A chemometric investigation of the selectivity of multisolvent mobile phase systems in RP-HPLC

Summary The selectivity of multisolvent mobile phase systems consisting of water, methanol, acetonitrile and tetrahydrofuran was studied when the solvent strength decreases at a constant ratio of the modifiers. From the retention measurements of six benzene derivatives on an octylsilica column at eleven different mobile phase compositions the relation between the logarithm of the capacity factor of each solute and the mobile phase composition was modeled by a quadratic equation. From the models the capacity factors of the solutes were predicted for 3 binary, 3 ternary and 7 quaternary mobile phase systems when the fraction of water decreases and the ratio of the organic modifiers is kept constant for the tenary and quaternary solvent systems. The selectivity factors, alpha, of five pairs of solutes were calculated from the capacity factors and plotted against the solvent strength of the mobile phase systems. The selectivity remained not constant, but varied with the solvent strength: if the water fraction of a multi-solvent system is changed at a constant ratio of the modifiers, not only the solvent strength, but also the selectivity changes. The consequence of this result for optimization strategies is discussed.     

Optimization of a multisolvent composition in RPHPLC: Generalization to non- ideal peaks

Summary When samples contain solutes at very different concentrations, the use of the classical resolution function to express the quality of the separation does not provide a true measure of peak overlap and is therefore unsuitable for optimization. Apart from this case, the validity of the optimum is highly dependent on the accuracy of the predicted retention surfaces and it is essential to restrict the parameter space in which optimum performance is sought. As a result, most interpretive optimization procedures become unreliable for optimizing the composition of multisolvent systems in RPHPLC, particularly for non-ideal peaks. These difficulties can be overcome by the method proposed here: the ability, by means of a criterion, called CRIT, to select a restricted range of composition in order to enhance the accuracy of the response surface is developed for non-ideal peaks. The best composition within this selected range is then determined by using a modified resolution function. The performance of this procedure is discussed with some illustrative examples.     

计算机辅助高效液相色谱梯度洗脱的优化

本文介绍了计算机辅助反相高效液相色谱梯度洗脱优化分离的方法。首先采用混合设计法，通过９个预试验建立目标函数，然后运用双因素（初始流动相组成Ｃ和梯度时间Ｔ）扫描技术，由计算机给出优化分离条件，实验结果和预示完全一致。     

Optimization by isochronal analysis I. Changes in mobile phase composition and velocity

Summary The theory of isochronal analysis (time normalization) has been extended to reversed-phase liquid chromatography. Of the possible manipulation of experimental parameters, the present paper develops a framework for optimizing the resolution by the simultaneous change of mobile phase velocity and composition. For that purpose the resolution equation was written in terms of these two parameters and under the constraint of constant analysis time. This treatment allows plotting the resolution versus the mobile phase composition and velocity. The surfaces thus obtained show the changes that the analyst must perform in order to optimize the resolution. The treatment deals with three possible α dependencies on the amount of organic modifier: constant, increasing and decreasing. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Use of molecular connectivity indices to predict LC retention of dansylamides in six different eluent systems

Summary Molecular connectivity indices (x) through the sixth order were calculated and compared with measured reversed-phase liquid chromatographic retention data of sixteen dansyl derivatives of amines (dansylamides). Retention measurements were performed in the capacity factor, k′, range 1–30 with organic solvent-water eluents using acetone, acetonitrile, ethanol, methanol, 1-propanol and 2-propanol. Mainly valence level indices were selected to describe retention. Indices of different order were selected in the different eluents, suggesting that there are differences in the retention mechanism in these eluents. Retention can be predicted with great accuracy by the molecular connectivity indices, as the high correlations between the calculated and observed retention indicate. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

The role of mobile-phase pH in the determination of catecholamine-related compounds by reversed-phase liquid chromatography with amperometric detection

Summary A study was conducted for the optimization of the reversed-phase high performance liquid chromatographic separation of different types (acidic, basic, amphoteric and neutral) of catechol-related compounds by varying the pH. For pH to be used as the optimizing parameter, its effects on retention, peak width and peak shape had to be investigated. Therefore, the pH dependence of the amperometric detector response, as measured by the oxidation peak height or peak area, was taken into account.     

Effects of first dimension eluent composition in two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC×LC) has received a great deal of attention during the past few years because of its extraordinary resolving power. The biggest advantage of this technique is that very high peak capacities can be generated in a relatively short time. Numerous approaches to maximize the peak capacity in LC×LC have been employed. In this work we investigate the impact of the first dimension mobile phase on selectivity. LC×LC has several potential advantages over one-dimensional LC (1DLC) in that unconventional solvents, at least in reversed-phase LC, can be used. For example, solvents which strongly adsorb in the UV in the first dimension are not problematic in LC×LC. This so because the UV detector is placed after the second dimensional column, as pulses of the first dimension eluent arrive at the second dimensional column, they elute well before the solutes of interest and therefore do not interfere at all with detection of solute peaks. So far, the most widely used solvents in reversed-phase 1DLC are methanol and acetonitrile. However, the "UV advantage" of 2DLC allows us to employ UV active solvents, such as acetone. We compare their differential selectivities to that of acetonitrile for the separation of 23 indole acetic acids of interest in plant biology. We also apply them to the separation of a maize seed extract, a very complex sample. In both sample sets, mobile phase composition can be an important parameter to increase the orthogonality of the two dimensions and thus, to increase the effective peak capacity of LC×LC.     

RP-HPLC as an evaluation method for the behaviour of urinary trypsin inhibitors

Summary Human urine contains a family of trypsin inhibitors. Procedures for their purification and characterization involve laborious techniques and the conclusions are different in the identification of the separated compounds. We report results obtained by applying our RP-HPLC method to some procedures adopted by different authors.     

Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition

Summary An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.     

Computer-assisted optimization of selectivity (mobile phase,pH, and ion concentration) in high-perfomance liquid chromatography

A computer-assisted method is presented for optimization of mobile phase composition in reverse-phase and normal-phase HPLC. The method is based on window diagrams, but only three preliminary tests are required. The method is successfully applied to two examples and there is good agreement between predicted and experimental results. Optimal values for ion concentration and pH in ion chromatography from a published optimization method are compared to values calculated using the computer-assisted method presented in this paper. The same results are obtained, but the method presented here is simpler and faster than previously published one.     

Statistical scanning method for the optimization of gradient elution high performance liquid chromatography

Summary A computer-assisted method is presented for the optimization of separation in gradient elution reversed-phase HPLC. The method is based on a polynomial estimation from nine preliminary experiments according to a two-factor (initial solvent composition C and gradient time T) rectangular design. This is followed by a two-dimension computer scanning technique. Resolution is used as the selection criterion. Good agreement was obtained between predicted data and experimental results.     

Dependence of the methylene selectivity on the composition of hydro-organic eluents for reversed-phase liquid chromatographic systems with alkyl bonded phases

Summary A theoretical treatment is presented which considers differences between the composition of the mobile phase and solvents that are incorporated into the bonded phase via preferential sorption. Equations are derived and used to analyze retention data for various homologs chromatographed under reversed-phase conditions using alkyl bonded phases and combinations of water-methanol, water-acetonitrile and watertetrahydrofuran as mobile phases. In the case of water-methanol the surface phase and bulk mobile phase compositions are similar. However, significant differences in composition between the two phases are observed when binary combinations of water-acetonitrile and water-tetrahydrofuran are used as the cluents.     

A Validated Stability Indicating RP-LC Method for Nitazoxanide, a New Antiparasitic Compound

The present paper describes stability indicating reverse phase high-performance liquid chromatography (RP-HPLC) assay method for nitazoxanide in bulk drugs. The developed method is also applicable for the related substances determination in bulk drug. The drug substance was subjected to stress conditions of hydrolysis, photolysis and thermal degradation. The considerable degradation of nitazoxanide was observed under base and peroxide hydrolysis. The drug was found to be stable in other stress conditions attempted. The chromatographic separation of the drug was achieved on reversed-phase C-18 column. Eluents were monitored on photo-diode array detector at a wavelength of 240 nm. The mobile phase was aqueous 0.005 M tetra butyl ammonium hydrogen sulphate and acetonitrile (45:55, v/v). In the developed HPLC method, resolution between nitazoxanide and its potential impurities, namely Imp-A (5-nitro-1,3-thiazol-2-amine), Imp-B (N-(5-nitro-1,3-thiazol-2-yl) acetamide) and Imp-C (2-{[(5-nitro-1,3-thiazol-2-yl) amino] carbonyl} phenyl 2-(acetyloxy) benzoate) was found greater than three. The developed RP-HPLC method was validated with respect to response function, accuracy, precision, specificity, stability of analytical solutions and robustness. Also to determine related substances and assay determination of nitazoxanide that can be used to evaluate the quality of regular production samples. The developed method can also be conveniently used for the assay determination of nitazoxanide in pharmaceutical formulations.     

Optimization of experimental variables in the dabsyl chloride derivatization of biogenic amines for their determination by RP-HPLC

Summary In the last few years special attention has been paid to the pre-column derivatization of biogenic amines with dabsyl chloride because proper experimental conditions for this reaction are very important. In this study, an experimental design (Doehlert design) was used to optimize the variables involved in the dabsylation of the following amines: histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermidine, and spermine. The optimum experimental conditions for forming the dabsyl derivatives are: reagent concentration, 1.75.10⁻³ M; pH, 8.2; temperature, 70°C; heating time (t _h ), 21 min. Under these conditions good chromatographic repeatability is obtained.     

On the mechanism of solute retention in RP-HPLC systems with B+AB1+AB2 type eluents

Summary Intermolecular interactions between the solute and the moieties constituting the mobile phase significantly contribute to the overall retention pattern of a given solute in a given chromatographic system. In this paper retention of solute is discussed in the case of the B+AB₁+AB₂ type mobile phase, which, in a quasithermodynamic way, can be divided into seven individual moieties. One evaluates the influence of each moiety on solute retention, and refers these regularities to the polarity of the solutes.     

Modelling chromatographic behaviour as a function of pH and solvent composition in RPLC

Summary In this work we establish the basic layout of IONICS, an expert system for optimizing the separation of ionogenic solutes in Reversed-Phase Liquid Chromatography, using the pH and the organic-modifier concentration of the mobile phase as parameters. We also present REMO, a front-end system that automates the retention modelling stage, based on a 9-parameter model. This system uses a scale transformation to suppress several numerical problems previously observed and features a strategy for automatic calculation of an initial approximation to the model optimum. The successful application of this system to a set of seven drugs is described. The final models are accurate and have smaller numerical problems. We also describe the use of a genetic algorithm instead of classical non-linear least-squares for fitting the model to the experimental data. Results indicate that genetic algorithms are a valuable, complementary tool for retention modelling.     

Prediction of Retention in Liquid-Solid Chromatography with Ternary Mobile Phases

The method for the prediction of capacity factors in ternary mobile phases is presented. The adsorption mechanism of retention is considered. The simple theoretical equations are proposed for mobile phases for which the ratio of mole fractions of the weaker solvents remains fixed. The relations between parameters characterizing retention in ternary and binary mobile phases are discussed. The theoretical model is verified for numerous solutes and different mobile phases.     

Ethylammonium Acetate as a Mobile Phase Modifier for Reversed Phase Liquid Chromatography

We have synthesized the low melting hygroscopic ionic liquid ethylammonium acetate (EAA) and characterized a concentrated solution of EAA as a reversed phase mobile phase replacement for organic solvents like methanol. This solution of EAA acts like an organic solvent with a polarity parameter (P=6.0) similar to methanol (P=5.1) but less than water (P=10.2). A test mixture of salicylate, nitrofurantoin, and acetophenone was separated in this order on C1, C4, and C18 columns using an EAA modified aqueous mobile phase. On a C-18 column, the reduction in retention factor for acetophenone from about 20 to 2 as the % EAA is raised from 20–80% is similar to the same trend found using methanol. However, the retention order using methanol is first nitrofurantoin, then salicylate, and finally acetophenone. Log retention factor (k) versus the volume fraction of either EAA or methanol plots were linear indicating the solvent strength parameter for EAA was about 85% of that for methanol. Despite the relatively high viscosity of EAA, plate count values averaged only about 15% less for EAA as compared to methanol using the same mobile phase composition and could be raised by either working at a temperature above ambient or reducing the flow rate. EAA as a modifier allowed for faster separations of water-soluble vitamins on a reversed phase column designed for totally 100% aqueous mobile phases.Acknowledgements This work was supported by a grant from the Miami University Committee on Faculty Research. We thank ES Industries for the gift of the AquaSep column.     

Computer-assisted transposition of conditions in high-performance thin-layer chromatography to high-performance liquid chromatography for the separation of pesticides

Summary A computer-assisted method is presented for mobile phase selection for the optimal separation of pesticides by HPTLC and HPLC. The system is based on a plot of solute retention value and separation criterion vs. binary mobile phase composition for graphic optimization. The result of HPTLC can be transposed to HPLC for optimal separation. The transposition equation is given.