Preparation of a New Chiral Stationary Phase Based on Macrocyclic Amide Chiral Selector for the Liquid Chromatographic Chiral Separations

A new chiral stationary phase (CSP) based on macrocyclic amide receptor was prepared starting from (1R,2R)‐1,2‐diphenylethylenediamine. The new CSP was successfully applied to the resolution of various N‐(substituted benzoyl)‐α‐amino amides with reasonably good separation factors and resolutions (α = 1.75 ~ 2.97 and R_S = 2.89 ~ 6.82 for 16 analytes). The new CSP was also applied to the resolution of 3‐substituted 1,4‐benzodiazepin‐2‐ones and some diuretic chiral drugs including bendroflumethiazide and methylchlothiazide and metolazone. The resolution results for 3‐substituted 1,4‐benzodiazepin‐2‐ones and some diuretic chiral drugs were also reasonably good. Chirality 28:253–258, 2016. © 2016 Wiley Periodicals, Inc.     

A surface molecularly imprinted polymer as chiral stationary phase for chiral separation of 1,1′‐binaphthalene‐2‐naphthol racemates

Acrylamide (AM) was copolymerized with ethylene glycol dimethacrylate (EGDMA) in the presence of (R )‐1,1′‐binaphthalene‐2‐naphthol (BINOL) as the template molecules on the surface of silica gel by a free radical polymerization to produce a chiral stationary phase based on the surface molecularly imprinted polymer (SMIP‐CSP). The SMIP‐CSP showed a much better separation factor (α = 4.28) than the CSP based on the molecularly imprinted polymer (MIP‐CSP) without coating on the silica gel (α = 1.96) during the chiral separation of BINOL enantiomers by high‐performance liquid chromatography. The influence of the pretreatment temperature and the content of the template molecule ((R )‐BINOL) of the SMIP‐CSP, and the mobile phase composition on the separation of the racemic BINOL were systematically investigated.     

Cellulose type chiral stationary phase based on reduced graphene oxide@silica gel for the enantiomer separation of chiral compounds

The graphene oxide (GO) was covalently coupled to the surfaces of silica gel (SiO₂) microspheres by amide bond to get the graphene oxide@silica gel (GO@SiO₂). Then, the GO@SiO₂ was reduced with hydrazine to the reduced graphene oxide@silica gel (rGO@SiO₂), and the cellulose derivatives were physically coated on the surfaces of rGO@SiO₂ to prepare a chiral stationary phase (CSP) for high performance liquid chromatography. Under the optimum experimental conditions, eight benzene‐enriched enantiomers were separated completely, and the resolution of trans‐stilbene oxide perfectly reached 4.83. Compared with the blank column of non‐bonded rGO, the separation performance is better on the new CSP, which is due to the existence of rGO to produce special retention interaction with analytes, such as π‐π stacking, hydrophobic effect, π‐π electron‐donor–acceptor interaction, and hydrogen bonding. Therefore, the obtained CSP shows special selectivity for benzene‐enriched enantiomers, improves separation selectivity and efficiency, and rGO plays a synergistic effect with cellulose derivatives on enantioseparation.     

Evaluation of the Edman degradation product of vancomycin bonded to core‐shell particles as a new HPLC chiral stationary phase

A modified macrocyclic glycopeptide‐based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide‐based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide‐based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core‐shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography‐mass spectrometry with EDP was performed in approximately 3 minutes. Other highlights include simultaneous liquid chromatography separations of rac‐amphetamine and rac‐methamphetamine with VancoShell, rac‐pseudoephedrine and rac‐ephedrine with NicoShell, and rac‐dichlorprop and rac‐haloxyfop with TeicoShell.     

Study on the HPLC‐based separation of some ezetimibe stereoisomers and the underlying stereorecognition process

The enantioseparation of ezetimibe stereoisomers by high‐performance liquid chromatography on different chiral stationary phases, ie, 3 polysaccharide‐based chiral columns, was studied. It was observed that cellulose‐based Chiralpak IC column exhibited the best resolving ability. After the optimization of mobile phase compositions in both normal and reversed phase modes, satisfactory separation could be obtained on Chiralpak IC column, especially in normal phase mode. The use of prohibited solvents as nonstandard mobile phase gave rise to better resolution than that of standard mobile phases (n‐hexane/alcohol system). In addition, the presence of ethanol in nonstandard mobile phase has played an important role in enhancing chromatographic efficiency and resolution between ezetimibe stereoisomers. Various attempts were made to comprehensively compare the chiral recognition capabilities of immobilized versus coated polysaccharide‐based chiral columns, amylose‐based versus cellulose‐based chiral stationary phases, reversed versus normal phase modes, and standard versus nonstandard mobile phases. Moreover, possible solute‐mobile phase‐stationary phase interactions were derived to explain how stationary and mobile phases affected the separation. Then the method validation with respect to selectivity, linearity, precision, accuracy, and robustness was carried out, which was demonstrated to be suitable and accurate for the quantitative determination of (RRS)‐ezetimibe impurity in ezetimibe bulk drug.     

Preparation and chromatographic performance of a multifunctional immobilized chiral stationary phase based on dialdehyde microcrystalline cellulose derivatives

A novel high‐performance liquid chromatography (HPLC) multifunctional immobilized chiral stationary phase was prepared by bonding dialdehyde microcrystalline cellulose to aminosilica via Schiff base reaction and then derivatized with 3,5‐dimethylphenyl isocyanate. The HPLC multifunctional immobilized chiral stationary phase could not only achieve chiral separation but also achieve achiral separation. Chiral separation evaluation showed that 1‐(1‐naphthyl)ethanol and mandelonitrile got separation in normal phase (NP) mode. Ranolazine, benzoin ethyl ether, metalaxyl, and diclofop were successfully separated in reversed phase (RP) mode. Aromatic compounds such as polycyclic aromatic hydrocarbons (PAHs), anilines, and aromatic acids were selected as analytes to investigate the achiral separation performance of the multifunctional immobilized chiral stationary phase in NP and RP modes. The achiral separation evaluation showed that six PAHs could get good separation within 10 minutes in NP mode. Four aromatic acids were well separated in RP mode. The retention mechanism of aromatic compounds on the stationary phase was discussed, founding that π‐π interaction, π‐π electron‐donor‐acceptor (EDA) interaction, and hydrogen bonding interaction played important roles during the achiral separation process. This multifunctional immobilized chiral stationary phase had the advantages of simple bonding steps, short reaction time, and no need for space arm.     

Separation of (R)- and (S)-tert-butyl 2-tert-butyl-4-methoxy-2,5-dihydro-1,3-imidazole-1-carboxylate (building block for amino acid synthesis) by preparative high performance liquid chromatography on a polysaccharide stationary phase

The preparative separation of the enantiomers of the title compound, a versatile chiral building block for the synthesis of unnatural amino acid esters, by high performance liquid chromatography on a chiral stationary phase (CSP), is reported for the first time. The CSP consists of amylose-(3,5-dimethylphenyl-carbamate), which has been coated onto the surface of macroporous aminopropyl-functionalized silica gel. The effect of mobile phase composition and the amount of amylose derivative on the silica gel has been thoroughly investigated. Using 2-propanol as organic modifier in hexane as mobile phase, on a semi-preparative column (200 mm × 40 mm ID, containing 192 g of stationary phase) about 200 mg of the racemate was separated per injection. Running the equipment under automatic conditions with repetitive injection mode allowed for the separation of 30 g per day. Both enantiomers were obtained with enantiopurities >99.75:0.25. Chirality 10:217–222, 1998. © 1998 Wiley-Liss, Inc.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Preparation and evaluation of biselector bonded-type multifunctional chiral stationary phase based on dialdehyde cellulose and 6-monodeoxy-6-monoamino-β-cyclodextrine derivatives

A novel biselector bonded-type multifunctional chiral stationary phase (MCSP) was prepared by covalently crosslinking dialdehyde cellulose (DAC) with 6-monodeoxy-6-monoamino-β-cyclodextrine (CD) via Schiff base reaction. The biselector bonded-type MCSP had good chiral and achiral chromatographic performance in normal phase (NP) and reversed phase (RP) modes. Seven and eight enantiomers were successfully separated on the prepared biselector bonded-type MCSP in NP and RP modes, respectively. The biselector bonded-type MCSP showed enhanced chiral resolution ability compared with single selector chiral stationary phases due to the simultaneous introduction of DAC and 6-monodeoxy-6-monoamino-β-CD on the surface of silica gel. Aromatic compounds including polycyclic aromatic hydrocarbons, anilines, phenols, phenylates, and aromatic acids were choosed as analytes to investigate the achiral chromatographic performance of the biselector bonded-type MCSP in NP and RP modes. Chromatographic evaluation results showed that the above aromatic compounds were essentially capable of achieving baseline separation by hydrophobic interaction, π-π interaction, and π-π electron-donor-acceptor interaction. Moreover, the host-guest inclusion effect of 6-monodeoxy-6-monoamino-β-CD and the multiple interactions made the biselector bonded-type MCSP have good steric selectivity. The preparation method of the biselector bonded-type MCSP was simple and provided a new idea and strategy for the preparation of the subsequent novel biselector MCSP.     

Synthesis of new C3 symmetric amino acid‐ and aminoalcohol‐containing chiral stationary phases and application to HPLC enantioseparations

We recently reported a new C3‐symmetric (R)‐phenylglycinol N‐1,3,5‐benzenetricarboxylic acid‐derived chiral high‐performance liquid chromatography (HPLC) stationary phase (CSP 1) that demonstrated better results as compared to a previously described N‐3,5‐dintrobenzoyl (DNB) (R)‐phenylglycinol‐derived CSP. Over a decade ago, (S)‐leucinol, (R)‐phenylglycine, and (S)‐leucine derivatives were used as the starting materials of 3,5‐DNB‐based Pirkle‐type CSPs for chiral separation. In this study, three new C3‐symmetric CSPs (CSP 2, 3, and 4) were prepared by combining the ideas and results mentioned above. Here we describe the synthetic procedures and applications of the new C3‐symmetric CSPs (CSP 2–CSP 4).     

Reliable HPLC separation,vibrational circular dichroism spectra,and absolute configurations of isoborneol enantiomers

Resolution of chiral compounds has played an important role in the pharmaceutical field, involving detailed studies of pharmacokinetics, physiological, toxicological, and metabolic activities of enantiomers. Herein, a reliable method by high‐performance liquid chromatography (HPLC) coupled with an optical rotation detector was developed to separate isoborneol enantiomers. A cellulose tris(3, 5‐dimethylphenylcarbamate)‐coated chiral stationary phase showed the best separation performance for isoborneol enantiomers in the normal phase among four polysaccharide chiral packings. The effects of alcoholic modifiers and column temperature were studied in detail. Resolution of the isoborneol racemate displayed a downward trend along with an increase in the content of ethanol and column temperature, indicating that less ethanol in the mobile phase and lower temperature were favorable to this process. Moreover, two isoborneol enantiomers were obtained via a semipreparative chiral HPLC technique under optimum conditions, and further characterized by analytical HPLC, and experimental and calculated vibrational circular dichroism (VCD) spectroscopy, respectively. The solution VCD spectrum of the first‐eluted component was consistent with the Density Functional Theory (DFT) calculated pattern based on the SSS configuration, indicating that this enantiomer should be (1S , 2S , 4S )‐(+)‐isoborneol. Briefly, these results have provided reliable information to establish a method for analysis, preparative separation, and absolute configuration of chiral compounds without typical chromophoric groups.     

Enantioseparation of α‐Hydroxyallylphosphonates and Phosphonoallylic Carbonate Derivatives on Chiral Stationary Phases Using Sequential UV,Polarimetric, and Refractive Index Detection

Chromatographic separation of the enantiomers of parent compounds dimethyl α‐hydroxyallyl phosphonate 1a and 1‐(dimethoxyphosphoryl) allyl methyl carbonate 1b was demonstrated by high‐performance liquid chromatography (HPLC) using Chiralpak AS‐H and ad ‐H chiral stationary phases (CSP), respectively, using a combination of UV, polarimetric, and refractive index detectors. A comparison was made of the separation efficiency and elution order of enantiomeric α‐hydroxyallyl phosphonates and their carbonate derivatives on commercially available polysaccharide AS, ad , OD, IC‐3, and Whelk‐O 1 CSPs. In general, the α‐hydroxyallyl phosphonates were resolved on the AS‐H CSP, whereas the carbonate derivatives 1b and 2b were preferentially resolved on the ad ‐H CSP. The impact of aryl substitution on the resolution of analytes 1d , 1e , 1f and 2 , 3 , 4 , 5 , 6 , 7 , 8 was evaluated. Thermodynamic parameters determined for enantioselective adsorption hydroxyphosphonates 1a and 4 on the AS‐H CSP and carbonate 1b on the ad ‐H CSP demonstrated enthalpic control for separation of the enantiomers. Chirality 28:656–662, 2016. © 2016 Wiley Periodicals, Inc.     

Preparation and characterization of a new open‐tubular capillary column for enantioseparation by capillary electrochromatography

In order to use the enantioseparation capability of cationic cyclodextrin and to combine the advantages of capillary electrochromatography (CEC) with open‐tubular (OT) column, in this study, a new OT‐CEC, coated with cationic cyclodextrin (1‐allylimidazolium‐β‐cyclodextrin [AI‐β‐CD]) as chiral stationary phase (CSP), was prepared and applied for enantioseparation. Synthesized AI‐β‐CD was characterized by infrared (IR) spectrometry and mass spectrometry (MS). The preparation conditions for the AI‐β‐CD‐coated column were optimized with the orthogonal experiment design L₉(3⁴). The column prepared was characterized by scanning electron microscopy (SEM) and elemental analysis (EA). The results showed that the thickness of stationary phase in the inner surface of the AI‐β‐CD‐coated columns was about 0.2 to 0.5 μm. The AI‐β‐CD content in stationary phase based on the EA was approximately 2.77 mmol·m^?2. The AI‐β‐CD‐coated columns could separate all 14 chiral compounds (histidine, lysine, arginine, glutamate, aspartic acid, cysteine, serine, valine, isoleucine, phenylalanine, salbutamol, atenolol, ibuprofen, and napropamide) successfully in the study and exhibit excellent reproducibility and stability. We propose that the column, coated with AI‐β‐CD, has a great potential for enantioseparation in OT‐CEC.     

Enantioselective potential of polysaccharide‐based chiral stationary phases in supercritical fluid chromatography

The enantioselective potential of two polysaccharide‐based chiral stationary phases for analysis of chiral structurally diverse biologically active compounds was evaluated in supercritical fluid chromatography using a set of 52 analytes. The chiral selectors immobilized on 2.5 μm silica particles were tris‐(3,5‐dimethylphenylcarmabate) derivatives of cellulose or amylose. The influence of the polysaccharide backbone, different organic modifiers, and different mobile phase additives on retention and enantioseparation was monitored. Conditions for fast baseline enantioseparation were found for the majority of the compounds. The success rate of baseline and partial enantioseparation with cellulose‐based chiral stationary phase was 51.9% and 15.4%, respectively. Using amylose‐based chiral stationary phase we obtained 76.9% of baseline enantioseparations and 9.6% of partial enantioseparations of the tested compounds. The best results on cellulose‐based chiral stationary phase were achieved particularly with propane‐2‐ol and a mixture of isopropylamine and trifluoroacetic acid as organic modifier and additive to CO₂, respectively. Methanol and basic additive isopropylamine were preferred on amylose‐based chiral stationary phase. The complementary enantioselectivity of the cellulose‐ and amylose‐based chiral stationary phases allows separation of the majority of the tested structurally different compounds. Separation systems were found to be directly applicable for analyses of biologically active compounds of interest.     

Separation of enantiomers by open capillary electrochromatography on polysiloxane-bonded permethyl-beta-cyclodextrin

The separation of enantiomers by open capillary electrochromatography (o-CEC) using Chirasil-Dex as chiral stationary phase (CSP) is reviewed. In Chirasil-Dex, permethylated beta-cyclodextrin is linked via a single octamethylene spacer to polydimethylsiloxane. The CSP is coated and thermally immobilized onto the internal surface of a fused-silica column (i.d. 50 microm). Employing a single open-tubular column coated with Chirasil-Dex, a unified enantioselective approach can be realized using the four common chromatographic techniques: o-GC, o-SFC, o-LC and o-CEC. The chiral stationary phase Chirasil-Dex can be combined with a charged cyclodextrin derivative, which is added into the mobile phase. In the resulting dual chiral recognition system, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) are observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand. The feasibility to couple chiral o-CEC and ESI/MS is demonstrated for trace analysis of enantiomeric drugs in body fluids.     

Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phase

In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host-guest interactions might contribute to this enantiomer separation.     

HPLC enantioseparation on cellulose tris(3,5-dimethylphenylcarbamate) as a chiral stationary phase: Influences of pore size of silica gel,coating amount,coating solvent,and column temperature on chiral discrimination

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) was coated on large-pore silica gels and used as a chiral stationary phase (CSP) for high-performance liquid chromatographic separation of enantiomers. The influences of pore size of silica gel, coating amount of CDMPC, coating solvent, and column temperature on chiral discrimination were investigated. CSPs prepared with a large-pore silica gel having a small surface area showed higher chiral recognition. The amount of CDMPC adsorbed on the silica gel influenced the chiral recognition of some racemates. Loading capacity of racemates increased with an increase of the amount of CDMPC supported on the silica gel, and a CSP coated with 45% CDMPC by weight can be used for both analytical and semi-preparative scale separations. The CDMPC, coated using acetone as the coating solvent, exhibited, in many cases, higher enantioselectivity than that obtained with tetrahydrofuran F as the coating solvent. © 1996 Wiley-Liss, Inc.     

HPLC with cellulose Tris (3,5‐DimethylPhenylcarbamate) chiral stationary phase: Influence of coating times and coating amount on chiral discrimination

Coating cellulose tris (3,5‐dimethylphenylcarbamate) (CDMPC) on silica gels with large pores have been demonstrated as an efficient way for the preparation of chiral stationary phase (CSP) for high‐performance liquid chromatography (HPLC). During the process, a number of parameters, including the type of coating solvent, amount of coating, and the method for subsequent solvent removing, have been proved to affect the performance of the resultant CSPs. Coating times and the concentration of coating solution, however, also makes a difference to CSPs' performance by changing the arrangement of cellulose derivatives while remaining the coating amount constant, have much less been studied before, and thereby, were systematically investigated in this work. Results showed that CSPs with more coating times exhibited higher chiral recognition and column efficiency, suggesting that resolution was determined by column efficiency herein. Afterwards, we also investigated the effect of coating amount on the performance of CSPs, and it was shown that the ability of enantio‐recognition did not increase all the time as the coating amount; and four of seven racemates achieved best resolution when the coating amount reached to 18.37%. At the end, the reproducibility of CDMPC‐coated CSPs were further confirmed by two methods, ie, reprepared the CSP‐0.15‐3 and reevaluated the effect of coating times.     

Enantioselective and diastereoselective separation of synthetic pyrethroid insecticides on a novel chiral stationary phase by high-performance liquid chromatography

A novel chiral packing material for high-performance liquid chromatography (HPLC) was prepared by connecting (R)-1-phenyl-2-(4-methylphenyl) ethylamine (PTE) amide derivative of (S)-isoleucine to aminopropyl silica gel through 2-amino-3,5-dinitro-1-carboxamido-benzene unit. This chiral stationary phase was applied to the enantioselective and diastereoselective separation of five pyrethroid insecticides by HPLC under normal phase condition. To achieve satisfactory baseline separation an optimization of the variables of mobile phase composition was required. The two enantiomers of fenpropathrin and four stereoisomers of fenvalerate were baseline separated using hexane-1,2-dichloroethane-2-propanol as mobile phase. The results show that the enantioselectivity of CSP is better than Pirkle type 1-A column for these compounds. Only partial separations for the cypermethrin and cyfluthrin stereoisomers were observed. Seven peaks and eight peaks were observed for cypermethrin and cyfluthrin, respectively. The elution orders were assigned by using different stereoisomer-enriched products.     

Functionalities tuned enantioselectivity of phenylcarbamate cyclodextrin clicked chiral stationary phases in HPLC

The mixed chloro‐ and methyl‐ functionalities can greatly modulate the enantioselectivities of phenylcarbamate cyclodextrin (CD) clicked chiral stationary phases (CSPs). A comparison study is herein reported for per(4‐chloro‐3‐methyl)phenylcarbamate and per(2‐chloro‐5‐methyl)phenylcarbamate β‐CD clicked CSPs (i.e., CCC4M3‐CSP and CCC2M5‐CSP). The enantioselectivity dependence on column temperature was studied in both normal‐phase and reversed‐phase mode high performance liquid chromatography (HPLC). The thermodynamic study revealed that the stronger intermolecular interactions can be formed between CCC4M3‐CSP and chiral solutes to drive the chiral separation. The higher enantioselectivities of CCC4M3‐CSP were further demonstrated with the enantioseparation of 17 model racemates in HPLC.