Aqueous Sample Solution Research Articles

Development of High Performance Liquid Chromatography with Electrochemical Detection for Alcohols and Its Application to the Determination of Ethanol in Detergents and Alcohol Beverage

We have developed a determination method for alcohol using high-performance liquid chromatography by using a diamond electrode-electrochemical detector. At this time, 4 alcohols (methanol, ethanol, 2-propanol and 1-butanol) were separated on a Shodex® ODP2 HP-4E column with an acetonitrile-25 mM disodium hydrogen phosphate buffer (pH 11.0, 1 : 99, v/v) as the mobile phase. The electrochemical detector, equipped with a diamond electrode, was set at 2000 mV (applied voltage). The detection limits of the four alcohols were 20-25 ng/mL. The proposed liquid chromatography was fifty-times more sensitive compared to reported gas chromatography – flame ionization method, and was found to be suitable for large-volume aqueous sample solutions compared to gas chromatography. In the case of poor absorbance compounds, an easy-to-set additional detector in wide using the liquid chromatography – ultraviolet detection system. It was meaningful economical work to use an existing apparatus in the laboratory. With this method, one could determine volatile alcohols, e.g., ethanol, which could not be determined by other universal-type detectors (corona-CAD and ELSD). The proposed method was applied to the alcohol in detergents (contains ethanol and 2-propanol) and alcoholic beverages. Good relationships were obtained compared to the enzymatic method.

Dynamic three-phase hollow fiber microextraction based on two immiscible organic solvents with automated movement of the acceptor phase

Dynamic three-phase hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) based on two immiscible organic solvents, with automated movement of organic acceptor phase to facilitate mass transfer was introduced for the first time. Polycyclic aromatic hydrocarbons were used as model compounds and extracted from water and soil samples. The extraction involved filling an 8 cm length of hollow fiber with 25 μL of organic acceptor solvent using a microsyringe, followed by impregnation of the pores in the fiber wall with n-dodecane. The fiber was then immersed in 20 mL of aqueous sample solution. During extraction, the organic acceptor phase was repeatedly moved in the lumen of the hollow fiber by movement of the syringe plunger controlled by programmable syringe pump. Following this microextraction, 2 μL of organic acceptor phase was injected into gas chromatography-flame ionization detector. This new technique provided up to 554-fold preconcentration of the analytes under the optimized conditions. Good repeatabilities (with RSDs ≤8.4%) were obtained. Detection limits were in the range of 0.2-0.5 μg/L. The utilization of the proposed method for extraction of the polycyclic aromatic hydrocarbons from different real samples (such as water and soil samples) also gave good precision and recovery.

Determination of chlorophenols in environmental water samples using directly suspended droplet liquid-liquid-liquid phase microextraction prior to high-performance liquid chromatography

A new sample preparation method named directly suspended droplet liquid-liquid-liquid phase microextraction was used in this research for determination of three chlorophenols in environmental water samples. The analytes (2-chlorophenol, 3-chlorophenol and 4-chlorophenol) were extracted from 4.5 mL acidic donor phase, (pH 2, P1) into an organic phase, 350 µL of benzene/1-octanol (90 : 10 v/v, P2) and then were back-extracted into a 7 µL droplet of an basic (pH 13) aqueous solution (acceptor phase, P3). In this method, contrary to the ordinary single drop liquid-phase microextraction technique, an aqueous large droplet is freely suspended on the surface of the organic solvent, without using a microsyringe as supporting device. This aqueous microdroplet is delivered at the top-centre position of an immiscible organic solvent which is laid over the aqueous donor sample solution while the solution is being agitated. Then, the acceptor phase containing chlorophenols was withdrawn back into a HPLC microsyringe and neutralised by adding of 7 µL HCl 0.1 M. The total amount was eventually injected into the HPLC system with UV detection at 225 nm for further analysis. Parameters such as the organic solvent, phases volumes, extraction and back-extraction times, stirring rate and pH values were optimised. The calibration graphs are linear in the range of 10–2000 µg L−1 with r ≥ 0.9973. The enrichment factors were ranged from 115 to 170, and the limit of detection (LOD, n = 7) varied from 5 to 10 µg L−1. The relative standard deviations (RSDs, n = 5) were found 6.8 to 7.4 at S/N = 3. All experiments were carried out at room temperature, (22 ± 0.5°C).

Determination of nicotine, anabasine, and cotinine in urine and saliva samples using single-drop microextraction

A simple, sensitive, and inexpensive singe-drop microextraction (SDME) followed by gas chromatography and flame-ionization detection (GC-FID) was developed for determination of nicotine, anabasine, and cotinine in human urine and saliva samples. The target compounds were extracted from alkaline aqueous sample solution into an organic acceptor drop suspended on the tip of a 25-μL GC microsyringe in the aqueous sample solution. This microsyringe was also used for direct injection after extraction. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.5–25.0, 0.5–65.0, and 0.5–45.0 mg L −1 for nicotine, anabasines and cotinine, respectively. The method detection limit values were in the range of 0.33–0.45 mg L −1. Intra-day and inter-day precisions for peak area ratios were in the range of 1.3–9.2% and 2.0–7.0%, respectively. The proposed procedure was successfully applied to the determination of analytes in spiked urine and saliva samples with satisfactory results. The mean relative recoveries of spiked water samples ranged over 71.2–111.0%, with relative standard deviations varying from 2.3% to 10.0%.

Ionic Liquid-Based Submerged Single Drop Microextraction: a New Method for the Determination of Aromatic Amines in Environmental Water Samples

A novel microextraction method termed ionic liquid-based submerged single drop microextraction combining liquid chromatography (LC) was developed for the determination of aromatic amines in environmental water samples. This method is simple and rapid for the determination of three aromatic amines (2-nitroaniline, 3-chloroaniline and 4-bromoaniline) which were selected as model compounds for validation of this new mode of microextraction. This technique combines extraction and preconcentration of the analytes in one step before determination by LC-UV, and the ionic liquid was used instead of the organic solvent as extractant. Several important parameters influencing the extraction efficiency such as the volume of the aqueous sample solution (donor phase), extraction time, pH of the donor phase and salt effect were investigated. Under the optimized conditions, acceptable enrichment factors (132–186) and relative recoveries (82–98%) were obtained for the extraction of the target analytes in real water samples. The calibration curves were linear with correlation coefficients ranging from 0.9983 to 0.9989 and RSDs (n = 5) of 4.9–6.8%. The LODs for the three aromatic amines were 1–2.5 μg L−1 at a signal-to-noise ratio (S/N) of 3.

Functional Fluorinated Modifications on a Polyelectrolyte Coated Polydimethylsiloxane Substrate for Fabricating Antibody Microarrays

Fluorinated compounds exhibit hydrophobic, nonstick, and self-cleaning properties, making them attractive for use as the coating material for biochips. In this study, we copolymerized the fluorinated compound 1H,1H,2H-perfluoro-1-decene (FD) with acrylic acid (AA) and bonded the resulting copolymer with protein G on the surface of polyelectrolyte coated polydimethylsiloxane (PDMS) to form a functional surface that captures antibodies. We demonstrated that the modified PDMS surface remained hydrophobic while becoming resistant to nonspecific protein binding. Thus, aqueous sample solutions formed the droplets (4 μL) on the surface without spreading and drying during the sample printing. Contact angle measurements showed that this functionalized surface was as hydrophobic as the native PDMS with a virtually constant contact angle over seven days of the study under dried condition at 4 °C. Spectroscopic measurements revealed that FD/AA copolymerization formed a homogeneous and highly dense multilayer (50 mg/mm(2)) with a fluorine coverage of 35.4%. Moreover, protein G was shown to covalently bind to AA molecules on the surface at a binding density of 0.24 μg/mm(2). We demonstrated that the fluorinated coating withstood nonspecific binding with extremely low background emission, leading to bioassays that, without the need of blocking agents, exhibited six times more sensitivity than PEG coatings. The fluorinated PDMS antibody microarrays were further applied to accurately determine the absolute concentration of ERα in MCF-7 cells. In conclusion, the unique properties of fluorinated compounds, such as withstanding wetting, nonspecific binding and contamination, make them an excellent coating material for use in sensitive and simple on-chip assays.

Liquid-phase microextraction with solidification of the organic floating drop for the preconcentration and determination of mercury traces by electrothermal atomic absorption spectrometry

A procedure for the determination of traces of mercury by liquid-phase microextraction based on solidification of a floating organic droplet for separation and electrothermal atomic absorption spectrometry for final measurement has been developed. For this purpose, 50 microL of pre-heated (50 degrees C) undecanoic acid (UA), are added to 25 mL of aqueous sample solution at pH 5. The mixture, maintained at 50 degrees C, is stirred for 10 min using a high stirring rate in order to fragment the UA drop into droplets, thus favoring the extraction process. Next, the vial is immersed in an ice bath, which results in the solidification of the UA drop that is easily separated. Injection into the atomizer is carried out after gentle heating. The pyrolytic atomizers are coated with electrolytically reduced palladium that acts as an effective chemical modifier for more than 500 firings. Under the optimized conditions, the detection limit was 70 ng L(-1) mercury with an enrichment factor of 430. The relative standard deviation of the measurements was in the 2.1-3.5% range. Recovery studies applied to the determination of mercuric ions in bottled and tap water samples were in the 92-104% range.

An oxo-bacteriochlorin derivative for long-wavelength fluorescence ratiometric alcohol sensing

A fluorescent ratiometric optical chemical sensor (optode) for alcohol detection is presented. A lipophilized bacteriochlorin derivative possessing a trifluoroacetyl group at the 3-position was synthesized for alcohol detection. When embedded in a plasticized poly(vinyl chloride) (PVC) membrane, the fluororeceptor shows a significant signal change on exposure to aqueous ethanol solutions both in the absorbance and the fluorescence emission. This optode allows the determination of ethanol concentrations in aqueous sample solutions by ratiometric fluorometry. The fluorescent indicator responds with increasing fluorescence around 701 nm and decreasing fluorescence around 751 nm towards the presence of ethanol. The optode response was found to be fully reversible in a dynamic measurement range from 0.3% to at least 25% (v/v) ethanol. The limit of detection (LOD) and the limit of quantification (LOQ) of the sensor for ethanol were estimated to be 0.1% and 0.4% (v/v), respectively. These results indicate that the new optode can be employed as an ethanol-sensing device with high sensitivity for beverage and industrial analyses.

Microextraction based on solidification of a floating organic drop followed by electrothermal atomic absorption spectrometry for the determination of ultratraces of lead and cadmium in waters

A procedure for the determination of ultratraces of lead and cadmium by liquid-phase microextraction based on solidification of a floating organic droplet (LPME-SFO) separation and electrothermal atomic absorption spectrometry (ETAAS) has been developed. For this purpose, 50 μL of pre-heated (50 °C) undecanoic acid (UA) are added to 25 mL of aqueous sample solution at pH 5. The mixture, maintained at 50 °C, is stirred at 1000 rpm for 10 min and then the vial is immersed in an ice-bath, which results in the solidification of the UA drop that is easily separated. Injection into the atomizer is carried out after a gentle heating. By impregnating the atomizer with tungstate as a permanent chemical modifier, the detection limits were 10 and 0.5 ng L−1 with enrichment factors of 380 and 420, for lead and cadmium, respectively. The relative standard deviation was in the 2.8–3.2% range (n = 5, 25–400 ng L−1 Pb(II) and 1–15 ng L−1 Cd(II)). The proposed method has been applied to the determination of lead and cadmium in bottled, tap and sea water samples, the reliability of the results being verified by means of recovery tests and by using ICP-MS.

Determination of hippuric acid in biological fluids using single drop liquid–liquid-liquid microextraction

A simple, sensitive, and inexpensive single drop liquid–liquid-liquid microextraction (LLLME) followed by isocratic reverse phase high performance liquid chromatography (RP-HPLC) and UV detection was developed for determination of hippuric acid in human urine and serum samples. The analyte was extracted from an acidic aqueous sample solution (pH 3) through a thin layer of organic solvent membrane and back-extracted to a basic acceptor drop (pH 11) suspended on the tip of a 10-μL HPLC syringe in the organic layer. The influence of several important parameters on extraction efficiency of hippuric acid was evaluated. Under optimized experimental conditions, the calibration graph was linear in the concentration range of 1–400 μg L−1 with coefficient of determination 0.998. The limits of detection and quantification were 0.3 and 1.0 μg L−1, respectively. Intra-day and inter-day precision were in the range of 1.1–2.7% and 1.1–3.1%, respectively. This procedure was successfully applied to the determination of hippuric acid in spiked urine and serum samples with satisfactory results. The relative recoveries of urine and serum samples ranged from 91.4 to 99.3%, with relative standard deviations varying from 1.6 to 4.2%.

Separation and preconcentration system based on ultrasonic probe-assisted ionic liquid dispersive liquid–liquid microextraction for determination trace amount of chromium(VI) by electrothermal atomic absorption spectrometry

A novel method of ultrasonic probe-assisted ionic liquid dispersive liquid-liquid microextraction combined with electrothermal atomic absorption spectrometry (ETAAS) was developed for the determination of chromium(VI) species in water samples. In this procedure, the hydrophobic chelate of chromium(VI) with ammonium pyrrolidinedithiocarbamate (APDC) was extracted into the fine droplets of 1-hexyl-3-methylimidazolium hexafluorophosphate ([Hmim][PF(6)]), which was dispersed into the aqueous sample solution by ultrasonication using an ultrasonic probe. Several variables such as the volume of [Hmim][PF(6)], sample pH, concentration of APDC, and extraction time were investigated in detail. Under the optimum conditions, the limit of detection of the proposed method was 0.07 ng mL(-1) for Cr(VI) and the relative standard deviation for five-replicated determination of 2.0 ng mL(-1) Cr(VI) was 9.2%. The proposed method has been also successfully applied to the determination of chromium(VI) species in lake and tap water samples.

Fast analysis of multiple pesticide residues in apple juice using dispersive liquid–liquid microextraction and multidimensional gas chromatography–mass spectrometry

A method for the rapid trace analysis of 24 residual pesticides in apple juice by multidimensional gas chromatography–mass spectrometry (MD-GC/MS) using dispersive liquid–liquid microextraction (DLLME) was developed and optimized. Several parameters of the extraction procedure such as type and volume of extraction solvent, type and volume of dispersive solvent and salt addition were evaluated to achieve the highest yield and to attain the lowest detection limits. The DLLME procedure optimized consists in the formation of a cloudy solution promoted by the fast addition to the sample (5 ml) of a mixture of carbon tetrachloride (extraction solvent, 100 μl) and acetone (dispersive solvent, 400 μl). The tiny droplets formed and dispersed among the aqueous sample solution are further joined and sedimented (85 μl) in the bottom of the conical test tube by centrifugation. Once extracted, all the 24 pesticides were directly injected and separated by a dual GC column system, comprising a short wide-bore DB-5 capillary column with low film thickness connected by a Deans switch system to a second chromatographic narrower column, with identical stationary phase. The instrumental setting used, in combination with carefully optimized operational fast GC and MS parameters, markedly decreased the retention times of the targeted analytes. The total chromatographic run was 8 min. Mean recoveries for apple juice spiked at three concentrations ranged from 60% to 105% and the intra-repeatability ranged from 1% to 21%. The limits of detection of the 24 pesticides ranged from 0.06 to 2.20 μg/L. In 2 of a total of 28 analysed samples were found residues of captan, although at levels below the maximum limit legal established.

Commercial polymeric fiber as sorbent for solid-phase microextraction combined with high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbons in water

A novel microextraction method making use of commercial polymer fiber as sorbent, coupled with high-performance liquid chromatography-fluorescence detection for the determination of polycyclic aromatic hydrocarbons (PAHs) in water has been developed. In this technique, the extraction device was simply a length (8 cm) of a strand of commercial polymer fiber, Kevlar (each strand consisted of 1000 filaments, each of diameter ca. 9.23 μm), that was allowed to tumble freely in the aqueous sample solution during extraction. The extracted analytes were desorbed ultrasonically before the extract was injected into HPLC system for analysis. Extraction parameters such as extraction time, desorption time, type of desorption solvent and sample volume were optimized. Each fiber could be used for up to 50 extractions and the method showed good precision, reproducibility and linear response within a concentration range 0.05–5.00 μg L −1 with correlation coefficients of up to 0.9998. Limits of detection between 0.4 and 4.4 ng L −1 for seven PAHs could be achieved. The relative standard deviations ( n = 3) of this technique were between 2.9% and 12.1%.

Determination of traces of lead and cadmium using dispersive liquid-liquid microextraction followed by electrothermal atomic absorption spectrometry

A procedure is presented for the determination of very low concentrations of lead and cadmium in water samples. Dispersive liquid-liquid microextraction with ammonium pyrrolidine dithiocarbamate is applied. The aqueous sample solution (adjusted to pH 6) is based on the rapidly mixed with of a small volume of a mixture of carbon tetrachloride and methanol. The two phases are separated by centrifugation, and the metals determined in the organic layer using electrothermal atomic absorption spectrometry. An effective chemical modification during the heating cycle is achieved by impregnating the graphite atomizer with a sodium tungstate solution. This allows well defined absorbance time profiles and low background values to be obtained during the atomization stage. The detection limits for lead and cadmium are 10 and 4 ng L−1, respectively. The procedure is applied to the determination of both analytes in bottled water, tap waters, and seawaters.

Graphite furnace atomic absorption spectrometric determination of cadmium after solid-liquid extraction with dithizone

A method for the determination of trace amount of cadmium ion after preconcentration by extracting its dithizone complex into molten naphthalene was developed. Several experimental conditions such as pH of the solution, stirring time, the amounts of naphthalene, standing time and volume of the solution were optimized. Trace amount of cadmium ion in aqueous solution of sample was chelated with 5 mL of 0.001 M dithizone at pH 8. After addition of 0.15 g naphthalene, the solution was heated to about 85 oC and stirred (800 rpm) for 2 min to reproduce the microcrystalline naphthalene. Cadmium ion was determined by a graphite furnace atomic absorption spectrometer. The interfering effects of diverse concomitant ions (cations and anions) were investigated. Artificial sea water and a standard reference material (SRM) were analyzed by this method. The sensitivity and detection limit of 1.2 ngL -1 and 1.5 ng L -1 were found, respectively.

Preconcentration of environmental waters by agar for XRF analysis

We have developed a convenient and effective XRF analysis procedure for trace amount of K, Ca, V, Mn, Fe, Ni, Cu, Zn, Cd, and Pb in environmental waters by using a preconcentration using the natural polymer (agar). The thin agar film was prepared by drying a homogeneous agar gel after mixing the aqueous sample solution with the agar powder. XRF analysis of the preconcentrated thin agar films containing trace metals showed a good repeatability because agar films were homogeneous enough. SB (signal to noise) ratios of the XRF intensity of the analytes were improved drastically. The linear calibration curves of K, Ca, V, Mn, Fe, Ni, Cu, Zn, Cd, and Pb showed a good linearity within the calibration ranges. The lower limits of detection (LLD) were 1.4 μg/mL for K, 0.26 μg/mL for Ca, 0.088 μg/mL for V, 0.029 μg/mL for Mn, 0.11 μg/mL for Fe, 0.016 μg/mL for Ni, 0.030 μg/mL for Cu, 0.017 μg/mL for Zn, 0.20 μg/mL for Cd, and 0.066 μg/mL for Pb, respectively. The proposed preconcentration method was applied to several environmental water samples.

Determination of 3-nitroaniline in water samples by directly suspended droplet three-phase liquid-phase microextraction using 18-crown-6 ether and high-performance liquid chromatography

Liquid–liquid–liquid microextraction (LLLME) with directly suspended droplet in high-performance liquid chromatography (HPLC) has been applied as a new, rapid and easy method for the determination of 3-nitroaniline in environmental water samples. The target compound was extracted from the aqueous sample solution (donor phase, pH 13) into an organic phase and then was back-extracted into a directly suspended droplet of an acidic aqueous solution (acceptor phase, pH 0.3). In this method, without using a microsyringe as supporting device, an aqueous large droplet is freely suspended at the top-center position of an immiscible organic solvent, which is laid over the aqueous sample solution while being agitated. Then, the droplet was withdrawn into the microsyringe and directly was injected into the HPLC system with UV detection at 227 nm. Up to 148-fold enrichment of the analyte could be obtained under the optimal conditions [i.e. donor phase: 0.1 M sodium hydroxide solution (4.5 mL); organic phase: o-xylene/1-octanol (90:10, v/v; 250 μL); acceptor phase: 0.5 M hydrochloric acid and 500 mM 18-crown-6 ether (6 μL); extraction time: 60 s; back-extraction time: 6 min and stirring rate: 600 rpm]. The limit of detection was 1 μg/L ( n = 7) and the relative standard deviation (RSD, n = 5) was 4.9 at S/N = 3. The calibration graph was linear in the range of 5–1500 μg/L with r = 0.9983. All experiments were carried out at room temperature (22 ± 0.5 °C).

A new high-speed hollow fiber based liquid phase microextraction method using volatile organic solvent for determination of aromatic amines in environmental water samples prior to high-performance liquid chromatography

A new and fast hollow fiber based liquid phase microextraction (HF-LPME) method using volatile organic solvents coupled with high-performance liquid chromatography (HPLC) was developed for determination of aromatic amines in the environmental water samples. Analytes including 3-nitroaniline, 3-chloroaniline and 4-bromoaniline were extracted from 6 mL basic aqueous sample solution (donor phase, NaOH 1 mol L −1) into the thin film of organic solvent that surrounded and impregnated the pores of the polypropylene hollow fiber wall (toluene, 20 μL), then back-extracted into the 6 μL acidified aqueous solution (acceptor phase, HCl 0.5 mol L −1) in the lumen of the two-end sealed hollow fiber. After the extraction, 5 μL of the acceptor phase was withdrawn into the syringe and injected directly into the HPLC system for the analysis. The parameters influencing the extraction efficiency including the kind of organic solvent and its volume, composition of donor and acceptor phases and the volume ratio between them, extraction time, stirring rate, salt addition and the effect of the analyte complexation with 18-crown-6 ether were investigated and optimized. Under the optimal conditions (donor phase: 6 mL of 1 mol L −1 NaOH with 10% NaCl; organic phase: 20 μL of toluene; acceptor phase: 6 μL of 0.5 mol L −1 HCl and 600 m mol L −1 18-crown-6 ether; pre-extraction and back-extraction times: 75 s and 10 min, respectively; stirring rate: 800 rpm), the obtained EFs were between 259 and 674, dynamic linear ranges were 0.1–1000 μg L −1 ( R > 0.9991), and also the limits of detection were in the range of 0.01–0.1 μg L −1. The proposed procedure worked very well for real environmental water samples with microgram per liter level of the analytes, and good relative recoveries (91–102%) were obtained for the spiked sample solutions.

Is a “Cloud-Point Curve” in Aqueous Poly(N-isopropylacrylamide) Solution Binodal?

Transmittance of light passing through an aqueous solution of a linear poly(N-isopropylacrylamide) (PNIPA) sample synthesized by living anionic polymerization was examined in detail in the vicinity of its cloud point. It is found that the transmittance approaches a constant value between 0 and 100% even at a temperature slightly higher than the cloud point, indicating that macroscopic phase separation does not take place at the cloud point in the solution and therefore the cloud-point curve dose not correspond to the binodal. Static and dynamic light scattering measurements were then carried out for aqueous solutions of the linear sample and also of another one synthesized by radical polymerization, which has random-branched structure, at some temperatures considerably lower than the cloud point. It was found that both the PNIPA samples in aqueous solutions form aggregates even at such temperatures, and the number, size, and density profile of the aggregates depend on the kind of chain end group and also on the primary structure.

Sensing method based on impedance variation of minicolumn packed with cation-exchanger under electric field

Voltage-induced impedance variation of the minicolumn (i.d. 0.53 mm, length 2 mm) packed with cation exchanger was investigated to develop a sensing method. An aqueous sample solution containing the metal cations was continuously supplied to the minicolumn during the impedance measurement with the simultaneous application of both alternating current voltage (amplitude, 1.0 V; frequency, 200 kHz to 6 Hz) and direct current (DC) offset voltage (0.1 to 1.0 V). On a complex plane plot, the profile of the column impedance consisted of a semicircle (200 kHz to 100 Hz) and a straight line (<100 Hz), of which slope varied with the magnitude of the applied DC offset voltage (V(DC)). The slope-V(DC) relation depended on the kind of the metal cation and its concentration; in particular, the slope-V(DC) relations of monovalent cations (Na(+) and K(+)) and divalent ones (Mg(2+) and Ca(2+)) were significantly different. With the change in the concentration of minor divalent salt of MgCl(2) or CaCl(2) (60 to 140 microM) in the sample solution containing 10 mM NaCl, the slopes showed almost linear relationships between those with application of V(DC) = 0.1 V and 1.0 V both for magnesium and calcium additions. In the case of plural addition of both MgCl(2) and CaCl(2) to the solution, the data points in the slope(0.1 V)-slope(1.0 V) plot were located between the two proportional lines for single additions of magnesium and calcium, reflecting both the mixing ratio and net concentrations of the divalent cations. Thus, simulations determination of Mg(2+) and Ca(2+) can be attained on the basis of the slope(0.1 V)-slope(1.0 V) relation obtained by the impedance measurements of the minicolumn. Actually, the contents of both magnesium and calcium cations in the bottled mineral waters determined simultaneously using the proposed method were almost equivalent to those obtained by the atomic absorption spectrometric measurement.