Determination of 90Sr in highly radioactive aqueous samples via conversion to a kinetically stable 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex followed by concentration-separation-fractionation based on capillary electrophoresis-liquid scintillation.

BACKGROUND: The Fukushima Daiichi Nuclear Power Plant accident (2011) released large amounts of radioactive substances into the environment and generated highly radioactive debris. Post-accident countermeasures are currently in the phase of fuel debris removal, which requires the analysis of radioactive contaminants in the environment and fuel. The spectra of solely ß-emitting nuclides, such as 90Sr, overlap; thus, an effective method for nuclide separation is desired. Since conventional methods for high-dose sample analysis pose substantial exposure risks and generate large amounts of secondary radioactive waste, faster procedures allowing for decreased radiation emission are highly desirable. RESULTS: In this study, we developed a 90Sr2+ quantitation technique based on liquid scintillation counting (LSC)-coupled capillary transient isotachophoresis (ctITP), along with two-point detection and relying on the rapid concentration, separation, and fractionation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-complexed 90Sr2+ in a single run. The applicability of our method for the analysis of real-world samples was verified by conducting addition-recovery experiments using a seawater reference material and radioactive liquid waste obtained from the radioactive waste treatment facility at the Japan Atomic Energy Agency. The recovery determined by LSC was 95-113%, indicating successful quantitative analysis. 90Sr recovery was determined to be 90.1% from a contaminated water sample obtained from the Fukushima Daiichi Nuclear Power Plant, which was analyzed using the standard addition of 90Sr. The sensitivity (detection limit = 0.016 Bq) of the proposed method on a radioactivity basis was equal to or higher than that of the conventional method using ion exchange-LSC (0.012-0.07 Bq). SIGNIFICANCE AND NOVELTY: Our method allows for the handling of high-dose radioactive samples at the microliter level and is substantially faster than conventional ion exchange protocols, whereas ctITP has not been used for practical applications due to inaccurate collection and lack of a suitable chemical system. The concentration-separation-fractionation protocol in ctITP is successful due to the existence of a rare inert Sr2+ complex and precise fractionation. This study establishes a pathway toward safer and more practical analysis of radionuclides.

Multistep pH-peak-focusing liquid chromatography with a hydrophilic polymer gel column for separation of rare earth elements.

Multistep pH-peak-focusing liquid chromatography with a column packed with a hydrophilic polymer gel (a cross-linked hydroxylated methacrylic polymer gel) was developed for separation of rare earth metal ions. Metal ions in a sample solution introduced to the column are chromatographically extracted into the stationary gel phase at the top of the column equilibrated with a basic solution used as the first mobile phase containing acetylacetone and 1,10-phenanthroline by synergistic extraction effect. After the sample solution is introduced, the mobile phases are delivered into the column by stepwise gradient elution in order of decreasing pH. Each metal ion is concentrated at a pH border formed between the zones of different pH in the column and moves toward the outlet of the column with the pH border. Mutual separation of La(III), Ce(III), Nd(III), Eu(III), Y(III), Tb(III), and Yb(III) was achieved by the present method for an 1-mL sample injection with the column of which the inner volume is 11.8 mL. The multistep pH-peak-focusing liquid chromatography with a hydrophilic polymer gel column developed in this study has great potential as a useful method for the separation of rare earth metal ions on a preparatory scale.

Holo/apo conversion two-dimensional urea PAGE for speciation of Fe3+-bound transferrin in serum.

This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe3+-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe3+ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe3+ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe2-Tf, FeN-Tf, and FeC-Tf (holo-Tf, Fe3+-bound Tf attached to N-lobe, and Fe3+-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe3+ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe3+ ions from the gel medium to ensure accurate determination of the Fe3+ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe3+ concentration in the serum was estimated.

High-pH mobile phase in reversed-phase liquid chromatography-tandem mass spectrometry to improve the separation efficiency of aminoglycoside isomers.

In chromatography, the use of extreme conditions can often lead to unique separation selectivity. In this study, a highly basic mobile phase (pH > 11), which is not typically employed for reversed-phase liquid chromatography (RPLC), was utilized in RPLC-tandem mass spectrometry (MS/MS) to achieve effective separation between electrically neutral bases of aminoglycosides (AGs). A mixture of AGs was simultaneously analyzed using 500 mmol L-1 ammonia aqueous solution (pH 11.8) as the mobile phase. A total of 11 AGs, including 2 stereoisomers of neomycin (B and C) and 5 structurally similar components of gentamicin (C1, C1a, C2, C2a, and C2b), were completely separated for the first time. The high separation performance for AGs was mainly due to two factors: First, slight differences in hydrophobicity among the AGs were significantly enhanced at a high pH by the complete acid dissociation of amines. Second, the high pH of the mobile phase minimized any electrostatic interactions between the AGs and residual silanol groups in the stationary phase, resulting in extremely sharp peaks for the AGs. The sensitivity of spectinomycin decreased by more than 20% when using the highly basic mobile phase (pH 11.8) due to its degradation, therefore, a mixture of 10 AGs was analyzed with 250 mmol L-1 ammonia aqueous solution (pH 11.5) with less degradation as the optimum condition. The developed analytical method could be used to determine the concentrations of trace AGs in milk with high accuracy and precision. Thus, RPLC-MS/MS using a high-pH mobile phase has great potential for the efficient separation of basic compounds containing amino sugars such as AGs.

Unusually Kinetically Inert Monocationic Neptunyl Complex with a Fluorescein-Modified 1,10-Phenanthroline-2,9-dicarboxylate Ligand: Specific Separation and Detection in Gel Electrophoresis.

We found a singly charged Np(V)O2+ complex with unprecedented kinetic inertness in aqueous solution, one million times slower than the widely accepted fast kinetics of neptunyl complexes. An inert NpO2+ complex with a fluorescent 1,10-phenanthroline-2,9-dicarboxylate derivative was found by kinetic selection using polyacrylamide gel electrophoresis (PAGE) from a small chemical library. Autoreduction from Np(VI)O22+ to Np(V)O2+ via complexation was observed. A remarkably small spontaneous dissociation rate constant of 8 × 10-6 s-1 (half-life of 23 h) was determined using PAGE. Selective detection of Np(V)O2+ was achieved in PAGE with a detection limit of 68 pmol dm-3 (17 fg). This system was successfully applied to simulated radioactive waste samples. Our finding that electron-rich NpO2+ forms a uniquely inert complex with no strong electrostatic interaction reveals a new aspect of actinide chemistry for developing a novel separation system of real radioactive material samples.

Effect of Catastrophic Thinking on the Analgesic Effect of Electroacupuncture.

Introduction: Patients with chronic pain and high-level catastrophic thoughts often do not respond to acupuncture. This may be related to hypofunctioning of the dorsolateral prefrontal cortex and the descending pain inhibitory system. Therefore, we examined the relationship between the level of catastrophic thinking and the analgesic effect of electroacupuncture using the pain catastrophizing scale (PCS). We also evaluated the descending pain inhibitory system using conditioned pain modulation (CPM) and offset analgesia (OA). The relationship between catastrophic thinking and the descending pain inhibitory system was also examined. Materials and Methods: After testing the hospital anxiety and depression scale and the PCS in 14 healthy adults, the current pain threshold (CPT), CPM, and OA were measured, in order, before the intervention. Thereafter, electroacupuncture was applied to 3 limbs (the dominant hand and both lower extremities) at 4 Hz, and to the scalp at 100 Hz, for 30 minutes, and the CPT was measured again immediately after the intervention. The difference in the CPT before and after the intervention was taken as the analgesic effect. Results: The participants were divided into 2 groups, the H-PCS group (≥16 points) and the L-PCS group (≤15 points), according to the PCS score, and the analgesic effects of electroacupuncture were significantly different (P = 0.04). However, no relationship was found between the PCS score and the CPM (r = -0.02, P = 0.94) and OA effects (r = -0.19, P = 0.49). Conclusion: It was suggested that people with high-level catastrophic thinking may find it difficult to obtain the analgesic effects of electroacupuncture.

Effects of Acupuncture Therapy on Drug-Resistant Fibromyalgia: An Exploratory Single-Arm Nonrandomized Trial.

Introduction: Fibromyalgia is a chronic illness that causes symptoms such as pain. In Japan, although pregabalin and duloxetine are the drugs of choice for fibromyalgia, they may be ineffective or may cause side-effects. Studies have reported on the efficacy of acupuncture against fibromyalgia. However, acupuncture is not always effective in clinical practice, and the reason for this is thought to be the dysfunction of the descending pain control system. This study aimed to determine whether the combined use of electro-scalp acupuncture and conventional electroacupuncture reduces fibromyalgia symptoms and drug dosage requirements. Methods: Patients with intractable fibromyalgia (visual analog scale [VAS] score ≥50 mm; Japanese version of the Fibromyalgia Impact Questionnaire [JFIQ] score ≥50) receiving pregabalin were recruited in this single-arm nonrandomized uncontrolled study. They underwent electroacupuncture on four limbs plus electro-scalp acupuncture once a week for 5 weeks. Drug intake, pain (as determined using VAS), quality of life (QOL; as determined using JFIQ), anxiety, depression, catastrophic thoughts, and sleep were assessed. Results: Although there was no increase in drug intake, 42.8% (3/7) of patients reduced pregabalin intake by approximately 10% (a moderate though insignificant effect). Pain levels significantly declined (VAS 75.4 ± 11.7 mm to 64.3 ± 17.3 mm; P = 0.05) and QOL significantly improved (JFIQ 67.0 ± 13.4 to 50.9 ± 18.3; P = 0.02). The parameters for anxiety, depression, catastrophic thoughts, and sleep did not significantly change. Conclusion: The combination of conventional electroacupuncture plus electro-scalp acupuncture may effectively alleviate pain, improve QOL, and reduce pregabalin dosage requirements in patients with fibromyalgia.

Characterization of the Interfacial Liquid Layer Formed on Hydrophobic Packing Material Surfaces by Liquid Chromatographic Analysis of the Distribution of Ions and Molecules.

We determine the bulk liquid phase volumes in octadecyl-bonded silica (C18 silica) columns equilibrated with acetonitrile-water and methanol-water (0-19%(v/v)) binary mixed solvents by a liquid chromatographic method with inorganic ions used as probes. The solvent composition and the thickness of the interfacial liquid layer formed on the C18-bonded silica surface are then determined from the bulk liquid phase volume, the total liquid phase volume, the surface area of the C18 silica packing material, and the retention volumes of the isotopically labeled eluent components for the columns. We used two C18 silica packing materials having identical bonding structures but different pore sizes and surface areas. Our results show that various hydrophilic organic compounds as well as inorganic ions recognize the interfacial liquid layer as being different from the bulk phase. The behavior of the solute compounds exhibiting substantially weak retention in reversed-phase liquid chromatography or the so-called negative adsorption, such as urea, sugars, and inorganic ions, can rationally be interpreted with a partition between the bulk liquid phase and the interfacial solvation liquid layer. The structural properties of the solvent layer on the C18-bonded layer determined by liquid chromatography are consistent with the molecular dynamics simulation results that have been obtained by other researchers.

A chromatographic approach for studying the adsorption of polar small molecules on tetrabutylammonium bromide semiclathrate hydrate.

A new liquid chromatography system in which tetrabutylammonium bromide semiclathrate hydrate (TBAB-SCH) was used as the column-packing material is proposed to evaluate the adsorption mechanisms of polar small molecules on a TBAB-SCH surface. It has been revealed that the more hydrophilic molecules tend to be more strongly retained on the TBAB-SCH/hexane-diethyl ether interface, and the retentions of aromatics (ARs) and crown ethers (CEs) are differently controlled. CEs are likely retained on the TBAB-SCH interface via hydrogen-bond formation as well on water-ice, whereas the retention mechanism of ARs is unique to TBAB-SCH and has not previously been observed on water-ice. This difference between CEs and ARs may arise from differences in the types of polar groups or molecular size. This chromatographic approach will provide useful information for evaluating the adsorption mechanisms of inhibitors for clathrate hydrate agglomeration, which is a common agglomeration problem experienced by industries.

Stoichiometry between Humate Unit Molecules and Metal Ions in Supramolecular Assembly Induced by Cu2+ and Tb3+ Measured by Gel Electrophoresis Techniques.

Humic acid (HA), a fraction of humic substances, can strongly complex with metal ions to form a supramolecular assembly via coordination binding and other intermolecular forces. However, determining the supramolecular size distribution and stoichiometry between small HA unit molecules constituting HA supramolecules and metal ions has proven to be challenging. Here, we investigated the changes in the size distributions of HAs induced by Cu2+ and Tb3+ ions using unique PAGE for the separation and quantification of HA complexes and metal ions bound, followed by UV-vis spectroscopy and excitation-emission matrix-parallel factor analysis. By determining the concentrations of HA and metal ions, it was possible to estimate the stoichiometry of the HA unit molecule to metal ions in supramolecular complexes. It was found that the supramolecular behaviors of Cu2+ and Tb3+ complexes with HA collected from peat (PAHA) and deep groundwater (HHA) differed. For example, two HHA unit molecules form a supramolecule via cross-linking by a Cu2+ ion in the case of Cu2+-HHA. Our results suggest that this supramolecular stoichiometry is related to the abundance of sulfur atoms in the elemental composition of HHA. Our experimental results and analysis provide new insights into HA supramolecules formed via metal complexation.

Alkali Metal Ion-exchange in a Metal-Organic Framework Based on Lanthanum and 1,4-Phenylenebis(methylidyne)tetrakis(phosphonic acid).

The ion-exchange selectivity of four metal-organic frameworks (denoted as MLaL), formed by alkali metal ions (M+), La3+, and 1,4-phenylenebis(methylidyne)tetrakis(phosphonic acid) (L), was examined. Unusual selectivity for the alkali metal ions was observed, which did not follow the previously proposed mechanism that was explained based on the ion-size similarity in the framework. The changes in the crystal structures after ion-exchange reactions were observed by powder X-ray diffraction analysis. The change in the lattice energy in a mixed-metal framework is likely to be one of the significant parameters to affect ion-exchange selectivity.

Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers.

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a "DNA Aptaomics" approach to systematically design functional aptamers as well as to obtain high affinity aptamers.

SELEX-based DNA Aptamer Selection: A Perspective from the Advancement of Separation Techniques.

DNA aptamers, which are short, single-stranded DNA sequences that selectively bind to target substances (proteins, cells, small molecules, metal ions), can be acquired by means of the systematic evolution of ligands by exponential enrichment (SELEX) methodology. In the SELEX procedure, one of the keys for the effective acquisition of high-affinity and functional aptamer sequences is the separation stage to isolate target-bound DNA from unbound DNA in a randomized DNA library. In this review, various remarkable advancements in separation techniques for SELEX-based aptamer selection developed in this decade, are described and discussed, including CE-, microfluidic chip-, solid phase-, and FACS-based SELEX along with other methods.

Intrinsic difference between phenyl hexyl- and octadecyl-bonded silicas in the solute retention selectivity in reversed-phase liquid chromatography with aqueous mobile phase.

For choosing an optimal column for a particular separation by reversed-phase liquid chromatography (RPLC), it is essential to quantitatively understand the effects of the chemical structure of hydrophobic bonded layer derived onto silica particles on the distribution equilibrium of a solute compound at the interface between the aqueous mobile phase and the packing material. However, there is still a lack of understanding of the solute distribution equilibrium in RPLC separation due to the complexities of the chemistry at the interface between the mobile phase and the bonded layer. We successfully determined the distribution coefficients of various organic compounds concerning to their accumulation onto the water/bonded layer interface and into the bonded layer from bulk water using surface-bubble-modulated liquid chromatography with octadecyl- and phenyl hexyl-bonded silica columns. The water/phenyl hexyl-bonded layer interface accumulates organic compounds much less than the water/octadecyl-bonded layer interface due to its lower interfacial tension, and this result suggests that phenyl hexyl group orient their benzene ring facing toward water. On the other hand, aromatic moiety of phenyl hexyl group enhances partitioning of the organic compounds into the bonded layer. Experimental findings in the present work demonstrated that the water/bonded layer interface and the bonded layer itself have independent contributions to the solute distribution and the water/phenyl hexyl-bonded layer interface shows quite different solute retention selectivity from the water/octadecyl-bonded layer interface.

Transmetalation in a Ce(III)-phosphoester Crystalline Coordination Polymer with an Exceptionally High Selectivity for Yb(III) and Lu(III).

A novel crystalline coordination polymer containing Ce3+ and bis(4-nitrophenyl) phosphate (L), CeL3 , was synthesized and its unique transmetalation selectivities toward Yb3+ and Lu3+ in the lanthanide series were evaluated. The relatively large difference in transmetalation selectivity between the neighboring Tm3+ and Yb3+ species is noteworthy because the reactivities of heavy lanthanides are generally considerably similar. The structural strain of the polymeric framework is likely responsible for this unusual trend. Powder X-ray diffraction analysis indicated that, in the cases of only Yb3+ and Lu3+ , large differences in their ionic sizes compared to that of Ce3+ in the parent framework may induce a structural strain after solid solution formation, while cleavage of the relatively weak Ce-O bond allows the formation of new Yb-O and Lu-O bonds with the incoming Yb3+ and Lu3+ , respectively. Structural phase transitions likely caused by the heterogeneous nucleation of the Yb- (or Lu-) type phase were also observed.

Purification of anionic fluorescent probes through precise fraction collection with a two-point detection system using multiple-stacking preparative capillary transient isotachophoresis.

A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF.

Advanced Gel Electrophoresis Techniques Reveal Heterogeneity of Humic Acids Based on Molecular Weight Distributions of Kinetically Inert Cu2+-Humate Complexes.

Humic acids (HAs) play important roles for the fate of metal ions in the environment. Most chemical speciation models involving HAs assume heterogeneous metal ion binding. However, these models also assume that the binding affinities of metal ions with HAs are the same regardless of the molecular weight (MW) ranges of the HAs involved. Here, we develop new polyacrylamide gel electrophoresis (PAGE) techniques to investigate the MW distributions of HAs with strongly complexed Cu2+ ions. By combining contaminant metal-free and high-resolution PAGE for HAs, this work was able to provide accurate MW distributions for the complexed metal ions. The MW distribution of Cu2+ binding ability per quantity of HA indicates that strong metal-binding moieties in HAs are heterogeneous in terms of MW. Coupling of the PAGE techniques with UV-vis and excitation-emission matrix (EEM) spectrometry-parallel factor analysis (PARAFAC) methods revealed new insights into kinetically inert interactions between HAs and Cu2+ ions. By this method, we found that the protein-like fluorescence components in the high- and low-MW regions cooperatively responded through Cu2+ binding. Thus, the advanced gel electrophoresis techniques developed herein are able to shed new light on the heterogeneity of metal binding affinities of HAs in terms of MW.

Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye.

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein.

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a 'bottom-up' method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis.