Enantioseparation of amino acids on a polysaccharide-based chiral stationary phase

Sulfonic acids have been shown to be more effective than the commonly used trifluoroacetic acid (TFA) in the chiral resolution of underivatized aromatic amino acids on an amylosic column. Sulfonic acid additives give a more UV transparent mobile phase, possibly allowing the detection of non-aromatic analytes. Work presented demonstrates that through the combination of sulfonic acid mobile phase additives, amine mobile phase additives and solvent modifier variations, the enantiomers of 20 of 25 probe amino acids are fully resolved, four are partially resolved with only one failing to be separated on a common amylosic column.     

Enantioseparation of planar chiral ferrocenes on cellulose-based chiral stationary phases: Benzoate versus carbamate pendant groups

In this study, the enantioseparation of 14 planar chiral ferrocenes containing halogen atoms, and methyl, iodoethynyl, phenyl, and 2-naphthyl groups, as substituents, was explored with a cellulose tris(4-methylbenzoate) (CMB)-based chiral column under multimodal elution conditions. n-Hexane/2-propanol (2-PrOH) 95:5 v/v, pure methanol (MeOH), and MeOH/water 90:10 v/v were used as mobile phases (MPs). With CMB, baseline enantioseparations were achieved for nine analytes with separation factors (α) ranging from 1.24 to 1.77, whereas only three analytes could be enantioseparated with 1.14 ≤ α ≤ 1.51 on a cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC)-based column, used as a reference for comparison, under the same elution conditions. Pendant group–dependent reversal of the enantiomer elution order was observed in several cases by changing CMB to CDMPC. The impact of analyte and chiral stationary phase (CSP) structure, and MP polarity on the enantioseparation, was evaluated. The two cellulose-based CSPs featured by different pendant groups were also compared in terms of thermodynamics. For this purpose, enthalpy (ΔΔH°), entropy (ΔΔS°) and free energy (ΔΔG°) differences, isoenantioselective temperatures (T_iso), and enthalpy/entropy ratios (Q), associated with the enantioseparations, were derived from van ’t Hoff plots by using n-hexane/2-PrOH 95:5 v/v and methanol/water 90:10 v/v as MPs. With the aim to disclose the functions of the different substituents in mechanisms and noncovalent interactions underlying analyte–selector complex formation at molecular level, electrostatic potential (V) analysis and molecular dynamics simulations were used as computational techniques. On this basis, enantioseparations and related mechanisms were investigated by integrating theoretical and experimental data.     

Zwitterionic chiral stationary phases based on cinchona and chiral sulfonic acids for the direct stereoselective separation of amino acids and other amphoteric compounds

An extensive series of free amino acids and analogs were directly resolved into enantiomers (and stereoisomers where appropriate) by HPLC on zwitterionic chiral stationary phases (Chiralpak ZWIX(+) and Chiralpak ZWIX(?)). The interaction and chiral recognition mechanisms were based on the synergistic double ion‐paring process between the analyte and the chiral selectors. The chiral separation and elution order were found to be predictable for primary α‐amino acids with apolar aliphatic side chains. A systematic investigation was undertaken to gain an insight into the influence of the structural features on the enantiorecognition. The presence of polar and/or aromatic groups in the analyte structure is believed to tune the double ion‐paring equilibrium by the involvement of the secondary interaction forces such as hydrogen bonding, Van der Waals forces and π–π stacking in concert with steric parameters. The ZWIX chiral columns were able to separate enantiomers and stereoisomers of various amphoteric compounds with no need for precolumn derivatization. Column switching between ZWIX(+) and ZWIX(?) is believed to be an instrumental tool to reverse or control the enantiomers elution order, due to the complementarity of the applied chiral selectors.     

Enantioseparation of racemic N-acylarylalkylamines on various amino alcohol derived tau-acidic chiral stationary phases

Five tau-acidic chiral stationary phases (CSPs), CSP 4, CSP 5, CSP 6, CSP 7 and CSP 8, were prepared by connecting the N-(3,5-dimethylbenzoyl) derivative of (R)-alaninol, (S)-leucinol, (1S,2R)-ephedrine and (S)-tert-leucinol and the O-(3,5-dinitrobenzoyl) derivative of (R)-phenylglycinol to silica gel through a carbamate or urea linkage. The CSPs were applied to the resolution of various racemic N-acyl-1-naphthylaminoalkanes by chiral HPLC, and the chromatographic resolution results were compared with those of previously reported CSPs (CSP 2, CSP 3), which are derived from N-(3,5-dinitrobenzoyl)-(1S,2R)-norephedrine and N-(3,5-dinitrobenzoyl-(R)-phenylglycinol. Based on a comparison of the resolution results for each CSP, the role of each functional group on the five chiral selectors is explained.     

High-performance ligand-exchange chromatography of amino acids on chiral stationary phases

Three chiral stationary phases, obtained by grafting silica gel with (-)-trans-1,2-cyclohexanediamine, were studied for the resolution of α-amino acids by ligand-exchange chromatography. The packings were prepared by bonding the chiral ligand to silica gel via different hydrocarbon spacers. Separation of the optical isomers was accomplished by eluents containing a constant concentration of copper(II) acetate (0.05mM). The elution sequence of amino acids was found to be dependent on the grafting reaction selected to prepare the chiral packings.     

Chiralpak AD-H和Chiralcel OJ-H手性固定相拆分扁桃酸系列化合物

对比了商品化的淀粉型手性固定相Chiralcel OJ-H和纤维素型手性固定相Chiralpak AD-H柱在正相条件下对扁桃酸系列8个化合物的拆分,结果表明Chiralcel OJ-H柱对扁桃酸系列化合物具有更强的手性识别能力,8个外消旋扁桃酸化合物在36 min内都得到了基线分离。研究发现,扁桃酸苯环上的取代基对其拆分的难易程度影响很大,其电子诱导效应影响扁桃酸类化合物在固定相上的保留时间,其空间位阻效应是扁桃酸在固定相上被拆分成败的决定因素。通过对比分析扁桃酸和手性柱的结构,探讨了可能的手性拆分机理是基于Chiralpak AD-H(Chiralcel OJ-H)手性固定相和扁桃酸系列化合物之间的氢键-氢键、偶极-偶极、π-π电子相互作用以及空间适应性等诸多因素的综合影响,其中空间适应性起到至关重要的作用。本研究可为一些实际光学活性扁桃酸及其类似物的对映体纯度测定与拆分研究提供参考。     

键合型手性固定相拆分麻醉药JM-1232

使用Chiralpak IA色谱柱,实现了麻醉药JM-1232的对映体分离,并进一步考优化了流动相组成、柱温和流速等条件.得到最优色谱条件:流动相为正己烷:异丙醇= 65:35(V/V)、柱温301、流速1.0 mL/min,分离度达2.31.该方法显示出JM-1232对映体的足够分离度.     

手性固定相AD、AS和OD的拆分性能

手性固定相-高效液相色谱法在手性药物、手性农药等的分离分析中应用广泛。本文采用3种多糖衍生物的手性固定相(即EnantioPak AD、AS和OD)对20种手性化合物开展手性分离研究,进而探讨样品分子结构、多糖骨架和衍生基团对手性分离的影响。结果表明,除化合物13外,其余化合物在EnantioPak AD上均实现基线分离,分离度多在2.0以上,在正己烷-醇流动相中加入酸碱添加剂可改善和优化酸性或碱性化合物的分离效果;芳香醇(化合物13~16)随着侧链碳数增加在色谱柱上的保留减弱,其分离度呈现增加的趋势;对比8种化合物在3种手性固定相上的分离结果可知,EnantioPak AD表现出更优的分离性能。这为深入研究和了解多糖手性固定相、拓展其手性分离应用提供了参考。     

Enantioseparation characteristics of the chiral stationary phases based on natural and regenerated chitins

Natural and regenerated chitins were derivatized with 3,5‐dimethyphenyl isocyanate. The corresponding chiral stationary phases were prepared by coating the resulting chitin derivatives on 3‐aminopropyl silica gel. The swelling capacity of the chitin derivatives, enantioseparation capability, as well as eluents tolerance of the chiral stationary phases were evaluated. The results demonstrated no remarkable difference in enantioseparation capability between natural and regenerated chitins based chiral stationary phases. The similar enantioseparation characteristics of two chiral stationary phases could be understood by comparing the IR spectra of related chitin derivatives. The one of the two chiral stationary phases prepared by coating the chitin derivative with a lower molecular weight generally provided better enantioseparations. All chiral stationary phases can work in 100% chloroform, 100% ethyl acetate, 100% acetone, and the mobile phases containing a certain amount of tetrahydrofuran. The chiral stationary phase prepared from the chitin derivative with the highest swelling capacity exhibited better enantioseparations than others. This chiral stationary phase was damaged by flushing with 100% tetrahydrofuran, however, the enantioseparation capability was recovered again after the column was allowed to stand for 1 month. Furthermore, the recovered chiral stationary phase provided better enantioseparations for some chiral analytes than before.     

High-performance liquid chromatographic enantioseparation of amino compounds on newly developed cyclofructan-based chiral stationary phases

Direct separation of the enantiomers of amino acid amines, amino alcohols, and diamines was performed on recently developed chiral stationary phases containing isopropyl carbamate-cyclofructan 6 (IP-CF6), (R)-naphthylethylcarbamate cyclofructan 6 (RN-CF6), or dimethylphenylcarbamate cyclofructan 7 (DMP-CF7) as chiral selectors, using n-hexane/alcohol/TFA as mobile phase. The effects of the mobile phase composition, the nature and concentrations of the alcoholic and acidic modifiers, and the structures of the analytes on the retention and resolution were investigated. In some cases, separations were carried out at constant mobile phase composition in the temperature range 5-40 °C. Thermodynamic parameters and T(iso) values were calculated from plots of lnk versus 1/T. It was found that the enantioseparations were enthalpy driven. The sequences of elution of the stereoisomers were determined but no general rule could be established.     

Determination of soil amino acids by high performance liquid chromatography-electro spray ionization-mass spectrometry derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

Determination of amino acids by mass spectrometry (MS) is an important technique to investigate soil nitrogen transformation and cycling as amino acids being the major nitrogen-containing compounds in soil organic matter. However, researchers have long faced a critical problem in coupling an efficient separation technique to a sensitive MS detection system simultaneously. In this context, we established a new method of liquid chromatography coupled to mass spectrometry based on the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization method for convenient and accurate quantification of amino acids in soil samples. Baseline separation of 17 amino acid AQC-derivatives was achieved on an XTerra^R MS C₁₈ column using ammonium formate as a mobile phase modifier. The concentration of ammonium formate and the pH of the mobile phase were optimized in order to obtain sensitive MS signals. The response curves were linear over the range of 50-800 μmol L⁻¹ amino acids. The detection limits were 0.20-0.60 pmol μL⁻¹ on column and 0.07-0.24 μg g⁻¹ soil under the optimized conditions. The method has been applied successfully for the first time to determine amino acids in 4 types of soil samples, in which 15 amino acids were quantified by MS detector but methionine and cystine were below the detection limits. Both the recovery and the precision were satisfactory. Hence, this proposed technique shows a potential for the identification of amino acids in soil as well as tracing the transformation of soil amino acids with isotope dilution technique in nitrogen cycling investigation.     

Determination of amino acids by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high performance liquid chromatography with ultraviolet detection

A precolumn derivatization method for the determination of amino acids using 6-aminoquinolyl-N-hyroxy-succinimidyl carbamate (AQC) followed by high-performance liquid chromatography is described. Ultraviolet detection was used for the assay of AQC derivatives of amino acids with the detection wavelength set at 248 nm. The reagent peak interference was minimized by optimizing the pH of the eluent and the gradient elution profile to improve the resolution between the reagent peak and amino acid derivatives. All nineteen amino acids were separated in 35 min with resolutions 1.6. The correlation coefficients of the calibration graphs for seventeen amino acids were fairly good (r 0.9999) at concentrations of 25–500 μM. The detection limits for all common amino acids including cystine and trytophan were at the range of 0.07–0.3 pmol. Good reproducibility and accuracy of the method were demonstrated by the determination of amino acids in three typical kinds of samples (protein, peptide and feed.) The average relative standard deviations for bovine serum albumin (BSA) and neuromedin were 0.86% and 1.36, respectively, and the average relative errors were 3.2% and 2.3%, respectively. The results of the analysis of feed hydrolysates agreed with those obtained by an ion-exchange method and the average recovery of the method for feed hydrolysates was 98%.     

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.     

Analysis of neuroactive amino acids from microdialysate samples by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method

A sensitive and rapid reversed-phase high-performance liquid chromatographic method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and fluorescence detection is reported. By directly derivatizing microdialysate samples with AQC, an automatic and rapid simultaneous measurement of aspartate, serine, glutamate, glycine and histidine was developed. Excellent linearity (r2 > or = 0.998) was achieved for the standard mixture used for the validation experiments. Within-day and between-day precision was less than 6.2%, and the accuracy ranged from 95 to 105.2% in standards. This method is suitable for single run analysis of a high number of small volume microdialysate samples from rat hippocampus. Amino acids from microdialysate samples were quantified with RSD for reproducibility below 2%, and at approximately 0.1% for retention time.     

含磷手性化合物在多聚糖类手性固定相上的手性分离

在纤维素 三(3,5 二甲基苯基氨基甲酸酯)(ChiralcelOD)和直链淀粉 三(3,5 二甲基苯基氨基甲酸酯)(ChiralpakAD H)手性固定相上,采用高效液相色谱正相条件,分离了系列含磷手性化合物。考察了流动相中有机改性剂的种类及浓度对手性分离的影响;研究了化合物的结构与保留及对映体选择性的关系;并探讨了手性识别机理。     

手性有机磷农药对映体在衍生化环糊精类手性固定相上的分离

正相色谱条件下,首次在两种新型衍生化β-环糊精键合硅胶手性固定相上,对系列2-(噻唑基)-α-氨基膦酸酯化合物进行了有效的手性拆分研究,对映异构体选择性α达1.812,Rs高达4.072,为该类化合物的制备型拆分提供了一条行之有效的方法.探讨了化合物结构、流动相组成及流速对拆分的影响及其相应的手性识别机理.     

Enantioseparation of protonated primary arylalkylamines and amino acids containing an aromatic moiety on a pyridino-crown ether based new chiral stationary phase

This paper reports the preparation and testing of a new pyridino-18-crown-6 ether based chiral stationary phase (CSP). The chiral crown ether was covalently bound to silica gel. Circular dichroism (CD) spectroscopy was used for probing the complex formation of the chiral crown ether with the enantiomers of protonated primary arylalkylamines. The (S,S)-dimethylpyridino-18-crown-6 ether selector having a terminal double bond was first transformed to a triethoxysilyl derivative by regioselective hydrosilylation, and then heated with spherical HPLC quality silica gel to obtain the CSP. The discriminating power of the HPLC column filled with the above CSP was tested by using the hydrogenperchlorate salts of racemic α-(1-naphthyl)ethylamine (1-NEA), α-(2-naphthyl)ethylamine (2-NEA) and the hydrochloride salts of aromatic α-amino acids and α-amino acids containing different aromatic side-chain protecting groups.     

Enantioseparation of selected chiral sulfoxides using polysaccharide-type chiral stationary phases and polar organic, polar aqueous-organic and normal-phase eluents

HPLC enantioseparation of selected chiral sulfoxides was studied using cellulose and amylose phenylcarbamate derivatives as chiral stationary phases (CSPs). The contributions of various functional groups of a chiral analyte as well as the polysaccharide derivatives in the analyte retention and chiral recognition were evaluated. A very high enantioseparation factor exceeding 110 was observed in the enantioseparation of 2-(benzylsulfinyl)benzamide (BSBA) on cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) CSP by using 2-propanol as a mobile phase. The enantiomer elution order was opposite on cellulose and amylose phenylcarbamates. For the polysaccharide-type CSPs, pure alcohols such as methanol, ethanol and 2-propanol represent a valuable alternative to more common alcohol-hydrocarbon and reversed-phase eluents.     

Liquid chromatographic enantiomer separations of novel quinazolone derivatives on quinine carbamate based chiral stationary phases using hydro-organic mobile phases

Quinine carbamate-type weak chiral anion-exchange selectors (SOs) and the respective chiral stationary phases (CSPs) have been used for the direct liquid chromatographic enantiomer separation of a wide range of chiral acids. In the present work, we demonstrate that these CSPs can also be extended to chiral discrimination of a set of neutral polar potential NMDA (N-methyl-D-aspartic acid) and/or AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) antagonist imidazo-quinazoline-dione derivatives (selectands, SAs) using acetonitrile and methanol containing hydro-organic and buffered mobile phases. The influence of mobile phase composition, column temperature and structure variation of the SAs and SOs on retention and enantioselectivity was systematically investigated to gain insight into the overall chiral recognition mechanism. As was expected for the reversed-phase mode, acetonitrile has a stronger eluotropic effect compared to methanol. Except for two analytes, the acetonitrile containing mobile phases provided baseline resolution (R(S)) of the enantiomers with R(S) values ranging between 1.68 and 2.76. Using methanol as the organic modifier enhanced the enantioselectivity. The enthalpic and entropic terms for the SO-SA association were calculated from the linear van't Hoff plots. Data reveal that the enantiomer separations are predominantly enthalpically driven.