Determination of Trace Proteins by Rayleigh Light Scattering Technique with Indophenol Blue

The interaction of indophenol blue (IPB) with proteins in aqueous solution has been studied by optical absorption and Rayleigh light scattering (RLS) spectroscopy. At pH 3.8, the weak RLS of IPB is enhanced by proteins. Based on this phenomenon, a novel method for the determination of proteins at nanogram levels using the RLS technique is developed. The method is simple, practical and sensitive. The linear range is 0.25–20.9µgmL^–1 for BSA, and 0.25–17.6µgmL^–1 for HSA. The detection limits (S/N=3) are 23ngmL^–1 for BSA and 22ngmL^–1 for HAS. The results for the determination of proteins in human serum samples are very close to those obtained by an established clinical method. There is very little interference from amino acids, metal ions or other coexisting compounds.     

Determination of Proteins at Nanogram Levels Using the Resonance Light Scattering Technique with a Novel PVAK Nanoparticle

A novel functionalized polyvinyl alcohol keto-derivative nanoparticle (PVAK) has been prepared in a one-step method using oxidation and degradation under ultrasonic irradiation. The nanoparticle is water-soluble, chemically stable, non-toxic and biocompatible. The surface of the nanoparticle is covered with abundant hydroxyl, carbonyl and carboxyl. At pH 3.0, the interactions of PVAK with different proteins can result in obviously enhanced RLS signals at 380nm. Under optimal conditions, the calibration graphs are linear over the range of 0.024.0µgmL^–1 for human serum albumin (HSA), 0.023.5µgmL^–1 for bovine serum albumin (BSA), and 0.053.5µgmL^–1 for human -globulin (-G), respectively. Detection limits were 6.4ngmL^–1 for HSA, 9.2ngmL^–1 for BSA, and 12.5ngmL^–1 for -G, respectively. The method was employed for the determination of total proteins in human serum with satisfactory results.     

Determination of Volatile Sulfur Compounds in Beverage and Coffee Samples by Purge-and-Trap On-Line Coupling with a Gas Chromatography-Flame Photometric Detector

A sensitive and fast analytical method using purge-and-trap on-line coupling with gas chromatography was developed for the determination of trace volatile sulfur compounds including dimethyl sulfide (DMS), ethyl-methyl sulfide (EMS), and dimethyl disulfide (DMDS) in beverage and coffee samples. The analytes were purged for 12min from the sample by high purity nitrogen at a flow rate of 35KPa and preconcentrated in the cooled fused-silica capillary trap at –75°C. The NaCl content in the samples was maintained at 10%. The volatile sulfur compounds were separated with an Agilent-6890 gas chromatograph by a suitable temperature program and detected by means of a flame photometric detector (FPD). The detection limits were 80ngL^–1 for DMS, 80ngL^–1 for EMS, and 100ngL^–1 for DMDS, respectively. This method was successfully applied to the determination of volatile sulfur compounds in different beverage and coffee samples.     

Determination of the Antibacterial Drug Enrofloxacin by Solid-Phase Spectrofluorimetry

A method for the determination of trace amounts of enrofloxacin based on solid-phase spectrofluorimetry has been developed. The relative fluorescence intensity of enrofloxacin fixed on Sephadex SP C-25 gel was measured directly after packing the gel beads in a 1-mm silica cell, using a solid-phase attachment. The wavelengths of excitation and emission were 277 and 448nm, respectively. The linear concentration range of application was 0.2–4.0ngmL^–1 of enrofloxacin, with a relative standard deviation of 1.2% (for a level of 2.0ngmL^–1) and a detection limit of 0.04ngmL^–1. The method was applied to the determination of enrofloxacin in commercial pharmaceutical formulations and spiked canine serum samples. It was validated using HPLC as a reference method and applying the standard addition methodology. Recovery levels of the method reached 100% in all cases.     

Determination of Nucleic Acids Based on their Resonance Light Scattering Enhancement Effect on Metalloporphyrin Derivatives

A new method for the determination of nucleic acids at nanogram per mL level is proposed based on the enhanced resonance light scattering (RLS) signal resulting from the interaction of metalloporphyrins with nucleic acids. Under optimum conditions, the weak RLS signal of metalloporphyrin is enhanced by nucleic acids, and the enhanced RLS intensity is proportional to the concentration of nucleic acids. The detection limits of calf thymus DNA were 3.5ngmL^–1, 2.9ngmL^–1 and 1.0ngmL^–1 for three metalloporphyrins, respectively. Synthetic samples were determined with satisfactory results.     

Determination of Heavy Metal Ions in Chinese Herbal Medicine by Microwave Digestion and RP-HPLC with UV-Vis Detection

A new method for the simultaneous determination of heavy metal ions in Chinese herbal medicine by microwave digestion and reversed-phase high-performance liquid chromatography (RP-HPLC) has been developed. The Chinese herbal medicine samples were digested by microwave digestion. Lead, cadmium, mercury, nickel, copper, zinc, and tin ions in the digested samples were pre-column derivatized with tetra-(4-chlorophenyl)-porphyrin (T₄-CPP) to form the colored chelates which were then enriched by solid phase extraction with C₁₈ cartridge and eluted from the cartridge with tetrahydrofuran (THF). The chelates were separated on a Waters Xterra RP₁₈ column by gradient elution with methanol (containing 0.05molL^–1 pyrrolidine-acetic acid buffer salt, pH=10.0) and THF (containing 0.05molL^–1 pyrrolidine-acetic acid buffer salt, pH=10.0) as mobile phase at a flow rate of 0.5mLmin^–1 and detected with a photodiode array detector in the range of 350–600nm. In the original samples the detection limits of lead, cadmium, mercury, nickel, copper, zinc and tin are 4ngL^–1, 3ngL^–1, 6ngL^–1, 5ngL^–1, 2ngL^–1, 6ngL^–1, and 4ngL^–1, respectively. This method was applied to the determination of lead, cadmium, mercury, nickel, copper, zinc and tin in Chinese herbal medicine samples with good results.     

Determination of Total Selenium Content in Cereals and Bakery Products by Flow Injection Hydride Generation Graphite Furnace Atomic Absorption Spectrometry Applying in-situ Trapping on Iridium-Treated Graphite Platforms

A flow injection hydride generation graphite furnace atomic absorption spectrometric (FI-HG-GFAAS) method was applied to the determination of Se in Se-doped and undoped cereals and bakery products. For the purpose of doping, the soils used for the cultivation of the cereals were dosed with Se-doped foliar fertilizers. The samples were dissolved in a mixture of HNO₃ and H₂O₂ solutions using microwave-assisted digestion. The decomposition of H₂Se generated from the sample solutions and the trapping of elemental Se were performed at a temperature of 300°C on an Ir-pretreated integrated graphite platform of a transversally heated graphite atomizer (THGA). For release of the trapped Se within a fairly short atomization time (5s), an atomization temperature of 2200°C was observed to be optimal. The overall efficiency of hydride generation, transport and trapping was 86%.The upper limit of the linear dynamic range of calibration was 10µgL^–1, which corresponds to 0.5µgg^–1 for solid samples. Recovery of the samples spiked with Se^VI solutions was found to be 93±6% on average. The relative standard deviation of the determinations was less than 8%. The limit of detection was found to be 0.06µgL^–1, corresponding to 3ngg^–1 for solid samples. The accuracy of the method was verified with the use of IAEA-155 (whey powder) certified reference material. End-capped THGA tubes resulted in an extension of the linear calibration range compared to that of standard THGAs.The Se content in bakery products made of undoped cereals ranged from 7.7 to 68ngg^–1 (wet weight) in 18 samples, whereas the Se content of the corresponding cereals was found to be below 100ngg^–1 (wet weight). The Se level of cereals grown on soils treated with Se-doped fertilizers ranged from 128 to 1046ngg^–1 (wet weight), and it depended linearly on the Se concentration of the corresponding foliar fertilizer.     

Flame Atomic Absorption Spectrometric Determination of Gold and Palladium Using Microcolumn On-line Preconcentration and Separation

A microcolumn on-line preconcentration and separation system was developed for the flame atomic absorption spectrometric (FAAS) determination of trace levels of gold and palladium. The analytes were selectively adsorbed onto the microcolumn packed with 2-mercaptothiazole immobilized silica gel (MBTSG) in an acidity range of 0.1 to 6.0M HCl at a sampling flow rate of 4.0mLmin^–1. The analytes adsorbed could be desorbed by a thiourea solution with a flow rate of 2.0mLmin^–1. Most of the common coexisting metal ions at a concentration of 25.0mgmL^–1 and anions at a concentration of 50.0mgmL^–1 did not interfere with the preconcentration and determination of Au and Pd. The limits of detection (LOD), defined as three times the standard deviation of the blank (3), of Au and Pd are 10ngmL^–1 and 26ngmL^–1, respectively, with a preconcentration time of 60s. The relative standard deviation (RSD) is about 2.0% for 0.20µgmL^–1 Au and 0.30µgmL^–1 Pd. With a sample loading time of 30min, 6.7ngmL^–1 Au and 10ngmL^–1 Pd can be preconcentrated quantitatively. A geological sample, an anode slime and a secondary nickel alloy were successfully determined with the proposed method, and the results obtained showed good agreement with the certified values.Received December 23, 2002; accepted May 14, 2003 Published online August 8, 2003     

Development of an FI-ICP Method for On-Line Preconcentration and Determination of Platinum

This paper presents a new simple and rapid procedure for the preconcentration and determination of platinum. It is based on the adsorption of the metal ion and preconcentration on a micro-column (3cm×3mm) placed in the injection valve of a flow injection (FI) manifold and packed with 1,5-bis[(2-pyridyl)-3-sulphophenyl-methylene]thiocarbonohydrazide (PSTH) immobilised on an anion-exchange resin (Dowex 1X8-200). The metal was eluted from the column using a solution of 2M HNO₃. Various parameters and chemical variables affecting the preconcentration and determination of this metal by ICP-AES were evaluated. Five variables (sample flow rate, eluent flow rate, nebulizer flow rate, buffer concentration and mixing coil length) were considered as factors in the optimisation process. Interactions between analytical factors, and their optimal levels were investigated using two level factorial and central matrix designs. The optimum conditions established were applied to the determination of platinum by flow injection inductively coupled plasma atomic emission spectrometry (FI-ICP-AES). The method has a linear calibration range of 25 to at least 200ngmL^–1 with a detection limit of 7.4ngmL^–1 (S/N=3) and a throughput of 10 samples h^–1 using 5min. preconcentration time. The precision of the method (RSD) was 3.06% ngmL^–1 at the 50ngmL^–1 level of Pt(IV) and 2.93% at the 150ngmL^–1 level. The accuracy of the method was examined by determining the analyte content in spiked waters and by analysing an automobile catalyst standard reference material. The results show good agreement with the certified value and sufficiently high recoveries.     

Magnetic Separation Immunoassay for Digoxin in Plasma with Flow Injection Fluorescence Detection

Immunoassay (IA) is a sensitive and selective approach for low level quantitation of drugs. Magnetic separation immunoassays use magnetic beads to facilitate the separation of bound labeled antigens from free antigens in solution. Digoxin was chosen for this study because low level analysis (ngmL^–1) in biological samples isrequired, antibodies to digoxin were commercially available and derivatization procedures for fluorescence labeling were well established. A competitive immunoassay format was used in this study. Streptavidin coated magnetic beads were attached to biotinylated anti-digoxin antibodies for the separation. The inhibition curve for off-line magnetic separation immunoassay of digoxin in spiked plasma was characterized and the dynamic range of the curve was 0.25–2.5ngmL^–1. A power fit weighted by the inverse of concentration was found to provide the best fit to the data (r=0.9934). The percent RSDs for the two controls, 0.8 and 2.2ngmL^–1, were 9.95% and 20.62% (n=6) and the percent errors were 11.75% and 22.63% (n=6), respectively. The limit of detection (LOD) in plasma is 0.14ngmL^–1. The dynamic range of the inhibition curve for on-line magnetic separation immunoassay of digoxin was 0.5–15ngmL^–1 of digoxin. A quadratic fit was found to provide the best fit to the data (r=0.9937). The percent RSDs for the two controls, 4.0 and 12ngmL^–1, were 14.1% and 10.7% (n=6) and the percent errors were 5.8% and 3.3% (n=6) from the spiked value, respectively. The LOD was estimated to be 0.44ngmL^–1 (determined as two times the standard deviation of the blank, n=6). The on-line method has the advantages of being relatively easy to automate in the continuous flow mode and is adaptable for use in conjunction with HPLC separations.     

Kinetic Spectrophotometric Determination of Trace Aluminum by Catalytic Activity of Al<Superscript>3+</Superscript> on the Oxidation of Methylene Blue by Hydrogen Peroxide

A novel kinetic spectrophotometry for the determination of trace aluminum is described based on the catalytic activity of Al³⁺ on the redox reaction between methylene blue (MB) and hydrogen peroxide in acetate buffer (pH 3.8). The reaction is monitored spectrophotometrically by measuring the decrease in absorbance of the dye at the maximum absorption wavelength of 670nm. The variables that affected the reaction rate were fully investigated, and the optimum conditions were established. Aluminum can be determined in the range of 0–80ngmL^–1 with a detection limit of 1.0ngmL^–1. The relative standard deviation for 10 replicate determinations of 40ngmL^–1 aluminum is 0.9%. The method was used to determine aluminum in tap water and biological samples and produced satisfactory results.Received December 18, 2002; accepted May 5, 2003 published online August 22, 2003     

Voltammetric Behavior of Dopamine at ct-DNA Modified Carbon Fiber Microelectrode

A sub-micrometer thin-layer DNA modified carbon fiber microcylinder electrode was prepared by electrodeposition of ct-DNA at 1.5V (vs. Ag/AgCl). The voltammetric behavior of dopamine (3-hydroxytyramine) was investigated at the modified electrode. It was found that the modified electrode exhibits a highly electrocatalytic activity toward dopamine oxidation. Differential pulse voltammetry was used for determination of dopamine in pH 7.4 phosphate buffer solution. A linear response of the peak current versus the concentration was found in the range of 4×10^–6 to 10^–4molL^–1 at 10^–4molL^–1 AA (ascorbic acid) coexistence (R=0.9959) and the range of 6×10^–5 to 10^–3molL^–1 at 10^–3molL^–1 AA (R=0.9960). The presence of a high concentration of ascorbic acid did not interfere with the determination. The proposed method exhibited good recovery and reproducibility. This method can be applied to the detection of DA in real samples.     

Use of 2,4-dinitrophenylhydrazone of glyoxylic acid for the determination of glyoxylic acid by the chromatographic-spectrophotometric method and by differential pulse polarography

Two different methods (one based on chromatography and spectrophotometry and the other on polarography) have been developed for the determination of glyoxylic acid in the form of a derivative with 2,4-dinitrophenylhydrazine (DNPHGA). The TLC method allows the separation of two DNPHGA isomers (trans and cis). Spectrophotometric measurements of the eluents of the separated compounds (=360 nm) allow the determination of GA within the range from 4 to 30 g. Using differential pulse polarography, the conditions of DNPHGA formation were examined. The reduction peak of this derivative (E_P=–0.430 V), observed by dpp, was used to develop a polarographic determination of GA within the concentration range from 110^-4 to 710^–4 mol/l.     

Investigation of Medium Power Radiofrequency Capacitively Coupled Plasmas and Their Application to Atomic Emission Spectrometry for the Determination of Aluminium in Water Samples

A medium power radiofrequency capacitively coupled plasma (275W, 27.12MHz) with low argon consumption (0.5Lmin^–1) was investigated to be used for the determination of aluminium in water samples pneumatically nebulised by atomic emission spectrometry. The plasma torch was operated in two geometric configurations, coaxial and coaxial-annular with single (SRTr.f.CCP) or double ring electrodes (DRTr.f.CCP), respectively. The optimum experimental parameters (observation height, Ar flow rate, distance between ring electrodes, matrix interference of Na⁺ and Ca²⁺ and anion nature) for Al 396.152nm emission were established. Under optimum operating conditions of the DRTr.f.CCP torch (6cm distance between annular electrodes), a detection limit of 70ngmL^–1 aluminium was found. Both operating parameters and several figures of merit of the investigated plasmas were compared to those of microwave plasmas and inductively coupled plasma used in atomic emission spectrometry. The method was used to control the quality of drinking and industrial water. Samples were mineralised with 2mL of 1+1 HNO₃ and 10mL of 1+1 HCl. In the range of 0.15–3.5mgL^–1 Al³⁺ the relative standard deviation varied between 3.7 and 8.6% (n=3). For two certified drinking water samples containing 0.206 and 0.218mgL^–1 Al³⁺ the recovery (n=5) was 97.6±6.8% and 98.6±7.3%, respectively.     

Determination of Na,K, Rb and Cs Distribution in Human Brain Using Neutron Activation Analysis

This study aims to investigate the distribution of Na, K, Rb and Cs in human brains (5 individuals, 12 brain parts, mean age: 75 years). Distribution of the trace metals between lipid fraction and brain tissue was investigated in solvent extraction experiments. Determinations were carried out by instrumental neutron activation analysis. The present results show a rather non-homogeneous distribution for Na and a relatively uniform distribution for K, Rb and Cs. The mean concentrations found are 7440µgNag^–1 dry weight, 12800µgKg^–1, 14µgRbg^–1 and 50ngCsg^–1. A highly significant positive correlation was found between Rb and Cs. Solvent extraction experiments showed that 19% of Rb and 26% of Cs of the total content is located in lipid fraction.     

Determination of Iodine in Enriched Chlorella by ICP-OES in the VUV Region

A method of determining iodine in the vacuum ultraviolet region by inductively coupled plasma optical emission spectrometry in enriched Chlorella is established and evaluated. Alga Chlorella samples enriched with iodine were solubilized with tetramethylammonium hydroxide. The iodine content was quantified by inductively coupled plasma optical emission spectrometry using the 182.980nm emission line of iodine to avoid the spectral interference from phosphorus which occurs in Chlorella at the 178.218nm emission line of iodine. The methods accuracy was confirmed by a modified Sandell and Kolthoff spectrophotometric method (As³⁺, Ce⁴⁺ and brucine) after alkaline ashing, and the agreement between the methods was about 96%. The limit of detection in real samples was 3mgkg^–1 for enriched Chlorella, and the iodine content in the samples varied between 100 and 1300mgkg^–1.     

Determination of Nucleic Acids Based on Shifting the Association Equilibrium between a Heptamethine Cyanine Dye and Poly-Lysine

A near-infrared (near-IR) fluorescence recovery method for the determination of nucleic acids is presented. This method employs a two-reagent system composed of anionic heptamethines cyanine (HMC) and polycationic poly-lysine. The fluorescence of HMC, with maximum excitation and emission wavelengths at 778 and 804nm, respectively, was quenched by poly-lysine in proper concentration, but recovered by adding nucleic acids. Under optimal conditions, the recovered fluorescence was proportional to the concentration of nucleic acids. The calibration graphs are linear over the range of 5–300ngmL^–1 for herring sperm DNA (FS DNA), 2–100ngmL^–1 for calf thymus DNA (CT DNA) and 5–500ngmL^–1 for snake ovum RNA (SO RNA). The corresponding detection limits are 1.49ngmL^–1 for FS DNA, 0.7ngmL^–1 for CT DNA and 1.61ngmL^–1 for SO RNA, respectively. Four synthetic and three real nucleic acid samples were determined with satisfactory results.     

Simple and Rapid Method for Determination of Selenium in Breast Milk by HG-AFS

A digestion procedure using H₂SO₄/HNO₃/H₂O₂ was found to be effective for destruction of human milk samples. In conjunction with a sensitive hydride generation atomic fluorescence spectrometry detection system, it is suitable for determination of selenium in those samples where the available mass of breast milk and the low selenium concentration are limiting factors. Only 1g of milk sample is needed. The procedure is simple, rapid and of low contamination potential since it is performed in the same Teflon tube from weighing to measurement. The digestion of 20 samples is completed in three hours. The detection limit is 0.25±0.04ngg^–1 of a measured solution of sample or 2.5ngg^–1 of milk. The relative uncertainty is 10% (coverage factor of 2.3, 95% probability). Because of these advantages the method is particularly suitable for epidemiological studies. The mean concentration of selenium in 62 samples of human milk from lactating women residing in the North East of Italy was 12±3ngg^–1, which is in the range of reference data.     

Determination of Iron in Tap and Waste Water Using Liquid–Liquid Extraction in a Flow-Injection System

A flow-injection procedure for the determination of iron(III) in water is described. The procedure is based on the formation of an ion pair between the tetraphenylarsonium (Ph₄As⁺) (TPA) or tetrabutylammonium (But₄N⁺) (TBA) cations and the tetrathiocyanatoferrate(III) complex (TTF). This ion pair is extracted with chloroform, and the absorbance of the organic phase is measured at 503nm (for Ph₄As⁺) or 475nm (for But₄N⁺). Iron concentrations higher than 0.9×10^–6molL^–1 (50µgL^–1) can be detected in the first case, with a relative standard deviation of 1.9% (n=12), a linear application rangeof between 1.34 and 54.0×10^–6molL^–1 (75–3015µgL^–1), and a sampling frequency of 30h^–1. For the ion pair with But₄N⁺, the detection limit is 0.52×10^–6molL^–1 (29µgL^–1), with a relative standard deviation of 1.6% and a linear application range between 0.73 and 54.0×10^–6molL^–1. Under the proposed working conditions, only Pd(IV), Cu(II) and Bi(III) interfere. With the application of the merging zones technique, considerable amounts of organic reagent can be saved. The TBA method was applied to the analysis of iron(III) in tap and industrial waste waters.     

Determination of Attogram Quantities of Silver via Quenching of the Solid Substrate Room-Temperature Phosphorescence of Rhodamine B Based on the Catalysis of its Oxidation by Potassium Persulfate

A new method of SS-RTP for the determination of trace silver has been established. This method is based on the fact that Ag⁺, when activated by ,-bipyridyl (bipy) in a media of HAc–NaAc (pH=4.9), can catalyze the reaction of Rhodamine B (RhoB) oxidized by K₂S₂O₈, thus causing the Solid Substrate Room-Temperature Phosphorescence (SS-RTP) of RhoB to be quenched. The activating efficiency of bipy is 6.7 times higher than that of o-phenanthroline (phen). The reduction of the phosphorescence intensity (I_p) of RhoB is directly proportional to the concentration of Ag⁺ ions in the range of 1.6016.0agspot^–1 (0.40µLspot^–1). The regression equation of the working curve can be expressed as I_p=18.78+5.100m_Ag+ (agspot^–1) (r=0.9994, n=6), the detection limit is 0.28agspot^–1. This rapid, accurate and sensitive method has been successfully applied to the determination of trace silver in tea and human hair samples, and the results agree well with the Atomic Absorption Spectroscopy (AAS) method. The mechanism of the catalyzing reaction is also discussed.