Enantioselective separation of indole derivatives by liquid chromatography using immobilized cellulose (3,5-dimethylphenylcarbamate) chiral stationary phase

A new chiral stationary phase of 3,5-dimethylphenylcarbamates of cellulose chemically bonded to 3-aminopropyl silica gel was prepared, which may be used with a wide range of solvents including standard and non-standard ones. Several racemic indole derivatives have been resolved using standard and non-standard solvents on the immobilized chiral column (15 cm × 0.46 cm) at a flow rate of 1.0 mL/min or 0.5 mL/min with a UV detection at 230 nm. Separation of indole derivatives on immobilized and coated chiral stationary phases (CSP) in HPLC using a mixture of hexane/2-propanol as mobile phase was compared. The resolution factors for immobilized and coated chiral column were 0.57–2.02 and 0.61–4.03, respectively. It was found that both coated and immbolized chiral stationary phases were suitable for the separation of indole derivatives; however, the coated CSP possesses a higher resolving power than the immobilized one. The article is published in the original.     

Per(3-chloro-4-methyl)phenylcarbamate cyclodextrin clicked stationary phase for chiral separation in multiple modes high-performance liquid chromatography

In this work, a detail study has been performed on the enantioselectivity of per(3-chloro-4-methyl)phenylcarbamate-β-CD clicked chiral stationary phase (CSP) in high-performance liquid chromatography. Both normal phase and polar organic mobile phases have been explored for the enantioseparation of 39 model racemic pairs including aromatic alcohols, flavonoids, β-blockers and amino acids. Chiral resolution values over 10 were achieved for flavonoids. The comparison study with reference perphenylcarbamate-β-CD clicked CSP reveals the significance of 3-chloro-4-methyl functionality in improving the enantioselectivities. This study may provide insight on the multiple interactions between functionalized CD and analytes.     

键合型纤维素类手性固定相高效液相色谱法拆分对映异构体

制备了一种键合型纤维素-三苯基氨基甲酸酯手性固定相。在正相色谱条件下，拆分了九种结构不同的外消旋对映体，实验发现，在流动相中添加四氢呋喃有利于手性化合物拆分，而衍生化官能团可参与手性识别过程，在反相色谱条件下，此固定相同样具有手性拆分能力。研究结果表明，随着流动相中乙腈浓度的增加，对映体的保留值明显减小，但对映异构体的选择性却变化不大。在流动相中使用低pH值，能有效抑制酸性化合物的解离，从而显著增强其手性识别能力；对于中性化合物，流动相中pH对手性分离影响不大。     

An amylopectin tris(phenylcarbamate) chiral stationary phase for high performance liquid chromatography

Amylopectin tris(phenylcarbamate) has been evaluated as a chiral stationary phase for HPLC; the influence on its cptical resolving capabilities of mobile phase composition and nature of the alcohol used as modifier has been studied. Separation and resolution of twelve arylalcohol racemates were examined. In most instances, the stationary phase exhibited high optical resolving capacity.     

纤维素手性固定相拆分阿替洛尔对映体

建立了阿替洛尔对映体的高效液相色谱分析方法。使用Chiralpak OD-H手性色谱柱,检测波长275nm。确定了最佳拆分条件:流动相为V(正己烷)∶V(异丙醇)∶V(三乙胺)=80∶20∶0.4;流速1.0 mL/min,柱温30℃。在该实验条件下,得到阿替洛尔对映体的出峰顺序为:先R体,后S体。所建立的方法简单准确,重复性好,可用于阿替洛尔的质量研究和控制。     

Enantioselective separation of racemic secondary amines on a chiral crown ether-based liquid chromatography stationary phase

The first general enantioselective separation of racemic secondary amines on a crown ether-based liquid chromatography chiral stationary phase (CSP) is presented. The CSP is based on (+)- or (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid covalently bonded to silica gel. A mobile phase containing methanol, acetonitrile, triethylamine and acetic acid was employed in these separations of secondary amines with crown ether CSPs. The separation mechanism is believed to be the secondary amine forming a complex which includes crown ether coordination and electrostatic interaction of the positively charged amine with a carboxylate anion of the immobilized crown ether.     

Enantioselective high-performance liquid chromatographic separation of N-methyloxycarbonyl unsaturated amino acids on macrocyclic glycopeptide stationary phases

This paper describes the enantiomeric resolution of a series of unsaturated N-methyloxycarbonyl-alpha-H-alpha-amino acids (N-MOC-alpha-amino acids) on macrocyclic glycopeptide stationary phases by means of high-performance liquid chromatography (HPLC). Three types of glycopeptide phases, i.e. Chirobiotic T, V and R, were evaluated in both reversed-phase (RP) and polar ionic mode (PIM). The best results in terms of enantioselectivity and resolution were obtained on Chirobiotic R phase, with the PIM mobile phase giving the highest resolution per min. Investigation of the pH of the reversed-phase mobile phase in the pH range 4.1-5.9 showed little effect on enantioselectivity. The method was applied for monitoring the conversion and product enantiomeric excess of an enzymatic hydrolysis reaction using N-MOC-alpha-H-alpha-amino acid esters as substrate.     

Chiral separation of selected proline derivatives using a polysaccharide type stationary phase by high-performance liquid chromatography

Proline derivatives, such as Boc-proline, Boc-2-methylproline, Boc-2-methylproline benzyl ester and Boc-2-methyl-4-hydroxy-proline benzyl ester, have been widely used as a building block leading to a variety of pharmaceutical compounds. Therefore, there is a wide interest in the chiral separation of these compounds. High-performance liquid chromatography (HPLC) methods were developed using a Chiralpak AD-H column to separate enantiomers of these proline derivatives. The effect of mobile phase composition and column temperature was studied. For the proline derivatives studied in this work, good resolution was achieved using a mobile phase composition of hexane, ethanol and 0.1% TFA. For prolines containing carboxyl or hydroxy group, resolution was changed dramatically corresponding to changes as little as 1% of ethanol in the mobile phase, suggesting that the dominant chiral recognition is from hydrogen bonding interactions. On the other hand, for prolines containing a benzyl ester instead of hydroxy group next to the chiral center, resolution was not affected as significantly with the changes of ethanol content in the mobile phase, indicating a different leading chiral recognition mechanism, such as inclusion, steric effect, or possible pi-pi interaction. Linearity, precision and limit of detection were also measured for Boc-2-methylproline and Boc-2-methylproline benzyl ester.     

Unusually high enantioselectivity in high-performance liquid chromatography using cellulose tris(4-methylbenzoate) as a chiral stationary phase

The cellulose tris(4-methylbenzoate) chiral stationary phase (CSP) (commercially known as Chiralcel OJ-H) exhibited an extremely high enantioselectivity when used in the HPLC resolution of N-thiocarbamoyl-3-(4′-prenyloxy)-phenyl-5-phenyl-4,5-dihydro-(1H) pyrazole (Compound 1), in both normal-phase and polar organic conditions. Using n-hexane–ethanol (80:20, v/v) as a mobile phase, an enantioseparation factor value of 138.5 was found. In order to modulate the elution time of the longer retained enantiomer, a simple HPLC procedure was developed. The optimized analytical protocol was based on the stopped flow technique and did not involve any change in mobile phase composition. The stronger interaction energy of the (S) enantiomer compared to that of the (R) enantiomer was mainly attributed to the formation of a hydrogen bonding between the amino group of the thiocarbamoyl moiety and the carbonyl oxygen of the CSP.     

Chiral separations of pesticide enantiomers by high-performance liquid chromatography using cellulose triphenylcarbamate chiral stationary phase

The direct chiral separations of pesticide enantiomers by high-performance liquid chromatography by applying self-prepared cellulose triphenylcarbamate chiral stationary phase are performed. The mobile phase is n-hexane modified by isopropanol as a polar modifier. Nine chiral pesticides (benalaxyl, vinclozolin, diclofop-methyl, tebuconazole, quizalofop-ethyl, hexaconazole, lactofen, isocarbophos, and paclobutrazol) show enantioselectivity on the chiral stationary phase. An online circular dichrorism detector is used for identifying the pesticide enantiomers. The influences of the volume content of isopropanol and column temperature on the separations are investigated. The thermodynamic parameters related to the chiral distinguish mechanisms are also calculated.     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl--amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (ln ) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparaton for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Δ_R,SΔH° and Δ_R,SΔS° afforded by Van’t Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP, remarkable increases in enanselectivity were observed for all the compounds, as the result of a “synergistic” effect.     

Enantiomeric separation of various lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters by high-performance liquid chromatography

Lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters were separated by chiral high-performance liquid chromatography (HPLC) with a Chiralcel OD-H column in the normal-phase mode. Major lipoxygenase derivatives of linoleic, -linolenic and arachidonic acids are well resolved by this column, provided they have been individually purified. Our method allows an easy and rapid determination of lipoxygenases enantioselectivity. In all cases tested the R enantiomer is eluted first.     

Analysis of enantiomeric flavanones in plant extracts by high-performance liquid chromatography on a cellulose triacetate based chiral stationary phase

Summary Flavanones ofArachis hypogaea, Hemizonia increscens, Eriodictyon glutinosum andThymus vulgaris extracts have been stereospecifically analysed by gradient elution on a column of cellulose triacetate supported on silica gel diol. The stereochemistry of naringenin, eriodictyol and homoeriodictyol was in favour of the 2S-configurated flavanones.     

Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined. The choice of modifier depended on the nature of the stationary phase. The more polar solvents were better suited for stationary phases with higher percentage of free silanol groups. Isomeric species were eluted according to chain length, number of double bonds, and the position of the unsaturated acyl chain in the glycerol molecule. Retention increases in the order 20:5 < 22:6 < 18:3 < 20:4 < 18:2. Within each isomeric pair, the species with unsaturated acyl chain occupying either the sn-1- or the 3-position were retained preferentially. Complete simultaneous regioselective separation of 10 isomeric triacylglycerols in a single isocratic run on a single ODS column was demonstrated.     

Fullerenes separation with monomeric type C30 stationary phase in high-performance liquid chromatography

The temperature effect on the separation of fullerenes in LC was examined using monomeric type C30, C18 and C8 alkyl bonded stationary phases. It appears that the C30 phase exhibits superior separation ability for fullerenes. It is observed that the maximum retention temperature of fullerenes on the C30 phase is around 20 degrees C. A strong correlation between the changes in NMR spectra and the retention behavior of the solutes was found. The interpretation of the retention behavior of fullerenes on the alkyl bonded stationary phases, including the behavior in subambient temperature, is discussed using the information obtained by CP-MAS solid-state NMR spectroscopy and LC.     

Micropreparative separation of transfer ribonucleic acids by high-performance liquid chromatography

A method was developed for the micropreparative separation of individual species of tRNA using reversed-phase high-performance liquid chromatography on large pore spherical silica bonded with C3 alkyl chains. Columns were eluted with linear gradients of decreasing sodium chloride and increasing methanol concentrations. The decreasing salt gradient gradually abolished hydrophobic interactions and a significantly higher selectivity was thus obtained when compared with increasing gradients of salts usually employed in reversed-phase separations of tRNA. The acceptance of tRNA fractions was tested by charging them with fifteen different amino acids. Significantly different separations were obtained with tRNA from Escherichia coli and from rat liver. tRNAGlu and tRNATyr from E. coli were obtained in a pure form, all other tRNAs were more or less contaminated by adjoining tRNAs for other amino acids. Rechromatography under suitable isocratic conditions was required to obtain pure tRNA species from rat liver. Isoaccepting tRNAs for several amino acids were separated from rat liver. The method described seems suitable for preliminary fractionations of complex mixtures of tRNA and for a simple purification of isoaccepting species if the presence of tRNAs for other amino acids is not an hindrance.