Preparative separation of fullerene adducts by liquid chromatography on polystyrene gel

Summary The preparative separation of numerous fullerene adducts by liquid chromatography on polystyrene gel is described. These adducts can be resolved on a scale of 10–40 g/day on a 600×20 mm column. Both toluene and chloroform can be used as mobile phases; toluene offers better solubility for the adducts and chloroform better peak resolution.     

Separation of alkylnaphthalenes by preparative liquid chromatography using column switching systems

Summary The paper describes the separation of the mixture of alkynaphthalenes from distillation cuts of a pyrolysis oil, by preparative liquid chromatography on silica. The design of the system permits the connection of the columns to form multicolumn systems.The samples were first separated on a single column. The mixtures were further separated using two-column chromatography systems.The obtained fractions were analyzed by capillary gas chromatography. In most cases a substantial increase in the concentrations of the individual components was achieved. In several cases, pure compounds have been obtained. Separation efficiency increases in the following order: single column, two directly coupled collumns, two-step switching chromatography, heartcutting.     

An expert system for the optimization of columns,operating conditions and instrumentation for high-pressure liquid chromatography

Summary Given the mobile and the stationary phase and values for the physical parameters such as temperature and pH, a separation can be optimized by varying the so-calledchromatographic parameters. These include the column dimensions, particle size, operating conditions (e.g. flow rate, attenuation) and instrumentation (e.g. detector cell, time constant). Optimization of the chromatographic parameters implies finding the best possible set of values, which we define as yielding (i) sufficient separation and (ii) sufficient sensitivity in (iii) the shortest possible time. Finding the best possible conditions (the global optimum) is very difficult for chromatographers in practice.An expert system is described that allows chromatographic optimization to be performed for isocratic separations. An initial chromatogram is required to consult the system. In return, the system provides a complete set of chromatographic parameters, which represents the global optimum within the limits set by the required resolution and signal-to-noise ratio specified by the user. The tolerated flow and pressure ranges, the volume of the available detector cells and the time constant of the detection system are constraints during the optimization. A separate module of the system concerns the sample preparation for pharmaceutical formulations in solid dosages and aqueous solutions.Prototype expert systems have been successfully implemented in the expert-system shell Knowledge Craft on a MicroVAX workstation.     

Preparative liquid chromatography: Choice of column dimensions and working conditions for occasional preparative separation

Summary This paper deals with a theoretical study which makes it possible to calculate column dimensions (length and inner diameter) and working conditions (particle diameter of the stationary phase, elution flow rate, run number) with regard to purity, recovery ratio and the amount of pure substance that may be isolated. This determination takes into account the pressure limitation and pumping capacity of the available chromatograph. The isolation of microgram amounts of a component is shown in order to illustrate the above-mentioned calculation method.Presented at the 14th International Symposium on Chromatography London, September, 1982     

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

Summary 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.     

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.     

Preparative Liquid Chromatography and Centrifugal Partition Chromatography for Purification of New Anticancer Precursors

In organic synthesis, the purification of reactional intermediates or final products is generally carried out by normal phase flash chromatography. However, for dihydrodipyridopyrazines, a new family of antitumor agents, the efficiency of this technique is too low to achieve the purification of the isomer mixture. Purification of the DHDPP isomer mixture has therefore been studied using preparative liquid chromatography. With the stationary phase used in flash chromatography, PLC provides greater efficiency and allows to increase the mobile phase flow rate. A complete study of preparative purification was performed, including that of compounds solubility and analytical optimization. This work has allowed to lower the overlap between the two DHDPP isomers, to greatly reduce the total duration of the process, to increase the purified quantity per run and consequently to greatly improve the throughput of the purification. In addition, this technique can be easily and totally automated. Concurrently, another purification method (centrifugal partition chromatography ), based on acidic constants difference of the two components in two immiscible liquids, was developed. CPC has demonstrated its ability to separate the two DHDPP isomers. Finally, the economic aspects of PLC and CPC results are compared.     

Purification of thymine dimer from monomer solution in preparative liquid chromatography

Summary Cyclobutane pyrimidine dimers and monomers of thymine were separated on a C₁₈ reversed-phase high performance liquid chromatographic column. Using two mathematical models, the effect of the sample sizes on peak shapes in preparative liquid chromatography was investigated. One of these approaches is through a linear kinetic model, and the other is based on the non-linear adsorption isotherm. With the injection of small samples good agreement between the calculated value by the linear kinetic model and experimental, data were achieved. With increased sample size, this model is defective in its prediction of large concentration profiles of sample. However, the nonlinear model permits the accurate prediction of the location of the component band and the determination of the appropriate time to start and stop collection of the enriched fraction at higher concentrations of monomer and the lower concentrations of dimer. Therefore, extremely small amounts of dimer can be extracted from monomer solutions.     

Uptake of hydrophobic amino acids and sodium chloride by a strong cation exchanger: Application to desalting and separation by preparative liquid chromatography

Summary The uptake of sodium chloride, leucine and phenylalanine by a strong cation-exchange resin in the sodium form have been compared. Sodium chloride was excluded from the resin, as indicated by its parabolic upwards curved isotherm, and the corresponding desorption edge was sharpened (constant pattern). Isotherms for both amino acids constructed from a single elution experiment displayed slightly downwards curvature; the corresponding desorption edges spread in contrast to the breakthrough ones. The affinities of both amino acids for the resin were higher (from 15 to 40 %) in the presence of 1 M sodium chloride, indicating that hydrophobic interactions played a significant role in the mechanism of sorption. As expected from the isotherms, a mixture of 1 M sodium chloride, leucine and phenylalanine was separated with satisfactory recovery of both amino acids for a column loading of 10 % of the bed volume. Real mother liquors have also been separated under the same conditions.     

Computer simulation of non-linear preparative chromatography processes

Summary A theory of non-linear chromatography based on the mass balance equation is presented. The model is a system of partial differential equations and it includes axial dispersion in the mobile phase, flow in the mobile phase and mass transfer between phases. In the case of the injection of a mixture competition between components for places in the stationary or the mobile phase is taken into account. In the paper are discussed: numerical calculations of concentration and volume overloading and the effect of the mutual influence of the components of the injected mixture on the process of mass transfer between phases. Additionally, results of experiments for convex and concave adsorption isotherms are presented. In both cases the results obtained qualitatively confirm the theoretical analyses.     

A simplex optimization of the experimental parameters in preparative liquid chromatography

Summary A study of the optimization of the experimental conditions for the purification or extraction of pure compounds by liquid chromatography is presented. Optimum values of the parameters of overloaded elution are derived for maximum production rate, using a Simplex algorithm and the procedure previously described for the simulation of the elution profiles of binary mixtures. The mobile phase flow velocity and the sample size have been optimized together in a first step, simulating the procedure followed in practice, when a column is available. In a second part, the influence of the column length and the average particle size of the packing material on the column performance as well as the trade-offs between the production rate and the yield are discussed.There are three major conclusions in this work. First, the optimum experimental conditions are often very different, depending whether one is primarily interested in the first or in the second eluted component of a mixture. Second, the column efficiency under analytical conditions is very important: it is traded-off for high flow rates, hence short cycle time and increased production rate. Third, the production rate depends strongly on the maximum pressure at which the equipment can be operated. Finally, the optimum production rate varies rather smoothly with the mobile phase velocity and the sample size, so a high yield (70% or more) can usually be obtained with a limited loss in production rate (30 to 60%).     

Evaluation of loop and pump injection systems in preparative liquid chromatography

The usefulness of pump or loop injectors in preparative liquid chromatography was evaluated. The concentration distribution of samples flowing through a UV detector cell, directly connected to the injector, was recorded at various flow velocities, at various diameters of loop tubing, and using various methods of injection [injection with sample volumes equal to a fraction or total loop volume, etc.]. The most advantageous methods were found to be either use of a loop injector to inject only a fraction of its total volume or use of a pump. Both of these methods ensure a almost rectangular concentration distribution.     

Process scale high performance liquid chromatography part IV—Examples of reversed phase separations

Summary The flexibility of process scale high performance liquid chromatography is demonstrated by three examples of reversed-phase separations (a) a standard purification (b) isolation of trace compounds, and (c) recovery from crystallisation mother liquors. It was shown that a material, thought to be acceptably pure, contained a toxic impurity, and a remaining impurity, a previously unseen component of potential therapeutic interest. Favourable cost data were indicated for example (3). This work was conducted in the Chemical Technology Unit, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M6O 1OD, UK.     

Process scale high-performance liquid chromatography Part I: An optimisation procedure to maximise column efficiency

Summary Scale up of analytical High-Performance Liquid Chromatography (HPLC) to the process scale offers promise as a preferred separation method for many high value fine chemicals, both for development purposes and continuous production. It's early adoption is curtailed by the widely held perception that preparative HPLC is a low efficiency high cost technique of limited application. This view has largely beeen generated by the generalised use of large particle size, low quality angular silica supports, and difficulties with packing methodologies. A systematic study of a new silica, in new axially compressed equipment using Plackett-Burman statistics, has demonstrated that very high efficiences, leading to high capacities, can be attained. This work was conducted in the Chemical Technology Unit, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester, M60 1QD, UK.     

Optimization of throughput in preparative column luquid chromatography by column overloading and partial fractionation of the effluent

Summary The criteria for the optimization of preparative chromatography are sample throughput and product purity. It is shown for column liquid chromatography that the throughput has been maximized for a given purity by column overloading and appropriate fractionation. The size and position of the fraction must be determined by chemical analysis, since overlapping peaks under overloading conditions are usually distorted compared to the peaks of the same amounts of pure compounds. With overloading of the column larger particles can be used as column packings without reduction of the separation effect.     

Determination of ephedra alkaloids by high-performance liquid chromatography

Summary Two simple methods were developed for the simultaneous determination of six alkaloids (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine and methyl-pseudoephedrine) inEphedrae Herba by high-performance liquid chromatography. The first method was carried out by using a Cosmosil 5C₁₈-MS column with a gradient solvent system consisting of a phosphate buffer and acetonitrile, and detection at 210 nm. The contents of alkaloids in non-pretreated ephedra herb extracts could be determined easily in 50 min. Alternatively, the alkaloids could be determined within 35 minutes by using a Cosmosil 5C₁₈-MS column with an isocratic solvent system of a sodium dodecyl sulfate-acetonitrile solution. The two methods are compared and discussed.     

Liquid chromatography of gramicidin

Summary A simple, selective method is described for separation of more than 10 gramicidin components on Spherisorb ODS B, 5 μm,250×4.6 mm I.D. column, maintained at 50°C. The mobile phase comprised methanol-water (71:29) at a flow rate of 1.0 mL min⁻¹. Detection was by UV at 282 nm. Valine gramicidins A, B and C were very well separated from the isoleucine gramicidins A, B and C. Four new gramicidin components were also resolved and their structures determined by liquid chromatography/ mass spectrometry. The names 10-methionine valine gramicidin C, 4-methionine valine gramicidin A, valine gramicidin A hydroxypropyl and isoleucine gramicidin A hydroxypropyl were proposed. Robustness of the liquid chromatography method was evaluated by performing a full factorial design experiment. The method also showed good repeatability, linearity and sensitivity.     

An expert system for the selection of criteria for selectivity optimization in high-pressure liquid chromatography

Summary Systematic procedures for the optimization of chromatographic selectivity require objective criteria to characterize the quality of separation in a chromatogram. Numerous criteria have been suggested. Different criteria yield different results and the choice will depend on a large number of factors. It is genuinely difficult to select the most suitable criterion in a particular situation. For these reasons, an expert system has been developed to assist chromatographers in the selection of optimization criteria. A structured representation of the required knowledge and its implementation in an expert-system shell are presented in this paper.     

Direct preparative resolution of racemic amino-acids using ligand exchange chromatography (chirallec)

Summary Preparative resolutions of racemic amino-acids by ligand-exchange chromatography are described. Base-line resolution of multigram quantities of many racemic solutes was obtained using very simple equipment (open tubular column).     

Preparative LC separation and isolation of enantiomerically pure olefins

Summary Diastereomer formation by the reaction of chiral olefins with optically active platinum complexes followed by LC separation of the diastereomers, and on-line or off-line release of the olefins from the complexes by ligand exchange yield optically pure enantiomers.The 100% dextrostetatory enantiomer of exo-2-vinylbicyclo[2.2.1]heptane (vinylnorbornane) could be isolated by an off-line method and was used for polarimetric measurements before and after preparative scale gas chromatographic clean-up.The initial valuable platinum complex could be easily recovered without loss.