Fluorimetric determination of phytic acid in urine based on replacement reaction

A sensitive fluorimetric method for determination of phytic acid in human urine samples was described. The method was based on a fluorimetric replacement reaction, in which the added phytic acid replaced the Cu²⁺ ion from Cu²⁺-gelatin complex, liberating the fluorescent gelatin molecule. The fluorescence of the solution was accordingly recovered proportionally to the amount of the foreign phytic acid. The excitation wavelength was 273.5 nm and the characteristic emission wavelength was 305.0 nm, respectively. The calibration graph was obtained by plotting the recovered fluorescent intensity at maximum 305.0 nm against the added standard phytic acid, and was divided into two sections. One section was linear over the range of 0.40-2.40 mg L⁻¹ with a linear regression equation of I_f = −0.895 + 15.146c (R² > 0.9993), and the other over the range of 2.40-9.20 mg L⁻¹ with a linear regression equation of I_f = −29.526 + 26.113c (R² > 0.9996), respectively. The relative standard deviation (R.S.D.) at 95% confidence degree for a 2.0 mg L⁻¹ of standard phytic acid within 1 month was less than 1.26% (n = 5), indicating the procedure is reproducible. The detection and the quantification limits of phytic acid were estimated to be 0.23 and 0.40 mg L⁻¹, respectively. The proposed method was applied to the determination of phytic acid in urine samples and the found concentrations of phytic acid in urine were in the range of 0.49-0.75 mg L⁻¹ with recoveries of 96.2-108.8%. Comparison of the obtained results with the reported HPLC was performed, indicating the proposed method was reliable.     

A green analytical procedure for flow-injection determination of nitrate in natural waters

Nitrate determination in waters is generally carried out with cadmium filings and carcinogenic reagents or by reaction with phenolic compounds in highly concentrated sulfuric acid medium. In this work, it was developed a green analytical procedure for nitrate determination in natural waters based on direct spectrophotometric measurements in ultraviolet, using a flow-injection system with an anion-exchange column for separation of nitrate from interfering species. The proposed method employs only one reagent (HClO₄) in a minimum amount (equivalent to 18 μL concentrated acid per determination), and allowed nitrate determination within 0.50-25.0 mg L⁻¹, without interference of up to 200.0 mg L⁻¹ humic acid; 1.0 mg L⁻¹ NO₂⁻; 200.0 mg L⁻¹ PO₄³⁻; 75.0 mg L⁻¹ Cl⁻; 50.0 mg L⁻¹ SO₄²⁻ and 15.0 mg L⁻¹ Fe³⁺. The detection limit (99.7% confidence level) and the coefficient of variation (n = 20) were estimated as 0.1 mg L⁻¹ and 0.7%, respectively. The results obtained for natural water samples were in agreement with those achieved by the reference method based on nitrate reduction with copperized cadmium at the 95% confidence level.     

Sensitive sequential injection determination of naproxen based on interaction with beta-cyclodextrin

A sensitive sequential injection analysis (SIA) methodology for the fluorimetric determination of naproxen is proposed. The developed automatic analytical procedure is based on the complexation of naproxen with β-cyclodextrin (β-CD) yielding an enhanced fluorimetric signal (λ_ex = 280 nm, λ_em = 356 nm).Linear calibration plots were obtained for naproxen concentrations up to 1 × 10⁻⁵ mol l⁻¹. The developed methodology exhibited a good precision, with a R.S.D. < 2.1% (n = 15). The detection limit of the determination was 1.9 × 10⁻⁷ mol l⁻¹ with a sampling rate of about 70 h⁻¹. The automatic method was applied to the determination of naproxen in pharmaceutical formulations. The obtained results were compared with those furnished by the reference procedure and the relative deviations were lower than 3.6%. No interference was found from the excipients usually used in solid pharmaceutical formulations.     

On-line analysis of volatile fatty acids in anaerobic treatment processes

In this paper, an on-line spectrofluorimetric system is proposed for a simple, rapid and accurate measurement of volatile fatty acids (VFA) in anaerobic treatment processes. The determination method is based on the derivatization of VFA with N-(1-naphthyl)ethylenediamine (EDAN) followed by a spectrofluorimetric detection of the corresponding amide. The analytical procedure is automated with a flow analysis technique, coupling multisyringe (MSFIA) and multi-pumping (MPFS) methods. Operative conditions have been investigated with a special attention paid to the activation and amidation steps and to the liquid-liquid extraction of the derivatized final product. Fluorescence intensities (λ_em = 335 nm, λ_ex = 395 nm) were found to be proportional to the concentration of VFA, expressed as acetic equivalent, in the range 19-1000 mg L⁻¹, with a detection limit (3σ) of 5.1 mg L⁻¹. Our results showed a good selectivity for VFA as compared to other organic and inorganic compounds usually found in sewage sludges. Validation of the on-line system developed has been assessed by application of the procedure to aqueous samples originating from sewage sludge treatment plants. The results were in good agreement with ion chromatography measurements.     

A flow injection procedure based on solenoid micro-pumps for spectrophotometric determination of free glycerol in biodiesel

A flow system designed with solenoid micro-pumps is proposed for fast and greener spectrophotometric determination of free glycerol in biodiesel. Glycerol was extracted from samples without using organic solvents. The determination involves glycerol oxidation by periodate, yielding formaldehyde followed by formation of the colored (3,5-diacetil-1,4-dihidrolutidine) product upon reaction with acetylacetone. The coefficient of variation, sampling rate and detection limit were estimated as 1.5% (20.0 mg L⁻¹ glycerol, n = 10), 34 h⁻¹, and 1.0 mg L⁻¹ (99.7% confidence level), respectively. A linear response was observed from 5 to 50 mg L⁻¹, with reagent consumption estimated as 345 μg of KIO₄ and 15 mg of acetylacetone per determination. The procedure was successfully applied to the analysis of biodiesel samples and the results agreed with the batch reference method at the 95% confidence level.     

A multi-pumping flow-based procedure with improved sensitivity for the spectrophotometric determination of acid-dissociable cyanide in natural waters

An analytical procedure with improved sensitivity was developed for cyanide determination in natural waters, exploiting the reaction with the complex of Cu(I) with 2,2′-biquinoline 4,4′-dicarboxylic acid (BCA). The flow system was based on the multi-pumping approach and long pathlength spectrophotometry with a flow cell based on a Teflon AF 2400^® liquid core waveguide was exploited to increase sensitivity. A linear response was achieved from 5 to 200 μg L⁻¹, with coefficient of variation of 1.5% (n = 10). The detection limit and the sampling rate were 2 μg L⁻¹ (99.7% confidence level), and 22 h⁻¹, respectively. Per determination, 48 ng of Cu(II), 5 μg of ascorbic acid and 0.9 μg of BCA were consumed. As high as 100 mg L⁻¹ thiocyanate, nitrite or sulfite did not affect cyanide determination. Sulfide did not interfere at concentrations lower than 40 and 200 μg L⁻¹ before or after sample pretreatment with hydrogen peroxide. The results for natural waters samples agreed with those obtained by a fluorimetric flow-based procedure at the 95% confidence level. The proposed procedure is then a reliable, fast and environmentally friendly alternative for cyanide determination in natural waters.     

Multi-pumping flow system for the determination of dissolved orthophosphate and dissolved organic phosphorus in wastewater samples

A multi-pumping flow system (MPFS) for the spectrophotometric determination of dissolved orthophosphate and dissolved organic phosphorus in wastewater samples is proposed. The determination of orthophosphate is based on the vanadomolybdate method. In-line ultraviolet photo-oxidation is employed to mineralise organic phosphorus to orthophosphate prior to detection. A solenoid valve allows the deviation of the flow towards the UV-lamp to carry out the determination of organic phosphorus.Calibration was found to be linear up to 20 mg P L⁻¹, with a detection limit (3s_b/S) of 0.08 mg P L⁻¹, an injection throughput of 75 injections h⁻¹ and a repeatability (R.S.D.) of 0.6% for the direct determination of orthophosphate. On the other hand, calibration graphs were linear up to 40 mg P L⁻¹, with a detection limit (3s_b/S) of 0.5 mg P L⁻¹, an injection throughput of 11 injections h⁻¹ and a repeatability (R.S.D.) inferior to 2.3% for the procedures involving UV photo-oxidation.     

A reagent-free method based on a photo-induced fluorimetry in a sequential injection system

According to the current demands of environmentally friendly analytical chemistry and with a view to achieving lower reagent consumption with improved analytical performance, an automatic methodology composed of a photoreactor and fluorimetric detection (λ_exc = 287 nm, λ_em = 378 nm) was developed. To this end, a sequential injection analysis (SIA) system was developed for indomethacin determination using ultra-violet (UV) light which promotes an increase in the fluorescence of indomethacin. This increase in sensitivity makes it possible to apply this methodology to a dissolution test and to determine indomethacin in pharmaceutical formulations.The calibration graph for indomethacin was linear between 4.10 × 10⁻⁶ and 9.00 × 10⁻⁵ mol L⁻¹and the detection limit was 1.23 × 10⁻⁶ mol L⁻¹. The method was proven to be reproducible with a R.S.D. < 5% and sampling rate of approximately 20 per hour. The potential effect of several compounds commonly used as excipients on analytical signals was studied and no interfering effect was observed. Statistical evaluation at the 95% confidence level showed good agreement between the results obtained for the pharmaceutical samples with both the SIA system and comparison batch procedures.     

Hybrid sequential injection-flow injection manifold for the spectrophotometric determination of total sulfite in wines using o-phthalaldehyde and gas-diffusion

A new automated spectrophotometric method for the determination of total sulfite in white and red wines is reported. The assay is based on the reaction of o-phthalaldehyde (OPA) and ammonium chloride with the analyte in basic medium under SI conditions. Upon on-line alkalization with NaOH, a blue product is formed having an absorption maximum at 630 nm. The parameters affecting the reaction - temperature, pH, ionic strength, amount concentration and volume of OPA, amount concentration of ammonium chloride, flow rate and reaction coil length - and the gas-diffusion process - sample and HCl volumes, length of mixing coil, donor flow rate - were studied. The proposed method was validated in terms of linearity (1-40 mg L⁻¹, r = 0.9997), limit of detection (c_L = 0.3 mg L⁻¹) and quantitation (c_Q = 1.0 mg L⁻¹), precision (s_r = 2.2% at 20 mg L⁻¹ sulfite, n = 12) and selectivity. The applicability of the analytical procedure was evaluated by analyzing white and red wine samples, while the accuracy as expressed by recovery experiments ranged between 96% and 106%.     

Reversed flow injection spectrophotometric determination of chlorate

An interfacing has been developed to connect a spectrophotometer with a personal computer and used as a readout system for development of a simple, rapid and sensitive reversed flow injection (rFI) procedure for chlorate determination. The method is based on the oxidation of indigo carmine by chlorate ions in an acidic solution (dil. HCl) leading to the decrease in absorbance at 610 nm. The decrease in absorbance is directly related to the chlorate concentration present in the sample solutions. Optimum conditions for chlorate were examined. A linear calibration graph over the range of 0.1-0.5 mg L⁻¹ chlorate was established with the regression equation of Y = 104.5X + 1.0, r² = 0.9961 (n = 6). The detection limit (3σ) of 0.03 mg L⁻¹, the limit of quantitation (10σ) of 0.10 mg L⁻¹ and the RSD of 3.2% for 0.3 mg L⁻¹ chlorate (n = 11) together with a sample throughput of 92 h⁻¹ were obtained. The recovery of the added chlorate in spiked water samples was 98.5 ± 3.1%. Major interferences for chlorate determination were found to be BrO₃⁻, ClO₂⁻, ClO⁻ and IO₃⁻ which were overcome by using SO₃²⁻ (as Na₂SO₃) as masking agent. The method has been successfully applied for the determination of chlorate in spiked water samples with the minimum reagent consumption of 14.0 mL h⁻¹. Good agreement between the proposed rFIA and the reference methods was found verified by Student's t-test at 95% confidence level.     

Development of a novel flow injection liquid-liquid microextraction method for the on-line separation and preconcentration for determination of zinc(II) using 5-(8-hydroxy-2-quinolinylmethyl)-2,8-dithia-5-aza-2,6-pyridinophane as a sensitive and selective fluorescent chemosensor

A novel flow injection analysis (FIA) system based on liquid-liquid microextraction and fluorimetric determination was developed for the determination of traces of the Zn²⁺ ion using 5-(8-hydroxy-2-quinolinylmethyl)-2,8-dithia-5-aza-2,6-pyridinophane (L) as a sensitive and selective fluorimetric sensor, with λ_ex = 373 nm and λ_em = 530 nm, and hexanol as the extracting organic solvent. In the designed FIA system, the phase separation takes place via gravitation forces in the absence of any segmenter. The influence of pH and ionic strength of the solution, amount of ligand, nature of counter ion, volume of organic solvent, extraction time and coil length was investigated. Under optimized experimental conditions, the calibration curve found to be liner over a concentration range of 0.025-4.53 μg mL⁻¹ (R² = 0.9951) with a limit of detection of 2.3 ng mL⁻¹. The enrichment factor was 45 and relative standard deviation for 7 replicate determinations was 2.43%. The method is very fast and uses low levels of organic solvents. The proposed method was applied successfully to the determination of zinc(II) in human hair, human serum and two inorganic sludge samples.     

Spectrofluorimetric determination of tannins based on their activative effect on the Cu(II) catalytic oxidation of rhodamine 6G by hydrogen peroxide

A simple and highly sensitive kinetic fluorimetric method is proposed for the determination of trace tannins, based on the activation of tannins on the oxidation of rhodamine 6G (Rh 6G) by hydrogen peroxide catalyzed by Cu(II) ion. The calibration graph was rectilinear in the range 0.08-1.28 mg l⁻¹ for tannin, the 3σ detection limit for tannin is 0.0455 mg l⁻¹. The relative standard deviation for 11 determinations of 0.4 mg l⁻¹ tannin is 0.96%. The proposed method has been successfully used to determine tannins in tea and Chinese gall. The results obtained were compared with those provided by the Folin-Ciocalteu method. This is the first procedure to be reported for the determination of tannins based on fluorimetric measurements.     

Fluorimetric determination of aminocaproic acid in pharmaceutical formulations using a sequential injection analysis system

A sequential injection analysis (SIA) methodology for the fluorimetric determination of aminocaproic acid in pharmaceutical formulations is proposed. The developed analytical procedure is based on the derivatisation reaction of the aminocaproic primary amine with o-phthalaldehyde (OPA) and N-acetylcysteine (NAC) and fluorimetric detection of the formed product (λ_ex = 350 nm; λ_em = 450 nm). The implementation of a SIA flow system allowed for the development of a simple, fast and versatile automated methodology, which exhibits evident advantages regarding the US Pharmacopoeia 24 (USP 24) reference procedure. By combining the SIA time-based sample insertion with a subsequent zone sampling approach, which permitted to select for detection of a well-defined sample zone, it was possible to implement an on-line dilution strategy that enabled the expansion of the analytical working range of the methodology, and thus its application in dissolution studies, without manifold re-configuration.Linear calibration plots were obtained for aminocaproic acid concentrations up to 6 × 10⁻⁵ mol l⁻¹. The developed methodology exhibit a good precision, with a R.S.D. < 2.0% (n = 15) and the detection limit was 2.5 × 10⁻⁷ mol l⁻¹. The obtained results complied with those furnished by the reference procedure with a relative deviation lower than 1.2%. No interference was found.     

Selective stopped-flow sequential injection method for the spectrophotometric determination of titanium in dental implant and natural Moroccan phosphate rock

The present work reports the first sequential injection (SI) method for the spectrophotometric determination of Ti(IV). The method is based upon the reaction of Ti(IV) with chromotropic acid (CA) in acidic medium to form a water-soluble complex (λ_max = 420 nm). The chemical and instrumental variables of the system that affected the reaction were studied. Selectivity was greatly enhanced using ascorbic acid. A linear calibration graph was obtained in the range 0.2-10.0 mg l⁻¹ Ti(IV) at a sampling frequency of 24 h⁻¹. The precision was satisfactory (s_r = 1.5% at 5.0 mg l⁻¹ Ti(IV), n = 12) and the 3σ limit of detection, c_L, was 0.7 mg l⁻¹ (n = 10). The developed method proved to be adequately selective and was applied successfully to the analysis of real samples (dental implant and natural Moroccan phosphate rock) giving accurate results based on recovery studies (98-105%).     

Fluorescence and terbium-sensitised luminescence determination of garenoxacin in human urine and serum

Fluorescence and terbium-sensitised luminescence properties of new quinolone garenoxacin have been studied. The fluorimetric method allows the determination of 0.060-0.600 μg ml⁻¹ of garenoxacin in aqueous solution containing HCl/KCl buffer (pH 1.5) with λ_exc=282 nm and λ_em=421 nm. Micellar-enhanced fluorescence was also studied, leading to a higher than 400% increase in analytical signal in presence of 12 mM sodium dodecyl sulphate (SDS), allowing the determination of 0.020-0.750 μg ml⁻¹ of garenoxacin. The terbium-sensitised luminescence method allows the determination of 0.100-1.500 μg ml⁻¹ of garenoxacin in 12 mM SDS solution containing 0.08 M acetic acid/sodium acetate buffer (pH 4.1) and 7.5 mM Na₂SO₃ (chemical deoxygenation agent), with λ_exc=281 nm and λ_em=546 nm. Relative standard deviation (R.S.D.) values for the three methods were in the range 1.0-2.0%. The proposed procedures have been applied to the determination of garenoxacin in spiked human urine and serum.     

Sequential injection methodology for carbon speciation in bathing waters

A sequential injection method (SIA) for carbon speciation in inland bathing waters was developed comprising, in a single manifold, the determination of dissolved inorganic carbon (DIC), free dissolved carbon dioxide (CO₂), total carbon (TC), dissolved organic carbon and alkalinity. The determination of DIC, CO₂ and TC was based on colour change of bromothymol blue (660 nm) after CO₂ diffusion through a hydrophobic membrane placed in a gas diffusion unit (GDU). For the DIC determination, an in-line acidification prior to the GDU was performed and, for the TC determination, an in-line UV photo-oxidation of the sample prior to GDU ensured the conversion of all carbon forms into CO₂. Dissolved organic carbon (DOC) was determined by subtracting the obtained DIC value from the TC obtained value. The determination of alkalinity was based on the spectrophotometric measurement of bromocresol green colour change (611 nm) after reaction with acetic acid. The developed SIA method enabled the determination of DIC (0.24–3.5 mg C L⁻¹), CO₂ (1.0–10 mg C L⁻¹), TC (0.50–4.0 mg C L⁻¹) and alkalinity (1.2–4.7 mg C L⁻¹ and 4.7–19 mg C L⁻¹) with limits of detection of: 9.5 μg C L⁻¹, 20 μg C L⁻¹, 0.21 mg C L⁻¹, 0.32 mg C L⁻¹, respectively. The SIA system was effectively applied to inland bathing waters and the results showed good agreement with reference procedures.     

Catalytic activity of iron hexacyanoosmate(II) towards hydrogen peroxide and nicotinamide adenine dinucleotide and its use in amperometric biosensors

Hydrogen peroxide and nicotinamide adenine dinucleotide (NADH) may be determined amperometrically using screen-printed electrodes chemically modified with iron(III) hexacyanoosmate(II) (Osmium purple) in flow injection analysis (FIA). The determination is based on the exploitation of catalytic currents resulting from the oxidation/reduction of the modifier. The performance of the sensor was characterized and optimized by controlling several operational parameters (applied potential, pH and flow rate of the phosphate buffer). Comparison has been made with analogous complexes of ruthenium (Ruthenium purple) and iron (Prussian blue). Taking into account the sensitivity and stability of corresponding sensors, the best results were obtained with the use of Osmium purple. The sensor exhibited a linear increase of the amperometric signal with the concentration of hydrogen peroxide in the range of 0.1-100 mg L⁻¹ with a detection limit (evaluated as 3σ) of 0.024 mg L⁻¹ with a R.S.D. 1.5% for 10 mg L⁻¹ H₂O₂ under optimized flow rate of 0.4 mL min⁻¹ in 0.1 M phosphate buffer carrier (pH 6) and a working potential of +0.15 V versus Ag/AgCl. Afterwards, a biological recognition element - either glucose oxidase or ethanol dehydrogenase - was incorporated to achieve a sensor facilitating the determination of glucose or ethanol, respectively. The glucose sensor gave linearity between current and concentration in the range from 1 to 250 mg L⁻¹ with a R.S.D. 2.4% for 100 mg L⁻¹ glucose, detection limit 0.02 mg L⁻¹ (3σ) and retained its original activity after 3 weeks when stored at 6 °C. Optimal parameters in the determination of ethanol were selected as: applied potential +0.45 V versus Ag/AgCl, flow rate 0.2 mL min⁻¹ in 0.1 M phosphate buffer carrier (pH 7). Different structural designs of the ethanol sensor were tested and linearity obtained was up to 1000 mg L⁻¹ with a maximum R.S.D. of 5.1%. Applications in food analysis were also examined.     

A quick method for the simultaneous determination of ascorbic acid and sorbic acid in fruit juices by capillary zone electrophoresis

A method of quickly determining ascorbic acid and sorbic acid by capillary zone electrophoresis with ultraviolet detection was developed. The choice of background electrolyte, wavelength, injection time and applied voltage were discussed. Ascorbic acid and sorbic acid were well separated in 80 mmol L⁻¹ boric acid-5 mmol L⁻¹borax (pH = 8.0) in 5 min at the detecting wavelength of 270 nm. Under the optimum condition, the method has linear ranges of 2.54-352.00 mg L⁻¹ for ascorbic acid and 1.08-336.39 mg L⁻¹ for sorbic acid with the detection limit of 1.70 mg L⁻¹ for ascorbic acid and 0.54 mg L⁻¹ for sorbic acid, respectively. Other organic acids in fruit juices have no effect on the detection. This method is very feasible and simple and can be used to detect ascorbic acid and sorbic acid in fruit juices.     

The inner filter effects and their correction in fluorescence spectra of salt marsh humic matter

The inner filter effects in synchronous fluorescence spectra (Δλ = 60 nm) of sedimentary humic substances from a salt marsh were studied. Accordingly to their type and the influence of plant colonization, these humic substances have different spectral features and the inner filter effects act in a different manner. The fluorescence spectra of the humic substances from sediments with colonizing plants have a protein like band (λ_exc = 280 nm) which is strongly affected by primary and secondary inner filter effects. These effects were also observed for the bands situated at longer wavelengths, i.e., at λ_exc = 350 nm and λ_ex = 454 nm for the fulvic acids (FA) and humic acids (HA), respectively. However, they are more important for the band at 280 nm, causing spectral distortions which can be clearly seen when the spectra of solutions 40 mg L⁻¹ of different samples (Dissolved Organic Carbon – DOC ∼ 20 mg L⁻¹) are compared with and without correction of the inner filter effects. The importance of the spectral distortions caused by inner filter effects has been demonstrated in solutions containing a mixture of model compounds which represent the fluorophores detected in the spectra of sedimentary humic samples. The effectiveness of the mathematical correction of the inner filter effects in the spectra of those solutions and of solutions of sedimentary humic substances was studied. It was observed that inner filter effects in the sedimentary humic substances spectra can be mathematically corrected, allowing to obtain a linear relationship between the fluorescence intensity and humic substances concentration and preventing distortions at concentrations as high as 50 mg L⁻¹ which otherwise would obscure the protein like band.     

Determination of azoxystrobin residues in grapes, musts and wines with a multicommuted flow-through optosensor implemented with photochemically induced fluorescence

In this paper, the conversion of azoxystrobin in a strongly fluorescent degradation product by UV irradiation with quantitative purposes and its fluorimetric determination are reported for the first time. A multicommuted flow injection-solid phase spectroscopy (FI-SPS) system combined with photochemically-induced fluorescence (PIF) is developed for the determination of azoxystrobin in grapes, must and wine. Grape samples were homogenized and extracted with methanol and further cleaned-up by solid-phase extraction on C₁₈ silica gel. Wine samples were solid-phase extracted on C₁₈ sorbent using dichloromethane as eluent. Recoveries of azoxystrobin from spiked grapes (0.5-2.0 mg Kg⁻¹), must (0.5-2.0 μg mL⁻¹) and wine (0.5-2.0 μg mL⁻¹) were 84.0-87.6%, 95.5-105.9% and 88.5-111.2%, respectively. The quantification limit for grapes was 0.021 mg Kg⁻¹, being within European Union regulations, and 18 μg L⁻¹ and 8 μg L⁻¹ for must and wine, respectively.