Evaluation of chromatographic columns packed with semi‐ and fully porous particles for benzimidazoles separation

The behavior of 15 benzimidazoles, including their main metabolites, using several C₁₈ columns with standard or narrow‐bore diameters and different particle size and type were evaluated. These commercial columns were selected because their differences could affect separation of benzimidazoles, and so they can be used as alternative columns. A simple screening method for the analysis of benzimidazole residues and their main metabolites was developed. First, the separation of benzimidazoles was optimized using a Kinetex C₁₈ column; later, analytical performances of other columns using the above optimized conditions were compared and then individually re‐optimized. Critical pairs resolution, analysis run time, column type and characteristics, and selectivity were considered for chromatographic columns comparison. Kinetex XB was selected because it provides the shortest analysis time and the best resolution of critical pairs. Using this column, the separation conditions were re‐optimized using a factorial design. Separations obtained with the different columns tested can be applied to the analysis of specific benzimidazoles residues or other applications.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

Fast separation of flavonoids by supercritical fluid chromatography using a column packed with a sub‐2 μm particle stationary phase

The purpose of this study was to compare the effects of different chromatographic columns for the separation of seven flavonoids. Four different stationary phases are available, including bridged ethyl hybrid, BEH and the same hybrid phase modified with 2‐ethylpyridine, CSH fluorophenyl, and HSS C₁₈ SB. The analytes included calycosin, genistein, medicarpin, calycosin‐7‐O‐β‐d ‐glucoside, formononetin, formononetin‐7‐O‐β‐d ‐glucoside, and liquiritigenin. The CSH fluorophenyl column was determined to be the most suitable and provided the fastest separation within 17 min using gradient elution with carbon dioxide as the mobile phase and methanol as the co‐solvent. Good peak shapes were obtained, and the values of the peak asymmetry were close to 1.0 for all of the flavonoids. The resolution was more than 1.41 for all of the separated peaks. Baseline separation on the optimal columns was achieved by changing the co‐solvent type and adjusting the temperature and pressure. Quantitative performance was evaluated under optimized conditions, and method validation was accomplished. The validation parameters, such as linearity, sensitivity, precision, and accuracy, were satisfactory. Good repeatability of both peak area (relative standard deviation <1.02%) and retention time (relative standard deviation <0.88%) was observed. The optimized chromatographic methods were successfully used for the determination of seven flavonoids in Radix astragali . The sensitivity was sufficient for the analysis of real samples.     

Fast reversed‐phase liquid chromatographic separation of proteins by flow‐through poly(styrene‐co‐divinylbenzene) microspheres

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High‐performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow‐through poly(styrene‐co‐divinylbenzene) microspheres characteristic of the binary pores, i.e. flow‐through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene‐co‐divinylbenzene) microspheres were suitable as the reversed‐phase stationary phase for separation of proteins. For the high permeability of the poly(styrene‐co‐divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ～2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0–99.4%. The proposed column had good pH stability of 1–13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio‐sample separation. The flow‐through poly(styrene‐co‐divinylbenzene) microspheres were promising for fast separation of large molecules.     

Liquid chromatographic enantioseparation of limonene‐based carbocyclic β‐amino acids on zwitterionic Cinchona alkaloid‐based chiral stationary phases

The eight stereoisomers of limonene‐based carbocyclic β‐amino acids containing three chiral centers have been directly separated on chiral stationary phases containing Cinchona alkaloid‐based zwitterionic selectors. The effects of bulk solvent composition of the mobile phase, the nature of base additives, counterion concentration, and the structure of selector on the enantiorecognition were studied. Experiments were performed at constant mobile phase composition in the temperature range 5–40°C to study the effect of temperature. Thermodynamic parameters were calculated on the basis of the plots of ln α versus 1/T curves. The enthalpically or entropically driven enantioseparations were found to depend strongly on the structures of analyte and selector. The eight stereoisomers of limonene‐based carbocyclic β‐amino acids could be differentiated as well‐separated peaks in a traditional 1D chromatographic system in two runs by applying the two complementary ZWIX(+)™ and ZWIX(–)™ columns.     

Micellar and aqueous‐organic liquid chromatography using sub‐2 μm packings for fast separation of natural phenolic compounds

The objective of the present work was to investigate the chromatographic behavior of natural phenolic compounds in micellar and aqueous‐organic LC using a short column packed with 1.8 μm particles. Firstly, the effect of ACN and SDS on elution strength and selectivity was examined by isocratic submicellar (0–30% ACN/5% 1‐butanol/1–6 mM SDS) and micellar (0–30% ACN/5% 1‐butanol/40–60 mM SDS) systems. The varied concentrations of two modifiers in the mobile phases revealed different eluting power. Then, the application of organic modifier gradient was discussed in both submicellar and micellar LC using mobile phases of 4 mM SDS/5% 1‐butanol or 50 mM SDS/5% 1‐butanol containing ACN gradient from 0 to 30%, respectively. For micellar system, the separation was found to be better in gradient than isocratic elution. Additionally, the sensitivity of aqueous‐organic LC was examined. The mobile phase was a mixture of ACN and water employing gradient elution at a flow rate of 0.5 mL/min, with analysis time below 9 min. It was found that separation efficiency was significantly better compared with micellar LC. Besides, the aqueous‐organic LC has been applied to separation of various phenolic compounds in Yangwei granule or Radix Astragali samples.     

Enantioselective determination of 1‐[4‐(2‐methoxyethyl)phenoxy]‐3‐[2‐(2‐methoxyphenoxy)ethylamino]‐2‐propanol hydrochloride,a novel antihypertensive agent,in rat plasma and tissues by liquid chromatography–tandem mass spectrometry

Enantioselective biodistribution studies of 1‐[4‐(2‐methoxyethyl)phenoxy]‐3‐[2‐(2‐methoxyphenoxy)ethylamino]‐2‐propanol hydrochloride (TJ0711), a novel antihypertensive agent, require the accurate and precise quantification of each TJ0711 enantiomer in biological fluids and tissues. Here we report a simple and sensitive liquid chromatography with tandem mass spectrometry method for simultaneous determination of (R )‐TJ0711 and (S )‐TJ0711 in rat plasma and tissue samples using protein precipitation. The influence of column type, temperature, mobile phase composition, and flow rate on the retention and enantioselectivity was evaluated. The separation of the TJ0711 enantiomers was ultimately achieved on a SUMICHIRAL OA‐2500 column in 15 min using isocratic elution with ethanol/hexane (40:60) at a flow rate of 0.8 mL/min. Good linearities of spiked analyte concentration from 5 to 2000 ng/mL were achieved and the correlation coefficients (R ) were greater than 0.99. The intra‐ and inter‐day accuracy and precision for both analytes were <15% at all concentration levels, and the extraction recoveries were consistent among the five quality control concentrations. This assay was successfully applied to quantify plasma and tissue concentrations of TJ0711 enantiomers in a preclinical study.     

Efficient preparative separation of 6‐(4‐aminophenyl)‐5‐methyl‐4, 5‐dihydro‐3(2H)‐pyridazinone enantiomers on polysaccharide‐based stationary phases in polar organic solvent chromatography and supercritical fluid chromatography

6‐(4‐Aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone is a key synthetic intermediate for cardiotonic agent levosimendan. Very few studies address the use of chiral stationary phases in chromatography for the enantioseparation of this intermediate. This study presents two efficient preparative methods for the isolation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone in polar organic solvent chromatography and supercritical fluid chromatography using polysaccharide‐based chiral stationary phases and volatile organic mobile phases without additives in isocratic mode. Under optimum conditions, Chiralcel OJ column showed the best performance (α = 1.71, Rs = 5.47) in polar organic solvent chromatography, while Chiralpak AS column exhibited remarkable separations (α = 1.81 and Rs = 6.51) in supercritical fluid chromatography with an opposite enantiomer elution order. Considering the sample solubility, runtime and solvent cost, the preparations were carried out on Chiralcel OJ column and Chiralpak AS column (250 × 20 mm i.d.; 10 µm) in polar organic mode and supercritical fluid chromatography mode with methanol and CO₂/methanol as mobile phases, respectively. By utilizing the advantages of chromatographic techniques and polysaccharide‐based chiral stationary phases, this work provides two methods for the fast and economic preparation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone, which are suitable for the pharmaceutical industry.     

Efficient preparative separation of β‐cypermethrin stereoisomers by supercritical fluid chromatography with a two‐step combined strategy

An efficient two‐step method has been developed for the separation of β‐cypermethrin stereoisomers by supercritical fluid chromatography with polysaccharide chiral stationary phases. With respect to retention, selectivity, and resolution of β‐cypermethrin, the effects of chiral stationary phases, cosolvents, mobile phases, and column temperature have been studied in detail. Through a two‐step separation, β‐cypermethrin was firstly separated by using a cellulose‐derived chiral stationary phase to obtain two stereoisomeric pairs, and further resolved on an amylose‐based chiral stationary phase to produce four enantiopure stereoisomers. The electronic circular dichroism patterns of the first‐ and the third‐eluted isomers in methanol solution showed the mirror image of each other in the wavelength range 200∼300 nm, indicating that they were a pair of enantiomers. Moreover, the second‐ and the fourth‐eluted isomers were also enantiomers. This proposed two‐step strategy showed low solvent consumption, fast separation speed, and high‐purity, which may provide an effective approach for preparative separation of compounds with multiple chiral centers and difficult‐to‐separate multicomponent samples.     

Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector

A recycling high‐speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing β‐cyclodextrin derivatives as a chiral selector. The two‐phase solvent system of n‐hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two‐phase system (e.g., the type and concentration of β‐cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl‐β‐cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)‐brompheniramine and 85% for (–)‐brompheniramine, respectively.     

Liquid chromatographic resolution of proline and pipecolic acid derivatives on chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

Two liquid chromatographic chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid were applied to the resolution of the amide derivatives of cyclic α‐amino acids including proline and pipecolic acid. Among the five amide derivatives of proline, aniline amide was resolved best on the first chiral stationary phase, which contains two N–H tethering amide groups, with the separation factor of 1.31 and the resolution of 2.60, and on the second chiral stationary phase, which contains two N–CH₃ tethering amide groups, with the separation factor of 1.57 and the resolution of 5.50. Among the five amide derivatives of pipecolic acid, 2‐naphthyl amide was resolved best on the first chiral stationary phase with the separation factor of 1.30 and the resolution of 1.75, but 1‐naphthylmethyl amide was resolved best on the second chiral stationary phase with the separation factor of 1.30 and the resolution of 2.26. In general, the second chiral stationary phase was found to be better than the first chiral stationary phase in the resolution of the amide derivatives of cyclic α‐amino acids. In this study, the second chiral stationary phase was first demonstrated to be useful for the resolution of secondary amino compounds.     

N‐methyl‐2‐pyrrolidonium methyl sulfonate acidic ionic liquid as a new dynamic coating for separation of basic proteins by capillary electrophoresis

A simple and economical CE method has been developed for the analysis of four model basic proteins by employing N‐methyl‐2‐pyrrolidonium methyl sulfonate ionic liquid (IL) as the dynamic coating material based on the interaction of both between electrostatic attraction and hydrogen bond, and between the organic cations of IL and the inner surface of bare fused‐silica capillary. The N‐methyl‐2‐pyrrolidonium‐based IL modified capillary not only generated a stable suppressed electroosmotic flow, but also effectively eliminated the wall adsorption of proteins. Several important parameters such as the IL concentration, pH values, and concentrations of the background electrolyte were optimized to improve the separation of basic proteins. Consequently, under the optimum separation conditions, a satisfied separation of basic proteins including lysozyme, cytochrome c, ribonuclease A, and α‐chymotrypsinogen A with theoretical plates ranging from 2.09 × 10⁵ to 4.48 × 10⁵ plates/m had been accomplished within 15 min. The proposed method first illustrated the effect of hydrogen bond between coating material and inner capillary surface on the coating, which should be a new strategy to design and select more effective coating materials to form more stable coatings in CE.     

Investigation of the chromatographic regulation properties of benzyl groups attached to bridging nitrogen atoms in a calixtriazine‐bonded stationary phase

Two reversed‐phase/anion‐exchange mixed‐mode stationary phases for high‐performance liquid chromatography using calixtriazines as chromatographic ligands were investigated with Tanaka test solutes, monosubstituted benzenes, aromatic positional isomers, and inorganic anions. Calixtriazine as a chromatographic ligand has been reported previously, but the benzylated nitrogen‐bridged calixtriazine‐bonded silica gel reported in this study is new. The experimental data showed that the calixtriazine platform is a unique chromatographic selector because its multiple active sites are available for different solutes and its chromatographic selectivity could be tuned by introducing substituent on the bridging nitrogen atoms present in the calixtriazine matrix. The synergistic effects of aromatic rings, nitrogen atoms, benzyl groups, and tunable cavity in the host molecule influenced the separation selectivity by multiple retention mechanisms. Such hybrid stationary phases provide more versatility and have great potential in the analysis of complex samples. Moreover, the synthetic protocols presented herein may provide an alternative understanding on macrocyclic host–guest chemistry, leading to new and selective separation media.     

Stereoisomer separation of flavanones and flavanone‐7‐O‐glycosides by means of nanoliquid chromatography employing derivatized β‐cyclodextrins as mobile‐phase additive

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7‐O‐glycosides has been proposed employing a C18 capillary column and a chiral mobile‐phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl‐β‐cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether‐β‐cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl‐β‐cyclodextrin and 10 mM of sulfobutyl ether‐β‐cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3‐2.5 and 7.5‐12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone‐7‐O‐glycosides in Citrus juices after solid‐phase extraction procedure.     

High‐performance liquid chromatographic and subcritical fluid chromatographic separation of α‐arylated ß‐carboline,N‐alkylated tetrahydroisoquinolines and their bioisosteres on polysaccharide‐based chiral stationary phases

New, pharmacologically interesting chiral amino compounds, namely, stereoisomers of α‐hydroxynaphthyl‐ß‐carboline, benz[d]azepine and benz[c]azepine analogs as well as N‐α‐hydroxynaphthylbenzyl‐substituted isoquinolines were enantioseparated by high‐performance liquid chromatographic and subcritical fluid chromatographic methods on polysaccharide‐based chiral stationary phases. Separation of the stereoisomers was optimized in both subcritical fluid chromatography and normal phase liquid chromatographic modes by investigating the effects of the composition of the bulk solvent, temperature, and the structures of the analytes and selectors. Both normal phase liquid chromatography and subcritical fluid chromatography exhibited satisfactory performance, albeit with somewhat different effectiveness in the separation of the stereoisomers studied. The optimized methods offer the possibility to apply preparative‐scale separations thereby enabling further pharmacological investigations of the enantiomers.     

Investigation of mass transfer properties and kinetic performance of high‐efficiency columns packed with C18 sub‐2 μm fully and superficially porous particles

Three columns packed with 2.0 μm superficially porous particles, 1.7 μm fully porous particles, and monodisperse 1.9 μm fully porous particles with narrow particle size distribution have been deeply characterized from a kinetic point of view. The 1.9 μm column showed excellent kinetic performance, comparable to that of the superficially porous one. These two columns also exhibit flatter c‐branches of the van Deemter curve compared to the 1.7 μm fully porous particles column, resulting in smaller loss of efficiency when they are operated at higher flow rates than the optimal ones. The independent evaluation of each contribution to band broadening has revealed that the difference in kinetic performance comes from the very small eddy dispersion contribution on the 1.9 μm column, surprisingly even lower than that of the superficially porous one. This finding suggests a very good packing of the monodisperse 1.9 μm column. On the other hand, the potential of 1.7 μm fully porous particles is completely broken down by the strong frictional heating effect already arising at relatively low flow rates.     

Polystyrene‐graft‐poly(ethylene oxide) copolymers prepared by macromonomer technique in dispersion. I. Liquid chromatographic separation of product mixtures

Products of the radical dispersion copolymerization of methacryloyl‐terminated poly(ethylene oxide) (PEO) macromonomer and styrene were separated and characterized by size exclusion chromatography (SEC), full adsorption‐desorption (FAD)/SEC coupling and eluent gradient liquid adsorption chromatography (LAC). In dimethylformamide, which is a good solvent for PEO side chains but a poor solvent for polystyrene (PS), amphiphilic PS‐graft‐PEO copolymers formed aggregates, which were very stable at room temperature even upon substantial dilution. The aggregates disappeared at high temperature or in tetrahydrofuran (THF), which is a good solvent for both homopolymers and for PS‐graft‐PEO. FAD/SEC procedure allowed separation of homo‐PS from graft‐copolymer and determination of both its amount and molar mass. Effective molar mass of graft‐copolymer was estimated directly from the SEC calibration curve determined with PS standards. Presence of larger amount of the homo‐PS in the final graft‐copolymer products was also confirmed with LAC measurements. The results indicate that there are at least two or maybe three polymerization loci; namely the continuous phase, the particle surface layer and the particle core. The graft copolymers are produced mainly in the continuous phase while PS or copolymer rich in styrene units is formed mostly in the core of monomer‐swollen particles. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2284–2291, 2000     

Simultaneous determination of quinolones in fish by liquid chromatography coupled with fluorescence detection: Comparison of sub‐2 μm particles and conventional C18 columns

A simple and effective multi‐residue analysis method is presented for the extraction and determination of eleven quinolones (pipemidic acid, enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, gatifloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in fish tissues. In this study, multi‐residue separations on four columns packed with 5 μm or sub‐2 μm particles were simultaneously developed for the purpose of comparison. Various gradients were optimized and best resolutions were achieved on each column. A short and sub‐2 μm particle‐sized HPLC column was chosen for its advantages in analysis time and column performance. Additionally, considering the matrix effect of the complex crude fish tissue, an effective extraction protocol was also established for sample pre‐treatment procedure. Good recoveries (71–98%) were obtained from samples fortified with a mix of eleven quinolones at three levels, with satisfactory relative standard deviations and limits of detection. As a result, the sub‐2 μm HPLC column and proposed analytical procedures have been evaluated and applied to the analysis of different fish tissues. Detectable residues were observed in 8 of 30 samples, at concentrations ranging from 4.74 to 23.27 μg/kg.     

Die Kristallstrukturen von PPh4[MCl5(NCMe)] · MeCN (M = Ti,Zr), zwei Modifikationen von PPh4[TiCl5(NCMe)] und von cis‐TiCl4(NCMe)2 · MeCN

The Crystal Structures of PPh₄[MCl₅(NCMe)] · MeCN (M = Ti, Zr), two Modifications of PPh₄[TiCl₅(NCMe)] and of cis ‐TiCl₄(NCMe)₂ · MeCN The title compounds were obtained by reactions of TiCl₄ or ZrCl₄, respectively, with PPh₄Cl and acetonitrile in the presence of S₂Cl₂. PPh₄[TiCl₅(NCMe)] · MeCN is unstable and emanates the incorporated acetonitrile. PPh₄[TiCl₅(NCMe)] forms the two modifications aP114 and mP228, the latter being more stable. The crystal structures were determined by X‐ray diffraction. Triclinic PPh₄[TiCl₅(NCMe)]‐(aP114) crystallizes in a distorted variety at the tetragonal AsPh₄[RuNCl₄] type, i. e. with PPh₄⁺ ions that are piled to columns in the c direction; the [TiCl₅(NCMe)]^– ions are tilted vs. this direction and thus cause the symmetry reduction from P4/n to P1. PPh₄[TiCl₅(NCMe)] · MeCN and PPh₄[ZrCl₅(NCMe)] · MeCN also have the same packing principle as in AsPh₄[RuNCl₄] with a symmetry reduction from P4/n to P112₁/n and a doubled c axis. Instead, PPh₄[TiCl₅(NCMe)]‐(mP228) has a packing with (PPh₄⁺)₂ pairs. Orthorhombic TiCl₄(NCMe)₂ · MeCN contains molecules having two acetonitrile ligands attached to the Ti atom in a cis configuration.