Ligand-exchange Liquid Chromatography Research Articles

Simple and robust determination of monosaccharides in plant fibers in complex mixtures by capillary electrophoresis and high performance liquid chromatography

Carbohydrates partially liberated by acid hydrolysis of plant fiber can be separated by hydrophilic interaction liquid chromatography (HILIC), ligand-exchange liquid chromatography or other forms of LC with ion-exchange columns. However, the robust hydrogen-exchange columns show co-elution of galactose, xylose and mannose. Free solution capillary electrophoresis (CE) can be used without derivatization at pH 12.6 and was found to provide a higher resolution of galactose and xylose than common LC with no sample pre-treatment required, other than dilution, within 26min. CE was able to provide resolution higher than 0.79 for all separated carbohydrates, and the RSDs of determined concentrations lower than 10% for concentrations above 1.3gL−1. A quantitative comparison between CE and HPLC revealed that up to 22% more carbohydrates are quantified with CE. Direct UV detection in CE of mono- and disaccharides is unexpectedly possible at 270nm. NMR analysis shows that alkaline degradation is too slow to explain this detection. This CE detection sensitivity is increased by the electric field and our CE and NMR analyses are consistent with a photo-oxidation process.

Simultaneous measurement of proline and related compounds in oak leaves by high-performance ligand-exchange chromatography and electrospray ionization mass spectrometry for environmental stress studies

A mass spectrometer was coupled to high-performance ligand-exchange liquid chromatography (HPLEC) for simultaneous analysis of stress associated solutes such as proline, hydroxyproline, methylproline, glycine betaine and trigonelline extracted from leaves of drought stressed oaks and an internal standard namely N-acetylproline. Methanol/chloroform/water extracts were analyzed using an Aminex HPX-87C column and specifically quantified by the positive ion mode of an electrospray ionisation-mass spectrometry (ESI-MS) in single ion monitoring (SIM) mode. The recovery of N-acetyl proline added to oak leaf extracts ranged from 85.2 to 122.1% for an intra-day study. Standard calibration curves showed good linearity in the measured range from 0.3125 to 10 μmol L −1 with the lowest correlation coefficient of 0.99961 for trigonelline. The advantages of this alternative procedure, compared to previously published methods using fluorescence or amperometric detections, are the simultaneous and direct detection of osmoprotectants in a single chromatographic run, a minimal sample preparation, a good specificity and reduced limits of quantification, ranging from 0.1 to 0.6 μmol L −1. Fifty-six days of water deficit exposure resulted in increased foliar free proline levels (2.4-fold, P < 0.001, 155 μmol g −1 FW) and glycine betaine contents (2.5-fold, P < 0.05, 175 μmol g −1 FW) of drought stressed oak compared to control.

Enantiomeric Separation of Moxifloxacin and Its (R,R)-Isomer by Ligand-Exchange Chiral Chromatography

A new stereospecific LC method for the separation and quantification of moxifloxacin and its (R,R)-enantiomer in bulk drug was developed and validated by ligand-exchange liquid chromatography on a reversed phase column using aqueous mobile phase containing the chiral reagent l-isoleucine-Cu(II). The UV detector was operated at 293 nm. The flow rate of mobile phase was set at 0.9 mL min−1. The achiral ODS column offers good separation of the two enantiomers in less than 20 min. The test concentration was 1,000 μg mL−1 in the mobile phase. This method was capable of detecting the (R,R)-enantiomer of moxifloxacin up to 0.1 μg mL−1 for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with the (R,R)-enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the (R,R)-enantiomer in bulk drug samples ranged from 98.1 to 104.4%. The test solution was found to be stable in the mobile phase for 48 h after preparation.

Copper(II) complexes of lipophilic aminoglycoside derivatives for the amino acid enantiomeric separation by ligand-exchange liquid chromatography

In this paper, a new class of ligand-exchange chiral stationary phase (LE-CSP) based on the copper complexes of lipophilic aminoglycoside derivatives was reported. Different stationary phases were developed by coating reversed-phase liquid chromatography supports with three neamine derivatives carrying a lipophilic octadecyl chain at the 4′, 5 and 6 positions, respectively. The enantioselective ability of these LE neamine-based CSPs was evaluated and the 4′-derivative coated column was found to be the most interesting one for the amino acid resolution. The effects of the variation of several chromatographic parameters on the enantioseparation were evaluated in order to identify the analysis optimal conditions.

Optimum operational conditions for chiral separation of tryptophan enatiomers using ligand exchange liquid chromatography

A simple and precise method for chiral separation of tryptophan enantiomers using high performance liquid chromatography with aligand exchange mobile phase was developed. Chiral separation was performed on a conventional C18 column, using a mobile phase that consisted of a water-methanol solution (88∶12, v/v) containing 10 mmol/Ll-leucine and 5 mmol/L copper sulfate as a chiral ligand additive at a flow rate of 1.0 mL/min. This method allowed baseline separation of two enantiomers with a resolution of 1.84 in less than 30 min. The effect of various conditions, including concentration, type of ligand, organic modifier, pH, flow rate, and temperature, on enantioseparation were evaluated and chiral recognition mechanisms were investigated. Thermodynamic data (ΔΔH and ΔΔS) obtained by van't Hoff plots revealed that enantioseparation is an enthalpy-controlled process.

Modeling of Selected Enantioseparation Mechanisms in the Gas and Liquid Chromatography with the Aid of Molecular Mechanics

Three types of diastereomeric complexes formed between low molecular chiral selectors and enantiomeric analytes were modeled in order to simulate enantioseparation of these analytes via the complexation gas chromatography, chiral ligand exchange liquid chromatography and charge transfer complexation liquid chromatography. The diastereomeric complexes were submitted to optimization of geometry and to sequential search of low energy conformations. The differences of the total energy of interaction obtained for the two enantiomeric solutes, R and S, were correlated with the experimental values of the differences in the change in Gibbs free energy. The application of molecular mechanics could facilitate predicting of separation and the elution order of enantiomeric solutes on the accessible, and promote designing of new chiral stationary phases.

Elimination of amino acid interferences in the chiral ligand-exchange chromatographic analysis of lactic acid enantiomers in wine.

Chiral ligand-exchange liquid chromatography is used to identify and quantitate lactic acid enantiomers in wines that have or have not undergone malolactic fermentation. The stationary phase is (R)-penicillamine, which is bound lipophilically to a C18 bonded silica matrix. The mobile phase is 1mM copper sulfate, and the detection mode is ultraviolet. Serious interference from (S)-aspartic acid and other amino acids is eliminated by the use of propanesulfonic acid-type cation exchange solid-phase extraction cartridges prior to chromatographic analysis. Lactic acid enantiomers in wine are quantitated in the range of 10 to 500 mg/L. The detection limit is 3 mg/L. The method is also successful in the determination of lactic acid enantiomers in certain beers (e.g., lambic beers), kim-chi, sauerkraut, and various yogurts.

High-performance ligand-exchange liquid chromatography of fluoro derivatives of alanine

The separation of enantiomers of 3-fluoroalanine, 3,3-difluoroalanine and 3,3,3-trifluoroalanine on ChiralProCu, ChiralValCu and Chiral-1 Nucleosil columns was studied. The substitution of hydrogen for fluorine atoms results in a significant increase in the selectivity of separation of the enantiomers. The relationship between the number of fluorine atoms and the retention of enantiomers on different columns was studied. Optimum conditions for the separation of enantiomers were found.

Chromatographic separation of polyols by ligand exchange: Effects of the ion-exchange resin cross-linking and size

The influence of the degree of ion-exchange resin cross-linking and size on the resolution and purity of products separated by ligand-exchange liquid chromatography was studied with mannitol-sorbitol and arabitol-xylitol mixtures. The experiment was carried out with seven calcium resins. The best results (90% recovery of pure mannitol and sorbitol and 80% recovery of pure arabitol and xylitol) were obtained with an average size of 25 μ and 7% cross-linking.

High-performance ligand-exchange liquid chromatography (HPLEC) of aromatic amines with copper(II), nickel(II) and zinc(II) loaded chelating silica gel columns

An 8-hydroxyquinoline, 8-hydroxy-5-sulphonic-acid-quinoline and a 2-amino-1-cyclopentene-1-dithiocarboxylic acid silica gel, loaded with copper(II), nickel(II) or zinc(II) were examined for the separation of some aromatic amines in the normal phase mode. All phases studied interact strongly with the sample compounds, but the presence of cations increases the selectivity of the columns drastically in some cases. Nickel(II) or zinc(II) loading proved to be better than copper(II) loading, because with these ions strong peak broadening and disturbances of the baseline are avoided. Endcapping of the chelating silica gels was indispensable in order to obtain reasonable efficiencies.

Carbohydrate separation by ligand-exchange liquid chromatography: Correlation between the formation of sugar-cation complexes and the elution order

Carbohydrate separation (hexoses, pentoses and corresponding polyols) was studied by liquid chromatography using ligand exchange on cation-exchange resin column with water as eluent. Seven cations (Ca 2+, Sr 2+, Ba 2+, Pb 2+, Y 3+, La 3+ and Pr 3+) were tested. The carbohydrate elution order is considered in connection with the complexing sites likely to be involved for each sugar or polyol molecule and with the exclusion processes.

Liquid Chromatographic Determination of Cyclopiazonic Acid in Poultry Meat

A liquid chromatographic procedure has been developed for the determination of cyclopiazonic acid (CPA) in poultry meat. CPA is extracted from ground meat with chloroform-methanol (80 + 20), partitioned into 0.1N sodium hydroxide, acidified, and extracted into dichloromethane. An interfering component of meat is removed by transferring the dichloromethane extract to a minicolumn containing silica gel and washing the column with petroleum ether and chloroform. CPA is eluted with methanol-acetic acid (99 + 1), and subjected to ligand-exchange liquid chromatography. Recovery of CPA from 40 separate samples of meat spiked with CPA at levels from 0.016 to 15.6 ppm was 70.4 +/- 14.1%. Analysis of meat from a chicken orally dosed with 10 mg CPA/kg body weight revealed that 14.5% of the dose was in muscle 48 h after administration.

Ligand-exchange high-performance liquid chromatography of aliphatic amines

The separation and retention behaviour of some aliphatic amines in ligand-exchange liquid chromatography has been studied using copper(II)-modified bonded silica gels as the stationary phase. Hexane containing ammonia and methanol as the mobile phase showed good efficiency for separating sample amines. Ammonia has been found to be very effective for improving both the separation and the peak shape. Sample selectivity and the effect of additives in the mobile phase are discussed.

Determination of the two-phase equilibrium constants of copper(II)-modified silica gels used in liquid chromatography

Silica gels modified with copper(II) are currently used in ligand-exchange liquid chromatography. The main species present at the surface of the stationary phase is shown to be (SiO) 2Cu(NH 3) 2(H 2O) x. Measurements of the ammonia concentration in the mobile and stationary phases are combined with computer-assisted calculations to determine the following apparent equilibrium constants: for SiOH + NH 3 ⇌ SiO −NH + 4, log K = −0.2; for (SiO) 2Cu + NH 3 ⇌ (SiO) 2Cu(NH 3), log β 1 = 2.8; and for (z.tbnd;SiO) 2Cu + 2NH 3 ⇌ (SiO) 2Cu(NH 3) 2, log β 2 = 4.0. A distribution diagram of the different species present at the surfac of copper-modified silica gel is given. Under the usual operating conditions for ligand-exchange chromatography (pNH 3 < 1), more than 60% of the silanol groups which fix copper(II) have a silicate structure with two moles of ammonia per mole of copper(II).