Simultaneous spectrophotometric determination of phosphate and silicate ions in river water by using ion-exclusion chromatographic separation and post-column derivatization

The simultaneous spectrophotometric determination of phosphate and silicate ions in river water was examined by using ion-exclusion chromatography and post-column derivatization. Phosphate and silicate ions were separated by the ion-exclusion column packed with a polymethacrylate-based weakly acidic cation-exchange resin in the H⁺-form (TSKgel Super IC-A/C) by using ultra pure water as an eluent. After the post-column derivatization with molybdate and ascorbic acid, so-called molybdenum-blue, both ions were determined simultaneously by spectrophotometry. The effects of sulfuric acid, sodium molybdate and ascorbic acid concentrations and reaction coil length, which have relation to form the reduced complexes of molybdate and ions, on the detector response for phosphate and silicate ions were investigated. Under the optimized conditions (color-forming reactant, 50 mM sulfuric acid-10 mM sodium molybdate; reducing agent, 50 mM ascorbic acid; reaction coil length, 6 m), the calibration curves of phosphate and silicate ions were linear in the range of 50-2000 μg L⁻¹ as P and 250-10,000 μg L⁻¹ as Si. This method was successfully applied to water quality monitoring of Kurose-river watershed and it suggested that the effluent from a biological sewage treatment plant was significant source of phosphate ion in Kurose-river water.     

Simultaneous determination of silicate and phosphate in environmental waters using pre-column derivatization ion-pair liquid chromatography

A highly sensitive HPLC method for the simultaneous determination of soluble silicate and phosphate in environmental waters was developed, using ion-pair liquid chromatography preceded by the formation of their yellow α-heteropolymolybdates. The moderate-pH mobile phase enabled to use a highly efficient reversed-phase silica column. The pre-column coloring reactions at moderate-pH were reproducible for both silicate and phosphate in all quantification ranges with R.S.D.s less than 2% and 5%, respectively. The linear calibration lines between concentrations (mg-SiO₂/L and mg-PO₄/L) and peak area intensities were obtained for silicate and phosphate both with acceptable determination coefficients (r²) of 0.9999. The limits of determination for both analytes were 0.007 mg-SiO₂/L and 0.003 mg-PO₄/L, which were calculated theoretically using 10σ/slope. The four-digit dynamic ranges were obtained for 0.007-10 mg-SiO₂/L and 0.003-20 mg-PO₄/L. The developed method was applied for the analysis of tap water, river water, coastal seawater, well water, hot-spring water, commercial mineral water, and laboratory water. The results were very reasonable and acceptable from the environmental viewpoints, which were well correlated with those confirmed by the molybdenum-blue spectrophotometry.     

Selective determination of ammonium ions by high-speed ion-exclusion chromatography on a weakly basic anion-exchange resin column

This paper describes an ion-exclusion chromatographic system for the rapid and selective determination of ammonium ion. The optimized ion-exclusion chromatographic system was established with a polymethacrylate-based weakly basic anion-exchange resin column (TSKgel DEAE-5PW) as the separation column, an aqueous solution containing 0.05 mM tetramethylammonium hydroxide (pH 9.10) as eluent with conductimetric detection for the analyte determination. Under the optimum chromatographic conditions, ammonium ion was determined within 2.3 min with a detection limit (S/N=3) better than 0.125 microM. Ammonium ion in rain and river waters was precisely determined using this ion-exclusion chromatographic system.     

Fluorimetric differential-kinetic determination of silicate and phosphate in waters by flow-injection analysis

A flow-injection analysis (FIA) method for simultaneous determination of silicate and phosphate, based on the different rates of formation of their molybdate heteropoly acids is suggested. The fluorimetrically monitored product is thiochrome, formed by oxidation of thiamine by the heteropoly acid. The FIA configurations designed allow performance of two measurements at different times on each sample injected. The method permits the determination of these anions in the range 30-600 ng ml in ratios from 1:10 to 10:1 and can be applied to samples of running and bottled water with good results. The sampling frequency achievable is 60 hr .     

On-line preconcentration and simultaneous determination of heavy metal ions by inductively coupled plasma-atomic emission spectrometry

A flow injection analysis system for on-line preconcentration and simultaneous determination of Bi³⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Ni²⁺, Pb²⁺ and Zn²⁺ in aqueous samples by inductively coupled plasma (ICP)-atomic emission spectrometry with a charge coupled detector is described. The preconcentration of analytes is accomplished by retention of their chelates with sodium diethyldithiocarbamate in aqueous solution on a solid phase containing octadecyl silica in a minicolumn. Methanol, as eluent, is introduced into the conventional nebulizer of the ICP instrument. The effects of different parameters, including preconcentration flow rate (equal to sample flow rate (SR)), eluent flow rate (ER), weight of solid phase (W) and eluent loop volume (EV), were optimized by the super-modified simplex method. The optimum conditions were evaluated to be SR 7.2 ml min⁻¹, ER 3.5 ml min⁻¹, W of 100 mg and EV of 0.8 ml. An enrichment factor of 312.5 for each analyte was obtained. The detection limits of the proposed method for Bi³⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Ni²⁺, Pb²⁺ and Zn²⁺ were evaluated as 1.3, 1.0, 0.8, 0.3, 14.7, 0.5, 5.5 and 0.1 ng l⁻¹, respectively. The effect of several metal ions on percent recovery was also studied. The method was applied to the recovery of these heavy metals from real matrices and to the simultaneous determination of these cations in different water samples.     

Separation and determination of sugars by reversed-phase high-performance liquid chromatography after pre-column microwave-assisted derivatization

In this study the possibility of derivatizing sugars using microwave irradiation was investigated. The amount of reagent, irradiation intensity, and derivatization time were optimized. In the derivatization of sugars with p-nitroaniline the reaction is complete within 5 min at 600 W when the p-nitroaniline-to-sugar and NaBH₃CN-to-sugar mole ratios were above 1.4 and 3.1, respectively. A Doehlert design was used to optimize the mobile phase for separation of p-nitroaniline-labeled sugars; and the best separation was obtained by use of 0.01 mol L⁻¹ acetate buffer at pH 4.40 containing 11.0% acetonitrile. Analysis using this method was highly sensitive and analysis time was short. Finally, a food sample was analyzed using the proposed method.     

Interlaboratory comparison of an analytical method for the determination of semduramicin in poultry feed at the authorized level using high-performance liquid chromatography coupled to postcolumn derivatization and spectrophotometric detection

The performance characteristics of a method based on HPLC with postcolumn derivatization and spectrophotometric detection for the quantification of semduramicin in poultry feedingstuffs have been determined via a collaborative study. Semduramicin is a feed additive that is authorized for fattening chickens within the European Union at a minimum and maximum content of 20 and 25 mg/kg in feedingstuffs, respectively. The target concentration of semduramicin in the test samples ranged from 11.5 to 45.0 mg/kg. The study has been conducted with two different types of test material, namely, feedingstuff samples that have been previously ground in our laboratory and pelleted feedingstuffs. In the latter case, the laboratories participating in the study had to grind the samples prior to analysis. The obtained RSD for repeatability (RSD(r)) ranged from 2 to 10% for the ground materials, and from 2 and 7% for the pelleted materials. The RSD for reproducibility (RSDR) varied between 11 and 16% for the ground materials, and between 12 and 15% for the pelleted materials. These data indicated that grinding as an additional step in the analytical procedure did not influence the precision profile of the method. In addition, the HorRat values for all test materials were below or equal to 1.5, thus demonstrating that the obtained precision data were acceptable for the purpose of the method. Furthermore, an estimation of trueness based on statistical treatment of the results reported from the laboratories for spiked samples revealed acceptable mean recovery values of 88 +/- 4%. Based on the obtained performance profile, the method can be considered fully validated and transferable to control laboratories to be used within the framework of official control.     

Fluorometric determination of khellin in human urine and serum by high-performance liquid chromatography using postcolumn photoirradiation.

For the determination of khellin in urine and serum, fluorometry using HPLC-postcolumn photoirradiation has been developed. Khellin and visnagin of similar structure were separated on a column of Capcell Pak C8. The mobile phase consisted of 40%(v/v) ethanol containing 75 mmol l(-1) H2O2. The postcolumn reagent, 70 mmol l(-1) KH2PO4-NaOH buffer (pH 12.7) containing 50%(v/v) ethanol, were mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The fluorescence was monitored with excitation at 378 nm and emission at 480 nm. The calibration graph for khellin was linear over the range of 65 - 2620 ng ml(-1) using an injection volume of 20 microl. The pretreatment of the urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively.     

柱前衍生-高效液相色谱法同时测定血清中氨基酸类及单胺类神经递质

以邻苯二甲醛（o-phthalaldehyde，OPA）为衍生试剂，建立了柱前衍生-高效液相色谱（HPLC）同时测定血清中氨基酸类神经递质牛磺酸（Tau）、谷氨酸（Glu）、甘氨酸（Gly）、γ-氨基丁酸（γ-GABA）和单胺类神经递质多巴胺（DA）含量的分析方法。血清与乙醇以1：2的体积比混合，进行蛋白质沉淀后离心，取其上清液，氮吹至近干。前处理后的样品与OPA进行柱前衍生，衍生化产物采用Luna 5u C18色谱柱（250 mm×4.6 mm，5 μm）分离，以柠檬酸-乙酸钠缓冲溶液（pH 3.73）为流动相A、乙腈为流动相B进行梯度洗脱，流速为1.0 mL/min，柱温为30℃，检测波长为338 nm。5种神经递质在各自范围内线性关系良好（r²≥0.9866），检出限为0.10~0.40 μmol/L，不同加标水平下目标物的加标回收率为87.57%~115.31%，相对标准偏差均低于7.80%。方法操作简单，灵敏度高，精密度、线性关系和回收率等方法学指标较好，可实现血清中氨基酸类及单胺类神经递质的同时检测。     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

Selective determination of trace arsenic and antimony species in natural waters by gas chromatography with a photoionization detector

Traces amounts of arsenic and antimony in water samples were determined by gas chromatography with a photoionization detector after liquidnitrogen cold trapping of their hydrides. The sample solution was treated with sodium hydroborate (NaBH₄) under weak-acid conditions for arsenic(III) and antimony(III) determination, and under strong-acid conditions for arsenic(III+V) and antimony(III+V) determination. Large amounts of carbon dioxide (CO₂) and water vapor obscured determination of arsine and stibine. Better separation from interference could be achieved by removing CO₂ and water vapor in two tubes containing sodium hydroxide pellets and calcium chloride, respectively. The detection limits of this method were 1.8 ng dm^?3 for arsenic and 9.4 ng dm^?3 for antimony in the case of 100-cm³ sample volumes. Therefore, it is suitable for determination of trace arsenic and antimony in natural waters.     

Selective separation and simultaneous determination of trace levels of five types of fluorinated quinolone drugs by thin-layer chromatography/fluorescence densitometry.

This paper reports the determination of trace levels of 5 types of fluorinated quinolone drugs, i.e., ciprofloxacin, norfloxacin, enoxacin, pefloxacin, and ofloxacin, by thin-layer chromatography (TLC)/fluorescence densitometry. The new analytical method uses 2-step TLC development, selective separation, and simultaneous determination of the 5 drugs. The method was also applied to the determination of recoveries of standards of the 5 drugs in plasma and urine samples. The results show that the method has a wide linear range, high repeatability, and good stability.     

Method development for the determination of residual fluorotelomer raw materials and perflurooctanoate in fluorotelomer-based products by gas chromatography and liquid chromatography mass spectrometry

The methodology for the determination of perfluorooctanoate (C(7)F(15)COO-, PFO), fluorotelomer alcohols (FTOHs: 6-2, 8-2, and 10-2), perfluorooctyl iodide (PFOI), and 8-2-8 fluorotelomer alcohol ester in complex fluorotelomer-based commercial products has been demonstrated and validated. Sample preparation procedures allowing determination of residual levels of these compounds were developed. The analytes were detected either by LC/MS/MS (PFO), LC/MS (FTOHs), or GC/MS (PFOI, 8-2-8 ester). The methods were validated by investigating the recoveries of analytes spiked at multiple levels to authentic sample matrices. The recoveries generally were between 70 and 130%. The limits of detection were in sub-microg/g range and the limits of quantitation were in the mug/g range. The methods were applied to fluorotelomer-based raw materials and fluorotelomer-based surfactants and polymeric products and represent methods useful for the determination of higher carbon chain length homologs as well.     

Single laboratory validation of a method for determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by high-performance liquid chromatography with FMOC-Su derivatization

Single laboratory validation of a method for determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by with high-performance liquid chromatography FMOC-Su derivatization. Tests with 2 blank matrixes containing SAMe, vitamin C, citric acid, chondroitin sulfates, methylsulfonylmethane, lemon juice concentrate, and other potential interferents showed the method to be selective and specific. Eight calibration curves prepared over 7 working days indicated excellent reproducibility with the linear range at least over 2.0-150 microg/mL, and determination coefficients >0.9999. Average spike recovery from the blank matrix (n = 8 over 2 days) was 93.5, 99.4, and 100.4% at respective spike levels of 15, 100, and 150%, and from the sample matrix containing glucosamine (n = 3) was 99.9 and 102.8% at respective levels of 10 and 40%, with relative standard deviations <0.9%. The method was also applied to 12 various glucosamine finished products and raw materials. The stability tests confirmed that glucosamine-FMOC-Su derivative once formed is stable at room temperature for at least 5 days. Limit of quantitation was 1 microg/mL and limit of detection was 0.3 microg/mL. The method is ready to proceed for the collaborative study.     

Quantitative determination of quinic acid and derivatives by high-performance liquid chromatography after derivatization with p-bromophenacyl bromide

Quantitative determinations of quinic, shikimic and glycolic acide have been performed by reversed-phase high-performance liquid chormatography using p-bromophenacyl bromide as a visualizing reagent. The reactions are carried out in N,N-dimethylformamide (DMF) with KF as a catalyst. Linear calibration curves are obtained in the concentration range 0.1–2 mg acide per ml DMF. Spectroscopic and chromatographic properties of the pbromophenacyl bromide derivatives of quinic, shikimic, dehydroshikimic and glycolic acid are given. A quantitative analysis of quinic acid in plant material is demonstrated.     

Method for the determination of beta-carotene in supplements and raw materials by reversed-phase liquid chromatography: single laboratory validation

A single laboratory validation (SLV) study was conducted for a liquid chromatography (LC) method for the determination of total and all-trans-beta-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of beta-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane-ethanol. Oily suspensions were directly dissolved in dichloromethane-ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of alpha-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1-50 microg beta-carotene/mL. The main geometrical isomers of beta-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as a-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total beta-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2-4.4%. Recoveries determined for supplements and beadlet raw material spiked with beta-carotene levels of 10 microg to 100 mg/test portion and 0.2-40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-beta-carotene in dietary supplements.     

Rapid and simultaneous determination of piperidinium and pyrrolidinium ionic liquid cations by ion pair chromatography coupled with direct conductivity detection

A method of ion-pair chromatography with direct conductivity detection was developed on a silicabased monolithic column for the fast and simultaneous determination of piperidinium and pyrrolidinium ionic liquid cations. The effects of the mobile phase, column temperature and flow rate on the retention of the cations were investigated. The retention rules were discussed. As an ion-pair reagent, sodium heptanesulfonate is more suitable than sodium pentanesulfonate for the separation and determination of piperidinium and pyrrolidinium cations. The increase of ion-pair reagent concentration led to the increased retention time of the cations. When acetonitrile content and mobile phase flow were increased, the retention time of the cations became shorter. The retention of piperidinium and pyrrolidinium cations is an exothermic process, and the retention of the cations conforms to the carbon number rule. The chromatographic analysis was performed using the Chromolith Speed ROD RP-18e column, 0.5 μmol/L sodium heptanesulfonate-5% acetonitrile as the mobile phase at a flow rate of 3.0 mL/min and column temperature of 30℃. Separation of N-methyl-N-ethyl piperidinium, N-methyl-N-propyl piperidinium, N-methyl-N-butyl piperidinium and N-methyl-N-ethyl pyrrolidinium, N-methyl-N-propyl pyrrolidinium, N-methyl-N-butyl pyrrolidinium cations were achieved within 10 min. The detection limits (S/N=3) were between 0.19 and 3.08 mg/L. Relative standard deviations (n=5) for peak areas were less than 1.2%. The method has been applied to the determination of piperidinium and pyrrolidinium cations in ionic liquid samples. The spiked recoveries of ionic liquid cations were between 96% and 111%. The method is accurate, reliable, rapid, and has a better practicability.     

Simultaneous determination of dimethylarsinic acid and monomethylarsonic acid after derivatization with thioglycol methylate by gas chromatography/mass spectrometry

A gas chromatography/mass spectrometry (GC/MS) method has been developed to determine two methylated arsenic species in human urine samples. The yield of derivatization for dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) using thioglycol methylate (TGM) was measured. The detection limit for the derivatized DMA and MMA using the GC/MS method are 0.95 and 0.8 ng cm-3, respectively. This simple and rapid method has good precision and accuracy. Fragmentation routes of derivatized MMA and DMA are suggested on accurate mass measurements.