Studies on the analytical performance of a non-covalent coating with N,N-didodecyl-N,N-dimethylammonium bromide for separation of basic proteins by capillary electrophoresis in acidic buffers in 25- and 50-microm capillaries.

Capillaries (25- and 50-microm inner diameter) coated with a double-alkyl-chain cationic surfactant N,N-didodecyl-N,N-dimethylammonium bromide (DDAB) were used for the separation of four basic standard proteins in buffers of pH 4 at various ionic strengths. The choice of buffer is critical for the analytical performance. Ammonium ions must be avoided in the buffer used in the non-covalent coating procedure owing to competition with the surfactant. Phosphate buffer gave a better separation performance than some volatile buffers; the reason seems to be a complex formation between the proteins and dihydrogenphosphate ions, which decreases tendencies for adsorption to the capillary surface. The DDAB coating was easy to produce and stable enough to permit, without recoating, consecutive separations of the proteins for up to 100 min with good precision in migration times and peak areas. A strong electroosmotic flow gives rapid separations, which is of special importance when commercial instruments are used, since the choice of the length of the capillary is restricted.

Enantioseparation of hydroxy acids on easy-to-prepare continuous beds for capillary electrochromatography.

This paper deals with the enantioseparation of hydroxy acids by ligand-exchange capillary electrochromatography. A chiral continuous bed was easily prepared by in situ polymerization of monomers, including an L-4-hydroxyproline derivative. This phase showed chiral recognition for several hydroxy acids, in addition to amino acids.

Capillary electrochromatography of hydrophobic amines on continuous beds.

The capillary electrochromatographic separation performance of hydrophobic amines and a related quaternary ammonium compound on continuous beds based on polymers of acrylamide has been studied. The chromatographic bed is polymerized in situ and the character of the polymers with regard to hydrophobicity and charge has been systematically changed by regulating its content of isopropyl and sulfonate ligands, respectively. The best performance was obtained for columns with a molar ratio of 1:80 for the sulfonate and isopropyl groups, and resulted in efficiencies up to 200000 plates per meter. The effects on retention, resolution and elution order by ionic strength, pH, and content of acetonitrile in the mobile phase have been investigated. The quaternary ammonium compound was always the least retained irrespective of pH. By increasing the pH, a reversal of the migration order between the tertiary and secondary amine was obtained. The results indicate a complex migration/retention mechanism where ion-exchange, adsorption and electrophoretic mobilities play a role. The concentration limit of detection could be lowered from 1.3 microg/mL to 50 pg/mL by using a high content of 2-propanol (96%) in the sample compared to dissolving the analytes in the mobile phase.

A new easy-to-prepare homogeneous continuous electrochromatographic bed for enantiomer recognition.

Completely homogeneous polyacrylamide-based gels were used for capillary electrochromatography (CEC) of drug enantiomers. Like continuous beds (also called continuous polymer rods, silica rods, monoliths) they do not require frits to support the bed because it is covalently linked to the capillary wall. A long lifetime is an important feature of the beds. The gel matrices can be prepared in any laboratory and for specific interactions they can be derivatized with appropriate ligands. The application range is, therefore, broad. For chiral electrochromatography, negatively and positively charged polyacrylamide gels copolymerized with 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) were prepared. The latter monomer was synthesized from beta-CD and allylglycidyl ether by a very simple one-step procedure. Eight acidic, neutral and basic drug compounds were resolved into their enantiomers, most of them with baseline separation. Interestingly, the resolution is independent of the electroendosmotic velocity, i.e., rapid analyses will not give low resolution. Upon increasing this velocity, the plate height for the fast enantiomer did not change (or decreased slightly), whereas that for the slow enantiomer increased. Only the last term in the van Deemter equation contributed significantly to the total plate height. The composition of the gel was chosen such that the "pores" became large enough to guarantee a satisfactory electroendosmotic flow (EOF). This open gel structure explains why acetone diffused as in free solution, i.e., independently of the presence of the gel matrix. This finding also indicates that the separation of small molecules in polyacrylamide gels cannot be explained by "molecular-sieving", but rather by some type of adsorption ("aromatic adsorption"?).

Chiral separation of amino acids by ligand-exchange capillary electrochromatography using continuous beds.

A chiral ligand-exchange phase for capillary electrochromatography based on continuous bed technology was developed. The chiral stationary phase is prepared by a one-step in situ copolymerization procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline. These chiral continuous beds are inexpensive and easy to prepare. They also have several advantages over silica-based packed capillaries. Since the bed is covalently attached to the capillary wall, no frit is required. The applicability of this new approach to the chiral separation of underivatized amino acids is demonstrated.

Electroosmosis- and pressure-driven chromatography in chips using continuous beds.

The application range of microchips can be extended to any mode of chromatography by filling the narrow channels with continuous polymer beds, exemplified by electrochromatography and ion-exchange chromatography. "Wall effects" are eliminated by anchoring the bed to the wall of the channel, an arrangement which has the additional advantage that no frits to support the bed are required. The design of the equipment is based on a quartz chip with all auxiliary pieces (for example, electrode vessels and fluid transfer fittings) placed in a rack, which permits a flexibility of great importance for automation. The same resolution and van Deemter plots were obtained in experiments performed in fused-silica capillaries and in chips for both low-molecular-weight (alkyl phenones, antidepressants) and high-molecular-weight substances (proteins). A sample of uracil, phenol, and benzyl alcohol was separated by electrochromatography in less than 20 s.

Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis.

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.

Continuous beds for microchromatography: chromatofocusing and anion exchange chromatography.

A method was developed for the preparation of continuous beds derivatized with polyethyleneimine (PEI) for chromatofocusing and anion exchange chromatography in the capillary mode. First, a continuous bed activated by epoxy groups was synthesized inside a fused silica capillary and became at the same time covalently attached to the inner wall of the capillary. A PEI solution was then pumped through the continuous bed to allow the imine groups in PEI to react with the epoxy groups in the bed. Efficient immobilization of PEI was indicated by the high-resolution separation of standard proteins (hemoglobins C, S, F, and A) in both chromatofocusing and anion exchange chromatography on a capillary column prepared by this method.

An approach to ideal separation media for (electro)chromatography.

In packed chromatographic beds, both Eddy diffusion and the relatively long time the analytes stay in the mobile phase until they collide and interact with the ligands attached to the beads (the residence time in the mobile phase) contribute considerably to zone spreading. One should not expect Eddy diffusion to occur in macroscopically homogeneous separation media, such as gels or polymer solutions, and residence time should be shorter, since the pore size of these media is much smaller than the average distance between the beads in a packed bed. Accordingly, these separation media (which can be regarded as homogeneous continuous beds [monoliths]) should theoretically give very high resolution, which has been verified experimentally: Frontal analysis of a neutral marker, acetone, showed that electroendosmosis in a homogeneous gel displaced the boundary without any distortion except that caused by diffusion (the marker was selected not to interact with the gel). All other common disturbing phenomena in chromatography (for instance, Eddy diffusion and nonspecific adsorption) were, accordingly, negligible, indicating that electrochromatography in homogeneous gels may be the ideal chromatographic method. However, to fully utilize these desirable chromatographic properties of homogeneous continuous beds, one has to choose analyte/bed interactions with sufficiently high association-dissociation rate constants (i.e., the residence time of the analytes in the stationary phase must be kept very short), which will be the subject of forthcoming studies. The electrophoretic counterpart of capillary electrochromatography (CEC), electrophoresis of noncharged analytes in a solution of charged polymers, seems to give a somewhat larger zone broadening, probably due to their high viscosity and conductivity, with attendant longer analysis (= diffusional) times compared to gels. Since the mobile phase is propelled through the gels by electroendosmosis, the theoretical and experimental requirements for high electroendosmotic flow in gels, i.e., short analysis times, are discussed. The electroendosmotic velocity v(x) can be estimated by the simple equation v(x) = Vmax (1-e-kappa x) (1/kappa = the thickness of the double layer; Vmax = the plug flow velocity) when the distance from the channel wall, x, << the radius R of the channel (pore). For kappa R > or = 5, the equation obtains with good approximation for all R values. An initially straight zone in a gel pore should be heavily distorted by the electroendosmotic flow, according to this equation (see Figure 1). However, due to rapid diffusion and other leveling effects, the zone is transported as in perfect plug flow, as is shown experimentally. A plot of electroendosmotic mobility obtained by frontal analysis against 1/(1 + square root of mu) can be used to estimate roughly the pore size in a gel and permits quantitative examination of the current theory of electroendosmosis. It is not a trivial problem to synthesize ligand-containing gels with pores large enough to allow a high electroendosmotic flow. Therefore, we have described a universal method: a polymer containing phenylboronate and acrylic acid groups was synthesized and entrapped in a standard agarose gel (for automated runs, replaceable methoxylated agarose should be used). Both of these charged groups serve to generate the electroendosmotic flow required in electrochromatography. This gel was designed to have the dual property of separating compounds that contain vicinal OH groups in the cis-configuration (exemplified by ribonucleosides) by reaction with the boronate groups, and aromatic substances by virtue of the acrylic acid residues (and perhaps also the phenyl groups) in the polymer and the agarose chains. The latter interaction, the so-called aromatic adsorption, has the advantage that it does not require a time-consuming attachment of ligands. (ABSTRACT TRUNCATED)

The effect of beta-cyclodextrins as buffer additives on the (capillary) electrophoretic separation of peptides and proteins.

Electrophoretic and chromatographic experiments performed under straightforward conditions do not always provide satisfactory resolution. An obvious approach, then, is to manipulate the magnitude of relevant separation parameters, such as charge (zeta potential), size, and hydrophobicity, all of which can be accomplished by complex formation. This alternative has been studied with both a neutral, an anionic, and a cationic derivative of beta-cyclodextrin in an attempt to increase the resolution of peptides and proteins in free-zone electrophoresis utilizing the capillary format. The investigation showed that charged beta-cyclodextrins are suitable for this purpose. As expected, the effect seems to be most significant for substances with a small net surface charge, i.e., low mobility. Consequently, it may be advantageous to choose a pH of the buffer that is not far from the isoelectric point of the solutes. It should be emphasized that changes in the electropherograms observed upon addition of any complexing agent to the buffer may involve improvement or worsening of the resolution. Only by experimentation can one determine whether complexation with cyclodextrins favors resolution, since our knowledge about the interactions taking place is limited. However, if a positively (negatively) charged beta-cyclodextrin decreases the resolution of an acidic (basic) protein, one can expect theoretically, a negatively (positively) charged beta-cyclodextrin to increase the resolution, as was verified experimentally. The difference in mobility between two peaks caused by the complexation with cyclodextrins need not be larger than 2-3% for satisfactory resolution because the peaks are sharp. We have introduced a new definition for the resolution of two very adjacent peaks--the most common and interesting case in real-world analyses--that does not require measurement of peak widths.

Capillary zone electrophoresis for the study of the binding of antithrombin to low-affinity heparin.

When low-affinity interactions between glycosaminoglycans and precious proteins are studied, it is imperative to design an experimental set-up that consumes as little material as possible. To evaluate the applicability of the CZE technique to this problem, we explored the interaction between antithrombin and low-affinity heparin. In a series of CZE experiments we demonstrated that the mobility of antithrombin increases gradually as increased concentrations of low-affinity heparin were added to the electrolyte. The results were, as expected, consistent with the general algorithm for monovalent binding. The binding constant was estimated at 20+/-6 microM in excellent agreement with the value reported in the literature.

Immobilized liposome chromatography of drugs on capillary continuous beds for model analysis of drug-membrane interactions.

Liposomes were immobilized in capillary continuous beds with covalently linked C4 or C8 alkyl ligands for chromatographic analysis of drug interaction with phospholipid bilayers, as reflected by drug retention volumes and calculated differences in interaction free energies. This procedure is a high-resolution micro-scale version of immobilized liposome chromatography for prediction of diffusion of drugs across biological membranes. The logarithm of the specific capacity factors of several structurally unrelated drugs showed a linear correlation with the logarithm of known apparent drug permeabilities through Caco-2 epithelial cell monolayers. The latter values are used for prediction of absorption of orally administered drug doses.

Continuous beds for microchromatography: detection of proteins by a blotting membrane technique.

Continuous beds have been used as matrices for cation- and anion-exchange chromatography of proteins on columns with an i.d. in the range of 0.005-0.015 mm. On-tube uv detection is not feasible at low protein concentrations with these narrow-bore columns. Therefore, a more sensitive detection system has been developed based on blotting technique: as the protein zones leave the microcolumn chromatographically they become adsorbed onto a rotating polyvinylidene difluoride blotting membrane. The protein spots can then be visualized by means of Coomassie brilliant blue, immunomethods, and other standard techniques. By using an immunomethod 0.015 ng of human transferrin can easily be detected. The blotting membrane can be washed with water without loss of adsorbed protein. This is an attractive feature because the presence of salts, etc., diminishes the accuracy in the determination of molecular weights of proteins by mass spectrometry. The microcolumns are easy to prepare. A solution of appropriate monomers is sucked into a piece of fused silica tubing. The rod formed upon polymerization contains channels through which the eluent can pass. No supporting frit is required because the polymer rod is anchored by covalent bonds to the tubing wall.

High-performance field inversion capillary electrophoresis of 0.1-23 kbp DNA fragments with low-gelling, replaceable agarose gels.

Field inversion capillary gel electrophoresis (FICGE) has been used for the separation of 0.1-23 kbp DNA fragments in a low-melting, low-gelling agarose gel. The influence of the amplitude of the voltage pulses, the pulse times and gel concentrations on the separation factor has been studied and found to be similar to that of polyacrylamide gels. Very high resolution can be obtained by altering continuously the pulse times and/or voltage according to a program tailor-made for the size of the DNA molecules to be separated (programmed FICGE). The advantage of these agarose gels in comparison with polyacrylamide gels is that they are nontoxic, easy to prepare and they have high UV transmission. They give a resolution that is equivalent to or better than that of polyacrylamide gels, and the risk of bubble formation in the gel is small. In addition, they are replaceable and, therefore, very convenient for automated analyses.

The resolution of DNA fragments in capillary electrophoresis in replaceable agarose gels.

The resolution of DNA restriction fragments in CE in replaceable agarose gels was investigated as a function of the concentration and pH of Tris-acetate ethylenediaminetetraacetic acid (EDTA) buffer both at constant migration time and at constant field strength. The resolution increased for all fragments when the buffer concentration was increased from 40 to 200 mM and the pH was lowered from 8.5 to 7.5. The resolution was lower in a Tris-borate-EDTA buffer in this pH range and lowest at pH 7.5.

Capillary-zone electrophoresis in agarose gels using absorption imaging detection.

A simple home-built electrophoretic unit and a commercially available charge-coupled device (CCD) camera with image acquisition and analysis software were used to study the separation process in zone electrophoresis experiments in 4 cm long, round capillaries (inside diameter 0.2 mm). Several capillaries could be investigated simultaneously. The absorption imaging system was used not only to follow the course of the separation but also to study the interaction between biologically active substances (proteins, detergents, enzymes and-substrate). Since the system allows visual on-line observation of the separation one can rapidly decide when the analysis is finished, which often shortens the analysis time. The electrophoresis method presented is suitable also for preparative runs, since direct visualizations of a solute zone allows it to be excised and then used for further studies.

Adherence of human prostasomes to mouse spermatozoa and their displacement by monoclonal antibodies as revealed by free zone electrophoresis.

Prostasomes, small corpuscular organelles derived from the prostate gland, were isolated from human seminal plasma by means of ultracentrifugation and Sephadex G 200 chromatography to examine their interactive ability with mouse spermatozoa as assessed by free zone electrophoresis. During the run, human prostasomes and mouse spermatozoa approached each other and finally fused into one single peak that was not further dissociated. In some cases the fusion of prostasomes and spermatozoa failed to appear following pretreatment of prostasomes with monoclonal antibodies raised against these organelles.

Continuous beds for microchromatography: reversed-phase chromatography.

Simple and cost-effective methods for the preparation of microcolumns (i.d. 0.025-0.32 mm) for reversed-phase chromatography are described. The procedure includes (1) synthesis in the column tube of a continuous bed matrix from a monomer solution (piperazine diacrylamide, methacrylamide) containing allyl glycidyl ether and 2-hydroxyethyl methacrylate and (2) linking of C18 ligands by reacting 1,2-epoxyoctadecane with the epoxy and hydroxy groups in the matrix. The derivatization can be accomplished within 20 min. The columns prepared in this way showed high performance in the separation of proteins and peptides and permitted short analysis times (100 s).