Choice of procedures for preparative chromatography

Quantitative criteria, necessary and sufficient, were developed basing on the analysis of asymtotic solutions of border equations in elution sorption dynamics for realization of selectivity inversion for components in a chromatographic system due to the effect of kinetic selectivity. Analysis is suggested for experiments, where the new approach to realization of chromatographic processes using effect of kinetic selectivity allows optimization of preparative separation of biologically active substances. The approach suggested implies shortening of the experiment duration for separation processes, which can be crucial in most systems where components to be isolated are labile, or the process economics suffers considerably due to mobile phase, or energy consumption.     

Selectivity in preparative separations of inorganic electrolytes by size-exclusion chromatography on hypercrosslinked polystyrene and microporous carbons

Preparative-scale separation of concentrated solutions of simplest mineral electrolytes by size-exclusion chromatography was performed on three samples of commercially available microporous hypercrosslinked polystyrene sorbents "Macronet Hypersol" and two experimental samples of activated carbons. Selectivity of separation of a pair of electrolytes was found to be determined by the largest ions in each pair. Fortunately, selectivity rises at higher concentrations of electrolytes, which was explained by exclusion of smaller species from the concentrated solution, i.e., mobile phase, into small pores of the column packing that are inaccessible to large species. The separation of concentrated mixtures revealed another remarkable advantage of the new process - self-concentrating of each of two separated components in the corresponding fractions. Self-concentration is more pronounced for the minor component that occupied less space in the initial mixture. The new method may prove productive in processing pickle bath solutions.     

Long-acting androgens: Analytical and preparative HPLC of testosterone esters

Procedures are described for the normal phase and reverse phase HPLC analysis of testosterone esters having diverse side chains: examples are given of the gram-scale purification of these compounds using two commercial preparative liquid chromatography and an economical and efficient laboratory-assembled preparative liquid chromatograph.     

S-trityl-(R)-cysteine, a powerful chiral selector for the analytical and preparative ligand-exchange chromatography of amino acids

S-trityl-(R)-cysteine [(R)-STC] is the new selector of a dynamically coated, chiral ligand-exchange stationary phase which proved to be highly effective in both analytical and preparative-scale separation of enantiomers of some natural and unnatural underivatized amino acids, with good separation and resolution factors. With the aim of identifying the best chromatographic conditions suitable for the preparative-scale separations, some parameters controlling retention, separation and resolution factors (such as the type and amount of cupric salt and the eluent pH) were investigated. The relatively easy removal of the Cu(II) ions renders this technique suitable for obtaining small amounts of enantiomerically pure samples for preliminary biological evaluations.     

Large volume injection for preparative supercritical fluid chromatography

Summary A large volume injection system for preparative supercritical fluid chromatography is described. The method which is based on the solvent venting technique coupled with dilution of the sample solution consists of three steps. The first step is continuous dilution of the sample solution with liquid carbon dioxide at a controlled flow rate. The second step is solvent removal and solute trapping in a packed trap column. Combination of these two steps results in efficient solvent removal and the volume of sample which can be injected in a single injection becomes virtually unlimited. The third step is transfer and re-concentration of the solutes from the trap column on to the separation column with the pressures of both columns controlled independently; the final step is the separation. With this method, mass overloading behavior has been investigated and preparative separations performed.     

Analytical and preparative methods for β-exotoxin fromBacillus Thuringensis

Summary Theβ-exotoxin fromBacillus thuringensis is a well known insecticide. The bioassay usually employed for its analysis is time-consuming and subject to many disturbances. The previously reported HPLC analysis has been shown to give erroneous results in some cases. A new analysis system has how been designed. The system is based on separation ofβ-exotoxin from the culture broth, and from various preparations containingβ-exotoxin by ion-pairing. For sample preparation in more complex matrices an method employing solid phase extraction was developed. In order to obtain a standard for quantitative analysis a preparative method for the isolation ofβ-exotoxin from concentrated culture broth after successive precipitations was also developed.     

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     

Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds

A fully automated countercurrent chromatography system has been constructed to rapidly screen the commonly used heptane/ethyl acetate/methanol/water solvent system series and translate the results to preparative scale separations. The system utilizes “on‐demand” preparation of the heptane/ethyl acetate/methanol/water solvent system upper and lower phases. Elution‐extrusion countercurrent chromatography was combined with non‐dynamic equilibrium injection reducing the screening time for each heptane/ethyl acetate/methanol/water system to 17 min. The result enabled solvent system development to be reduced to under 2 h. The countercurrent chromatography system was interfaced with a mass spectrometer to allow selective detection of target components in crude medicinal chemistry reaction mixtures. Mass‐directed preparative countercurrent chromatography purification was demonstrated for the first time using a synthetic tetrazole epoxide derived from a routine medicinal chemistry support workflow.     

Some aspects of the preparative separation of enantiomers on chiral stationary phases

Summary In preparative column liquid chromatography, it is necessary to work with conditions of mass overload to obtain high throughput per unit time. The influence of sample mass and of eluent composition on the separation of R,S-1-(1-naphthylethyl)propylamide on chiral 3,5-dinitrobenzoylphenylglycine-silica (Pirkle type stationary phase) was investigated. At a sample load of 200μg of racemate per gram of stationary phase, the column becomes overloaded. At higher sample loads the peaks become triangular; therefore the first peak is always pure up to loads of 10mg g⁻¹, although resolution is low. The purity of the second peak depends on sample mass, but not on the strength (polarity) of the mobile phase. This is due to the effect that peak width, as well as resolution, decrease as the polarity of the eluent increases. In certain cases the polarimeter, used as a detector, can give more information on peak purity than the UV detector.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.     

Improvement of separation in zonal preparative thin-layer chromatography by gradient elution

Summary Complex extracts of the plants Azulan and Hemorigen were separated by zonal micropreparative thin-layer chromatography in sandwich chambers of the ES and DS type which permitted zonal application of large volumes of sample, without auxiliary equipment. Application from the edge of the layer, in the frontal chromatography mode, markedly improved the separation efficiency and capacity owing to displacement effects which narrow the initially broad zones. Further improvement of separation efficiency and purity of fractions, revealed by densitometry, was observed using stepwise gradient elution. This was confirmed by extraction of some of the separated fractions from the layer and rechromatography; the composition of these fractions were generally simpler than for the corresponding isocratic chromatograms.     

Separation of acid whey proteins on the preparative scale by hyperdiffusive anion exchange chromatography

Summary The eluent flow through a fixed bed of a strong anion-exchanger Q Hyper D/F packing has been characterized by mean of the residence time distribution and the separation conditions of acid whey proteins have been established. Myoglobin under non-retaining conditions was used as a test protein because its molecular weight was close to that of α-lactalbumin, the target protein of this study. In the interstitial velocity range of 44–350 cm h⁻¹ a constant reduced height equivalent to a theoretical plate of 13 was observed. Nearly pure fractions of the five main acid whey proteins were obtained on the preparative scale for a gradient slope of NaCl 1 mM mL⁻¹, in the pH range of 6–8 and an interstitial velocity of 127 cm h⁻¹ (flow rate of 2 mL min⁻¹). A separation focused on a pure fraction of α-lactalbumin was achieved at pH 7.5 and was effective up to an interstitial velocity of 500 cm h⁻¹ (flow rate of 8 mL min⁻¹). An indepth characterization of α-lactalbumin by electrospray ionization—mass spectrometry showed that 15% of α-lactalbumin was lactosylated both in the collected fraction and in the acid whey protein concentrate used as feed.     

Methodological aspects of an off‐line combination of preparative isotachophoresis and high‐performance liquid chromatography with mass spectrometry in the analysis of biological matrices

This work reports on some methodological aspects of an off‐line combination of preparative ITP and HPLC with mass spectrometric detection (pITP‐HPLC‐MS) and its potential applications to the analysis of high molecular mass compounds present in complex biological matrices from the analytical chemistry perspective. Lysozyme served as the model analyte and human saliva as the complex biological matrix in this study. A mixture of five low‐molecular mass compounds was found and successfully used in the pITP experiments as discrete spacers to isolate the analyte from the interferents present in the complex biological matrix and to minimize their disturbance effect on the final MS analysis. The experiments at the pITP stage were performed in the cationic mode. On‐column conductivity detectors were used for the detection of ITP zones. Lysozyme was found in the human saliva samples using just deconvolution of the MS data after background correction. The MS data obtained from HPLC‐MS analysis of pITP fractions exhibited the great analytical potential of the combination of pITP‐HPLC‐MS resulting from the ITP clean‐up effect as well as the ITP preconcentration of the analyte present at low concentration levels in complex biological matrices.     

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.     

Fundamental challenges and opportunities for preparative supercritical fluid chromatography

The use of supercritical fluids as mobile phases in chromatography was suggested nearly fifty years ago. In spite of some major potential advantages, this mode of chromatography, generally known as SFC, is only now beginning to be considered by the mainstream community but it still does not yet enjoy a popularity comparable to those of gas or liquid chromatography. This seems to be largely due to a combination of (1) the serious instrumental difficulties that took many years to solve; (2) the complexity of the behavior of supercritical fluids in chromatographic systems when their temperature, pressure, or composition changes; (3) the long-lasting absence of any substantial incentive to use more complex systems, when the simpler and more robust approaches provided by HPLC are available. This situation, however, has begun to significantly change during recent years. The incentive of employing green, sustainable technologies in industrial processes as well as in analyses is increasing. Because mobile phases generally used in SFC tend to be less environmentally harmful and less expensive than those used in HPLC, SFC presents strong economical and regulatory advantages over the latter technique. Added to that, steady advancements in LC techniques in the last three decades has solved many instrumental difficulties related to SFC, which is now taking full advantages of many of these advances. One factor, however, has remained mostly unresolved. A clearer understanding of the physico-chemical behavior of supercritical fluids in preparative chromatographic columns under nonlinear conditions is still needed. This seems to be the main obstacle to the establishment of SFC as a sustainable separation tool. One aim of this review is to highlight these issues in more detail through a survey of the state-of-the-art techniques available for the design and operation of SFC. Another aim is to outline a possible series of investigations, which are necessary to develop a better physical understanding of SFC.     

Enantiomeric separation of racemic sulphoxides on chiral stationary phases by gas and liquid chromatography

Summary The enantiomeric resolution of seven racemic sulphoxides on chiral stationary phases has been investigated by gas and liquid chromatography. In gas chromatography the separations were performed on octakis-(2,6-di-O-pentyl-3-O-butyryl)-γ-cyclodextrin (FS Lipodex-E) and heptakis-(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin (DMP-β-CD). Both stationary phases were suitable for separation of the enantiomers of the sulphoxides. With one exception for each series all racemetes could be resolved on both stationary phases; FS Lipodex-E was more enantioselective than DMP-β-CD, whereas the latter seemed more generally applicable. Liquid chromatographic separations with Chiralcel-OB as stationary phase were significantly improved by optimization of mobile phase composition and temperature. Resolution factors up to R_s=6 were achieved indicating that the improved separations could now be easily used for preparative purposes.     

Improvement in the solvent evaporation injection technique for preparative supercritical fluid chromatography

Summary In supercritical fluid chromatography, the sample load that can be injected using the sample solvent evaporation technique is limited by the volume of the loading precolumn. A multiple sample loading procedure is investigated in order to increase the injectable amount and to optimize injection conditions. This work also deals with optimization of solvent removal conditions.     

Isolation and purification oil glucuronides with preparative anion exchange chromatography in methanol

890 ml of urine of rats treated with [¹⁴C] 205–734 was desalted over an Amberlite XAD-2 adsorption column. The methanol extract dissolved in 10 ml methanol was injected onto an anion exchange column (Nucleosil 10 SB 250 × 16 mm i.d.) equilibrated in methanol. A gradient of decreasing methanol proportion versus a 0.2 M ammonium hydrogen carbonate buffer eluted a pure glucuronide of 205–734. Its structure was established by NMR, MS, UV, and IR analyses. This procedure is simple, rapid, and very selective for the compound of interest.     

One-step multiple component isolation from the oil of Crinitaria tatarica (Less.) Sojak by preparative capillary gas chromatography with characterization by spectroscopic and spectrometric techniques and evaluation of biological activity

Gas chromatographic analysis revealed that the oil of Crinitaria tatarica was rich in sabinene (32.1%), β-pinene (8.8%), and two unknown (M+200) compounds (I) and (II) (21.4% and 3.4%). One-step multiple fractionation of the oil and separation of two unknown constituents were performed using preparative capillary gas chromatography connected to preparative fraction collector system. This combination allowed separation and recover of sufficient quantities of two unknown compounds with high purity from complex oil matrix. Separation conditions (column temperature, cooling temperature, flow rate, injection volume, cut time) were optimized to achieve the best isolation and successful collection. The target compounds were separated from the oil using a HP Innowax (Walt & Jennings Scientific, Wilmington, DE, USA) preparative capillary column in rapid one-step manner with 95.0% purity. Trapping of the isolated compounds in collector system was facilitated by cooling with liquid nitrogen. Structure determination was accomplished by spectral analysis including ultraviolet, nuclear magnetic rezonance, and high-resolution electrospray ionization mass spectrometry. Z- (I) and E-artemidin (II) were isolated for the first time from this species. Crinitaria tatarica oil and Z- (I) and E-artemidin (II) were evaluated for biological activity.     

Microcalorimetric study of the adsorption of PEGylated lysozyme on a strong cation exchange resin

Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl Gigacap S-650M in sodium phosphate buffer is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements at pH 6 and 25°C. The production and separation of PEGylated lysozyme is discussed. Two different PEG sizes are used (5 kDa and 10 kDa) which leads to six different forms of PEGylated lysozyme which were systematically studied. The sodium chloride concentration is varied according to the elution conditions in the production process. The specific enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It was found to be exothermal and constant with increasing adsorber loading for native lysozyme. For all PEGylated forms there is an influence of the adsorber loading on Δh(p)(ads) which is found to become more important with increasing PEGylation degree (total molecular weight of conjugated PEG). At low loadings the adsorption of all PEGylated lysozyme forms is exothermal. With increasing loading the adsorption becomes less exothermal and for the species with higher PEGylation degree also endothermal effects are observed at higher loadings. A thermodynamic analysis is carried out by which the enthalpic and entropic contributions to the binding constants are quantified. The findings are discussed on a molecular level. The results provide insight into the adsorption mechanisms of polymer-modified proteins on chromatographic resins.