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PubMed1997

[The combinations of separation modes in multidimensional high performance liquid chromatography (MDHPLC)].

MDHPLC is a new chromatographic technique, in which fractions from a separation system are selectively transferred to one or more secondary chromatographic systems with different separation mechanism to enhance resolution and sensitivity, or to decrease analysis times. In this review, the principle, characteristics and developments of MDHPLC are discussed. The various possible combinations of separation modes in HPLC [such as, size exclusion chromatography/reversed-phase chromatography (SEC/RPC), ion exchange chromatography/reversed-phase chromatography (IEC/RPC), normal bonded phase chromatography/reversed-phase chromatography (NPC/RPC), size exclusion chromatography/ion exchange chromatography (SEC/IEC), liquid-solid chromatography/reversed-phase chromatography (LSC/RPC), affinity chromatography/reversed-phase chromatography (AC/RPC) and achiral column/chiral column] and their applications are systematically reviewed with 63 references.

English Abstract↗

PubMed2000

Hydrophobic displacement chromatography of proteins.

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.

Biotechnology↗

PubMed2003

Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow-through mode.

The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.

Animals↗

PubMed1994

Reduced-scale large-zone analytical gel filtration chromatography for measurement of protein association equilibria.

The method of large-zone analytical gel filtration chromatography (AGFC) for determination of the energetics and stoichiometries of protein association processes was adapted for use with a semicompressible hydrophilic sieving resin and high-performance liquid chromatography (HPLC) instrumentation. Chromatography was performed using a 2.3-ml glass column, providing a 10-fold reduction in column volume over the conventional AGFC low-pressure chromatography format. Equipment consisted of a Teflon injection valve, HPLC pump, and uv-vis spectrophotometric detector. Method validation included preparation of calibration curves using data obtained from small- and large-zone chromatography of monomeric protein standards. Following confirmation of the agreement of small- and large-zona data obtained for monomeric proteins, the method was further tested by measurement of the dimer-tetramer equilibrium constant for the well-characterized oxygenated human hemoglobin system. Excellent agreement between the experimentally determined equilibrium association constant and the literature value was obtained, as well as a dramatic savings in material and time required to perform the experiment.

Calibration↗

PubMed1979

[Thin layer and high pressure liquid chromatography investigations with cefaclor in urine and serum (author's transl)].

After oral administration of 500 mg cefaclor, antibacterially active metabolites could not be detected in human urine using thin layer chromatography followed by bioautography. Degradation products of cefaclor could also not be detected in the serum of human volunteers (n = 10) using high pressure liquid chromatography with a reversed phase system. Cefaclor was eluated as a single and homogenous peak with a retention period of 2.9 min. High pressure liquid chromatography for the measurement of cefaclor serum levels and a technique for preparation of serum samples are described. After administration of 500 mg cefaclor to volunteers (n = 10), the average peak serum concentration of 9.8 mg/l, determined by high pressure liquid chromatography, was observed after one hour. Four hours later the serum level was 0.3 mg/l. Using microbiological methods no statistically significant difference was obtained in comparison with the chromatography results. Some of the sera stored at -75 degrees C for four weeks showed a substantial loss of activity of cefaclor.

Administration, Oral↗

PubMed1987

Analysis of estrogen sulphate mixtures in pharmaceutical formulations by reversed-phase chromatography.

The relative retention times of estrogen sulphates for reversed-phase, ion-pair and argentous chromatography have been determined. Detector wavelengths of 200 nm, 200 nm and 270 nm were used, respectively. Samples are prepared by dissolution of pharmaceutical formulations in the high-performance liquid chromatography (HPLC) eluent. With reversed-phase chromatography, the estrone, equilin and 17 alpha-dihydroequilin estrogen sulphates are resolved from other estrogen sulphates in 20 min. Any traces of free estrogens which might be present are eluted too slowly for the formation of chromatographic peaks. With ion-pair chromatography, any hydrolyzed estrogens would be eluted before the sulphates with the formation of resolved chromatographic peaks. With argentous chromatography the retention times of the estrogen sulphates varied by nearly a factor of four. The utility of HPLC for the analysis of estrogen sulphates in pharmaceuticals is discussed.

Chromatography, High Pressure Liquid↗

PubMed1992

Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA.

Capillary zone electrophoresis and high-performance liquid chromatography were compared with regard to the separation of oligonucleotides and double-stranded DNA. Both anion-exchange and reversed-phase high-performance liquid chromatography on non-porous particles are considered to be superior to capillary electrophoresis in terms of speed and selectivity in the analysis of oligonucleotides up to 30 bases in length. Moreover, reversed-phase chromatography allows the simultaneous purification of detritylated oligonucleotides with recoveries > 90%. Compared with anion-exchange chromatography, there is no need for a subsequent desaltation step because the volatile buffer system can be readily evaporated. With regard to dsDNA, however, the resolving power of capillary electrophoresis cannot be matched by anion-exchange chromatography at present. Moreover, the combined use of hydroxyethylcellulose and ethidium bromide not only yielded a separation efficiency equal to that achieved by means of gel-filled capillaries but also avoids some of their limitations such as the destruction of the gel matrix at high current densities and the bias involved in electrokinetic injection.

Base Sequence↗

PubMed1988

One-step purification of monoclonal IgG antibodies from mouse ascites. An evaluation of different adsorption techniques using high performance liquid chromatography.

Monoclonal antibodies were purified in one step from mouse ascites by four different high performance liquid chromatography (HPLC) adsorption techniques. The antibodies were of the IgG1, IgG2a, IgG2b and IgG3 subtypes, with isoelectric points between 6.4 and 7.6. Similarly pretreated samples were applied to columns for anion exchange (Mono Q), cation exchange (Mono S), chromatofocusing (Mono P) and hydrophobic interaction chromatography (Alkyl Superose), respectively. Highly purified IgG fractions, as judged by electrophoresis, were obtained in all these techniques. The yields of immunoreactive material were above 80%. A strategy for single step HPLC purification of monoclonal IgG antibodies from mouse ascites by adsorption techniques is proposed, as a complement to the well-established technique of affinity chromatography on immobilized protein A. The strategy involves (i) cation exchange chromatography as a first choice for antibodies with high isoelectric points (greater than 7.2), and (ii) hydrophobic interaction chromatography as a first choice when the isoelectric point is below 7.2.

Animals↗

PubMed1988

Gas chromatography of retinol and alpha-tocopherol without derivatization.

An improved gas chromatographic method for the analysis of retinol and alpha-tocopherol in biological samples is described. The use of cold on-column injection in combination with wall coated open tubular column gas chromatography eliminates thermal decomposition of vitamin A and yields efficient separations of fat-soluble vitamins (A, D2, D3, and E) without derivatization. Peak tailing was judged to be minimal. Vitamins were quantified by flame ionization detection responses down to 3.5 ng injected, and their identities were confirmed using gas chromatography-mass spectrometry. Extracts of biological samples were saponified, and sterols were removed using digitonin-impregnated celite chromatography before analysis by gas chromatography and gas chromatography-mass spectrometry. Recoveries of vitamins from a test diet ranged from 89 to 103%.

Animals↗

PubMed1996

Two-step chromatographic purification of human plasma alpha(1)-acid glycoprotein: its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent.

Alpha1-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (FI*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A*AD, S/A*X0 and F1/A*C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X0 and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.

Chelating Agents↗

PubMed2004

Determination of binding constants by affinity chromatography.

This review summarizes developments in the use of affinity chromatography to characterize biospecific interactions in terms of reaction stoichiometry and equilibrium constant. In that regard, the biospecificity incorporated into the design of the experiment ensures applicability of the method regardless of the sizes of the reacting solutes. By the adoption of different experimental strategies (column chromatography, simple partition equilibrium, solid-phase immunoassay and biosensor technology protocols) quantitatiative affinity chromatography can be used to characterize interactions governed by an extremely broad range of binding affinities. In addition, the link between ligand-binding studies and quantitative affinity chromatography is illustrated by means of partition equilibrium studies of glycolytic enzyme interactions with muscle myofibrils, an exercise which emphasizes that the same theoretical expressions apply to naturally occurring examples of affinity chromatography in the cellular environment.

Biosensing Techniques↗

PubMed2004

Ion-exchange chromatography method for the purification of genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin.

The goal of this study was to provide practical strategies for purifying genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin (BCG-DNA) by ion-exchange chromatography. A multistep process was developed to purify BCG-DNA. The process consisted of sonication, heating, trypsin digestion, ion-exchange chromatography, gel-filter chromatography, and lyophilization. After ion-exchange chromatography, BCG-DNA was highly purified and possessed potent biological effects. The methods described were efficient and had good reproducibility. Further, this was the first reported chromatography method to purify BCG-DNA.

Chromatography, Gel↗

PubMed1984

High-performance chromatofocusing and size-exclusion chromatography: separation of human uterine estrogen-binding proteins.

We have employed high-performance chromatofocusing (HPCF) and high-performance size exclusion chromatography (HPSEC) to separate and identify radiolabelled estrogen binding proteins present in human uterine cytosol. Results obtained using these high-performance methods are compared to results of similar analyses by conventional isoelectric focusing procedures and open-column size exclusion chromatography. By HPCF, descending pH gradients (pH 8-4) allow us to discern four to five estrogen binding proteins with elution pH values typically between pH 4.5 and 7.2. However, when using HPCF, significant quantities of estrogen binding proteins are rarely detected between pH 7.2 and 8.0. This observation has been confirmed by open-column chromatofocusing of these proteins on Polybuffer Exchanger 94. In contrast, preparative isoelectric focusing by electrophoresis in polyampholytes reveals substantial quantities of estrogen binding activity eluted between pH 7.5 and 8.0. Several possible explanations for this disparity are discussed. Apparent differences are also observed when the size heterogeneity of estrogen binding proteins is analyzed simultaneously by size exclusion chromatography in open-column (Sephacryl S-300) and high-performance (TSK-3000SW) modes. HPSEC of these estrogen binding proteins on TSK-3000SW columns demonstrates a predominant 80-85 A species, whereas size exclusion chromatography on conventional Sephacryl S-300 columns reveals two to three distinct regions of estrogen binding proteins with Stokes radii of ca. 85, 60 and 30 A (major species). The larger form of receptor, whether a non-specific aggregate or a multisubunit complex, is stable in unfractionated cytosol and becomes more labile only during size exclusion chromatography.

Carrier Proteins↗

PubMed1983

Two-dimensional high-performance liquid chromatography and chemical modification in the strategy of sequence analysis. Complete amino acid sequence of the lambda light chain of human immunoglobulin D.

Rapid high-performance liquid chromatographic procedure forms part of the strategy for determining the amino acid sequence of proteins. It was applied to the light chain of an immunoglobulin D, and the result, together with previous data on the sequence of the heavy chain, established the complete covalent structure of a human IgD immunoglobulin. We aimed for rapid purification of both small and large peptides. The two-step chromatography system ("two-dimensional chromatography") consists of a combination of macroreticular cation-exchange chromatography as the first dimension and reversed-phase chromatography as the second. This procedure was used for the systematic separation of the tryptic peptides of the light chain. In order to obtain overlapping of the tryptic peptides, chemical modifications of the light chain and reversed-phase chromatography were found to be the most reliable path toward the sequence analysis and greatly accelerated the determination of the light chain.

Amino Acid Sequence↗

PubMed1988

Comparison of mono Q, superose-6, and ABx fast protein liquid chromatography for the purification of IgM monoclonal antibodies.

Nine immunoglobulin M (IgM) monoclonal antibodies (MAbs) produced in ascites fluids or in cell culture supernatants, have been purified on a fast protein liquid chromatography (FPLC) system using anion-exchange, size-exclusion, or mixed-mode chromatography matrices. The use of a mixed-mode ABx column provided an IgM that had a purity of greater than 99% after a single purification step. Anion-exchange chromatography using a Mono Q column, provided a partial purification of the IgM which could subsequently be purified to a product of ca. 90% purity (determined from sodium dodecyl sulfate polyacrylamide gel electrophoresis) by size-exclusion chromatography on a Superose-6 column. Alternatively, the ascites containing the IgM was ammonium sulfate precipitated and chromatographed on the Superose-6 column under normal- as well as high-ionic strength conditions, which also yielded a product of ca. 90% purity. The purification of IgM from concentrated cell culture supernatants was evaluated using the Superose-6 or the ABx column. IgM purified from this source was greater than 99% pure when chromatographed on the mixed-mode column and ca. 60% pure on the size-exclusion column. MAbs from each of the procedures retained their immunoreactivity, as shown by indirect immunofluorescence staining of fixed cell preparations. A comparison of these methods revealed that mixed mode chromatography was simple, efficient, and yielded a product of high purity. The optimization of these methods facilitates the large-scale purification of mouse IgM MAbs and provides practical procedures for generating IgMs for use as diagnostic and therapeutic reagents.

Anion Exchange Resins↗

PubMed2001

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.

Blood Proteins↗

PubMed1989

Profiling cells and body fluids--chromatography and two-dimensional electrophoresis as complementary techniques.

Recent results on the use of gas chromatography-mass spectrometry, high-performance liquid chromatography with diode array detector, amino acid analysis and high-resolution two-dimensional (2-D) electrophoresis to study body fluids and cells in health and disease are surveyed. The chromatography profiling techniques are particularly suitable for the diagnosis of inborn errors of metabolism, with DNA technology as a complementary tool for prenatal diagnosis. Both chromatography and electrophoresis were utilized to study pre-diagnostic sera from the JANUS serum bank and to classify certain bacteria. Protein profiling by 2-D electrophoresis was employed to identify marker proteins associated with the metastatic properties of cloned cancer cells. The electrophoretic technique is also appropriate as a preparative tool for isolating sufficient amounts of marker proteins for microsequencing of amino acids. Chromatography and protein profiling were complementary tools for evaluating the toxicity and mutagenicity of environmental samples in a new test utilizing living human leukocytes and fibroblasts.

Amino Acids↗

PubMed1997

Temperature-responsive liquid chromatography. 2. Effects of hydrophobic groups in N-isopropylacrylamide copolymer-modified silica.

We recently reported the new concept of temperature-responsive liquid chromatography using temperature-responsive poly(N-isopropylacrylamide)-modified surfaces as high-performance liquid chromatography media with aqueous mobile phases. Incorporation of hydrophobic sites is an important factor to improve the efficacy (selectivity and retention) of temperature-responsive chromatography. Toward this goal, we have synthesized semitelechelic copolymers of N-isopropylacrylamide (IPAAm) and butyl methacrylate (BMA) having reactive terminal functional groups using telomerization. The lower critical solution temperatures of the copolymers shift to lower temperatures with increasing hydrophobic BMA content in the poly(IPAAm-co-BMA) relative to that of the IPAAm homopolymer. This temperature-responsive semitelechelic copolymer was grafted to the surface of (aminopropyl)silica through the reaction of activated ester-amine coupling. The polymer-modified silica was used as a column packing material. Separation of a mixture of five steroids having various hydrophobicities was investigated. Retention of steroids on poly(IPAAm-co-BMA)-modified columns is increased with an increase in column temperature. The capacity factors for steroids on the copolymer-modified silica beads was much larger than that on homopolymer PIPAAm-modified columns. The capacity factor for testosterone at 50 degrees C was 33.8 for poly(IPAAm-co-BMA) containing 5 mol% BMA, while that for the PIPAAm homopolymer was 15.0 at the same temperature. The influence of column temperature on steroid retention behavior on copolymer-modified stationary phases was significant compared with the case of homopolymer-modified columns. Furthermore, retention times for steroids increased remarkably with increasing BMA composition. The temperature-responsive elution behavior for the steroids was strongly affected by the hydrophobicity of the grafted polymer chains on silica surfaces. Possible protein separation in temperature-responsive liquid chromatography was explored using insulin chains A and B, and beta-endorphin fragment 1-27. On IBc-3.2-modified silica column, these three peptides were successfully separated at 30 degrees C with 0.5 M NaCl aqueous solution (pH 2.1) as mobile phase. The retention times of these peptides were related to the number of hydrophobic amino acid residues in the peptides. In the proposed chromatography system, elution of target substances is controlled only by a small change in column temperature without any further modification of the aqueous mobile phase.

Acrylamides↗