Ternary Mobile Phase Research Articles

Separations of tocopherols and methylated tocols on cyclodextrin-bonded silica

α-, β-, γ-, and δ-tocopherols (methyl-substituted tocols) and 5,7-dimethyltocol were separated by normal-phase HPLC on β- or γ-cyclodextrin-bonded silica (CDS) with fluorescence detection. The HPLC behavior of the tocol components was studied under various mobile phase conditions. Hexane or cyclohexane was used in combination with oxygen-containing solvents (alcohol, ether, and esters) in binary and ternary mobile phases. Capacity factors ( k′) and separation factors (α) for adjacent tocol components were determined. Incorporation of non-polar ethers in the hydrocarbon (hexane or cyclohexane) mobile phases favored the separation of β- and γ-tocopherols with improved α values, which enabled trace analysis of the β-isomer present in soybean oil. Analyte solutes tended to be more strongly adsorbed in mobile phases containing branched-chain alcohols and ethers than in those containing the corresponding straight-chain solvents. Generally, the k′ values obtained with hexane mobile phases or with the β-CDS phase were greater than those observed in HPLC with cyclohexane mobile phases or with the γ-CDS phase.

High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C 8 cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C 18 stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% ( n = 10) for plasma (0.2 μg/ml) and lower than 12.4% ( n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.

Experimental Conditions for HPLC Analysis of Ethoxylated Alkyl Phenol Surfactants in Microemulsion Systems. Part I. Isocratic Mode with Mixed Solvents

Abstract In surfactant–oil–water phase behavior studies, especially those related to enhanced oil recovery, each of the oligomers of commercial polyethoxylated non-ionic surfactants partitions in a different pattern into the two or three phases in equilibrium, and a simple, fast, and accurate method is required to analyze all phases. Up to now, only oil-phase GC was relied upon to determine the partitioning without closing the surfactant inventory. Isocratic normal phase HPLC with a ternary mobile phase (n-heptane–chloroform–methanol 70–10–20) is shown to comply with these requirements to analyze the oligomer distribution in microemulsion, excess oil, and excess water phases of an optimum formulation system.

Liquid chromatographic studies for essential fatty acids on a commercial alkyl phenyl bonded silica column

Retention studies on arachidonic acid 5, 8, 11, 14 all cis eicosatetraenoic acid, C20:4ω6), eicosapentaenoic acid (5, 8, 11, 14, 17 all cis eicosapentaenoic acid, C20∶5ω3) and docosahexaenoic acid (4, 7, 10, 13, 16, 19 all cis docosahexaenoic acid C22∶6ω3) were performed on a commercial μBondapak free fatty acid analysis column. The ternary mobile phase consisting of acetonitrile, water and tetrahydrofuran was used in an isocratic mode with differential refractometry detection. Retention data were mesured at various flow rates with two different, mobile-phase compositions. Capacity factors and enthalpy of adsorption were calculated from the retention data. Finally the retention mechanism is explained.

Retention and peak shape of acidic substances in capillary supercritical fluid chromatography with binary and ternary mobile phases

AbstractAcidic model compounds were chromatographed on a cyanopropyl capillary column using binary and ternary mobile phases with supercritical carbon dioxide as the primary mobile phase component. When a binary mobile phase was used, large amounts (> 15%) of methanol in carbon dioxide were needed to elute the most polar analytes. Formic and citric acid were tested as modifier additives. The addition of formic acid to the eluent resulted in virtually no positive effects on the chromatographic behavior of the acidic substances. Addition of small amounts of citric acid to the modifier did, on the other hand, significantly improve peak shapes of all acidic compounds tested. Retention could then be adjusted by changing the concentration ratio between carbon dioxide and the methanol‐citric acid solution. The effect of the ternary citric acid additive is probably caused by deactivation of stationary phase active sites and possibly also facilitation of solute mass transfer across the boundary between the chromatographic phases. Finally, methanol could be replaced by less toxic ethanol as a modifier in binary and ternary mobile phases, without noticeable effects on the chromatographic behavior.

Use of methyl and ethyl acetate as organic modifiers in reversed-phase high-performance liquid chromatography: Application to impurity control in bulk drug steroids

We assayed methyl and ethyl acetate as organic modifiers for mobile phases used in reversed-phase high-performance liquid chromatography for the removal of impurities from steroids of pharmaceutical interest. By using ternary mobile phases consisting of the esters, water and a third component that was miscible with both and applying systematic experimental methodology, we developed an optimal procedure for the isolation of the compound of interest (9α-fluoroprednisolone acetate) from the impurities resulting from its synthesis in a minimum time. Inclusion of an acetic ester in the reversed-phase high-performance liquid chromatographic mobile phase was found to improve the resolution of chemically similar subtances.

A Rapid Method For Phospholipid Class Separation by HPLC using an Evaporative Light-Scattering Detector

Abstract A high-performance liquid chromatographic method for the separation and quantification of major natural phospholipid classes using an evaporative light-scattering detector is described. Separation and detection of cholesterol, palmitic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin was achieved in less than 15 minutes using a Spherisorb 10 cm long silica column. A simplified ternary mobile phase gradient of hexane, isopropanol and water was used to separate these six standards reproducibly and easily. A crude sample of egg yolk was also analyzed using this method and found to contain mostly cholesterol with small amounts of phosphatidylethanolamine, phosphatidylcholine and sphingomyelin. The evaporative detector gave a stable flat baseline with detection limits for most compounds in the low nanogram range.

Predictive Optimization of the Separation of Phenylurea Pesticides using Ternary Mobile Phase Gradients in Reversed-Phase HPLC

Abstract The method for optimization of ternary mobile phase gradients in HPLC developed earlier is illustrated on a practical example of reversed-phase separation of phenylurea pesticides. Predictive calculation methods were used to optimize subsequently “solvent strength”, “selectivity” and “combined solvent strength - selectivity” gradients. The merits of the three types of ternary gradients for this specific separation problem are discussed to elucidate the basic principles of the optimization strategy. The predicted retention data are compared with the experimental results and the limitations of the approach are shown. The present method makes it possible to reduce significantly the number of experiments and calculations necessary to perform the optimization in comparison to a systematic search of the optimized parametr space.

Adsorption of Solvents and Molecular Interactions in LSC with a Ternary Mobile Phase

Abstract A modification of Jaroniec equation [1] is proposed. Adsorption of all solvents is taken into account. A physical meaning of the slope of the equation is analysed. The effects of solvents adsorption, solvent-solvent and solute-solvent molecular interactions on the retention in LSC with ternary mobile phase are discussed.

The application of capillary SFC, packed column SFC, and capillary SFC‐MS in the analysis of controlled drugs

AbstractMulti–component mixtures of controlled drugs, drug impurities, and adulterants have been analyzed by capillary SFC‐FID, packed column SFC‐UV, and capillary SFC‐MS. Isocratic packed column SFC has been performed with binary and ternary mobile phases using a single syringe pump. The combination of capillary SFC and double focusing MS is described with reference to MS source pressures and the spectra obtained. The use of negative temperature programming in SFC is described.

On Application of the Snyder-Soczewinski Equation for Describing of LSC Process with a Ternary Mobile Phase

Abstract A possibility of an application of the Snyder-Soczewinski relationship for describing of chromato-graphic systems with ternary mobile phases is analysed. the Snyder-Soczewinski type equation is derived for experimental data measured at constant ratio of mole fractions of less polar solvents. A physical meaning of the slope of the relationship is discussed.

Mechanism of liquid adsorption chromatography in thin-layer chromatography with ternary mobile phases

A model of the chromatographic process which takes into account molecular association in the mobile phase is discussed. This model is applied to the study of the association effects in liquid adsorption chromatography with ternary mobile phases. Jaroniec's equation was used to describe the molecular interactions in the systems investigated. The assumed model of the molecular mechanism of the chromatographic process significantly broadens the possibility of intepretation of experimental chromatographic data

Application of HPLC mobile phase optimization methods to the detection of impurities in bulk drug steroids

Some mobile phase optimization methods have been used in developing a Reversed-Phase High-Performance Liquid Chromatography methodology for the determination of related impurities in bulk pharmaceutical steroids. Using two different approaches with binary and ternary mobile phase, the complete separation of 9-α-fluoroprednisolone acetate and its main potential impurities, at low concentration levels, has been achieved. This methodology may be applicable to other steroidal drugs and it is presented as an alternative to the thin-layer chromatography currently prescribed in official methods.

A refined physico-chemical model of solute retention in reversed-phase high-performance liquid chromatography with ternary mobile phases

In our previous publication we have introduced a new model of solute retention in RP-HPLC systems with ternary mobile phases of the B+AB1+AB2 type (B: acetonitrile or tetrahydrofuran; AB1: methanol; AB2: water). That model proposed no stoichiometric differentiation between acetonitrile and tetrahydrofuran, alternatively present in the solvent system; moreover, it made some very rough assumptions only as to the intermolecular interactions among the mobile phase constituents.

Quantitative Determination of Intermediary Metabolism of Heme Biosynthesis

Abstract A sensitive procedure has been developed for the determination of the intermediary metabolites of heme biosynthesis by high-performance liquid chromatography. The method requires a pre-treatment of the reversed phase C18 column with a binary mobile phase containing 0.1 M NaH2PO4 in methanol (18:30, v/v, pH 5.3). The separation of the intermediates including Uro-, Hepta-, Hexa-, Penta-, Copro-, Meso-, Proto- and Zinc-protoporphyrin, is complete with a ternary mobile phase containing 0.1 M NaH2PO4 in methanol and tetrahydrofuran (18:30:16, v/v/v, pH 5.3). The detection limit of the method is in the subpicomole range for the porphyrins. It is an efficient and reproducible method for the simultaneous determination of Zn-protoporphyrin and the intermediary metabolites of heme biosynthesis. The method was applied to study the intermediary metabolites of heme biosynthesis in blood.

Comparison of various stationary phases for normal-phase high-performance liquid chromatography of ethoxylated alkylphenols

Unmodified silica and chemically bonded diol, nitrile and amino phases were tested as column packings for normal-phase liquid chromatographic separations of ethoxylated alkylphenols in mobile phases consisting of aliphatic alcohol(s) and a non-polar hydrocarbon. The performance of unmodified silica is improved when ethanol is used instead of propanol in binary mobile phases or when ethanol—propanol—aliphatic hydrocarbon ternary mobile phases are applied. Diol and nitrile bonded phases show regular retention behaviour only in mobile phases with a low content of propanol, where 6–8 ethoxymers can be resolved in a reasonable time. In propanol-rich mobile phases, a mixed retention mechanism causes a non-linear increase in log k′ with increasing number of oxyethylene units, which hinders the prediction of retention by calculation and deteriorates the separation of the individual oligomers in propanol— n-alkane mobile phases. The amino-bonded stationary phase shows regular retention behaviour even for higher ethoxymers in mobile phases containing high proportions of propanol in n-alkane, so that the capacity factors can be predicted by calculation. The amino-bonded phase offers better separation possibilities for the individual ethoxymers than the other stationary phases tested, in both the isocratic and gradient modes of elution.

Predictive calculation methods for optimization of gradient elution using binary and ternary solvent gradients

Predictive optimization methods in gradient elution liquid chromatography are reviewed. The optimization approach is based on calculations of elution volumes and band widths in various modes of gradient elution liquid chromatography. The calculation makes use of the parameters of retention-mobile phase composition equations determined in a few preliminary isocratic experiments. In liquid chromatography with binary solvent gradients, either a direct calculation method may be used to optimise the gradient profile in order to achieve a desired resolution of a “critical pair” of sampls solutes, or “maps” of the dependence of resolution of the individual pairs of compounds on gradient steepness and initial mobile phase composition can be constructed for an assumed constant gradient volume (time). Calculation methods are also applicable to ternary mobile phase gradients in reversed-phase systems. There, ternary “solvent strength”, “selectivity”, or “combined selectivity-solvent strength” gradients are chosen, depending on the separation problem to be solved. In these systems, construction of resolution “maps” is the method of choice of predictive optimization. The precision of predicted gradient elution data depends on the instrumentation used and on its ability to reproduce accurately the gradient profile programme and on possible deviations of the experimental retention-mobile phase composition plots from the theoretically expected forms of these dependences. The effect of the choice of gradient shape (curvature) and volume on the optimized separation is also addressed.

Physico-chemical modelling of solute retention in reversed-phase HPLC with temary mobile phases

In our previous publications we have established a new physico-chemically grounded retention model valid for the adsorption and partition mechanism of retention using multicomponent mobile phases. Moreover, we have demonstrated its practical usefulness for predicting solute retention in several different chromatographic modes (i.e. in TLC, normal-phase and reversed-phase HPLC) employing binary mobile phases. In this paper we give an ultimate check to the physicochemical correctness of our new retention model, using it for the description of solute retention in reversedphase HPLC systems with ternary mobile phases of the B+AB1+AB2 type. The complexity of these mobile phases and of the possible intermolecular interactions among the consitituent solvents was supposed to rigidly scrutinize the premises of the assumed new approach. We have observed a good agreement between the experimental retention parameter for a number of solutes chromatographed with the use of two different mobile phases of the B+AB1+AB2 type, and the retention parameter calculated according to the assumed model. Therefore, we believe to have sufficiently tested our new approach and confirmed its physico-chemical correctness.

Molecular interactions in liquid adsorption chromatography with mixed mobile phase. Solvation of the chromatographed substance in a ternary mobile phase

The linear Jaroniec equation has been utilized for the description of chromatographic systems in which the test substance molecule is solvated by the solvent molecules. A possibility of the formation of multimolecular solvates in the bulk phase has been confirmed. The measurements were made using liquid adsorption chromatography with ternary mobile phase.

Analysis of Chromatographic Parameters in the Systems with Ternary Mobile Phases. II. Two Polar Solvents in Mobile Phase

Abstract The experimental data are presented on Gibbs triangles in the form of isolines. The effect of change of the type of two polar mobile phase components on the chromatographic parameters of model substances has been investigated. The presence of alcohol in ternary mobile phase leads to formation double or “closed” RF isolines on Gibbs triangles. Such presentation of the experimental data allowed to complex analysis of the chromatographic process in systems with ternary mobile phase. The measurements were made by adsorption thin layer chromatography on silica gel at temperature 293 K.