A test to determine the nature and presence of the memory effect columns packed with the amylose tris(3,5-dimethylphenylcarbamate) stationary phase

Acid/base mobile phase modifiers affect enantioseparations in ways that are not fully understood yet, for the lack of systematic studies. This makes chiral analysis of some pharmaceuticals difficult to reproduce. Once a column has been exposed to a modifier, the selectivity of certain pairs of enantiomers may change, for the better or the worse. We study the behavior of five enantiomeric pairs, three which are highly sensitive to the addition of certain modifiers and two that have little sensitivity to these modifiers. Their use permits the determination of the extent of the memory effect response on individual columns. The selectivity of 4-chlorophenylalanine methyl and ethyl ester, and of ketoprofen improve as a solution of ethanesulfonic acid is percolated through the column. As a result, these pairs are most useful for the determination of the extent of acid memory effect on a column. The selectivity of propranolol HCl and, to a lesser degree, Tröger's base increases as a solution of diisopropylethylamine is percolated through the column. The separation of each one of these five pairs is inversely affected by the percolation of the opposite acid/base solution. We used trans-stilbene oxide (TSO) as a ‘standard’ to determine the column stability because no memory effect is observed for it (its retention, enantioselectivity, and resolution remain constant). Understanding whether a column is under the influence of the memory effect is critical to both the analysis of pharmaceutical ingredients and to the development of preparative purification techniques for racemic mixtures. Thus, columns that were unreliable for method development and method transfer, due to the memory effect and a lack of proper solvent exposure records, can now be used.     

The influence of the memory effect on preparative separations using the amylose tris(3,5-dimethylphenylcarbamate) stationary phase

Acid/base mobile phase modifiers affect enantioseparations in ways that are not yet understood for the lack of systematic studies, which makes the scale-up of preparative separations difficult to predict. Shifts of the selectivity of certain pairs of enantiomers upon exposure of the column to these modifiers is amply documented. Furthermore, once the modifier has been removed from the mobile phase, the improved selectivity remains, this phenomenon has been named the memory effect. We selected four enantiomeric pairs for a systematic study of this memory effect. The selectivity of 4-chlorophenylalanine ethyl ester (4CPEE) improves after a solution of ethanesulfonic acid (ESA) is percolated through the column. The selectivity of propranolol HCl and Tröger's base increases after a solution of diiospropylethylamine is percolated through the column. The selectivity of these three pairs of enantiomers is inversely affected by percolation of the opposite acid/base solution. Each of these four compounds reached an equilibrium concentration that maintained the separation of the enantiomeric pairs. In contrast, the selectivity of trans-stilbene oxide (TSO) is not affected by either acid/base modifier. Preparative separations can be used to detect changes in the active surface of the chiral polymer stationary phase by measuring the change in selectivity and resolution when modifiers are used. Preparative method development was carried out on analytical columns and scale-up to 1 cm ID columns were performed in this study.     

The influence of water on the memory effect of the amylose tris(3,5-dimethylphenyl carbamate) stationary phase

Acid/base modifiers are sometimes used as additives in normal phase elution on columns packed with CHIRALPAK^® AD^®. These modifiers affect enantioseparations in ways that are not yet fully understood for the lack of systematic studies. Shifts of the selectivity of certain pairs of enantiomers upon exposure of the column to these modifiers is amply documented. Furthermore, once the modifier has been removed from the mobile phase, the modified selectivity remains, which has been named the Memory Effect. After a column has been exposed to an eluent stream containing acidic/basic modifiers, this particular column no longer separates certain enantiomeric pairs with the same selectivity as a modifier naive column. This makes the transfer of developed methods from one to other CHIRALPAK AD columns difficult to predict, if the selectivity needs to be similar between the two columns. We selected four enantiomeric pairs for a systematic study of this Memory Effect. The selectivity of 4-chlorophenylalanine ethyl ester improves after a solution of ethanesulfonic acid (ESA) is percolated through the column. The selectivity of Propranolol and Tröger's base increases after a solution of Diiospropylamine is percolated through the column. The selectivity of Propranolol and Tröger's base enantiomers is inversely affected by percolation of the acid solution. The 4-chlorophenylalanine ethyl ester enantiomers is inversely affected by percolation of the base solution. In contrast, the selectivity of trans-stilbene oxide (TSO) is not affected by either modifier. Analytical studies of the stationary phase suggest that slow protonation/deprotonation of water molecules attached to the carbamate moiety may be responsible for the acid/base Memory Effect. To further the understanding of the effect of water on the Memory Effect, mobile phases – spiked with water (0.01–0.43%) – were used to measure changes in the Memory Effect. Finally, we showed that the influence of water on the Memory Effect can be minimized by percolating through the column a sufficiently concentrated solution of the appropriate base while using dried mobile phases.     

Reversed-phase separations on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase

Summary The chiral separation of eight racemic compounds has been investigated on a cellulose tris(3,5-dimethylphenylcarbamate) chiral column under reversed-phase conditions. The discrimination mechanism under reversed-phase conditions is discussed. Addition of acid to the mobile phase is necessary for resolution of acidic racemic compounds. The presence of ion-pair reagent in the mobile phase is a key factor in the resolution of basic racemic compounds. Retention of the racemates is also affected by addition of acid or salt. The anion in buffer not only interacts with the racemete, but also with the polysaccharide derivative on the silica gel surface.     

Enantioseparation of 15 organic phosphonate esters on the cellulose tris(3,5-dimethylphenyl carbamate) chiral stationary phase by HPLC

A series of 15 organic phosphonate esters enatiomers containing a carbon atom as a chiral center have been separated on the cellulose tris(3,5-dimethylphenyl carbamate) chiral stationary phase (CSP) in the normal phase by high performance liquid chromatography (HPLC). Both the capacity factor (k) and separation factor () of all solutes are presented. The influence of the substitutional group on the benzene ring attached to the chiral carbon atom and the steric hindrance of alkoxyl of the phosphonate ester on the chiral separation are discussed. Based on and different structure parameters, good agreement between the predicted values and the experimental ones is obtained. The most characteristic parameter influencing the chromatographic separation is chosen from many structure parameters by linear regression method of QSAR software. The probable mechanism of the chiral recognition is proposed.     

纤维素-三（3,5-二甲苯基氨基甲酸酯）手性固定相拆分氨鲁米特对映体

以纤维素-三（3,5-二甲苯基氨基甲酸酯）为手性固定相（Chiralcel OD-H）在高效液相色谱上拆分了氨鲁米特对映体。通过测定氨鲁米特在正己烷/乙醇和正己烷/异丙醇中的溶解度,优选了对样品溶解度大的流动相体系,并考察了流动相添加剂乙醇胺对拆分效果的影响。在此基础上进一步研究了流动相中乙醇含量、柱温和进样量对分离因子、分离度、不对称因子和理论板数的影响,从而确定了最佳的拆分条件：固定相为Chiralcel OD-H,流动相为正己烷/乙醇/乙醇胺（体积比为30：70：0.1）,柱温25℃。本文所得结果可为工业放大提供基础数据。     

Enantioseparation of novel chiral tetrahedrane-type clusters on an amylose tris (Phenylcarbamate) chiral stationary phase

Summary Amylose tris(phenylcarbamate) (ATPC) coated on a small particle silica gel was prepared. This ATPC chiral stationary phase (ATPC-CSP) was found to be useful for the enantiomeric separation of some novel chiral tetrahedrane-type clusters. Moreover, the influence of mobile phase modifier and of the structure of chiral tetrahedrane-type clusters on the chiral separation and retention were investigated. The results suggest that not only the structure and concentration of alcohol in mobile phase, but also the subtle structural differences in racemates can have a pronounced effect on enantiomeric separation and retention.     

Enantioseparation of Binaphthol and Its Derivatives on Cellulose Tris(3,5-dimethylphenyl carbamate)

A Chiralcel OD-H column was used for the normal phase enantioseparation of binaphthol and its derivatives. Unexpectedly, binaphthol and its dibromo-substituted analogue could not be enantioseparated fully, whereas its ether and ester derivatives gave complete or partial resolution. Aspects of the chiral recognition are discussed further through calculated thermodynamic parameters.     

氟环唑外消旋体在纤维素-三(3,5-二甲基苯基氨基甲酸酯)手性固定相上的高效液相色谱法拆分

在纤维素-三(3,5-二甲基苯基氨基甲酸酯)（CDMPC）手性固定相上,分别采用正相、反相及极性有机相色谱模式对氟环唑外消旋体进行了拆分,并考察了流动相组成在手性识别中对手性分离的影响。氟环唑在Chiralcel OD-H手性色谱柱（填充CDMPC手性固定相）上采用反相色谱模式,以甲醇-水(体积比为80∶20)为流动相,获得了最佳的拆分,其两对对映异构体的分离度Rs分别为1.64和6.50。     

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 × 4.6 mm, 3 μm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min, column temperature of 35°C, injection volume of 10 μL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859–4.524 μg/mL was found to be R² = 0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 μg/mL, 0.859 μg/mL, and 96.0, respectively.     

Study of enantioselectivity on an immobilized amylose carbamate stationary phase under subcritical fluid chromatography

In this paper, we describe the enantiomeric separation of a chiral alcohol under subcritical fluid chromatography conditions using a 3 μm particle size bonded amylose carbamate stationary phase. Linear and branched alcohols were used as polar modifiers in CO₂. The studies with linear alcohols showed a decrease in selectivity factor as the number of carbons in the linear chain increased. For branched alcohols, as the bulk of substituents at the α carbon atom increases the separation factor decreases. Thermodynamic studies showed that in the presence of the alcohols studies, except methanol and ethanol, a positive ΔΔS was observed. Molecular mechanics simulation brought more insights into the mechanism of enantiomeric separation on this stationary phase under subcritical fluid chromatography.     

直链淀粉手性固定相法分离利阿唑对映体

建立了利阿唑对映体的高效液相色谱拆分方法。采用Chiralpak AD-H手性柱在正相条件下直接拆分利阿唑对映体,考察了流动相中有机极性调节剂的种类和浓度、酸碱的种类和含量、柱温及流速等对利阿唑对映体分离的影响。确定了最佳的拆分条件:流动相为正己烷-乙醇(含0.3%二乙胺和0.1%冰醋酸)(体积比为80∶20),流速0.6 mL/min;检测波长254 nm;柱温20 ℃。在此条件下利阿唑对映体的分离度为3.4。该法简单快速,重现性好。     

手性离子液体和纤维素三(3,5-二甲基苯基氨基甲酸酯)混合气相色谱手性固定相研究

合成了手性离子液体[C7H17NO] [(CF3SO2)2]-和纤维素三(3,5-二甲基苯基氨基甲酸酯),将其混合作为气相色谱固定相。研究结果表明:该混合固定相麦氏常数的平均极性值为760;在容量因子大于2的前提下,新型柱的塔板数可达1680块/m;其对手性化合物、特别是对氨基酸类具有良好的分离效果。有10对氨基酸的分离因子大于1.10,展示了该类色谱柱良好的手性应用前景。     

Selectivity of amylose tris(3,5-dimethylphenyl-carbamate) chiral stationary phase as a function of its structure altered by changing concentration of ethanol or 2-propanol mobile-phase modifier

In a previous publication, solid-state NMR data showed that the structure of Chiralpak AD chiral stationary phase (CSP) was altered by changing the concentration of ethanol or 2-propanol modifier in the chromatographic mobile phase. This present paper reports the effect of the CSP structural change on chiral selectivity alpha. The enantiomers of a series of compounds were chromatographed using ethanol or 2-propanol in various concentrations as mobile-phase modifier and the alpha values were determined. Changes of alpha were observed for some enantiomeric pairs when ethanol and 2-propanol concentrations were varied. These data correlate with previous findings on the structural changes of the CSP. Not every enantiomeric pair showed changes in alpha as the alcohol concentration was varied, indicating that the chiral selectivity depends not only on the CSP's structure, but also on the structures of the analytes.     

Computational study of enantioseparation by amylose tris(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase

The mechanism of chiral separation on amylose tris(3,5‐dimethylphenylcarbamate) is studied with docking simulations of enantiomers by molecular dynamics. All‐atom models of amylose tris(3,5‐dimethylphenylcarbamate) on the modified silica gel surface were constructed for the docking simulations of metalaxyl and benalaxyl. The elution orders and energetic differences were also predicted based on the intermolecular interactions, which were in agreement with the experimental results. The radial distribution function was employed to analyze the structural features of the enantiomer‐chiral stationary phase complex and used to elucidate the mechanism of chiral separation. The separation of metalaxyl and benalaxyl is mainly controlled by the hydrogen bond. And the binding sites had slight differences for the pair of enantiomers, but obvious differences between different chemicals.     

Enantiomer separation on amylose tris(n-butylcarbamate) by gas chromatography

Summary Seven aromatic and alkyl amylose and cellulose carbamates have been tested as chiral stationary phases in gas chromatography. One of them, amylose tris(n-butylcarbamate) can be used for enantiomer separation.     

淀粉及纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相的制备及其性能评价

为了扩展多糖类手性固定相的种类,制备了基于淀粉及纤维素三(3-三氟甲基苯基氨基甲酸酯)的涂敷型手性固定相,以正己烷-异丙醇混合液为流动相,对8种手性化合物进行了高效液相色谱拆分。研究表明: 虽然与应用最广泛的分别以淀粉及纤维素三(3,5-二甲基苯基氨基甲酸酯)为手性选择因子的商品化手性柱Chiralpak AD和Chiralcel OD相比,所制备的手性固定相的手性分离能力较低,但纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相显示出特异的手性识别能力,一些手性化合物在此固定相上得到了比在Chiracel OD上更好的分离;所制备的手性固定相的手性识别能力随流动相中异丙醇含量的降低而变好,当流动相中正己烷与异丙醇的体积比为95:5时所制备的手性固定相显示出相对较高的手性识别能力;总体来说,淀粉三(3-三氟甲基苯基氨基甲酸酯)手性固定相的手性识别能力稍强于纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相,同时两种手性固定相的手性识别能力具有一定的互补性。     

Resolution of naftopidil and bufuralol enantiomers by high-performance liquid chromatography on cellulose tris-(3,5-dimethylphenyl carbamate) column

The liquid Chromatographic resolution of the racemic cardiovascular drugs naftopidil and bufuralol to their corresponding enantiomers was achieved on cellulose tris-(3,5-dimethylphenyl carbamate) chiral stationary phase known as Chiralcel OD. The chiral recognition mechanism(s) involved between the chiral stationary phase and these drugs, which include hydrogen bonding, intercalative interactions, and steric interactions, among other factors, were discussed.     

Enantiomeric separation of chiral pesticides by high performance liquid chromatography on cellulose tris-3,5-dimethyl carbamate stationary phase under reversed phase conditions

Twenty chiral pesticides were tested, of which seven samples were directly separated by HPLC using cellulose tris-3,5-dimethyl carbamate (CDMPC) chiral stationary phase under RP conditions. The influence of mobile phase composition and column temperatures from 0 degrees C to 40 degrees C on the separations were investigated. The mobile phases were methanol/water or ACN/water at a flow rate of 0.8 mL/min with UV detection at 230 or 210 nm. Epoxiconazole, terallethrin, benalaxyl, and diclofopmethyl were observed to obtain the baseline separation under suitable conditions and other pesticides pyriproxyfen, lactofen, and quizalofop-ethyl were separated partially. The retention factors (k) and selectivity factor (alpha) for the enantiomers of most investigated pesticides decreased upon increasing the temperature except for the selectivity factors (alpha) of pyriproxyfen in methanol/water. The ln alpha - 1/T plots for racemic chiral pesticides were linear at the range of 0-40 except for that of pyriproxyfen enantiomers in methanol/water and the chiral separations were controlled by enthalpy. Better separations were not always at low temperature. The elution orders of the eluting enantiomers were determined by a circular dichroism (CD) detector.