The combination of partition, size exclusion, and hydrodynamic models in chromatography, and application to bonded phases on porous supports.

Liquid-liquid partition chromatography has been used for many years as a model and teaching introduction to column chromatography. However, the partition model does not describe separations on bonded phases with porous supports particularly well, especially regarding the thermodynamics controlling solute distribution. Further difficulties arise when more than one mechanism is involved in solute retention. Nomenclature is not perfectly aligned with the underlying thermodynamic descriptors and is inconsistently applied over various chromatographic techniques. Presented here is a general description of retention that spans partition, size exclusion, and hydrodynamic separation processes, and is then applied to bonded-phase separations on porous supports. The model provides a general description applicable to adsorption, reversed-phase, hydrophilic interaction, size-exclusion, hydrodynamic chromatography, and any combination of these techniques including liquid chromatography at the critical condition. Further expansion to include retention by ion-exchange and field-flow fractionation appears to be possible. Recommendations on retention factor definition and evaluation are given.

Maximizing the speed of separations for industrial problems.

Recent improvement efforts in chromatography have provided great improvements in the rate of plate production, but less attention has been spent on optimizing the kinds of problems that are most often encountered in industry. When factors are not independent in their effects on the responses of a chromatographic separation, all adjustable factors must be considered in concert in seeking the best or optimum condition that solves the problem. This requires careful attention to specifying the goals, the adjustable factors, and the constraints required to make sure the outcome can actually be implemented. Strategies for optimizing assay and screening methods in the context of industrial needs are presented. Expanding the factor space of the system being investigated can lead to better outcomes. The prospect of adding column-outlet pressure control and expanding the mobile phase composition to include condensed gases or supercritical fluids is explored. Reversed-phase liquid chromatography, hydrophilic interaction chromatography, electrostatic repulsion hydrophilic interaction chromatography, and supercritical fluid chromatography are contiguous with regard to mobile phase characteristics. Adjustment of selectivity through instrument-controlled factors can benefit method development. Opportunities obtained by blending modifiers, varying temperature and pressure with compressible mobile phases, and controlling pH are discussed in the context of optimizing methods.

Interactions between minimum run time, modifier concentration, and efficiency parameters in a high performance liquid chromatography separation.

We modeled and studied the separation of uracil, nicotinamide, resorcinol, theobromine, theophylline, and caffeine on four C-18 columns of different lengths packed with the same stationary phase using water/methanol mobile phase at one temperature. Predictions of retention times and peak widths were compared with experimental results and were found to be sufficiently accurate for performing optimization calculations. With limits set on the required resolution and on maximum values for pressure and flow rate, calculations were performed for numerous virtual column lengths seeking the smallest possible analysis time for each length while allowing methanol concentration and flow rate to vary as required to minimize run time. Predictions were experimentally verified for the column lengths actually available. These calculations revealed the dependence of best-possible analysis time on column length, modifier concentration, flow rate, and pressure for the real system that was modeled, and provided insight into parameter interactions with respect to analysis times meeting the needs and limits specified. We show that when these parameters are considered in concert, rather than individually, conventional guidelines regarding setting their values may not always lead to the optimum.

A virtual-modeling and multivariate-optimization examination of HPLC parameter interactions and opportunities for saving analysis time.

The interrelations of parameters in HPLC are very complicated, even in a simple problem. Optimization requires considering all the adjustable parameters in concert, but the amount of work required to do this experimentally is prohibitive. However, if we first choose the selectivity parameters, we can then successfully and rapidly perform a multivariate optimization of the efficiency parameters within a numerical model. By examining this process with a level of detail not normally necessary in routine work, we reveal the complexity of parameter interactions in a simple separation, and the potential for large savings of analysis time by properly balancing parameter values. We show how to reduce a 13 min experimental separation to less than 2 min without utilizing ultra-small particles or pressure beyond the capabilities of an ordinary HPLC instrument. Ultra-small particles will often improve analysis times when the separation is plate-number-limited, but if the particles are smaller than optimal for the required separation, then larger particles will require less analysis time.

Determination of pressure-temperature coordinates of liquid-vapor critical loci by supercritical fluid flow injection analysis.

Knowledge of phase behavior as sample is transferred through a chromatograph is necessary for the user to either take advantage of desirable effects, such as peak focusing possibilities, or to avoid disastrous peak broadening. Users staying within the norms of conventional chromatographic techniques may not realize the phase behavior events that might be happening or that might be avoided by virtue of the parameter values they use. However, users working with unconventional conditions or with unconventional fluids, such as near-critical or supercritical fluids, must have an awareness of phase behavior through their chromatograph to ensure success. Complete phase diagrams of binary fluids are rare. However, most chromatographic parameters can be set using only knowledge of the temperature and pressure coordinates of the appropriate critical locus. These coordinates can be quickly determined for Type I binary mixtures using chromatographic equipment and a peak-shape observation technique to perform a simple flow injection experiment. Results and chromatographic applications of this knowledge will be summarized.

Business-objective-directed, constraint-based multivariate optimization of high-performance liquid chromatography operational parameters.

The goal of a separation can be defined in terms of business needs. One goal often used is to provide the required separation in minimum time, but many other goals are also possible. These include maximizing resolution within an analysis-time limit, or minimizing the overall cost. The remaining requirements of the separation can be applied as constraints in the optimization of the goal. We will present a flexible, business-objective-based approach for optimizing the operational parameters of high performance liquid chromatography (HPLC) methods. After selecting the stationary phase and the mobile-phase components, several isocratic experiments are required to build a retention model. Multivariate optimization is performed, within the model, to find the best combination of the parameters being varied so that the result satisfies the goal to the fullest extent possible within the constraints. Interdependencies of parameters can be revealed by plotting the loci of optimal variable values or the function being optimized against a constraint. We demonstrate the concepts with a model separation originally requiring a 54 min analysis time. Multivariate optimization reduces the predicted analysis time to as short as 8 min, depending on the goals and constraints specified.

Techniques for increasing the throughput of flow injection mass spectrometry.

Improvements to the design and operation of a Gilson 215 multiprobe liquid-handling system have resulted in a significant increase in the throughput for flow injection molecular weight characterization of combinatorial chemistry libraries. The rapid injection sequence, and subsequent increased sample throughput, is effected by directing the entire mobile-phase flow through each of the injection loops sequentially while isolating or "dead-ending" the remaining nonactive loops. This mode of operation was accomplished by incorporating column-switching valves prior to and following the set of eight parallel injectors. Analysis rates are achieved without sacrificing the integrity of the flow injection peak profile as baseline resolution is maintained for all samples. Using this system, the total analysis time for a 96-well microtiter plate has been reduced to approximately 5 min.

Books and Software: SFC for all stages.

A review of Supercritical Fluid Chromatography with Packed Columns-Techniques and Applications.

Estimation of liquid-vapor critical loci for CO2-solvent mixtures using a peak-shape method.

Critical-mixture curves for 13 CO2-solvent binary mixtures were estimated using the peak-shape method. Mixture critical points were determined within 1 degrees C and 1 atm. The results for CO2-toluene and CO2-methanol were compared to previously reported data from high-pressure view cell studies. No more than a 3% difference was observed in the data generated by the two different techniques. A few abnormalities encountered while using the peak-shape method are also discussed.

Investigation of carbon dioxide modified supercritical and near supercritical carrier streams for flow injection systems.

Since flow injection (FI) is a dilution technique, efforts have been undertaken to minimize online dilution or dispersion. Solutes in supercritical fluids exhibit increased diffusion coefficients which have been shown to decrease dispersion of the sample zone. This work investigates the use of supercritical fluids (or CO(2) modified fluids) as carrier streams for FI. Both a non-reacting tracer and an online chemical reaction were employed to investigate the behavior of solutes in supercritical and near critical systems. Further, these results are compared to those obtained in the system studied with a conventional carrier stream. Plots of peak response vs% CO(2) modifier increase with a sharp break at moderate modifier composition (20-30%). Plots of peak variance vs% CO(2) modifier show decreased variance with increasing % modifier. The system was also optimized with regards to temperature and pressure. The optimized system displayed improved limits of detection and decreased variance relative to 0% CO(2) modifier carrier streams.

Determination of (dichloromethylene) diphosphonate in physiological fluids by ion-exchange chromatography with phosphorus-selective detection.

An analytical method is presented for the determination of (dichloromethylene) disphosphonate (Cl2MDP) in serum and urine. Cl2MDP is isolated from biological samples by adsorption onto precipitated calcium phosphate. Orthophosphate is separated from Cl2MDP by anion-exchange chromatography using AG 1-X8 resin. Detection is accomplished on-line using a flame photometric detector. Potentially interfering condensed phosphates are removed by acid hydrolysis. Sample handling losses are corrected by monitoring the recovery of a [14C] Cl2MDP spike added to the samples. Determinations of Cl2MDP to concentrations as low as 2 mumol/l are possible. Extension of the method to determine other diphosphonates is discussed.