Single-column Chromatography Research Articles

Micro-SPE method for sample introduction in capillary HPLC/MS.

A new solid-phase extraction on-line device for micro-HPLC is presented. This device optimizes the injection of very dilute samples into a packed capillary column. It consists of two capillary, reversed-phase, HPLC columns of different length that can be linked together as a single chromatographic column. The first segment, only 2 cm long is connected to the HPLC injector. When disconnected from the longer column, several milliliters of an aqueous sample can be passed through at a high flow rate for fast trapping. On the basis of the retention mechanism, all suitable compounds are focused on the short column head in a sharp band. As soon as the chromatographic column is recomposed, the trapped analytes are eluted and separated at the optimal flow rate and gradient conditions. Due to the high preconcentration factor, trace-level analysis can be performed successfully. Different classes of analytes of various polarities and molecular weights can be determined, depending on the stationary phase and on the detector used. Some pesticides belonging to different classes were chosen to evaluate the performance of the device using an electron ionization mass spectrometer as HPLC detector. A fungicide in an irrigation canal water was determined at a concentration level of 4.5 microg x L(-1).

Modeling and computationally efficient simulation of chromatographic separation processes

Chromatographic separation processes are an emerging technology, especially in the field of fine chemicals and pharmaceutical products. As an alternative to conventional batch chromatography, the simulated moving bed (SMB) process gained more and more impact recently. Because of the complex dynamics, the automatic control of chromatographic separation processes is a challenging task, the solution of which requires reliable and computationally efficient simulation models. The purpose of this contribution is to give an overview about the generation of dynamic models both for single chromatographic columns and SMB processes. We here distinguish chromatographic processes by the type of adsorption isotherm and proceed from the ideal model for chromatographic columns while increasing the model complexity as far as necessary in order to build a simulation model which on the one hand is computationally effective and on the other hand correctly describes the dynamics of the process which is relevant for on-line optimization and control purposes.

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry.

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was </=5 min per sample.

Pollution Models May Need More Carbon

Urban air pollution models may not accurately reflect levels of volatile organic compounds (VOCs) in the atmosphere, a new research report suggests. VOCs play a role in the formation of ground-level ozone and secondary organic aerosols. Alastair C. Lewis of the University of Leeds, England, and his coworkers there and at Royal Melbourne Institute of Technology in Victoria, Australia, use gas chromatography to analyze urban air samples collected in Melbourne [ Nature , 405 , 778 (2000)]. The authors write that they can isolate more than 550 individual compounds using this method, which employs two chromatographic columns with different properties. Many compounds with more than six carbon atoms, the authors note, can only be isolated by using comprehensive chromatography. The peaks for many of these compounds are hidden in the baseline of conventional single-column chromatography analyses. If you start to look at the baseline in detail with this technique, you can see ...

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was </=5 min per sample.

Modelling and simulation of nonlinear chromatographic separation processes: a comparison of different modelling approaches

Chromatographic processes provide a powerful tool for the separation of multicomponent mixtures in which the components have different adsorption affinities. As an alternative to conventional batch chromatography as a discontinuous process, the simulated moving bed (SMB) process as a continuous chromatographic separation process gained more and more impact recently. Mathematical modelling and simulation of batch chromatographic processes is widely documented in the literature. For the modelling of SMB chromatographic processes two different strategies exist. The first one considers the corresponding true moving bed process neglecting the cyclic switching and thus the dominating dynmics of the process. The second alternative which is considered within this framework is to connect dynamic models of single chromatographic columns while considering the cyclic port switching in order to represent the dynamics of the real process correctly. We here describe three different approaches for the modelling and simulation of chromatographic separation processes with nonlinear adsorption thermodynamics described by the Langmuir isotherm. The first one is a rigorous modelling and numerical simulation approach, the second one is based on the equilibrium theory and proceeds from the analytical solution for binary separation processes based on nonlinear wave propagation. The third approach only includes nonidealities caused by the limited mass transfer rates. All approaches are analyzed and compared with respect to complexity, accuracy and computational time.

Experimental analysis of a chiral separation through simulated moving bed chromatography

An eight column HPLC-Simulated Moving Bed (SMB) laboratory unit for the separation of enantiomers has been built and operated. The separation of the enantiomers of the Tröger’s base compound on triacetylcellulose has been considered as model separation. First the thermodynamics of adsorption has been characterised on a single analytical chromatographic column, then the separation has been scaled- up to the SMB laboratory unit. SMB experiments with different operating parameters, i.e., switch time and flow rates, have been run in order to analyse the separation performance of the laboratory unit. The experimental results are discussed in the light of the triangle theory for SMB operation.

An improved, inexpensive method for the large-scale purification of human nerve growth factor.

Human nerve growth factor (hNGF) was purified to near homogeneity on a large scale from human term placenta with an improved and inexpensive method. The purification procedure included tissue homogenization, ultrafiltration and single CM-cellulose column chromatography. The purified hNGF was a 14.4-kDa protein with an isoelectric point of approximately 9.3. The specific activity of the purified hNGF was approximately 38000 units/mg, and the activity was completely inhibited by the monoclonal antibody against recombinant hNGF (rhNGF). Western-blot analysis showed that the purified hNGF could interact with the monoclonal antibody against rhNGF.

A novel stationary phase for the high performance liquid chromatographic separation and determination of phenols

A novel type of hyper-crosslinked polystyrene resin has been used as a stationary phase in the high performance liquid chromatographic separation of phenol and its derivatives; a simplified procedure for their quantitative determination on a ppb level, based on preconcentration and separation using a single chromatographic column, is proposed.

D-Galactoside-Specific Lectins from the Body Wall of an Echiuroid (Urechis unicinctus) and Two Annelids (Neanthes japonica and Marphysa sanguinea)

Lectins recognizing d-galactosides were purified from the body wall of an echiuroid; Urechis unicinctus and two annelids; Neanthes japonica and Marphysa sanguinea, with single step lactosyl-agarose affinity column chromatography. SDS-PAGE under reduced and non-reduced conditions showed that U. unicinctus lectin had a major (36 kDa) and two minor (40 and 14 kDa) proteins, and that N. japonica lectin and M. sanguinea lectin had single 33 and 35 kDa proteins, respectively. Lectins were solubilized in the presence of lactose from tissues, and all polypeptides were shown to have sugar binding activity. The antisera raised against U. unicinctus lectin and N. japonica lectin crossreacted with each other but did not crossreact with bull frog (Rana catesbeiana) egg galectin-1 or a d-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs. These echiuroid and annelid lectins are immunologically similar, but distinct from members of the vertebrate galectin family.

Purification and characterization of isocitrate dehydrogenase from a hyperthermophilic archaebacterium, Caldococcus noboribetus

Isocitrate dehydrogenase from a hyperthermophilic archaebacterium Caldococcus noboribetus produced in Escherichia coli was purified. The purification was performed by heat treatment at 80°C followed by single column chromatography. N-terminal amino acid sequencing analysis revealed that the N-terminal methionine is removed from the purified enzyme. Gel filtration analysis suggests that the enzyme has a homodimeric structure with a molecular weight of 90 000. The isoelectric point of the enzyme was estimated to be 5.6 by isoelectric focusing electrophoresis. The circular dichroism spectrum suggests that the enzyme has a secondary structure consisting of 23% α-helix and 34% β-sheet. Enzymatic activity was observed under neutral pH, and the highest specific activity was obtained using cacodylic acid–KOH (pH 7.0) buffer. MgCl 2 or MnCl 2 was essential for the activity, and KCl concentrations higher than 0.33 M had an inhibitory effect on it. Apparent K m values were 72 and 43 μM for d, l-isocitrate and NADP, respectively. The enzyme showed extremely high stability against heat treatment, and no activity loss was observed by the treatment at 80°C. The specific activity of the enzyme increased as temperature rose. Nearly no activity was observed at 40°C or lower.

Method for increasing the yield of properly folded recombinant human gamma interferon from inclusion bodies

A strategy is described for improved refolding and purification of recombinant human gamma interferon (rh-IFN γ), which could warrant a higher yield and specific activity than reported previously. The optimal conditions of refolding are obtained by addition of a labilizing agent, l-arginine, in the refolding buffer. A 10-fold increase in the yield was observed with 0.5 M l-arginine, compared with renaturation in its absence. By varying renaturation parameters, the conditions that allow functional refolding of ∼25–30% of the recombinant protein have been standardized. A simple process is also described for the purification of rh-IFN γ. The purification involves a single-column chromatography on S-Sepharose, after refolding of rh-IFN γ in arginine containing buffer. This procedure has consistently produced rh-IFN γ having a purity of at least 97%, the rest being the aggregated form of gamma interferon. The purified protein is a dimer under non-denaturing conditions and has a specific activity of 2×10 8 IU mg −1 protein, as measured by viral cytopathic assay.

Overproduction and Purification of σ 32, the Escherichia coli Heat Shock Transcription Factor

This paper reports the overproduction and the details of a rapid method to purify active σ 32 that is free of core RNA polymerase enzyme. Maximal overproduction of σ 32 in a T7 RNA polymerase-based expression system is achieved only in the presence of rifampicin. This 2-day procedure involves solubilizing inclusion bodies in Sarkosyl, removal of Sarkosyl hy dialysis, and a single S-Sepharose column chromatography step. The final yield of σ 32 is about 4.1 mg of approximately 95% purity from 1 g of wet weight cells.

Taxol and related taxanes. I. Taxanes of Taxus brevifolia bark.

The published procedures for the isolation of taxol from the Pacific yew (Taxus brevifolia) and other species of Taxus are cumbersome, and the yields of taxol are in the range of 0.0075-0.01%. This paper describes a simple and efficient procedure for the isolation of taxol and its major natural analogues from the bark of T. brevifolia consisting of a single chromatographic column (using silica gel, Florisil, or a reverse-phase C18-silica), followed by crystallization. Isolated yields of taxol from five "pooled" bark samples (blended from many different batches by the supplier) were in the range of 0.02-0.04%, and from bark collected from a more restricted locale, yields reached 0.06%. The procedure also yielded taxol analogues, such as 10-deacetylbaccatin III (0.02-0.04%), 10-deacetyltaxol-7-xyloside (0.06-0.1%), taxol-7-xyloside (0.005-0.01%), 10-deacetyltaxol (0.01-0.02%), 10-deacetylcephalomannine-7-xyloside (0.006-0.01%), and cephalomannine (0.005-0.007%). Of these, 10-deacetyltaxol-7-xyloside is the most abundant taxane in the Pacific yew bark.

Determination of d-alanine and d-glutamic acid in biological samples by coupled-column chromatography using β-cyclodextrin as mobile phase additive

A high-performance liquid chromatographic assay for monitoring traces of d-alanine and d-glutamic acid in peptide and protein hydrolysates is presented, which uses a two-column set-up with column switching. The main advantage of the proposed method is the higher reliability of the results, compared with the analysis by derivatization and single-column chromatography of the diastereomers. A non-chiral alkylsilica reversed-phase column is combined with a second column, in which β-cyclodextrin is used as a chiral mobile phase additive. The amino acids of the hydrolysate are dansylated, and the amino acid of interest is separated from the others on the first column and transferred to the second column where chiral resolution is performed. The transfer volume of ca. 200 μl is small enough not to cause any peak distortion or dilation in the second column.

Simplex optimization of the analysis of polychlorinated biphenyls: Application to the resolution of a complex mixture of congeners of interest on a single gas chromatographic column

The modified sequential simplex method was applied to optimize the oven temperature programming of a single-column gas chromatography procedure developed to determine polychlorinated biphenyl congeners in a mixture of technical Aroclors. Both the overall resolution of fourteen congeners of interest and the time of analysis were taken into account in the optimization. Successful seperations of some clusters usually unresolved with a single column have been obtained.

Expression and purification of human angiotensinogen in Chinese hamster ovary cells.

We have produced human angiotensinogen in Chinese hamster ovary (CHO) cells. The expression products were purified to homogeneity by a single column chromatography and its 17 amino-terminal sequences were identical to those of the native protein. We demonstrated the recombinant human angiotensinogen to be a substrate for human renin.

Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: General characteristics and active site studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase has been isolated from Penicillium funiculosum cultural medium by a single hydrophobic column chromatography. The enzyme is a glycoprotein composed of a single polypeptide chain with a molecular mass of about 37,000 Da as analyzed by denatured sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by native gel filtration on Sephadex G-100. Its optimum activity occurs at pH 6.0. It has an isoelectric point of 5.8 and has a K m for PHB (average molecular weight = 45,000 Da) of 0.17 mg/ml. Various nonionic detergents competitively inhibit the enzyme with K i values of 0.56 and 0.014% for Tween 80 and Triton X-100, respectively. The enzyme is extremely sensitive to diisopropyl fluorophosphate, mercuric ion, and dithiothreitol (DTT). However, sulfhydryl reagents have little or no effect on its activity. The inactivation by mercuric ion and DTT is reversible by mercaptoethanol and hydrogen peroxide, respectively. These data suggest that the enzyme may be a serine esterase and may contain an important disulfide bond. The enzyme is also inactivated by diazoacetyl and epoxide compounds at low pH, which can be prevented by PHB, indicating the presence of a critical carboxyl group at the active site. These characteristics of the enzyme are compared to other extracellular polymerases isolated from bacterial culture media.

Amplification and substantial purification of cardiolipin synthase of Escherichia coli.

A simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed. This measures the radioactivity of glycerol formed from phosphatidyl [2-3H]glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme. Cardiolipin synthase was amplified 760-fold upon induction with isopropyl beta-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter. Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency. The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10,000-times higher than that of wild-type whole cell lysate. This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography. This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E. coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.

Overproduction of glutamate racemase of Pediococcus pentosaceus in Escherichia coli clone cells and its purification

We previously isolated a 6.0-kb DNA fragment that specifies glutamate racemase activity from the chromosomal DNA of Pediococcus pentosaceus by digestion with HindIII ( N. Nakajima, K. Tanizawa, H. Tanaka, and K. Soda, 1986), Agric. Biol. Chem. 50, 2823–2830). We digested it further with EcoRI to obtain a fragment of 1.8 kb, which was blunt-ended and ligated into the SmaI site of vector plasmid pKK223-3. The recombinant plasmid showed a high glutamate racemase activity upon transformation of Escherichia coli W3110 cells with it; the plasmid was named pICR223. Glutamate racemase was overproduced in the clone cells and occurred in inclusion bodies in the cells. The enzyme was solubilized with 6 M urea, renatured by dialysis to remove urea, and purified to homogeneity with an overall yield of about 70% after a single DEAE-cellulose column chromatography. The amount of enzyme produced by the clone cells corresponded to about 38% of the total insoluble protein.