Showing papers in "Chromatographia in 1996"

A novel support with artificially created recognition for the selective removal of proteins and for affinity chromatography

Abstract: Acrylamide and N,N′-methylenebisacrylamide were copolymerized in the presence of a protein to form a gel which was pressed through a sieve. The gel particles obtained were packed into a chromatographic tube. The experimental conditions for the polymerization are such that the pores of the gel particles are large enough to permit the protein to diffuse out of the particles, so that the entrapped protein can be removed from the bed by washing with an aqueous solution. However the interaction with the matrix is so strong that the protein can be desorbed only by a buffer containing 0.5 M sodium chloride or by a 10% solution of acetic acid containing 10% SDS. When a sample containing the protein present during the polymerization was applied to the column along with other proteins this protein was the only one adsorbed. The technique worked selectively with hemoglobin, cytochrome C and transferrin.

Molecularly imprinted polymer as chiral selector for enantioseparation of amino acids by capillary gel electrophoresis

Abstract: A capillary electrophoresis method for the enantioseparation of D,L-aromatic amino acids using molecularly imprinted polymer as chiral selector has been developed Methacrylic acid was used as functional monomer and ethylene glycol dimethacrylate (EDMA) as crosslinking monomer The molecularly imprinted polymer was packed in capillaries by incorporating with acrylamide gel The composition of the polymerization mixtures affects the separation factor to some extent The molar ratio of cross-linker to functional monomer to print molecule was 20∶4∶1, which gives the best separation for aromatic amino acid enantiomers by using L-phenylalanine anilide (L-pheAN) as print molecule Under the same conditions, however, separation of D,L-phenylalanine using L-phenylalanine as print molecule is poorer than that using L-pheAN Polymer particles (≤5 μm) were supported by 6% acrylamide and 5% bisacrylamide gel in the capillary The effects of organic solvents, electrophoretic buffer and pH on separation were also investigated

Enantiomer separation by open-tubular liquid chromatography and electrochromatography in cellulose-coated capillaries

Abstract: The feasibility of enantiomer separations by normal-phase and reversed-phase open-tubular liquid chromatography as well as by capillary electrochromatography in 50 μm i.d. fused silica capillaries coated with cellulose derivatives is demonstrated for a number of chiral pharmaceuticals. The excellent enantioselectivity of 3,5-dimethylphenylcarbamoyl cellulose andpara-methylbenzoyl cellulose has been transferred from HPLC to a miniaturized, open-tubular system. A comparison of HPLC, OTLC and CEC is made and the influence of retention and temperature on efficiency, selectivity and resolution is discussed.

Instrumental developments in capillary electrochromatography

Abstract: An instrumental device for capillary electrochromatography (CEC) is described that permits conditioning of packed columns at pressures up to 100 bar, in or on-column fluorescence detection and electrokinetic sample injection. Capillaries with varying inner diameters (75, 100, 150 μm) packed with octadecyl silica gel (3 μm) are compared for efficiency and detection sensitivity. The influence of counter pressure on electroosmotic velocity and plate number is discussed.

Polycyclic aromatic hydrocarbons in waste waters and sewage sludge: Extraction and clean-up for HPLC analysis with fluorescence detection

Abstract: A modified solid-phase extraction technique using sonication of the adsorbent material instead of the elution normally applied has been compared with two conventional liquid-liquid extraction procedures for the determination of the 16 EPA PAHs in municipal waste waters by means of HPLC coupled with fluorescence detection. Liquid-liquid extraction with cyclohexane proved to be the most efficient and simplest procedure. Clean-up of the waste-water extracts was not considered necessary, because of the high chromatographic resolution of the column and the selectivity of the fluorescence detector. Different organic solvents were also compared for ultrasonic extraction of PAHs from sewage sludge. The best results were obtained by use of acetonitrile. Clean-up of sewage-sludge extracts was not found necessary for accurate quantification of the major PAH components with fluorescence detection. The precision of the whole analytical procedure from extraction to the final determination of PAHs was satisfactory for both waste-water and sewage-sludge samples.

Native and modified alumina, titania and zirconia in normal and reversed-phase high-performance liquid chromatography

Abstract: Chromatographic properties of silica, alumina, titania and zirconia have been investigated in normal phase mode in the separation of test mixtures of basic, neutral and acidic compounds. In contrast to silica the chromatographic behaviour revealed the basic properties of the alumina, titania and zirconia surfaces. Therefore, separation of basic compounds on these packings seems very promising. Lypophilic packings have been synthesized by modification of titania, zirconia and alumina with organosilanes and polymers and tested for the separation of basic compounds and proteins. High hydrolytic stability of the modified packings was observed during separations with strong alkali and acidic eluents.

Detection of organophosphate pesticides in human body fluids by headspace solid-phase microextraction (SPME) and capillary gas chromatography with nitrogen-phosphorus detection

Abstract: Organosphosphate pesticides have been found extractable by headspace solid-phase microextraction (SPME), and the best conditions of their extraction from human whole blood and urine samples have been investigated. The body fluid samples containing nine pesticides (IBP, methyl parathion, fenitrothion, malathion, fenthion, isoxathion, ethion, EPN and phosalone) were heated at 100°C in a septum-capped vial in the presence of various combinations of acid and salts, and SPME fiber was exposed to the headspace of the vial to allow adsorption of the pesticides before capillary gas chromatography (GC) with nitrogen-phosphorus detection. The heating with distilled water/HCl/(NH4)2SO4/NaCl and with distilled water/HCl gave the best results for urine and whole blood, respectively. Recoveries of the nine pesticides were 0.8–10.6% except for phosalone (0.03%) for whole blood, and 3.8–40.2% for urine. The calibration curves for the pesticides showed linearity in the range of 50–400 ng/0.5 mL for whole blood except for malathion (100–400 ng/0.5 mL whole blood) and 7.5–120 ng/0.5 mL for urine except for phosalone (15–120 ng/0.5 mL urine) with detection limits of 2.2–40 ng/0.5 mL for whole blood and 0.8–12 ng/0.5 mL for urine.

The use of a short-end injection procedure to achieve improved performance in capillary electrophoresis

Abstract: Minimum capillary lengths on commercial instruments are fixed and cannot be decreased further. To effectively reduce the capillary length used for separation the sample can be injected from the end of the capillary nearest the detector. This procedure is known as a ‘short-end’ injection and can reduces analysis times by at least two-thirds compared to conventional injections. The time reduction benefits are shown in rapid separations of basic drugs, drug-related impurities and chiral compounds. Short-end injections, in combination with both increased electrolyte strength and reduced voltage are an effective approach to reducing the detrimental impact of high sample solution ionic strength. They can also lead to improved resolution by increasing stacking effects and reducing peak tailing. Peak area and migration time precision obtained are shown to be equivalent to those obtained for conventional injection procedures. It is concluded that short-end injections should be considered for routine operation as they are a useful means of reducing analysis time, increasing sensitivity, decreasing buffer depletion effects. They also allow use of higher electrolyte strengths which can improve resolution and reduce peak tailing, and can overcome significant problems which occur when analysing samples containing high salt contents.

Supported liquid membrane work-up in combination with liquid chromatography and electrochemical detection for the determination of chlorinated phenols in natural water samples

Abstract: A sample preparation method has been developed for the determination of chlorinated phenols in water. The method is based on a supported liquid membrane extraction system connected on-line to liquid chromatography with electrochemical detection. The supported liquid membrane technique utilizes a porous PTFE membrane. The membrane is impregnated with an organic solvent which forms a barrier between two aqueous phases and enables selective extraction. The technique can easily be coupled in a flow system. In this investigation five chlorinated phenols (1–5 chlorine atoms) were extracted from natural water samples. Extraction for 30 minutes resulted in detection limits of approximately 25 ng L−1.

Capillary electrochromatography using columns packed with a supercritical fluid carrier

Abstract: Columns for capillary electrochromatography may be prepared by packing reversed phase silica-based material using a supercritical fluid carbon dioxide carrier. Procedures for the in-situ manufacture of frits and UV detection windows, and the wetting of columns are described. The columns were employed in two commercial instruments (and a home-built system), and their properties investigated during the separation of standard mixtures of test compounds. The columns are highly efficient and durable, with reduced plate heights of 1.0–1.4. The repeatability of retention time, peak area and peak height was measured. The influence of applied voltage and column temperature and of electrokinetic injection parameters was explored, along with other practical considerations.

Dextran and dextrin as chiral selectors in capillary zone electrophoresis

Abstract: Polysaccharides such as dextrins, which are mixtures of linear α-(1,4)-linked D-glucose polymers, and dextrans, which are polymers of D-glucose units linked predominantly by α-(1,6) bonds, have been employed as chiral selectors in the capillary electrophoretic (CE) separation of enantiomers. Because these polymers are electrically neutral, the method is applicable to ionic compounds. Among the compounds tested the enantiomers of ibuprofen, naproxen, warfarin and the diltiazem synthetic intermediate 1,1′-binaphthyl-2,2′diyl hydrogen phosphate, which are anionic compounds, were successfully recognized by employing dextran or dextrin under neutral conditions. The enantiomers of basic or cationic drugs such as timepidium, primaquine, sulconazole, trimetoquinol, diltiazem and clentiazem were also successfully separated under acidic conditions. The effects of molecular mass and polysaccharide concentration on enantioselectivity were investigated. High concentrations of these polymers, especially dextrin, led to delayed migration times after repeated injections of the solutes into an uncoated capillary under neutral conditions. Consequently the capillary washing process was found to be very important to obtaining reproducible migration times in the system. A coated capillary was also investigated to overcome this problem. Finally, the method using electrically neutral polysaccharides was applied to the testing of the optical purity of the drug substances.

Normal and reversed-phase HPLC for more complete evaluation of tocopherols, retinols, carotenes and sterols in dairy products

Abstract: We have previously described a normal-phase HPLC method for the simultaneous determination of α, β, γ and δ tocopherol, thecis andtrans isomers of retinol, and total carotenes in different Italian cheeses and milk. In this paper we describe a reversed-phase HPLC method using solely methanol as the mobile phase for the determination of α-tocopherol, β-+γ-tocopherols, total retinols, cholesterol, β-sitosterol, campesterol and stigmasterol, and α- and β-carotene. The method developed has been applied to some new dairy products such as natural Quark, with olive, basil or banana and to processed cheese with tomato and the results have been compared with the corresponding results obtained by the normal-phase method. The conclusion is that more complete evaluation of the tocopherol, retinol, carotene and sterol content of dairy products, especially when vegetables and fruits are present, is necessary and can be obtained using both the normal- and reversed-phase HPLC methodologies.

Simultaneous gas chromatographic determination of methanol and free glycerol in biodiesel

Abstract: The methanol and free glycerol content of vegetable oil methyl esters used as diesel fuel (biodiesel) is very important in describing the quality of this fuel and is therefore limited by specifications. A previously described GLC method for the determination of free glycerol in biodiesel has been further developed and also allows the simultaneous determination of methanol. Sample preparation includes dissolving in dimethylformamide, silylation with bis-trimethylsilyltrifluoracetamide (BSTFA) and separation on a methylsilicone fluid, coated-capillary column using either FID or MS-detection. Ethanol and 1,4-butanediol were used as internal standards. Both detection systems show sufficient sensitivity for concentrations relevant to biodiesel samples. The recovery was tested using a RME-sample containing known amounts of methanol and glycerol.

Analysis of contact allergenic compounds in oxidized d-limonene

Abstract: A detailed chemical analysis and quantification of oxygen-containing monoterpenes in auto-oxidizedd-limonene was performed. Some of these, e.g. the cis- and trans-isomers of limonene-2-hydroperoxide, have previously been shown to exhibit strong contact allergenic properties. GC-MS with chemical ionization in negative ion mode was shown to be a successful method for the identification and determination of the molecular weight of chemically unstable limonene hydroperoxides. An HPLC method for isolation of individual compounds in auto-oxidizedd-limonene is presented. Two different stationary phases were used in normal phase mode, cyanopropyl- and diamino-modified silica. The method described can be used for the isolation of individual contact allergens in sensitization experiment vehicles, such as petrolatum and olive oil. This makes it possible to study whether test compounds, such as hydroperoxides, are chemically stable during a sensitization experiment.

Polyethylene-coated silica and zirconia stationary phases in view of quantitative structure-retention relationships

Abstract: Retention properties of two new high-performance liquid chromatographic (HPLC) stationary phases, polyethylene-coated silica (PECSiO2) and polyethylene-coated zirconia (PECZrO2), were compared chemometrically. Reversed phase HPLC capacity factors were determined isocratically for a carefully designed series of 25 structurally diverse solutes. Linear free energy relationship (LFER)-based solute parameters (excess molar refraction, dipolarity-polarizability, hydrogen-bond acidity and basicity, McGowan volume) and solute structural descriptors determined by physico-chemical methods (water accessible van der Waals volume, dipole moment, atomic electron excess charge) were regressed as independent variables against log capacity factors. The quantitative structure-retention relationships (QSRR) obtained show the predominating partition mechanism of separation on both phases. Both the LFER parameters and the structural parameters obtained by molecular modelling produced QSRR equations of high and practically identical retention prediction ability. It has been concluded that intermolecular interactions of the dipolarity-polarizability type manifest themselves more strongly on the silica-based phase than on the zirconia-based one. This can be due to a role of active free silanols on the silica support.

Classification of the botanical origin of cinnamon by solid-phase microextraction and gas chromatography

Abstract: The conditions which affect the reproducibility of characteristic chromatographic profiles of semivolatile compounds from true cinnamon and cassia by headspace solid-phase microextraction are determined. Optimum conditions for sample amount, vial size, temperature, sampling time, and fiber type for steady state sampling conditions are identified. Vial size was an unexpected critical parameter possibly related to the build up of internal ternal pressure during sample heating followed by expulsion of a portion of the vapor phase through the septum as it was punctured by the syringe. Gas chromatography with ion trap mass spectrometry was used to identify the major volatile compounds in cassia and true cinnamon. Reasonable semi-quantitative agreement (r 2>0.87 and generally greater than 0.93) was observed for the major semivolatile compounds isolated by headspace solid-phase microextraction and solvent-assisted supercritical fluid extraction. The extracts isolated by solid-phase microextraction contained relatively high concentrations of terpene-type compounds in low abundance in the solvent-assisted supercritical fluid extracts, but these compounds were of little value in distinguishing the botanical origin of authentic cinnamon and cassia samples. The latter were easily distinguished by the presence of eugenol and benzyl benzoate in true cinnamon, that was absent in cassia, and the presence of coumarin and δ-cadinene, in cassia, that was either absent or in low concentration in true cinnamon. Headspace solid-phase microextraction provides a rapid and simple method for establishing the botanical origin of the principal cinnamons of commerce.

Detection of ethanol in human body fluids by headspace solid-phase micro extraction (SPME)/capillary gas chromatography

Abstract: Ethanol has been found extractable from human whole blood and urine samples by headspace solid-phase micro extraction (SPME) with a Carbowax/divinylbenzene-coated fiber. After heating a vial containing the body fluid sample with ethanol, and isobutanol as internal standard (IS) at 70°C in the presence of (NH4)2SO4, a Carbowax/divinylbenzene-coated SPME fiber was exposed in the headspace of the vial to allow adsorption of the compounds. The fiber needle was then injected into a middle-bore capillary gas chromatography (GC) port. The headspace SPME-GC gave intense peaks for both compounds; a small amount of background noises appeared, but did not interfere with the detection of the compounds. Recoveries of ethanol and IS were 0.049 and 0.026% for whole blood, respectively, and 0.054 and 0.085% for urine, respectively. The calibration curves for ethanol showed excellent linearity in the range of 80–5000 mg L−1 for whole blood and 40–5000 mg L−1 for urine; the detection limits for both samples were 20 and 10 mg L−1, respectively. The data on actual determination of ethanol after the drinking of beer are also presented for two subjects.

Chiral separation of dansyl amino acids by capillary electrophoresis: Comparison of formamide and N -Methylformamide as background electrolytes

Abstract: Chiral separation of 12 dansyl amino acids has been achieved by capillary electrophoresis using β-cyclodextrin dissolved inN-methylformamide or formamide. The viscosity and the dielectric constants of these two solvents are very different, giving them disimilar electrophoretic properties. Intense electroosmotic flow, high electrophoretic mobilities and high efficiency were achieved inN-methylformamide, but high chiral selector concentration was needed for sufficient resolution. In contrast, the separation of dansyl amino acids in formamide was characterized by longer analysis time and lower efficiency, but the resolution and selectivity of the separation were better and baseline separation could be achieved at lower β-cyclodextrin concentrations.

Bioactivity screening by chromatography-bioluminescence coupling

Abstract: Because luminescent microorganisms change their light emission in the presence of bioactive substances, they provide an effective way of monitoring toxicity. Here we report the direct coupling of bioluminescence to chromatography which is capable of detecting single toxic components in complex mixtures. Two approaches were investigated, utilizing thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The TLC-bioluminescence imaging technique was developed into a versatile and rugged method and proved to be superior to the HPLC-based design. Employing genetically engineered luminescent bacteria gives customized selectivity of bioactivity detection. The combination of analytical separation technology with the biospecific sensing ability of living cells provides a novel screening tool for targeting bioactive components in complex samples.

Identification and determination of fumonisin FB1 and FB2 in corn and corn products by high-performance liquid chromatography - electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS-MS)

Abstract: A new method has been developed for qualitative and quantitative analysis of fumonisin FB1 and FB2 in corn and corn products. After extractive sample preparation followed by solid phase extraction using a strong anionexchanger, highest selectivity and sensitivity was achieved by high-performance liquid chromatography-electrospray-ionization, tandem mass spectrometry (HPLC-ESI-MS-MS) analysis combined with selected reaction monitoring (SRM). With deuterated fumonisin FB1-d6 as internal standard the routine quantitation limit for FB1 was set at 400 pg (0.8 ng g−1 sample) with a signal-to-noise ratio of 10∶1. Almost all corn grit and corn meal samples under study were found to be contaminated with fumonisin FB1 and FB2 in concentrations ranging from 1 to 200 ng g−1. Lower fumonisin levels were found in cornflakes, popcorn and baby food, while no fumonisins were detected in canned sweet corn.

Microwave assisted solvent extraction (MASE) for the efficient determination of triazines in soil samples with aged residues

Abstract: The use of microwave-assisted solvent extraction (MASE) as an alternative for conventional solvent extraction procedures for the determination of some triazine herbicides in soil samples has been investigated. In this study MASE method development was focused on the selection of a suitable extraction solvent prior to the instrumental analysis of uncleaned extracts with gas chromatography and nitrogen-specific detection. A mixture of dichloromethane-methanol (90∶10, v/v) yielded recoveries ranging from 89 to 103 (spiked level 200 μg/kg) with RSDs ranging from 2.1 to 5.3%. This solvent mixture is also very convenient for further procedure. The selected MASE procedure was tested by analyzing freshly spiked soil samples and samples with aged residues of atrazine, desethylatrazine, desisopropylatrazine and simazine. The results were compared with those obtained by a conventional liquid extraction method. The comparative study indicated that MASE yields recoveries at least as good as those obtained by the conventional method. Moreover, the MASE procedure provides low solvent consumption in combination with a high sample throughput.

Use of methylamino-β-cyclodextrin in capillary electrophoresis. Resolution of acidic and basic enantiomers

Abstract: β-cyclodextrin derivatives, namely 6A methylamino-β-CD and hepta-methylamino-β-CD have been used as chiral selectors for the enantiomeric separation of a number of acidic and basic compounds by capillary electrophoresis employing either coated or uncoated capillaries. The effects of the CD type and concentration and the pH of the background electrolyte on the mobility and chiral resolution of the analytes have been studied. The use of monomethylamino-β-CD in a coated capillary allowed the enantiomeric resolution of phenyl lactic acid, warfarin and acenocoumarol but was not successful for tiaprofen and its 3-isomer. The heptamethylamino derivative, under the same experimental conditions, was a better chiral selector than the monosubstuted CD toward the arylpropionic acids and phenyl lactic acid while the anticoagulant drugs showed poor or no chiral resolution. Inversion of migration order was obtained for phenyl lactic acid, warfarin and acenocoumarol enantiomers. The enantiomers of basic compounds of pharmaceutical interest, namely propranolol, terbutaline, ketamine, chlorpheriramine and isoproterenol were only resolved using monomethylamino-β-CD dissolved in a phosphate buffer containing tetramethylammonium ions.

Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry

Abstract: An analytical method for the simultaneous determination of the pyrethroid metabolites cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine samples is described. The urine is subjected to acid-induced hydrolysis followed by exhaustive solvent extraction, covering both conjugated and free acids, followed by a common derivatisation step yielding the corresponding methyl esters. Quantitation was by diastereomeric, capillary gas chromatography-mass spectrometry. It appears that 4-fluoro-3-phenoxybenzoic acid is a characteristic urinary marker for cyfluthrin exposure. The limits of determination are 0.5–1.0 μg L−1 urine depending on the metabolites concerned. The applicability of the method was tested on urine samples from pest control operators exposed occupationally to cypermethrin and cyfluthrin.

Cross validation of capillary electrophoresis and high-performance liquid chromatography for cefotaxime and related impurities

Abstract: A micellar electrokinetic chromatography method is presented which permits separation of cefotaxime and its major related impurities. Separation was carried out at 15 kV, using 30 mM sodium dihydrogen phosphate adjusted to pH 7.2 with 5 M NaOH and which contained 165 mM sodium dodecylsulfate as electrolyte. Results obtained by capillary electrophoresis were in good agreement with those of high-performance liquid chromatography with respect to the level of the major known impurities, total impurity content and cefotaxime purity.

Rapid identification of volatile compounds in aromatic plants by automatic thermal desorption — GC-MS

Abstract: Thermal desorption is a valuable method for the fractionation of plant volatile components, which can be carried out on-line with GC analysis. The use of coupled GC-MS affords additional qualitative information, of special interest for plant species whose composition has not been previously studied. Some examples of the application of automatic thermal desorption, coupled to GC-MS to the identification and characterization of volatile components of plants of different families are given.

Detection of cow's milk in ewe's or goat's milk by HPLC

Abstract: High-pressure liquid chromatography (HPLC) was used to detect added cows' milk in goat and ewe's milk. Analysis of the whey proteins enabled detection of mixtures in proportions of less than 1%. The method does not distinguish between goat and ewe's milk.

High performance liquid chromatography —Electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) for the analysis of heterocyclic aromatic amines (HAA)

Abstract: Sensitive and selective detection of the sixteen most abundant heterocyclic aromatic amines (HAA) has been achieved by application of high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) in combination with selected reaction monitoring (SRM). Detection limits between 0.1 and 50 ng mL−1 were established by use of HAA model solutions.

Combined chiral crown ether and β-cyclodextrin for the separation of o-, m-, and p-fluoro-D,L-phenylalanine by capillary gel electrophoresis

Abstract: A combination of (β-CD) with a chiral crown ether, 18-crown-6-tetracarboxylic acid (18C6TA), has been used for the chiral capillary gel electrophoretic (CGE) separation of the six enantiomers and positional isomers in a mixture ofo-, m-, andp-fluoro-D,L-phenylalanine; the isomers were successfully resolved in 0.3% agarose −2×10−2 mol L−1 18C6TA −2×10−2 mol L−1 β-CD. The method has also been successfully extended to the separation of the enantiomers of 13 other amino acid or primary amine substrates. The role of the chiral crown ether was studied. The effects of the concentrations of 18C6TA, β-CD and agarose, and of pH and applied field strength on the migration time and separation factors of the analytes are reported.

Enantiomer separation by capillary electrophoresis using DEAE-dextran and aminoglycosidic antibiotics

Abstract: Diethylaminoethyl (DEAE) dextran hydrochloride and three kinds of aminoglycosidic antibiotics; fradiomycin sulfate, kanamycin sulfate and streptomycin sulfate, were employed as chiral selectors in capillary electrophoresis, enantiomer separation. These selectors are cationic or basic because of amino functionality and therefore used for enantiomer separation of acidic compounds. To avoid adsorption of the basic or cationic selectors on the capillary inner surface, a coated capillary was employed. Among those tested, enantiomers of binaphthyl compounds and synthetic intermediates of diltiazem analogues were separated. Methanol addition was effective for the improvement of peak shape and resolution.