Continuous bed for conventional column and capillary column chromatography.
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Chromatography, Liquid↗
Biomedical subjects
J L Liao
Publications and source records attributed to J L Liao.
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↗
Biochemical separations by continuous-bed chromatography.
Innovations in column-packing media for biomolecule purification have progressed from large spherical, porous polysaccharide beads to advanced polymeric supports. Continuous-bed technology is a radical new technology for chromatography based on the polymerization of advanced monomers and ionomers directly in the chromatographic column. The polymer chains form aggregates which coalesce into a dense, homogeneous network of interconnected nodules consisting of microparticles with an average diameter of 3000 A. The voids or channels between the nodules are large enough to permit a high hydrodynamic flow. Due to the high cross-linking of the polymer matrix, the surface of each nodule is nonporous yet the polymeric microparticles provide a very large surface area for high binding capacity. This paper will demonstrate the properties and advantages of using a continuous bed support for high resolution biomolecule separations at high flow-rates without sacrificing capacity.
Animals↗
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↗
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↗
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↗
Continuous beds for microchromatography: cation-exchange chromatography.
Microcolumns (i.d. 10-320 microns) for cation-exchange chromatography can be prepared simply by polymerization of an aqueous solution of appropriate monomers, including the desired ligand, directly in the chromatographic tube (fused-silica tubing) in the presence of salt. The beds thus prepared are in the form of rods traversed by channels through which the eluent can pass. The walls of the channels are composed of very small particles and are impermeable to peptides and proteins, which is important for rapid mass transfer and thus for high resolution at high flow rates. The bed becomes attached covalently to the tube wall during synthesis. A complicated column tube design with a supporting frit at the bottom is thus eliminated. The absence of a frit reduces the flow resistance and facilitates interfacing to mass spectrometers. The covalent linkage of the bed to the tube wall also serves to suppress the zone-broadening "wall effect." A homogeneous "packing" of a continuous bed column with an inner diameter as small as 10 microns is easily obtained. The resolution, binding capacity, and flow rate (i.e., run time at a given pressure) can be varied by changing the composition of the monomer solution. One can thus tailor the beds to each separation problem. The chromatographic properties of the microcolumns are demonstrated by separations of model proteins.
Chromatography, Ion Exchange↗
Mitogenic properties of two distinct forms of toxic shock syndrome toxin-1 separated on hydroxyapatite by high-performance liquid chromatography.
The homogeneity of a purified staphylococcal toxic shock syndrome toxin-1 (TSST-1) was tested by high-performance methods. This preparation was homogenous in ion-exchange chromatography and isoelectric focusing (pI = 7.4), but was resolved into two distinct peaks by high-performance hydroxyapatite chromatography. Both components, TSST-1hA and TSST-1hB had similar molecular weights (22 kD) and amino acid compositions. TSST-1 did not dimerize or polymerize upon heating at 60 degrees C for 30 min or in solutions with pH varying from 4.0 to 8.5. TSST-1hA and TSST-1hB showed similar immunological reactivity to native TSST-1 goat polyclonal antibodies. TSST-1hA and TSST-1hB as well as staphylococcal enterotoxin A and staphylococcal exfoliative toxin were potent mitogens in lymphocyte proliferation assays. The lymphocyte proliferative response to 10 pg of TSST-1hB was comparable to a response elicited by 10 ng of TSST-1hA, suggesting that the former component is a more potent mitogen. Rabbit or goat polyclonal antibodies to native TSST-1 efficiently neutralized both TSST-1 components. Heat treatment at 80 degrees C for 15 min had minimal or no effect on the mitogenic properties of TSST-1hA and TSST-1hB.
Amino Acids↗
High-performance liquid chromatography of proteins on compressed, non-porous agarose beads. I. Hydrophobic-interaction chromatography.
Macroporous agarose beads were converted into non-porous beads by shrinkage and cross-linking in organic solvents. These beads could be used for high-performance hydrophobic-interaction chromatography without derivatization with non-polar ligands, because the 1,4-butanediol diglycidyl ether, used as cross-linker, gives relatively hydrophobic bridges. The resolution for compressed columns packed with these beads was determined as a function of gradient time at constant flow-rate, flow-rate at constant gradient volume and flow-rate at constant gradient time and as a function of loading capacity. Interestingly, the resolution is virtually independent of flow-rate at constant gradient volume even when the column is packed with relatively large beads (diameter 30 microns). The beads have the advantage of being stable up to pH 14.
Butylene Glycols↗
High-performance liquid chromatography of proteins on compressed, non-porous agarose beads. II. Anion-exchange chromatography.
Macroporous agarose beads were rendered impermeable to proteins by shrinkage and cross-linking in organic solvents. The chromatographic properties of compressed beds of these non-porous beads derivatized for high-performance ion-exchange chromatography were studied, e.g., the resolution as a function of gradient time, flow-rate (at constant gradient volume) and loading capacity. The columns permit high flow-rates and the resolution is about the same at low and high flow-rates. The beads are stable up to pH 14.
Animals↗
Carrier-free zone electrophoresis, displacement electrophoresis and isoelectric focusing in a high-performance electrophoresis apparatus.
A characteristic feature of high-performance electrophoresis (HPE), the electrophoretic counterpart of high-performance liquid chromatography (HPLC), is that the separation chamber is a thin-walled, narrow-bore (0.05-0.3 mm) glass or fused-silica capillary tube for rapid dissipation of the Joule heat in order to minimize thermal zone deformation even at high field strengths. This paper is centered around the usefulness of HPE for separation in a carrier-free medium (i.e., in buffer alone) and deals with both zone electrophoresis, isoelectric focusing and displacement electrophoresis. Examples are given of analytical and micropreparative separations of inorganic and organic ions, proteins, viruses and bacteria. The run times are 5-30 min. Discontinuous buffer systems have up to now been used exclusively for the separation of proteins by electrophoresis in polyacrylamide gels ("disc electrophoresis"). However, the Ornstein and Davis discontinuous buffer system has been modified to adapt it to carrier-free zone electrophoresis in order to achieve automatic sharpening of the starting zone. Very high resolution of serum proteins was obtained when they were subjected to free high-performance disc electrophoresis in such a modified buffer system. To show that the HPE apparatus permits electrophoresis also in a gel medium, a polyacrylamide electrophoresis in SDS is presented. This experiment illustrates the difference between electropherograms obtained in free solution and in a molecular-sieving medium. Detection can be performed both on- and off-tube. The latter technique permits the rapid identification of the solutes by photodiode array spectrophotometry and the collection of fractions for further studies. The former detection method is simpler but mainly useful for analytical purposes. Non-UV-absorbing ions can be monitored with the aid of an on-tube UV detector if the run is performed in a UV-absorbing buffer.
Blood Proteins↗
Application of high-performance chromatographic and electrophoretic methods to the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum.
The high resolving power of the preparative and analytical high-performance chromatographic and electrophoretic methods recently developed in this laboratory for the separation of biopolymers has been demonstrated by the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum. Crude glucose oxidase was purified to homogeneity in one step by high-performance hydrophobic-interaction chromatography (HIC) on a pentylagarose column. Crude catalase was purified by a combination of HIC and high-performance anion-exchange chromatography on 3-diethylamino-2-hydroxypropylagarose. The homogeneity of the enzymes was monitored by high-performance electrophoresis and free zone electrophoresis. The pI values of these two enzymes determined by isoelectric focusing in the high-performance electrophoresis apparatus were 4.2 and 6.5, respectively. Their molecular weights were determined by high-performance molecular sieve chromatography on an agarose column. Glucose oxidase has a molecular weight of 175,000 and probably consists of two identical subunits, as sodium dodecyl sulphate polyacrylamide gel electrophoresis gave a molecular weight of around 72,000. The molecular weight of catalase, which is probably composed of non-identical subunits, as indicated by sodium dodecyl sulphate electrophoresis, is around 320,000. Some other characteristics of these two enzymes were also investigated, e.g., electrophoretic mobility, pH stability and optimum pH.
Amino Acids↗
Theoretical and experimental study of high-performance electrophoretic mobilization of isoelectrically focused protein zones.
In an earlier paper we showed that it is possible to mobilize a train of isoelectrically focused proteins and thus detect them on-tube or off-tube. The mobilization was performed in different ways, for instance electrophoretically by exchanging the anolyte for the catholyte or vice versa. In this paper we treat the electrophoretic mobilization theoretically, originating from the conditions of electroneutrality. The information thus gained was used to design anolytes and catholytes of appropriate compositions for mobilization of focused proteins. The usefulness of these electrode solutions is illustrated by focusing-mobilization experiments performed in free solution in a glass tube of length 110 mm. Since the inside diameter of the tube and its wall thickness were only 0.05 mm, the Joule heat was efficiently removed, which allowed the use of high field strengths (270 V/cm). The focusing time was therefore as short as 6 min. The time required for mobilization was about 15 min (360 V/cm). The mobilized protein zones were detected on-tube by absorbance measurements at 280 nm. The glass tube was treated with non-cross-linked polyacrylamide to eliminate electroendosmosis and adsorption of proteins onto the tube wall. The following conclusions drawn from the theoretical studies were experimentally verified: mobilization toward the anode (cathode) can be accomplished by selecting an anolyte (catholyte) containing a cation (anion) other than the proton (hydroxyl ion); the cation (anion) will then electrophoretically migrate into the separation tube and continuously increase (decrease) the pH from the anodic (cathodic) end of the tube. The pH of the electrode solution toward which the mobilization takes place is critical for off-tube, but not for on-tube detection. With the aid of the electroneutrality condition that applies in isoelectric focusing, one can easily explain the generation of the so-called plateau phenomenon.
Animals↗
A highly stable methyl cellulose coating for capillary electrophoresis.
A highly stable capillary-coating has been created by coupling covalently a hydroxyl group of methyl cellulose to the epoxy group of alpha-glycidoxypropyltrimethoxysilane attached to a piece of fused silica tubing. This coating strongly suppresses electroendosmotic flow (EOF) and minimizes sample adsorption onto the capillary wall for several weeks or months. The stability of the coating has been tested with isoelectric focusing and free zone electrophoresis. The coupling procedure is easy to perform, and the coating withstands at least 135 consecutive runs under extreme pH conditions (2-12). EOF is still reduced after placing the coated capillary in 0.01 M NaOH for 30 days. A detailed description of the coating procedure is provided.
Electrophoresis, Capillary↗