Enantiomeric separation of 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs by HPLC with hydroxypropyl‐β‐cyclodextrin as chiral mobile phase additive

The enantio‐separations of eight 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (2‐APA NSAIDs) were established using reversed‐phase high‐performance liquid chromatography with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral mobile phase additive for studying the stereoselective skin permeation of suprofen, ketoprofen, naproxen, indoprofen, fenoprofen, furbiprofen, ibuprofen and carprofen. The effects of the mobile phase composition, concentration of HP‐β‐CD and column temperature on retention and enantioselective separation were investigated. With 2‐APA NSAIDs as acidic analytes, the retention times and resolutions of the enantiomers were strongly related to the pH of the mobile phase. In addition, both the concentration of HP‐β‐CD and temperature had a great effect on retention time, but only a slight or almost no effect on resolutions of the analytes. Enantioseparations were achieved on a Shimpack CLC‐ODS (150 × 4.6 mm i.d., 5 μm) column. The mobile phase was a mixture of methanol and phosphate buffer (pH 4.0–5.5, 20 mM) containing 25 mM HP‐β‐CD. This method was flexible, simple and economically advantageous over the use of chiral stationary phase, and was successfully applied to the enantioselective determination of the racemic 2‐APA NSAIDs in an enantioselective skin permeation study. Copyright © 2009 John Wiley & Sons, Ltd.     

Protonation of polyaniline‐coated silica stationary phase affects the retention behavior of neutral hydrophobic solutes in reversed‐phase capillary liquid chromatography

Because of its high conductivity when acid doped, polyaniline is known as a synthetic metal and is used in a wide range of applications, such as supercapacitors, biosensors, electrochromic devices, or solar and fuel cells. Emeraldine is the partly oxidized, stable form of polyaniline, consisting of alternating diaminobenzenoid and iminoquinoid segments. When acidified, the nitrogen atoms of emeraldine become protonated. Due to electrostatic repulsion between positive charges, the polarity and morphology of emeraldine chains presumably change; however, the protonation effects on emeraldine have not yet been clarified. Thus, we investigated these changes by reversed‐phase capillary liquid chromatography using a linear solvation energy relationship approach to assess differences in dominant retention interactions under a significantly varied mobile phase pH. We observed that hydrophobicity dominates the intermolecular interactions under both acidic and alkaline eluent conditions, albeit to different extents. Therefore, by tuning the mobile phase pH, we can even modulate the retention of neutral hydrophobic solutes, such as aromatic hydrocarbons, because the pH‐dependent charge and structure of polymer chains of the emeraldine‐coated silica stationary phase show a mixed‐mode separation mechanism.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

The HPLC Resolution of Nucleic‐Acid Bases and An a Log Hypoxanthine on R,S‐2‐Hydroxypropyl Derivatized β‐Cyclodextrin Bonded Chiral Stationary Phase Using a Water‐Based Mobile Phase

A HPLC approach using R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin packed column as the stationary phase was developed to resolve five nucleic‐acid bases and an a log hypoxanthine in the reversed‐phase mode. These bases are not only similar in structure but also very close in basicity. However, the resolution can be completed in less than ten minutes and is considered to be better carried out on the R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin phase than that obtained on the native β‐cyclodextrin phase under the same chromatographic conditions. The mechanism involved in the resolution is believed to be inclusion complexation between the analyte and the cavity of cyclodextrin in the reversed‐phase mode. The retention time was found relevant to the size of the analyte. The number of groups on analyte that is available to form hydrogen bonding with hydroxyl groups on CDs also affects the retention scale. Factors of introducing organic acid and base or organic modifier such as methanol to the water‐based mobile phase or increasing their percent ages in the mobile phase decreases the retention time without de grading the resolution significantly.     

Analysis of the constituents in rat plasma after oral administration of Shexiang Baoxin pill by HPLC‐ESI‐MS/MS

A valid method using liquid chromatography coupled with electrospray ionization (ESI) and ion trap mass spectrometry was established for the study of the absorbed components in rat plasma after oral administration of a traditional Chinese medicine (TCM) Shexiang Baoxin pill. The plasma was deproteinated by adding methanol prior to liquid chromatography, in which separation was carried out on a Symmetry C₁₈ column (5 µm, 250 × 4.6 mm). A linear gradient with 0.5% formic acid–water–acetonitrile was used as mobile phase. Mass spectra were acquired in both negative and positive modes. Twenty‐one components including 17 components from Shexiang Baoxin pill and four metabolites were observed from a comprehensive analysis of the chromatography of Shexiang Baoxin pill, controlled plasma and dosed plasma. All of the 17 prototype compounds and three of the metabolites were identified by comparing their retention behaviors and MS and MS/MS spectra with reference compounds and literature data. This study developed an integrated method for screening the bioactive constituents in plasma after oral adminstration of Chinese herbal medicine and provided helpful chemical information for further pharmacology and active mechanism research on TCM. Copyright © 2009 John Wiley & Sons, Ltd.     

Porous silica particles grafted with an amphiphilic side‐chain polymer as a stationary phase in reversed‐phase high‐performance liquid chromatography

The amphiphilic polymer‐grafted silica was newly prepared as a stationary phase in high‐performance liquid chromatography. Poly(4‐vinylpyridine) with a trimethoxysilyl group at one end was grafted onto porous silica particles and the pyridyl side chains were quaternized with 1‐bromooctadecane. The obtained poly(octadecylpyridinium)‐grafted silica was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and Brunauer–Emmett–Teller analysis. The degree of quaternization of the pyridyl groups on the obtained stationary phase was estimated to be 70%. The selective retention behaviors of polycyclic aromatic hydrocarbons including some positional isomers were investigated using poly(octadecylpyridinium)‐grafted silica as an amphiphilic polymer stationary phase in high‐performance liquid chromatography and results were compared with commercially available polymeric octadecylated silica and phenyl‐bonded silica columns. The results indicate that the selectivity toward polycyclic aromatic hydrocarbons exhibited by the amphiphilic polymer stationary phase is higher than the corresponding selectivity exhibited by a conventional phenyl‐bonded silica column. However, compared with the polymeric octadecylated silica phase, the new stationary phase presents similar retention behavior for polycyclic aromatic hydrocarbons but different retention behavior particularly for positional isomers of disubstituted benzenes as the aggregation structure of amphiphilic polymers on the surface of silica substrate has been altered during mobile phase variation.     

Preparation of a silica‐based high‐performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation

In this work, based on the structural characteristics of bio‐membrane molecules, a novel type of high‐performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one‐step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.     

Monolithic metal–organic framework MIL‐53(Al)‐polymethacrylate composite column for the reversed‐phase capillary liquid chromatography separation of small aromatics

A monolithic capillary column containing a composite of metal–organic framework MIL‐53(Al) incorporated into hexyl methacrylate‐co‐ethylene dimethacrylate was prepared to enhance the separation of mixtures of small aromatic compounds by using capillary liquid chromatography. The addition of 10 mg/mL MIL‐53(Al) microparticles increased the micropore content in the monolithic matrix and increased the Brunauer–Emmett–Teller surface area from 26.92 to 85.12 m²/g. The presence of 1,4‐benzenedicarboxylate moieties within the structure of MIL‐53(Al) as an organic linker greatly influenced the separation of aromatic mixtures through π–π interactions. High‐resolution separation was obtained for a series of alkylbenzenes (with resolution factors in the range 0.96–1.75) in less than 8 min, with 14 710 plates/m efficiency for propylbenzene, using a binary polar mobile phase of water/acetonitrile in isocratic mode. A reversed‐phase separation mechanism was indicated by the increased retention factor and resolution as the water percentage in the mobile phase increased. A stability study on the composite column showed excellent mechanical stability under various conditions. The higher resolution and faster separation observed at increased temperature indicated an exothermic separation, whereas the negative values for the free energy change of transfer indicated a spontaneous process.     

Competitional hydrophobicity driven separations under RP‐LC mechanism: Application to sulfonylurea congeners

Retention of a model set of sulfonylurea compounds has been studied under RP‐LC conditions, considering competitional effects brought by different alcohols (ethanol, 1‐propanol, 2‐propanol, 1‐butanol, 1‐pentanol, and 1‐octanol) used as additives in the organic component of the mobile phase (methanol). The capacity factors determined for the model compounds decreased with the increase of the hydrophobic character of the organic additive in the mobile phase. The amount of the additive within the organic component of the mobile phase was kept constant (1% as volumetric ratio). Retention was studied at different mobile phase compositions (aqueous to organic component ratios). Different functional fitting models were used to correlate retention to the content of the organic component in the mobile phase. Extrapolation of retention expressed as capacity factor to a mobile phase composition free of organic component is well correlated to the hydrophobic characteristics of the organic additives. The adsorption model was used for tuning the experimental find‐outs. The possibility of controlling retention through the competitive effects induced by hydrophobic additives in the mobile phase is highlighted.     

Enantioseparation of tryptophan and its unnatural derivatives by nano‐LC on CSP‐teicoplanin silica based

This work deals with the potentiality of nano liquid chromatography (Nano‐LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory‐assembled nano‐LC–UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.     

Intramolecular hydrogen bond in 3‐imino‐propenylamine isomers: AIM and NBO studies

The molecular structure and intramolecular hydrogen bond energy of 18 conformers of 3‐imino‐propenyl‐amine were investigated at MP2 and B3LYP levels of theory using the standard 6‐311++G** basis set. The atom in molecules or AIM theory of Bader, which is based on the topological properties of the electron density (ρ), was used additionally and the natural bond orbital (NBO) analysis was also carried out. Furthermore calculations for all possible conformations of 3‐imino‐propenyl‐amin in water solution were also carried out at B3LYP/6‐311++G** and MP2/6‐311++G** levels of theory. The calculated geometrical parameters and conformational analyses in gas phase and water solution show that the imine–amine conformers of this compound are more stable than the other conformers. B3LYP method predicts the IMA‐1 as global minimum. This stability is mainly due to the formation of a strong N? H···N intramolecular hydrogen bond, which is assisted by π‐electrons resonance, and this π‐electrons are established by NH2 functional group. Hydrogen bond energies for all conformers of 3‐imino‐propenyl‐amine were obtained from the related rotamers methods. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

The HPLC Enantioresolution of Selected Native or Derivatized Amino Acids on Teicoplanin Bonded Chiral Stationary Phase Using a Methanol‐Based Mobile Phase

The facile HPLC enantiomeric resolution of a variety of selected native or derivatized amino acids is carried out on the glycopeptide antibiotic teicoplanin bonded chiral stationary phase using a methanol‐based mobile phase and found very sensitive to the structural variations. This mobile phase is mainly composed of methanol. Organic additives such as acetic acid and triethylamine are introduced to the mobile phase in small percentages to control the analyte's retention time. Additive of low viscosity such as ethyl ether or petroleum ether is incorporated in the mobile phase as well to improve the resolution. Further increasing its percentage in the mobile phase deteriorates the resolution slightly; however, it extends the retention scale of enantiomers. The change in enantioselectivity is found to be insignificant under these circumstances. The hydrogen bonding and π‐π complexation in the hydrophobic pocket of teicoplanin chiral selector is believed to be the mechanism mainly responsible for the enantioresolution observed in this report.     

Effect of mobile phase composition on the liquid chromatographic enantioseparation of bulky monoterpene‐based β‐amino acids by applying chiral stationary phases based on Cinchona alkaloid

Stereoselective HPLC separations of five sterically constrained monoterpene‐based 2‐aminocarboxylic acid enantiomers were carried out by using the newly developed zwitterionic chiral stationary phases Chiralpak ZWIX(+)? and ZWIX(?)? based on Cinchona alkaloid. In order to optimize the retention and enantioselectivity parameters, the ratio of the different organic solvents in the mobile phase and the nature of the acid and base additives (counter‐ and co‐ions) were systematically varied. The effects of structure variants of the analytes on the resolution were investigated. The elution sequence was determined in all cases and observed to be opposite on ZWIX(+)? and ZWIX(?)?.     

Ultra‐high performance separation of basic compounds on reversed‐phase columns packed with fully/superficially porous silica and hybrid particles by using ultraviolet transparent hydrophobic cationic additives

The use of the tetrabutylammonium additive was investigated in the ultra‐high performance reversed‐phase liquid chromatographic elution of basic molecules of pharmaceutical interest. When added to the mobile phase at low pH, the hydrophobic tetrabutylammonium cation interacts with the octadecyl chains and with the residual silanols, thus imparting a positive charge to the stationary phase, modulating retention and improving peak shape of protonated basic solutes. Two sources of additive were tested: a mixture of tetrabutylammonium hydroxide/trifluoroacetic acid and tetrabutylammonium hydrogen sulfate. Retention and peak shape of 11 basic pharmaceutical compounds were evaluated on commercially available ultra‐fast columns packed with octadecyl stationary phases (Ascentis Express C18 2.0 µm, Acquity BEH C18 1.7 µm, Titan C18 1.9 µm). All columns benefit from the use of additive, especially tetrabutylammonium hydrogen sulfate, providing very symmetric peaks with reasonable retention times. Focusing on the probe compounds amitriptyline and sertraline, efficiency and asymmetry values were investigated at increasing retention factor. The trend is very different to that obtained in reversed‐phase conditions and the effect lies in the complex molecular interaction mechanisms based on hydrophobic and ion exchange interactions as well as electrostatic repulsion.     

Evaluation of an amide‐based stationary phase for supercritical fluid chromatography

A relatively new stationary phase containing a polar group embedded in a hydrophobic backbone (i.e., ACE ® C18‐amide) was evaluated for use in supercritical fluid chromatography. The amide‐based column was compared with columns packed with bare silica, C₁₈ silica, and a terminal‐amide silica phase. The system was held at supercritical pressure and temperature with a mobile phase composition of CO₂ and methanol as cosolvent. The linear solvation energy relationship model was used to evaluate the behavior of these stationary phases, relating the retention factor of selected probes to specific chromatographic interactions. A five‐component test mixture, consisting of a group of drug‐like molecules was separated isocratically. The results show that the C₁₈‐amide stationary phase provided a combination of interactions contributing to the retention of the probe compounds. The hydrophobic interactions are favorable; however, the electron donating ability of the embedded amide group shows a large positive interaction. Under the chromatographic conditions used, the C₁₈‐amide column was able to provide baseline resolution of all the drug‐like probe compounds in a text mixture, while the other columns tested did not.