Determination Of Mexacarbate And Five Of Its Derivatives By High-Performance Liquid Chromatography

Abstract

A simple, sensitive and reliable analytical method has been developed and reported for mexacarbate (4-ditnethylamino-3,5-xylyl N-methylcarbamate) and five of its possible degradation products likely to be found in environmental samples using reversed-phase high-performance liquid chromatography with isocratic and gradient solvent systems. All chromatogram peaks were Identified through comparison to standards. The method has been used to identify and separate the six compounds from a mixture of the standards. It has been evaluated under different column conditions and with different mobile phases. Best resolution of the analytes was obtained by using a gradient solvent system consisting of CH₃CN and H₃O detecting at 200 nm and 30°C using a HP-RP8, 10 μ m, 20 cm 4.6 mm column.     

High Performance Liquid Chromato-Graphic Method For The Determination Of Permethrin Deposits In A Forestry Spray Trial

Abstract

A convenient and sensitive reversed-phase high performance liquid Chromatographie method has been developed for the determination of perraethrin [3-phenoxybenzyl (±)- cis,trans -3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate] insecticide. Various isocratic and gradient mobile phases, consisting of acetonitrile:water (CH₃CN:H₂O) and methanol:water (CH₃OH:H₂O) solvent systems at two flow rates, were tested to separate and quantify the Isoners of permethrin using octadecylsilyl (ODS) (Regis 5μ m) and octylsilyl (OS) (RP-8, 10 μ m) bonded columns. The optimal mobile phase for perraethrin using the ODS column was 70:30 (v/v) CH₃CN:H₂O mixture at flow rates of either 1.0 or 1.5 mL/min. The measurement was done with a UV detector at 200 nm and 50°C. The OS column gave a less satisfactory separation than the ODS. Gradient elution systems examined did not improve the iso-meric separation of perraethrin. Using the method developed, deposit levels obtained on various sampling units during a perraethrin spray trial were analyzed after elution or extraction followed by necessary column cleanup. Minimum levels of detection for permethrin varied from 1 to 3 ng depending on the nature of the sampler used.     

A Simple Solvent Programmer for Liquid Chromatography. I

Abstract

Equations have been derived from which the dimensions of a solvent gradient generator, coupled to open tubular, micro-packed, semi-micro packed or conventional HPLC columns, may be calculated for a desired gradient volume. Packed and open-tubular generators have been considered. Calculations, using the derived equations, predict that a generator of particular dimensions is needed for each column type. These dimensions are practically feasible for all column types except the conventional column.     

HPLC Laser Fluorometric Determination of Amines in Beer

Abstract

A method has been developed for the rapid and accurate determination of the predominant aliphatic amines in beer. Chromatography was performed on a reverse phase C₁₈ column using an acetonitrile-water solvent gradient. Chromatographic detection was facilitated by pre-column derivatization with 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) which fluoresces under visible light (in this case an argon ion laser operating at 488 nm) after reaction with an amine. Response was linear over four decades of concentration with detection limits at approximately five picograms injected.     

Improved Separation of Closely Related Metal Ions by Centrifugal Partition Chromatography

Abstract

Centrifugal Partition Chromatography (CPC) has been used for the analytical scale separations of adjacent trivalent lanthanides. The stationary phase was 0.1 M Cyanex 272 (bis(2,4,4-trimethylpentyl)phosphinic acid) in heptane and the mobile phase was water at the appropriate pH. Baseline separations of the adjacent lanthanides were achieved with an observed column efficiency of 320 /pm 40 theoretical plates. The column efficiency decreased with flow rate, i.e., the normal Van Deemter behavior was observed. The distribution ratios (D) of selected trivalent lanthanides at different pH values were in general in good agreement with the values determined by batch solvent extraction method. The D obtained with CPC differed markedly from the solvent extraction values inn certain cases resulting in a dependence of separation factor (α) of adjacent lanthanides on pH. This anomaly is under further investigation. The number of theoretical plates, selectivity and resolution of adjacent lanthanides obtained with the current system is ssignificantly better than previously reported. We have demonstrated for the first time, that a mixture of light and heavy lanthanides can been efficiently separated in a single run by CPC, by using gradient pH elution.     

Separtion of Menthol Isomers by Normal Phase High Performance Liquid Chromatography (1)

Abstract

An HPLC method has been developed for the separtion of the four isomers of methol using isocaratic and normal phase ethyl acetate/isooctane systems. This method has used to detect and measure these isomers in peppermint techniques. It is more rapid than GC which in addition requires unstable columns for similar analysis. Beacause solvent and column are normal phase and isocratic, the method and sample preparation are very simple.     

A Novel Reverse Phase HPLC Method to Separate and Quantify Androstenedione and Its Stereospecific Hydroxyaromatic Derivatives

Abstract

Reverse - phase High Pressure Liquid Chromatography with a gradient elution on a LiChrocart 250-4 LiChrospher 100 RP-18 column has been used to separate and quantify 7 α-hydroxyandrostenedione (7α-OHA), 6β-hydroxyandrostenedione (6β- OHA), 16α-hydroxyandrostenedione (16α - OHA), 2 β - hydroxyandrostenedione (2 β - OHA), testosterone (T) and androstenedione (A). These steroids are the major products of androstenedione hydroxylation by adult rat liver microsomes. Separation was achieved in 30 minutes by using a linear gradient of acetonitrile (CH³CN) and water in increasing amounts from 30% to 60% of the first solvent (CH₃CN).

This new method compares very favourable with other methods already reported for studying microsomal hydroxylations of androstenedione in different positions of the steroidal skeleton.     

The Effects of an Ionic Mobile Phase Modifier and Thermal History on a Reversed-Phase Liquid Chromatographic System

Abstract

The addition of millimolar quantities of potassium chloride to a totally aqueous mobile phase increased retention and reduced band broadening with an RP-8 column and polar solute as compared to pure water. This effect was minimized after cycling the solvent-conditioned column through a temperature gradient. These effects are explained by a dynamic surface model in which entrapped conditioning solvent as well as imbibed ionic species all play a part in structuring the interfacial region where retention occurs.     

The Use of Perfluorinated Carboxylic Acids in the Reversed-Phase HPLC of Peptides

Abstract

The relative effectiveness of trifluoroacetic acid (TFA), pentafluoropropanoic acid (PFPA), heptafluorobutyric acid (HFBA) and undecafluorocaproic acid (UFCA) as hydrophobic counter-ions in the reversedphase high performance liquid chromatography (RP-HPLC) of peptides was assessed. Four solvent systems were compared each containing 0.01M of a perfluorocarboxylic acid throughout. Twelve standard peptides and proteins were loaded onto the RP-HPLC column which was eluted with a linear gradient of 20-58.4% aqueous acetonitrile over 90 minutes. The retention times of the peptide standards were different in each solvent system. In progressing from TFA to PFPA, HFBA and UFCA all the peptides showed greater retention times. However, the effect was most marked with peptides having the greatest number of basic groups. By exploiting this behaviour a different type of chromatography can be introduced into the RP-HPLC purification of peptides. For instance, column fractions obtained from the use of the TFA solvent system can be re-chromatographed in a solvent system containing HFBA. It is possible by this procedure to purify naturally occurring peptides on the basis of their overall positive charges. At 0.01M each acid solution is sufficiently U.V. transparent to permit monitoring of column effluents at 210 nm. TFA, PFPA, HFBA and UFCA solvent systems are also completely volatile and this property facilitates the bioassay, radioimmunoassay and amino acid analysis of column fractions.     

An Improved Method for the Purification of Hepatic Proliferation Inhibitor by Anion-Exchange HPLC

Abstract

An improved method for the purification of hepatic proliferation inhibitor from rat liver by means of anion-exchange HPLC has been developed. The inhibitor can be purified on an anion exchange HPLC column by using a linear sodium phosphate gradient. The HPLC method allows repeated use of one column and is both rapid and reproducible. The hepatic proliferation inhibitor isolated by this method retains all of its biological activity and is homogeneous as revealed by reverse-phase HPLC.     

Rapid and Simple Technique for the Quantitation of Polyamines in Biological Samples

Abstract

A rapid and simple technique has been developed to quantify putrescine, spermidine, and spermine in biological tissue. The method, based upon several published procedures, involves protein precipitation with perchloric acid followed by dansylation with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride). After extraction on a Waters C₁₈ Sep-Pak cartridge, the samples are analyzed by high pressure liquid chromotography using a step solvent change and a 3μ C₁₈ reverse phase column. The chromotographic conditions allowed complete analysis of the three polyamines within 10 min with a total run time of 13 min (sample injection and re-equilibrium of column). Standard curves were linear up to 1 μg polyamine and the coefficient of variation for the assay ranged from 4% at l μg polyamine per sample to 11% at 50 ng polyamine per sample. The assay is therefore both rapid and simple. Moreover, unlike other available methods, the present technique does not require duel pumps, ion pairing agents, solvent extraction or a gradient control system. The concentrations of putrescine, spermidine and spermine in rat lung, liver and kidney are reported.     

Wine Phenolics: Optimization Of HPLC Analysis

Abstract

Without previous extraction wine phenolics could be analysed by RP-HPLC via direct injection of the wine samples into the column. In order to optimize the analytical procedure the results obtained with two different columns of slightly different polarity and three different gradient elution systems have been compared.

The separated phenolics were further tentatively identified by means of their retention times and UV spectra which were recorded with a Photodiode Array detector     

A Rapid Isolation of Human Chorionic Gonadotropin and Its Subunits by Reversed-Phase High Performance Liquid Chromatography

Abstract

A rapid isolation of human chorionic gonadotropin and its subunits from a commercially available concentrate of human urine has been achieved using reversed-phase high performance liquid chromatography. With μBondapak C₁₈ columns and a gradient employing aqueous trifluoroacetic acid as one solvent and dilute trifluoroacetic acid in acetonitrile as the other, complete separation can be accomplished in one day whereas standard column chromatographic procedures take about two weeks. Specific radioimmunoassays, polyacrylamide gel electrophoresis, and amino acid analyses were used to identify and characterize chromatographic peaks.     

Estimation of Adsorption Layer Capacity by Sandwich Thin-Layer Chromatography

Abstract

The use of sandwich tanks with a capillary solvent delivery system permits the determination of the volume of the developing solvent in the adsorbent layer as well as the position of the solvent demixing front. Therefore, the adsorption isotherms of polar solvents from solutions in nonpolar diluents and the adsorption layer capacities can be determined in a simple manner, analogous to the column technique: Instead of determination of breakthrough volume, the solvent demixing front on the thin layer is localized by means of a series of test dyes whose spots flatten and merge on the steep solvent composition gradient. The method is illustrated for nine aliphatic ketones adsorbed from heptane and benzene solutions. The experimental results indicate different modes of adsorption from solutions in the two diluents; the surface areas corresponding to one solute molecule are also different for symmetrical dialkyl ketones and isomeric methyl-alkyl ketones.     

Rapid HPLC Methods for the Separation and Quantitation of a Mono-, Di-, and Tri-Saccharides Mixture and Applications

Abstract

A simple, rapid method has been developed for the separation and quantitation of mono-, di-, and tri-saccharides. The method utilizes a 30cmx 3.9mm i.d. Microbondapak NH₂ column, refractive index detection and water-acetonitrile elution. Two chromatographic systems are described. The isocratic mode was necessary to develop a procedure. 20 Carbohydrate's retention times were evaluated. To optimize the separation of nine water-soluble sugars, a gradient mode flow-programming was used. Separation was achieved within 28 minutes. The low detection limit (4 micrograms) of the above chromatographic procedure and its different possibilities could be of great interest to the analyst. The method has been successfully applied to quantify the major carbohydrates found in two types of commercial honey.     

HPLC Determination of the Sun-Screen Agent Uvinul T-150 in Cosmetic Products Using Diode-Array Detection

Abstract

A method is described for the quantitative determination of the sun-screen agent Uvinul T-150 in cosmetic products. It is based on a simple extraction into acidic THF and a HPLC determination with gradient elution and diode array detection, using a column packed with 5-μm Lichrosorb RP-18. The detection limit is 10 ng and linearity is observed up to 5μg injected.

Other sun-screen agents, commonly used in the finished products, have been analyzed and their chromatographic parameters are reported. The method has been applied for determiningOvinu1 T-150 and all the compounds considered in some cosmetic samples of different polarity.     

Simultaneous Determination of Maprotiline,Desipramine, and Moclobemide by Reversed-Phase High-Performance Liquid Chromatography and Statistical Optimization

Abstract

A method for separation of three antidepressants, maprotiline, desipramine, and moclobemide, by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated. To find optimal conditions and estimate the impact of individual parameters on the separation, a complete set of 2³ interdependent relationships of the mobile phase composition, temperature, and the volume flow rate were examined. Full separation of the investigated components from a laboratory mixture was achieved on a Supelcosil LC-18 (120 mm × 4.6 mm, 5 µm) column, using two solvent systems, 3% ammonium ion in water/ethanol and acetonitrile, and alternating isocratic gradient–isocratic elution modes. Relevance of the proposed method for therapeutic drug monitoring is anticipated.     

Adsorption of Tetralkyl Ammonium Ions on Reversed-Phase HPLC Columns

Abstract

A systematic study of the influence of several chromatographic parameters upon adsorption of tetralkyl ammonium ions on reversed-phases has been made. Results show that organic solvent content in mobile phase, tetralkyl ammonium hydrophobicity and tetralkyl ammonium concentration in eluent are the most influent variables. A mathematical expression to calculate the adsorbed ion concentration for different combinations of these variables is deduced. Results from this study can be applied in Ion Pair Reversed-Phase Chromatography to calculate column equilibration times, for setting gradient elution separation methods and for the study of retention mechanisms.     

Amino Acid Profiling of Protein Hydrolysates Using Liquid Chromatography and Fluorescence Detection

Abstract

A liquid chromatography procedure is described for separating the amino acids in protein hydrolysates. The proteins are hydrolyzed with hydrochloric acid and an aliquot of the hydrolysate is derivatized with dansyl chloride reagent. The derivatization procedure takes only 2 minutes using a reaction temperature of 100°C. The dansylated amino acids are chromatographed using a reversed-phase C₈ column and a multi-step, nonlinear gradient elution solvent program which is readily achieved using a microprocessor-controlled liquid chromatograph. Chromatography is complete in approximately 40 min. The procedure is useful for characterizing proteins and may also be used to analyse intact dansylated polypeptides. Chromatograms showing the amino acid profile of chymotrypsin, albumin and histone are given.     

High Performance Liquid Chromatographic Determination of Amino Acids in Biological Samples by Precolumn Derivatization with O-Phthaldialdehyde

Abstract

A suitable gradient system has been developed for rapid analysis of amino acids in biological samples using O-phthaldialdehyde as a precolumn derivatizing agent and fluorescence detection. Resolution of 21 amino acids has been accomplished with 3 μm Ultrasphere ODS column by using a multi-step gradient system of two solvents (0.1M sodium acetate, pH 7.2/methanol:tetrahydrofuran) in less than 1 hour. Within-assay and between-assay coefficients of variation of retention times and fluorescence yield show good reproducibility. The fluorometric detection response is linear from 25 to 500 pmoles with a minimum detection limit of less than 1 pmol. High resolution, rapid analysis and high sensitivity of this method facilitates amino acid analysis in samples of less than 1 mg of tissue.