Jozef Lehotay

Two-dimensional high performance liquid chromatography for determination of homocysteine, methionine and cysteine enantiomers in human serum

A two-dimensional HPLC system with electrochemical detection was used for determination of homocysteine, methionine and cysteine enantiomers in biological samples. The amino acid separations were not possible only by using a chiral column. The compounds were separated from each other on an achiral column (Purospher RP-18 endcapped 250-4mm, 5μm) and their enantiomers were separated on Chirobiotic TAG (250-4.6mm, 5μm) column in an on-line system. The mobile phase composition and a choice of electrode potentials for detection were investigated. The l-enantiomers always eluted before the d-enantiomers. The proposed method was applied to the analysis of human serum of healthy volunteers and patients with multiple sclerosis. The limit of detection (LOD) and quantitation (LOQ) were defined as the concentration that produced a signal-to-noise ratio (S/N) of 3 and 10. The method LOD values were found to be between 0.05 and 0.50μgmL(-1). The range of LOQ values were between 0.17 and 1.67μgmL(-1), respectively.

Using of molecularly imprinted polymers for determination of gallic and protocatechuic acids in red wines by high performance liquid chromatography

The sorption capacities of gallic- and protocatechuic acid-molecularly imprinted polymers (GA-MIP and PCA-MIP, respectively) and non-imprinted polymer (NIP) have been determined on the piston columns by the frontal analyses (FAs). Mobile phases consisted of MeOH, MeOH/H2O (1:1), 12.5% EtOH or ACN. Solutes concentrations used in FAs were 1μg/mL and 50μg/mL. All sorption capacities were depended on analyte and solvent used. Results obtained from the FAs have shown that both imprinted polymers almost always were preferentially recognized PCA molecule. Only in MeOH, the GA-MIP had ability to recognize its template molecule positively. Surprisingly, in some cases, also the NIP exhibited higher sorption capacities than the MIPs for their templates, e.g. in ACN for GA or in MeOH for PCA. This behaviour indicates that in some solvents, the low affinity sites of the blank polymer can act as strong interacting sites. In the next, prepared MIPs were successfully used as the SPE-sorbents for the extraction and purification of chosen phenolic acids from red wine samples. The recoveries both of MIPs were the highest for PCA, what was in agreement with the experiments carried out in 12.5% EtOH during FAs. Prepared MIP-beads allowed the purification of chosen red wine samples with satisfactory selectivities and high recoveries. The linearity of the method was in the range from 10μg/mL to 70μg/mL and 0.1μg/mL to 4.5μg/mL for GA and PCA, respectively, with the determination coefficients ranging from 0.996-0.999. The LODs (S/N=3) ranged from 0.1μg/mL to 0.4μg/mL. The RSDs for the recoveries varied from 4.0% to 8.1%. The PAs-MIPs and corresponding NIP were also characterized by attenuated total reflectance analysis Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron analysis (SEM).

STUDY OF THE MECHANISM OF ENANTIOSEPARATION. I. CHIRAL ANALYSIS OF ALKYLAMINO DERIVATIVES OF ARYLOXYPROPANOLS BY HPLC USING MACROCYCLIC ANTIBIOTICS AS CHIRAL SELECTORS

The enantiomeric separation of alkylaminoderivatives of aryloxypropanols using macrocyclic bonded chiral stationary phases was studied. Teicoplanin and vancomycin chiral stationary phases were used to separate a large number of derivatives of aryloxypropanol enantiomers by HPLC in the polar-organic mode. By comparison of chromatographic parameters obtained by using both chiral stationary phases (CSPs), aspects of the enantioselective separation mechanism could be discerned. Originally, the polar organic mode was developed for chiral compounds that contained a minimum of two hydrogen bonding groups, resulting in a minimum of two hydrogen bonding interactions to the CSP. This work demonstrated that a combination of one hydrogen bonding interaction and one electrostatic interaction is equally effective. The environment (i.e., functional groups) nearest to the stereogenic center of the aryloxypropanols had the greatest effect on the enantioresolution. Teicoplanin CSPs produced the greatest ΔΔG°s and the best enantiomeric separations of these compounds. The site of a possible electrostatic interaction of these compounds is different from that found for amino acids.

STUDY OF MECHANISM OF ENANTIOSEPARATION. III. THE INFLUENCE OF CARBOHYDRATE MOIETIES OF TEICOPLANIN-BONDED CHIRAL STATIONARY PHASE ON THE SEPARATION OF SOME DERIVATES OF PHENYLCARBAMIC ACID

ABSTRACT Enantiomeric separation of alkoxysubstituted esters of phenylcarbamic acid (local anesthetic drugs) is carried out using high performance liquid chromatography (HPLC) with two chiral stationary phases. The teicoplanin chiral stationary phase—chirobiotic T and the teicoplanin chiral stationary phase without the pendant carbohydrate moieties—chirobiotic teicoplanin aglycone (TAG) were used. The influence of the sugar units, the concentration of the ionic modifiers (diethylamine), the influence of the position and of number of carbon atoms in the analytic alkoxychain substituent, and the composition of the mobile phase; on retention factors and resolution of enantiomers were investigated. By comparison of chromatographic parameters obtained by using both chiral stationary phases, the influence of carbohydrate moieties on the mechanism of chiral separation could be evaluated. The interaction mechanism of the enantiomeric separations is discussed. Better separation, in terms of—greater values of R ij of enantiomers—was achieved with the chirobiotic TAG column.

Determination of Methyl- and Dimethylamine in Waste Water by HPLC

Abstract Two liquid chromatographic methods have been developed for the selective determination of methyl and dimethylamine in waste water after derivatization. The derivatization was made in aqueius medium (pH = 11.2) by phenylizothiocyanate and p-toluenesulphochloride. Examples for the determination of methyl- and dimethylamine in waste waters are shown. The presented methods were proposed for the control of the performance of the purification plant.

Comparison of Several Extraction Methods for the Isolation of Benzoic Acid Derivatives from Melissa officinalis

Abstract Several extraction techniques, such as Soxhlet extraction, solid phase extraction using molecularly imprinted polymer, matrix solid phase dispersion, and supercritical fluid extraction were evaluated for the isolation and purification of phenolic compounds, e.g., benzoic acids from natural samples of Melissa officinalis. The extracts of benzoic acids were analyzed by high performance liquid chromatography (HPLC) in reversed phase modus (C18 column) and under gradient elution (acetonitrile/0.074 mol/L formic acid). The results showed that the recovery rates of gallic acid, p‐hydroxybenzoic acid, protocatechuic acid, gentisic acid, vanillic acid, and syringic acid from biological materials by MSPD was equivalent with and, in fact, higher than that of conventional extraction methods.

MODIFICATION OF THE CHIRAL BONDING PROPERTIES OF TEICOPLANIN CHIRAL STATIONARY PHASE BY ORGANIC ADDITIVES. HPLC SEPARATION OF ENANTIOMERS OF ALKOXYSUBSTITUTED ESTERS OF PHENYLCARBAMIC ACID

The behaviour of teicoplanin-based chiral stationary phase (CHIROBIOTIC T) towards changes in organic and ionic modifiers in mobile phase was investigated in order to deduce suitable conditions for the liquid chromatographic enantioseparations of a series of alkoxysubstituted esters of phenylcarbamic acid. Methanol and acetonitrile were the non-ionic modifiers tested in the mobile phase, while different aliphatic carboxylic acids (formic acid, acetic acid, propionic acid, hexanoic acid) and bases (triethylamine, trimethylamine, diethylamine) were used as ionic modifiers. The influence of the nature and concentration of the modifiers on retention, selectivity, and resolution of enantiomers was investigated. Under these conditions, enantiomeric separations could be obtained for 3- and 4-alkoxysubstituted derivatives. The elution order of enantiomers was also determined.

Selectivity tuning in an HPLC multicomponent separation

Abstract The optimisation of the selectivity of two columns coupled in series has been investigated in HPLC. Two columns with different polarities (PEG and CN) were coupled in series via a T connector and the retention of analytes chromatographed on the column system was tuned by varying the individual mobile phase flows in the coupled columns. The flow rate ratio necessary for receiving an optimum selectivity was calculated on the basis of the measured retention factors on the individual columns. The performance of this method has been studied by the separation of a model mixture of 17 analytes with various functionalities.

Study of the Mechanism of Enantioseparation. IV. Study of Enantioseparation of Some Derivatives of Phenylcarbamic Acid Using π‐Complex Stationary Phase in HPLC

Abstract The Whelk‐O 1 chiral stationary phase (CSP) is an useful column for the high performance liquid chromatographic (HPLC) resolution of enantiomers of 2‐methoxy‐1‐[(4‐methylpiperazino)methyl]ethyl esters of N‐(2‐,3‐, and 4‐alkoxyphenyl)carbamic acid (group of the local anaesthetic drugs) in the polar organic mode and the reversed mode. According to the results of enantiomeric separations in different mobile phases [containing organic modifiers (methanol, acetonitrile, water), with different additives of acetic acid and trimethylamine], it can be postulated that the piperazino part of the phenylcarbamic acid compound is essential because the separation of piperidino, pyrrolidino, and perhydroazepino esters of alkoxy phenylcarbamic acid were not observed. Also the influence of the environment near the analytes stereogenic is important for enantioseparation. A comparison of the enantiomeric elution order and the configuration of the chiral selector shows that R‐(−) enantiomers are more retained on the (R,R) Whelk‐O 1 column and S‐(+) enantiomers are more retained on the (S,S) Whelk‐O 1 column.

Use of a threshold criterion in the computer-assisted optimization of chromatographic separations

A threshold criterion based on the calculation of the degree of overlap of adjacent peaks was used for the computer-based optimization of the binary mobile phase composition in HPLC. The procedure was applied to the chromatographic separation of selected phenylurea herbicides and some of their aniline degradation products. The optimization procedure was based on the modelling of retention, peak widths and peak shapes by the general exponential function, which allows non-symmetrical tailed peaks also to be described. The results from the optimization were presented as a contour map, which gives a good impression of the quality of separation over the whole range of two-dimensional space.