Optimization of ion-exchange displacement separations. II. Comparison of displacement separations on various ion-exchange resins

A variety of stationary-phase materials are currently available for the chromatographic purification of biomolecules. However, the effect of various resin characteristics on the performance of displacement chromatography has not been studied in depth. In Part I, a novel iterative scheme was presented for the rapid optimization of displacement separations in ion-exchange systems. In this article, the optimization scheme is employed to identify the optimum operating conditions for displacement separations on various ion-exchange resin materials. In addition, the effect of different classes of separation problems (e.g., diverging, converging or parallel affinity lines) on the performance of displacement separations is also presented. The solid film linear driving force model is employed in concert with the Steric Mass Action isotherm to describe the chromatographic behavior in these systems. The results presented in this article provide insight into the effects of resin capacity and efficiency as well as the type of separation problem on the performance of various ion-exchange displacement systems.     

Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems

A general rate model was employed in concert with a preferential interaction quadratic adsorption isotherm for the characterization of HIC resins and the prediction of solute behavior in these separation systems. The results indicate that both pore and surface diffusion play an important role in protein transport in HIC resins. The simulated and experimental solute profiles were compared for two model proteins, lysozyme and lectin, for both displacement and gradient modes of chromatography. Our results indicate that a modeling approach using the generate rate model and preferential interaction isotherm can accurately predict the shock layer response in both gradient and displacement chromatography in HIC systems. While pore and surface diffusion played a major role and were limiting steps for proteins, surface diffusion was seen to play less of a role for the displacer. The results demonstrate that this modeling approach can be employed to describe the behavior of these non-linear HIC systems, which may have implications for the development of more efficient preparative HIC separations.     

Screening approach for chiral separation of pharmaceuticals. Part III. Supercritical fluid chromatography for analysis and purification in drug discovery

High-throughput and performance analysis and purification of enantiomers are important parts of drug discovery and provide high-quality compounds for pharmacological testing. We have previously reported two parts describing chiral chromatographic screens using normal-phase (NPLC) and reversed-phase (RPLC) liquid chromatography, in order to cope with increasing numbers of new compounds generated by chemistry programs. We present in this part the development and implementation of a third faster screen using supercritical fluid chromatography (SFC) to maximize chance in achieving rapid enantiomer resolution of large numbers of compounds in a minimum of time. The SFC screen utilizes a narrow combination of only four columns (Chirlapak AD and AS, and Chiralcel OD and OJ) and two solvent modifiers (methanol and isopropanol). A modifier and column-switching setup was employed to allow the entire screening process to be serially run in the order AD> OD > OJ > AS and methanol > isopropanol, so that the screening for a given molecule can be stopped when separation is achieved. The switching system was fully automated for unattended operation of multiple compounds. An optimization procedure was also defined, which can be performed if needed for unsuccessful separations in the screening step. The chiral SFC strategy proved its performance and robustness in resolution of hundreds proprietary chiral molecules generated by drug discovery programs, with a success rate exceeding 95%. In addition, the generic capability of the strategy was evaluated by applying the screen and optimization methodology to a test set comprising 40 marketed drugs differing from proprietary compounds in terms of chemical diversity, revealing a similar high success rate of 98%. Chiral separations developed at the analytical scale work easily and equally well at the semi-preparative level, as illustrated with an example. The SFC screen allows resolution of compounds that were partially separated by NPLC or not separated at all by RPLC, demonstrating the utility of implementing complementary chromatographic techniques. The SFC screen is currently an integral part of our analytical support to discovery chemical programs and is considered the first try for chiral separations of new compounds, because it offers a higher success rate, performance and throughput.     

Preparative liquid chromatography

The status of the theory and the main methods of implementation of preparative liquid chromatography are reviewed. On the theory front, the focus has recently shifted. The theory of non-linear, non-ideal chromatography has given rise to numerous models whose advantages, disadvantages and ranges of application are now well understood. Interest now resides in investigating the equilibrium thermodynamics of complex new systems, in the study of the kinetics of mass transfers in conventional chromatographic systems, and in the application of the various models of chromatography to optimize the experimental conditions. Progress in computer technology allows the use of sophisticated models, provided their parameters can be measured. This allows the detailed investigation of separations for which the mass transfer kinetics is slow such as chiral separations, the purification of basic compounds, and the extraction of recombinant proteins. On the applied front, in addition to numerous incremental improvements in reliability and economic performance, a few essential new features should be noted, i.e. the availability of instruments for simulated moving bed separations at the scale needed for preparative chiral separations, the use of expanded beds for the extraction of recombinant proteins from fermentation broths, and the attention given to improvements in the performance of packed beds. A survey of the literature dealing with practical applications and recent meetings shows that preparative chromatography is becoming a well established separation and purification method in the pharmaceutical industry.     

A binary-like approach for the computer assisted method development of isocratic and programmed ternary solvent elutions in reversed-phase liquid chromatography

A specific tool to enable exploration of multisolvent isocratic and programmed elutions for the computer assisted method development of reversed-phase liquid chromatography separations is described. The tool is purposely identical to those used in the optimization of binary solvent systems, which are by far the most commonly used by chromatographers. Existing data from failed binary solvent optimization processes are reused to explore ternary solvent systems with a few additional isocratic and programmed runs. This allows the development of efficient retention models for ternary systems, although the work of the chromatographer remains identical to that for optimization of binary systems. The retention models are used to develop an unattended optimization process and finally, the chromatographer selects the most satisfactory solution for testing and implementing in routine analysis. The process is exemplified with a mixture of 12 compounds that cannot be separated satisfactorily in aqueous binary solvent systems with methanol and acetonitrile as modifiers.     

Estimation of human blood plasma 5-hydroxyindoleacetic acid and homovanillic acid.

A method for simultaneous quantification of plasma homovanillic acid and 5-hydroxyindoleacetic acid has been developed, permitting more efficient neurochemical examinations of these often interrelated biogenic amine systems. Zinc sulphate and sodium hydroxide solutions were used for precipitating the protein in plasma prior to injection on the column. This technique allows for cleaner chromatography, greater sensitivity and high precision. The method uses high performance liquid chromatographic separations of these compounds on C18 reversed phase columns with electrochemical detection. The detailed results from controls and untreated parasuicide patients are given.     

High-speed ion-pair partition chromatography in pharmaceutical analysis.

Ion-pair chromatography offers attractive possibilities in pharmaceutical analysis. The specificity of the separation systems can be varied over a wide range by appropriate selection of the stationary phase. The choice of a suitable counter-ion can also drastically improve the detection limit, permitting the determination of drug substances in low dosage and possibly of by-products or breakdown products. Ion-pair chromatography of tropane and ergot alkaloids has been investigated using picrate as counter-ion. Alumina, Kieselguhr and various grades of silica gel have been tested as supports. Partition properties studied in a batch procedure have been compared with the actual chromatographic conditions. Columns (10 cm) filled with silical gel (particle size, 5 mum; pore size, 1000 A) show the best performance in the separation of hyoscyamine, scopolamine and ergotamine as picrate ion-pairs. Close control of pH and temperature is essential for reproducible separations. Improvements in detection limits between 100 and 300 times have been observed with these systems. Ion-pair extractions of these alkaloids from dosage forms can be used for sample preparation prior to injection on the the column. This provides an added degree of selectivity and sensitivity.     

Integration of supercritical fluid chromatography into drug discovery as a routine support tool. Part I. fast chiral screening and purification

Supercritical fluid chromatography (SFC) has been implemented within our group as a purity assessment and purification tool to complement high performance liquid chromatography (HPLC) for diastereomer and chiral separations. Using a novel strategy, rapid chiral screening has been implemented using short columns, high flow rates and fast gradients. A primary screen delivers a separation assessment using one solvent modifier (methanol) and four columns (Chiralpak AD-H and AS-H, and Chiralcel OD-H and OJ-H) run serially in a total of 24 min. A secondary screen then uses ethanol and isopropanol (IPA) modifiers across the same columns. The screens can be combined to run a sequence of samples overnight where each racemate is analysed over 80 min. The fast analytical screening and optimisation process enables rapid identification of the purification method. Furthermore, subsequent preparative chiral SFC has decreased the overall sample turnaround time for the Medicinal Chemist, delivering high fraction purities and acceptable recoveries, substantial operational cost savings and increased flexibility with respect to large scale purification feasibility in comparison to HPLC. SFC has been so successful it is now used as the primary method for chiral analysis and purification within our laboratory.     

Ion-exchange displacement chromatography of proteins Dextran-based polyelectrolytes as high affinity displacerss

Dextran-based polyelectrolyte displacers were successfully employed for the displacement purification of proteins in ion-exchange displacement systems. The effect of molecular mass was investigated by examining the efficacy of DEAE-dextran and dextran sulfate displacers of various molecular masses in cation- and anion-exchange systems, respectively. Induced salt gradients produced during these displacement experiments were measured in order to study their effect on the protein separations. The unique characteristics of these displacements were well predicted by simulations obtained from a steric mass action (SMA) ion-exchange model. These displacements differ from the traditional vision of displacement chromatography in several important ways: the isotherm of the displacer does not necessarily lie above the feed component isotherms; the concentration of the displaced proteins can sometimes exceed that of the displacer; higher-molecular-mass displacers are not necesarily more efficacious than lower-molecular-mass compounds; and the salt gradients induced by the adsorption of the displacer produce different salt micro-environments for each displaced protein.     

Thermotropic Liquid Crystalline Side Group Polymers as Stationary Phases in High Performance Liquid Chromatography. II. the Mechanism of Separation

Abstract

Liquid crystalline side group polymers support coated on silica gels have been applied as stationary phases in high performance liquid chromatography. It has been possible to show that also in liquid chromatography, separations based on the mesophase structure can be observed in analogy to gas chromatography. From results of separations in which temperature, flow rate, sample concentration and the solvent strength of the mobile phase were varied, this work derives views on the fundamental mechanisms involved. In addition, it will be shown that different mechanisms are probably involved in the separation of steroids and dinitrobenzene isomers on these stationary phases.     

Separation and identification of illicit heroin samples by liquid chromatography using an alumina and C18 coupled column system and photodiode array detection

The analysis of illicit heroin and opium samples on a coupled alumina and C18 column system is described. The compounds to be analysed can be divided into two groups: those with low pKa values, such as caffeine, papaverine and noscapine, and those with high pKa values, such as heroin, acetylcodeine, O6-monoacetylmorphine, procaine, codeine, morphine and strychnine. The first group can best be separated on a C18 column, whereas alumina is more suitable for the second group. Previously reported criteria for choosing proper buffer systems for ion-exchange separations on alumina were used together with an iterative regressive optimization procedure developed in our laboratory. The system can be used with and without valve-switching, depending on the sample type. The peak purity of the judicially important components heroin and O6-monoacetylmorphine has been checked with a photodiode array detector and by use of advanced software.     

Systematic optimization of micellar electrokinetic chromatographic separation of flavonoids

Summary A simple and rapid systematic optimization scheme was described for the micellar electrokinetic chromatographic separation of a group of flavonoids. The scheme employed an interpretative optimization approach to predict the optimum conditions for the separation of a group of flavonoids by micellar electrokinetic chromatography. By performing a set of nine pre-planned experiments conducted over the maximum working range for the system, global optimum separation conditions could be determined. To validate the optimization procedure, additional experiments were performed using the optimum experimental conditions derived from the optimization scheme. The results showed that satisfactory separation of all the peaks could be obtained. In addition, the application of the method in micropreparative micellar electrokinetic chromatography of the flavonoids was demonstrated.     

Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

A detailed study was carried out to combine the unique selectivity of ceramic hydroxyapatite (CHA) with the separation power of selective displacement chromatography. A robotic liquid handling system was employed to carry out a parallel batch screen on a displacer library made up of analogous compounds. By incorporating positively charged, metal chelating and/or hydrogen bonding groups into the design of the displacer, specific interaction sites on CHA were targeted, thus augmenting the selectivity of the separation. The effect of different mobile phase modifiers, such as phosphate, sulfate, lactate and borate, were also investigated. Important functional group moieties and trends for the design of CHA displacers were established. Selective batch separations were achieved between multiple protein pairs which were unable to be resolved using linear gradient techniques, demonstrating the applicability of this technique to multiple protein systems. The specific interaction moieties used on the selective displacer were found to dictate which protein was selectively displaced in the separation, a degree of control not possible using a mono-interaction type resin in displacement chromatography. Mobile phase modifiers were also shown to play a crucial role, augmenting the selectivity of a displacer in a synergistic fashion. Column separations were carried out using selective displacers and mobile phase modifiers identified in the batch experiments, and baseline separation of the previously unresolved protein pairs was achieved. Further, the elution order in these systems was able to be reversed while still maintaining baseline separations. This work establishes a new class of separations which combine the selectivities of multi-modal resins, displacers/eluents, and mobile phase modifiers to create unique selectivity windows unattainable using traditional modes of operation.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Using computer modeling to predict and optimize separations for comprehensive two-dimensional gas chromatography

In order to fully realize the separation power of comprehensive two-dimensional gas chromatography (GC x GC), a means of predicting and optimizing separations based on operating variables was developed. This approach initially calculates the enthalpy (DeltaH) and entropy (DeltaS) for the target compounds from experimental input data, and then uses this information to simultaneously optimize all column and runtime variables, including stationary phase composition, by comparing the performance of large numbers of simulated separations. This use of computer simulation has been shown to be a useful aid in conventional separations. It becomes almost essential for GC x GC optimization because of the large number of variables involved and their very complex interaction. Agreement between experimental and predicted values of standard test samples (Grob mix) using GC x GC separation shows that this approach is accurate. We believe that this success can be extended to more challenging mixtures resulting in optimizations that are simpler and transferable between GC x GC instruments.     

Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

 Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection     

Affinity monolith chromatography

The combined use of monolithic supports with selective affinity ligands as stationary phases has recently given rise to a new method known as affinity monolith chromatography (AMC). This review will discuss the basic principles behind AMC and examine the types of supports and ligands that have been employed in this method. Approaches for placing affinity ligands in monoliths will be considered, including methods based on covalent immobilization, biospecific adsorption, entrapment, and the formation of coordination complexes. Several reported applications will then be presented, such as the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, and biomimetic chromatography. Other applications that will be discussed are chiral separations and studies of biological interactions based on AMC.     

Applications of silica supports in affinity chromatography

The combined use of silica-based chromatographic supports with immobilized affinity ligands can be used in many preparative and analytical applications. One example is the use of silica-based affinity columns in HPLC, giving rise to a method known as high-performance affinity chromatography (HPAC). This review discusses the role that silica has played in the development of affinity chromatography and HPAC and the applications of silica in these methods. This includes a discussion of the types of ligands that have been employed with silica and the methods by which these ligands have been immobilized. Various formats have also been presented for the use of silica in affinity chromatographic methods, including assays involving direct or indirect analyte detection, on-line or off-line affinity extraction, and chiral separations. The use of silica-based affinity columns in studies of biological systems based on zonal elution and frontal analysis methods will also be considered.     

Recent advances in counter-current chromatography

During the past several years, counter-current chromatography (CCC) technology has been advanced to cover a broad spectrum of applications, from large-scale preparative to analytical-scale separations. These advances include liquid-liquid dual CCC, foam CCC and partition of macromolecules with aqueous-aqueous polymer phase systems. For these developments the synchronous coil planet centrifuge scheme has been used, which relies on a relatively simple mechanical design. Future developments in CCC may be focused on the improvement of the more intricate non-synchronous coil planet centrifuge scheme which has a greater potential for the separation of biopolymers and cell particles.