. However, to the best of our knowledge, so far no one has used MOCs as chiral stationary phase to separate chiral compounds in CEC. In this study, three MOCs were developed as the stationary phase for CEC separation of enantiomers. The MOCs coated capillary column showed good chiral recognition ability for some chiral compounds, including amine, alcohols, ketone, etc. The influence of buffer concentration, applied voltage, pH of buffer solution on the chiral separations was also investigated. The RSDs of run-to-run, day-to-day, and column-to-column for retention time were 2.1-4.67%, 1.2-4.36%, and 3.62-6.43%, respectively. This work reveals that the chiral MOCs material is feasible for the enantioseparation in CEC.