High-performance Ligand-exchange Chromatography Research Articles

Chromatographic diagnosis of maple syrup urine disease by measuring the l-alloisoleucine/ l-phenylalanine ratio in dried blood spots

A high-performance ligand-exchange chromatography with ultraviolet detection method for confirmation diagnosis of maple syrup urine disease (MSUD) was developed that relies on the determination of branched-chain amino acids (BCAAs) and Phe levels in blood. The dynamic ranges for the BCAAs and Phe were 50–1000 μM ( r 2 = 0.9982–0.9996) and 74–873 μM ( r 2 = 0.9992) from a dried blood spot, and the BCAA detection limits (S/N = 3) were 0.43–1.91 μM. The mean recoveries of BCAA for intra- and inter-day assays were 92.1–103.0%. The ranges of alloisoleucine (Allo-Ile)/Phe ratio were ND–0.04 and 1.5–2.4 for PKU and MSUD patient samples, respectively. The lowest ratio (1.5) of the MSUD samples was 37.5 times higher than the highest ratio (0.04) of the PKU samples. Therefore, the Allo-Ile/Phe ratio was very useful biomarker for confirmation diagnosis of MSUD.

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.

Analysis of sugars in bee pollen and propolis by ligand exchange chromatography in combination with pulsed amperometric detection and mass spectrometry

The analysis of sugars in the natural products propolis and bee pollen was carried by high-performance thin layer chromatography (HPTLC), high-performance ligand exchange chromatography with pulsed amperometric detection (HPLEC-PAD) and HPLEC coupled to liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) in the positive ion mode. Eleven samples of propolis were analysed, most of which contained no sugars or only traces of sugars. Similar analysis was performed on five samples of bee pollen which were found to contain glucose, fructose and sucrose. These sugars were quantified by HPLEC-PAD. LC–ESI-MS analysis also indicated small amounts of a trisaccharide in the samples. Calibration curves were linear in the range 0.005–0.5 mg/ml and the LOD for all the sugars was <0.005 mg/ml. The positive ESI-MS of the sugars extracted from bee pollen yielded mainly Ca 2+ adducts of sugars, which afforded good sensitivity for their detection.

Molecularly imprinted polymers on silica: selective supports for high-performance ligand-exchange chromatography

Thin coatings of molecularly imprinted, metal-complexing polymers have been grafted to activated silica beads suitable for high-performance liquid chromatography (HPLC). Propylmethacrylate-activated silica particles were coated by copolymerization with a metal-chelating monomer, Cu 2+-[N-(4-vinylbenzyl)-imino]diabetic acid, a metal-coordinating (imidazole) template, and ethylene glycol dimethacrylate. After extraction to remove the template and re-loading with metal, the composite materials re-bind the templates with which they were prepared and exhibit selectivities comparable to bulk-polymerized imprinted materials. The strong Cu 2+-imidazole interaction, desirable for creating a high-fidelity imprint, leads to excessive retention in elution chromatography. By replacing the copper in the imprinted metal-complexing polymers with weaker-binding Zn 2+, these novel ligand-exchange supports can effect partial to complete chromatographic separation of their bis-imidazole templates from other, highly similar imidazole-containing substrates. This “bait-and-switch” approach can significantly enhance the performance of molecularly imprinted materials. Scatchard plots of equilibrium binding data show a significant degree of heterogeneity in the imprinted binding sites of material prepared with a bis-imidazole template, but not with a mono-imidazole template. The best chromatographic separations are observed with small sample sizes, where the substrates occupy the strongest (highest-fidelity) sites.

High-performance ligand-exchange chromatography of some amino acids containing two chiral centres

The separation of 2 S,3 S-, 2 R,3 R-, 2 S,3 R- and 2 R,3 S-isomers of threonine, phenylserine and the o- and p-fluoro derivatives of the latter on chiral sorbents was studied. The sorbent ChiralProCuSi100 gave a complete separation of the four isomers of the amino acids studied.

Efficiency in chiral high-performance ligand-exchange chromatography : Influence of the complexation process, flow-rate and capacity factor

Chromatographic separations of enantiomers by ligand-exchange mechanisms are performed either by using chiral stationary phases (CSPs) or chiral mobile phase additives (CMAs). The two modes differ significantly with respect to their efficiency. It is shown that the process of chelate formation and dissociation at the CSP is the main source of enhanced plate height values when employing CSPs. When using highly soluble CMAs as selector ligands, the rate-determining ligand-exchange process takes place mainly in the mobile phase and therefore does not contribute much to the plate heights. These fundamental differences in the kinetics of the two modes of ligand-exchange chromatography are reflected in different dependences of the plate height data on the flow-rates. It is shown that for CSPs the plate height depends on the structure and configuration of the analyte and the composition of the mobile phase. Within a series of homologous amino acids a pronounced positive correlation is found between the kinetic plate height contribution and the capacity factor. However, this positive correlation is not a general dependence: on changing the capacity factor by changing the eluent composition, a negative correlation is found.

Optical resolution of 2-hydroxy acids by high-performance ligand exchange chromatography

Using an MCl GEL CRS10W column, which was packed with octadecyl silica (ODS) coated with N,N-dioctyl-L-alanine, optical resolution of 2-hydroxy acids was performed. The optical resolution of mandelic acid derivatives and C2 to C5 2-hydroxy acids was carried without any pretreatment. The MCl GEL CRS10W column could resolve amino acids and 2-hydroxy acids. For their selective detection, a post-column method was employed in which the specific color reaction of 2-hydroxy acids with iron(III) ion was utilized. For the analysis of foodstuffs, in which amino acids and 2-hydroxy acids often coexist, the present method was found to be effective in distinguishing between these enantiomers.

Optical resolution of RS-malic acid by high performance ligand exchange chromatography.

Direct chromatographic optical resolution of RS-malic acid without pre-derivatization was successfully achieved for the first time on a ligand exchange chiral column, MCI GEL CRS 10W. Addition of organic solvent, particularly acetonitrile, was effective for obtaining a small capacity factor as malic acid was found to be strongly adsorbed on the column. The degree of resolution (Rs) was raised by lowering the column temperature; however, resolution at room temperature was more suitable as column pressure simultaneously increased. Optical resolution of malic acid in commercial apple juice and soft drinks was also effectively carried out, predicting a high possibility for quality control usage.

High-performance ligand exchange chromatography on chiral diamine-bonded silica gel

The resolution of racemates on chiral chemically bonded silica gel by high-performance ligand-exchange chromatography is described. The stationary phase was prepared by bonding (−) trans 1,2 cyclohexanediamine to silica gel through the coupling agent 3-glycidoxypropyl silane. The resolution of several α-amino acids and of compounds of pharmaceutical interest is reported.

Direct Separation of Enantiomers by High Performance Ligand Exchange Chromatography on Chemically Bonded Chiral Phases

Abstract The application of chemically bonded chiral phases, prepared by binding various chelating ligands to silica gel via 3-glycidoxipropyltrimethoxysilane as spacer, for the separation of enantiomers, is described. The influence of the nature of the matrix, the fixed ligand, the metal ion and the mobile phase on the enantioselectivity is discussed. Examples are given for the application of the system to the resolution of amino acids, amino acid derivatives, dipeptides and hydroxy acids.

Monomeric vs. polymeric bonded iminodiacetate silica supports in high-performance ligand-exchange chromatography

The synthesis of monomeric and polymeric bonded iminodiacetate-groups on silica supports is described. The polymeric support offers advantages over the monomeric, such as (i) preparation is much simpler, (ii) higher surface concentration of functional groups can be achieved without loss of efficiency and (iii) hydrolytic stability. Cu2+ loaded supports were examined for their kinetic performance, in particular for the C-parameter of the Knox equation, and for their hydrolytic stability. Calculation of the surface coverage shows that only a rather thin polymeric layer is formed; this is in agreement with the results of the kinetic performance. The retention of α-amino acids on the Cu2+ loaded polymeric support demonstrates its use in HPLEC.

Enantiomeric resolution of amino acids by high-performance ligand-exchange chromatography using histidine-bonded silica gel

The preparation of histidine-bonded silica gel is described. The formation of an amide bond between aminopropyl silica gel and Boc-Histidine ( imTos) and cleavages of the protecting groups were characterized by IR spectroscopy. This histidine-bonded silica gel was able to resolve enantiomers of many amino acids, irrespective of the type of amino acids. In addition, d, l-mandelic acid was well resolved.

Direct separation of amino enantiomers by high performance ligand exchange chromatography on chemically bonded chiral phases

Various chiral chemically bonded stationary phases for the separation of amino acid enantiomers by high performance ligand exchange chromatography are described. The phases were synthesized by treating silica gel with 3-glycidoxypropyltrimethoxysilane and bonding L-amino acids such as L-azetidine carboxylic acid, L-pipecolic acid or L-phenylalanine to the product. The best results were obtained with L-pipecolic acid as a fixed ligand. Nearly all common amino acid enantiomers could be separated.

Hydrolytically stable chemically bonded silica supports with metal complexating ligands: Synthesis, characterization and use in high-performance ligand-exchange chromatography (HPLEC)

A silica of 14 nm pore size was reacted according to two procedures with the following silanes: γ-aminopropyltriethoxysilane (1), N-aminoethyl-N′-aminopropyltrimethoxysilane (2), N-aminoethyl-N′-aminoethyl-N″-aminopropyltrimethoxysilane (3), N-(3-triethoxysilanepropyl)-N, N-diacetic dimethylester (4), N-(3-trisodiumsilanolatepropyl)-N, N-diacetic acid disodium salt (5) and N-(3-trisodiumsilanolatepropyl)-ethylenediamine-N, N′, N′-triacetic acid trisodium salt (6). The reaction of silanes 1–4 with the silica was carried out under anhydrous conditions (procedure A). Silanes 1–6 were subjected to surface modification under essentially hydrous conditions applying a two-step procedure (procedure B). Procedure B is much simpler to perform and allows binding of high and definite amounts of silane onto the surface. Complexation of all bonded phases with Cu2+ was examined by measuring the sorption isotherms at constant pH. The pH-dependence of Cu2+ uptake at constant Cu2+ concentration was evaluated. Tests on the chemical and chromatographic stability of the packings showed that only the silicas with bonded iminodiacetate and ethylenediaminetriacetate groups remain hydrolytically stable. Retention of aliphatic and α-amino acids on these two packings was found to be mainly controlled by the pH and the ionic strength of the eluent.

Enantiomeric resolution of amino acids by high-performance ligand-exchange chromatography using a chemically modified hydrophilic porous polymer gel

Enantiomeric resolution of amino acids by high-performance ligand-exchange chromatography using a chemically modified hydrophilic porous polymer gel