The importance of dead volume in the optimization of separations in reversed-phase liquid chromatography using temary solvents

Summary Described in this paper is a simple method for optimization of separations in reversed-phase liquid chromatography using ternary solvents. The graphical procedure is based on the linearity of the plots of log k against solvent composition. This relationship was found to depend critically both on the relative composition of the binary solvents used to prepare the ternary mixtures and the method chosen for dead volume determination.     

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

Summary 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.This paper introduces a significant refinement to the already established retention model, which is based on the simple quantitative relationships between acetonitrile and tetrahydrofuran, and the remaining components of the ternary liquid system. The refined model is tested with same experimental data.     

A method for characterization and optimization of reversed-phase liquid chromatographic separations based on the retention behaviour in homologous series

Summary A method based on the use of the members of homologous series as the calibration standards is suggested for prediction of retention in reversed-phase liquid chromatography. The method respects the dependence of retention on the composition of the mobile phase and makes possible distinguishing between the contributions of non-specific (hydrophobic) and specific (polar) interactions to the retention. The selection of calibration homologous series is described, the method is verified for a number of compounds and compared to the calculations based on the model of interaction indexes. The method suggested can be used for prediction of retention in mobile phases with different compositions and for the optimization of separation conditions. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Evaluation of ternary mobile phases for reversed-phase liquid chromatography: Effect of composition on retention mechanism

The effect of varying mobile phase composition across a ternary space between two binary compositions is examined, on four different reversed-phase stationary phases. Examined stationary phases included endcapped C8 and C18, as well as a phenyl phase and a C18 phase with an embedded polar group (EPG). Mobile phases consisting of 50% water and various fractions of methanol and acetonitrile were evaluated. Retention thermodynamics are assessed via use of the van’t Hoff relationship, and retention mechanism is characterized via LSER analysis, as mobile phase composition was varied from 50/50/0 water/methanol/acetonitrile to 50/0/50 water/methanol acetonitrile. As expected, as the fraction of acetonitrile increases in the mobile phase, retention decreases. In most cases, the driving force for this decrease in retention is a reduction of the enthalpic contribution to retention. The entropic contribution to retention actually increases with acetonitrile content, but not enough to overcome the reduction in the enthalpic contribution. In a similar fashion, as methanol is replaced with acetonitrile, the v, e, and a LSER system constants change to favor elution, while the s and c constants change to favor retention. The b system constant did not show a monotonic change with mobile phase composition. Overall changes in retention across the mobile phase composition range varied, based on the identity of the stationary phase and the composition of the mobile phase.     

Computerized optimization of gradient elution conditions with ternary solvent mobile phases in RPLC

Summary In this work, an optimization procedure for gradient RPLC separation, using ternary mobile phases, is described. This procedure requires eight preliminary experiments in gradient elution mode to predict the retention surface for each solute over the whole triangular space. This is followed by computerized calculations to determine the best ternary gradient elution profile with respect to both selectivity and analysis time. The efficiency of this procedure from the point of view of rapidity and of accuracy, is illustrated for the specific separation of twelve phenyl urea herbicides.     

A chromatographic response function for automated optimization of separations

Summary A chromatographic response function is studied that is sensitive to important criteria of analytical performance: selectivity, accuracy, precision and time required to perform the separation. This function can be applied to mixtures of unknown solutes and little computational effort is needed to evaluate the numerical value of the function from the chromatogram. It shows well defined optima at elution strengths that give minimal elution times simultaneously with good separations in high-performance liquid chromatography. No prior chromatographic information characterizing the expected performance of a system is required. It is possible to use this function without a mechanistic model describing the separation process.     

The determination of starting conditions for modifier optimization in reversed-phase HPLC

Summary After a brief introduction of the subject, the paper focusses on the first step in any optimization procedure: the delineation of the parameter space, wherein the global optimum is to be found. For organic modifier optimization in reversed-phase liquid chromatography it is shown that the necessary information can be derived from a single water-methanol gradient. It first yields an estimate of the total number of solutes in the sample, which is vital to define the peak capacity needed to achieve separation at a certain confidence level. Next, the gradient allows the prediction of suitable isocratic methanol binary solvents, and transfer rules formulate the iso-eluotropic composition of the common binary solvents (tetrahydrofuran and acetonitrile). Because all predictions are based on a statistical analysis of a limited data base, attention is given to the practical situation where an actual sample deviates from the average solute behaviour. Such deviations are revealed in the first isocratic run and can be used to arrive at a better estimate of solute retention.     

Convenient estimation of the mobile phase volume for water-rich eluents in reversed-phase liquid chromatography

Summary Oxalodihydroxamic acid is proposed as a UV-detectable substitute for D₂O for the convenient estimation of the mobile phase volume (V_m) of water-rich hydro-organic eluents in reversed-phase liquid chromatography. The retention volume of oxalodihydroxamic acid deviates less than 6% from that of D₂O in 0 to 50% methanol-and acetonitrile-water eluents on C₁, C₈, and C₁₈ reversed-phase supports; it is independent of the amount injected from 10ng to 20μg when monitored at 254nm, although the sensitivity is about four-fold greater at 219 nm. The pH should be maintained between about 2 and 4. The deviation from the D₂O retention volume on supports of typical porosity is attributed, at least in part, to a size-exclusion effect. Simple hydroxamic acids can be used as a homologous series for the estimation of V_m over most of the methanol- and acetonitrile-water concentration ranges by the linearization of retentions of homologous series method. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Retention prediction of large and condensed polycyclic aromatic hydrocarbons in reversed-phase microcolumn liquid chromatography with diphenylsilica stationary phase

The retention behavior of condensed large polycyclic aromatic hydrocarbons has been investigated with diphenylsilica stationary phases in reversed-phase microcolumn liquid chromatography. The results were correlated with two parameters which indicate size and shape of the molecules. Since the resulting equation can be used for retention prediction of large polycyclic aromatic hydrocarbons, computer-assisted “standardless” identification is accomplished for “unknown” compounds contained in the standard.     

A novel pseudo simulated moving bed with solvent gradient for ternary separations

A novel pseudo simulated moving bed was suggested to separate a ternary mixture. A solvent gradient was created to make the solvent strength decreasing from zone II to zone III. Under suitable conditions, the least retained solute A moved forward and the most retained solute C moved backward in zones II and III whereas the medium retained solute B moved forward in zone II but backward in zone III to be trapped in the two zones consequently. Once the columns in zones II and III were saturated with solute B, the solvent dissolving the feed was introduced at the feed port to remove solute A from the raffinate-port and solute C from the extract-port. Finally, solute B was recovered from the extract port by stopping the liquid flow in zone II. This scheme was validated by the successful separation of dihydrocapsaicin from capsaicinoids.     

Computer simulation for the convenient optimization of isocratic reversed-phase liquid chromatographic separations by varying temperature and mobile phase strength

The review summarizes the most recent developments in the field of enantioseparation of chiral drugs using capillary electromigration techniques. The basic principles of enantioseparations in CE are discussed. Recent developments in sample introduction, separation and detection in capillary electrophoresis and capillary electrochromatography are summarized. The applications are arbitrarily divided into the following three groups: (a) racemates and artificial mixtures of enantiomers, (b) drug forms and (c) chiral drugs and their metabolites in biological fluids. Among the various techniques involved the relatively new developments such as CEC in aqueous and nonaqueous buffers, on-line CE-MS coupling, etc. are emphasized.     

An improved optimization procedure for the selection of mixed mobile phases in reversed phase liquid chromatography

Summary The stepwise procedure described previously [1] for the selection of optimal mobile phase compositions in reversed phase liquid chromatography (RPLC) is improved in two ways. The optimization criterion is modified to account for variations in analysis time. The iterative approach to the optimal solvent composition makes a more efficient use of previous data. An example illustrates the resulting gain in speed and accuracy.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Adsorption interaction parameter of polyethers in ternary mobile phases: The critical adsorption line

It is shown that in LC of polymers, the interaction parameter in ternary mobile phases can be described by a plane, which is determined by the dependencies in binary mobile phases. Instead of a critical adsorption point, critical conditions are observed along a straight line of composition between the two critical points in binary mobile phases. Consequently, a separation of block copolymers under critical conditions for one block by an adsorption mechanism for the other block can be achieved in ternary mobile phases of different compositions, which allows an adjustment of the retention of the adsorbing block.     

Mobile phase effects in reversed-phase chromatography VI. Thermodynamic models for retention and its dependence on mobile phase composition and temperature

Summary The physico-chemical framework is examined by comparing the predictions of three models for the combined effects of the composition of the hydroorganic mobile phase and the column temperature on the retention ofn-alkylbenzenes on hydrocarbonaceous bonded stationary phases. The well-mixed model leads to expressions for the dependence of retention on three factors which are equivalent to those derived previously from linear extrathermodynamic relationships. The diachoric model stems from the assumption that the mobile phase is microscopically heterogeneous and the displacement model is identical to the retention model most widely used in chromatography with polar sorbents and less polar solvents. Over limited ranges of mobile phase composition and temperature, each model does describe retention behavior. However, only the wellmixed model describes retention well over the entire range of mobile phase composition and temperature studied here. The success of the well-mixed model, and its limits, give insight into the role of the organic solvent in determining the magnitude of chromatographic retention on non-polar stationary phases with hydro-organic eluents.Dedicated to Professor S. R. Lipsky on the occasion of his 60th birthday.     

Structure-retention and mobile phase-retention relationships for reversed-phase high-performance liquid chromatography of several hydroxythioxanthone derivatives in binary acetonitrile-water mixtures

The reversed-phase high-performance liquid chromatographic (RP-HPLC) behavior of some newly synthesized hydroxythioxanthone derivatives using binary acetonitrile-water mixtures as mobile phase has been examined. First, the variation in the retention time of each molecule as a function of mobile phase properties was studied by Kamlet-Taft solvatochromic equations. Then, the influences of molecular structure of the hydroxythioxanthone derivatives on their retention time in various mobile phase mixtures were investigated by quantitative structure-property relationship (QSPR) analysis. Finally, a unified model containing both the molecular structure parameters and mobile phase properties was developed to describe the chromatographic behavior of the systems studied. Among the solvent properties, polarity/polarizability parameter (π^*) and hydrogen-bond basicity (β), and among the solute properties, the most positive local charge (MPC), the sum of positive charges on hydrogen atoms contributing in hydrogen bonding (SPCH) and lipophilicity index (log P) were identified as controlling factors in the RP-HPLC behavior of hydroxythioxanthone derivatives in actonitrile-water binary solvents.     

Selectivity in reversed-phase separations: general influence of solvent type and mobile phase pH

The influence of the mobile phase on retention is studied in this paper for a group of over 70 compounds with a broad range of multiple functional groups. We varied the pH of the mobile phase (pH 3, 7, and 10) and the organic modifier (methanol, acetonitrile (ACN), and tetrahydrofuran (THF)), using 15 different stationary phases. In this paper, we describe the overall retention and selectivity changes observed with these variables. We focus on the primary effects of solvent choice and pH. For example, transfer rules for solvent composition resulting in equivalent retention depend on the packing as well as on the type of analyte. Based on the retention patterns, one can calculate selectivity difference values for different variables. The selectivity difference is a measure of the importance of the different variables involved in method development. Selectivity changes specific to the type of analyte are described. The largest selectivity differences are obtained with pH changes.     

Optimization of the mobile phase composition in gradient elution reversed-phase HPLC by stochastic prediction

Summary Quantitative analysis of more than ten compounds in a sample generally requires complex mobile phases to optimize the separation of the analytes by gradient elution reversed-phase HPLC. For this purpose, CHEOPS, a software package has been developed from the fully stochastic Computer Chromatogram Simulation Method. Calculation principles and optimization criteria are described. Experimental validation is presented with amino acids and steroids.