Preparation and characterization of methacrylate-based semi-micro monoliths for high-throughput bioanalysis

Hexyl methacrylate (HMA)-based monolithic semi-micro columns were prepared by in situ polymerization within the confines of 1.02-mm-i.d. silicosteel tubing for reversed-phase and/or precipitation–redissolution liquid chromatography. Practically useful monolithic columns with adequate separation efficiency, high permeability, and good mechanical strength were successfully obtained using a polymerization mixture comprising 24% hexyl methacrylate (HMA), 6% ethylene dimethacrylate (EDMA), 44.5% 1-propanol, and 25.5% 1,4-butanediol. The column performance was evaluated through the separations of a series of alkylbenzenes. At a normal flow rate of 50 μL min⁻¹, the produced HMA-based monolithic columns typically exhibited 3,000 theoretical plates for a 20-cm-long column, and the pressure drop was generally less than 1 MPa per 20 cm. The monolithic columns were resistant to at least 15 MPa, and could be properly operated at 15–20 times higher flow rate than normal, reducing the separation time to 1/15–1/20. The HMA-based monolithic columns were applied to rapid and efficient separations of proteins such as ribonuclease A, cytochrome c, transferrin, and ovalbumin in the precipitation–redissolution mode. Using a CH₃CN gradient elution at a flow rate of 1,000 μL min⁻¹, four proteins were baseline separated within 20 s.     

Migration and sorption of <Superscript>137</Superscript>Cs and <Superscript>152,154</Superscript>Eu in crushed crystalline rocks under dynamic conditions

In migration experiments, sorption of ¹³⁷Cs and ^152,154Eu in the columns of crushed crystalline rocks of 0.25–0.8 mm grain size under dynamic flow conditions from the synthetic groundwater (SGW) has been studied. Five samples of crystalline rocks from Cavernous Gas Reservoir near Příbram were taken. Plastic syringes of 8.8 cm length and 2.1 cm in diameter were used as columns. The water phase was pumped downward through the columns, using a multi-head peristaltic pump, with a seepage velocity of about 0.2 cm/min. The radioactive nuclides, containing chemical carriers, were added into the water stream individually in the form of a short pulse. Desorption experiments were carried out with 2:1 (v/v) mixture of H₂SO₄ and HNO₃. In the columns the longitudinal distribution of the residual ¹³⁷Cs and ^152,154Eu activities was also determined. By the evaluation of respective breakthrough and displacement curves, the experimental and theoretical retardation factors, distribution coefficients and hydrodynamic dispersion coefficients were determined using the integrated analytical form of a simple advection-dispersion equation (ADE). Dynamic sorption experiments were also compared with the results of static sorption experiments. The paper was presented in part as a poster No. PB1-1 at the 11th International Conference Migration’ 07, held in Munich, Germany, August 26–31, 2007, Abstracts, p. 212.     

Biosorption of Copper and Cadmium in Packed Bed Columns with Live Immobilized Fungal Biomass of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Biosorption of copper (II) and cadmium (II) by live Phanerochaete chrysosporium immobilized by growing onto polyurethane foam material in individual packed bed columns over two successive cycles of sorption–desorption were investigated in this study. Initial pH and concentrations of the metals in their respective solutions were set optimum to each of those: 4.6 and 35 mg·l⁻¹ in case of copper and 5.3 and 11 mg·l⁻¹ for cadmium. The breakthrough curves obtained for the two metals during sorption in both the cycles exhibited a constant pattern at various bed depths in the columns. The maximum yield of the columns in removing these metals were found to be, respectively, 57% and 43% for copper and cadmium indicating that copper biosorption by the immobilized fungus in its column was better than for cadmium. Recovery values of the sorbed copper and cadmium metals from the respective loaded columns by using 0.1 N HCl as eluant was observed to be quite high at more than 65% and 75%, respectively, at the end of desorption in both the cycles. Breakthrough models of bed-depth service time, Adams–Bohart, Wolborska, and Clark were fitted to the experimental data on sorption of copper and cadmium in the columns, and only the Clark model could fit the sorption performance of the columns well over the entire range of ratios of concentrations of effluent to influent, i.e., C/C ₀ for both copper and cadmium biosorption. The kinetic coefficients of mass transfer and other suitable parameters in the system were determined by applying the experimental data at C/C ₀ ratios lower than 0.5 to the other three models.     

Migration of239Pu in soluble and insoluble forms in soil

A field experiment study was performed at the rural site of South-East Lithuania. The main tasks of the study included an evaluation of the peculiarities of partition of²³⁹Pu in soluble (²³⁹Pu(NO₃)₄,²³⁹PuCl₃) and insoluble (²³⁹PuO₂) forms in soddy and forest soil horizons. The results of durable experiments (418 and 326 days) have shown that from 44.1% to 92.2% of²³⁹Pu of investigated chemical forms were accumulated in the top (0–5 cm) soil layer. Some share (5.7–39.2%) of plutonium from studied chemical forms was found in the 5–20 cm layers of studied soil samples (columns). Obtained distribution of plutonium in soil layers may be attributed to the consideration that the migration rate to the soil depth for plutonium is 0.1–1.0 cm·y⁻¹ but for some part of plutonium 10 times higher migration rate is characteristic as well. Plutonium transfer factor (TF) to the grassland plants was calculated, the values ranged from 10⁻² to 10⁻¹.     

Capillary electrochromatography of 1-phenyl-3-methyl-5-pyrazolone derivatives of some mono-and disaccharides

Summary A packing procedure was adopted for capillary electrochromatography (CEC) that produces capillary columns with high separation efficiencies. The columns were fully packed, 50 cm long, with UV detection being performed through the packed section 30 cm from the inlet end. The CEC experiments were run at ambient pressure, with no additional pressure applied to the ends of the column. The stationary phase (octadecyl silica (ODS), 5 μm) promoted a high velocity electroosmotic flow (EOF), enabling rapid and efficient separation of a hydrocarbon test mixture. Some 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of mono- and disaccharides were baseline separated, using a 5 mM NaH₂PO₄ in 80% acetonitrile and 20% water (v/v) buffer solution. CEC shows promise for future applications in carbohydrate analysis. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Carbon dioxide-methane mixture adsorption on activated carbon

In this work, we report new experimental data of pure and binary adsorption equilibria of carbon dioxide and methane on the activated carbon RB2 at 273 and 298 K. The pressure range studied were 0–3.5 MPa for pure gases and 0–0.1 MPa for mixtures. The combination of the generalized Dubinin model to describe the pure CO₂ and CH₄ isotherms with the IAST (Ideal Adsorbed Solution Theory) for the mixtures provide a method for the calculation of the binary adsorption equilibria. This formulation predicts with acceptable accuracy the binary adsorption data and can easily be integrated in general dynamic simulation of PSA (pressure swing adsorption process) adsorption columns. It involves only three parameters, independent of the temperature, and directly determined with only one adsorption isotherm of CO₂.     

Characterization of different RP-HPLC columns by a gradient elution technique

Summary The literature contains no unified testing procedure for evaluation and characterization of reversed-phase (RP) columns of different pore size (surface area) and containing different types of ligand. In our laboratory a testing procedure has been developed using gradient elution under standardized conditions independent of column dimensions and applicable to both narrow-pore and wide-pore columns. Six wide-pore and four narrow-pore columns were investigated and compared. The test solutes were selected to cover a wide range of chemical properties. The evaluation and characterization of the columns was performed on the basis ofk _app retention factors. Principal components analysis (PCA) was performed to reveal similarities and differences among the columns and test substances. The factors obtained characterize the columns according to the extent of various interactions such as hydrophobicity, HB (hydrogen bond)-donor acidity and HB-acceptor basicity. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Depletion of human lymphocytes from peripheral blood and bone marrow by affinity ligands conjugated to agarose—polyacrolein microsphere beads

Protein-A or goat anti-mouse-Ig (GAMIg) covalently bound to agarose-polyacrolein microsphere beads (APAMB) were employed for the removal of T cells from human peripheral blood leukocytes (PBL) and bone marrow (BM). The cell suspensions were treated with a monoclonal anti-T cell antibody (Leu-1) or monoclonal antilymphocyte antibody (CAMPATH-1) and passed through the conjugated APAMB columns. Cell separation efficacy was determined by assaying the number and function of T cells in the final cell preparation in comparison with a sample of unseparated cells. The number of cells that form rosettes (E-RFC) with sheep red blood cells (SRBC) in a sample of PBL treated with anti-Leu-1 antibodies and subsequently passed once through GAMIg-conjugated APAMB dropped from a range of 41.5–86.0% to a range of 1.6–13.3%. The in vitro response to concanavalin-A (Con-A) dropped to a range of 0.7–27.2% (GAMIg) and a range of 1.2–21.8% (protein-A column) of the response of untreated PBL. Treatment with CAMPATH-1 antibody and passage through a protein-A-conjugated APAMB reduced E-RFC from a range of 55.6–57.4% to a range of 3.2–3.9% and abolished the Con-A induced proliferative responsiveness to background levels. Treatment of BM cells with CAMPATH-1 and passage of the cells through either GAMIg or protein-A conjugated APAMB columns resulted in reduction of E-RFC from a range of 12.4–17.7% to a range of 0–1% and from a range of 17.7–19% to a range of 1.6–3.2%, respectively. Viability of BM precursors, determined by the CFU-GM assay in semisolid medium, was not affected by these cell separation procedures. The data suggest that protein-A or GAMIg-conjugated APAMB columns may be a useful tool for separation of BM cell suspensions into specific cell subsets that can be defined by monoclonal antibodies.     

Measurement of Henry coefficients by capillary columns coated with slightly polar solvents

Summary The specific retention volumes and Henry coefficients measured on packed columns are compared with those calculated via retention indices determined on wall coated open tubular capillary columns. The stationary phase liquids used were derivatives of a branched paraffin, C₇₈H₁₅₈, where a methyl or ethyl group of the paraffin was replaced by OH, CN, OCH₃ and SH groups. The retention indices of a series of molecular probes of varying polarity (aromatics, 1-chloroalkanes, 1-acetoxy-alkanes, 1-alkanols, 2-alkanones) were determined atT=403.15 K on fused-silica open tubular columns prepared by static coating that gave stable and uniform solvent films. After obtaining the absolute data on packed columns forn-alkanes, the specific retention volumes and Henry coefficients of the solutes were calculated. The retention data obtained on both columns showed good agreement. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Dimethylditetradecylammonium bromide (2C14DAB) as a self-assembled surfactant coating for detection of protein–dye complexes by CE-LIF

A self-assembled column coating for capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF) has been evaluated for the separation and quantitation of protein–dye complexes. This semi-permanent coating, composed of dimethylditetradecyl-ammonium bromide (2C₁₄DAB), is inexpensive and easily assembled onto the column and it allows for better peak resolution and greater control over electroosmotic flow. The versatility of long-chained surfactant coatings was determined particularly with respect to their use with fluorescent probes, different pH buffers, and different proteins. Studies were performed to determine the stability of the coating under various pH and buffer conditions. Red-1c, a red luminescent squarylium dye, was used for on-column protein labeling concurrently with the surfactant coating and LIF detection. Protein–Red-1c complexes were excited with a 650-nm diode laser and their emission detected by a photomultiplier tube with a 664-nm filter. A comparison of pre-column labeling and on-column labeling of a two-model protein system (human serum albumin and β-lactoglobulin A) revealed higher efficiencies and greater sensitivities for both proteins using on-column labeling and coated columns. A linear relationship between peak height and protein concentration was obtained by CE-LIF for this on-column labeling method with 2C₁₄DAB-coated columns and the Red-1c probe.     

Analysis of mismatched DNA by mismatch binding ligand (MBL)–Sepharose affinity chromatography

Mismatch binding molecules (MBLs), strongly and selectively bound to the mismatched base pair in duplex DNA, were immobilized on Sepharose. Three MBL–Sepharose columns were prepared with three MBLs, naphthyridine dimer (ND), naphthyridine–azaquinolone (NA), and aminonaphthyridine dimer (amND), which exhibited different binding profiles to the mismatched base pairs. These three MBL–Sepharose columns showed characteristic elution profiles for DNA duplexes containing mismatched base pairs. The ND–Sepharose column separated the G–G and G–A mismatched DNA from fully matched duplexes. The NA–Sepharose column separated the A–A and G–A mismatched DNA from other DNA duplexes. The amND–Sepharose column separated the C–C mismatched DNA. These chromatographic profiles were very consistent with the binding preference of each MBL. By changing the elution conditions from sodium hydroxide to sodium chloride, MBL–Sepharose columns were also able to separate the mismatched DNA that weakly bound to the MBL from fully matched DNA duplex. Figure MBL-Sepharose affinity chromatography successfully separates the mismatched duplex DNA from fully matched duplex.     

2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS

A 2D liquid chromatography (LC) system using hydrophilic interaction chromatography (HILIC) and reversed phase columns has been employed for comprehensive (LC × LC) separation of rat muscle tissue micro-dialysate. Incorporation of an on-line reverse-phase solid phase extraction (SPE) enrichment column in front of the first dimension enabled aqueous samples with high salt concentrations to be injected directly without compromising the chromatographic performance of the HILIC column. Since the SPE enrichment column allowed injection of large sample volumes (e.g. 450 μL), a capillary HILIC column (inner diameter 0.3 mm) could be employed instead of a larger column which is often used in the first dimension to load sufficient amounts of sample. The two chromatographic dimensions were connected using a column selector system with 18, 1.0 mm I.D. C₁₈ “transition” SPE columns. A PLRP C₁₈ column was used in the second dimension. The 2D LC system’s performance was evaluated with a tryptic digest mixture of three model proteins. Good trapping accuracy (HILIC→transition SPE→RP recovery >95%) and repeatability (within-and between day retention time RSDs of first and second dimension chromatography >1%) was achieved. A dialysis sample of rat muscle tissue was separated with the 2D system, revealing complexity and large differences in concentrations of the various compounds present, factors which could potentially interfere with the quantification and monitoring of two target analytes, arg-bradykinin and bradykinin. Subsequently, “Heart-cut” 2D LC-electrospray–mass spectrometry (ESI–MS) with post-column on-line standard injection was employed to monitor arg-bradykinin and bradykinin levels as a function of various muscle conditions. The method’s quantification precision was RSD = 3.4% for bradykinin.     

Separation of chiral pharmaceuticals using ultrahigh pressure liquid chromatography

Summary Ultrahigh pressure liquid chromatography was demonstrated for fast and efficient chiral separations. Capillary columns approximately 13–24 cm in length packed with nonporous 1.0μm C₆-modified silical particles were used. β-Cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were added to the mobile phase as modifiers to produce transient diastereomeric complexes with the analytes. Pressures up to ≈42,000 psi were applied, and efficiencies in excess of 200,000 plates m⁻¹ were obtained for separations that were accomplished in less than 2 minutes.     

Multicapillary columns with a porous layer based on the divinylbenzene copolymer

A method for preparing a multicapillary column with a porous layer based on the styrene-divinylbenzene copolymer has been developed. The column makes it possible to quickly separate C₁–C₄ hydrocarbons and oxygen-containing compounds. The main chromatographic properties of the columns were studied.     

Recent advances in micro-scale and nano-scale high-performance liquid-phase chromatography for proteome research

High-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS–MS) is regarded as one of the most powerful techniques for separation and identification of proteins. Recently, much effort has been made to improve the separation capacity, detection sensitivity, and analysis throughput of micro- and nano-HPLC, by increasing column length, reducing column internal diameter, and using integrated techniques. Development of HPLC columns has also been rapid, as a result of the use of submicrometer packing materials and monolithic columns. All these innovations result in clearly improved performance of micro- and nano-HPLC for proteome research.     

An improved method for assay of vitamin C in fish feed and tissues

Summary A method was devised to assay four forms of vitamin C: L-ascorbic acid (AA), dehydroascorbic acid (DHA), ascorbate-2-mono- and polyphosphate (AMP, APP), as well as ascorbate-2-monosulphate (AMS), in sample series of different fish tissues and feed. Direct and indirect detection were combined. Sample extractions were carried out with 0.2 mol L⁻¹ sodium acetate buffer (pH 4.8) and extracts were deproteinized after different chemical or enzymatic reactions, with perchloric acid. The DHA was reduced to AA with dithioerythritol (DTE). Ascorbate oxidase enzyme was used for the detection of background and an acidic phosphatase enzyme for the hydrolysis of different phosphate esters. Ascorbate-2-sulphate was detected directly with help of coinjection of the compound. Chromatographic analysis was carried out with a single column isocratic reverse phase method. The mobile phase was an aqueous buffer of 0.04 M sodium-acetate, 0.05 mM EDTA, 0.5 mM tetrabutylammonium dihydrogen phosphate (TBA) adjusted to pH 3.76 with 85% H₃PO₄ and with 24 mL methanol added to 1000 mL. C-18 columns were used with 0.6 mL min⁻¹ flow rate at 23°C. The vitamin C forms were detected by UV absorption at 250 nm. The determination limit was 1.0–5.0 μg g⁻¹ in AA equivalent. The standard deviations were between 1–6% and depended on the concentrations of vitamin C forms and tissues. Recoveries were between 90–96% in samples. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

Determination of naturally occurring uranium concentrations in seawater,sediment, and marine organisms in Japanese estuarine areas

For safety assessments of geological repositories of nuclear waste, understanding of uranium (U) fate in estuarine areas is important because U chemical behavior in the areas is expected to be complex. Environmental transfer parameters such as sediment–water distribution coefficients (K _d) and concentration ratios (CRs) for marine organisms are useful in mathematical models for the assessment. However, due to its low concentration in estuarine water, K _d and CF data for U are scarce. Thus we studied a rapid method for separation and concentration of U from estuarine water samples using NOBIAS-CHELATE PA1 resin columns followed by inductively coupled plasma mass spectrometry (ICP-MS) for U measurement. Chemical recovery was about 100% at pH of 5.7 ± 0.1 from the water samples and alkali and alkaline earth metals were removed. The method was used to measure U concentrations in estuarine water samples collected at eight Japanese estuarine areas; they ranged from 0.1 to 3.8 μg L⁻¹. We also measured U concentrations in sediment and marine organism samples by ICP-MS after acid digestion. Using these values, we observed K _d (range: 39–284 L kg⁻¹) and CRs (0.86–52 L kg⁻¹ for macroalgae, 0.087–15 L kg⁻¹ for crustaceans, and 0.52–93 L kg⁻¹ for molluscs).     

Purity testing of organometallic catalysts by micro liquid chromatography-electron ionization mass spectrometry

Summary A micro liquid chromatography-mass spectrometry system utilizing 100 μm i. d. packed capillary columns has been used for purity testing of organometallic catalysts. The total effluent, 1 μLmin⁻¹ from the column, was introduced directly into the ion source of a bench-top quadrupole mass spectrometer and electron ionization mass spectra were acquired. In full scan mode a mass limit of detection of 10–15 ng was achieved for the organometallic compounds investigated. The catalysts dimethyl pentamethylcyclopentadienyl iridium dimethylsulphoxide (Cp^*Ir(DMSO)Me₂) and di(pentamethylcyclopentadienyl dichloro iridium) ((Cp^*IrCl₂)₂) were purity tested and their electron ionization mass spectra recorded. Impurities present down to 1% of the main compound could be determined using large volume injection.     

Determination of calibration curves for <Superscript>131</Superscript>I in thyroid tumour metabolic radiotherapy and other radionuclides used in SPECT imaging

Large columns containing aluminum oxide (Al₂O₃) or gel (e.g. zirconium molybdate) are needed to prepare ⁹⁸Mo(n,γ)⁹⁹Mo→^99mTc column chromatographic generators that results in large elution volumes containing relatively high ⁹⁹Mo impurity and low concentrations of ^99mTc. The decrease in radioactive concentration or specific volume concentration of ^99mTc places a limitation on some pharmaceutical kits (DTPA, MIBI, ECD, etc.) or clinical procedures. We report on the post elution concentration of ^99mTc using in house prepared lead cation-exchange and alumina columns. Using these columns high bolus volumes (10–60 mL 0.02M sodium sulfate) of ^99mTc can conveniently be concentrated in 1 mL of physiological saline. This approach also works very effectively to prepare high specific volume solutions of ^99mTc-pertechnetate from a fission based ⁹⁹Mo/^99mTc generator in the second week of its normal working life.     

Use of short columns and high flow rates for rapid gradient reversed-phase chromatography

Summary Rapid analyses were performed using reversed-phase liquid chromatography with short (20–100 mm) columns swept by fast yet shallow gradients, and the results compared with those obtained with 150 mm columns and slow gradients. The resolution losses incurred with shorter columns were minimised by employing elevated flow rates, to ensure that comparable mean retention factors were experienced by individual analytes during gradients run on different columns. This conserves gradient steepness. High quality performance was obtained with turn-around times of 5–10 minutes. An overall 5-fold enhancement in the rate of information generation was obtained. The relevance of instrumental parameters and of column and packing dimensions, upon the potential for improved performance is discussed. Some implications for the rapidly developing technique of capillary electrochromatography are briefly indicated.