High-Speed Large Scale Chromatographic Purification of Plasmid DNA with a Novel Giant-Pore Stationary Phase

A rigid spherical giant-pore poly (glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension–polymerization on the basis of a novel porogenic mode using superfine particles of calcium carbonate as a solid porogen. Scanning electron microscopy reveals that the bead has pores as large as 10 μm. The hydrodynamic properties show that this polymeric material has good strength and a low back pressure of 1.0 MPa at a flow velocity of 3,000 cm h⁻¹. After being modified to be an anion-exchange material, high dynamic binding capacity of plasmid DNA of above 1,000 μg plasmid per mL of bed by a column of this material, could be obtained comparing to the 150 μg plasmid per mL of bed with a Q-Sepharose FF column at the same flow rate. Large-scale preparative plasmid separations (2–20 mL) from cell lysate were investigated. A 75% yield and 94.9% purity of SC plasmid DNA were obtained by a 20 mL column of giant-pore beads at a flow rate of 600 cm h⁻¹.     

Determination of Urinary Glucose by a Flow Injection Analysis Amperometric Biosensor and Ion-Exchange Chromatography

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 ±2.4 RU μM^-1 (RU or relative unit is defined as 2.86 μV at the detection output) for glucose with a minimum detection level of 10 μM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25–30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.     

Fast supercritical fluid chromatography using capillaries packed with nonporous particles

Summary Packed columns containing microparticles provide high column efficiency per unit time and strong retention characteristics compared with open tubular columns, and they are favored for fast separations. Nonporous particles eliminate the contribution of solute mass transfer resistance in the intraparticle void volume characteristic of porous particles, and they should be more suitable for fast separations. In this paper, the evaluation of nonporous silica particles of sizes ranging from 5 to 25 μm in packed capillary columns for fast supercritical fluid chromatography (SFC) using neat CO₂ is reported. These particles were first deactivated using polymethyl-hydrosiloxanes and then encapsulated with a methylphenylpolysiloxane stationary phase. The retention factors, column efficiencies, column efficiencies per unit time, separation resolution, and separation resolution per unit time for fast SFC were determined for various length capillaries packed with various sizes of polymerencapsulated nonporous particles. It was found that 15 μm nonporous particles provided the highest column efficiency per unit time and resolution per unit time for fast packed capillary SFC. Under certain conditions, separations were completed in less than 1 min. Several thermally labile silylation reagent samples were separated in times less than 5 min. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Detection of transferrin isoforms in human serum: comparison of UV and ICP–MS detection after CZE and HPLC separations

Two methods for separation of transferrin (Tf) sialoforms, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) with conventional UV absorbance detection, have been investigated and compared. First, conditions affecting the separation of the Tf isoforms by capillary zone electrophoresis and HPLC were carefully optimized. The use of 15 mmol L⁻¹ borate buffer (pH 8.4) containing 3 mmol L⁻¹ diaminobutane (DAB) as additive enabled good separation of the Tf isoforms by CE (75 cm×50 μm i.d. fused silica capillary) at 25 kV. In HPLC, a gradient of ammonium acetate (from 0 to 250 mmol L⁻¹ in 45 min) buffered at pH 6 (Tris-HCl) proved suitable for separation of Tf isoforms on a Mono-Q HR 5/5 anion-exchange column. On-line specific detection of the iron associated with the different Tf isoforms, after Fe saturation, by inductively coupled plasma mass spectrometry (ICP–MS) was studied in detail to compare its analytical performance with UV detection. For both CE and HPLC an octapole reaction system (ORS) ICP–MS instrument was used to minimize polyatomic interferences on the ⁵⁶Fe major isotope. Limits of detection of the different isoforms were in the range of 0.02–0.04 μmol L⁻¹ Tf for HPLC–ICP (ORS)–MS. This hybrid technique proved more selective and reliable detection of transferrin isoforms with 2, 3, 4, 5, and 6 sialic acid residues (S₂, S₃, S₄, S₅, and S₆) in real serum samples. Interesting results from iron speciation of Tf in serum from healthy individuals and from pregnant women are given.     

Development and Validation of an HPLC Method for Determination of Ceftiofur Sodium

An isocratic high-performance liquid chromatographic method has been developed for assay of ceftiofur sodium in drug substance and in sterile powder for injection. Chromatography was performed on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column with a 78:22 (v/v) mixture of 0.02 m disodium hydrogen phosphate buffer (pH adjusted to 6.0 with 85% orthophosphoric acid) and acetonitrile as mobile phase, at a flow rate of 1.0 mL min⁻¹. The separation was monitored by UV detection at 292 nm. Validation of the method for linearity and range, intra- and inter-day precision, accuracy, specificity, recovery, robustness, and limits of quantification and detection yielded good results. The calibration plot was linear from 20.0–120.0 μg mL⁻¹ and the correlation coefficient was 0.9999. It was shown that ceftiofur was degraded under acidic, alkaline, oxidative, and photolytic conditions. The method was found to be stability-indicating and could be used for routine analysis of ceftiofur sodium for injection.     

Optimization of a high-performance liquid chromatography method to quantify bilirubin and separate it from its photoproducts

A rapid reversed-phase (RP) high-performance liquid chromatography method for the isolation of bilirubin from its photoproducts (e.g., biliverdin) is reported. The method is based on isocratic elution using methanol:water as the mobile phase. A 2⁴ full-factorial experimental design approach was adopted. For the optimization, the best separation was obtained using a flow rate of 1.50 mL/min, a mobile phase of 99∶1 methanol:water (v/v) at pH 3.60, and a 150×4.6 mm id RP (C₁₈) column containing 5-μm particles. These conditions produced the fastest total retention time of 3.38±0.055 min, and other chromatographic parameters were acceptable. Under the optimum conditions, a linear calibration curve for bilirubin was obtained over the 1.0–40.0 μg/L concentration range studied. The limit of quantification was 0.79 g/L and the limit of detection was 0.24 μg/L. Bilirubin in solution was monitored by ultraviolet detection at 450 nm.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Determination of cisapride in human plasma by high-performance liquid chromatography with fluorescence detection

Summary A new sensitive HPLC-FLD method has been developed and validated for the determination of cisapride in human plasma for a bioequivalence study. A gradient method was used to remove late-eluting plasma components of no interest. The separation was performed on a Li-ChroCART 250-4 Purospher RP-18 (5 μm particle) analytical column fitted with a LiChroCART 4-4 Purospher RP-18 endcapped (5 μm particle) guard column. The excitation and emission wavelengths were 295 and 350 nm during fluorescence detection. The calibration plot was linear in the range of 5–200 ng mL⁻¹. A demethoxy analogue of cisapride was used as internal standard.     

Determination of Pyrethroid Insecticide Deltamethrin by Micellar Liquid Chromatography with Spectrophotometric Detection

A simple micellar liquid chromatographic technique for deltamethrin determination was developed and validated. The method provided to be suitable for deltamethrin determination in pediculicide shampoo. Kromasil C18 column (150 mm×4.6 mm, 5 μm) and mobile phase −0.12 M sodium dodecyl sulfate with 9% (v/v) 1-butanol were used for deltamethrin separation. Detection wavelength was 265 nm. The retention time was about 15 min. Different validation parameters were evaluated. The specificity of the method was demonstrated. Linearity was established in the range 10–40 μg L⁻¹. The limits of detection and quantitation were 1.06 and 3.22 μg mL⁻¹, respectively. The method showed excellent accuracy (100.6%) and precision (repeatability) gave a relative standard deviation of less than 1%. The influence of the various method parameters (robustness study) was also studied.     

Fast supercritical fluid chromatography of polymers using packed capillary columns

Summary Fast separations of perfluorinated polyethers and polymethylsiloxanes that are composed of 50–80 oligomers were demonstrated in packed capillary column supercritical fluid chromatography (SFC) using a carbon dioxide mobile phase. Separations were accomplished within 10 min using a 13 cm×250 μm i.d. column packed with 2 μm porous octadecyl bonded silica (ODS) particles. Effects of particle diameter of the packing material and pressure programming on separation were investigated, and packed column SFC was compared with open tubular column SFC. Results show that as the particle diameter was decreased from 5 to 3 to 2 μm and the column length was reduced from 85 to 43 to 13 cm, the separation time could be reduced from 70 to 20 to 10 min while still maintaining similar separation (resolution). Short columns packed with small porous particles are very suitable for fast SFC separations of polymers.     

A new affinity-HPLC packing for protein separation: Cibacron blue attached uniform porous poly(HEMA-<Emphasis Type="Italic">co</Emphasis>-EDM) beads

In this study, a new affinity high-performance liquid chromatography (HPLC) stationary phase suitable for protein separation was synthesized. In the first stage of the synthesis, uniform porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(HEMA-co-EDM), beads 6.2 μm in size were obtained. Homogeneous distribution of hydroxyl groups in the bead interior was confirmed by confocal laser scanning microscopy. The plain poly(HEMA-co-EDM) particles gave very low non-specific protein adsorption with albumin. The selected dye ligand Cibacron blue F3G-A (CB F3G-A) was covalently linked onto the beads via hydroxyl groups. In the batch experiments, albumin adsorption up to 60 mg BSA/g particles was obtained with the CB F3G-A carrying poly(HEMA-co-EDM) beads. The affinity-HPLC of selected proteins (albumin and lysozyme) was investigated in a 25 mm×4.0-mm inner diameter column packed with CB F3G-A carrying beads and both proteins were successfully resolved. By a single injection, 200 μg of protein was loaded and quantitatively eluted from the column. The protein recovery increased with increasing flow rate and salt concentration of the elution buffer and decreased with the increasing protein feed concentration. During the albumin elution, theoretical plate numbers up to 30,000 plates/m were achieved by increasing the salt concentration.     

Laser-induced breakdown spectroscopy (LIBS) for carbon single shot analysis of micrometer-sized particles

The purpose of this work is to study the ability of the laser-induced breakdown spectroscopy (LIBS) technique to perform in situ (without sample preparation) detection of graphite particles circulating in a gas loop used to simulate the cooling gas circuit of a helium-cooled nuclear reactor. Results obtained with a laboratory scale set up are presented. The experiments were performed in nitrogen with micrometer-sized particles containing carbon (glucose particles and sodium hydrogenocarbonate particles). Statistical shot to shot analysis was used to determine the concentration of the analyte. The variation of LIBS signal as a function of glucose particle diameter showed an underestimation of the signal of particles of diameters larger than 5 μm. This phenomenon is likely to be correlated to an incomplete vaporization in the laser-induced plasma of particles of sizes above 5 μm. Analytical measurements were performed with glucose particles and sodium hydrogenocarbonate particles, and the concentration-based limit of detection of carbon was evaluated to be about 60 μg m⁻³.     

Determination of thorium and uranium in activated concrete by inductively coupled plasma mass spectrometry after anion-exchange separation

A sensitive analytical method was established for the determination of Th and U in activated concrete samples. The method combines an anion-exchange separation step with an ICP-MS determination technique. In the ICP-MS measurement, a few μg mL^–1 of Al and Ca, a few ng mL^–1 of Mn, La, Ce, Nd and Pb and pg mL^–1 amounts of Li, Zr, Nb and Ba coexisting in the anion-exchange fraction of Th and U did not interfere. No adverse interference effects were observed in real sample analyses. The obtained detection limits (3σ, n = 10) of Th and U were 2.3 and 1.8 pg mL^–1, respectively. The analytical precisions for ca. 5 μg g^–1 Th and ca. 1 μg g^–1 U in real activated concrete samples were equally less than 7% RSD. The accuracies obtained by the analysis of GSJ rock standard samples were –18.1 to 0.4% for the Th determination and –14.0 to –5.7% for the U determination. The method uses the conventional absolute calibration curve. The internal standard calibration is unnecessary. Received: 14 March 1999 / Revised: 13 July 1999 / Accepted: 15 July 1999     

Preparation of strong anion-exchange chromatographic packings based on monodisperse polymeric beads and their application in the separation of biopolymers

A new hydrophilic strong anion-exchange (SAX) stationary phase for HPLC has been synthesized by chemical modification of macroporous 8.0-m monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads (P_GMA/EDMA). The stationary phase was evaluated in detail to determine its ion-exchange properties, separability, reproducibility, hydrophilicity, and the effect of column loading and pH on the separation and retention of proteins. It was found to have an ion-exchange chromatographic (IEC) retention mechanism. The highest dynamic protein loading capacity of the synthesized SAX packing for BSA was 22.6 mg g^–1. Five proteins were separated within 6.0 min using the synthesized SAX resin. The SAX resin was also used for rapid separation and purification of recombinant human stem cell factor (rhSCF) from a crude extract solution in only one step. The purity of the purified of rhSCF was >92.4%.     

Simple HPLC method for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in tablet formulations

Summary A simple, rapid and accurate, routine-HPLC method is described for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in a new tablet formulation Chromatographic separation of the three pharmaceuticals was achieved on a Hypersil CN column (150×5.0 mm, 5 μm) using a mobile phase comprising a mixture of acetonitrile, an ion-pair solution and tetrahydrofuran (13:14:87, v/v,pH4.5). The flow-rate was changed from 1.0 mL min⁻¹ (in 0≈7.5 min) to 1.8 mL min⁻¹ (after 3.5 min). was complete in <10 min. The method was validated for system suitability, linearity, accuracy, precision, limits of detection and quantitation, and robustness. Linearity, accuracy and precision were found to be acceptable over the ranges 31.6≈315.8 μg mL⁻¹ for acetaminophen, 9.5≈94.6 μg mL⁻¹ for caffeine and 1.4≈13.8 μg mL⁻¹ for chlorpheniramine maleate.     

Ion-exchange chromatography with an oxalic acid-α-hydroxyisobutyric acid eluent for the separation and quantitation of rare-earth elements in monazite and xenotime

Summary An oxalic acid-α-hydroxyisobutyric acid eluent has been used for the separation and determination of rare-earth elements by high-performance ion-exchange chromatography. Fifteen rare-earth elements were separated within less than 25 min on a 150×4.6 mm i.d. column packed with 5-μm sulfonic acid-bonded silica particles by elution with a combined gradient of 0.60–9.0 mM oxalic acid and 19.0–5.0 mM α-hydroxyisobutyric acid at pH 4.6. Detection and quantitation of the separated rare-earth elements was accomplished by visible-absorbance measurements at 600 nm after postcolumn reaction with arsenazo I. The gradient of the two complexing agents was optimized to enable the separation of yttrium(III) without interference from other elements, especially dysprosium(III) and terbium(III). Mass detection limits of the elements were in the range of 2–4 ng. Finally, the chromatographic system was applied to the quantitative analysis of rare-earth elements in monazite and xenotime.     

Capillary gas chromatography of metal chelates of diethyl dithiocarbamates

Summary Capillary GC of metal chelates of diethyl dithiocarbamate (DDTC) was examined on a methylsilicone DB-1 column, (25 meter, 0.2 mm. i.d) with a film thickness of 0.25 μm. Elution was carried out at the initial column temperature of 180°C and programmed at 5°C min⁻¹ to 260°C. Detection was by FID or ECD. Symmetrical peaks with base line separation were obtained with the metal chelates of copper(II), nickel(II), cobalt(III), manganese(II) and chromium(III). The ECD gave better sensitivity than the FID with a linear calibration range of 5–50 μg mL⁻¹ and detection limits 2.0–6.0 μg mL⁻¹, corresponding to 111–333 pg of metal ion reaching the detector. The method was applied to the determination of metal ions in water and pharmaceutical preparations with a coefficient of variation (CV) within 4.0%. When compared with a standard flame AAS method the results revealed no significant difference.     

Application of micropellicular poly-styrene/divinylbenzene stationary phases for high-performance reversed-phase liquid chromatography electrospray-mass spectrometry of proteins and peptides

Summary Short columns packed with highly crosslinked 2.3 μm poly-styrene/divinylbenzene (PS/DVB) particles were used for rapid and efficient separation of proteins and peptides by reversed-phase high-performance liquid chromatography at elevated temperatures. Enhancement of the diffusivities of the sample components at elevated temperatures together with the short diffusion pathlength with the micropellicular polymeric stationary phases were responsible for high efficiency, high speed of analysis, and short column regeneration times. Underivatized PS/DVB beads as well as PS/DVB microspheres which have been modified with polyvinylalcohol or octadecyl chains on the surface were synthesized, employed, and compared to HY-TACH-C18, a commercially available micropellicular octadecyl-silica stationary phase, for the separation of proteins, octapeptides and tryptic protein digests. Highest performance was obtained with the silica- and PS/DVB-based octadecyl stationary phases, which exhibited similar column efficiencies but different selectivities for proteins and peptides. The minimum detectability at 214 nm and the maximum loading capacity for ribonuclease A using analytical 30×4.6 mm I.D. columns were 10 ng (0.6 pmol) and 1 μg, respectively. Finally, reversed-phase HPLC with a 60×2 mm I.D. narrow-bore column packed with micropellicular octadecyl PS/DVB was coupled successfully to electrospray mass spectrometry at a flow-rate of 0.15 mL min⁻¹ and on-line full-scan mass spectra for molecular mass determination and identification of proteins in the lower picomol range were obtained.     

Preconcentration of traces of rhenium in geological matrix by NAA

A simple preconcentration approach for quantitative separation and determination of traces of rhenium in geological matrix using neutron activation analysis is presented in this work. Anionic species of rhenium were collected on small amounts of anion-exchange resin beads from a large-size geological sample solution followed by neutron irradiation of the resin beads and measurements of rhenium activity for its quantification. ∼200 mg of resin beads were used to concentrate rhenium from 0.5 to 5 gram of the borehole samples. Radioactive tracers of the analyte directly and in the presence of the matrix were used to establish the separation process. During the present work the detection limit of rhenium was found to be 10 ng/g.     

Magnetic molecularly imprinted polymer beads prepared by microwave heating for selective enrichment of β-agonists in pork and pig liver samples

Novel magnetic molecularly imprinted polymer (MIP) beads using ractopamine as template for use in extraction was developed by microwave heating initiated suspension polymerization. Microwave heating, as an alternative heating source, significantly accelerate the polymerization process. By incorporating magnetic iron oxide, superparamagnetic composite MIP beads with average diameter of 80 μm were obtained. The imprinted beads were then characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and vibrating sample magnetometer. Highly cross-linked porous surface and good magnetic property were observed. The adsorption isotherm modeling was performed by fitting the data to Freundlich isotherm model. The binding sites measured were 3.24 μmol g⁻¹ and 1.17 μmol g⁻¹ for the magnetic MIP beads and the corresponding non-imprinted magnetic beads, respectively. Cross-selectivity experiments showed the recognition ability of the magnetic MIP beads to analytes is relative to degree of molecular analogy to the template. Finally, this magnetic MIP bead was successfully used for enrichment of ractopamine, isoxsuprine and fenoterol from ultrasonically extracted solution of pork and pig liver followed by high performance chromatography with fluorescence detection. The proposed method presented good linearity and the detection limits was 0.52-1.04 ng mL⁻¹.The recoveries were from 82.0% to 90.0% and from 80.4% to 86.8% for the spiked pork and pig liver, respectively, with the RSDs of 5.8-10.0%. Combination of the specific adsorption property of the MIP material and the magnetic separation provided a powerful analytical tool of simplicity, flexibility, and selectivity.