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PubMed2003

Analytical exclusion chromatography.

This review summarizes the development of exclusion chromatography, also termed gel filtration, molecular-sieve chromatography and gel permeation chromatography, for the quantitative characterization of solutes and solute interactions. As well as affording a means of determining molecular mass and molecular mass distribution, the technique offers a convenient way of characterizing solute self-association and solute-ligand interactions in terms of reaction stoichiometry and equilibrium constant. The availability of molecular-sieve media with different selective porosities ensures that very little restriction is imposed on the size of solute amenable to study. Furthermore, access to a diverse array of assay procedures for monitoring the column eluate endows analytical exclusion chromatography with far greater flexibility than other techniques from the viewpoint of solute concentration range that can be examined. In addition to its widely recognized prowess as a means of solute separation and purification, exclusion chromatography thus also possesses considerable potential for investigating the functional roles of the purified solutes.

Biopolymers↗
PubMed1999

Quantitative competitive reverse transcription-PCR as a method to evaluate retrovirus removal during chromatography procedures.

Chinese hamster ovary cells used for pharmaceutical protein production express non-infectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing process to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for validation studies. Some chromatography procedures used for pharmaceutical protein purification utilize low pH (< pH 4.0) elution buffers which readily inactivate X-MuLV. Therefore, cell-based infectivity assays are unable to evaluate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by chromatography, a quantitative competitive reverse transcription PCR method capable of quantifying both infectious and non-infectious X-MuLV has been developed. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amount of X-MuLV pRNA in the load pool and the product-containing elution pool represents the extent of X-MuLV removal. This method is an extremely powerful complement to cell based-infectivity assays as it allows physical removal of X-MuLV by chromatography and filtration procedures to be distinguished from X-MuLV inactivation when buffers with the ability to inactivate retrovirus are used.

Animals↗
PubMed1986

Analytical affinity chromatography in studies of molecular recognition in biology: a review.

Measuring macromolecular and cellular interactions remains central to the study of recognition in biology and its application in biotechnology. Analytical affinity chromatography provides a versatile methodology to detect and quantitate such interactions. Both zonal and frontal elution approaches have been developed, essentially in parallel, for analytical affinity chromatography. A close quantitative relatedness of chromatographically obtained equilibrium constants and analogous constants determined fully in solution has been found for a growing number of proteins. This consistently observed correlation has formed the basis for extending theoretical treatments in order to evaluate not only monovalent molecular systems of varying types but also multivalently interacting macromolecules, including those which exhibit cooperativity. The potential to measure chemical rate constants by affinity chromatography also has been recognized, and experimental tests of the available theory are being made. As a micromethod, the quantitative use of affinity chromatography has important applicability for biochemical analysis of an increasing array of biologically active molecules being discovered and isolated but available in only relatively small amounts. Analytical affinity chromatography thus provides a means to use matrix--mobile interactant systems to study mechanisms of biomolecular interactions and therein to attain an understanding of such interactions which often is not easily achieved by solution methods alone.

Animals↗
PubMed1990

Chelation ion chromatography as a method for trace elemental analysis in complex environmental and biological samples.

The development and evaluation of a new method for the determination of trace transition and rare-earth elements based on the combination of chelation and ion chromatography are described. The new method, chelation ion chromatography (Chelation IC), uses a chelating column to concentrate and separate transition and rare-earth elements from the common alkali and alkaline-earth metals, as well as other matrix components, prior to analysis by ion chromatography. The sample fraction from the chelating column contains only the concentrated analyte ions, thus eliminating interfering matrix components from complex matrices such as seawater and digested biological, botanical, and geological materials. This combination of chelation and ion chromatography provides a technique that makes possible the determination of trace elements in complex matrices that have proven to be difficult or impossible to analyze by ion chromatography or conventional atomic spectroscopy techniques.

Animals↗
PubMed2003

Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition.

A protocol combining immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. Beta-elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to beta-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin.

Amino Acid Sequence↗
PubMed2003

On-chip hydrodynamic chromatography separation and detection of nanoparticles and biomolecules.

For the first time, on-chip planar hydrodynamic chromatography is combined with UV absorption detection. This technique is suitable for size characterization of synthetic polymers, biopolymers, and particles. Possible advantages of an on-chip hydrodynamic chromatography system over conventional techniques, such as size exclusion chromatography, and field-flow fractionation are fast analysis, high efficiency, reduced solvent consumption, and easy temperature control. The hydrodynamic separations are performed in a planar configuration realized in fused silica using a mixture of fluorescent and nonfluorescent polystyrene particles with sizes ranging from 26 to 155 nm. The planar chip configuration consists of a 1-microm-high, 0.5-mm-wide, and 69-mm-long channel, an integrated 150-pL injection structure, and a 30-microm-deep and 30-microm-wide detection cell, suitable for UV absorption detection. By combination of the separation data obtained in the new fused-silica chip with those obtained using a previously presented planar hydrodynamic chromatography chip, which was realized using silicon and glass microtechnology, a description of the retention and dispersion behavior of planar hydrodynamic chromatography is obtained. Especially the influence of the sidewalls on the dispersion is investigated. Furthermore a hydrodynamic separation within 70 s of several biopolymers is shown in the glass-silicon chip.

Chromatography, Gel↗
PubMedDate not supplied

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale.

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.

Biotechnology↗
PubMedDate not supplied

Continuous separation of multicomponent protein mixtures by annular displacement chromatography.

Displacement chromatography is a predominantly nonlinear mode of chromatography, which has certain advantages over the elution mode for preparative bioseparations. Whereas continuous production (and separation) processes have their theoretical benefits in this context, protein displacement chromatography has up to now only been performed in the batch mode. In this contribution, we demonstrate that the principle of continuous annular chromatography can be adapted to displacement chromatography. Separations of up to three standard proteins (two whey proteins, soybean trypsin inhibitor) were developed and optimized using a small (4 x 250 mm) batch column. These separations were subsequently transferred directly to the continuous system (500-mL column). Separations of similar quality in terms of final product purity and recovery yield were obtained using the continuous system.

Adsorption↗
PubMed1999

Purification of catechol siderophores by boronate affinity chromatography: identification of chrysobactin from Erwinia carotovora subsp. carotovora.

Catechols are co-planar cis-diols known to form stable, isolable complexes with borate under weakly basic conditions. We exploited this chemistry and developed a boronate affinity chromatography for isolating catechol siderophores. The method was applied to the isolation of chrysobactin, enterobactin, and an unknown catechol siderophore produce by Erwinia carotovora subsp. carotovora W3C105. Yields of chrysobactin and enterobactin purified by boronate affinity chromatography were at least two-fold greater than those achieved through alternate methods. The unknown catechol produced by E. carotovora subsp. carotovora W3C105 was isolated by boronate affinity chromatography and shown to be identical to chrysobactin. Boronate affinity chromatography enabled separation of catechol from its rust-colored decomposition products, and simultaneous isolation of catechol and hydroxamate siderophores. Boronate affinity chromatography is a rapid and efficient method for purifying catechol siderophores from bacterial culture supernatants.

Borates↗
PubMed1989

Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography.

We recently found that many RNA polymerase II transcription factors are modified with N-acetylglucosamine residues. These sugar moieties confer upon transcription factors an ability to bind the lectin wheat germ agglutinin. We have taken advantage of this interaction to devise a purification procedure for the "GC-box" binding transcription factor Sp1. Crude nuclear extracts are first subjected to wheat germ agglutinin affinity chromatography and then subjected to sequence-specific DNA affinity chromatography. The Sp1 protein purified by this procedure is at least 95% pure, and the overall recovery is greater than 80%. In addition to yielding larger quantities of Sp1 than conventional schemes, the new purification procedure is also simpler and more rapid. We show that wheat germ agglutinin affinity chromatography can also be used to purify the glycosylated forms of the CCAAT-binding transcription factor. Thus, wheat germ agglutinin affinity chromatography may aid the purification of other transcription factors that bear N-acetylglucosamine residues. Furthermore, the ability to separate glycosylated forms of transcription factors from their unglycosylated counterparts by wheat germ agglutinin affinity chromatography should facilitate investigations into the role of N-acetylglucosamine residues in the functioning of transcription factor proteins.

Acetylglucosamine↗
PubMed1990

Analysis of the oligosaccharides on rat androgen-binding protein using serial lectin chromatography.

The microheterogeneity seen when rat androgen-binding protein (rABP) is analyzed by two-dimensional polyacrylamide gel electrophoresis is attributable, at least in part, to the differential glycosylation of a single promoter. Further insight into the chemical nature of the oligosaccharide units on rABP was obtained by serial lectin chromatography. When rABP was chromatographed on immobilized Concanavalin A (Con-A), it was fractionated into three classes: (1) one that did not bind to the lectin (about 44% of the rABP), (2) one that was bound and could be eluted with 10 mM 1-O-methyl alpha-D-glucopyranoside (glucoside), about 34%, and (3) one that could be eluted with 0.5 M methyl alpha-D-mannopyranoside (mannoside), about 23%. Binding to Con-A indicates the presence of asparagine-linked oligosaccharides. Chromatography of the glucoside-eluted peak on lentil lectin (LcH) indicated that the rABP in that fraction contained a fucose residue on the chitobiose core. Chromatography of the mannoside-eluted peak on wheat germ agglutinin (WGA) indicated the presence of rABP with high mannose- (44%) and hybrid-type (56%) glycans attached. Chromatography on Ricinus communis I (RCA-I) lectin indicated a species containing galactosylated complex-type oligosaccharide chains. Treatment of rABP forms with exoglycosidases confirmed the presence of externally disposed fucose, sialic acid, mannose, and galactose residues. LcH chromatography indicated that about 30% of the rABP that did not bind to Con-A possessed triantennary oligosaccharides with fucose on the chitobiose core. About 28% of the rABP was retarded when it was chromatographed on Phaseolus vulgaris E lectin, suggesting the presence of bisected biantennary chains with terminal galactose residues. We were unable to detect rABP species with serine- or threonine-linked oligosaccharide chains in this fraction. Other forms of rABP in the nonretained fraction of Con-A were not resolved. Western blotting did not reveal major differences in relative molecular weight (Mr) among the rABP species; some differences in the ratio of the heavy to the light subunit of the molecule were detectable.

Androgen-Binding Protein↗
PubMed1985

Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains.

Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domain eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps; hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.

Chemical Phenomena↗
PubMed1979

Rapid differentiation of Streptomyces from Nocardia by liquid chromatography.

A liquid chromatographic method is described for analysis of aerobic actinomycetes for isomers of diaminopimelic acid. One or two colonies of organism were hydrolyzed with 6.0 mol of HCl per liter at 121 degrees C for 15 min. The hydrolysate was neutralized and buffered with an NaOH solution (3 mol/liter) containing 0.15 mol of sodium borate per liter. Precolumn derivatization with dansyl chloride was used to form a fluorescent product for detection. Analysis was performed by reversed-phase, ion-pair chromatography. The L-diaminopimelic acid isomer was detected in all 10 strains of Streptomyces tested, and the meso-diaminopimelic acid isomer was detected in all 10 strains of Nocardia tested. Liquid chromatography was compared simultaneously with thin-layer chromatography in the analysis of three strains of aerobic actinomycetes. Liquid chromatography required less growth of the organisms, and analysis was completed within 1 h, compared with the 3 to 5 days required by thin-layer chromatography.

Aerobiosis↗
PubMed1984

Determination of 25-hydroxyvitamin D3 in serum by high performance liquid chromatography and isotope dilution-mass spectrometry.

Four different HPLC methods for analysis of 25-hydroxyvitamin D3 in serum were evaluated with a method based on isotope dilution-mass spectrometry (ID-MS). Method I utilized Sephadex LH-20 chromatography as the only prepurification step. No correlation with the ID-MS method was obtained. Method II utilized Sephadex LH-20 chromatography and a subsequent reversed phase HPLC step as prepurification. The correlation coefficient was 0.99 (regression coefficient 1.2 and intercept - 3.9 micrograms/l). Method III included open silicic acid chromatography and straight phase HPLC as prepurification. The correlation when compared with the ID-MS method was 0.94 (regression coefficient 1.2 and intercept - 0.4 micrograms/l). In method IV Sep-pak C18 chromatography and open silicic acid chromatography were used as prepurification. The correlation coefficient when compared with the ID-MS method was 0.97 (regression coefficient 0.8 and intercept 0.1 microgram/l). It is concluded that a single Sephadex LH-20 step is not sufficient as prepurification and that method IV had an accuracy sufficient for its intended use to analyse 25-hydroxyvitamin D3 in serum from cattle.

Calcifediol↗
PubMed1989

Soluble complex formation of bovine immunoglobulin G2 with staphylococcal protein A studied by gel filtration chromatography.

The binding property of bovine IgG2 to staphylococcal Protein A was investigated by the methods of gel filtration chromatography and affinity chromatography. High performance gel filtration chromatography was carried out using TSK gel G3000SW and G2000SW columns, and immobilized Protein A column was used for affinity chromatography. Although bovine IgG2 did not form any precipitin lines with Protein A by double diffusion method on agar gel, IgG2 could bind to immobilized Protein A column. Moreover, by gel filtration chromatography, peaks of the complex between bovine IgG2 and Protein A were observed in addition to the IgG2 monomer peak. Thus, it is concluded that bovine IgG2 interacts with staphylococcal Protein A and forms "soluble complexes". Carbethoxylated IgG2 lost its affinity to Protein A indicating that histidyl residues in IgG2 is essential for the binding to Protein A.

Animals↗
PubMed2000

Advances in capillary chromatography.

Capillary columns are used in both capillary liquid chromatography and capillary electrochromatography. The design for capillary liquid chromatography is discussed in comparison with capillary gas chromatography. The difference of diffusion coefficient in gas and liquid phase is a key role. The study for obtaining a high performance capillary liquid chromatography is discussed. Capillary electrochromatography is recently interesting for its instinct ability to realize a high performance chromatography. Capillary electrochromatography with and without pressurized flow is reviewed briefly. Instrumentation for capillary electrochromatography with pressurized flow is discussed. The port of splitting, and gradient elution of both solution and potential are described. The new findings of both the variation of column resistance and capacity factor according to the value of applied electric voltage are also discussed.

Chromatography, Liquid↗
PubMed1999

[The advances of the study on the optical chromatography].

This review is mainly concerned with the recent developments in (1) the differences between the optical tweezer and optical chromatography; (2) the principle of optical chromatography separation; (3) the theory studies on the optical chromatography, especially the relationship between the particle radius with the intensity of the applied laser radiation force and the relationship between the particle retention distance with the particle radius; (4) the application of the optical chromatography technique to the separation of polymers, biological macromolecules and biological cells. The application of optical chromatography to the immunoassy and molecular biology is highlighted.

Chromatography↗
PubMed1986

Immunoaffinity chromatography of carcinogen DNA adducts with polyclonal antibodies directed against benzo[a]pyrene diol-epoxide-DNA.

Polyclonal antibodies specific for (+/-)-trans-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(BPDE) CAS: 58917-67-2]-DNA adducts were obtained from the sera of New Zealand White rabbits immunized with BPDE-DNA. These antibodies did not recognize benzo[a]pyrene [(BP) CAS: 50-32-8] or DNA alone or other carcinogen adducts, such as aflatoxin (CAS: 1402-68-2)-DNA or aminopyrene (CAS: 58-15-1)-DNA, up to the concentrations used. In a competitive enzyme-linked immunosorbent assay, 0.18 microgram BPDE-DNA/ml (single stranded), equivalent to 7 pmol BPDE adduct, caused 50% inhibition with this antibody. (When referring to the DNA content of BPDE-DNA, the authors gave the concentration in microgram/ml; when referring to the BPDE content of BPDE-DNA, the authors gave the concentration as pmol/ml.) Chemical and enzymic modifications of the BPDE-DNA substrate suggested that the epitope for the antibody is greater than that represented by a BPDE-nucleoside adduct. The specific BPDE-DNA antibodies were covalently bound to cyanogen bromide-activated Sepharose 4B, and the extent of ligand binding to the immunoaffinity column was measured with the use of [3H]BPDE-DNA as substrate. Maximum binding to the immunoaffinity column was obtained after DNase 1 digestion of [3H]BPDE-DNA: The bound adducts could be readily eluted from the column with 50 mM NaOH. The binding of DNase 1-digested [3H]BPDE-DNA to the immunoaffinity column was dose related and not affected by the addition of unmodified DNA. The columns have proven to be reusable. Samples of [3H]BP-DNA isolated from the skin of mice treated topically with either 0.75 mumol [3H]BP/mouse or 1.5 mumol [3H]BP/mouse were examined by immunoaffinity chromatography. Binding values of 6.0 and 12.2 pmol BP/mg DNA were obtained; these values from immunoaffinity chromatography were slightly lower than those determined by high-pressure liquid chromatography analysis (9 and 17 pmol BP/mg DNA). With chemically reacted BPDE-DNA, around 70% of that applied was retained by immunoaffinity chromatography, whereas with [3H]BP-DNA isolated from the in vivo treatment of mouse skin, only 40% was retained--a possible reflection of the greater heterogeneity of the in vivo BP-DNA adducts. This immunoaffinity chromatography technique should prove useful in the selective examination of levels of BPDE-DNA adducts present in biological samples.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗