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PubMed1981

Countercurrent chromatography.

Countercurrent chromatography is a newly developed versatile partition chromatography which totally eliminates the use of solid supports. The method utilizes the intriguing hydrodynamic behavior of two immiscible solvents in a rotating coiled tube. Various types of seal-free flow-through centrifuge schemes are introduced to facilitate a continuous elution process. The method yields high partition efficiencies comparable to liquid chromatography but without the complications arising from the use of solid supports. Countercurrent chromatography covers a wide spectrum of applicable samples ranging from small ions and molecules to macromolecules and even cell particles in both analytical and preparative scales.

Acceleration↗
PubMed1991

Purification of measles virus by affinity chromatography and by ultracentrifugation: a comparative study.

The purification of viruses by ultracentrifugation in regions with limited resources is often hampered by problems of instrument maintenance. Viral antigens must therefore be imported, which results in delays in obtaining epidemiological data. To address this problem, we undertook the development of purification procedures by affinity chromatography using measles virus (strain Y15) and monkey IgG (anti-measles) coupled covalently via their carbohydrate residues to glass spheres as a model system. The measles virus prepared in this way was compared with measles virus purified by ultracentrifugation using three criteria: the degree of purification, yield and the sensitivity in HA and ELISA. The purification of the antigen by ultracentrifugation (AgU) was undertaken using 10(9) infected cells, and by affinity chromatography (AgAc) using 10(7) infected cells. The total concentration of proteins before purification was 5000 mg for AgU and 11.3 mg for AgAc. After purification by AgU 21.5 mg protein was obtained which was assessed by specific hemagglutinating units (HAU/mg protein) and found to be 179 for AgU and 372-744 for AgAc. Based on total HAU, the yield by centrifugation was 2.4%, whereas the affinity chromatography produced essentially 100%. By ELISA, using reference sera to calibrate the antigens, maximum OD was obtained with 0.5 microgram/well of AgAc and with 2 micrograms/well of AgU. From the relatively small amount of starting material, good yields were obtained with increased specific activity economy in coating by ELISA. These results favour the adoption of affinity chromatography for purifying viral antigens.

Animals↗
PubMed1989

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence.

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.

Amino Acid Sequence↗
PubMed1993

Characterization of the covalent chromatography of thymidylate synthase on thiopropyl-Sepharose 6B.

We have examined the covalent chromatography of Lactobacillus casei thymidylate synthase on thiopropyl-Sepharose 6B resin. This enzyme is a dimer of identical subunits, each of which contains one active site that features a catalytic sulfhydryl group (Cys-198). Reversible coupling was achieved via the attack of one of the enzyme's two catalytic cysteine residues, causing displacement of 2-thiopyridone, the reactive moiety of the resin. To establish the usefulness of this matrix for immobilization and the conditions required for chromatography, model studies were conducted with 2,2'-dithiodipyridine. The chemical modification of thymidylate synthase with 2,2'-dithiodipyridine was shown to be specific for the catalytic sulfhydryl groups of the native dimer, titrating 1.51 sulfhydryl groups, while 2.93 cysteines were modified in the GnHCl-denatured protein. The former reaction, which resulted in total loss of enzyme activity, was reversible with complete recovery of control activity within 30 min after addition of 100 mM 2-mercaptoethanol. Characterization of the protein pools generated in the covalent chromatography procedure indicated that the enzyme fraction washing through the column without attachment had substantially lower catalytic and ligand binding activities than the original protein stock; conversely, the enzyme fraction eluted from the column by 2-mercaptoethanol exhibited higher levels of these activities. Gel electrophoresis studies further illustrated that the unique application of the covalent chromatography technique described herein fractionated homogeneous thymidylate synthase protein into enzyme pools exhibiting distinct biochemical properties. As immobilization reaction times were increased beyond 6 h, the coupling of thymidylate synthase was demonstrated to occur through more than one enzymic sulfhydryl group. Interestingly, no covalent coupling was detected in attempts using activated thiol-Sepharose 4B, a result underlining the importance of the structure of the resin linker arm in enzyme immobilization.

2,2'-Dipyridyl↗
PubMed1992

Purification of A-subtype pancreatic cholecystokinin receptor by immunoaffinity chromatography.

So far, no efficient affinity chromatography for CCK receptor purification has been reported that prevented obtention of sequenceable amounts of purified receptor. In this work, 10% of plasma membrane receptor sites were specifically cross-linked with the photoreactive cleavable agonist 125I-ASD-[Thr28, Ahx31]-CCK-25-33, solubilized by NP-40, chromatographied on immobilized wheat germ agglutinin and further immunopurified using anti-CCK antibodies to an overall rate of 3000-3600-fold. Analysis of eluted material demonstrated a protein migrating at Mr 85,000-100,000 and the absence of 35S-labeled impurity. This single and efficient affinity chromatography should provide enough homogeneous receptor protein for microsequence determination and leads to consider immunoaffinity chromatography on immobilized anti-ligand antibodies as a potential tool for purification of membrane receptors.

Affinity Labels↗
PubMed1985

Comparison between inhibin from human and bovine ovarian follicular fluid using fast protein liquid chromatography.

Inhibin from human and bovine ovarian follicular fluid was purified 700-900-fold using affinity chromatography on immobilized Procion Red 3B, desalting on Sephadex G-25, ion-exchange chromatography on the fast protein liquid chromatography (FPLC) columns Mono Q and Mono P and chromatography on immobilized lectins. Isoelectric points for inhibin from human and bovine origin were between 5.1-5.7 and 4.75-5.25, respectively. Inhibin from both sources was retained by immobilized lectins, indicating its association with a glycoprotein. Overall recoveries of inhibin activity after these chromatographic procedures were approximately 1%.

Animals↗
PubMed1991

Carrier membrane as a stationary phase for affinity chromatography and kinetic studies of membrane-bound enzymes.

The use of membrane supports as stationary phase, coupled with ligands of choice, allows all kinds of chromatography [Dj. Josić, K. Zeilinger, Y. Lim, M. Raps, W. Hofmann and W. Reutter, J. Chromatogr., 484 (1989) 327] and offers a powerful alternative to both soft gel chromatography and high-performance liquid chromatography. In this work we present affinity membrane chromatography for purification of the enzyme carbonic anhydrase from haemolysates of human erythrocytes. Furthermore, the coupling of the enzymes to the membrane support allows kinetic investigations. As an example, kinetic experiments were carried out by means of carbonic anhydrase coupled to the membrane support using 4-nitrophenyl acetate and 2-chloro-4-nitrophenyl acetate as substrates.

Carbonic Anhydrases↗
PubMed1991

Comparative study on the determination of the anti-neoplastic drug teniposide in plasma using micellar liquid chromatography and surfactant-mediated plasma clean-up.

The potential of micellar liquid chromatography and of an on-line surfactant-mediated sample cleanup, which involves column-switching prior to conventional reversed-phase high-performance liquid chromatography, has been evaluated for the determination of the anti-neoplastic drug teniposide in plasma by using electrochemical detection. A major advantage of surfactant-mediated techniques is that they allow fully automated processing of plasma samples, because protein precipitation is prevented by the addition of the surfactant sodium dodecylsulphate. With the automated column-switching technique, a degree of sample enrichment and of selectivity can be attained, which is similar to that for the conventional procedure which, however, involves a labour-intensive off-line isolation of teniposide, using liquid-liquid extraction prior to chromatography. An inherent drawback of automated micellar liquid chromatography is that no sample clean-up or preconcentration can be carried out, which results in only a moderate detection limit and selectivity. The linearity, reproducibility and recovery of the surfactant-mediated techniques are similar to those of the conventional procedure. Based on the presented results, it was concluded that the surfactant-mediated column-switching technique is a highly attractive sample enrichment technique with respect to simplicity, speed and cost.

Chromatography, High Pressure Liquid↗
PubMed1996

Inhibition of hepatic lipase by m-aminophenylboronate. Application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases.

Phenylboronates are competitive inhibitors of serine. hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1,1.3). m-Aminophenylboronate inhibited hepatic lipase activity with a Ki value of 55 microM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h-1 mg-1 protein with a total purification factor of 14,400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h-1 mg-1. The purification factor was 23,400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.

Boronic Acids↗
PubMed1996

Chromatography of hydroxysteroid dehydrogenases.

The structure-function study of hydroxysteoid dehydrogenases has stimulated the development of their chromatography, which in turn reveals more mechanisms of these enzymes. Due to the various membrane associations and mild hydrophobic nature of most of the enzymes studied up to now, hydrophobic interaction chromatography has played a crucial role in their purification, using media such as phenyl-Superose or Sepharose-PEG. At the same time, affinity chromatography, especially the dye-containing columns, proves very efficient for these dehydrogenases, as the latter utilizes adenylyl-containing cofactors. Elution by their specific ligand facilitates their purification. In this paper, the use of detergents in the purification of these enzymes is also reviewed. Hydroxysteroid dehydrogenase preparation is further improved by rapid purification which facilitates the elimination of protein microheterogeneity, caused in vitro by oxidation, reduction or partial proteolysis. This process was shown to increase the crystallizability of the enzymes [Lin et al., J. Cryst. Growth, 122 (1992) 242-245; Zhu et al., J. Mol. Biol., 234 (1993) 242-244]. The fast purification permitted a simpler procedure and better combination of various columns than conventional chromatography. This leads to even higher efficiency, yielding homogeneous and highly active preparations.

Animals↗
PubMed1995

Determination of true octanol-water partition coefficients by means of solvent generated liquid-liquid chromatography.

Compared to other methods for the determination of octanol-water partition coefficients chromatography offers a number of advantages: sample purification is unnecessary, the partition coefficients of the components of a mixture can be measured simultaneously and a minimum amount of sample is needed. In the past these determinations were almost exclusively carried out by liquid-solid chromatography (LSC) on alkyl bonded silica as stationary phase (conventionally described as 'reversed-phase liquid chromatography; RPLC). Such systems based on liquid-solid distribution are, however, a poor simulation of liquid-liquid partition. On the other hand liquid-liquid chromatographic columns loaded with high amounts of water-saturated octanol are unstable since they suffer from "column bleeding"--a loss of the stationary liquid octanol phase caused by erosion. It is shown that the technique of solvent generated liquid-liquid chromatography (SGLLC) leads to stable columns with liquid-liquid partition as the predominant retention mechanism, if systematic errors due to specific adsorption effects are avoided by the selection of an "inert" solid support. This is demonstrated by the comparison of LSC and SGLLC data. SGLLC significantly reduces the scattering of the retention data of calibration standards around the calibration line. The use of SGLLC thus significantly improves the accuracy and precision of the resulting octanol-water partition coefficients.

Adsorption↗
PubMed2005

Comparison of standard methods and gas chromatography method in determination of formaldehyde emission from MDF bonded with formaldehyde-based resins.

Formaldehyde emissions from MDF bonded with urea-formaldehyde resin (UF), melamine-formaldehyde resin (MF) and the co-polycondensation resin of urea-melamine-formaldehyde (UMF) and melamine-formaldehyde, measured by the Japanese standard method of determining formaldehyde emission with a desiccator (JIS A 5908) and the DIN EN 120 (European Committee For Standardization, 1991) method using the perforator value, were used as the typical standard methods. While the UF resin showed a desiccator value of 7.05 ppm and a perforator value of 12.1 mg/100 g panel, the MF resin exhibited a desiccator value of 0.6 ppm and a perforator value of 2.88 mg/100 g panel. According to the Japanese industrial standard and the European standard, the formaldehyde emission level of the MDF panels made with UF resin in this study was E(2) grade. The formaldehyde emission level was dramatically reduced by the addition of MF resin. This is because the addition of formaldehyde to melamine occurs more easily and completely than its addition to urea, even though the condensation reaction of melamine with formaldehyde is similar to that between urea and formaldehyde. These two methods, the desiccator method and the perforator method, produced proportionally equivalent results. Gas chromatography, a more sensitive and advanced method, was also used. The samples used for gas chromatography were gathered during the experiment involving the perforator method. The formaldehyde emission levels obtained from gas chromatography were similar to those obtained from the perforator method. The formaldehyde contents measured by gas chromatography were directly proportional to the perforator values.

Chemistry Techniques, Analytical↗
PubMed2004

Rapid simultaneous quantification of immunosuppressants in transplant patients by turbulent flow chromatography combined with tandem mass spectrometry.

BACKGROUND: Immunosuppressant therapeutic drug monitoring (TDM) is an important requirement in the management of post-transplant patients. Our aim was to develop and evaluate a robust high-throughput method using turbulent flow chromatography (TFC) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantification of cyclosporin A (CsA), tacrolimus (FK 506) and sirolimus. METHODS: A total of 1483 EDTA-blood pre-dosage samples from 147 kidney, 67 liver, 15 kidney/pancreas, and 48 bone marrow recipients were collected. After hemolysis and protein precipitation of 50 microl blood, fast and efficient on-line matrix elimination was achieved using turbulent flow chromatography. Tandem mass spectrometric detection and quantification was performed using multiple reaction monitoring (MRM). RESULTS: The total analysis time of the column switching method was 3 min. The method was linear from 4.5 to 1500 ng/ml for cyclosporin A, from 0.2 to 100 ng/ml for tacrolimus, and from 0.4 to 100 ng/ml for sirolimus. The accuracy was >95%. Within and between-run assay variation coefficients ranged from 2.4% to 9.3%. Excellent correlation with other standard methods (immunoassay, HPLC) was observed. CONCLUSIONS: The presented turbulent flow chromatography-tandem mass spectrometric platform offers a very fast, simple and economical method with an excellent validation profile and is well suited for daily pre- and post-dosage immunosuppressant monitoring.

Bone Marrow Transplantation↗
PubMed2004

Preparative and analytical chromatography of pegylated myelopoietin using monolithic media.

Monolithic media were compared with Q- and SP-Sepharose high performance chromatography for preparative purification and with Q- and SP-5PW chromatography for analysis of a pegylated form of myelopoietin (MPO), an engineered hematopoietic growth factor. The use of either monolithic or Sepharose based supports for preparative chromatography produced highly purified pegylated MPO with the monolithic media demonstrating peak resolution and repeatability at flow rates of 1 and 5 ml/min resulting in run times as much as five-fold shorter compared to Sepharose separations. The monolithic disks also resulted in 10-fold shorter run times for the analytical chromatography, however, their chromatographic profiles and peak symmetry were not as sharp compared to their Q-5PW and SP-5PW counterparts.

Chromatography, Gel↗
PubMed2004

Equivalence of the microscopic and macroscopic models of chromatography: stochastic-dispersive versus lumped kinetic model.

The microscopic model of chromatography is a stochastic model that consists of two fundamental processes: (i) the random migration of the molecules in the mobile phase, and (ii) the random adsorption-desorption of molecules on the stationary phase contained in a chromatographic column. The diffusion and drift of the molecules in the mobile phase is described with a simple one-dimensional random walk. The adsorption-desorption process is modeled by a Poisson process that assumes exponential sojourn times of the molecules in both the mobile and the stationary phases. The microscopic, or molecular model of chromatography studied here turns out to be identical to the macroscopic lumped kinetic model of chromatography, whose solution is well known in chromatography. A complete equivalence of the two models is established via the identical expressions they provide for the band profiles.

Chromatography↗
PubMed2004

Using ion chromatography to monitor haloacetic acids in drinking water: a review of current technologies.

A review of the application of ion chromatography to the determination of haloacetic acids in drinking water is given. As it requires no sample derivatisation, ion chromatography in its various modes, such as ion-exchange, ion-interaction and ion-exclusion chromatography, is increasingly being investigated as a simpler alternative to gas chromatographic methods for the determination of polar disinfection by-products (DBPs) in drinking waters. Detection limits quoted for the regulated haloacetic acids (HAA5), are commonly in the mid to low microg/L range, however, in most cases analyte preconcentration is still necessary for detection at concentrations commonly found in actual drinking water samples. The coupling of ion chromatography to electrospray mass spectrometry provides a potential future direction, with improved sensitivity and selectivity compared to conductivity based detection, however associated cost and complexity for routine analysis is currently relatively high.

Acetates↗
PubMed2004

Optimization of diagonal chromatography for recognizing post-translational modifications.

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents a formidable analytical challenge. This paper examines the potential of diagonal chromatography for recognizing post-translational modifications. Diagonal chromatography is the process of using the same chromatographic separation in two dimensions. Between the two dimensions, a chemical modification is applied to all fractions. Substances that have been modified are revealed by the change in their chromatographic properties between the two dimensions. When the modification is specific to a particular type of post-translational modification, peptides that carry the modification have the potential to be revealed. Changes in the retention time of modified peptides have to be large enough to be different from unmodified peptides. Tyrosine, serine, and threonine phosphorylation were identified with diagonal chromatography. Heptafluorobutyric acid was used as an ion-pairing agent to improve the selectivity between serine and threonine phosphorylated peptides and parent peptides after dephosphorylation. The diagonal chromatography method was also examined in the recognition of glycopeptides. However, changes of retention time after deglycosylation were considered to be too small to make this an unequivocal method for the study of glycosylation.

Chromatography↗
PubMed2005

Novel system for classifying chromatographic applications, exemplified by comprehensive two-dimensional gas chromatography and multivariate analysis.

For practical chromatographers it is extremely difficult to judge the merits and limitations of new technological developments. On the other hand, it is nearly impossible for those at the forefront of technology to judge the implications of their efforts for all specific applications of chromatography. Both chromatographers and researchers can be aided by a classification of the numerous specific applications into a few well-defined categories. In this paper, we propose such a classification of all chemical analysis by chromatography into three generic types of applications, viz. target-compound analysis, group-type separation, and fingerprinting. The requirements for each type are discussed in general terms. The classification scheme is applied to assess the benefits and limitations of comprehensive two-dimensional gas chromatography (GC x GC) and the possible additional benefits of using multivariate-analysis (MVA) techniques for each type of application. The conclusions pertaining to the generic types of applications are indicative for the implications of new developments for specific chemical analysis by chromatography.

Chromatography, Gas↗