Detection of traces of oxidized and reduced sulfur compounds in small samples by combination of different high-performance liquid chromatography methods

Depending on the sulfur species, picomoles of different inorganic sulfur compounds can be detected and separated by HPLC in one arrangement in a sample volume less than 50 μl. The combination of fluorescence labelling of reduced inorganic sulfur compounds such as sulfide (S²⁻), sulfite(SO₃²⁻ and thiosulfate (S₂O₃²⁻) with monobromobimane followed by an extraction of elemental sulfur (S°) by chloroform treatment enables the detection of all mentioned sulfur compounds as well as sulfate (remaining aqueous phase) in the same sample. While the derivatized sulfur compounds could be detected by their fluorescence emission at 480 nm, elemental sulfur is identified by its UV absorption at 263 nm. Sulfate in the remaining aqueous phase is detected by HPLC with indirect UV detection at 254 nm. Detection ranges for the different sulfur compounds examined are as follows: sulfide (5 μM to 1.5 mM), sulfite (5 μM to 1.0 mM), thiosulfate (1 μM to 1.5 mM), elemental sulfur (2 μM to 32 mM) and sulfate (5 μM to >1 mM).     

Low-molecular-mass anion screening for forensic environmental analyses by capillary zone electrophoresis with indirect UV detection

A method for low-molecular-mass anion screening is described using a buffer composed of 5-sulfosalicylate (SS) as a visualizing ion, hexadimethrine bromide as an electroosmotic flow modifier and Tris as a pH buffer component, at pH 8.6. All ions with effective mobility higher than 2610⁻⁹ m² s⁻¹ V⁻¹ can be separated within 7.5 min under −30 kV. By using the moderately mobile SS (5410⁻⁹ m² s⁻¹ V⁻¹), not only the sensitivity of the detection is improved due to its high UV absorptivity, but also a smaller overall overloading effect is achieved. Meanwhile, the resolution of the high mobility ions, which is normally critical, remains almost the same as compared to a chromate buffer. With an electrokinetic injection, the limit of detection (LOD) of the common ions is 2–13 nM and the detection range is linear up to 0.5–3 μM. With a hydrostatic injection the LOD is 0.15–1 μM and the detection range is linear up to 25–200 μM. The identification of ions is performed by comparing the mobility of the ions with that of standards, taking the apparent and effective mobility of HCO₃⁻, which is normally present in the sample solution, as a reference.     

Ion-pair chromatography of polythionates and thiosulfate with detection based on their catalytic effects on the postcolumn azide–iodine reaction

The reduction of iodine with azide, catalyzed by polythionates (tri-, tetra-, penta- and hexathionate) and thiosulfate, has been utilized as a postcolumn reaction for chromatographic determination of these sulfur oxyanions. The method is based on the separation of polythionates and thiosulfate on an octadecylsilica column with an acetonitrile–water (20:80, v/v) mobile phase (pH 5.0) containing 3 mM tetrapropylammonium hydroxide and 6 mM acetic acid, followed by photometric measurement of the residual iodine (as triiodide) from the catalytic postcolumn azide–iodine reaction after mixing a reaction solution containing azide and iodine with the column effluent. Chromatograms obtained for the sulfur oxyanions showed negative peaks as a result of the decrease in absorbance of background. The conditions for the catalytic postcolumn reaction of the sulfur oxyanions in the column effluents were established by varying the concentrations of azide, iodine, iodide and acetic acid in the reaction solution, and varying the flow-rate, reaction temperature and length of the reaction tube. The detection limits (defined as S/N=3) were 4.3 μM for trithionate, 0.10 μM for tetrathionate, 2.7 nM for pentathionate, 5.0 nM for hexathionate and 1.1 nM for thiosulfate. When compared with earlier methods, the proposed method gave a much higher sensitivity for the determination of two polythionates (penta- and hexathionate) and thiosulfate. This method was applied successfully to the analysis of polythionates and thiosulfate added to hot-spring water samples.     

Determination of fluoride in feed mixtures by capillary isotachophoresis

An isotachophoretic (ITP) method for the determination of fluoride in feed mixtures was developed. A sample of feed mixture, after extraction with 1 M HCl, was analysed using a ZKI 02 column-coupling isotachopherograph. Leading electrolytes for presentation and analytical capillaries consisted of 0.008 M HCl-0.022 M -aminocaproic acid (EACA)-0.001 M CaCl₂-0.05% hydroxypropylmethyl cellulose (HPMC) and 0.002 M HCl-0.005 M EACA-0.05% HPMC, respectively. The terminating electrolyte was 0.01 M tartaric acid. The fluoride released from samples by microdiffusion in 25% perchloric acid was determined using an Ionosep 900.1 single capillary isotachopherograph with 0.002 M HCl-0.005 M EACA-0.05% HPMC as the leading electrolyte and 0.002 M tartaric acid as the terminating electrolyte. The detection limit, depending on the sample treatment, was as low as 4 μg/g as fluoride. A comparison of the developed ITP method with ion- selective electrode method was carried out.     

Microchannel-assisted thermal-lens spectrometry for microchip analysis

We present a new method for homocysteine quantitation in human plasma based on in-capillary reaction of homocysteine with 2,2′-dipyridyl disulfide. Homocysteine is in this so-called thiol-exchange reaction quantitatively transformed in mixed disulfide concomitantly with formation of an equimolar amount of 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined specifically at 343 nm. The concentration of homocysteine is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay was from 0.03–3 mM (correlation coefficient 0.994) with a detection limit of 6 μM and a limit of quantitation 20 μM. The inter-day reproducibility of the peak area and the migration time were 1.37% and 0.05%, respectively. The method is simple, relatively rapid and can be easily automated. Moreover the common capillary electrophoresis apparatus with a UV detector can be used to distinguish between normal and pathological hyperhomocysteinemia plasma samples.     

Post-column oxidative derivatization for the liquid chromatographic determination of phenothiazines

A first post-column chemical derivatization method for the liquid chromatographic determination of phenothiazines is presented. Peroxyacetic acid is introduced as a derivatizing agent for phenothiazines, yielding the colored radical cations or fluorescent sulfoxides, depending on reaction conditions. Both reaction products were successfully employed for the detection of the phenothiazines after their liquid chromatographic separation. The fluorescence spectroscopic detection of the sulfoxides proved to be the more robust and sensitive method. Limits of detection ranged from 4 nM for triflupromazine and trimeprazine to 300 nM for phenothiazine for the fluorescence spectroscopic detection of the sulfoxide and from 0.3 μM for phenothiazine and triflupromazine to 2 μM for trifluperazine for the UV–Vis spectroscopic detection of the radical cation. The calibration functions for the fluorimetric sulfoxide determination ranged from two to more than three decades, starting at the limit of quantification.     

Rapid determination of drugs in biofluids by capillary electrophoresis Measurement of antipyrine in saliva for pharmacokinetic studies

A micellar electrokinetic capillary chromatography method was developed that permitted the resolution of antipyrine from endogenous compounds and its quantitation in neat saliva in as little as 1 min. Final conditions were: SpectraPhoresis 1000, 30(23) cm × 50 μm silica capillary, 50 mM sodium phosphate pH 9.6, 50 mM SDS, 10 s hydrodynamic load, detection scanning 200–300 nm or 260 nm, run 25 kV. To overcome the effects of Joule heating the capillary was cooled to 15°C. Sensitivity was <10 μM and linearity extended to 350 μM. Comparison with an HPLC assay demonstrated that hydrodynamic injection gave a loading bias unless samples and standards were of equal viscosity. For 75 samples from five subjects the correlation of CE vs. HPLC was then r = 0.99.     

Spectrophotometric determination of H(2)O(2) in marine waters with leuco crystal violet

H₂O₂ in marine waters may be determined by measuring the absorbance of crystal violet (CV⁺), formed by the oxidation of leuco crystal violet (LCV) by H₂O₂ in the presence of the enzyme horseradish peroxidase, at 592 nm at pH 4. The detection limit and the precision of the method are about 0.02 μM and ± 1% (at a concentration of 0.03 μM), respectively. The results obtained by this method and by a widely used fluorimetric method agreed with each other well. After color development, samples together with the sub-samples for constructing the calibration curve may be stored for up to 5 days before their absorbances are measured without significantly changing the estimated concentration of H₂O₂.     

Reversed-phase ion-interaction chromatography of Cu(I)-cyanide complexes

It has been established that during the separation of Cu(I)-cyanide complexes, the cyanide:Cu(I) molar ratio, R of the eluted complex remained constant irrespective of the R value injected onto the column, and there was considerable tailing of the unretained cyanide peak and fronting of the Cu(I)-cyanide peak in an eluent containing no cyanide. The addition of small amounts of cyanide (100 μM) to the eluent resulted in the elimination of these effects on peak shape and a significant increase in the retention of the Cu(I)-cyanide species. These results suggested that more than one Cu(I)-cyanide complex may be present in the Cu(I)-cyanide peak in an eluent containing no cyanide. Three different detection systems [Fourier transform Infrared (FTIR) and photodiode array spectrophotometry and a post-column reaction (PCR)], were used to determine changes that occurred to the Cu(I)-cyanide complexes during the separation with eluents containing from 0 to 100 μM cyanide. The FTIR approach was unsuccessful due to a lack of sensitivity. The UV spectrum of the Cu(I) peak in any one eluent remained constant, irrespective of the composition of the injected sample, but there were distinct changes in this spectrum among eluents. Similarly, the R value of the Cu(I) peak determined by PCR remained the same in any one eluent but ranged from about 2.5 to about 3.4 for these eluents. The R value was found to vary within the eluted Cu(I)-cyanide peak, especially in an eluent containing no added cyanide. These results show that more than one Cu(I)-cyanide complex is present in the eluted peak and that in the absence of cyanide in the eluent, the eluted peak consists of a mixture of the di- and tricyano complexes of Cu(I).     

Applications of enzyme-catalysed reactions in trace analysis-II Determination of cyanide, sulphide, and iodine with invertase

Methods are described for the determination of cyanide (10⁻⁸–10⁻⁵M and sulphide (10⁻⁷–10⁻⁵ M) based on the de-inhibitory effect of these ions on invertase inhibited by mercury(II) or by silver. Iodine (0.1–3 μg) may be determined by its inhibition of invertase.     

Improved determination of tributyl phosphate degradation products (mono- and dibutyl phosphates) by ion chromatography

Tributyl phosphate (TBP) is a very important compound in the nuclear industry, particularly in the area of nuclear fuel reprocessing. This compound is used in the PUREX (plutonium and uranium refining extraction) process which consists of the extraction of uranium and plutonium from an aqueous nitric acid phase, for the purpose of recycling. But TBP may be degraded to dibutyl phosphate (DBP) and monobutyl phosphate (MBP) by dealkylation of one or two butoxy groups, respectively. We have compared and evaluated the capacity of two resins manufactured by Dionex (AS11 and AS5A) in the separation and measurement of these two degradation products. AS11 generates two interferences: nitrite/DBP and carbonate/MBP. The first one is the most serious. So, we have developed a method for oxidising nitrite ions to nitrate ions which have no trouble over the measurement. The second resin tested, AS5A, allows a very efficient separation between DBP and NO₂⁻ ions and a good separation between MBP and CO₃²⁻ in comparison with the AS11. The detection limits for the AS5A column are 0.13 μM for MBP and 0.71 μM for DBP (injection LOOP=50 μl).     

Critical study of fluorimetric determination of selenium in urine

Different steps for the fluorimetric determination of Se in urine have been investigated. A HNO₃---HClO₄ (4:1) mixture is useful for urine digestion, and reduction of Se(VI) to Se(IV) is effectively carried out with HCl (6M). Selenium(VI) present after the digestion process constitutes 14.5–36.6% of total Se. An optimum pH of 1.80±0.05 and the addition of 1 ml of 2,3-diaminonaphthalene (DAN) (0.1%, w/v) are established in the formation of Se—DAN complex. Heating to 60°C, a time of incubation of 15 min is recommended to assure the complete formation of Se—DAN complex. A volume of 5 ml of cyclohexane and vigorous shaking for 45 sec is necessary for the extraction process. With this optimized method, the detection limit of selenium was 0.82 μg/l., within-day precision for a 50.0 μg/l. standard solution and urine (27.3 μg/l.) were 2.4 and 2.7% and between-day for the urine was 3.9% (33.9 μg/l.). Analytical recovery of 0.5 ml of Se standard (250 μg/l.) added to 1 ml of urine was 99.9±2.9% (95.8–104.4, n = 12). Normal levels of selenium excretion in urine obtained from healthy people were 27.9±8.7 μg/day (13.2–44.1), not observing significant differences (P < 0.05) between sexes.     

Sensitive and selective ion chromatographic method for the determination of trace beryllium in water samples

A selective and sensitive ion chromatographic method has been developed for the determination of beryllium in a number of water samples at low-μg/l concentrations. The separation was performed on a 250×4.0 mm I.D. iminodiacetic acid functionalised silica gel column. Chromatographed Be(II) was detected using visible detection at 590 nm following post-column reaction with chrome azurol S (CAS). The optimum separation and derivatisation conditions were studied in detail. The optimum eluent conditions were found to be 0.4 M KNO₃, adjusted to pH 2.5 using HNO₃, with optimum post-column detection being achieved using a solution containing 0.26 mM CAS, 2% Triton X-100, 50 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.0. Under the above conditions, the concentration detection limit for Be(II) was found to be 3 μg/l in a standard solution and 4 μg/l in a typical tap water sample, using a 250 μl injection. The method was linear over the investigated range of 10 μg/l to 10 mg/l and highly reproducible. The method was successfully applied to a number of water samples of varying matrix complexity, including simulated seawater, and also to a natural freshwater certified reference material NIST 1640.     

Extraction and electrochemical quantification of the active ingredient (diazepam) in pharmaceutical products

A method for the extraction and separation of the active ingredient diazepam (7-chloro-di-hydro-1-methyl-5-phenyl-2H, -1,4-benzodiazepam-2-one) in pharmaceutical tablets is discussed. This method avoids the hydrolysis of diazepam. Polarographic studies with direct current (DCP), alternating current (ACP) and differential pulse polarography (DPP) were used under different sets of conditions. The pH value most suitable for these studies was found to be 4.6. The detection range for ACP was found to be from 17.4 to 167 μM, whereas for DPP, the range was 4.8–96 μM.

Commercial tablets of diazepam labelled to contain 10 mg per tablet were assayed. For five replicate samples, the relative standard deviations were lower than 1% and the contents were determined with errors lower than 4%.     

Continuous monitoring for cyanide in waste water with a galvanic hydrogen cyanide sensor using a purge system

A continuous monitoring system for cyanide with a galvanic hydrogen cyanide sensor and an aeration pump for purging was developed. Hydrogen cyanide evolved from cyanide solution using a purging pump was measured with the hydrogen cyanide sensor. The system showed good performance in terms of stability and selectivity. A linear calibration curve was obtained in the concentrating range from 0 to 15 mg dm³ of cyanide ion with a slope of −0.24 μA mg⁻¹ dm⁻³. The lower detection limit was 0.1 mg dm⁻³. The 90% response time of the sensor system was within 3.5 min for a 0.5 mg dm⁻³ cyanide solution, when the flow rate of the purging air was 1 dm³ min⁻¹. The system maintained the initial performance for 6 months in the field test. The developed galvanic sensor system was not subject to interference from sulfide and residual chlorine, compared with a potentiometric sensor system developed previously. The analytical results obtained by the present system were in good agreement with those obtained by the pyridine pyrazolone method. The correlation factor and regression line between both methods were 0.979 and Y=2.30×10⁻⁴+1.12X, respectively. This system was successfully applied for a continuous monitoring of cyanide ion in waste water.     

Amperometric detection of cytochrome c by capillary electrophoresis at a sol–gel carbon composite electrode

Capillary electrophoresis (CE) was employed for the determination of cytochrome c using a wall-jet amperometric detector consisting a copper(I) oxide-modified sol–gel carbon composite electrode (CCE), which exhibits a sensitive electrocatalytic response for the oxidation of cytochrome c. The optimum conditions of separation and detection are 0.08 M NaOH for the separation solution, 12 kV for separation voltage and +0.60 V versus saturated calomel electrode (SCE) for the detection potential. Calibration was linear over the concentration range 1–600 μM with the limit of detection of 3.4 μM, based on a signal-to-noise ratio (S/N) of 3.     

Differential-pulse voltammetric determination of low μg l cyanide levels using EDTA, Cu(II) and a hanging mercury drop electrode

The proposed method for cyanide determination at the ultratrace level by differential pulse voltammetry is based in the sensitivity enhancement obtained when both Cu(II) and EDTA are present in the background electrolyte. Comparison of the detection limits and linear dynamic ranges using the conventional borate (pH 9.75), and the proposed borate-EDTA–Cu(II) background electrolytes was carried out. Best results have been obtained with the addition of 0.5 mmol l⁻¹ EDTA and 0.02 mmol l⁻¹ of Cu(II), which allow a detection limit of 1.7 μg l⁻¹ CN⁻ (65 nmol l⁻¹ — absolute detection limit 34 ng) with a precision better than ±2% for a 40 μg l⁻¹ level. Calibration range extended from detection limit up to 100 μg l⁻¹. Cyclic voltammetry indicates that the measured cyanide peak is obtained when the electrogenerated CuCN adsorbed onto the hanging mercury drop electrode surface, is oxidised at positive going potential scan. The method has been successfully applied to various industrial waste waters such as metal-finishing waste waters, water/sand mixtures from cleaning processes of coke production, leachates from wastes obtained from electrolytic cells of aluminium production, and liquors from gold extraction industry. Results obtained by the proposed method showed good agreement with those obtained by the standard methods (ion-selective potentiometry and the spectrophotometric pyridine method).     

Extractive spectrophotometric and fluorimetric determination of boron with 2,2,4-trimethyl-1,3-pentanediaol and carminic acid

Boric acid at μg/ml or ng/ml level can be extracted from 1-6M hydrochloric acid into 2,2,4-trimethyl-1,3-pentanediol solution in chloroform and thus separated from many ions which interfere in the usual spectrophotometric methods. The boron is determined directly in the organic phase without back-extraction into water, by adding a solution of carminic acid in a mixture of sulphuric and glacial acetic acids (1+2 v/v) and measuring the absorbance at 549 nm. The molar absorptivity is 2.58 × 10⁴ l.mole⁻¹.cm⁻¹ and Beer's law is valid for the 0.05–0.4 μg/ml boron range. In the fluorimetric method, 509 or 547 nm can be used as the excitation wavelength and 567 nm for emission measurement, giving a linear response in the 8–120 ng/ml boron range. Both methods have been applied to determination of boron in plants and natural waters with good precision and accuracy.     

Purification of DNA-derived deoxynucleotides from leukocytes involving nuclease elution of an ion-exchange column

A method has been developed in which the DNA of leukocytes (as the buffy coat from blood) is isolated in the form of its constituent deoxynucleotides. The steps in this method are as follows: (1) lyse the leukocytes with sodium dodecyl sulfate (SDS) and enzymatically digest the proteins and RNA, (2) remove the SDS on a non-polar adsorbent (Bio-Beads SM-4) and then trap the DNA on a quaternary amine silica cartridge, (3) wash the column with 1 M NaCl-buffer, (4) digest the DNA on the column with staphylococcal nuclease and (5) elute the digested DNA with 0.5 M NaCl-buffer and digest it further with bovine spleen phosphodiesterase II to deoxynucleotide-3′-monophosphates. From a 40-μl sample of butty coat was obtained 126 ± 14 μg (two experiments, eight sample total) of deoxynucleotides. Reversed-phase high-performance liquid chromatography, which removed the added enzymes, showed only peaks for deoxynucleotides. For comparison, the amount of deoxynucleotides obtained from the leukocytes by an automated phenol extraction procedure was 101 ± 5.4 μg (one experiment in triplicate).     

Determination of trimethylselenonium iodide, selenomethionine, selenious acid, and selenic acid using high-performance liquid chromatography with on-line detection by inductively coupled plasma mass spectrometry or flame atomic absorption spectrometry

An analytical method has been developed for the determination of selenious acid, selenic acid, trimethylselenonium ion, and selenomethionine. The four selenium compounds were separated by HPLC on a column (25 cm×4 mm I.D.) of the anion-exchanger ESA Anion III with a mobile phase (1.5 ml/min) of 0.0055 M ammonium citrate (pH 5.5). Detection was carried out using an on-line inductively coupled plasma mass spectrometer (ICP-MS) or a flame atomic absorption spectrometer (FAAS) as the selenium-specific detector. The chromatographic parameters and the chemical factors affecting the separation of the selenium species were optimized. The four selenium compounds could be separated within 8 minutes. The detection limits of the coupled HPLC–FAAS system were approximately 1 mg Se/l for each compound (100 μl injection), estimated as three times the base-line noise of the chromatograms. More powerful selenium detection was achieved with an ICP-MS. Selenium was measured at m/z 78. To increase the nebulization efficiency, the Meinhard concentric glass nebulizer was replaced by an ultrasonic nebulizer. The ICP-MS signal intensity was increased with the ultrasonic nebulization by a factor of 7 times for selenious acid and 24 to 31 times for trimethylselenonium ion, selenomethionine, and selenic acid compared to that with the Meinhard nebulization. The detection limits achieved by the HPLC–ICP-MS with the ultrasonic nebulization were 0.08 μg Se/l for trimethylselenonium ion, 0.34 μg Se/l for selenious acid, 0.18 μg Se/l for selenomethionine, and 0.07 μg Se/l for selenic acid, respectively.