Synthesis and application of mono-6-ammonium-6-deoxy-beta-cyclodextrin chloride as chiral selector for capillary electrophoresis

A facile synthetic approach for mono-6-amino-6-deoxy-beta-cyclodextrin (beta-CD-NH2) was proposed. Its hydroxy chloride salt, mono-6-ammonium-6-deoxy-beta-cyclodextrin chloride (beta-CD-NH3Cl) was further prepared and used for the enantioseparation of various anionic and ampholytic analytes by capillary electrophoresis (CE). The effect of background electrolyte (BGE) pH and selector concentration on the enantioseparation was studied. Results showed that beta-CD-NH3Cl displayed powerful chiral resolution ability towards anionic analytes. In addition, baseline separation of a standard mixture consisting of eight acids was achieved within 35 min.     

Enantiomer separation of N-protected amino acids by non-aqueous capillary electrophoresis and high-performance liquid chromatography with tert.-butyl carbamoylated quinine in either the background electrolyte or the stationary phase

A non-aqueous CE method was developed for evaluating the chiral discrimination potential of cinchona alkaloids and different kinds of carbamoylated derivatives of quinine and quinidine type chiral selectors towards acidic analytes, in particular a series of various Bz (benzoyl), DNB (3,5-dinitrobenzoyl) and DNZ (3,5-dinitrobenzyloxycarbonyl) amino acid derivatives. In this study, the enantioselectivity values obtained in non-aqueous CE with tert.-butyl carbamoylated quinine as chiral additive have been compared with the values found for the same series of selectands in HPLC using the same selector immobilized onto silica as chiral stationary phase. Similarly to the background electrolyte used in CE an ethanol-methanol mixture (60:40, v/v) containing 100 mM octanoic acid and 12.5 mM ammonia has been selected as HPLC mobile phase. Under these conditions, a good correlation (r = 0.954) between the enantioselectivities observed with the two techniques has been obtained. Thus the non-aqueous CE method can be applied as a screening tool for the rapid evaluation of the chiral discrimination potential of a large set of newly developed chiral selectors derived from quinine and related alkaloids.     

A concise synthesis of protected diethyl 1-amino-2-hydroxyalkylphosphonates

An efficient diastereoselective synthesis of 5-substituted (2-thioxo-oxazolidin-4-yl)phosphonic acid diethyl esters from metallated diethyl isothiocyanomethylphosphonate and aldehydes has been developed. The three-step transformation of oxazolidine-2-thione derivatives into N-Boc 1-amino-2-hydroxyalkylphosphonic acid diethyl esters is also described.     

Azithromycin as a new chiral selector in capillary electrophoresis

In capillary electrophoresis (CE), separation of enantiomers of a chiral compound can be achieved through the chiral interactions and/or complex formation between the chiral selector and the enantiomeric analytes on leaving their diastereomeric forms with different stability constants and hence different mobilities. A great number of chiral selectors have been employed in CE and among them macrocyclic antibiotics exhibited excellent enantioselective properties towards a wide number of racemic compounds. The use of azithromycin (AZM) as a chiral selector has not been reported previously. This work reports the use of AZM as a chiral selector for the enantiomeric separations of five chiral drugs and one amino acid (tryptophan) in CE. The enantioseparation is carried out using polar organic mixtures of acetonitrile (ACN), methanol (MeOH), acetic acid and triethylamine as run buffer. The influences of the chiral selector concentration, ACN/MeOH ratio, applied voltage and capillary temperature on enantioseparation are investigated. The results show that AZM is a viable chiral selector in CE for the enantioseparation of the type of chiral drugs investigated.     

Ketoprofen enantioseparation with a Cinchona alkaloid based stationary phase: Enantiorecognition mechanism and release studies

With the present contribution, we demonstrate that the baseline separation of ketoprofen enantiomers can be successfully achieved (α = 1.09; R_S = 1.60) in the reversed‐phase mode of elution with a commercially available anion‐exchange‐based chiral stationary phase, incorporating the quinine 2,6‐diisopropylphenyl carbamate derivative as the enantioresolving unit. Focused modification of the eluent composition indicated a stereoselective role of hydrophobic and π–π interactions between the selector and selectand units, besides the prime ionic intermolecular interaction. The mechanistic hypotheses based on the chromatographic data were confirmed by in silico molecular dynamic simulations, which allowed us to establish the network of selector–selectand interactions underlying the stereorecognition process at a molecular level. The validated method was successfully used to evaluate the drug content and release profile of ketoprofen‐loaded polymeric film, showing drug homogeneous distribution into the film and no preferential interactions between the polymer and one of the enantiomers, with the racemate released at each time point.     

Separation of aminoalkanephosphonic acid enantiomers by indirect UV detection capillary electrophoresis with application of cyclodextrins

Indirect UV detection capillary electrophoresis (CE) was used for the separation of aminoalkanephosphonic acid (AP) enantiomers by applying commercially available cyclodextrins as chiral discriminators. The results show that the separation of the enantiomers depends on pH of the background electrolyte, the molar ratio of cyclodextrin to aminophosphonic acid, and on the type of the applied chiral selector. Optimization of process conditions allowed enantiomeric baseline separation or partial separation of 12 out of 14 alpha-aminophosphonic acids studied. This type of CE might therefore be successfully used for routine determination of enantiomeric purity of aminophosphonic acids.     

Determination of pKa values of 2-amino-2-oxazolines by capillary electrophoresis

The dissociation constants of new 2-amino-2-oxazolines were determined by capillary electrophoresis (CE) as a new technique. A method based on a linear model has been used in the CE determination. A series of eight 2-amino-2-oxazolines are investigated to determine their ionization constant. Among them, three new oxazolines synthesized are presented. The Ka values were obtained from the plots of reciprocal effective mobility against inverse concentrations of protons. The potentiometric method (PM) was performed as a comparative method. No significant differences were observed between the determined dissociation constants using both methods. Thus, the pKa values have been found to vary between 8.55 and 8.68.     

Monolithic silica-based capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography

A silica-based monolithic stationary phase prepared by the sol-gel process in a 100 microm I.D. fused-silica (FS) capillary has been modified chemically with 3-mercaptopropyl trimethoxysilane followed by immobilization of a strong cation-exchange (SCX) type chiral selector, (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutane phosphonic acid, by radical addition reaction onto the reactive sulfhydryl surface. After a fine-tuning of the mobile phase composition, the enantioselective capillary column was evaluated for the separation of various chiral basic drugs by enantioselective non-aqueous capillary electrochromatography (CEC), in comparison to capillary column analogs packed with 3.5 microm silica particles having attached the same selector. The performance of the monolithic silica column was further compared to corresponding polymethacrylate-based organic polymer monoliths. The study indicated that strong counter-ions such as 2-aminobutanol or N,N,N',N'-tetramethylethylenediamine are needed, although they reduce the electroosmotic flow velocity and separation factors in comparison to less efficient counter-ions, in order to allow the elution of the oppositely charged solutes in the ion-exchange retention mode within reasonable run time and as sharp zones. In contrast, weak counter-ions such as N,N-diisopropylethylamine (Huenig base) provided stronger electroosmotic flow and much better separation factors, but relatively poor peak efficiencies. Overall, with the chemically functionalized monolithic silica column the high quality separations of packed column analogs could be approximated, with regards to both separation factors and peak performances. On the other hand, the monolithic capillary column certainly outperformed the packed column in terms of system robustness under capillary electrochromatography conditions and showed excellent column longevity. The enantioselective strong cation-exchange-type monolithic silica column performed also well in comparison to the organic polymer monolith.     

Non-aqueous capillary electrophoretic enantioseparation of N-derivatized amino acids using cinchona alkaloids and derivatives as chiral counter-ions

A non-aqueous capillary electrophoretic method developed with quinine and tert.-butyl carbamoylated quinine as chiral selectors for the enantioseparation of N-protected amino acids was applied to the investigation of other quinine derivatives as chiral additives. The optimum composition of the background electrolyte was found to be 12.5 mM ammonia, 100 mM octanoic acid and 10 mM chiral selector in an ethanol-methanol (60:40, v/v) mixture. Under these conditions, a series of chiral acids, as various benzoyl, 3,5-dinitrobenzoyl and 3,5-dinitrobenzyloxycarbonyl amino acid derivatives were investigated with regards to selectand-selector relationships and enantioselectivity employing quinine, quinidine, cinchonine, cinchonidine, tert.-butyl carbamoylated quinine, tert.-butyl carbamoylated quinidine, dinitrophenyl carbamoylated quinine and cyclohexyl carbamoylated quinine as chiral selector.     

Development of stereoselective nonaqueous capillary electrophoresis system for the resolution of cationic and amphoteric analytes

A stereoselective ion-pair nonaqueous capillary electrophoresis (NACE) method employing the partial filling technique with N-derivatized amino acids, e.g., (R)- and (S)-3,5-dinitrobenzoyl-leucine (DNB-Leu), as chiral selector for the separation of "pseudoenantiomeric" cinchona alkaloid derivatives and other structurally related basic compounds like the enantiomers of mefloquine is presented. Originating from NACE with cinchona alkaloid derivatives as chiral counterions, this method was developed by application of the reciprocity principle of chiral recognition, which was proven to be valid for stereoselective ion-pair capillary electrophoresis (CE). A variety of basic and amphoteric selectands (SAs) could be well resolved. Thereby, the separation was primarily based on stereoselective ion-pair formation of corresponding SA stereoisomers and mobility differences of free and complexed (ion-paired) SAs. Additionally, in the case of diastereomeric SAs, naturally existing mobility differences between the diastereomers played also a role, but was shown by control experiments with racemic DNB-Leu and without selector (SO) to be of minor contribution to overall separation selectivity. Due to its simplicity, speed, and good reproducibility, the established method can be utilized for fast screening of cationic as well as amphoteric chiral compounds, and therefore is a valuable tool in the development of new chiral selectors and chiral stationary phases. Small sample amounts of the SO (4-5 mg) and only analytical amounts of SAs are needed, and about 20-50 compounds per day can be tested.     

Estimation and comparison of zeta-potentials of silica-based anion-exchange type porous particles for capillary electrochromatography from electrophoretic and electroosmotic mobility

Mobilities of different chromatographic particles obtained from two electrokinetic methods were determined and compared. The particles were all based on porous silica, between 3 and 15 microm diameter, and were either native, or derivatized. As intermediate of chemical modification 3-mercaptopropyl-modified silica particles (TP-silica) are obtained. These particles were finally transformed into weakly basic anion exchangers with O-9-(tert-butylcarbamoyl)quinine (tBuCQN) as chiral selector. The electrophoretic mobility of the particles was determined from their migration velocity in an electric field using microelectrophoresis. Electrokinetic chromatography with a capillary column packed with the same particles was used to measure the electroosmotic flow generated. All measurements were carried out in background electrolytes of equal ionic strength (10(-2) mol/L), at pH varying between 3.5 and 9.5. From these data a rough estimation of the zeta-potential was made, taking Helmholtz-Smoluchowski conditions into consideration. With both methods the zeta-potential of the native silica particles is negative throughout, and its value increases with pH. The weakly basic tBuCQN particles have positive zeta-potentials at pH lower than about 7.5, but exhibit a negative zeta-potential above this pH, indicating the dominating effect of residual silanol groups at the silica surface. The zeta-potential for these anion-exchange particles ranged between +30 and -40 mV. The zeta-potentials derived with electrophoresis and electroosmosis agree, showing the adequacy of the approach, although many limitations must be taken into account in the treatment of the electrokinetic phenomena in such porous systems. These restrictions in interpreting mobility and zeta-potential were discussed.     

Enantiopurity analysis of new types of acyclic nucleoside phosphonates by capillary electrophoresis with cyclodextrins as chiral selectors

CE methods have been developed for the chiral analysis of new types of six acyclic nucleoside phosphonates, nucleotide analogs bearing [(3‐hydroxypropan‐2‐yl)‐1H‐1,2,3‐triazol‐4‐yl]phosphonic acid, 2‐[(diisopropoxyphosphonyl)methoxy]propanoic acid, or 2?(phosphonomethoxy)propanoic acid moieties attached to adenine, guanine, 2,6‐diaminopurine, uracil, and 5‐bromouracil nucleobases, using neutral and cationic cyclodextrins as chiral selectors. With the exception of the 5‐bromouracil‐derived acyclic nucleoside phosphonate with a 2‐(phosphonomethoxy)propanoic acid side chain, the R and S enantiomers of the other five acyclic nucleoside phosphonates were successfully separated with sufficient resolutions, 1.51–2.94, within a reasonable time, 13–28 min, by CE in alkaline BGEs (50 mM sodium tetraborate adjusted with NaOH to pH 9.60, 9.85, and 10.30, respectively) containing 20 mg/mL β‐cyclodextrin as the chiral selector. A baseline separation of the R and S enantiomers of the 5‐bromouracil‐derived acyclic nucleoside phosphonate with 2‐(phosphonomethoxy)propanoic acid side chain was achieved within a short time of 7 min by CE in an acidic BGE (20:40 mM Tris/phosphate, pH 2.20) using 60 mg/mL quaternary ammonium β‐cyclodextrin chiral selector. The developed methods were applied for the assessment of the enantiomeric purity of the above acyclic nucleoside phosphonates. The preparations of all these compounds were found to be synthesized in pure enantiomeric forms. Using UV absorption detection at 206 nm, their concentration detection limits were in the low micromolar range.     

Chiral counter-current chromatography of gemifloxacin guided by capillary electrophoresis using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector

(+)-(18-crown-6)-tetracarboxylic acid (18C6H4) has been known as a highly efficient chiral selector for resolving primary amine enantiomers in capillary electrophoresis (CE). We investigated the chiral separation of gemifloxacin using 18C6H4 in analytical counter-current chromatography (CCC). The separation conditions for CE, including the binding constant, pH, and run buffer constituents, provided a helpful guideline for chiral CCC. A successful separation of gemifloxacin enantiomers could be achieved using a two-phase solvent system composed of 1-butanol-ethyl-acetate-bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane acetate buffer with a small amount of 18C6H4. The hydrophobicity of the solvent system and the 18C6H4 concentration were varied to optimize the chiral separation.     

Capillary electrophoretic chiral separation of Cinchona alkaloids using a cyclodextrin selector

A new capillary electrophoretic method for the chiral separation of four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) was developed using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin as the chiral selector. The inner walls of the separation capillary were modified with a thin polyacrylamide layer, which substantially reduced the electroosmotic flow and improved the chiral resolution and the reproducibility of the migration time of the analytes. Various operation parameters were optimised, including the pH, the capillary temperature, the concentration of the background electrolyte, and the concentration of the chiral selector. Baseline separation of the two diastereomer pairs was achieved in 12 minutes in ammonium acetate background electrolyte pH 5.0 with addition of cyclodextrin in a concentration of 3 mM or higher.     

Electrolyte and additive effects on enantiomer separation of peptides by nonaqueous ion-pair capillary electrophoresis using tert.-butylcarbamoylquinine as chiral counterion

Nonaqueous ion-pair capillary electrophoresis separations of N-protected (all-R)/(all-S) alanine peptide enantiomers with up to six amino acid residues using tert.-butylcarbamoylquinine as selector and employing the partial filling technique are presented. The effects of various conditional parameters on separation were studied, namely chemical nature of the capillary wall, solvent composition of the background electrolyte (BGE), acid-base-ratio (equivalent to apparent pH), ionic strength and selector concentration. The influence of the solvent composition (methanol-ethanol ratios) on resolution turned out to be rather complex. The separation of the peptide enantiomers was strongly altered by small changes in pH and ionic strength. An increase of the selector concentration was found to offer an easy way for enhancing enantioselectivity, although some drawbacks, e.g., elongation of run times, have to be considered. A method was developed that allowed the separation of N-3,5-dinitrobenzoyl oligoalanine enantiomers containing 1-6 amino acid residues in one run. Like in a recent high-performance liquid chromatography (HPLC) study, separation selectivity thereby decreased from 1.541 (Ala), 1.340 (Ala(2)), 1.054 (Ala(3)), 1.029 (Ala(4)), 1.024 (Ala(5)) to 1.020 (Ala(6)). In addition, all four stereoisomers of N-2,4-dinitrophenyl- and N-3,5-dinitrobenzyloxycarbonyl-protected alanylalanine could be baseline-resolved.     

On the use of dispersed nanoparticles modified with single layer beta-cyclodextrin as chiral selecor to enhance enantioseparation of clenbuterol with capillary electrophoresis

A new strategy for chiral separation by capillary electrophoresis employing modified-nanoparticles as chiral selector is described for clenbuterol analysis. Nanoparticles modified with β-cyclodextrin (β-CD) form a large surface area platform to serve as a pseudostationary chiral phase, which can be applied for the enhancement of the enantioseparation. The application of four kinds of nanoparticles was investigated (multi-walled nanotubes (MWNTs), polystyrene (PS), TiO₂ and Al₂O₃) modified with single layer β-CD as chiral selector in the enantioseparation of clenbuterol by capillary electrophoresis (CE). Successful clenbuterol enantioseparation could be achieved with the β-CD-modified MWNTs as chiral selector. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) confirmed the β-CD modification of the nanoparticles. The effects of nanoparticles, surfactant, chiral selector (β-CD) and run buffer were studied in relation to the enantiomeric separation of clenbuterol. This study opens attractive perspectives for the use of modified nanoparticles for chiral separational purposes in CE.     

Stereoselective separations of chiral phosphinic acid pseudodipeptides by CEC using silica monoliths modified with an anion-exchange-type chiral selector

A nonaqueous CEC method for the simultaneous separation of the four stereoisomers of the N-benzyloxycarbonyl phosphinic pseudodipeptide methyl ester benzyloxycarbonyl-homophenylalanine Z-hPhepsi(PO2HCH2)Phe-OCH3 as well as of the corresponding N-2,4-dinitrophenyl (DNP)-derivative with free C-terminal carboxylic group DNP-hPhepsi(PO2HCH2)Phe-OH was developed. For this purpose, a monolithic silica capillary column modified with a cinchona alkaloid-derived anion-exchange-type chiral selector, namely O-9-(tert-butylcarbamoyl)quinidine (tBuCQD) was prepared. The mobile phase composition (ACN/methanol ratio, counterion type) was thoroughly optimized to end up with baseline resolution of all four stereoisomers with critical resolution of as high as about 2. The CEC method proved to be superior over the corresponding HPLC separations primarily due to significantly enhanced plate numbers (between 200,000 and 600,000 m(-1) in CEC). Diastereoselectivity contributions arising from electrophoretic mobility differences of the diastereomers facilitated the separation of the later eluted diastereomeric peak pair (peaks III and IV), but had a negative influence on the selectivity of the earlier eluted diastereomeric peak pair (peaks I and II). The stereoselective CEC assay allowed the assessment of the stereoisomeric purity of the individual isomers which were obtained by preparative HPLC on a CHIRALPAK QD-AX column that is based on the same tBuCQD selector. The present study demonstrates that there exist problems which are hard to solve by HPLC, yet can be conveniently solved by CEC. Moreover, it was intended to prove by this practical application that CEC with monolithic columns is robust enough to be used for solving real-life problems.     

Principle and applications of the partial filling technique in capillary electrophoresis

Summary The partial filling technique (PFT) in capillary electrophoresis (CE) is an efficient system where, only 50–800 nanolitres of a chiral selector solution needs to be added to each run. PFT is especially applicable when these additives to the background electrolyte (BGE) are expensive or absorb UV light. The selector dissolved in the BGE is applied to the capillary as a plug, shorter than the effective length of the capillary, prior to application of the analyte. During the run both ends of the capillary are connected to the BGE. The applied plug and the analyte may move in opposite directions or in the same direction at different velocities depending on their electrophoretic mobilities. Thus the final plug length is either longer or shorter than the original length. The technique has been successfully applied in a number of studies including enantiomeric separation with a variety of selectors, and for the determination of conditional association constants. Taken from Dr. Pharm. Sc. Thesis, A. Amini, Uppsala, 1998.     

Separation of racemic 2,4-dinitrophenyl amino acids on zirconia-immobilized quinine carbamate in reversed-phase liquid chromatography

Zirconia is known to be one of the best chromatographic support materials due to its excellent chemical, thermal, and mechanical stability. A quinine carbamate-coated zirconia was prepared as a chiral stationary phase for separation of enantiomers of DNP-amino acids in reversed-phase liquid chromatography. Retention and enantioselectivity of this phase were compared to those for quinine carbamate bonded onto silica. Most amino acids studied were separated on the quinine carbamate-zirconia CSP although retention was longer and chiral selectivity was somewhat lower than on the corresponding silica CSP. Increased retention and decreased selectivity are probably due to strong non-enantioselective Lewis acid-base interactions between the amino acid molecule and the residual Lewis acid sites on the zirconia surface.     

Chiral ligand exchange capillary electrophoresis using borate anion as a central ion

Native DL-pantothenic acid, having a 1,3-diol structure, was chirally resolved by ligand exchange capillary electrophoresis using (S)-3-amino-1,2-propanediol as a chiral selector and the borate anion as a central ion. The optimum conditions for both high resolution and short migration time of DL-pantothenic acid were found to be 200 mM (S)-3-amino-1,2-propanediol and 200 mM borate buffer (pH 9.2) containing 15% methanol with an applied voltage of +25 kV at 20 degrees C, using direct detection at 200 nm. With this system, the resolution (Rs) of racemic pantothenic acid was approximately 1.7. When (S)-1,2-propanediol, (S)-1,2,3-propanetriol, (S)-1,3-butanediol or (S)-1-amino-2-propanol were used as chiral ligand instead of (S)-3-amino-1,2-propanediol, DL-pantothenic acid was not enantioseparated. When borate was replaced with Tris or butylborate, no chiral separation was achieved. Therefore, the ionic interaction between the amino and carboxyl groups of the ternary complex may play an important role in the enantioseparation of DL-pantothenic acid by the proposed CE system.