Methanol-water Eluents Research Articles

Evaluation of a transfer technique for direct coupling of reversed-phase liquid chromatography with gas chromatography

A method that allows direct transfer of liquid chromatographic aqueous eluents into capillary gas chromatography, using a programmed temperature vaporizer as interface, was investigated. The method involved both the evaporative and nonevaporative modes of solvent elimination and allowed large fractions of methanol–water eluents to be transferred from LC to GC. The speed of sample transfer was 1800 μl/min and the internal diameter of the column used in the preseparation step was 4.6 mm. Working rules of the method were investigated to improve sensitivity for the direct analysis of trace compounds. Reproducibility was achieved and detection limits ranging from 0.1 to 1.5 μg/ml of ethyl esters covering a wide range of boiling points were obtained.

Prediction of Partition Coefficient and Toxicity for Phenylthio, Phenylsulfinyl and Phenylsulfonyl Acetates

The log K ow values of 27 phenylthio, phenylsulfinyl, and phenylsulfonyl acetates were determined by the shake-flask method. Their capacity factors were determined by RP-HPLC with C18 column and methanol-water eluent. Partial molecular connectivity indices were calculated for these compounds and used to describe their log K ow values and toxicities to Photo-bacterium phosphoreum. Results demonstrated that while both the HPLC results and the molecular connectivity indices (MCls) could be used to predict the K ow for all of the compounds, the MCI method was more accurate. The correlation between toxicity and molecular connectivity index was also significant, and the correlation was better than a previous a and E s analysis.

Solid state NMR and extraction studies on “phenyl”-bonded stationary phases used for solid phase extraction

The solid phase extraction (SPE) and elution of [ 14C]-propranolol from aqueous buffer has been studied for a range of phenyl-bonded SPE materials. Differences were noted in the recovery of the analyte using methanol-water eluents depending upon the manufacturer and whether or not phase had been endcapped. Efficient recoveries of [ 14C]-propranolol were only achieved when triethylamine was added to the eluting solvent as a competing base. Solid state cross polarisation/magic angle spinning (CP/MAS) NMR spectroscopy was used to characterise the phases further, which revealed differences in endcapping between materials as well as differences in the type and extent of cross-linking.

Simultaneous ion-exclusion chromatography-cation-exchange chromatography with conductimetric detection of anions and cations in acid rain waters

A simple, selective and sensitive method was investigated for simultaneously determining anions (Cl −, NO 3 −, SO 4 2−) and cations (Na +, NH 4 +, K +, Mg 2+, Ca 2+) in acid rain and related environmental waters in central Japan. The method involves simultaneous ion-exclusion-cation-exchange chromatography with conductimetric detection on a polyacrylate weakly acidic cation-exchange resin column with a weak-acid eluent. With the weak-acid eluent (tartaric acid) both anions and cations were separated simultaneously, based on ion-exclusion and cation-exchange mechanism. Owing to the presence of H + ions in the tartaric acid eluent, the detector response was positive for the anions and negative for the cations. Using a 5 m M tartaric acid-7.5% methanol-water eluent, good simultaneous separation and detection were achieved in about 30 min. The results indicated an ionic balance of about 100% between the anions (including HCO 3 −) and the cations (including H +).

The calculation of retention and selectivity in reversed-phase liquid chromatography II. Methanol-water eluents

To calculate retention in reversed-phase, high-performance liquid chromatography a method based on the molecular structure of the analyte and the characteristics of sorbents and mobile phases has been employed. Characteristics of different ODS-columns in water-methanol eluents have been determined.

Porous graphitized carbon and octadecylsilica columns in the separation of some monoamine oxidase inhibitory drugs

The retentions of seventeen monoamine oxidase inhibitory drugs (propargylamine derivatives were determined on a porous graphitized carbon (PGC) column using unbuffered methanol-water and acetonitrile-water eluents and on an octadecylsilica column with methanol-water eluents using constant and varying buffer concentrations. Principal component analysis showed that the retention characteristics of the PGC and octadecylsilica columns differ considerably. A marked deviation between the selectivities of methanol and acetonitrile organic modifiers was observed on PGC. The buffer concentration has a significant impact on the retention of drugs on the octadecylsilica column. The type of substitution (heterocyclic, condensed ring, substituted benzene) accounts for the retention differences irrespective of the type of column, eluent and buffer concentration.

Hydrophobicity Parameters Determined by Reversed-Phase Liquid Chromatography. V. Relationship between the Capacity Factor and the Octanol-Water Partition Coefficient for Simple Heteroaromatic Compounds and Their Ester Derivatives.

The log k' (k' : capacity factor) values of various heteroaromatic compounds were measured by reversed-phase high performance liquid chromatography (RPLC) using a Capcell pack C18 column and methanol-water eluents, and the relationship with the log P values (P : 1-octanol-water partition coefficient) was examined as a function of the mobile phase composition. Furans having nonhydrogen-bonding or weakly hydrogen-accepting substituents exhibited a good linear relationship at 50% MeOH concentration. Amphiprotic sibstituents, CONHR, showed an acceleration effect (less retained than nonhydrogen-bonders and hydrogen-acceptors under the condition of equivalent log P), as is often observed in heterocyclic systems. The relationship was more complicated at other methanol contents, due to different retention behavior demonstrated by the ester-type substituents, CO2R and CONR2 (ester effect), To gain more insight into this ester effect, the retention behavior of ester derivatives of various heteroaromatics such as furan, benzofuran, N-Me pyrrole and indole was also studied. The factors affecting the log P-log k' relationship were discussed, and the optimum RPLC condition for predicting the log P value was described.

Simultaneous determination of otilonium bromide and diazepam by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic method for the simultaneous determination of otilonium bromide and diazepam in tablets has been developed. Owing to the residual free silanol groups on the modified silica surface, otilonium bromide eluted from the reversed-phase column without retardation effects, using a methanol-water eluent containing tetramethylammonium bromide and acetic acid. The effects of the tetramethylammonium bromide concentration on the capacity and symmetry factors of otilonium bromide and diazepam were investigated.

Measurement of lipophilicity indices by reversed-phase high-performance liquid chromatography: comparison of two stationary phases and various eluents

Twenty-eight benzene derivatives spanning a broad range of lipophilicities were used as model compounds to examine the optimum stationary phase and eluent conditions for the determination of lipophilic indices by reversed-phase high-performance liquid chromatography. This was assessed by linear regressions comparing published octanol-water partition coefficients with isocratic capacity factors and capacity factors extrapolated to 100% water in the eluent. Methanol-water eluents are always to be preferred to acetonitrile-water and tetrahydrofuran-water eluents. The octadecylsilane (ODS) pahse yielded good correlations especially when a masking agent was added to the eluent, but this introduced an additional experimental variable. The octadecyl-polyvinyl copolymer (ODP) phase was just as satisfactory as the ODS phase without the need for a masking agent, and thus appears to be a valuable alternative.

Hydrophobicity Parameters Determined by Reversed-Phase Liquid Chromatography. II. Dependence of Retention Behabior of Pyrazines on Mobile Phase Composition.

The retention behavior of monosubstituted pyrazines was investigated by comparing the hydrophobic contribution of each substituent (κ) to the capacity factor obtaiend from reversed-phase high-performance liquid chromatography (HPLC), using a C18 capsule-type column and methanol-water eluents of various compositions. The mobile phase dependency of κ showed that the retention mechanism varied with the substituent, especially in mobile phases with very high water content, due to the intervention of the selective solute-solvent interactions. Under such circumstances, the capacity factor (log k') obtaiend from HPLC methods correlated very poorly with the octanol-water partition coefficient (log P). The log k' for pyrazines with only alkyl and/or alkoxy groups was measured and the correlation with log P was examined. An excellent linear correlation was obtained with the eluent containing about 30% methanol. However, at other methanol concentrations, the log k' values of the alkyl and alkoxy derivatives correlated with log P by two separated linear equations. The use of log kW (log k' at 100% water), which has so far been considered to be the best index of hydrophobicity, failed to simulate log P.

On the mechanism of solute retention in reversed-phase HPLC systems with methanol-water eluent

In this paper we consider the possibility of gaining a better insight in the retetion mechanisms of solutes chromatographed in reversed-phase (RP) HPLC systems with the use of methanol-water binary mixtures as the mobile phase. This is done with help of the new retention model presented earlier as the relationship for the retardation coefficient (RF) of solute. The equation constants A, B and C were defined as proportional to the hypothetical retardation coefficients of the solute chromatographed separately in the three theoretically extracted moieties of the mobile phase (i.e. in pure methanol, pure water and the H-bonded associative methanol-water mixture); therefore the message imbedded in their numerical values can refer to the different and often opposite forces acting separately on the solute in a given RP-HPLC system from the side of each mobile phase moiety. An effort to decipher this message was undertaken upon the empirical results obtained for three groups of compounds, i.e., for dithia[3.3]phanes, and mono- and dihydroxyaromatics.

High-performance liquid chromatographic analysis of the new antitumour drug SK&F 104864-A (NSC 609699) in plasma

A high-performance liquid chromatographic (HPLC) assay is described for the determination of the new, investigational antitumour drug SK&F 104864-A and its lactone ring opened form (SK&F 105992). The analytical methodology reported here involves a protein precipitation step with methanol as sample pretreatment procedure. The instability of the drug necessitates that the plasma fraction is obtained within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (-30 degrees C). The methanolic extract can be stored at -30 degrees C for several days without deterioration of the analyses. Stability data of the drug and its lactone ring opened metabolite in plasma and after methanolic extraction are discussed. The parent drug and the metabolite are separated by reversed-phase ion-pair liquid chromatography on a LiChrosorb RP-18 column, using methanol-water eluent (pH 6.0) with sodium dioctylsulphosuccinate (DOSS) as ion-pairing agent and fluorescence detection. The proposed method has been validated and, subsequently, implemented in a phase I clinical trial for pharmacokinetic evaluation of the new cytotoxic agent.

Relationships between the chemical structure and the HPLC capacity factor of gyrase inhibitors and their metabolites in different methanol-water eluents

Relationships between the chemical structure and the HPLC capacity factor of gyrase inhibitors and their metabolites in different methanol-water eluents

Comparison of octadecyl-bonded silica and styrene—divinylbenzene copolymer sorbents for trace enrichment purposes : Fundamental aspects I. Calibration of the stationary phases in pure water

An octadecyl-bonded silica (Partisil ODS-3) and a styrene-divinylbenzene copolymer (PRP-1) with comparable particle sizes, average pore diameters and specific pore volumes were investigated for trace-enrichment purposes. The organic resin leads to systematically higher values of the logarithm of the capacity factor, log k′, whatever the composition of the methanol-water eluent and whatever the solute investigated. Lower retention differences were observed for two alcohols. Accordingly, the correlation of experimental log k′ values obtained for a given eluent composition with calculated hydrophobic log P coefficients leads to one regression line for the C 18 material and two lines (for hydroxylated and non-hydroxylated solutes respectively) for the organic resin. This also applies to the calibration graphs of log P with the weighted linearly extrapolated log k′ w values in pure water. A weaker affinity of the copolymer for hydroxylated compounds can generally account for all the experimental results.

Separation of isomeric compounds as cyclodextrin inclusion complexes on a cyanopropylsilica stationary phase

The retentino behaviour of isomeric compounds (enantiomers, conformers, steroisomers and stuctural isomers) on a cycanopropylsilica stationary phase was investigated using a methanol—water eluent containing α-, β- and γ-cyclodextrins as mobile phase additives. A stronger inclusion effect was observed than on an octadecylsilica. The selectivity of the separation depends on the size of the cavity of cyclodextrins and on the concentration of organic solvent and cyclodextrin in the eluent.

Lipophilicity measurements of protonated basic compounds by reversed-phase high-performance liquid chromatography : I. Relationship between capacity factors and the methanol concentration in methanol-water eluents

The retention behaviour of protonated basic compounds in reversed-phase high-performance liquid chromatography, using methanol-water mixtures as the eluent, is reported. A minimum is found in the relationship between the logarithm of the capacity factor (log k) and the percentage of methanol ( x) in the eluent. The deviation from linearity is postulated to be caused by a dual retention mechanism, namely polar interactions between the solute and eluent molecules in water-poor eluents, and hydrophobic expulsion in water-rich ones. The influence of the pH, p K a and lipophilicity on retention behaviour is also investigated.

Lipophilicity measurements of protonated basic compounds by reversed-phase high-performance liquid chromatography : II. Procedure for the determination of a lipophilic index measured by reversed-phase high-performance liquid chromatography

The lipophilic properties of a series of basic neuropharmacological compounds were determined by reversed-phase high-performance liquid chromatography. Apparent capacity factors (log k x, pH = 7.5) were measured at different compositions of the methanol-water eluent and extrapolated to 100% water as the eluent. While parabolic extrapolation was feasible only for a limited number of compounds, linear extrapolation to log k w values was possible for all 25 drugs investigated. Correction for solute ionization yielded log ▪ values which correlate well with published water-octanol partition coefficient (log P oct) values. The best procedure to obtain log K w values is discussed.

227 Steroid and gonadgtropins levels in artificial menopause

The pathological steroid biosynthesis of a virilizing ovarian tumor was examined via high performance liquid chromatography–radioimmunoassay (HPLC–RIA) determination of the intratissular steroid concentrations. Sex cord-stromal tumor of the ovary was obtained surgically from an 18-year-old female patient with extremely high androst-4-ene-3,17-dione (4-en-dione) and testosterone (Test) blood serum levels. The tissue specimen was extracted with ethyl acetate and the extract was then purified on a C18 mini-column with methanol–water eluents. Steroids were isolated by reversed-phase HPLC on a C18-silica gel column with 51%, 55% and 64% v/v methanol–water eluents. Steroids in the collected eluent fractions were detected by the radioactivity of tritiated internal standards and then quantified by specific RIAs. In the tumor specimen, very high 17α-hydroxyprogesterone (17-OH-Prog; 6300 fmol/g), dehydro-epiandrosterone (2870 fmol/g), androst-4-ene-3,17-dione (3000 fmol/g), testosterone (5700 fmol/g) concentrations, and less progesterone (PROG; 320 fmol/g) and androst-5-ene-3β,17β-diol (5-en-diol; 320 fmol/g), were determined. Tissue levels of 5α-dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α-diol), 5α-androstane-3β,17β-diol (3β-diol), and 17β-estradiol were found to be 71, 20, 28, and 12 fmol/g, respectively. Steroid profile analysis verified a pathological steroid biosynthesis in the ovarian tumor and suggested that the 17α-hydroxylase (17α-H), 17,20-lyase (17,20-L), and 3β-hydroxysteroid dehydrogenase/Δ5–4-isomerase (Δ5-3β-HSD) activities were particularly elevated in this tumorous tissue. Present data demonstrate that the analysis of intratissular steroid profile by a HPLC–RIA method may valuably contribute to the steroidal pathophysiology of endocrine tumors.

228 Ovarian insufficiency as risk factor for endometrial carcinoma

The pathological steroid biosynthesis of a virilizing ovarian tumor was examined via high performance liquid chromatography–radioimmunoassay (HPLC–RIA) determination of the intratissular steroid concentrations. Sex cord-stromal tumor of the ovary was obtained surgically from an 18-year-old female patient with extremely high androst-4-ene-3,17-dione (4-en-dione) and testosterone (Test) blood serum levels. The tissue specimen was extracted with ethyl acetate and the extract was then purified on a C18 mini-column with methanol–water eluents. Steroids were isolated by reversed-phase HPLC on a C18-silica gel column with 51%, 55% and 64% v/v methanol–water eluents. Steroids in the collected eluent fractions were detected by the radioactivity of tritiated internal standards and then quantified by specific RIAs. In the tumor specimen, very high 17α-hydroxyprogesterone (17-OH-Prog; 6300 fmol/g), dehydro-epiandrosterone (2870 fmol/g), androst-4-ene-3,17-dione (3000 fmol/g), testosterone (5700 fmol/g) concentrations, and less progesterone (PROG; 320 fmol/g) and androst-5-ene-3β,17β-diol (5-en-diol; 320 fmol/g), were determined. Tissue levels of 5α-dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α-diol), 5α-androstane-3β,17β-diol (3β-diol), and 17β-estradiol were found to be 71, 20, 28, and 12 fmol/g, respectively. Steroid profile analysis verified a pathological steroid biosynthesis in the ovarian tumor and suggested that the 17α-hydroxylase (17α-H), 17,20-lyase (17,20-L), and 3β-hydroxysteroid dehydrogenase/Δ5–4-isomerase (Δ5-3β-HSD) activities were particularly elevated in this tumorous tissue. Present data demonstrate that the analysis of intratissular steroid profile by a HPLC–RIA method may valuably contribute to the steroidal pathophysiology of endocrine tumors.

Correlation of hammett substituent constants with reversed-phase “ion-pair” liquid chromatographic adsorption parameters of charged organic compounds

Capacity factors for 28 trans-cinnamic, hydrocinnamic, phenylalkylcarboxylic and benzoic acids were determined at nine combinations of pH (3.4, 4.7, 6.0) and ion-interaction reagent concentration (0,2.0, 4.0 m M octylamine hydrochloride) in methanol-water (30:70) eluents at 25.o°C. An ion-interaction model was fit to capacity factor data for each of the solutes. Correlation coefficients were calculated among estimated model parameters, carbon number, σ p,m and π values. For the 28 aromatic monocarboxylic acids studied, the Hammett σ p,m values correlate with estimated p K a values only. Other correlations sugges that the increased retention resulting from the addition of surface-active ion-interaction reagent to th eluent is caused by electrostatic effects at the stationary phase surface.