Evaluation of an on-line coupled continuous flow liquid membrane extraction and precolumn system as trace enrichment technique by liquid chromatographic determination of bisphenol A

An on-line coupled continuous flow liquid membrane extraction (CFLME) and C₁₈ precolumn system was developed for sample preconcentration in liquid chromatography determination. After preconcentration by CFLME, which is based on the combination of continuous flow liquid–liquid extraction and supported liquid membrane, bisphenol A (BPA) was enriched in 960 μl of 1 mol l⁻¹ NaOH used as acceptor. This acceptor was on-line neutralized and transported onto the C₁₈ precolumn where analytes were absorbed and focused. Then the focused analytes were injected onto a C₁₈ analytical column for separation and detected at 220 nm with a diode array detector. CFLME related parameters such as flow rates, pH of donor and acceptor, and enrichment time were optimized. The proposed method presents a detection limit of 0.03 μg l⁻¹ (S/N=3) when 60 ml samples was enriched with an enrichment time of 30 min. Compared with C₁₈ based column-switching procedure, this proposed procedure presents similar sample throughput and lower detection limits. The proposed method was successfully applied to determine BPA in tap water, river water, and municipal sewage effluent samples.     

On-line focused microwave digestion-hydride generation of inorganic and organic selenium: Total determination and inorganic selenium speciation by atomic absorption spectrometry

An on-line FIA pretreatment with HBr/KBrO₃, assisted by on-line focused microwave-induced digestion, has been coupled with hydride generation-atomic absorption spectrometry (HG-AAS) for final detection for total selenium determination. This total selenium determination is virtually independent of the different Se species investigated (selenite, selenate, selenomethionine, selenoethionine and selenocystine). Detection limits of 0.8 μg l⁻¹ of Se can be achieved by AAS with precisions better than 5%. This continuous flow system for selenium determination allows a high sample throughput (about 30 samples h⁻¹ can be analyzed) in which high automation can be achieved and constitutes a convenient real-time continuous detector for the different selenocompounds tested. Direct non-chromatographic speciation of inorganic selenium (selenite and selenate in their mixtures) is demonstrated by simple on-off operation of the focused microwaves connected in the flow system.

Validation of this simple on-line FIA system has been carried out by analyzing total Se recovered from spiked tap waters and by speciating mixtures of Se(IV) and Se(VI) spiked to the same samples. The fast conversion of Se compounds into volatile selenium could be considered as a sort of specific “general” detector for Se compounds which can be extremely useful for Se speciation by hybrid chromatographic techniques.     

An on-line flow-injection microwave-assisted mineralization and a precipitation/dissolution system for the determination of molybdenum in blood serum and whole blood by electrothermal atomic absorption spectrometry

An on-line flow injection (FI) precipitation–dissolution system with microwave-assisted sample digestion has been developed for the electrothermal atomic absorption spectrometry (ETAAS) determination of trace or ultratrace amounts of molybdenum in human blood serum and whole blood samples. After the exposure of the sample to microwave radiation, the on-line precipitation of molybdenum was achieved by the merging-zone of a 0.5-ml plug of sample with a plug of potassium ferrocyanide, which were carried downstream with a solution of 0.5 mol l⁻¹ of HNO₃. The interfering effects of iron and copper were minimized by the introduction of a flow of a 5% (w/v) sodium potassium tartrate (for iron) and 2% (w/v) of thiourea (for copper and zinc) in a 5% (v/v) ammonia and 2% (v/v) ammonium chloride solution previous to the precipitation reaction. The reddish-brown precipitate of molybdenyl ferrocyanide was collected on the walls of a knotted reactor. The precipitate was dissolved with the introduction of 1 ml of a 3.0 mol l⁻¹ NaOH solution and the best performance in terms of detection limit and precision was achieved when a sub-sample of 140 μl was collected in a capillary of a sampling arm assembly, to introduce 20 μl volumes into the atomizer by means of positive displacement with air through a time-based injector. A detection limit (3σ) of 0.1 μg Mo l⁻¹ using an aqueous standard solution was obtained. The method is quantitative and is applied over the range 0.2–20.0 μg Mo l⁻¹. The precision of the method evaluated by ten replicate analyses of aqueous standard solutions containing 0.5 and 1.0 μg Mo l⁻¹ was 2.8 and 3.1% (relative standard deviation, RSD) (for n=5), respectively. Whereas, the precision evaluated by five replicate analysis of a serum and a whole blood sample were 3.3 and 3.8% RSD. An enrichment factor of ca. 3.5 was achieved with the introduction of 0.5 ml aqueous standard solutions at a sample flow rate of 1.0 ml min⁻¹. Recoveries of spiked molybdenum in blood serum and whole blood were in the ranges 96–102 and 94–98%, respectively. The results obtained for two human whole blood certified reference materials were in good agreement with the indicative values.     

Investigation of on-line coupling electrothermal atomic absorption spectrometry with flow injection sorption preconcentration using a knotted reactor for totally automatic determination of lead in water samples

A flow injection on-line sorption preconcentration electrothermal atomic absorption spectrometric system for fully automatic determination of lead in water was investigated. The discrete non-flow-through nature of ETAAS, the limited capacity of the graphite tube and the relatively large volume of the knotted reactor (KR) are obstacles to overcome for the on-line coupling of the KR sorption preconcentration system with ETAAS. A new FI manifold has been developed with the aim of reducing the eluate volume and minimizing dispersion. The lead diethyldithiocarbamate complex was adsorbed on the inner walls of a knotted reactor made of PTFE tubing (100 cm long, 0.5 mm i.d.). After that, an air flow was introduced to remove the residual solution from the KR and the eluate delivery tube, then the adsorbed analyte chelate was quantitatively eluted into a delivery tube with 50 μl of ethanol. An air flow was used to propel the eluent from the eluent loop through the reactor and to introduce all the ethanolic eluate onto the platform of the transversely heated graphite tube atomizer, which was preheated to 80°C. With the use of the new FI manifold, the consumption of eluent was greatly reduced and dispersion was minimized. The adsorption efficiency was 58%, and the enhancement factor was 142 in the concentration range 0.01–0.05 μg l⁻¹ Pb at a sample loading rate of 6.8 ml min⁻¹ with 60 s preconcentration time. For the range 0.1–2.0 μg l⁻¹ of Pb a loading rate of 3.0 ml min⁻¹ and 30 s preconcentration time were chosen, resulting in an adsorption efficiency of 42% and an enhancement factor of 21, respectively. A detection limit (3σ) of 2.2 ng l⁻¹ of lead was obtained using a sample loading rate of 6.8 ml min⁻¹ and 60 s preconcentration. The relative standard deviation of the entire procedure was 4.9% at the 0.01 μg l⁻¹ Pb level with a loading rate of 6.8 ml min⁻¹ and 60 s preconcentration, and 2.9% at the 0.5 μg l⁻¹ Pb level with a 3.0 ml min⁻¹ loading rate and 30 s preconcentration. Efficient washing of the matrix from the reactor was critical, requiring the use of the standard addition method for seawater samples. The analytical results obtained for seawater and river water standard reference materials were in good agreement with the certified values.     

On-line dilution and determination of the amount of concentrated hydrochloric acid in the final products from a hydrochloric acid production plant using a sequential injection titration system

An on-line sequential injection titration system for the determination of the concentration of concentrated hydrochloric acid as final product from a hydrochloric acid production plant is described. The system involves on-line dilution of the concentrated hydrochloric acid solution to an acceptable range for direct measurement by merging the sample stream with a de-ionized water diluent stream, followed by mixing in a dilution coil, before aspiration into the sequential injection system. Concentrated standard solutions were treated in exactly the same way as the samples. The system was evaluated for reproducibility, linearity, accuracy, and sample throughput. A linear relationship between peak width and logarithm of acid concentration was found in the range 5.934–8.995 mol l⁻¹ and a concentration of 0.005 mol l⁻¹ NaOH solution was used as titrant. Samples from the production plant showed excellent agreement when compared with the manual and automated batchwise titrations. The relative standard deviation was found to be less than 0.4% with a sample frequency of 30 samples per hour.     

On-line sample treatment and FT-IR determination of doxylamine succinate in pharmaceuticals

A low solvent consumption method for Fourier transform infrared spectroscopy (FT-IR) determination of doxylamine succinate in pharmaceuticals has been developed. The analyte was continuous and selectively extracted with a 13% (v/v) ethanol:chloroform solvent mixture, recirculating the solvent through the sample and monitoring the process by FT-IR. Doxylamine succinate was determined by on-line standard addition measuring the peak area in the regions 1730–1710 and 1485–1462 cm⁻¹ corrected with a two-point baseline established between 2000 and 1800 cm⁻¹. This new method implies low volumes of chloroformic solvent mixture, only 2.6 mL per sample, in front of classical batch FT-IR methods, improving analytical efficiency and reducing waste generation. The on-line extraction and standard addition determination of doxylamine succinate allowed a throughput of 10 h⁻¹.     

Design of a composite amperometric enzyme electrode for the control of the benzoic acid content in food

A graphite–Teflon–tyrosinase composite biosensor for the determination of benzoic acid in foodstuffs is reported. The biosensor functioning is based on the inhibition effect of benzoic acid on the biocatalytic activity of the enzyme in a reversed micelle working medium formed with ethyl acetate as the continuous phase, a 0.05 mol l⁻¹ phosphate buffer solution of pH 7.4 (5%) as the aqueous dispersed phase, and 0.10 mol l⁻¹ dioctyl sulfosuccinate (AOT) as the emulsifying agent. A potential value of −0.10 V, and a constant enzyme-substrate (phenol) concentration of 2.0×10⁻⁴ mol l⁻¹ were selected to carry out the amperometric inhibition measurements. The tyrosinase inhibition process by benzoic acid is reversible and of the competitive type, with an apparent inhibition constant of 0.016 mmol l⁻¹. The composite bioelectrodes allow the regeneration of the electrode surface by polishing and exhibit long-term operation and stability. A limit of detection of 9.0×10⁻⁷ mol l⁻¹ benzoic acid was obtained. An interference study from other substances which can be found in foodstuffs together with benzoic acid was performed. Taking advantage of the capabilities of reversed micelles as universal solubilization media, the composite tyrosinase electrode was used for the determination of benzoic acid in two different kind of samples: mayonnaise sauce, which is a highly hydrophobic matrix, and Cola soft drinks, a hydrophilic matrix for which practically no sample treatment is necessary.     

Flow injection for the determination of Se(IV) and Se(VI) by hydride generation atomic absorption spectrometry with microwave oven on-line prereduction of Se(VI) to Se(IV)

An on-line flow injection system has been developed for the selective determination of Se(IV) and Se(VI) in citric fruit juices and geothermal waters by hydride generation atomic absorption spectrometry with microwave-aided heating prereduction of Se(VI) to Se(IV). The samples and the prereductant solutions (4 mol l⁻¹ HCl for Se(IV) and 12 mol l⁻¹ HCl for Se(VI)) which circulated in a closed-flow circuit were injected by means of a time-based injector. This mixture was displaced by a carrier solution of 1% v/v of hydrochloric acid through a PTFE coil located inside the focused microwave oven and mixed downstream with a borohydride solution to generate the hydride. The linear ranges were 0–120 and 0–100 μg l⁻¹ of Se(IV) and Se(VI), respectively. The detection limits were 1.0 μg l⁻¹ for Se(IV) and 1.5 μg l⁻¹ for Se(VI). The precision (about 2.0–2.5% RSD) and recoveries (96–98% for Se(IV) and 94–98% for Se(VI)) were good. Total selenium values were also obtained by electrothermal atomic absorption spectrometry which agreed with the content of both selenium species. The sample throughput was about 50 measurements per hour. The main advantage of the method is that the selective determination of Se(IV) and Se(VI) in citric fruit juices and geothermal waters is performed in a closed system with a minimum sample manipulation, exposure to the environment, minimum sample waste and operator attention.     

Solvent impregnated hollow fibre for a selective preconcentration of Pb(II) in an on-line determination by flame atomic absorption spectrometry

A solvent impregnated hollow fibre (SIHF) module has been developed for the preconcentration of lead by using bis(2-ethylhexyl) phosphoric acid (DEHPA) dissolved in kerosene as extractant. The module has been designed for an on-line determination of trace amounts of lead(II) at mg l⁻¹ (ppm) level by flame atomic absorption spectrometry (FAAS).

The SIHF system is based on the metal liquid–liquid distribution between aqueous solutions of different acidity and the mentioned organic solution. The highest enrichment factor of Pb(II) was determined at pH=4.0 using a formic acid/formiate buffer solution.

Preconcentration experiments were carried out at low lead(II) concentration (mg l⁻¹ level) by using the SIHF module. This study includes the influence of hydrodynamic and chemical conditions on the loading and elution of Pb(II) on the SIHF, i.e., flow rate through the fibres, acidity of the eluent (as nitric acid concentration) and the chemical nature of the acid used in the elution. Breakthrough curves were determined for different sampling flow rates, 0.54 ml min⁻¹ was selected to minimise the loading volume of Pb(II) sample. 0.1 M nitric acid was chosen as eluent solution, and perchloric acid also shows appropriate elution characteristics. The degree of concentration obtained for Pb(II) are of 10 fold the original concentration. The quantification limit for Pb(II) achieved with this preconcentration system is 0.17 mg l⁻¹.

The results obtained indicate that the SIHF system can be applied for on-line determination of trace amounts of lead(II) by FAAS.     

Development of membrane inlet mass spectrometry for examination of fermentation processes

Membrane inlet mass spectrometry (MIMS) is useful for on-line monitoring of fermentation processes. However, readings are affected by the complex and dynamic matrix in which biological processes occur, making MIMS calibration a challenge. In this work, two calibration strategies were evaluated for measurement of typical products of acidogenic fermentation, i.e., ethanol, H₂, and CO₂ in the liquid phase, and H₂ and CO₂ in the gas phase: (1) “standard calibration”, which was performed independent of fermentation experiments with sterile standards in water with a N₂ headspace, and (2) “in-process calibration” whereby fermentation was monitored concurrent with off-line analysis. Fermentation was operated in batch and continuous modes. In-process calibration was shown to be most effective for measurements of H₂ and CO₂ in both gas and liquid phases; standard calibration gave erroneous results. In the gas phase, this was due to a lower sensitivity during experiments compared to the independent standard calibration, believed to be caused by formation of a liquid film on the surface of the probe. In the liquid phase, moving from the standard calibration environment to the fermentation caused the linear relationship between the H₂ concentration and MIMS signal to change in intercept, and the relationship for CO₂ to change in slope, possibly due to dissolved ions, and related non-ideality. For ethanol, standard calibration results were fairly consistent with in-process calibration results. The main limitation with in-process calibration is the potential for a lack of variability in target concentration. This could be addressed by spiking the targeted compound at the end of the experiment. Regardless, MIMS is an ideal instrument for analysing fermentation experiments, due to its ability to measure targeted compounds semi-continuously, and due to a lack of drift over long periods.     

Online monitoring of higher alcohols and esters throughout beer fermentation by commercial Saccharomyces cerevisiae and Saccharomyces pastorianus yeast

Higher alcohols and esters are among the predominant classes of volatile organic compounds (VOCs) that influence the quality of beer. The concentrations of these compounds are determined through a specific yeast strain selection and fermentation conditions. The effect of yeast strains on the formation of higher alcohols and esters throughout fermentations (at 20°C) was investigated. Flavour-relevant esters (ethyl acetate, isoamyl acetate, ethyl hexanoate and ethyl octanoate) and higher alcohols (isoamyl alcohol, isobutyl alcohol and phenylethyl alcohol) were monitored throughout the fermentation using proton transfer reaction–time of flight–mass spectrometry (PTR-ToF-MS) coupled with an automated sampling system for continuous measurements. Compound identification was confirmed by analysis of samples using gas chromatography–mass spectrometry (GC–MS). Results demonstrated the specific time points where variation in higher alcohol and ester generation between yeast strains occurred. In particular, the concentrations of isoamyl acetate, ethyl octanoate and isoamyl alcohol between yeast strains were significantly different over the first 2 days of fermentation; whereas, after Day 3, no significant differences were observed. The two Saccharomyces pastorianus strains produced comparable concentrations of the key higher alcohols and esters. However, the key higher alcohol and ester concentrations varied greatly between the two S. cerevisiae strains. The use of PTR–ToF–MS to rapidly measure multiple yeast strains provides new insights on fermentation for brewers to modify the sensory profile and optimise quality.     

On-line separation, simultaneous dilution and spectrophotometric determination of zinc in fertilisers with a sequential injection system and xylenol orange as complexing agent

A dialyser unit, equipped with a passive neutral membrane, was incorporated into the conduits of a sequential injection (SI) system for the on-line removal of suspended solids and simultaneous dilution of the analyte before reaction and detection of the analyte. The system was applied to the determination of zinc(II) in fertilisers. The fully automated system is able to analyse zinc at a sampling frequency of ten samples per hour at a %R.S.D. of better than 0.55. The calibration graph was linear between 10 and 50 mg l⁻¹. The detection limit was found to be 4.75 mg l⁻¹. The results obtained with the proposed SI analyser compared favourably with the standard manual flame atomic absorption spectrometric method.     

Continuous flow system for lead determination by faas in spirituous beverages with solid phase extraction and on-line copper removal

A continuous flow system for the determination of lead in home made spirituous beverages was developed. The determination was based on the formation of a neutral chelate of the element with ammonium pyrrolidine dithiocarbamate, its adsorption onto a minicolumn packed with sodium faujasite type Y synthetic zeolite, followed by elution with methyl isobutyl ketone and determination by flame atomic absorption spectrometry. Ethanol and copper interfere strongly in the determination and therefore, must be separated prior to the analysis. Copper is removed by precipitation with rubeanic acid, while ethanol is eliminated by rotaevaporation. Sample solutions containing Pb²⁺ in the concentration range from 5 to 120 μg l⁻¹ at pH 2.5 could be analyzed, by using a preconcentration time of 3 min. Preconcentration factors from 80 to 140 were achieved for a sample volume of 6 ml and the detection limit varied from 1.4 to 3.5 μg l⁻¹, depending on the matrix composition. The relative standard deviations for 60 μg l⁻¹ Pb was 3.2% (n = 10) and the recovery of spikes (20, 40, 60 and 80 μg l⁻¹) added to the samples was estimated within 92–105% range, suggesting that lead can be quantitatively determined in such samples. Determining lead in several samples by an alternative technique further checked the accuracy. Finally, the concentrations of Pb²⁺ determined in 28 samples of Venezuelan spirituous beverages were in 12.6–370.0 μg l⁻¹ range, depending on the fermenting material based on different mixtures of agave, raw sugar cane and white sugar.     

Repetitive determination of ascorbic acid using iron(III)-1.10-phenanthroline-peroxodisulfate system in a circulatory flow injection method

Ascorbic acid (AA) could be determined in large quantities of a co-existing oxidant. The incorporation of an on-line reagent regeneration step based on redox reaction eliminates the baseline drift in the procedure. This makes it possible to adopt a circulatory flow injection method (cyclic FIA) and to determine AA repetitively. The method is based on the reduction of iron(III) to iron(II) by the analyte, the reaction of the produced iron(II) with 1,10-phenanthroline (phen) in a weak acidic medium to form a colored complex, and the subsequent oxidation reaction of iron(II) to iron(III) by the co-existing peroxodisulfate. A solution (50 ml) of 3.0×10⁻⁴ mol l⁻¹ ferric chloride, 9.0×10⁻⁴ mol l⁻¹ phen and 5.0×10⁻² mol l⁻¹ ammonium peroxodisulfate in acetate buffer (0.2 mol l⁻¹, pH 4.5) is continuously circulated at a constant flow rate of 1.0 ml min⁻¹. Into this stream, an aliquot (20 μl) of the sample solution containing AA is quickly injected by means of a six-way valve. The complex formed is monitored spectrophotometrically (at 510 nm) in the flow system. The stream then returns to the reservoir after passing through a time-delay coil (50 m). The iron(II)–(phen)₃ complex is oxidized to iron(III)–(phen)₃ complex by peroxodisulfate which exists excessively in the circulating reagent solution. The proposed method allows as many as 300 repetitive determinations of 15 mg l⁻¹ AA with only 50 ml reservoir solution. The contents of AA in commercial pharmaceutical products were analyzed to demonstrate the capability of the developed system.     

Determination of mercury in saliva with a flow-injection system

A simple, fast and reliable method, with a low detection limit, has been developed for the determination of total mercury in saliva samples. The method uses a brominating reagent, followed by on-line addition of KMnO₄ at room temperature to convert organically bound mercury to inorganic mercury ions, and determines mercury by flow-injection cold-vapour atomic absorption spectrometry. Using the method described, complete recoveries of five mercury compounds from saliva were attained. Results obtained on real samples using the new method were comparable to that obtained using the established method with batch system. The detection limit of this method, based on three standard deviations of the blank, is 0.05 μg l⁻¹ Hg in a saliva sample of 500 μl. A sample throughput of 80 measurements per hour is possible with the method. The calibration curves are linear up to 20 μg l⁻¹ and the dynamic range extends to 40 μg l⁻¹ Hg. At a concentration of 1μg l⁻¹ mercury in saliva, the relative standard deviation is about 2% for 11 replicates; a total volume of 0.5 ml saliva is required for three replicates.     

Membrane-introduction mass spectrometry (MIMS)

Membrane-introduction mass spectrometry (MIMS) for chemical analysis involves directly sampling analytes in gaseous, liquid and solid samples through a semi-permeable membrane coupled to a mass spectrometer, yielding selective and sensitive quantitation. Because MIMS is an on-line technique, in which samples can be continuously flowed over a membrane interface, it can yield analytical results in real time without the need for sample clean-up and chromatographic separation. This review highlights trends and developments in MIMS over the past decade and describes recent studies that pertain to its use for on-site, in-situ and in-vivo chemical analysis. We report on advancements in instrumentation, including membrane materials, interface configurations and ionization techniques that have extended the range of analytes amenable to MIMS.We summarize the progress made in the miniaturization of mass spectrometers that have resulted in field-portable systems and review recent applications of continuous mobile monitoring and on-site environmental monitoring to yield both temporally and spatially resolved quantitative and semi-quantitative data. Finally, we describe recent work involving the use of MIMS for in-vivo chemical analysis.     

Field speciation of chromium with a sequential injection lab-on-valve incorporating a bismuthate immobilized micro-column

A fully automated and portable analyzer for field speciation of inorganic chromium in wastewater was developed. The instrument consists of a micro-sequential injection lab-on-valve (LOV) system and a miniature USB2000 spectrophotometer. A multi-purpose flow cell was incorporated on one side of the main body of the LOV, which offers vast potentials and versatilities in its compatibility with various detection modes. On-line oxidation of trivalent chromium was performed on a bismuthate immobilized silica micro-column reactor integrated in the LOV. When determining Cr(VI), its chromogenic reaction with 1,5-diphenylcarbazide (DPC) was facilitated in the flow cell and the absorbance was monitored in situ at 548 nm via optical fibers. While for the quantification of total chromium, Cr(III) was oxidized on-line by aspirating sample solution through the oxidizing column reactor, followed by chromogenic reaction with DPC and the absorbance was monitored in the flow cell. With a sampling volume of 200 μl, the detection limits of 5.6 μg l⁻¹ for Cr(VI) and 6.8 μg l⁻¹ for total chromium were achieved along with a sampling frequency of 60 h⁻¹. A R.S.D. value of 2.0% was recorded at 32 μg l⁻¹ of Cr(VI). The practical applicability of the speciation analyzer was validated by analyzing Cr(VI) and total chromium contents in two certified reference materials. The feasibility of performing rapid field speciation of chromium in wastewater samples was also demonstrated.     

气相色谱-质谱法分析啤酒中酒花香气成分

利用顶空固相微萃取-气相色谱质谱技术(HS-SPME/GC-MS)建立了定量分析啤酒中19种源自酒花的微量香气成分的方法。研究了不同萃取头、萃取时间、萃取温度对萃取效果的影响,最终确定HS-SPME最佳萃取条件为采用PDMS萃取头对啤酒样品在50℃下萃取60 min。在最佳萃取条件下,采用啤酒为基体以减少基体干扰,建立标准曲线,随后在SIM模式下以萜品烯-4-醇为内标定量测定了啤酒中酒花香气物质的含量。19种物质的回收率在81.2%～116.8%之间,相对标准偏差(RSD)低于9.8%,在5个加标浓度下,R2大于0.99。相比于传统方法,本方法所需样品量少、灵敏度高、操作过程简便,能准确的检测出啤酒中酒花香气物质的含量。     

Selective flow injection analysis of ultra-trace amounts of Cr(VI), preconcentration of it by solvent extraction, and determination by electrothermal atomic absorption spectrometry (ETAAS)

A rapid, robust, sensitive and selective time-based flow injection (FI) on-line solvent extraction system interfaced with electrothermal atomic absorption spectrometry (ETAAS) is described for analyzing ultra-trace amounts of Cr(VI). The sample is initially mixed on-line with isobutyl methyl ketone (IBMK). The Cr(VI) is complexed by reaction with ammonium pyrrolidine dithiocarbamate (APDC), and the non-charged Cr(VI)–PDC chelate formed is extracted into IBMK in a knotted reactor made from PTFE tubing. The organic extractant is separated from the aqueous phase by a gravity phase separator with a small conical cavity and delivered into a collector tube, from which 55 μl organic concentrate is subsequently introduced via an air flow into the graphite tube of the ETAAS instrument. The operations of the FI-system and the ETAAS detector are synchronously coupled. A significant advantage of the approach is that matrix constituents, such as high salt contents, effectively are eliminated. The extraction procedure was optimized by a simplex approach. A central composite design was subsequently employed to verify the estimated operational optimum. An 18-fold enhancement in sensitivity of Cr(VI) was achieved after preconcentration for 99 s at a sample flow rate of 5.5 ml min⁻¹, as compared to direct introduction of 55 μl of sample, yielding a detection limit (3σ) of 3.3 ng l⁻¹. The sampling frequency was 24.2 samples h⁻¹. The proposed method was successfully evaluated by analyzing a NIST Cr(VI)-reference material, synthetic seawater and waste waters, and waste water samples from an incineration plant and a desulphurization plant, respectively.     

HPLC determination of Vitamin D(3) and its metabolite in human plasma with on-line sample cleanup

A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D₃ and 25-hydroxyvitamin D₃ (25-OH-D₃) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1 ml of human plasma was treated with 2 ml of a mixture of ethanol–acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250 μL of a solution of methanol 5 mmol l⁻¹ phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200 μl aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5 min with a mobile phase constituted by a solution of 6% acetonitrile in 5 mmol l⁻¹ phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile–phosphate buffer 5 mmol l⁻¹, pH 6.5; 20:80 (v/v); solvent B: methanol–acetonitrile–tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265 nm. The method was linear in the range 3.0–32.0 ng ml⁻¹ with a limit of quantification of 3.0 ng ml⁻¹. Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was ≤2.80%. The proposed method permitted the simultaneous determination of Vitamin D₃ and 25-OH-D₃ in 16 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to five samples h⁻¹. The method was successfully applied for the determination of Vitamin D₃ and 25-OH-D₃ in plasma from 46 female volunteers, ranging from 50 to 94 years old. Vitamin D₃ and 25-OH-D₃ concentrations in plasma were found from 4.30–40.70 ng ml⁻¹ (19.74 ± 9.48 ng ml⁻¹) and 3.1–36.52 ng ml⁻¹ (7.13 ± 7.80 ng ml⁻¹), respectively. These results were in good agreement with data published by other authors.