Common Spectrophotometric Methods Research Articles

A New Voltammetric Approach for the Determination of Biomimetic Catalyst Kinetic Constants Based on Substrate Consumption

AbstractIn the present study, a new voltammetric method is introduced for the determination of kinetic parameters of artificial metalloenzymes (biomimetic complexes) mimicking the catecholase activity towards 3,5‐di‐tert‐butylcatechol (3,5‐DTBC) or 2,5‐di‐tert‐butylhydroquinone, and representing a promising alternative to common spectrophotometric method. To demonstrate the effectiveness of the electrochemical approach, two binuclear copper(II) complexes bearing the same pentadentate ligand and different ionic ligands were selected. Apparent kinetic parameters, such as maximum velocity (Vmax), Michaelis‐Menten constant (Km), and turnover number (kcat), obtained by the described method employing differential‐pulse voltammetry were in good agreement with the data evaluated from UV‐Vis spectrophotometric measurements.

Biochar impact on the estimation of the colorimetric‐based enzymatic assays of soil

AbstractThis study was carried out in order to assess the influence of biochar applications on the estimation of colorimetric‐based enzymatic assays and to verify the effectiveness of the most common methods. Since most methods used to determine enzymatic activities in the soil are based on colorimetry, biochar may absorb substrates and/or coloured products thereby distorting the analytical result. Biochar was added to two soils, with different textures and cation exchangeable capacities, at a rate of 2% (w/w), and seven enzyme activities were determined following standard methods. The biochar amendment lowered the spectrophotometer reading of the activity of FDAase and dehydrogenase in the sandy soil. In the three enzymatic activities based on p‐nitrophenol production (β‐glucosidase, phosphatase and arylsulphatase), the addition of biochar did not change the enzyme assays. The biochar led to an overestimation in terms of the protease and urease activities in the sandy soil. In the clay loamy soil, biochar did not change the response of any of the enzyme activities tested. A biochar dose of up to 2% only guarantees the effectiveness of the most common spectrophotometric methods for not excessively sandy soils.

Multi-wavelength spectrophotometric measurement of persulfates using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as indicator.

A spectrophotometric method using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the indicator was proposed for measuring persulfates, i.e., peroxymonosulfate (PMS) and peroxydisulfate (PDS). This method was based on the direct oxidation of the colorless ABTS by PMS/PDS to generate the green-colored ABTS+ at neutral pH and room temperature. The generated ABTS+ had four different characteristic absorption peaks at 820 nm, 732 nm, 650 nm and 415 nm. The absorption values at these wavelengths increased with the increase of PMS/PDS concentration, and there were well linear relationships (R2 > 0.998) between the corresponding absorption values and the concentrations of PMS/PDS in the range of 3-500 μM. Monitoring the change of PMS/PDS concentration by the proposed ABTS method was in good agreement with that monitored by the common spectrophotometric method using potassium iodide as the indicator. The green-colored waste solutions produced after PMS/PDS analysis with the ABTS method showed no bactericidal effect towards Escherichia coli. The proposed multi-wavelength spectrophotometric method using ABTS as the indicator could be employed as a candidate method for the accurate and convenient measurement of PMS/PDS in water.

Characterization of Oxidative Stress in Chicken Heart Tissue by High-Performance Liquid Chromatography with Ultraviolet‐Visible (HPLC–UV–Vis) Detection

Oxidative stress can lead to tissue damage in the body, resulting in conditions that may lead to heart failure. Carbonyl proteins and malondialdehyde are common markers for determining oxidative stress. In this study, a high-performance liquid chromatography with ultraviolet‐visible (HPLC–UV–Vis) method was developed for both carbonyl proteins and malondialdehyde and compared with common spectrophotometric methods. The HPLC–UV–Vis method for determining carbonyl proteins showed a linear correlation in a range of 0.05–1.6 µg.mL−1 (correlation coefficient: 0.9939). The limits of detection and quantitation were determined to be 1.7 and 5.1 pmol on-column, respectively. The HPLC–UV–Vis method for malondialdehyde exhibited a linear correlation in the range of 0.04–13.6 µg.mL−1 (correlation coefficient: 0.9982) with limits of detection and quantitation of 1.5 and 4.5 pmol on-column, respectively. As a proof of concept, the applicability of the methods for both marker substances was investigated in chicken heart tissue as a complex matrix sample. To simulate oxidative stress, the tissue was treated with hydrogen peroxide as the oxidizing agent. Samples prepared with hydrogen peroxide showed increased carbonyl protein and malondialdehyde levels when compared to non-oxidized samples and samples treated additionally with the antioxidant Trolox. Regarding oxidative stress, similar results were achieved when compared with common spectrophotometric methods. However, HPLC–UV–Vis presented higher reproducibility and accuracy. The developed methods for both markers enable rapid and simple investigation of possible antioxidant effects on tissue samples.

Multi-method approach to quantify uncertainties in the measurements of light absorption by particles.

Through technological and research advances, numerous methods and protocols have emerged to estimate spectral absorption of light by particles, ap, in an aquatic medium. However, the level of agreement among measurements remains elusive. We employed a multi-method approach to estimate the measurement precision of measuring optical density of particles on a filter pad using two common spectrophotometric methods, and the determination precision, or uncertainty, of the computational techniques for estimating ap for six ocean color wavelengths (412, 443, 490, 510, 555, 670 nm). The optical densities measured with the two methods exhibited a significant, positive correlation. Optical density measurement precision ranged from 0.061%-63% and exhibited a significant, positive correlation. Multi-method uncertainty ranged from 7.48%-119%. Values of ap at 555 nm and 670 nm exhibited the highest values of uncertainty. Poor performance of modeled ap compared to determined ap suggest uncertainties are propagated into bio-optical algorithms.

New Approach for Measuring Antioxidant Activity Via a Graphite Sensor

An ion selective membrane sensor from dioctyl phthalate as a plasticizer in a polymeric matrix of polyvinyl chloride (PVC) and �-cyclodextrin as an ionophore was constructed and evaluated according to IUPAC recommendations. Linear Nernstian response of DPPHwithin the concentration ranges of 10 �6 to 10 �2 mol L �1 was obtained with average recovery 99.87±0.617. Nernstian slope of 58.5 mV/decade with excellent selectivity over the pH range of 3-8 was observed. The suggested method was standardized using butylated hydroxyl anisole (BHA). The 50% radical scavenging activity (IC 50 ) determined by the proposed sensor correlated well with that of the common spectrophotometric method based on scavenging of 2,2-diphenyl-1- picrylhydrazyl (DPPH • ). An algorithm implemented in Microsoft Visual Basic ® 6.0 was used for calculating (IC 50 ) values which are 7.38 �g/mL ± 0.35, 89.98 �g/ mL ± 0.45 and 1.45 mg/ mL ± 1.50 for BHA, Paracetamol and Dipyridamole, respectively. The proposed sensor represents a simple and reproducible tool for measuring DPPH • scavenging activity of Paracetamol and Dipyridamole in bulk powder, pharmaceutical formulations and simulated intestinal fluid (SIF) without sophisticated separation techniques.

Comparison of North Italian and South Moravian wines on the base of their antioxidant activity, phenolic composition and sensory quality

In the present study, antioxidant capacity, phenolic composition and sensory evaluation of selected red and white wines originating from wine-growing regions of South Moravia and North Italy (wine-growing region Trident – Alto Adige) were investigated. The sensory analysis indicated that the evaluated wine samples were very similar. As far as basic the analytical parameters were concerned, concentrations of residual sugar were significantly higher in Moravian wines. Antioxidative characteristics were estimated by means of common spectrophotometric methods (total phenols, total anthocyanins, total flavanols, DPPH and FRAP) and thereafter compared. It was found out that the compared methods were highly significantly and positively correlated as far as their results were concerned. Individual phenolic compounds were detected by means of HPLC with DAD detection. In white wines, the content of GRP products was higher and this indicated that the quality of grapes used for making Moravian wines was lower. In red Moravian wines, the content of catechins (i.e. compounds responsible for the majority of phenolic substances and considered to be health-promoting compounds) was higher. This observation was corroborated also analytically, i.e. on the base of correlation with antioxidative characteristics.

Determination of NH4+ in Environmental Water with Interfering Substances Using the Modified Nessler Method

Nitrogen is an essential element in the environment. If excess nitrogen including is present in water, however, it can result in algae blooming and eventually the destruction of the aquatic ecosystem. Therefore, the determination of in streams, lakes, and effluents of the treatment facilities has long been carried out. The Nessler method is the most common spectrophotometric method to measure in water. However, the result of the method becomes inaccurate if there are interfering substances such as Cl2, Cl−, hardness‐causing compounds (e.g., Mg2+), and Fe2+ in target water samples. In this study, therefore, the traditional Nessler method has been modified to eliminate the effects of interfering substances; the so‐called MS was added to water samples. In addition, the polyvinyl alcohol reagent as a dispersing agent was added to water samples to increase the sensitivity and reproducibility of the method. The modified method could successfully analyze of water samples even with the interfering substance at high concentration.

DEVELOPMENT OF A SENSITIVE SPECTROFLUOROMETRIC-MULTIVARIATE CALIBRATION METHOD FOR ENZYME KINETIC OF ALDEHYDE OXIDASE

Attempts to obtain experimental values for the kinetic parameters of phenanthridine oxidation by guinea pig or rabbit liver aldehyde oxidase using common spectrophotometric methods have not been successful due to a lower limit of detection. In the present study, a new spectrofluorimetric assay in combination with a multivariate calibration method for enzymatic kinetic study of aldehyde oxidase activity, using phenanthridine as the substrate, has been developed and validated. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.025-1 µM and the emission flourimetric spectra of the solutions recorded at the excitation and emission wavelengths of 236 and 320-450 nm, respectively. The optimized calibration model of partial least squares (PLS) method was applied for the simultaneous determination of the concentration of each chemical in the prediction set. The limits of detection for phenanthridine and phenanthridinone were found to be 2.13 ± 0.33 and 3.41 ± 0.34 nM (mean ± SD, n = 5), respectively. This method was then used for kinetic study of phenanthridine oxidation using guinea pig and rat hepatic aldehyde oxidase. The results were compared with those obtained from a univariate spectroscopic method. Using this new spectrofluorimetric-multivariate calibration method, the K m value for the oxidation of phenanthridine with guinea pig and rat liver aldehyde oxidase were obtained as 0.83 ± 0.08 and 2.20 ± 0.40, µM (mean ± SD, n = 3), respectively.

Hydrogen sulphide-generating pathways in haemodialysis patients: a study on relevant metabolites and transcriptional regulation of genes encoding for key enzymes

Hydrogen sulphide, H(2)S, is the third endogenous gas with putative cardiovascular properties, after nitric oxide and carbon monoxide. H(2)S is a vasorelaxant, while H(2)S deficiency is implicated in the pathogenesis of hypertension and atherosclerosis. Cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (MPS) catalyze H(2)S formation, with different relative efficiencies. Chronic kidney disease (CKD) is characterized by elevation of both plasma homocysteine and cysteine, which are substrates of these enzymes, and by a high prevalence of hypertension and cardiovascular mortality, particularly in the haemodialysis stage. It is possible that the H(2)S-generating pathways are altered as well in this patient population. Plasma H(2)S levels were measured with a common spectrophotometric method. This method detects various forms of H(2)S, protein-bound and non-protein-bound. Blood sulphaemoglobin, a marker of chronic exposure to H(2)S, was also measured, as well as related sulphur amino acids, vitamins and transcriptional levels of relevant genes, in haemodialysis patients and compared to healthy controls. Applying the above-mentioned methodology, H(2)S levels were found to be decreased in patients. Sulphaemoglobin levels were significantly lower as well. Plasma homocysteine and cysteine were significantly higher; vitamin B(6), a cofactor in H(2)S biosynthesis, was not different. H(2)S correlated negatively with cysteine levels. CSE expression was significantly downregulated in haemodialysis patients. Transcriptional deregulation of genes encoding for H(2)S-producing enzymes is present in uraemia. Although the specificity of the method employed for H(2)S detection is low, the finding that H(2)S is decreased is complemented by the lower sulphhaemoglobin levels. Potential implications of this study relate to the pathogenesis of the uraemic syndrome manifestations, such as hypertension and atherosclerosis.

The Use of 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine as a Precolumn Derivatizing Reagent in HPLC Determination for Fe(II) in Natural Samples

3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine (PDT) was used for the first time as a precolumn derivatizing reagent in the high-performance liquid chromatography (HPLC) method with UV absorbance detection for the Fe(II) determination. The Fe(II) reacts with PDT to form a magenta colored chelate in the presence of sodium dodecyl sulfate (SDS) and acetic acid-sodium acetate buffer solution medium of pH 4.65. The selection of maximum absorbance detection wavelength and the optimum composition of the organic modifier in the mobile phase were investigated in detail for the quantitative determination of Fe(II) using HPLC system. The formed Fe(II)-PDT chelate was satisfactorily separated from PDT on an Agilent Shim-pack ODS column (Eclipse XDB-C8, 4.6 x 150 mm) by isocratic elution with acetonitrile and 0.02 mol L(-1) acetic acid-sodium acetate buffer solution (pH 4.65, containing 0.02% of SDS and 60 x 10(-3) mol L(-1) NaClO(4)) as mobile phase at a flow rate of 1 mL min(-1), and monitored with a multiple wavelength detector. The detection limit (S/N = 3) is 0.35 ng mL(-1). Due to the excellent separation ability of HPLC, the innovative introduction of PDT as the precolumn derivatizing reagent, and the proper selection of the detect wavelength, the sensitivity of our newly developed HPLC method was enhanced remarkably compared to those of the common spectrophotometric methods. The developed HPLC method was successfully applied to the determination of Fe(II) in lake water samples.

Direct rapid determination of traces of sulfide in environment samples

An improved ethylene blue method for determination of sulfide is developed. It has been adapted to a direct determination of sulfide by both common spectrophotometric method and total differential spectrophotometric method. In common spectrophotometric method, the calibration curve is A=1.69ρ+0.006 and the correlation coefficient is 0.9994. The apparent molar absorptivity is 5.42 × 104 L·mol−1·cm−1 and calibration curve is liner when ρ is in the range of 0–0.9 mg·L−1. In total differential spectrophotometric method, the calibration curve is A=9.25ρ + 0.004 and the correlation coefficient is 0.9996. The apparent molar absorptivity is 2.96 × 105 L·mol−1 L·mol−1·cm−1 and calibration curve is liner when ρ is in the range of 0–0.10 mg·L−1. The sensitivity of this method is increased significantly compared with the former ethylene blue method. The speed of reaction is also faster than the former one. The limit of detection is found to be 1.0 ng·mL−1 by both common spectrophotometric method and total differential spectrophotometric method. Ten replicate analyses of a sample solution containing 100 ng·mL−1 sulfide give a relative standard deviation of 1.8%. The effects of various cations and anions on the determination of sulfide are studied and procedures for removal of interference is described. The method is used for the determination of sulfide in environment samples with satisfactory results.

The nitrite methemoglobin complex—Its significance in methemoglobin analyses and its possible role in methemoglobinemia

Evidence consistent with the formation of a reversible complex between excess free nitrite ion and the ferric heme groups of methemoglobin is presented. The existence of this complex in nitrited red cells or in lysates in the presence of appropriate concentrations of free nitrite can lead to gross underestimation of methemoglobin levels by a common spectrophotometric method. Complex formation is manifested by a decrease in the absorption of methemoglobin solutions in the range of 600–650 mμ. This source of error can be eliminated in red cell suspensions simply by washing. Similarly, lysate solutions may be dialyzed or diluted to effect dissociation of the complex. Attempts were made to estimate the dissociation constant of this complex at 25°, pH 7.4, and physiologic ionic strength. Although unknown side reactions between methemoglobin and nitrite interfere in the spectrophotometric determination of this constant, its value is probably not greater than 3 mM. This estimate indicates that the complex must exist transiently in vivo when mice receive nitrite doses adequate to convert more than one-third of the circulating blood pigment to methemoglobin. Thus, complex formation may contribute to the unusually sustained methemoglobinemia produced in mice by nitrite.

Potentiometric determination of silver with dithiooxamide

Potentiometric titration with dithiooxamide solution can be used to determine silver in the 0.0100–50.0 p.p.m range with an average accuracy of ca. 0.5% and a relative standard deviation ranging from 1.26% to 0.03% The metals commonly associated with silver can be masked with fluoride and EDTA The advantages of the method over other potentiometric and common spectrophotometric methods are outlined.