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S Hjertén

Publications and source records attributed to S Hjertén.

At least 19 recordsLinked to original sources
PubMed2001

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.

Amines↗
PubMed2000

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.

Chromatography, High Pressure Liquid↗
PubMed2000

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"?).

Analgesics↗
PubMed2000

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.

Amino Acids↗
PubMed1999

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.

Crystallization↗
PubMed1999

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.

Buffers↗
PubMed1997

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.

Animals↗
PubMed1996

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.

Lipid Bilayers↗
PubMed1996

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.

Blotting, Western↗
PubMed1996

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).

Acrylamides↗
PubMed1996

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.

Electrophoresis, Agar Gel↗
PubMed1996

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.

Automation↗
PubMed1995

Capillary and rotating-tube isoelectric focusing of a transmembrane protein, the human red cell glucose transporter.

The human red cell glucose transporter (Glut1) is a transmembrane protein. Monomeric Glut1 was purified by ion-exchange chromatography in the presence of the non-ionic detergent n-dodecyl octaoxyethylene (C12E8). For focusing, the ionic strength of the solution of C12E8-Glut1 complexes with co-purified lipids was lowered by dialysis, the detergent concentration was increased and carrier ampholytes were added. Focusing was done for 5 min at 3000 V in a methyl cellulose-coated glass capillary (50 microns I.D.). The anolyte H3PO4 was then replaced by NaOH for mobilization towards the anode. Absorbance monitoring at 280 nm showed two groups of zones at pH 6 and 8. Similarly, isoelectric focusing in a rotating quartz tube (3 mm I.D.) gave Glut1 zones at pH 5.5 and 8.0. Phosphorus analysis revealed that the Glut1 zone at pH 8 contained more phospholipids than did the other one. The above results together with previously determined and calculated isoelectric points (pI) of Glut1 indicate that the Glut1 at pH 8 is monomeric and that the zone at pH 5.5-6 represents oligomeric materials. The pI 8.0 at 22 degrees C applies for monomeric Glut1 in the absence of urea. The results exemplify that capillary isoelectric focusing of hydrophobic membrane proteins is possible.

Chromatography, Ion Exchange↗
PubMed1995

Capillary liquid chromatography-fast atom bombardment mass spectrometry using a high-resolving cation exchanger, based on a continuous chromatographic matrix. Application to studies on neuropeptide peptidases.

Hyphenated mass spectrometric techniques such as LC-MS are advantageous over standard MS methods, because they provide increased sensitivity and minimize signal suppression by other compounds present in the reaction mixture. Recently, we have introduced so-called continuous beds, and applied this technique to prepare a 0.32 mm I.D. cation-exchange capillary column, in order to separate the reaction product substance P(1-7) after proteolytic cleavage of substance P by an endopeptidase recovered from human cerebrospinal fluid. The use of a volatile buffer for elution provides very good flow stability. Ion-exchange microcolumns may be particularly useful for the separation of those peptides that co-elute in reversed-phase chromatography because the separation mechanisms of these two methods are different.

Cation Exchange Resins↗
PubMed1995

Liposome capillary electrophoresis for analysis of interactions between lipid bilayers and solutes.

Liposomes, which mimic biomembranes, were used as a pseudostationary phase in capillary zone electrophoresis. The decrease in the mobility of an analyte owing to the presence of liposomes reflected interaction between the analyte and the liposomes. Equations were derived to calculate the specific capacity factor Ks (the capacity factor, k', normalized to the liposome concentration 1 M) from the migration times and to estimate the difference in free energy, delta(delta G0), of the weak analyte/liposome interactions. The order of Ks values for the drugs tested was aspirin < salicylic acid < warfarin << sulfasalazine. The peptide TyrGlySerThrProGlyCysCys interacted more strongly with the liposomes (Ks = 10.1 M-1) than did TyrGlySerThrProGlySerSer (Ks = 9.1 M-1). These results were similar to those obtained earlier by immobilized liposome chromatography.

Amino Acid Sequence↗
PubMed1995

Fast, high-resolution (capillary) electrophoresis in buffers designed for high field strengths.

Capillary electrophoresis in conventional buffers and in 50 microns capillaries permits field strengths as high as 300-500 V/cm with acceptably low thermal zone deformation. However, still higher field strengths (up to at least 2000 V/cm) can be applied without a decrease in resolution if the experiments are performed in the buffers described in this paper. Characteristic of these buffers is their low electrical conductivity and yet satisfactory buffering capacity accomplished either (i) by selecting buffer constituents of relatively high molecular weight and small net charge or (ii) by fractionation of carrier ampholytes (originally introduced for isoelectric focusing experiments) into a series of narrow pH range fractions and using these fractions as buffers, or (iii) by selecting an ampholyte with two acidic groups and one basic group (or one acidic group and two basic groups) and with a pI value close to two of its pK values. In such buffers, aromatic carboxylic acids and proteins used as model substances could be analyzed rapidly. For instance, albumin and transferrin were separated at 30,000 V (1.99 microA) in 15 cm long fused silica capillaries (50 microns ID) within 40 s and the carboxylic acids within 25 s. The resolution was similar to that obtained at standard voltage (5000 V; 0.33 microA), but the analysis time was reduced sixfold. Although not verified experimentally we also suggest the use of relatively high-molecular-weight polyoxyethylene derivatized with one acidic group (for instance, boric acid) and one basic group (an amine), both having the same pK value, which should afford both a very high buffering capacity and very low electrical conductivity (at low buffer concentrations).

Buffers↗