Application of Iodine-Azide Reaction as Postcolumn Reaction in HPLC for Determination of 2-Thiobarbituric Acid

The reaction between iodine and azide ion induced by 2-thiobarbituric acid (TBA) has been utilized as a postcolumn reaction for chromatographic determination of this sulphur compound. The method is based on the separation of thiobarbituric acid on an Nova-Pak® CN HP column with an acetonitrile–aqueous solution of sodium azide mobile phase. The separation stage is followed by spectrophotometric measurement of the residual iodine (λ=350 nm) from the postcolumn iodine-azide reaction induced by thiobarbituric acid after mixing iodine solution containing iodide with the column effluent containing azide ions and the inductor. Chromatograms obtained for thiobarbituric acid showed negative peaks as a result of the decrease in absorbance of background. The detection limit (defined as S/N=3) was 0.16 pmol (22.9 pg) for thiobarbituric acid. Calibration graphs, plotted as peak heights or peak area vs. concentrations, were linear up to 1 nM.     

Analysis of chlortetracycline by high performance liquid chromatography with postcolumn alkaline-induced fluorescence detection.

A high performance liquid chromatographic method for the analysis of chlortetracycline (CTC) using postcolumn fluorescence detection has been developed. After chromatographic separation of CTC on a polystyrene-divinylbenzene copolymer column, a highly fluorescent derivative isochlortetracycline (iso-CTC) was formed postcolumn in an on-line reaction coil with the addition of 25% NaOH (w/v). Chromatographic separation was achieved on a PRP-1 column, 15 cm x 4.6 mm, with 27:73 acetonitrile:0.2% perchloric acid (v/v), at 1.0 mL/min. Fluorescence derivatization was achieved by the on-line addition of 25% NaOH (w/v), at a flow rate of 0.2 mL/min, into the column eluant in a post-column reaction coil. The reaction coil was 9 m of teflon (1/16 in o.d., 0.3 mm i.d.) knitted into a six-sided coil. The fluorescent derivative was detected at lambda ex 355 nm and lambda em > 389 nm. Using this method after a simple sample cleanup, CTC can be detected in milk at 0.04 micrograms/mL, which is comparable to that obtained by microbiological assays. The detection method was linear between 0.02 micrograms/mL and 4 micrograms/mL. Because of the chromatographic separation, the method is more selective than microbiological assays and more sensitive than ultraviolet detection. With the chromatographic system described, the keto tautomeric forms of CTC and 4-epi-CTC are separated in a system which minimizes their formation on-column. In acidic aqueous organic solutions, the keto tautomer of CTC is the only product formed to any significant amount.     

Indirect fluorescence detection of amino sugars with the use of copper complexes of tryptophan and its analogues following high-performance liquid chromatographic separation

A simple, indirect fluorescence detection method has been developed for detecting specific mono-amino sugars (D-glucosamine, D-galactosamine, D-mannosamine) following chromatographic separation. The eluting amino sugars release L-tryptophan (L-Trp) from a copper-tryptophan complex which is introduced postcolumn. Analyte detection is based on measuring the increase in L-Trp fluorescence, which is quenched when complexed with copper. Two tryptophan analogues, 5-hydroxy-L-tryptophan (5-HTP) and DL-5-methoxytryptophan (5-MTP), were also evaluated as postcolumn reagents. 5-MTP was found to be a suitable alternative to L-Trp for the detection of these mono-amino sugars. Detection limits for D-glucosamine, D-galactosamine, and D-mannosamine are in the range of 0.15-0.30 nmol injected.     

Preconcentration based on paramagnetic microparticles for the separation of sarcosine using hydrophilic interaction liquid chromatography coupled with coulometric detection

Sarcosine has been identified as a potential prostate cancer marker. To provide determination of this compound, a number of methods are developing. In this study, we optimized a method for its separation by hydrophilic interaction LC with electrochemical detection (ED). Due to the fact that mobile phases commonly used for this type of separation altered the LODs measured by electrochemical detectors, we applied postcolumn dosing of buffer suitable for ED. The optimized conditions were mobile phase A acetonitrile, mobile phase B water in the ratio A/B 70:30, with postcolumn addition of mobile phase C (200 mM phosphate buffer pH 9). The optimal mixing ratio was A + B/C 1:1 with a flow rate of 0.80 mL/min (0.40 + 0.40 mL/min) and detection potential of 1000 mV. Due to the optimization of the parameters for effective separation, which had to meet the optimal parameters of ED, we reached a good resolution for separation also with a good LOD (100 nM). In addition, we successfully carried out sarcosine analysis bound on our modified paramagnetic microparticles with the ability to preconcentrate sarcosine isolated from artificial urine.     

Phycotoxins in seafood--toxicological and chromatographic aspects.

Two typical clinical types of algae-related seafood poisoning have attracted medical and scientific attention: paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP). Therefore, it became necessary to establish methods for the evaluation of possible hazards caused by contamination of seafood with these phycotoxins. Bioassays with mice or rats are the common methods for the determination of the toxin content of seafood. However, biological tests are not completely satisfactory because of a lack of sensitivity and pronounced variations. Additionally, there is growing opposition against animal testing. Therefore, many efforts have been undertaken to determine phycotoxins by chromatographic methods. PSP determination is mainly based on high-performance liquid chromatographic (HPLC) separation by ion-pair chromatography followed by postcolumn oxidation of the underivatized toxins in alkaline solution and fluorescence detection. HPLC methods for the determination of the DSP toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are characterized by precolumn derivatization with 9-anthryldiazomethane (ADAM) and/or 4-bromomethyl-7-methoxycoumarin (Br-Mmc), followed by chromatographic separation of the DSP esters formed and fluorescence detection. The chromatographic methods discussed in this review allow the rapid, sensitive and non-ambiguous determination of individual species of the two most important phycotoxins in seafood, PSP and DSP.     

Metal Ion Chromatography with Fluorescence Detection

Abstract

Nonpolar, agglomerated anion exchanger, and surface-sulfonated cation exchanger stationary phases have been used in conjunction with 8-hydroxyquinoline-5-sulfonic acid (HQS) in the eluent or as a postcolumn reagent for the separation and detection of a number of metals that form fluorescent HQS complexes. Several metals, notably those classified as transition metals, form nonfluorescent HQS chelates and quenches the fluorescence of other metal-HQS metal chelates. Such transition metals have been detected by introducing the fluorescent Al-HQS chelate postcolumn. Cation exchange stationary phases are the most useful for chromatographic applications involving HQS and are able to provide a variety of useful separations by tailoring elution conditions. Although not sensitive to Ba, the approach may be particularly good for the determination of the other alkaline earth metals. Fluorescence quenching resulting from Fe and Ni leaching from stainless steel chromatographic systems present a problem for trace analysis and accentuate the need for nonmetallic hardware. Subpicomole detection limits are attainable for Cd, Mg and Zn.     

Postcolumn HPLC detection of mono- and oligosaccharides with a chemosensor

Novel chromophoric compound 1 promotes the HPLC postcolumn detection of mono- and oligosaccharides. The detection of chromatographic peaks in the visible region for glucose, fructose, maltodextrins, sialic acid, and a ganglioside can be accomplished with a standard UV-vis detector. The use of selective, reversible binding agents in automated HPLC assays should allow for improved monitoring of specific analytes as well as material recovery. [structure: see text]     

还原性负离子的间接荧光检测离子色谱法

采用４个铈柱后反应和３价铈荧光检测的离子色谱法分离维生素Ｃ,亚硝酸根,硫代硫酸根,亚硫酸根,草酸根和碘离子６种还原性负离子,同时也给出了使用这种方法的一些最佳条件。     

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2‐(diethylamino)ethanol and triethanolamine. A buffer solution containing 2‐(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene‐2,3‐dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4–28) of proteins, compared with conventional absorbance detection.     

Ion-chromatographic screening method for monitoring arsenate and other anionic pollutants in ground waters of Northern Italy

A novel, rapid ion-chromatographic method for screening anionic pollutants in ground water, based on both conductivity and postcolumn spectrophotometric detection, has been developed. A relatively rapid separation of more than ten inorganic and polarizable anions was achieved by coupling an high capacity, hydroxide selective anion-exchange columns (Dionex IonPac AS16) supplied with an electrolytic eluent generator operating in gradient mode. The good control of the selectivity allowed the determination of polarizable anions including arsenate, thiocyanate, thiosulfate and perchlorate without interference from major components present at levels greater than 100 mg l⁻¹. This method was applied to the determination of arsenate in ground water samples collected in industrial and agricultural zones of Lombardia (Northern Italy). No traces of arsenate were detected in any sample, but added arsenate cannot be revealed by chromatographic analyses. This fact can be attributed to different causes, from reduction to the more reduced arsenic form to precipitation or dissolution in organic or inorganic based colloids. Oxidation with hydrogen peroxide seems to be useful for a partial recovery of added arsenate, but a stronger oxidation method, compatible with chromatographic separation, must be studied.     

Evaluation of Helically Coiled and Knitted Open Tubular Reactors for the Efficient Post-Column Determination of Tetrodotoxin by High-Performance Liquid Chromatography

A postcolumn fluorescence reaction system for the high-performance liquid chromatographic (HPLC) determination of tetrodotoxin in the silver-cheeked toadfish Lagocephalus sceleratus is discussed theoretically and investigated experimentally. Ion-pair chromatography with volatile ammonium perfluoroheptanoate was used for the separation of tetrodotoxin and 4,9-anhydrotetrodotoxin. The postcolumn reaction was based on the tetrodotoxin conversion to a quinazoline fluorescent compound under strong alkaline conditions. All postcolumn parameters were optimized that affected the sensitivity, dispersion, and stability. Helically coiled and knitted open tubular reactors composed of polyetheretherketone were constructed and characterized in detail. The performance of these reactors was evaluated on the basis of sensitivity and dispersion. Their optimal design is reported. The knitted reactors were more efficient than the relevant helically coiled reactors when higher reaction times are required. A 1500-µL polyetheretherketone knitted coil with 0.010″ internal diameter was optimum exhibiting higher pressure tolerances than Teflon coils. The HPLC postcolumn reaction method was evaluated in terms of linearity, sensitivity, accuracy, precision, and ruggedness. The linear dynamic ranges for tetrodotoxin and 4,9-anhydrotetrodotoxin were 40–3000 and 80–3000 ppb, respectively. The limits of detection and quantification were 12 and 41 ppb, respectively, for tetrodotoxin and 26 and 85 ppb, respectively, for 4,9-anhydrotetrodotoxin. The accuracy was evaluated by recovery measurements and the values for tetrodotoxin were between 90.7 and 93.6%. The use of a volatile perfuorocarboxylic acid as an ion-pair reagent allows confirmation of analytes in sample matrix by liquid chromatography tandem mass spectrometry using identical mobile phase conditions.     

Invetigations into the use of an auxiliary metal ion for indirect amperometri detection

Summary A method for determination of metal ions subsequent to separation by ion chromatography has previously been developed. The method is based on Indirect Amperometric Detection whereby the decrease in the oxidation current, due to a dithiocarbamate ligand added post column, is monitored. Upon elution from the chromatographic column the metal ions are complexed by the ligand. As the complexes formed are electroinactive at the applied potential, the background current decreases according to the metal ion concentration. The method developed in this work involves addition to the reagent of Zn(II) as a auxiliary metal ion to displace the analyte metals from the chromatographic eluent ligand complexes after separation. Sodium bis(2-hydroxy-ethyl) dithiocarbamate was used as the postcolumn derivatising reagent. The addition of Zn(II) to the reagent causes some unforeseen behaviour in the chromatographic system.     

Identification of products formed during UV irradiation of tamoxifen and their use for fluorescence detection in high-performance liquid chromatography

During the UV irradiation of tamoxifen, isomerization of the trans to the cis isomer takes place and consequently corresponding highly fluorescent phenanthrene derivatives are formed. Their formation can be used for the sensitive and selective detection of tamoxifen in high-performance liquid chromatography (HPLC). The structure of photoproducts was identified by 1H NMR spectroscopy, HPLC, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Owing to the variety of products formed and the higher selectivity and fluorescence response, on-line postcolumn photocyclization is preferred to the precolumn mode. A chromatographic system for the separation of isomers and photoproducts is suggested.     

Postcolumn determination of polyphenolic antioxidants in Cirsium vulgare (Savi) Ten. extracts

Novel methods for the determination of polyphenolic antioxidants present in extracts from inflorescences of Cirsium vulgare (Savi) Ten. based on ultra‐high performance liquid chromatography with photodiode array and chemiluminescence detection have been developed. Under the optimized conditions of chromatographic separation the analytical characteristic of the method was performed. The proposed method was successfully applied to the determination of ten polyphenols present in inflorescences of Cirsium vulgare . A comparison of the contents of analytes in extracts prepared by using various extraction media (methanol, ethanol, 70% methanol, 70% ethanol, and water) was carried out for the first time. For the postcolumn detection of scavenging activity of polyphenolic antioxidants against reactive oxygen species (H₂O₂, ^•OH, O₂^{• −}) three systems based on chemiluminescence of luminol were used. A review of the current scientific literature shows that this is the first report on the application of luminol‐based postcolumn detection for the on‐line investigation of ^•OH scavenging activity. The main compound determined in extracts from inflorescences of Cirsium vulgare was apigenin 7‐O‐glucuronide, whereas the highest antioxidant activity was observed for chlorogenic acid, luteolin 7‐O‐glucoside, and apigenin.     

间接荧光检测离子色谱法分析维生素C、亚硫酸根和硫代硫酸根

报道了一种用离子色谱分析维生素Ｃ、亚硫酸根和硫代硫酸根离子的新方法。在这种方法中采用了四价铈柱后氧化还原反应和三价铈荧光检测法。同时也给出了使用这种方法的一些最佳的实验条件。     

Liquid chromatographic analysis of glucosamine in commercial dietary supplements using indirect fluorescence detection

A method of using indirect fluorescence detection is evaluated for the analysis of glucosamine in commercial dietary supplements following chromatographic separation. In this method, the eluting analyte, glucosamine, was detected by monitoring an increase in the fluorescence signal for L-tryptophan (L-Trp) or DL-5-methoxytryptophan (5-MTP) after glucosamine complexed with a copper(II) ion and released either L-Trp or 5-MTP from a copper(II) complex, which is introduced postcolumn. The fluorescence of L-Trp and 5-MTP are quenched when complexed with the copper(II) ion. The results obtained using indirect fluorescence detection are compared with the results obtained for precolumn derivatization with phenylisothiocyanate. Statistical analysis is performed to compare the results obtained for the two postcolumn interaction components, Cu(L-Trp)2 and Cu(5-MTP)2, as well as the results obtained using the indirect fluorescence detection method and a precolumn derivatization method. The indirect fluorescence detection method provided an alternative to precolumn derivatization for determining the concentration of glucosamine in commercial dietary supplements. The stability of the glucosamine-o-phthalaldehyde-3-mercaptopropionic acid derivative is also evaluated to investigate the applicability of the popular precolumn derivatization reagent, o-phthalaldehyde-3-mercaptopropionic acid, for this analysis.     

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.     

A universal peroxyoxalate-chemiluminescence detection system for mobile phases of differing pH.

A universal peroxyoxalate-chemiluminescence detection system for high performance liquid chromatography, available for a variety of mobile phases, has been developed. The system consisted of a dual-head short-stroke pump and a chemiluminescence detector. The standard conditions using bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate were as follows. The first postcolumn solution was the mixture of 0.5 M imidazole-nitric acid (pH 7.5) and acetonitrile (1:4, v/v). The second was acetonitrile containing TCPO-hydrogen peroxide. These two solutions were delivered by the two pump-heads. After the pH of the column eluate was adjusted to the optimum range (6.5-7.5) by the first postcolumn solution, the solution was mixed with the second postcolumn solution. After flowing through a reaction coil, the chemiluminescence of the mixture was monitored. Using this system, a high sensitivity (fmol level) was obtained for perylene as an analyte with mobile phases having different pH values (2.0-8.0). Polycyclic aromatic hydrocarbons became detectable to a high sensitivity even after the column separation using an acidic mobile phase. The detection sensitivity of nitrated pyrenes after on-line electrochemical reduction using an acidic mobile phase was also increased. This system might be available for other aryl oxalates by some modifications of the postcolumn solutions.     

Postcolumn Iodine–Azide Reaction as a Detection System in HPLC for the Determination of Methylthiouracil in Urine

Abstract

This article describes a system for determining methylthiouracil in urine based on the sensitizing induction of methylthiouracil on the reaction between iodine and azide ions and combined techniques of high-performance liquid chromatography and simple postcolumn detection. The optimal conditions for iodine–azide reaction and high-performance liquid chromatographic separation were determined. The reproducibility, linearity, and recovery were evaluated under the optimal conditions. The methylthiouracil standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the postcolumn iodine–azide reaction), is linear within the concentration range of 0.4–5.0 nmol mL^?1 urine. The relative standard deviation values for precision and recovery within the calibration range varied from 0.9 to 4.8% and from 94 to 105%, respectively. Limits of detection (LOD) and quantitation (LOQ) were 0.3 and 0.4 nmol mL^?1 urine, respectively.     

Ion-exchange chromatography with an oxalic acid-α-hydroxyisobutyric acid eluent for the separation and quantitation of rare-earth elements in monazite and xenotime

Summary An oxalic acid-α-hydroxyisobutyric acid eluent has been used for the separation and determination of rare-earth elements by high-performance ion-exchange chromatography. Fifteen rare-earth elements were separated within less than 25 min on a 150×4.6 mm i.d. column packed with 5-μm sulfonic acid-bonded silica particles by elution with a combined gradient of 0.60–9.0 mM oxalic acid and 19.0–5.0 mM α-hydroxyisobutyric acid at pH 4.6. Detection and quantitation of the separated rare-earth elements was accomplished by visible-absorbance measurements at 600 nm after postcolumn reaction with arsenazo I. The gradient of the two complexing agents was optimized to enable the separation of yttrium(III) without interference from other elements, especially dysprosium(III) and terbium(III). Mass detection limits of the elements were in the range of 2–4 ng. Finally, the chromatographic system was applied to the quantitative analysis of rare-earth elements in monazite and xenotime.