Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein: V. Influence of immobilization method on chiral resolution

Ovoglycoprotein from chicken egg whites (OGCHI) was bound to aminopropyl-silica gels via an amino or carboxyl group(s) of OGCHI. In the former case, OGCHI was bound to N,N′-disuccinimidyl carbonate-activated aminopropyl-silica gels, while in the latter case OGCHI activated by a water-soluble carbodiimide and N-hydroxysulfosuccinimide was bound to aminopropyl-silica gels. The obtained OGCHI materials were compared with regard to the bound amounts, retentivity and enantioselectivity. The OGCHI materials prepared via a carboxyl group(s) of OGCHI are suitable for chiral resolution of acidic solutes, and those via an amino group(s) of OGCHI are suitable for chiral resolution of basic solutes. It is suggested that the electrostatic interaction between an amino or carboxyl group of OGCHI and a charged solute should play an important role in chiral recognition of the solute.     

Separation of basic drug enantiomers by capillary electrophoresis using ovoglycoprotein as a chiral selector: comparison of chiral resolution ability of ovoglycoprotein and completely deglycosylated ovoglycoprotein

Separations of basic drug enantiomers by capillary electrophoresis (CE) using ovoglycoprotein (OGCHI) as a chiral selector are described. The effects of running buffer pH and 2-propanol content on the migration times and resolution of basic drug enantiomers were examined using a linear polyacrylamide-coated capillary. High resolution of basic drug enantiomers was attained using a mixture of 50 mM sodium phosphate buffer (pH 4.5-6.0) and 2-propanol (5-30%) including 50 microM OGCHI. It was found that ionic and hydrophobic interactions could work for the recognition of basic drug enantiomers. Further, we compared the chiral resolution ability of OGCHI with that of completely deglycosylated OGCHI (cd-OGCHI) using them as chiral selectors in CE. OGCHI showed higher resolution for basic drug enantiomers tested than cd-OGCHI. The results suggest that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.     

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. III. Effect of aggregation of ovoglycoprotein on chiral resolution

Ovoglycoprotein from chicken egg whites (OGCHI) subjected to 80 degrees C has produced the associated OGCHI, which is a heat-induced aggregate of OGCHI. The molecular weight of the aggregate was estimated to be ca. 5.7 million daltons by a low-angle laser light-scattering detection. The heat-induced aggregate of OGCHI was found to dissociate reversibly to the OGCHI monomer. The OGCHI aggregate has little chiral recognition ability, or has much lower chiral recognition ability than the native OGCHI. Further, the OGCHI monomer from the reversibly dissociated OGCHI aggregate has chiral recognition ability comparable to that of the native OGCHI.     

pKa shift-associated effects in enantioseparations by cyclodextrin-mediated capillary zone electrophoresis.

Enantioselective migration of dansylated (Dns) amino acids in the presence of hydroxypropylated-beta-cyclodextrin under acidic conditions near the pI value of the analytes was investigated by means of capillary zone electrophoresis. Based on the migration data, the pH dependence of the complexation constants was evaluated, as well as the variation of the complex mobilities with pH. As a result of these data, the migration behavior in the pH region near the pI could be understood, which, in some instances, includes the reversal of migration order upon variation of selector concentration. The enantioselective pKa shifts upon complexation could be quantitated for the carboxylic and the amino group separately. pKa shifts were found in the order of 0.8 pI units, the differences between the enantiomers being up to 0.25 pH units. These data were in agreement with the pI shifts reported from isoelectric focusing experiments. The accurate determination of the pI values of the Dns amino acids makes it possible to calibrate the pI scale in isoelectric focusing in the presence of chiral selectors.     

Enantioseparation of vesamicol and novel vesamicol analogs by high-performance liquid chromatography on different chiral stationary phases

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.     

Enantioselective HPLC analysis of propafenone and of its main metabolites using polysaccharide and protein-based chiral stationary phases

HPLC on chiral stationary phases has been used for the enantioselective assay of propafenone (PPF), 5-hydroxypropafenone (PPF-50H) and N-despropylpropafenone (PPF-NOR) enantiomers. The results obtained on Chiralpak AD column showed that it is useful for the resolution of PPF and of its main metabolites, although the peaks obtained for PPF-NOR were not symmetrical under the conditions investigated. This column and circular dichroism-based detection system were used to determine the absolute configuration of the eluates. Furthermore, the influence of the mobile phase composition on the resolution of PPF and of its main metabolites was investigated on cellulose derivatives (Chiralcel OD-H and Chiralcel OD-R) and protein (Chiral AGP and Ultron ES-OVM)-based chiral stationary phases. The enantiomers of PPF were resolved on all the columns, except for the Ultron ES-OVM. This column, the Chiralpak AD and the Chiralcel OD-H columns were suitable for the resolution of the PPF-50H enantiomers. The PPF-NOR enantiomers were resolved on the Chiralpak AD, Chiral AGP and Chiralcel OD-R columns.     

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

Summary Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantios-electivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantioselectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.     

Dress-up chiral columns for the enantioseparation of amino acids based on fluorous separation

In this paper, we report a new type of chiral high-performance liquid chromatography (HPLC) column—a so-called dress-up chiral column—featuring a chiral stationary phase adsorbed reversibly in a commercial fluorous HPLC column through fluorous interactions. We synthesized perfluroalkylated proline derivatives as chiral stationary phase compounds and then adsorbed them reversibly in the fluorous HPLC column through the pumping of their solutions. By using this dress-up chiral column and fluorophobic elution of an aqueous copper(II) sulfate/MeOH mixture, we could enantioseparate seven racemic amino acids within 40 min. When we washed the dress-up chiral column with fluorophilic tetrahydrofuran or MeOH, the adsorbed chiral stationary phase compounds desorbed from the column, completely destroying its enantioseparation ability. The relative standard deviation of the retention times, the number of theoretical plates, and the resolution for each of four preparations of the dress-up columns were all less than or equal to 9.53 % in 20-times repeated analysis, and were all less than or equal to 18.7 % in four different preparations, respectively.     

Chiral comprehensive two-dimensional gas chromatography with electron-capture detection applied to the analysis of chiral polychlorinated biphenyls in food samples

The feasibility of comprehensive two-dimensional gas chromatography with electron-capture detection (GC x GC-ECD) for the enantioseparation of chiral PCBs from other possible interfering compounds has been evaluated. Three commercially available enantioselective beta-cyclodextrin-based capillary columns (Chirasil-Dex, BGB-172 and BGB-176SE) have been tested as first-dimension columns. Three non-enantioselective stationary phases (HT-8, BPX-50 and Supelcowax-10) were combined with the enantioselective columns to allow the unambiguous determination of the enantiomers of the target chiral PCBs. Each enantioselective first-dimension column tested was able to separate into enantiomers different PCB congeners, but in all cases, the use of Supelcowax-10 as second-dimension column provided the most satisfactory results. The Chirasil-Dex x Supelcowax-10 column combination allowed the determination of the enantiomeric fraction (EF) of PCBs 84, 91, 95, 132, 136, 149, 174 and 176 in the working standard solution, while that of congener 135 was hindered. The BGB-172 x Supelcowax-10 column set allowed a proper EF determination of congeners 45, 84, 131, 132, 135, 171, 174 and 183, while that of PCB 91 was interfered with co-elutants. The column combination BGB-176SE x Supelcowax-10 allowed the determination of all congeners that this enantioselective stationary phase was able to separate into enantiomers, i.e. PCBs 45, 91, 95, 136, 149 and 176. These column combinations have also been evaluated for the simultaneous determination of the 12 congeners with a toxic equivalency factor assigned by the WHO (PCBs 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169, 189) and the seven indicator congeners (PCBs 28, 52, 101, 118, 138, 153 and 180), and evaluated for the analysis of food samples.     

(3,5-二甲基苯基氨基甲酸酯)衍生的纤维素手性固定相

纤维素三（3,5-二甲基苯基氨基甲酸酯）是液相色谱中使用最广泛的手性柱。该文详细地研究了不同程度衍生的纤维素（3,5-二甲基苯基氨基甲酸酯）以及不同硅胶（粗制硅胶、氨丙基粗制硅胶、精制硅胶、氨丙基精制硅胶、大孔硅胶、氨丙基大孔硅胶）作为支撑体对该柱手性分离能力的影响。自制了13根手性色谱柱,分别考察了其对16种外消旋体的拆分,分离结果显示：三取代纤维素柱 > 二取代纤维素柱 > 纤维素柱;精制硅胶和大孔硅胶优于粗制硅胶,大孔硅胶的柱压更低;硅胶的氨丙基化对手性选择性有一定的影响;这些手性柱之间具有一定的互补性,尤其是纤维素柱。该文有助于人们更深刻地理解和更好地把握高效液相色谱手性柱的制备。     

Evaluation of a methacrylate-bonded cyclodextrins as a monolithic chiral stationary phase for capillary electrochromatography (CEC)-UV and CEC coupled to mass spectrometry

Glycidyl methacrylate-bonded β-cyclodextrin (GMA-β-CD) is synthesized as a new chiral monomer by direct chemical bonding with GMA using a fast and simple alternative procedure. Next, rigid and homogenous monolithic columns were prepared by polymerization of GMA-β-CD monomer with ethylene dimethacrylate (EDMA), in the presence of commonly used porogens and a charged achiral monomer to form a versatile chiral monolith. This is the first report in which a preparation procedure for a methacrylate-bonded CD is introduced for chiral separations in CEC. The degree of substitution of GMA-β-CD monomer and mobile-phase parameters were optimized to achieve the highest enantioselectivity and plate number. To evaluate the GMA-β-CD monolithic column, different classes of chiral compounds were screened. Under the optimized β-CD monolith phase and the optimum mobile-phase conditions, 30 neutral and basic chiral compounds and two acidic compounds could be separated. The high chemical and mechanical stability, homogenous microflow and no loss of material at the interface allows for the first time the feasibility of applying this polymer-based monolithic column for CEC coupled to ESI-MS. Compared with CEC-UV, CEC-ESI-MS showed higher sensitivity and lower resolution. However, resolution greater than 1.0 can still be obtained for majority of the select tested compound in CEC-ESI-MS with at least three out of seven compound providing Rs≥1.5. The results reinforce the potential of GMA-β-CD monolithic columns for chiral separations with high sensitivity in CEC-ESI-MS. Finally, using hexobarbital as the model chiral analyte, the monolithic column demonstrated excellent stability and reproducibility of retention time and enantioselectivity.     

Capillary electrophoresis and column chromatography in biomedical chiral amino acid analysis

Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of d-amino acids typically found in biological fluids and tissues. Chiral GC-MS analysis of heptafluorobutanol/pentafluoropropionanhydride amino acid derivatives on an Rt-gDEXsa column     

Ionic liquids as stationary phase solvents for methylated cyclodextrins in gas chromatography

Summary Room temperature ionic liquids (RTIL) are molten salts with melting points well below room temperature. 1-butyl-3-methylimidazolium chloride is a typical example of such RTIL. It was used as a solvent to dissolve permethylated-β-cyclodextrin (BPM) and dimethylated-?cyclodextrin (BDM) to prepare stationary phases for capillary columns in gas chromatography for chiral separation. The RTIL containing columns were compared to commercial columns containing the same chiral selectors. A set of 64 chiral compounds separated by the commercial BPM column was tested on the RTIL BPM column. Only 21 were enantioresolved. Similarly, a set of 80 compounds separated by the commercial BDM column was passed on the RTIL BDM column with only 16 positive separations. It is proposed that the imidazolium ion pair could make an inclusion complex with the cyclodextrin cavity, blocking it for chiral recognition. All the chiral compounds recognized by the RTIL columns had their asymmetric carbon that was part of a ring structure. The retention factors of the derivatized solutes were lower on the RTIL columns than those obtained on the commercial equivalent column. The peak efficiencies obtained with the RTIL capillary were significantly higher than that obtained with the commercial column. These observations may contribute to the knowledge of the mechanism of cyclodextrin-based GC enantioselective separations.     

Advanced dress-up chiral columns: New removable chiral stationary phases for enantioseparation of chiral carboxylic acids

This paper describes the preparation of new dress-up columns featuring reproducibly removable and replaceable chiral stationary phases. After synthesizing perfluroalkylated quinine and quinidine derivatives as chiral stationary phase compounds (F-CSPs), we adsorbed them reversibly onto a fluorous LC column through pumping of their solutions. Using this dress-up chiral column and fluorophobic elution of aqueous ammonium formate/MeOH mixtures, we could enantioseparate four racemic N-acetyl amino acids, dichlorprop, and sixteen fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized amino acids. Dressing and undressing of the coated F-CSPs could be controlled by varying the fluorophilicity and fluorophobicity of the eluent. The relative standard deviations of the retention times, the retention factors, the number of theoretical plates, the enantioseparation factors, and the resolutions of each of four preparations of such dress-up columns were all less than or equal to 5.26% (from 20 repeated analyses); the reproducibilities from four different preparations were all less than or equal to 10.6%. These columns also facilitated highly sensitive and selective analyses of AQC-amino acids when detected using LC–MS/MS.     

Capillary isoelectric focusing with UV-induced fluorescence detection

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.     

Enantioselective analysis of an asymmetric reaction using a chiral fluorosensor

An axially chiral 1,8-dipyridylnaphthalene N,N'-dioxide has been employed in enantioselective fluorescence analysis of the enzymatic kinetic resolution of trans-1,2-diaminocyclohexane. The procedure eliminates cumbersome purification and derivatization steps required by traditional methods. The results demonstrate the potential of fluorescence spectroscopy using suitable chiral chemosensors for real-time analysis of the enantiomeric composition of chiral compounds and for high-throughput screening of asymmetric reactions. [structure: see text]     

Monolithic silica columns functionalized with substituted polyproline‐derived chiral selectors as chiral stationary phases for high‐performance liquid chromatography

In this study, two polyproline‐derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead‐based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.     

In-line coupling of a reversed-phase column to cope with limited chemoselectivity of a quinine carbamate-based anion-exchange type chiral stationary phase

A chiral stationary phase based on tert-butylcarbamoyl quinine has shown remarkable enantiomer separation capability for the thyroid hormone thyroxine (T(4)) and its structural analogue triiodothyronine (T(3)) employing hydroorganic buffered mobile phases (typical RP conditions). To overcome the problem of a somehow limited chemoselectivity for the critical peak pair between adjacent L-thyroxine (L-T(4)) and D-thyroxine (D-T(4)) peaks on the chiral anion-exchanger CSP when all four compounds need to be analysed simultaneously like in impurity profiling of L-T(4 )drug products, an RP column (Gemini C18) was serially coupled with the chiral anion-exchanger column to add a hydrophobic selectivity increment and to improve thereby the critical resolution between L-T(3) and D-T(4). Various parameters such as organic modifier content, pH, buffer concentration and type, type of achiral column as well as sequence of achiral and chiral column have been investigated with individual and tandem columns. With the optimized conditions and use of the tandem column a significantly improved separation, as compared to the chiral anion-exchanger column alone, with a critical resolution as high as 3.7 and an almost equal band spacing of the four components of the test mixture could be obtained. The sequence of the columns (achiral-chiral or chiral-achiral) had no significant effect on the separation performance.     

Simulated moving columns technique for chiral liquid chromatography

Enantioselectivity of chiral selectors is often relatively low in chiral HPLC. For difficult chiral separations, often only partial resolution is obtained rather quickly by column and mobile phase screening, and, by trial-and-error, additional method optimization is required to achieve complete resolution. This paper describes the development of a novel column-switching technique called "simulated moving columns" (SMC) to quickly achieve complete chiral resolution on columns with limited enantioselectivity. The simulated moving columns (SMC) technique uses two (2) or three (3) short chiral HPLC columns connected in series, and forces the unresolved enantiomers to recycle exclusively through the columns until sufficient resolution is attained. In effect, SMC helps to achieve chiral resolution by virtually multiplying the column length, thus enhancing separation efficiency and resolution, without increasing backpressure. Comparison of the standard non-SMC approach with SMC, and selected applications of chiral separations of pharmaceutical drug molecules are presented. Through measurement and calculation, evaluation of off-column band broadening resulting from a two-column SMC system is provided. The results clearly indicate that SMC eliminates the significant band broadening that is inevitable in the closed-loop recycling techniques currently used in preparative chromatography. Furthermore, SMC is not only useful to enhance resolution for analytical and preparative chiral separation, but also has great potential to enhance recovery and purity for difficult chiral preparative chromatography.     

Enantioseparation of derivatized amino acids by capillary isoelectric focusing using cyclodextrin complexation

Enantioseparation of dansylated as well as 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC)-derivatized amino acids by means of capillary isoelectric focusing using various cyclodextrin derivatives is demonstrated. Separation is based on the enantioselective shift of the isoelectric points upon complexation with the chiral selectors. The zwitterionic, diastereomeric analyte-cyclodextrin complexes exhibited differences in the pI values up to more than 0.25 pI units. Enantioresolution was achieved for a number of derivatized amino acids and various selectors added to the carrier ampholyte solution. The hydroxypropyl-beta-cyclodextrin proved to be the best selector for this purpose. Enantioseparation as dependent on the selector concentration was evaluated in a range between 5 and 30 mM. Separation could be attained down to selector concentrations corresponding to a degree of complexation as low as 30%. The peaks appear according to the degree of complexation between the positions adopted without and with full complexation. The kinetics of complex formation and dissociation was fast enough in most instances to produce single peaks, even with complexation degrees near 0.5 and significant pI shifts. Peak widths were slightly enlarged in these instances. The method offers excellent perspectives for preparative applications.