Highly efficient analysis of underivatized carbohydrates using monolithic-silica-based capillary hydrophilic interaction (HILIC) HPLC

A polyacrylamide (PAAm)-modified monolithic silica capillary column of increased phase ratio, 200T-PAAm, for hydrophilic interaction liquid chromatography (HILIC) was prepared. The column showed high separation efficiency, with a theoretical plate height H = 7–20 μm at a linear velocity, u = 1–7 mm/s. From a kinetic plot analysis, it was expected that the monolithic column could provide three times faster separation than particle-packed HILIC columns under a pressure limit at 20 MPa. HILIC coupled with electrospray ionization (ESI)–mass spectrometry (HILIC-ESI-MS) using the 200T-PAAm column was employed for the analysis of underivatized carbohydrates to achieve fast and efficient separations of mixtures containing mono-, di-, and trisaccharides within 5 min. Under single MS full scan mode, 200 pg of oligosaccharides was detected by the system. The limit of detection (LOD) of the LC-ESI-MS/MS system was determined using selected reaction monitoring (SRM) to be as low as 3.2 ng/mL (attomol level) for nonreducing saccharides. The system was successfully applied to the detection of disaccharides in extracts of plant, such as corn, soybean, and Arabidopsis thaliana. Figure HILIC-ESI-MS provides a high-efficiency separation and sensitive detection of underivatized carbohydrate oligomers, e.g., the homologs of glucose (1) up to maltoheptaose (7)     

Purification and characterization of a biosurfactant produced by Pseudomonas sp. G11 by asymmetrical flow field-flow fractionation (AsFlFFF)

Three hundred and thirty two bacterial colonies were isolated from soil contaminated by an oil spill. All the bacteria were cultured in a liquid medium individually, and the surface tensions of the media were compared. The bacterium whose culture medium had the lowest surface tension was identified as Pseudomonas sp. G11. A biosurfactant was produced by cultivation of the Pseudomonas sp. G11 in the LB media. For extraction of the biosurfactant, two solvent systems were used (n-hexane and a 2:1 (v/v) mixture of chloroform/MeOH), and the results were compared. Various experimental conditions (solvent composition, flow rate, etc.) were tested to optimize the analysis of the biosurfactant by asymmetrical flow field-flow fractionation (AsFlFFF). The biosurfactant was successfully separated from the culture medium by AsFlFFF when pure water was used as the carrier. From the retention data, the hydrodynamic diameter (d _H) and molecular weight (M) of the biosurfactant were determined by AsFlFFF. The molecular weight was determined by using pullulans as the calibration standards. The d _H and M were 49 nm and 2.3 × 10⁵ Da when extracted with n-hexane, and 39 nm and 1.13 × 10⁵ Da when extracted with the 2:1 mixture of chloroform/MeOH, respectively. Figure Separation of biosurfactant from its culture medium by flow FFF     

Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay

Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (λ_max = 390 nm) and the mutant Y139F emits greenish light (λ _max = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones—follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca²⁺ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay. Figure Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained and applied in dual-color simultaneous immunoassay of two gonadotropic hormones.     

Catechol determination in compost bioremediation using a laccase sensor and artificial neural networks

An electrochemical biosensor based on the immobilization of laccase on magnetic core-shell (Fe₃O₄–SiO₂) nanoparticles was combined with artificial neural networks (ANNs) for the determination of catechol concentration in compost bioremediation of municipal solid waste. The immobilization matrix provided a good microenvironment for retaining laccase bioactivity, and the combination with ANNs offered a good chemometric tool for data analysis in respect to the dynamic, nonlinear, and uncertain characteristics of the complex composting system. Catechol concentrations in compost samples were determined by using both the laccase sensor and HPLC for calibration. The detection range varied from 7.5 × 10^–7 to 4.4 × 10^–4 M, and the amperometric response current reached 95% of the steady-state current within about 70 s. The performance of the ANN model was compared with the linear regression model in respect to simulation accuracy, adaptability to uncertainty, etc. All the results showed that the combination of amperometric enzyme sensor and artificial neural networks was a rapid, sensitive, and robust method in the quantitative study of the composting system. Figure Structure of the magnetic carbon paste electrode used in the electrochemical biosensor     

Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis

Comparative metabolomics of Leishmania species requires the simultaneous identification and quantification of a large number of intracellular metabolites. Here, we describe the optimisation of a comprehensive metabolite extraction protocol for Leishmania parasites and the subsequent optimisation of the analytical approach, consisting of hydrophilic interaction liquid chromatography coupled to LTQ-orbitrap mass spectrometry. The final optimised protocol starts with a rapid quenching of parasite cells to 0 °C, followed by a triplicate washing step in phosphate-buffered saline. The intracellular metabolome of 4 × 10⁷ parasites is then extracted in cold chloroform/methanol/water 20/60/20 (v/v/v) for 1 h at 4 °C, resulting in both cell disruption and comprehensive metabolite dissolution. Our developed metabolomics platform can detect approximately 20% of the predicted Leishmania metabolome in a single experiment in positive and negative ionisation mode.      

Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue

Many muscular diseases result from abnormal organization of connective tissue and/or collagen network formation. Only a few molecular imaging techniques are able to analyze this collagen network by differentiating collagen types. In this study, FT-IR spectroscopy was used to analyze type I and IV collagens, the most important compounds of which are perimysium and endomysium, respectively. Secondary structure of collagen types was determined by curve-fitting the 1,700–1,480 cm⁻¹ spectral interval. Type I collagen could be differentiated from type IV by its higher amounts of triple helix and α-helix, but lower amounts of β-sheets (P < 0.01). FT-IR imaging was then used to determine structural features of perimysium and endomysium collagen network in bovine Flexor carpi radialis muscle. Secondary structure of proteins contained in perimysium and endomysium was found to be very close to type I and IV collagens, respectively. FT-IR spectroscopy and imaging are thus analytical tools that might be used for investigating biodistribution and assembly of collagen types in connective tissues. Figure Visible (left) and full spectral FT-IR (right) images of skeletal muscle tissue section (16 μm) exhibiting a vertical arrangement of fibers. + and × in FT-IR image show selected positions to obtain FT-IR spectra of perimysium and endomysium, respectively     

Comparison of differential pulse and alternating current polarography in the soft-modelling study of the complexation of Cd(II) by the fragment Cys-Gly and by the phytochelatin (γ-Glu-Cys)<Subscript>2</Subscript>Gly

A comparison of a differential pulse polarographic with a phase sensitive alternating current polarographic study of the Cd-Cys-Gly and Cd-PC₂ systems [PC₂ being a phytochelatin of general structure (γ-Glu-Cys) _n -Gly, with n = 2] has been performed. The chemometric multivariate curve resolution method with alternating least squares was applied in the experimental data analysis. The results obtained by both polarographic techniques have made it possible to find out the formation sequences of the complexes and their final stoichiometries. The alternating current polarograms compared with the differential pulse ones show some differences (a new signal and an important shift of peak potentials), which anyway are consistent with some of the conclusions obtained by differential pulse polarography. This fact implies that although the alternating current polarography results need some corrections before data treatment, they provide extra information that complements the conclusions achieved by differential pulse polarography. Figure Voltammograms at ACP(−10°), ACP(−65°) and corrected ACP during the titration of a 10⁻⁵ mol L⁻¹ Cd(II) solution with PC₂ at pH 8.5 in 0.05 L⁻¹ Tris.     

FTICR-MS applications for the structure determination of natural products

Natural products are a source of unique chemical entities with specific biological activities of great value to the pharmaceutical industry. However, the determination of unknown structures is usually time consuming and often becomes a bottleneck in the effort to develop natural products into effective drugs. The high-performance features of high magnetic field FTMS have greatly alleviated the structural elucidation bottleneck to meet increasingly shorter discovery timelines for drug candidates based on natural products. The high-performance features of high field FTMS include unsurpassed mass measurement accuracy for elemental formula determination, ultra-high mass resolution for component separation, the ability to perform multiple levels of tandem mass spectrometry for structural elucidation, and moderate sensitivity for limited supply of isolates. A number of applications utilizing these properties of FTMS have been reported recently for the structural elucidation of novel natural product structures originating from terrestrial and marine microorganisms. In this review, FTMS methods and their applications for the structural elucidation and characterization of natural products will be reviewed. Figure Molecular structure and positive ion mode nanoelectrospray FTICR mass spectrum of methylspirastrellolide A (3). The inset shows the isotopic distribution with high abundance of the A + 2 peak, but less than the abundance of the A + 1 peak. The resolved isotopic fine structure of the A + 2 peak reveals the presence of one chlorine atom based on accurate mass assignment and the measured abundance ratio between the resolved ³⁷Cl peak and the monoisotopic peak     

Analytical approach for characterization of cadmium-induced thiol peptides—a case study using <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Phytochelatins (PC) were described earlier to play a role in metal detoxification in Chlamydomonas reinhardtii but were not clearly identified. The focus of this case study was to identify PC synthesized by C. reinhardtii exposed to Cd. Only low intracellular concentrations of cadmium (85 nmol g⁻¹ fresh weight) were sufficient to cause significant changes in thiol peptide pools. Thus, results showed a progressive decline of the glutathione content, accompanied by an induction of phytochelatins. Not only canonic phytochelatins but for the first time also the iso-phytochelatins CysPC _n and PC₂Ala were identified in this unicellular green alga using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Additionally, CysPC _n desGly, PC _n desGly, CysPC _n Glu, and PC₂Glu were found throughout MS analysis. Also, low abundant PCs could be detected due to the high sample preconcentration combined with little sample amounts (0.3 μL min⁻¹) necessary for electrospray. Identified PCs had a maximum number of 5 γ-glutamyl cysteine (γ-GluCys) units. Thiol peptides of higher molecular masses suggesting PC _n with n > 5 could be identified as intermolecular oxidation products of smaller PCs. Thiols may easily be oxidized. Therefore, PCs were reduced prior to MS analysis. Dithiothreitol and tris(2-carboxyethyl) phosphine were compared concerning their reduction effort.      

The potentiometric behavior of polymer-supported metallophthalocyanines used as anion-selective electrodes

Liquid polymer membrane electrodes based on nickel and manganese phthalocyanines were examined for use as anion-selective electrodes. The electrodes were prepared by incorporating the ionophores into plasticized poly(vinyl chloride) membranes, which were directly coated onto the surfaces of graphite electrodes. The resulting electrodes demonstrate near-Nernstian responses over a wide linear range of perchlorate anion (5 × 10⁻⁷ to 1 × 10⁻¹ M). The electrodes have a fast response time, submicromolar detection limits (5 × 10⁻⁷ M perchlorate), and could be used over a wide pH range of 3.5–10. The influences of lipophilic cationic and anionic additives on the response properties of the electrodes were investigated. The proposed sensors revealed high selectivity for perchlorate over a number of common inorganic and organic anions. The highest selectivity was observed for the electrode based on manganese phthalocyanine in the presence of the lipophilic anionic additive sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate. Application of the electrodes to determine perchlorate in tap water and human urine is also reported.      

Determination of glucosinolates in traditional Chinese herbs by high-performance liquid chromatography and electrospray ionization mass spectrometry

A reversed-phase HPLC method has been developed for determination of twelve intact glucosinolates—glucoiberin, glucocheirolin, progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin, and gluconasturtiin—in ten traditional Chinese plants. The samples were extracted with methanol and the extracts were cleaned on an activated Florisil column. A mobile phase gradient prepared from methanol and 30 mmol L⁻¹ ammonium acetate at pH 5.0 enabled baseline separation of the glucosinolates. Glucosinolate detection was confirmed by quadrupole time-of-flight tandem mass spectrometric analysis in negative-ionization mode. Detection limits ranged from 0.06 to 0.36 μg g⁻¹ when 5 g of dried plant was analyzed. Recoveries of the glucosinolates were better than 85% and precision (relative standard derivation, n = 3) ranged from 5.3 to 14.6%. Analysis of the glucosinolates provided scientific evidence enabling differentiation of three pairs of easily confused plants. Figure Glucosinolates Analysis for the Differentiation of Easily-Confusing Herbs     

Circular dichroism thermal lens microscope in the UV wavelength region (UV-CD-TLM) for chiral analysis on a microchip

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 × 10⁻⁴ mol L⁻¹ (ΔA = 5.2 × 10⁻⁶ Abs.) for (+)-CSA and 8.4 × 10⁻⁴ mol L⁻¹ (ΔA = 5.0 × 10⁻⁶ Abs.) for (−)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified. Figure Sensitive chiral analysis by thermal lens microscope (TLM)     

Application of glow discharge mass spectrometry to multielement ultra-trace determination in ultrahigh-purity copper and iron: a calibration approach achieving quantification and traceability

A new approach was developed for quantitative calibration in GD-MS which can afford reliable and metrologically traceable results for many trace elements and was exemplified for pure copper and pure iron. It can be assumed that the technique can be further improved and applied to the analysis of other pure metals. Pressed copper and iron powder samples were used to calibrate the glow discharge mass spectrometry applied to the analysis of pure copper and iron. The new type of glow discharge mass spectrometer—the Element GD (Thermo Electron Corporation)—was used with a Grimm-type discharge cell for flat samples. Two series of powder samples were prepared for each of the copper and iron matrixes. The powders were quantitatively doped with solutions of graduated and defined concentrations of 40 or 20 analytes, respectively. The mass fractions of the analytes in the dried and homogenized metal powder samples ranged from μg/kg levels up to 10 mg/kg levels. A special technique was developed to press the samples and to form mechanically stable pellets with low risk of contamination. Ion beam ratios of analyte ions to matrix ions were used as measurands. The calibration curves were determined and the linear correlation coefficients were calculated for different intervals of the curves. The linear correlation coefficients are very satisfactory for most of the calibration curves, which include the higher segments of mass fractions; however, they are less satisfactory for the lower segments of the calibration curves. Nevertheless, in many cases rather acceptable and rather promising values were achieved even for these lower segments, representing mass fractions of analytes at ultra-trace level. The comparison of the certified values of different reference materials with the measured values based on calibrations with the pressed powder samples led to deviations less than 30% for most of the considered examples.      

Laser ablation inductively coupled plasma mass spectrometry for direct analysis of the spatial distribution of trace elements in metallurgical-grade silicon

The spatial distribution and concentration of impurities in metallurgical-grade silicon (MG-Si) samples (97–99% w/w Si) were investigated by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The spatial resolution (120 μm) and low limits of detection (mg kg⁻¹) for quality assurance of such materials were studied in detail. The volume-dependent precision and accuracy of non-matrix-matched calibration for quantification of minor elements, using NIST SRM 610 (silicate standard), indicates that LA-ICP-MS is well suited to rapid process control of such materials. Quantitative results from LA-ICP-MS were compared with previously reported literature data obtained by use of ICP-OES and rf-GD-OES. In particular, the distribution of element impurities and their relationship to their different segregation coefficients in silicon is demonstrated. Dedicated to Professor Klaus G. Heumann     

Flow-injection solid surface lanthanide-sensitized luminescence sensor for determination of <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

A single optosensing device based on lanthanide-sensitized luminescence was developed for determination of p-aminobenzoic acid (PABA). The method is based on the formation of a complex between PABA and Tb(III) immobilized on the solid phase (QAE A-25 resin) placed inside the flow cell. NaCl (1 M) was used as carrier solution and HCl (0.05 M) as eluent. The sample solutions of PABA (100 μL) containing Tb(III) and buffered at pH = 6.0 were injected into the carrier stream and the luminescence was measured at λ _ex = 290 nm and λ _em = 546 nm. The method shows a linear range from 0.2 to 6.0 μg mL⁻¹ with an RSD of 1.2% (n = 10) and a sampling frequency of 22 h⁻¹. A remarkable characteristic of the method is its high selectivity which allows it to be satisfactorily applied to the analysis of PABA in pharmaceutical samples without prior treatment. Figure Typical emission bands of Tb(III) in a solid-phase PABA–Tb(III) luminescence spectrum     

Electrochemical behavior and differential pulse voltammetric determination of paracetamol at a carbon ionic liquid electrode

The electrochemical behavior of paracetamol in 0.1 M acetate buffer solution (pH 4.6) was investigated at a traditional carbon paste electrode (TCPE) and a carbon ionic liquid electrode (CILE) fabricated by replacing nonconductive organic binders with a conductive hydrophobic room temperature ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF₆). The results showed that the CILE exhibited better reversibility for the electrochemical redox of paracetamol. The oxidation potential of paracetamol at the CILE is +0.462 V, which is approximately 232 mV lower than that at the TCPE; the oxidation peak current response is nine times higher than that at the TCPE. The differential pulse voltammetric determination of paracetamol at the CILE was established based on this behavior. After optimizing several important parameters controlling the performance of paracetamol at the CILE, the oxidation peak current versus paracetamol concentration at the CILE showed linearity in the range from 1.0 μM to 2.0 mM (R ²  = 0.9992) with a detection limit of 0.3 μM (S/N = 3). The method has been applied to the determination of paracetamol in tablets and urine samples and the average recovery of paracetamol was 98.5% and 99.3%, respectively. The proposed CILE showed good sensitivity and reproducible response without influence of interferents commonly existing in pharmaceutical and urine samples. Figure CV curves of paracetamol illustrate the enhanced electrochemical behavior of paracetamol at the CILE (b), which forms the basis for the differential pulse voltammetric determination of paracetamol     

Surface modification of amine-functionalised graphite for preparation of cobalt hexacyanoferrate (CoHCF)-modified electrode: an amperometric sensor for determination of butylated hydroxyanisole (BHA)

A cobalt hexacyanoferrate (CoHCF)-modified graphite paraffin wax composite electrode was prepared by a new approach. An amine-functionalised graphite powder was used for the fabrication of the electrode. A functionalised graphite paraffin wax composite electrode was prepared and the surface of the electrode was modified with a thin film of CoHCF. Various parameters that influence the electrochemical behaviour of the modified electrode were studied by varying the background electrolytes, scan rates and pH. The modified electrode showed good electrocatalytic activity towards the oxidation of butylated hydroxyanisole (BHA) under optimal conditions and showed a linear response over the range from 7.9 × 10⁻⁷ to 1.9 × 10⁻⁴ M of BHA with a correlation coefficient of 0.9988. The limit of detection was 1.9 × 10⁻⁷ M. Electrocatalytic oxidation of BHA was effective at the modified electrode at a significantly reduced potential and at a broader pH range. The utility of the modified electrode as an amperometric sensor for the determination of BHA in flow systems was evaluated by carrying out hydrodynamic and chronoamperometric experiments. The modified electrode showed very good stability and a longer shelf life. The modified electrode was applied for the determination of BHA in spiked samples of chewing gum and edible sunflower oil. The advantage of this method is the ease of electrode fabrication, good stability, longer shelf life, low cost and its diverse application for BHA determination. Figure Cyclic Voltammogram of () CoHCF modified electrode, () in presence of 1.9 x 10⁻⁵ M of BHA and () bare electrode, () in the presence of 1.9 x 10⁻⁵ M of BHA in 1.0 M NaCl, pH 7.0     

Small-volume fiber-optic evanescent-wave absorption sensor for nitrite determination

A novel small-volume fiber-optic evanescent-wave absorption sensor based on the Griess–Ilosvay reaction has been developed and evaluated for nitrite determination. The sensor was constructed by inserting a decladded optical fiber into a transparent capillary to form an annular column microchannel. The Evanescent wave (EW) field produced on the optical fiber core surface penetrated into the surrounding medium and interacted with the azo dye, which was generated by the reaction of nitrite and nitrite-sensitive reagents. The detector was designed to be parallel to the axis of the optical fiber. The defined absorbance was linear with the concentration of nitrite in the range from 0.05 to 10 mg L⁻¹, and the detection limit was 0.02 mg L⁻¹ (3σ) with the relative standard deviation (RSD) of 2.6% (n = 8). The present sensor was successfully used to determine nitrite in real samples of mineral water, tap water, rain water, and seawater. The results were consistent with the data obtained by standard spectrophotometric method, showing potential of the proposed sensor for practical application.      

A catalytically active molecularly imprinted polymer that mimics peroxidase based on hemin: application to the determination of<Emphasis Type="Italic"> p</Emphasis>-aminophenol

Despite the increasing number of applications of molecularly imprinted polymers (MIP) in analytical chemistry, the synthesis of polymers with hemin introduced as the catalytic center to mimic the active site of peroxidase remains as a challenge. In the current work, a new type of molecularly imprinted polymer (MIP) was synthesized with 4-aminophenol (4-APh) as the template and two monomers: hemin, which acts as the catalytic center, and methacrylic acid (MAA), which is used to build the active sites. This work shows that MIP successfully mimics peroxidase. For this purpose, a flow injection analysis system coupled to an amperometric detector was investigated through multivariate analysis. The determination of 4-APh was not affected by the equimolar presence of structurally similar phenol compounds, including catechol, 4-chloro-3-methylphenol, 2-aminophenol, guaiachol, chloroguaiachol and 2-cresol, thus highlighting the good performance of the imprinted polymer. Under the optimized experimental conditions, an analytical curve covering a wide linear response range from 0.8 up to 500 μmol L⁻¹ (r > 0.999) was obtained, and the method gave satisfactory precisions (n = 8), as evaluated via the relative standard deviation (RSD), of 4.1 and 3.2% for solutions of 4-APh of 50 and 500 μmol L⁻¹, respectively. Recoveries of 96–111% from water samples (tap water and river water) spiked with 4-APh were achieved, thus illustrating the accuracy of the proposed system. Figure Schematic presentation of the synthesis of the MIP     

Evaluation of different calibration strategies for the analysis of pure copper and zinc samples using femtosecond laser ablation ICP-MS

Solution-doped metal powder pellets as well as aspirated liquids were used as calibration samples to analyze pure copper and zinc certified reference materials (CRMs) by femtosecond laser ablation ICP-MS. It was demonstrated that calibration by copper pellets resulted in relative deviations up to 20%, whereas fs-LA-ICP-MS among copper-based CRMs led to inaccuracies in the same range unless nominal mass fractions were chosen to be <3 mg/kg. Calibration by zinc pellets generally provided better accuracy. Depending on the analyte considered, deviations below 10% were obtained even for mass fractions close to the limit of quantification. Our data, therefore, indicate solution-doped metal powder pellets to be suitable as calibration samples for fs-LA-ICP-MS of metals. Furthermore, the utilization of liquid standards for calibration was found to result in stronger deviations of up to 50% for both copper and zinc samples which, in addition, turned out to be dependent on the plasma conditions.