Comparison of ion pair and buffered mobile phase chromatography of haloacetic acids using direct and indirect UV and fluorescence detection

Reversed-phase high performance liquid chromatographic methods for the analysis of Haloacetic acids have been developed and compared to conventional direct detection methods. Haloacetic acids commonly found in drinking water, including monochloro-, dichloro-, bromo-, iodo- and trichloroacetic acids- have been studied. The ion pairing agent benzyltributylammonium ion was studied in detail using indirect UV and indirect fluorescence detection. Five different competing ions were evaluated to decrease analysis times and lower the detection limit by this new method. The direct detection method utilized an ammonium sulfate buffer and UV detection yielding a detection limit of 100 ppb. The indirect method developed has the advantage of being able to simultaneously analyze UV and non-UV absorbing ions and molecules but requires long equilibration times and demonstrated lower sensitivity than the direct method. ^

Determination of tricyclazole in water using solid phase extraction and liquid chromatography

A method for determination of tricyclazole in water using solid phase extraction and high performance liquid chromatography (HPLC) with UV detection at 230nm and a mobile phase of acetonitrile:water (20:80, v/v) was developed. A performance comparison between two types of solid phase sorbents, the C18 sorbent of Supelclean ENVI-18 cartridge and the styrene-divinyl benzene copolymer sorbent of Sep-Pak PS2-Plus cartridge was conducted. The Sep-Pak PS2-Plus cartridges were found more suitable for extracting tricyclazole from water samples than the Supelclean ENVI-18 cartridges. For this cartridge, both methanol and ethyl acetate produced good results. The method was validated with good linearity and with a limit of detection of 0.008gL-1 for a 500-fold concentration through the SPE procedure. The recoveries of the method were stable at 80% and the precision was from 1.1-6.0% within the range of fortified concentrations. The validated method was also applied to measure the concentrations of tricyclazole in real paddy water.

Degradation studies of organic acids in commercially packed fruit juices: A reverse phase high performance liquid chromatographic approach

Organic acids are important constituents of fruit juices. They render tartness, flavour and specific taste to fruit juices. Shelf life and stability of fruit juices are important factors, which determine their nutritional quality and freshness. In this view, the effect of storage on the concentration of organic acids in commercially packed fruit juices is studied by reverse phase high performance liquid chromatography (RP-HPLC). Ten packed fruit juices from two different brands are stored at 30 C for 24, 48 and 72 hours. A reverse phase high performance liquid chromatographic method is used to determine the concentration of oxalic, tartaric, malic, ascorbic and citric acid in the fruit juices during storage. The chromatographic analysis of organic acids is carried out using mobile phase 0.5% (w/v) ammonium dihydrogen orthophosphate buffer (pH 2.8) on C18 column with UV-Vis detector. The results show that the concentration of organic acids generally decreases in juices under study with the increase in storage time. All the fruit juices belonging to tropicana brand underwent less organic acid degradation in comparison to juices of real brand. Orange fruit juice is found to be least stable among the juices under study, after the span of 72 hours. Amongst all the organic acids under investigation minimum stability is shown by ascorbic acid followed by malic and citric acid.

Monolithic column with zwitterionic stationary phase for capillary electrochromatography

A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by in situ polymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens. The stationary phases have zwitterionic functional groups, that is, both tertiary amine and acrylic acid groups, so the ionization of those groups on the zwitterionic stationary phase was affected by the pH values of the mobile phase, and further affects the strength and direction of the electroosmotic flow (EOF). Separations of alkylbenzenes and polycylic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various types of polar compounds, including phenols, anilines, and peptides, on the prepared column were performed under CEC mode with anodic and cathodic EOF, and different separation selectivities of those polar analytes were observed on the monolithic capillary column by using mobile phases with different pH values.

Separation of enantiomers by nanoliquid chromatography and capillary electrochromatography using a bonded cellulose trisphenylcarbamate stationary phase

A cellulose trisphenylcarbamate-bonded chiral stationary phase was applied to nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) with nonaqueous and aqueous solutions as the mobile phases. Several chiral compounds were successfully resolved on the prepared phase by nano-LC. The applicability of nonaqueous CEC on a cellulose derivative stationary phase was investigated with the organic solvents methanol, hexane, 2-propanol, and tetrahydrofuran (THF) containing acetic acid, as well as triethylamine as the mobile phases. Enantiomers of warfarin and praziquantel were baseline-resolved with plate numbers of 82 300 and 38 800 plates/m, respectively, for the first eluting enantiomer. The influence of applied voltage, concentration of nonpolar solvent, apparent pH, and buffer concentration in the mobile phase on the electroosmotic flow (EOF) and the mobility of the enantiomers was evaluated. Enantioseparations of traps-stilbene oxide and praziquantel were also achieved in aqueous CEC with plate numbers of 111 100 and 107 400 plates/m, respectively, for the first eluting enantiomer. A comparison between nonaqueous CEC and aqueous CEC based on a cellulose trisphenylcarbamate stationary phase was discussed. Pressure-assisted CEC was examined for the chiral separation of praziquantel and faster analysis with high enantioselectivity was acquired with the proper pressurization of the inlet vial.

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column

The mixed mode of reversed phase (RP) and strong canon-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for to and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.

A simple preparation of stationary phase of alkylamide reversed-phase liquid chromatograph for the separation of basic substances

A simple preparation process of alkylamide phase for reversed-phase HPLC (RP-HPLC) is described. The process includes aminopropyltrimethoxysilane firstly reacted with octanoyl chloride, then the intermediate was coupled onto porous silica. The resultant bonded silica has a reproducible ligand surface concentration and homogenous bonded ligand distribution on the porous silica. Characterization of prepared packing was carried out with elemental analysis, solid-state C-13 NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes under methanol/water as binary mobile phase. The results showed that the stationary phase have excellent chromatographic properties and can be efficiently used for the separation of basic compounds.

Detection of menadione sodium bisulfite (vitamin K-3) by reversed-phase high performance liquid chromatography with series dual-electrode amperometric detector

A reversed-phase high-performance liquid chromatography with series dual glassy carbon electrodes for the amperometric detection of water-soluble menadione is described. The complex post-column derivatization reaction and the high background currents were avoided. The menadione sodium bisulfite was reduced at -0.3 V vs. SCE at the upstream (generator) electrode and oxidized at +0.2V vs. SCE at the downstream (collector) electrode. The mobile phase was 0.2moll(-1) HAc-NaAc aqueous buffer (pH 5.50) and 40% (v/v) methanol. The linear response was in the range of 35 ng to 15 mu g, with a detection Limit of 15 ng (S/N=3). The correlation coefficient was 0.9997 (n=6). The electrochemical detection with series dual electrodes has a higher selectivity for menadione (vitamin K-3) compound than with UV detection.

SIMULTANEOUS DETERMINATION OF GUANINE, URIC-ACID, HYPOXANTHINE AND XANTHINE IN HUMAN PLASMA BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH AMPEROMETRIC DETECTION

A reversed-phase high-performance liquid chromatographic method with amperometric detection is described for the separation and quantification of uric acid, guanine, hypoxanthine and xanthine. The isocratic separation of a standard mixture of the compounds was achieved in 5 min on a Spherisorb 5 C-18 reversed-phase column, with a mobile phase of NaH2PO4 (300 mmol dm(-3) pH 3.0)-methanol-acetonitrile-tetrahydrofuran (97.8 + 0.5 + 1.5 + 0.2). Uric acid, guanine, hypoxanthine and xanthine were completely separated, with detection limits in the range 2-20 pmol per injection. The effect of pH and the composition of the mobile phase on the separation are described. The hydrodynamic voltammograms of these compounds were recorded at a glassy carbon electrode. The linear range of the calibration graph for each compound was: uric acid; 1-5000 mu mol dm(-3); guanine, 0.5-2000 mu mol dm(-3); hypoxanthine, 0.1-500 mu mol dm(-3) and xanthine, 0.5-5000 mu mol dm(-3). The within- and between-day precision was good. The uric acid and hypoxanthine content in human plasma was measured using the proposed method. Good recoveries of uric acid (97.9-103%), hypoxanthine (98.0-99.2%), guanine (96.0-98.3%) and xanthine (96.0-102%) were obtained from human plasma. The results of electrochemical detection were in good agreement with those of UV detection.

ELECTROCHEMICAL DETECTION OF COBALT IN HAIR FOLLOWING ON-COLUMN DERIVATIZATION WITH 1-(2-PYRIDYLAZO)-2-NAPHTHOL-6-SULFONIC ACID AND REVERSED-PHASE LIQUID-CHROMATOGRAPHY

A method for the specific determination of cobalt based on reversed-phase liquid chromatography with amperometric detection via on-column complex formation has been developed. A water-soluble chelating agent, 1-(2-pyridylazo)-2-naphthol-6-sulphonic acid (PAN-6S), is added to the mobile phase and aqueous cobalt solutions are injected directly into the column to form in situ the cobalt-PAN-6S chelate, which is then separated from other metal PAN-6S chelates and subjected to reductive amperometric detection at a moderate potential of -0.3 V. Because the procedure eliminates the interference of oxygen and depresses the electrochemical reduction of the mobile phase-containing ligand PAN-6S, by virtue of the quasi:reversible electrode process of the cobalt-PAN-6S complex, a low detection limit of 0.06 ng can be readily obtained. Interference effects were examined for sixteen common metal species, and at a 5- to 8000-fold excess by mass no obvious interference was observed. The feasibility of the method as an approach to the specific analysis of cobalt in a hair sample has been demonstrated.

A novel silica-based column packing for reversed-phase HPLC of basic compounds

A novel bonded phase for reversed-phase HPLC was synthesized in two steps. Octylamine was first reacted with beta-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (beta -ECTS) and then the intermediate product was coupled onto porous silica. The prepared packing was characterized by elemental analysis, solid-state C-13 NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes and methanol-water as binary mobile phase. The results showed that the stationary phase has excellent chromatographic properties and is resistant to hydrolysis between pH = 2 similar to 8. It can be used efficiently for the separation of basic compounds.

Fast analysis of capillary electrochromatography with non-porous ODS as the stationary phase

High-speed capillary electrochromatography was developed on both short and long packed columns with 2 mu m non-porous ODS as the stationary phase. Factors that affect the analysis time of samples, such as voltage, electrolyte concentration, pH and organic modifier concentration in the mobile phase, were studied systematically. Fast analysis of aromatic compounds within 13 seconds was realized with column efficiency of 573,000 plates/m and a R.S.D.% of the retention times of all components in 8 consecutive injections below 1.0%. which demonstrated the high efficiency and high reproducibility of such a technique. In addition, DNPH derived aldehydes and ketones in both standards and environmental samples were separated with high speed.

Capillary electrochromatography with monolithic poly(styrene-co-divinylbenzene-co-methacrylic acid) as the stationary phase

A new kind of monolithic capillary electrochromatography column with poly(styrene-co-divinylbenzene-co-methacrylic acid) as the stationary phase has been developed. The stationary phase was found to be porous by scanning electron microscopy and the composition of the continuous bed was proved by IR spectroscopy to be the ternary polymer of styrene, divinylbenzene, and methacrylic acid. The effects of operating parameters, such as voltage, electrolyte, and organic modifier concentration in the mobile phase on electroosmotic flow were studied systematically, The retention mechanism of neutral solutes on such a column proved to be similar to that of reversed-phase high performance liquid chromatography. In addition, fast analyses of phenols, chlorobenzenes, anilines, isomeric compounds of phenylenediamine and alkylbenzenes within 4.5 min were achieved.

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

Preparation of monolithic silica column with strong cation-exchange stationary phase for capillary electrochromatography

A monolithic silica based strong cation-exchange stationary phase was successfully prepared for capillary electrochromatography. The monolithic silica matrix from a sol-gel process was chemically modified by treatment with 3-mercaptopropyltrimethoxysilane followed by a chemical oxidation procedure to produce the desired function. The strong cation-exchange stationary phase was characterized by its substantial and stable electroosmotic flow (EOF), and it was observed that the EOF value of the prepared column remained almost unchanged at different buffer pH values and slowly decreased with increasing phosphate concentration in the mobile phase. The monolithic silica column with strong cation-exchange stationary phase has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). The column efficiencies for the tested beta-blockers varied from 210,000 to 340,000 plates/m. A peak compression effect was observed for atenolol with the mobile phase having a low phosphate concentration.