Towards multimodal HPLC separations on humic acid-bonded aminopropyl silica: RPLC and LEC behavior

In the present study, metal binding property of humic acid (HA) was successfully adapted to the ligand-exchange concept, and metal-loaded immobilized humic acid was used as a ligand exchanger stationary phase for separation of some nucleosides. Humic-acid bonded aminopropyl silica (EC-HA-APS) was turned into ligand exchanger forms by loading aqueous solutions of Cu²⁺, and Co²⁺ to the column (4.6 × 150; as mm) packed with EC-HA-APS. Metal ion solutions were loaded to the column in a stepwise manner where the concentration of metal ion solution being loaded to the column was increased gradually between 5 and 100 mM. The progress of metal loading process was monitored via the breakthrough curves propagated stepwise. Ligand-exchange chromatography (LEC) studies were performed on an HPLC system, and chromatographic behaviors of the studied nucleosides (i.e. uridine, Urd; thymidine, Tyd; cytidine, Cyd; adenosine, Ado; and guanosine, Guo) were investigated on Cu²⁺ and Co²⁺ loaded forms of the EC-HA-APS (Cu-EC-HA-APS and Co-EC-HA-APS). Effect of mobile phase composition, temperature, and the type of metal ion loaded to the column on the retentive behaviors of the compounds was studied, in detail. The studied solutes exhibited mixed-mode RPLC/LEC behavior on the stationary phase. Metal-loaded column (M-EC-HA-APS) was easily regenerated into its original form, EC-HA-APS, with 98 ± 2% metal recoveries, by using aqueous mixture of EDTA + NH₃ at pH = 7.5. Thus, the stationary phase exhibited a high flexibility between RPLC and LEC modes. This property, also, made it possible to convert the stationary phase into various ligand exchanger forms by loading different metal ions. Hence, capacity and selectivity of the stationary phase towards the studied species was manipulated easily by loading different metal ions to the stationary phase. Baseline separation for the studied species was achieved on Cu-EC-HA-APS and Co-EC-HA-APS and some differentiations were observed in capacity and selectivity, depending on the type of metal loaded. Thus, being as the first endeavor on usability of immobilized HA as a ligand exchanger stationary phase, the present study is believed to be useful to understand multifunctional character of HA-based solid/stationary phases.     

Purification and biochemical characterisation of acid phosphatase-I from seeds of Nelumbo nucifera

Acid phosphatase-I (Apase-I) from seeds of Nelumbo nucifera was purified to electrophoretic homogeneity by combination of ammonium sulfate precipitation, size-exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kDa under reducing and non-reducing conditions, indicating that the enzyme was a monomer. The purified enzyme showed maximum activity at 50°C and at pH 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 μM, 10 μmol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn²⁺, W²⁺; weakly inhibited by Cu²⁺, Mo²⁺ and Cr⁶⁺ and moderately activated by Mg²⁺. The enzyme was shown to be thermolabile as it lost 50% of its activity at 50°C after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase.     

Coordination properties of dehydroacetic acid – binary and ternary complexes

Complex formation equilibria of dehydroacetic acid with Cu²⁺, Ni²⁺, Co²⁺, Zn²⁺ and Mn²⁺ and the ternary complexes involving Cu²⁺, dehydroacetic acid and some amino acids containing different functional groups are investigated. Stoichiometry and stability constants for the complexes are estimated at 25°C and 0.1 M ionic strength in 25% dioxane-water mixtures. The concentration distribution diagrams of the complexes were evaluated. The effect of temperature and organic solvent on the acid dissociation constant of dehydroacetic acid and the formation constant of Cu²⁺ complex was studied and thermodynamic parameters calculated.     

Direct Measurement of Enantiomeric Excess of Synthetic Tert-Leucine with a Chiral-Ligand-Exchange Chromatography

Racemic tert-leucine can be completely resolved by ligand exchange chromatography (LEC) using a Chiralpak WH column. The retention time of the tert-leucine was decreased by increasing concentration of Cu²⁺, or decreasing pH of the eluent whereas it was unaffected by varying the temperature of eluent. Optimal conditions for complete separation of the D.L-tert-leucine were found to be (Cu²⁺, 5.0 mM, pH 5.5, 25 °C). The enantiomeric excess of synthetic tert-leucine, a chiral auxiliary reagent, was determined.     

Modeling the retention mechanism for high‐performance liquid chromatography with a chiral ligand mobile phase and enantioseparation of mandelic acid derivatives

The chromatographic retention mechanism describing relationship between retention factor and concentration of Cu²⁺(l ‐phenylalanine)₂ using chiral ligand mobile phase was investigated and eight mandelic acid derivatives were enantioseparated by chiral ligand exchange chromatography. The relationship between retention factor and concentration of the Cu²⁺(l ‐phenylalanine)₂ complex was proven to be in conformity with chromatographic retention mechanism in which chiral discrimination occurred both in mobile and stationary phase. Different copper(II) salts, chiral ligands, organic modifier, pH of aqueous phase, and conventional temperature on retention behavior were optimized. Eight racemates were successfully enantioseparated on a common reversed‐phase column with an optimized mobile phase composed of 6 mmol/L of l ‐phenylalanine or N,N‐dimethyl‐l ‐phenylalanine and 3 mmol/Lof copper(II) acetate or copper(II) sulfate aqueous solution and methanol.     

Complexation of 3-hydroxy-2,2′-iminodisuccinic acid (HIDS) with Mg2+, Ca2+, Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ ions in aqueous solution

In a search for environmental-friendly metal chelating ligands for industrial applications, the protonation and complex formation equilibria of 3-hydroxy-2,2′-iminodisuccinic acid with Mg²⁺, Ca²⁺, Mn²⁺, Fe³⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ ions in aqueous 0.1 mol L^?1 NaCl solution were studied at 25°C by potentiometric titration. The model for complexation and the stability constants of the different complexes were determined for each metal ion using the computer program SUPERQUAD. In all cases, complex formation was dominated by stable ML ^{n ?4} complexes.     

Enantioseparation of five racemic N-alkyl drugs by reverse phase HPLC using sulfobutylether-β-cyclodextrin as a chiral mobile phase additive

Analytical enantioseparations of five N-alkyl drugs, fluoxetine hydrochloride, labetalol, venlafaxine hydrochloride, trans-paroxol, and atropine sulfate, were investigated by reverse phase high-performance liquid chromatography with sulfobutylether-β-cyclodextrin as chiral mobile phase additive. Effects of various factors such as composition of mobile phase, concentration of cyclodextrins, and column temperature on retention and enantioselectivity were studied. Apparent formation constant between methanol, acetonitrile, and sulfobutylether-β-cyclodextrin were determined to be 2.90 × 10⁻³ and 1.00 × 10⁻⁴ L mmol⁻¹ under 25°C using UV-spectrophotometry. Van't Hoff plots were used to investigate thermodynamic parameters for enantiomers–stationary phase interaction and formation of inclusion complex. Two retention models were employed individually for evaluation of inclusion complexation between five racemates and sulfobutylether-β-cyclodextrin. The second model with complex adsorption was more accord with the retention behavior of fluoxetine hydrochloride, labetalol, and venlafaxine hydrochloride enantiomers, while the first model was more consistent with the retention behaviors of trans-paroxol and atropine sulfate. In the selected mobile phase, stoichiometric ratio for both of inclusion complex was found to be 1:1.     

Binding of metal ions by polyethylenimine and its derivatives

Measurements have been made of the binding of divalent metal ions, Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺ ions, by polyethylenimine (PEI) and its acetyl or alkyl derivatives by the equilibriumdialysis technique. These metal ions, in particular the Cu²⁺ ion, exhibited tremendously remarkable binding affinity toward PEI. The extent of complexation of the polymer with the metal ions was decreased markedly by acetylation or alkylation of the polymer. PEI with no primary amine showed an appreciable decrease in its affinity for the metal ion. These results indicate the participation of the primary amine of the polymer in the formation of the complex. A cooperative binding isotherm was observed in PEI–metal ion complex formation, suggesting swelling or conformational change of the polymer induced by this coordination process. Binding of the Cu²⁺ ion by PEI was found to be essentially independent of temperature over the range 5–35°C.     

C18柱串联冠醚手性柱高效分离3种芳香族氨基酸对映体

将C18柱与手性冠醚柱串联,建立了一种反相高效液相色谱法用于3种芳香族氨基酸对映体同时拆分的方法.考察了反相色谱流动相的组成、pH值、柱温、流速对对映体拆分的影响.实验结果表明,当流动相为HClO4-乙睛溶液(86:14,V/V,pH 2.0)、柱温20℃、流速0.4 mL/min时,3种氨基酸对映体可获得基线分离.进一步对比了C18柱、冠醚手性柱和串联顺序不同的4种分离模式,结果表明,C18柱不能拆分氨基酸对映体,仅能分离不同种类氨基酸;冠醚手性柱可分离氨基酸映体,但不同种类氨基酸色谱峰出现重叠;串联模式能实现3种氨基酸对映体的基线分离,实现双柱优势互补,而串联顺序对分离影响不大,仅影响色谱峰的峰形.     

Heavy-metal extraction capability of chalcogenoic aminophosphines derived from 1-amino-4-methylpiperazine

Aminophosphine of the type (Ph₂PNHR) derived from 1-amino-4-methylpiperazine and its chalcogen derivatives (Ph₂P(X)NHR X = S, Se) were used as ligands in solvent extraction of metal picrates such as Cu²⁺, Ni²⁺, and Pb²⁺ from the aqueous to the organic phase. Influence of parameters such as pH of the aqueous phase, ligand concentration in the organic phase, and concentration of the extractant extracted from the aqueous to the organic phase was investigated to determine the ligands’ ability to extract metal ions. Metal picrate extraction was investigated at 25°C using UV-VIS spectrophotometry in dichloromethane in the absence and in the presence of Ph₂PNHR and chalcogenides. The extraction results revealed that the extraction percentage of Cu²⁺, Ni²⁺, and Pb²⁺ metals was much higher at lower pH values, indicating an acidity dependent complexation equilibrium.     

High‐performance liquid chromatographic enantioseparation of isoxazoline‐fused 2‐aminocyclopentanecarboxylic acids on a chiral ligand‐exchange stationary phase

The application of a chiral ligand‐exchange column for the direct high‐performance liquid chromatographic enantioseparation of unusual β‐amino acids with a sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate‐Cu(II) complex as chiral selector is reported. The investigated amino acids were isoxazoline‐fused 2‐aminocyclopentanecarboxylic acid analogs. The chromatographic conditions were varied to achieve optimal separation. The effects of temperature were studied at constant mobile phase compositions in the temperature range 5–45°C, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Δ(ΔH°) ranged from –2.3 to 2.2 kJ/mol, Δ(ΔS°) from –3.0 to 7.8 J mol^?1 K^?1 and –Δ(ΔG°) from 0.1 to 1.7 kJ/mol, and both enthalpy‐ and entropy‐controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. The sequence of elution of the enantiomers was determined in all cases.     

Complexation of some environmentally friendly carboxylic acid ligands with La3+ ion in aqueous solution

In search for environmentally friendly metal chelating ligands for industrial applications, protonation, and complex-formation equilibria of iminodisuccinic acid (IDS), 3-hydroxy-2,2′ -iminodisuccinic acid (HIDS), N,N′-ethylenediaminedisuccinic acid (EDDS), and oxydisuccinic acid (ODS) with La³⁺ in aqueous 0.1?mol?L^?1 NaCl solution were studied at 25°C by potentiometric titration. The model for complexation and the stability constants of the different complexes were determined using the computer program SUPERQUAD. Complex formation was dominated by stable LaL^? complexes for all tested ligands.     

Thermodynamic study of HPLC enantioseparations of some sulfur-containing amino acids on teicoplanin columns in ion-pairing reversed-phase mode

Homocysteine is an essential amino acid produced in the metabolism of methionine. It can be regulated through two metabolic routes either to cysteine or back to methionine. The imbalance in the methionine or homocysteine metabolism is connected with many disorders. In this work, HPLC enantioseparations of cysteine, homocysteine, and methionine were performed. Enantiomers were separated on natural teicoplanin and teicoplanin aglycone chiral stationary phases when buffered mobile phase with the presence of an ion-interaction reagent was used. The column temperature effect on the enantiomeric retention, selectivity, and resolution was studied in the range of 5–45°C at 5°C increment. Thermal stability of homocysteine standard was investigated by X-ray powder-diffraction measurements at elevated temperature in the range of 25–100°C at 5°C increment in air. Moreover, the thermodynamic analysis was used to quantify contributions of entropic and enthalpic terms on separations of investigated amino acid enantiomers. The elution order of enantiomers was determined in all cases.     

New approaches to the preparation of highly efficient chromium-containing oxide catalysts for the water gas shift reaction

It was found experimentally that the solutions of Cr³⁺ nitrate and the nitrates of other metals that are the constituents of Cr-containing catalysts can be prepared by dissolving a corresponding metal (for example, cast iron and electrolytic copper) in a solution of chromic anhydride and nitric acid to reach the quantitative reduction of Cr⁶⁺ without the formation of nitrogen oxides. Analogously, the oxidation of Fe²⁺ cations to Fe³⁺ coupled with the reduction of hexavalent chromium can be performed. The precipitation of Fe³⁺, Cr³⁺, and Cu²⁺ ions at a ratio of Fe: Cr = 9 and a concentration of Cu²⁺ to 20 at % can result in the formation of a partially hydrated oxide with the hydrohematite structure—a dispersed and highly defective oxide structure with a high specific surface area more than 300 m²/g and a higher thermal stability, as compared with the goethite phase (α-FeOOH). The dehydration of hydrohematite occurred at a noticeable rate at temperatures higher than 400°C. Hydrohematite promoted with copper cations exhibited high activity below 400°C; this can decrease the starting temperature of the adiabatic high-temperature WGSR to 300°C or below.     

Time-dependent Cu-induced gelation of poly(ethylene-alt-maleic acid) in aqueous solution

The complexation of Cu²⁺ ions with an alternating copolymer of maleic acid (MAc) and ethylene in aqueous solution was followed through turbidimetry and absorption spectrophotometry as a function of the polymer concentration and the metal ion-to-polymer molar ratio, r. The introduction of Cu²⁺ ions was performed in aqueous solution through neutralization of the polyacid with Cu(OH)₂ powder. A gelation window between homogeneous and phase-separated solutions was observed in the phase diagram of the polymer/Cu²⁺ mixture and the viscoelastic properties of the hydrogels were evaluated through rheology measurements. It is found that the stiffness of the hydrogels can be tuned by the polymer concentration and the mixing ratio r. Moreover, the stiffness of the hydrogels increases substantially with time. In fact, this time evolution may be as long as one month or longer if the composition of the aqueous solution is close to the gelation threshold. The gelation properties can be qualitatively explained from the possible formation of binuclear polymer/Cu²⁺ complexes in aqueous solutions, as indicated from the absorption spectroscopy results.     

The Study of Separation and Thermodynamics of Adsorption of the Enantiomers of Ethofumasate on a Modified Cellulose

Abstract

Cellulose and cellulose derivatives are biopolymers that are often used as stationary phases for the separation of enantiomers. Describing the mechanism of such separations is a difficult task due to the complexity of these phases. In the present study, direct enantiomeric resolution of ethofumesate has been achieved, using hexane as the mobile phase with various alcoholic modifiers on cellulose tri(3,5‐dimethylphenylcarbamate) chiral stationary phase (CDMPC CSP). The influence of the mobile phase composition and the column temperature on the chiral separation was studied. It was found that at a constant temperature and within a certain range of alcohol modifier concentration, the conformation of the polymeric phase, and the selective adsorption sites were not affected by alcohol modifier concentration. The type and the concentration of the alcoholic modifiers influenced the retention factor and the separation factor. Ethofumesate gained the best enantioseparation using sec‐butanol as alcoholic modifier at 25°C with α‐value 1.70. And the separation factor decreased with the increase of the column temperature. The van't Hoff plots were linear (R ²>0.96) for ethofumesate from 25°C to 50°C. That showed the enantioselective interactions do not change over the temperature range studied. Furthermore the values of ΔH° and ΔS° were both negative, which indicated an enthalpy‐driven separation. And the possible chiral recognition mechanism of the analyte and CDMPC was discussed. It was found that hydrogen bonding plays an important role on enantioseparation of CDMPC CSP. The inclusion and fitness of solute shape in the chiral cavity significantly contributed to the enantioseparation of solute.     

Direct Enantiomeric Separation of Chiral Pesticides by LC on Amylose Tris(3,5-dimethylphenylcarbamate) Stationary Phase under Reversed Phase Conditions

Amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase was used by liquid chromatography under reversed-phase conditions for the chiral separation of 20 pesticides, of which ten samples were separated directly under suitable conditions. The influence of mobile phase composition and column temperature from 0 to 40 °C on the separation was investigated. The mobile phases were methanol/water or acetonitrile/water at a flow rate of 0.5 mL min^?1 with UV detection at 230 nm. The two enantiomers of fenamiphos, terallethrin, fenoxaprop-ethyl, benalaxyl and lactofen could obtain base separation under optimized conditions, while the enantiomers of quizalofop-ethyl, metalaxyl, napropamide, fluroxypyr-meptyl and 2,4-D-ethylhexyl got partial separation. The retention factors (k) and selectivity factor (α) for the enantiomers of most investigated pesticides decreased with increasing the temperature. The lnα–1/T plots for enantiomers of chiral pesticides were linear at the range of 0–40 °C except for that of metalaxyl, fenoxaprop-ethyl and 2,4-D-ethylhexyl enantiomers in methanol/water. The thermodynamic parameters calculated based on linear Van’t Hoff plots showed the chiral separation was controlled by enthalpy. Better separation was not always at low temperature. The chiral recognition mechanisms were discussed. The elution orders of the eluting enantiomers were determined by a circular dichroism detector.     

Electron spin resonance studies of manganese(II) and copper(II) salts of poly(ethylene-co-methacrylic acid)s

The ionic interaction of poly(ethylene-co-methacrylic acid) ionomer neutralized with Mn²⁺ or Cu²⁺ was studied by ESR spectroscopy to explore the local structure in the ionic aggregate. ESR spectra of the ethylene ionomer were obtained as functions of degree of neutralization and temperature. The existence of both isolated and aggregated cations in the ionomer was confirmed by ESR. In addition, the formation of a Cu²⁺?Cu²⁺ dimer structure similar to the crystal structure of copper acetate monohydrate was found in ethylene ionomer containing the Cu²⁺. Cation-cation interactions changed markedly around 70°C with increasing temperature, representing the onset of the motion of cations in the aggregated ionic structure.     

Separation of the Enantiomers of Thyroxine by HPLC with Chiral Mobile Phases

A high-performance liquid chromatographic (HPLC) method has been developed for separation of the enantiomers of thyroxine (T4). A silica gel column was used in conjunction with a chiral mobile phase (CMP) comprising 35:65 (v/v) acetonitrile–water containing 0.1 mM copper(II) acetate, 0.2 mM L-proline, and 0.5 mM triethylamine (TEA), pH 5.42. The flow rate was 1.0 mL min^?1 and the analysis temperature 40 °C. L-T4 was eluted before D-T4 by mobile phase containing L-proline copper complex. Inversion of the chirality of the mobile phase resulted in reversal of this order of elution. A racemic mobile phase containing DL-proline copper complex resulted in no separation. The enantioseparation phenomenon is discussed. When the method was used to determine the concentration of T4 enantiomers in the serum of a patients with thyroid disease, different concentrations of T4 enantiomers were found in different patents.     

Enantiomeric Separation of Non-Protein Amino Acids by Chiral Ligand-Exchange High-Performance Liquid Chromatography

Abstract

The enantiomeric separation by high-performance liquid chromatography of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Excellent to good separations of enantiomers were achieved with a variety of nonprotein amino acids carrying aliphatic or aromatic side-chains by optimizing the amount (0-20%, v/v) of the organic component (2-propanol) and the concentration (1-5 mM) of the complexing metal ion (Cu²⁺) in the hydro-organic eluent.