Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.     

Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.     

Monoliths as stationary phases for separation of proteins and polynucleotides and enzymatic conversion

Monoliths are considered as a novel generation of stationary phases. They were applied for capillary electrochromatography and liquid chromatography exploiting every action principle such as ion-exchange, affinity recognition, reversed-phase, and hydrophobic interaction. The fast separation was explained by convective transport of the solutes through the bed. The contribution of this mode of transport is similarly explained as done for the beds packed with particles with gigapores. For monolithic beds, the concept of an ultrashort bed was frequently used. This mode of operation allows very short separation time. In many cases a gradient elution is necessary to achieve separation. Examples of applications for protein and polynucleotide separation performed on monoliths are given. Enzymatic conversion was described showing the examples of several immobilzed enzymes.     

Analysis of acidic compounds using capillary electrochromatography

Capillary electrochromatography, CEC, is a hybrid of CE and HPLC and is rapidly gaining interest as a potential complementary technique. This paper provides an overview of literature concerning the separation of acidic compounds by CEC which fall into three distinct groups. These groups are those performed using capillaries packed with novel or unique stationary phases designed for CEC, and a smaller group where standard HPLC stationary phases packings such as ODS has been used. The third group involves the use of surface coated capillaries. This paper reviews the separation of acidic compounds by CEC and also includes a number of novel applications to illustrate the separation approaches and the analytical performance possible.     

Preparation and application of methylcalix[4]resorcinarene-bonded silica particles as chiral stationary phase in high-performance liquid chromatography

Two new types of methylcalix[4]resorcinarene-bonded stationary phases, (3-(C-methylcalix[4]resorcinarene)-2-hydroxypropoxy)-propylsilyl-appended silica particles (MCR-HPS) and bromoacetate-substituted MCR-HPS particles (BAMCR-HPS), have been synthesized and used as chiral stationary phases for high-performance liquid chromatography (HPLC) for the first time. The synthetic stationary phases are characterized by means of elemental analysis and Fourier-transform infrared spectroscopy. The chromatographic behavior of MCR-HPS and BAMCR-HPS was studied with several disubstituted benzenes and some chiral drug compounds under both normal phase and reversed-phase conditions. The results show that MCR-HPS has excellent selectivity for the separation of aromatic positional isomers and BAMCR-HPS exhibits excellent performance for separation of enantiomers of chiral compounds.     

The use of multilayered capillaries for chiral separation by electrochromatography

Fused-silica capillaries were modified by the successively multiple ionic-polymer layer (SMIL) coating technique for a capillary electrochromatography (CEC) analysis of binaphthyl enantiomers. The SMIL coating capillaries consisting of three different polymers (A(+)-B(-)-C(+) coating) were prepared by the alternative deposition of positively charged chiral or achiral polymers and negatively charged DNA. Previous studies have indicated that DNA-cationic polypeptide or synthetic polymer complexes immobilized onto the inner surface of the capillary worked as the chiral stationary phases for 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNP). In this study, to investigate the chiral recognition mechanism and optimize the CEC separation condition in the DNA-cationic polymer coating, effects of the chirality of the polymer unit, the strand of DNA, and the number of layer pairs on the separation were investigated. It should be noted that, since single stranded DNA (ssDNA) was more suitable to immobilize cationic polymers than double stranded DNA, the ssDNA-cationic polymer immobilized capillaries gave a stable electroosmotic flow and reproducible CEC analyses. As a result, a poly(ethyleneimine)-ssDNA-protamine (Prt) coating provided the best chiral separation of BNP. The high separation performance of the prepared capillary is discussed in terms of DNA/polycations interaction.     

Considerations on influence of charge distribution on determination of biomolecules and microorganisms and tailoring the monolithic (continuous bed) materials for bioseparations

The importance of continuous beds (monoliths) as separation materials is connected with their better chromatographic properties and easier preparation in comparison to particulate-packed columns. Moreover the tuning of porosity as well as surface chemistry can lead to obtaining of highly selective materials, especially useful in separation of biologically important compounds or even microorganisms. To obtain high selectivity for such analytes as e.g. proteins, it is often important to have a knowledge about their shape, size, charge and finally charge distribution. This article presents our considerations on the charge distribution on the monolithic stationary phase and surface of such species as proteins or microorganisms as well as its eventual influence on the separation or sample preparation processes and tuning of their selectivity.     

Chiral-recognition chromatography of β-blockers on continuous polymer beds with immobilized cellulase as enantioselective protein

Columns prepared by coupling cellulase as a chiral selector to silica beads are very efficient for the separation of enantiomers. In this paper we show that continuous polymer beds compete favorably with silica beads as chromatographic supports for such separations. The chiral stationary phase is prepared either by entrapment in and simultaneous covalent linkage of ally1 cellulase to the continuous beds during their preparation or by covalent immobilization of cellulase on an epoxy-activated continuous bed. Enantiomers of β-blockers were separated rapidly and with high resolution. The enantiomers of practolol were thus baseline resolved within 45 sec. The recognition center–or at least part of it—coincides with the active center of the enzyme, since the enantiomers could not be separated in the presence of the competitive enzyme inhibitors cellobiose and D-glucose and the separation was also impaired upon addition of the substrate carboxymethyl cellulose to the eluent. Similar observations have been reported for silica columns derivatized with cellulase. The capacity factor and the separation selectivity could be tuned by the pH and the concentration of the mobile phase, a phosphate buffer. No modifier was required, as is sometimes the case with silica-based supports. The continuous beds give faster enantiomer separations than do columns of silica and are more pH-stable and cost effective to prepare. © 1993 Wiley-Liss, Inc.     

Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Cellulose tris(3-chloro-4-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSPs) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the chiral selector loading onto silica, mobile phase composition and pH, as well as separation variables on separation of enantiomers was studied. It was found that CSPs based on cellulose tris(3-chloro-4-methylphenylcarbamate) can be used for preparation of very stable capillary columns useful for enantioseparations in nano-LC and CEC in combination with polar organic mobile phases.     

Considerations on influence of charge distribution on determination of biomolecules and microorganisms and tailoring the monolithic (continuous bed) materials for bioseparations

The importance of continuous beds (monoliths) as separation materials is connected with their better chromatographic properties and easier preparation in comparison to particulate-packed columns. Moreover the tuning of porosity as well as surface chemistry can lead to obtaining of highly selective materials, especially useful in separation of biologically important compounds or even microorganisms. To obtain high selectivity for such analytes as e.g. proteins, it is often important to have a knowledge about their shape, size, charge and finally charge distribution. This article presents our considerations on the charge distribution on the monolithic stationary phase and surface of such species as proteins or microorganisms as well as its eventual influence on the separation or sample preparation processes and tuning of their selectivity.     

New concepts in the chromatography of peptides and proteins

Although chromatography using a variety of novel bed configurations (e.g. fluidized beds, expanded beds, simulated moving beds, annular rotating beds, etc.) has been of recent interest, the majority of practical applications of analytical and preparative chromatography employ a stationary adsorbent bed into which a feed slug is charged periodically, similar to the technique first described by Mikhail Tswett over 100 years ago. However, new concepts in both the practice and theory of fixed-bed chromatography are continuing to expand the available range of applications for separating peptides and proteins.     

Continuous beds (monoliths): stationary phases for liquid chromatography formed using the hydrophobic interaction-based phase separation mechanism

The pioneering research work published by Hjertén et al. [J. Chromatogr. 473 (1989) 273] in 1989 dealing with development and application of the continuous bed (monolithic) technique as an attractive alternative for the classical packed columns in chromatography, stimulated further investigations in this direction. The research data published since that time on the development and application of the continuous beds formed using hydrophobic interaction-based phase separation mechanism are reviewed. Some innovative species of the beds, such as polyrotaxane beds or nonparticulate restricted-access materials for direct analysis of the biological fluids in the capillary format are also discussed. Characteristic features and practical details of the continuous bed technique are revealed. Due to many advantages, the continuous bed technique became a competitor with the traditional packings in capillary or chip-based microanalysis. The importance of the continuous bed morphology on the chromatographic characteristics is shown. The applicability of modern microscopic analysis to evaluate the morphology of the continuous beds is demonstrated.     

Analysis of liposomes by capillary electrophoresis and their use as carrier in electrokinetic chromatography

This contribution reviews work about liposomes in the context of electrically driven separation methods in the capillary format. The discussion covers four topics. The one broaches the application of liposomes as pseudo-stationary phases or carriers in vesicle or liposome electrokinetic chromatography (EKC) in the way as microemulsions and micelles are used; it includes the chromatographic use of liposomal bilayers as stationary phases attached to the wall for capillary electrochromatography (CEC). The second topic is the characterization and separation of liposomes as analytes by capillary electrophoresis (CE). Then the determination of distribution coefficients and binding constants between liposomes and ligands is discussed, and finally work dealing with peptides and proteins are reviewed with lipid bilayers as constituents of the electrically driven separation system.     

Conditional synthesis and utilization of 1,5-anhydroglucitol in Escherichia coli.

A cyclic polyol, 1,5-anhydro-D-glucitol (AG), is widely detected in most organisms, although little is known about its metabolism and physiological roles. The present study demonstrates the synthesis of AG in Escherichia coli C600. The major portion of the synthesized AG was indicated to be derived from glucose retaining all the six carbon atoms, and only 5% was attributed to AG synthesized from C3 compounds. AG synthesis is apparent in an early stage of the stationary phase, and accumulation is transient both in cells and in medium. Evidence is also presented for AG uptake and metabolism and for effects of cyclic AMP.     

Experimental design as a tool when evaluating stationary phases for the capillary electrochromatographic separation of basic peptides

Two different capillary electrochromatography (CEC) stationary phases, Hypersil phenyl and Hypersil C(18), have been characterised with respect to their ability to separate the four basic peptides H-Tyr-(D)Ala-Phe-Phe-NH(2) (TAPP), H-Tyr-(D)Ala-Phe-NH(2) (TAP), H-Phe-Phe-NH(2) (PP) and H-Phe-NH(2) (P). Optimal separation conditions were first established separately for the two phases by applying experimental design in a stepwise procedure. The first step comprised a study to acquire basic knowledge about the variables, their influence on the response and their respective experimental domains for each of the two stationary phases. The second step was screening the significant variables and the third step was an optimisation with response surface modelling (RSM) to locate the optimum separation conditions for each stationary phase. The experimental procedure was identical for both stationary phases, but their respective experimental domains were different. The response functions were peak resolution and peak efficiency. This procedure enables specific optimal experimental conditions to be identified for each of the two stationary phases. The optimal conditions identified for the separation on the phenyl stationary phase were to use 50% ACN, 20% 50 mM Tris(hydroxymethyl)aminomethane (TRIS) pH 7.5, 30% H(2)O as BGE, operating at 20 degrees C and 20 kV high voltage. For the C(18) stationary phase optimal separation was achieved using a BGE with 80% ACN, 20% 30 mM TRIS pH 8.5, again operating at 20 degrees C and 20 kV high voltage. Results show that the phenyl stationary phase is better suited for the separation of basic, hydrophilic peptides.     

New system for preparative electrochromatography of proteins

Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 degrees C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 degrees C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale-up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.     

Cell cycle operation during batch growth of fission yeast populations

Batch cultivation provides a continuous sequence of different environments useful for studying responses of cell cycle controls. Flow cytometry measurements have been made of the frequency functions for protein, RNA, and DNA at different times during batch growth of the fission yeast Schizosaccharomyces pombe. The mean cellular protein and RNA contents and their variances tend to increase with increasing population specific growth rates. Analysis of the mid-exponential phase DNA frequency function data indicates that DNA synthesis occupies 12% of the total cell cycle time and is completed at the same time as cell separation. Coordination of DNA synthesis and cell separation is less precise when population growth rate is low in late lag and early stationary phases.     

Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP)

The charged monoclonal antibody (mAb) variants of the commercially available therapeutics Avastin®, Herceptin® and Erbitux® were separated by ion‐exchange gradient chromatography in batch and continuous countercurrent mode (MCSGP process). Different stationary phases, buffer conditions and two MCSGP configurations were used in order to demonstrate the broad applicability of MCSGP in the field of charged protein variant separation. Batch chromatography and MCSGP were compared with respect to yield, purity, and productivity. In the case of Herceptin®, also the biological activity of the product stream was taken into account as performance indicator. The robustness of the MCSGP process against feed composition variations was confirmed experimentally and by model simulations. Biotechnol. Bioeng. 2010;107:652–662. © 2010 Wiley Periodicals, Inc.     

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

Proteins display interesting chiral discrimination properties owing to multiple possibilities of intermolecular interactions with chiral compounds. This review deals with proteins which have been used as immobilized chiral selectors for the enantioseparation of drugs in liquid chromatography and capillary electrophoresis. The main procedures allowing the immobilization of proteins onto matrices, such as silica and zirconia particles, membranes and capillaries are first presented. Then the factors affecting the enantioseparation of drugs in liquid chromatography, using various protein-based chiral stationary phases (CSPs), are reviewed and discussed. Last, chiral separations already achieved using immobilized protein selectors in affinity capillary electrochromatography (ACEC) are presented and compared in terms of efficiency, stability and reproducibility.     

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities. This revised version was published online in July 2006 with corrections to the Cover Date.