Ml Of Reagent Research Articles

Adaptation of the BCR sequential extraction procedure for fractionation of potentially toxic elements in airborne particulate matter collected during routine air quality monitoring

ABSTRACT The Community Bureau of Reference (BCR) four-step sequential extraction has been adapted for fractionation of potentially toxic elements (PTE) in simulant airborne particulate matter (APM) samples presented on 47 mm filter dynamics measurement system (FDMS) filters as used in routine air quality monitoring. Simulants were prepared from an urban soil reference material and from BCR CRM 701, which is certified for analytes extractable by the BCR procedure. Analysis was performed by inductively coupled plasma mass spectrometry using an Agilent 7700x instrument. Fractionation patterns similar to the full-scale protocol were obtained when test portions as small as 0.0625 g were extracted in 2–3 mL of reagents. However, changing the extraction vessel used was found to affect the outcome. This highlights the operational nature of sequential extraction procedures and the need carefully to evaluate the effects of procedural modifications. When the method developed was applied to blank FDMS filters, large amounts of Zn were detected, especially in step 3, the oxidisable fraction, and step 4, the residual fraction. Despite this, following blank-correction, fractionation patterns similar to certified values were obtained for BCR CRM 701, with overall recoveries (∑(steps 1–4)) of 84.2-113%. Given the increased awareness of public health risks associated with poor air quality, a sequential extraction procedure specifically designed for use with APM samples collected during routine air quality represents a valuable tool for use in source apportionment and to improve understanding of human exposure to PTE through inhalation.

ON-LINE MINI-COLUMN SOLID-PHASE EXTRACTION COUPLED WITH SEQUENTIAL INJECTION FOR THE DETERMINATION OF CADMIUM IN ENVIRONMENTAL SAMPLES

□Trace cadmium(II) in environmental samples was separated and enriched in an automated fashion using a diphenylcarbazone-functionalized silica gel mini-column coupling with sequential injection. Cadmium was determined using a multicomponent system involving the analyte, potassium iodide, ethyl violet, and polyvinyl alcohol. Under the optimum conditions, the linear range for cadmium(II) was 0.015–0.50 µ g mL−1, and the detection limit was 0.012 µ g mL−1. The relative standard deviation of 11 determinations for 0.1 and 0.2 µ g mL−1 standard solutions was smaller than 3.5%. This method was applied for the determination of cadmium(II) in water, plant, soil, and electroplating wastewater. No statistically significant differences in the results were observed between the reported method and those obtained by atomic absorption spectrometry. The analysis was performed within 13 min and a total reagent volume of only 0.14 mL (0.07 mL of eluent and 0.07 mL of reagents) was consumed in each determination of cadmium(II).

The development of paper microzone-based green analytical chemistry methods for determining the quality of wines

The colorimetric determination of the concentration of polyphenols, flavonoids, and anthocyanins in wine samples, using a mobile phone camera for sample spot capture on a paper microzone and a remote computer with dedicated software for quantification, is presented as an illustrative application of green analytical chemistry. A comparison of the results with conventional spectrophotometry demonstrates that both methods yield similar results. Developing the assay took approximately 2 months, and the use of chemicals, compared with spectrophotometry, was reduced by about two orders of magnitude: the paper assay consumed 0.4 mL of reagent for 100 samples, whereas the spectrophotometric assay required 100 mL. The relative testing times for 100 samples were 7 h by spectrophotometry and 2 h for paper-a savings on the order of 3.5. No analytical instrumentation was used for the colorimetry on paper microzones. Instead, the assay took advantage of the existing communication technology and free software. The assay was found to be effective, with a nonlinear response at the concentration range of 0.2-5 g/L. The detection limit of the proposed method is in sub-grams per liter.

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Threonine synthase (TS), which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the γ-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at Cβ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k(cat) and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, α-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the Cβ of the PLP-α-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the Cβ of the analog. These results indicated that the phosphate ion released from OPHS by γ-elimination acts as the base catalyst for the addition of water at Cβ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/deprotonation at Cγ.

Structural and Functional Fingerprint of the Mitochondrial ATP-binding Cassette Transporter Mdl1 from Saccharomyces cerevisiae

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 microm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.

Interference-free coulometric titration of water in lithium bis(oxalato)borate using Karl Fischer reagents based on N-methylformamide

A non-alcoholic coulometric reagent based on N-methylformamide (NMF) was shown to eliminate the severe interference effect caused by the alcohol component of the conventional Karl Fischer (KF) reagent on the battery electrolyte lithium bis(oxalato)borate (LiBOB). For sample amounts up to 240 μg of water, the stoichiometry of the KF reaction deviated only slightly from the ideal 1:1 ratio for the best reagent composition. Both solid and dissolved (in acetonitrile, tetrahydrofuran (THF), and ethylene carbonate/ethyl methyl carbonate) LiBOB were titrated successfully using a Metrohm 756 KF Coulometer with a diaphragm cell. The detection limit was estimated to be 0.5–1 μg of water using 100 ml of reagent in this system.

An electrochemical flow-cell for permanent modification of graphite tube with palladium for mercury determination by electrothermal atomic absorption spectrometry

An electrochemical procedure for palladium deposition on the inner surface of pyrolytic graphite-coated tubes for permanent chemical modification and a cold vapor generation system for the pre-concentration and determination of mercury trace levels in rain, potable, and non-potable water and lake sediment by electrothermal atomic absorption spectrometry is proposed. A tubular electrochemical flow-cell was assembled on the original geometry of the graphite tube, which operated as the working electrode. A stainless steel tube, positioned downstream from the working electrode, was used as the auxiliary electrode. The applied potential was measured against a micro Ag/AgCl (sat) reference electrode inserted in the auxiliary electrode. Palladium solution in acetate buffer (100 mmol l −1, pH=4.8), flowing at 0.5 ml min −1 for 60 min was used to perform the electrodeposition. A homemade cold vapor generation system composed of a peristaltic pump, an injector–commutator, a flow meter and a disposable polyethylene gas–liquid separator flask (approx. 4.0 ml volume) were used. Volumes of 1.0 ml of reagent (2.0% w/v NaBH 4 in 0.10 mol l −1 of NaOH) and 1.0 ml of reference or sample solution in 0.25 mol l −1 of HNO 3 were carried to the gas–liquid separator using the peristaltic pump. The mercury vapor was carried out to the modified graphite tube by argon flow (200 ml min −1), and pre-concentrated for 120 s. The characteristic mass for 1.0 ml of reference solution was 26 pg (R.S.D.=0.12%, n=5). The detection limit obtained was 93 pg ( n=20, 3δ). The reliability of the entire procedure was confirmed by addition and recovery tests and cold vapor atomic absorption spectrometry.

Assessment of the 1 M NH 4NO 3 extraction protocol to identify mobile forms of Cd in soils

Extraction of soils by leaching 20 g in 50 ml of unbuffered 1 M NH 4NO 3 for 2 h has become the standard German protocol (DIN 19730) to estimate mobile and potentially hazardous forms of trace elements. This protocol was examined using soil controls of chernozem and podzol character collected in the Canadian Prairies and Ontario. It was found that this procedure was not robust with respect to change in sample weight per unit volume of reagent or to contact time. A 3- to 15-fold increase in Cd extracted was produced by decreasing sample weight from 10 g to 1 g in 50 ml of reagent. This change in extractable-Cd was more pronounced with increasing pH of the soil, ranging from 5.2 to 8.1 amongst the control samples. Leach period was less significant in determining results but longer time (1–3 h) generally produced lower extractable-Cd, suggesting readsorption. Lack of stability of Cd in this reagent was further demonstrated by spiking experiments where Cd was added at the beginning of the leach. Recoveries of Cd at 50 and 200 ppb were below 20% for soils of pH 6.1 and 6.9. Such severe adsorption of Cd was not encountered with another unbuffered reagent, 1 M NH 4Cl, operating at a similar initial pH of 5. Cadmium levels extracted from soils by NH 4Cl and 0.1 M Na 4P 2O 7 were comparable and could be used to predict uptake in durum wheat on the Canadian Prairies, whereas data by the 1 M NH 4NO 3 leach were generally near the detection limit of 0.1 ppb (ng g −1) Cd. Readsorption of Cd and lack of equilibrium in the 1 M NH 4NO 3 extraction could lead to underestimation of plant-available Cd in neutral and alkaline soils.

Simple method for determining specific binding capacity of vitamin D-binding protein and its use to calculate the concentration of "free" 1,25-dihydroxyvitamin D

A quantitative method to measure the specific binding capacity for 25-hydroxyvitamin D (25D-binding capacity) is described that resembles the qualitative "T3-uptake" assay. Patient's serum or standard (10 microL) is mixed with 0.5 mL of reagent containing 0.5 mumol/L 25-hydroxyvitamin D [25(OH)D3] plus 3000 counts/min [3H]25(OH)D3. After 0.5 h at 37 degrees C, the samples are treated with dextran/charcoal on ice for 1 h and centrifuged. The radioactivity of the bound tracer in the supernate is counted. Calibration is linear to approximately 10 mumol/L. 25D-binding capacity in reference-group serum samples was 4.33 (0.58 SD) mumol/L. The relationship between the inverse of 25D-binding capacity and the free fraction of [3H]1,25-dihydroxyvitamin D3 [1,25(OH)2D3] measured by ultrafiltration isodialysis was essentially linear (r = 0.934, P < 0.0001). Given this relationship, the calculated free fraction of 1,25(OH)2D3 equals 4.88 x 10(-3)/25D-binding capacity. The 25D-binding capacity was significantly lower in newborn babies and in adults with liver disease, and was increased during pregnancy (P < 0.01 for each). This method is applicable to situations where the biologically available concentration of 1,25(OH)2D is of interest.

97 High-Performance Liquid Chromatographic method for Simultaneous Screening of the Deficiencies of APRT and HGPRT

A rapid and covenient screening method using high-performance chromatography(HPLC) for the simultanous detection of deficiencies of adenine phosphoribosyltransf erase (APUT, EC 2.4.2.7) and hypoxanthine guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) is presented.The enzyme reactions were started by the addition of 0.1 ml of the red cell lysates (2∼5g Hb/dl) treated with charcoal-dextran into 0.5 ml of the reaction mixture A(2 mM PRPP, 6 mM MgCl2, 1 mM HX and 0.2 mM adenine in 50 raM Tris-HCl pH7.4). After incubation at 37°C, 30 min, 0.5 ml of reagent B (20 U/ml ALP, 4 mM MgCl2 in 1 M Tris) were added and incubated for 30 min. at 37°C for the conversion of AMP and IMP into adenosine and inosine. ALP reaction was stopped by adding 1.0 ml of 0.5 M HCIO4. The mixture centrifuged at 3,000 x g for 10 min. The supernatants were used as samples for HPLC analysis. Normal ranges of APRT and HGPRT activities in erythrocytes obtained 28 healthy subjects were 0.40 ± 0.06 and 1.97 μ 0.19 μ mol/min/ g Hb. respectively. The method could detect down to 1 % of normal APRT activity and 0.3 % of normal HGPRT activity.

High resolution 31P NMR of extracted phospholipids.

The common phospholipids from biological sources were quantitated using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy in conjunction with an analytical reagent composed of two parts: 1) 2 ml of reagent chloroform in which was dissolved 0.01-100 mg of crude tissue lipid extracted from tissue sources using chloroform-methanol 2:1, the extract having been washed with 0.2 vol. of 0.1 M KCl; 2) 1 ml of an aqueous methanol reagent composed of one part 0.2 M (ethylenedinitrilo)-tetraacetic acid in D2O titrated to pH 6 with CsOH and four parts of reagent methanol. In a magnetic field of 11.75 Tesla, the extracted phospholipids yield narrow signals (1.8-3.2 Hz at half-height), corresponding to each generic species, e.g., phosphatidylcholines, phosphatidylethanolamines, etc., permitting resolution among the various phospholipid families and their lyso and plasmalogen derivatives. The reagent permits assays of high precision and accuracy using a modest amount of NMR spectrometer time (ca. 15 min/assay). The procedures described, which are compared to high-performance liquid chromatography, are convenient for the routine analysis of phospholipids from biological sources.

A rapid fluorometric assay for measurement of peptidase activity

A rapid method using orthophthaldialdehyde and mercaptoethanol to form a fluorescent adduct with released α-amino groups was developed and characterized as a means for assaying peptidase activity on physiological peptide substrates. A typical assay consisted of withdrawing a 10-μl aliquot of the substrate-enzyme solution at various time intervals, which was then added to 3 ml of reagent; the fluorescence was measured relative to 0.1 μg/ml quinine sulfate in 0.1 n H 2SO 4 following a standardized 2-min reaction time. The assay can be performed either in the presence or absence of sodium dodecyl sulfate; addition of the detergent can be a convenient means for stopping the reaction. Contrary to previous suggestions, addition of detergent was found to slightly decrease fluorescence of dipeptide adducts with this reagent rather than causing a substantial enhancement. However, enhancement was observed for cases where the spacing between isoindoles or between isoindole and indole was similar to that for the disubstituted derivative of lysine. This method was characterized by assaying porcine intestinal peptidase on the peptides Leu-Gly, Gly-Gly, Ala-Gly, and Gly-Leu-Tyr. Using an immobilized form of this enzyme, excellent agreement was found between amino acid determinations and fluorescence-derived calculated values for the fraction of bonds hydrolyzed for dipeptide substrates. It was shown that the fluorescence ratio (F t − F 0) (F c − F 0) corresponds to the fractional hydrolysis of dipeptides where F t, F 0, and F c are the relative fluorescences at time t, time zero, and complete hydrolysis, respectively.

The addition of SDS to the Bradford dye-binding protein assay, a modification with increased sensitivity to collagen

The standard Bradford protein assay is insensitive to collagen. But if a small, sub-threshold amount of SDS is added to the sample, the response to collagen in increased by at least an order of magnitude, while, on average, the sensitivity for non-collagens is decreased by approximately a factor of 2. As a result comparable color formation is achieved with both collagens and non-collagens. The addition of protein to a sub-threshold amount of SDS results in the formation of a green color measurable as an increase in absorbance at 700 nm, in contrast to the blue color measured at 595 nm in the standard assay. Depending upon the source, the threshold level for SDS varies from 30 to 50 μg. The response to protein is linear up to approximately 40 μg of protein per ml of reagent.

24] Colorimetric estimation of vitamin A with trichloroacetic acid

Publisher Summary This chapter discusses the use of trichloroacetic acid method for colorific estimation of vitamin A in serum and liver tissue. In this method, trichloroacetic acid crystals (50 g), freshly washed with solvent, are dissolved in alcohol-free distilled chloroform (25 ml). This gives about 50 ml of reagent that is stable for 5 h with daylight excluded. Optical density readings tend to decrease with storage for longer periods, up to 24 h. No color is given by vitamin A with reagent exposed to bright diffuse sunlight for several hours. It is observed with this method that the colors produced from vitamin A, its acetate, and palmitate with the Carr-Price antimony trichloride reagent and trichloroacetic acid reach the same intensity for any given amount of vitamin. The concentration of trichloroacetic acid controls the rate of formation and stability of anhydrovitamin, which may be correlated with loss of vitamin A or its acetate. It is important to observe that change occurs without appearance of blue color in the weaker acid medium. Formation of anhydrovitamin with fine-structure absorption follows protonation of vitamin A, and the slower formation from vitamin A acetate reflects differences in the rates of dehydration and deacetoxylation.

New enzymatic method for serum uric acid at 500 nm.

We describe a manual method, well suited to mechanization, for quantitating serum uric acid at 500 nm. In the assay mixture (0.10 ml of sample and 3.00 ml of reagent) the hydrogen peroxide produced from uric acid by uricase is coupled with p-hydroxybenzoate and 4-aminoantipyrine in the presence of peroxidase to form a colored complex, which is measured. A separate sample blank is obviated by taking an initial absorbance measurement 20 s after the sample is added. The reaction is complete within 5 min; its sensitivity is 0.001 deltaA/mg per liter. Absorbances are linearly related to uric acid concentrations up to 120 mg/liter. Many substances that may be present in normal serum do not interfere, but bilirubin in moderately above-normal concentrations will interfere. The procedure can be modified to largely correct for this, when necessary. The proposed method (y) correlated well (r = 0.979) with the uric acid 293 nm reference method (x) and the relation is described by the equation y = 0.998x + 2.42.

Design requirements for a high speed continuous flow analyser

In 1957 the original continuous flow analysers performed urea estimations at 20 samples/hr. Multichannel analysers were later introduced and by 1968 it was possible to analyse 60 samples/hr for 12 parameters, 4 of which were blanked. This paper described what was necessary to improve this technique to analyse 150 samples/hr, to perform 20 analyses on each sample, to require a maximum sample of 500 μl for these 20 tests and to require 6 ml of reagents for these 20 tests. The following improvements were noticed :— (1) Hydraulics (a) More extensive miniaturization, (b) variation in the use of bubbles, (c) flow cell changes (2) Modified chemical techniques (a) ion selective electrodes, (b) multipoint enzyme analysis. (3) The use of a controlling computer to monitor all estimations continuously.

A colorimetric method for the micro determination of 2-phenylphenol.

A colour reaction produced with the fungicide 2-phenylphenol is described. 2-Phenylphenol, its esters and sodium 2-phenylphenate give a pink colour with concentrated sulphuric acid and traces of formaldehyde and iron(III), which is specific for 2-phenylphenol. With 5 ml of reagents, 1 µg of these compounds produces colours that are discernible visually. In fixed conditions the reaction conforms to the Beer-Lambert law for up to 16 µg of 2-phenylphenol per 5 ml of reagents. A simple and rapid method has been evolved for its determination in disinfectant solutions containing sodium 2-phenylphenate, and in paper wrappers impregnated with 2-phenylphenol, its esters or its sodium salt. Recovery varies from 94·1 to 105·0 per cent.; the deviations remain within the limits of experimental error.

Differential microcalorimeter for biochemical reaction studies.

A differential solution microcalorimeter with a mixing system is described. Up to 1 ml of reagent A may be mixed with up to 3 ml of reagent B in less than 1 sec with a heating artifact of less than 0.5 mcal. A temperature range of 0 to 40°C has been utilized with a 2 h temperature equilibration time. The time course of biochemical reactions has been followed for up to 1 h. Computer simulation of the calorimeter permits data correction for heat conduction losses. For reaction heats greater than 25 mcal, ΔH and the rate constant of the reaction may be determined to ±2%. Detectivity is ±20 μcal. A digital computer simulation technique based on a finite-element analysis of heat conduction, which is of general applicability, was developed to correct the output data for heat conduction losses.

The Rapid Measurement of pH by the Glass Electrode: The Kinetics of Dehydration of Carbonic Acid at 25° and 37°

Abstract We have developed a comparatively simple and inexpensive apparatus that can be applied to the rapid determination of pH from 0.003 sec onward. This has been achieved by a combination of (a) a standard pH meter, (b), a driving-mixing arrangement of the Hartridge-Roughton type, and (c) a specially designed glass microelectrode. The glass electrode (2-mm diameter, 120-mm length) has a pH-sensitive tip about 0.5 mm long and has a resistance of 150 megohms at 20°. About 25 ml of reagents are needed for one observation. No significant physical effects (e.g. streaming potentials) have been observed. Thus a precision of ±0.005 pH unit is possible, which is essential for many biochemical studies. To test the performance of this apparatus we have studied the kinetics of dehydration of carbonic acid. The velocity constant for this reaction has been found to be 25.5 and 72.00 sec-1, respectively, at 25° and 37°.

Quantitative Determination of Piperine. I. The Komarowsky Reaction

SUMMARYA highly stable reagent consisting of para‐dimethylaminobenzaldehyde (I) or para‐hydroxybenzaldehyde (II) or other cylic aldehydes, plus thioureau in concentrated sulfuric acid, has been developed for use in determining piperine. The slightly yellow reagent, if stored under refrigeration in a brown bottle, maintains a constant chromogenic capacity for 4–6 weeks. Piperine when heated with this reagent for 15 min at 100°C develops a red color (I max. 490 mp) or purple color (II max. 570 mμ) whose intensity is proportional to the amount of piperine present. Water inhibits color development, but a small amount of methyl alcohol is necessary. Ethyl alcohol cannot be used, since it develops color with the reagent. Critical factors for reproducible, quantitative assay are the concentration of acid and thiourea in the reagent and the time and temperature of heating the reaction mixture. For I, the most sensitive of the aldehydes used, good precision, with a standard deviation of it ± 3470, was attained. Between the levels of 0.01 and 0.06μM of piperine per ml of reagent, there is a linear relation which may be described by the least‐squares equation.X = 0.088 Y + 0.00175 where X = concentration (μM) of piperine per ml of reagent Y = absorbance at 490 μM Percent piperine in sample = X (0.28533) (dilution factor)′ 100 Sample weight (mg)