Ion-interaction Reversed-phase Liquid Chromatography Research Articles

Sequence-dependent separation of trinucleotides by ion-interaction reversed-phase liquid chromatography—A structure-retention study assisted by soft-modelling and molecular dynamics

We studied sequence-dependent retention properties of synthetic 5′-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design–artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence.Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5′-NAN-3′, 5′-ANN-3′, 5′-TGN-3′, 5′-NTA-3′and 5′-NGA-3′ (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5′-NAN-3′ & 5′-ANN-3′ trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5′-TGN-3′, 5′-NTA-3′and 5′-NGA-3′ trinucleotides.

Toward reading the sequence of short oligonucleotides from their retention factors obtained by means of hydrophilic interaction chromatography and ion‐interaction reversed‐phase liquid chromatography

Retention characteristics of selected synthetic 5'-terminal phosphate absent penta-nucleotides containing adenine, guanine, and thymine were studied in relation to their sequence by hydrophilic interaction chromatography and ion-interaction reversed-phase liquid chromatography. The organic solvent content, pH, and buffer concentration in mobile phases were evaluated as influential separation conditions. Data demonstrate that both compared chromatographic modes can be used to separate synthetic penta-nucleotides according to their nucleotide composition. Moreover, reversed-phase liquid chromatography allows separation according to their sequence. We have found a simple linear additive model to describe the retention order in both separation modes in regard to their sequence. In hydrophilic interaction chromatography, the retention behavior is controlled primarily by the hydrophilicity of involved nucleotides and minimally by their sequence position. For reversed-phase liquid chromatography, the nucleotide hydrophobicity plays an important role in their retention properties and the influence of their location in sequence on the retention increases toward the center and decreases toward the termini. Our results show that the penta-nucleotide sequence, and thus its spatial arrangement induced by the surrounding environment, is highly related to the retention properties, so it may be hypothetically used to read the sequence from the retention properties acquired under particular separation conditions.

Determination of vanadium as 4-(2-pyridylazo)resorcinol–hydrogen peroxide ternary complexes by ion-interaction reversed-phase liquid chromatography

The separation and determination of the vanadium(V) ternary complex formed with 4-(2-pyridylazo)resorcinol (PAR) and hydrogen peroxide using ion-interaction reversed-phase high-performance liquid chromatography on a C 18 column has been investigated. The optimal mobile phase was a methanol–water solution (32:68, v/v) containing 3 m M tetrabutylammonium bromide, 5 m M acetic acid and 5 m M citrate buffer at pH 7, with absorbance detection at 540 nm. The stoichiometry of the ternary complex of vanadium at pH 6 in 10 m M acetate buffer using the mole ratio and Job’s method by HPLC indicated that the mole ratio of V(V):PAR:H 2O 2 was 1:1:1. The optimal conditions for precolumn formation of the ternary complex were 10 m M acetate, 7 m M H 2O 2, 0.3 m M PAR, and pH 6. The method gave relative standard deviations of retention time, peak area and peak height for the ternary complex of 0.187, 0.45 and 0.57%, respectively. The detection limit (at a signal-to-noise ratio of 3) for V(V) was 0.09 ng/ml in the digested sample using a 100-μl injection loop (or 0.09 μg/g in the solid fertiliser sample). The method was applied to the analysis of fertilisers (phosphate rocks and nitrogen, phosphorus and potassium fertiliser). The results for vanadium obtained by the HPLC method agreed well with those from magnetic sector inductively coupled plasma MS analysis.

Determination of trace cadmium in environmental water samples using ion-interaction reversed-phase liquid chromatography with fluorescence detection

An ion-interaction reversed-phase liquid chromatographic method has been developed for the determination of cadmium at low μg/l concentrations in environmental water samples. Cadmium and other matrix metals were separated through on-column complexation with 8-hydroxyquinoline sulphonate, using an octadecylsilica column and a mobile phase containing 15% acetonitrile, 10–13 m M tetrabutylammonium hydroxide, 5 m M 8-hydroxyquinoline 5-sulphonic acid and 10 m M acetic acid–acetate buffer (pH 4.8–5.4). Under the above conditions Cd(II) could be easily resolved from excess concentrations of matrix metals and could be detected at concentrations as low as 2 μg/l using fluorescence detection at 500 nm (based upon a 100-μl injection). The method showed a slightly curved detector response over the range of interest [up to 1 mg/l Cd(II)] and was successfully applied to the determination of trace Cd(II) in water samples containing large excesses of Mg(II) and Zn(II) and other matrix metals.

Stability of DTPA and iron(III)-DTPA under laboratory ecotoxicological conditions

The present study assessed the stability of uncomplexed DTPA and Fe(III)-DTPA over 24 h, under experimental conditions similar to those previously used to assess the aquatic toxicity of DTPA and Fe(III)-DTPA (MS synthetic medium, 20°C; wet laboratory water (WLW), 25°C). DTPA was measured using ion-interaction reversed-phase liquid chromatography at 0 h ( t 0) and 24 h ( t 24). DTPA consistently eluted at approximately 3.3 min. Actual DTPA concentrations corresponded well with nominal concentrations, justifying the use of nominal concentrations for previous ecotoxicological bioassays. There was no significant degradation of DTPA either when uncomplexed or ferric-complexed, over 24 h ( P>0.05). In addition, there was no difference in DTPA and Fe(III)-DTPA degradation between MS synthetic medium and WLW ( P>0.05). Therefore, the previously reported decrease in DTPA toxicity, when complexed with iron was concluded not to be due to enhanced DTPA degradation. However, the presence of what appeared to be a degradation product in all Fe(III)-DTPA samples after 24 h, indicated that DTPA might be more susceptible to degradation when complexed with iron, as reported by other researchers.

Monitoring of Aromatic Amine Exposures in Workers at a Chemical Plant With a Known Bladder Cancer Excess

In April 1991, an excess of bladder cancer cases among workers employed at a chemical manufacturing facility in Niagara Falls, NY, was reported. This excess was primarily confined to 708 workers who had ever been employed in the rubber chemicals manufacturing area of the plant, where the aromatic amines aniline and o-toluidine have historically been used. An environmental and biological monitoring survey was conducted to evaluate current exposures to aniline and o-toluidine in the rubber chemicals department. Personal air sampling for aniline and o-toluidine was conducted with the use of a modified Occupational Safety and Health Administration (OSHA) 73 method. Urine samples were collected before and after work (i.e., pre-shift and post-shift, respectively) and stored at -70 degrees C. Base hydrolysis was used to convert acetanilide and N-acetyl-o-toluidine, metabolites of aniline and o-toluidine present in the urine, to the parent compounds. The parent compounds were extracted from the alkaline urine into butyl chloride and then back-extracted from the butyl chloride into aqueous hydrochloric acid. An aliquot of each acidic extract was subjected to ion-interaction reversed-phase liquid chromatography with coulometric electrochemical detection. Hemoglobin (Hb) was extracted from blood and stored at -70 degrees C. For the measurement of adducts of aniline, o-toluidine, and 4-aminobiphenyl (4-ABP), precipitated Hb was dissolved in 0.1 M sodium hydroxide in the presence of recovery standards, and the hydrolysate was extracted with hexane, derivatized with pentafluoropropionic anhydride, and analyzed by gas chromatography-mass spectrometry with negative chemical ionization. A total of 73 workers, including 46 of 64 exposed workers who were employed in the rubber chemicals department and had the potential for exposure to aniline and o-toluidine and 27 of 52 unexposed workers employed in other departments where aniline and o-toluidine were not used or produced, had data available for both aniline and o-toluidine and Hb adducts; 28 of the workers in the former group also had personal air-sampling data. Personal air sample measurements showed that airborne concentrations of aniline and o-toluidine were well within the limits allowed in the workplace by OSHA. Urinary aniline and o-toluidine levels, however, were substantially higher among exposed workers than among unexposed control subjects. The most striking differential was for post-shift urinary o-toluidine levels, which averaged (+/- standard deviation) 2.8 micrograms/L (+/- 1.4 micrograms/L) in unexposed subjects and 98.7 micrograms/L (+/- 119.4 micrograms/L) in exposed subjects (P = .0001). Average aniline-Hb and o-toluidine-Hb adduct levels were also significantly higher (P = .0001) among exposed workers than among unexposed control subjects. Average levels of adducts to 4-ABP, a potential contaminant of process chemicals, were not significantly different (P = .48), although three exposed workers had 4-ABP levels above the range in unexposed workers. The adduct data suggest that, among current workers, o-toluidine exposure substantially exceeds aniline exposure and that 4-ABP exposure, if it occurs at all, is not widespread. These data support the conclusion that occupational exposure to o-toluidine is the most likely causal agent of the bladder cancer excess observed among workers in the rubber chemicals department of the plant under study, although exposures to aniline and 4-ABP cannot be ruled out.

Determination of diethylenetriaminepentaacetic acid in pulp mill effluent by ion-interaction reversed-phase liquid chromatography

A method for analysing diethylenetriaminepentaacetic acid (DTPA) in pulp and paper mill effluent using ion-interaction reversed-phase liquid chromatography is presented. The analysis is based on formation of the iron(III) complex of DTPA and its separation on a C 18 column using 4 m M octylamine in a water-acetonitrile mobile phase. A photodiode array detector is used in the analysis to enable peak purity of the Fe-DTPA peak to be compared between samples and standards. DTPA concentrations ranging from < 1 to 200 mg l −1 may be measured using the technique. Elimination of the FeCl 3 reagent in the sample-preparation stage enabled the measurement of DTPA complexed to iron(III). This feature has been used to demonstrate that a significant proportion of the DTPA present in effluent is present in the iron form. The method has been applied to the analysis of DTPA in pulp and paper mill effluent at concentrations ranging from 1 to 20 mg l −1. Recoveries of DTPA spiked into two sets of samples at levels of 2 and 4 mg l −1 averaged 102% with standard deviations of 12 and 16%, respectively. The use of the method to measure DTPA at various locations in the process has indicated that there was a major reduction in the DTPA concentration in pulp and paper mill effluent across the pulping and effluent treatment processes.

Separation of food-related biogenic amines by ion-interaction reversed-phase high performance liquid chromatography. Tyramine, histamine, 2-phenylethylamine, tryptamine and precursor aminoacids. Application to red wine

Methods for the separation of food-related biogenic amines (histamine, tyramine, 2-phenylethylamine and tryptamine) have been developed based on ion-interaction reversed-phase liquid chromatography.

Determination of nitrite and nitrate in Venice lagoon water by ion interaction reversed-phase liquid chromatography

Ion interaction reversed-phase liquid chromatography with octylammonium orthophosphate as the interacting reagent and a reversed-phase C 18 column was applied to the identification and determination of nitrite and nitrate in Venice lagoon water. Interference by the high chloride concentration was systematically studied and the results obtained with different column packings were compared. With spectrophotometric detection at 230 nm, nitrite at 0.005 mg 1 −1 can be detected and determined even in the presence of 0.70 M chloride. The dependence of the retention time of nitrite on the chloride concentration was studied for two reversed-phase columns with different packings. Concentrations of 0.30 ± 0.05 mg 1 −1 of nitrite and 0.20 ± 0.05 mg 1 −1 of nitrate were found in Venice lagoon water.

Ion-interaction reversed-phase chromatographic method for the determination of gold(I) cyanide in mine process liquors using automated sample preconcentration

Ion-interaction reversed-phase liquid chromatography is applied to the analysis of aurocyanide in liquors drawn from the carbon-in pulp (CIP) cyanidation process for the extraction and recovery of gold from its ores. Cyanide leach solutions containing low parts-per-million levels of gold may be directly analysed using a C 18 column with acetonitrile-water (23:77, v/v) containing tetrabutylammonium ions as the eluent. Tailings solutions containing parts-per-billion levels of gold require sample preconcentration and this may be achieved using a C 18 precolumn modified with the above ion-interaction reagent. An automated preconcentration system is described which permits matrix elimination on the concentrator column and use of a step-gradient to shorten the analysis time. The sample is loaded onto a precolumn equilibrated with acetonitrile-water (20:80, v/v) containing 5 m M tetrabutylammonium ions, and interfering cyano compexes are then selectively removed from the precolumn, after which the gold is eluted through an analytical C 18 column using acetonitrile-water (30:70, v/v) containing the same ion-interaction reagent. Analysis of a CIP tailings solution taken from a working gold mine is illustrated.

Determination of trace levels of gold(I) as its cyano complex by ion-interaction reversed-phase liquid chromatography with on-line sample preconcentration

Conditions chromatographies applicables a l'analyse d'ultratrace de complexes cyano d'or (I), de Paladium (II) et de platine (II) dans des solutions a forte concentration en ions cyanures libres

Induced peaks and the retention mechanism in ion-interaction reversed-phase liquid chromatography of inorganic anions

The mechanism of ion chromatography with a non-polar stationary phase and an hydrophobic ion-interaction reagent in the eluent, to augment the retention of inorganic anions, has been investigated. Ion pairing, ion exchange and double-layer ionic adsorption were examined as possible processes that may control the retention of the anions. The phenomenon of induced (system) peaks is qualitatively explained and related to the retention mechanism. The merits of benzyltributylammonium acetate as an ion-interaction reagent for the analysis of inorganic anions are briefly evaluated and compared with those of some other reagents used for anion analysis.