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ABSTRACT: The derivatization reactions commonly used to enhance the analytical signal in the HPLC and CE determination of compounds with hydroxyl functional groups are revised. Focus is placed on the determination of compounds having aliphatic alcohols and phenols while lacking other reactive functional groups. The derivatization with acyl chlorides, organic anhydrides, isocyanates and a variety of other approaches, including oxidation of primary and secondary alcohols, sulfonation, esterification with carboxylic acids, and the use of azides, sulfonyl chlorides and other reagents having miscellaneous leaving groups, is covered. Reactions mainly addressed to introduce a chromophore or a fluorophore in the analyte molecule, or to introduce a charge to enhance sensitivity in MS detection, or to enable CE separation are included. Applications related to the industrial quality control of raw materials and manufactured products, and to the evaluation of their environmental impact are emphasized. The problem of the different response factors of the derivatives when complex mixtures of oligomers are derivatized, as occurs with non-ionic surfactants (mainly fatty alcohol ethoxylates) and soluble synthetic polymers, is discussed. Other applications related to the biochemical, biomedical, pharmaceutical, nutritional and toxicological fields are also reviewed. The reactions, the criteria to be applied to select the reagent, and the characteristics of the derivatives in relation to separation and detection, are discussed.
Journal of chromatography. A 04/2013; · 4.19 Impact Factor
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ABSTRACT: A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater.
Journal of chromatography. A 11/2011; 1218(47):8511-8. · 4.19 Impact Factor
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ABSTRACT: Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 μm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 μm or 3 μm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners.
Journal of chromatography. A 08/2011; 1218(41):7275-80. · 4.19 Impact Factor
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ABSTRACT: A method for the determination of triacylglycerols (TAGs) in vegetable oils from different botanical origins by HPLC with UV-vis detection has been developed. Using a core-shell particle packed column (C18, 2.6 μm), TAG separation was optimized in terms of mobile phase composition and column temperature. Using isocratic elution with acetonitrile/n-pentanol at 10 °C, excellent efficiency with good resolution between most of the TAG peak pairs, within a total analysis time of 15 min, was achieved. Using mass spectrometry detection, a total of 15 peaks, which were common to oils of six different botanical origins (corn, extra virgin olive, grapeseed, hazelnut, peanut and soybean) were identified. These peaks were used to construct linear discriminant analysis (LDA) models for botanical origin prediction. Ratios of the peak areas selected by pairs were used as predictors. All the oils were correctly classified with assignment probabilities higher than 95%.
Journal of chromatography. A 07/2011; 1218(42):7521-7. · 4.19 Impact Factor
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ABSTRACT: Using capillary zone electrophoresis in nonequilibrium conditions, the complexes of poly(vinylpyrrolidone) (PVP) with anionic azo-dyes dissociate following a first-order kinetics. Two peaks due to the remaining PVP-dye complexes and the equilibrium concentration of the free dye, plus an exponential region due to the dye liberated by the complexes during the electrophoretic run, are obtained. This behaviour was closely similar to that described in the literature for protein-probe and DNA-protein mixtures, upon application of the technique known as nonequilibrium capillary electrophoresis of equilibrium mixtures or NECEEM. Using Congo Red and Acid Blue 113, information about the maximal stoichiometry and average stability of the PVP-dye complexes was obtained. The procedure was also useful to predict the average molecular mass of PVP and to determine PVP in cleaning products and pharmaceutical preparations. By using an appropriate probe, the procedure should be also useful to characterize and determine many other synthetic or natural nonionic polymers, and to study polymer-probe interactions.
Journal of chromatography. A 11/2009; 1216(51):9014-21. · 4.19 Impact Factor
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ABSTRACT: The enzymes present in raw materials of the cleaning industry (enzyme industrial concentrates) and in household cleaners were isolated by precipitation with acetone and hydrolyzed with HCl. The resulting amino acids were derivatized with o-phthaldialdehyde, and the derivatives were separated by HPLC. The peaks of 14 amino acids were observed using a C18 column and a multi-segmented gradient of acetonitrile-water in the presence of a 5 mM citric/citrate buffer of pH 6.5. Using either normalized peak areas (divided by the sum of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables, linear discriminant analysis models, capable of predicting the enzyme class, including proteases, lipases, amylases and cellulases, were constructed. For this purpose, both enzyme industrial concentrates and detergent bases spiked with them were included in the training set. In all cases, the enzymes of the evaluation set, including industrial concentrates, spiked detergent bases and commercial cleaners were correctly classified with assignment probabilities higher than 99%.
Talanta 08/2009; 79(2):275-9. · 3.79 Impact Factor
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ABSTRACT: A method for the determination of fatty alcohol ethoxylates (FAEs) using diphenic anhydride as derivatization reagent and RP-HPLC separation with UV-vis detection is presented and compared to derivatization with maleic, phthalic, and other cyclic anhydrides. With these anhydrides, the reaction rates increased when urea was added to the reaction medium, and the yields did not decrease when the samples contained moderate amounts of water. Gradient elution on a C8 column was performed with water/acetonitrile in the presence of 0.1% acetic acid. The use of diphenic anhydride was advantageous for both the chromatographic separation and the detection. Specifically, sensitivity at 200 and 220 nm was significantly better for the FAE diphenates, resulting in lower limits of detection at both wavelengths for the diphenates than for the maleates and phthalates (up to 30 and 4.3 times lower at 220 nm, respectively). Response factors for the diphenates decreased less than those of the phthalates when the number of ethylene oxide units, m, increased, reaching a constant value of ca. 0.62 when m>3. Peak symmetries and efficiencies were also better than those found for the other anhydrides. The optimized procedure was applied to the characterization and determination of FAEs in the effluent of a wastewater treatment plant and in sea water.
Journal of chromatography. A 03/2009; 1216(15):3023-30. · 4.19 Impact Factor
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ABSTRACT: Aliphatic and triterpene alcohols present in vegetable oils have been identified and determined by HPLC using UV-vis and MS detection after previous derivatization with diphenic anhydride. The alcoholic fraction was obtained by saponification, extraction and TLC (according to the European Union official procedure). Derivatization was performed in tetrahydrofuran in the presence of suspended grinded urea, which increases the reaction rate and yield. Derivatized extracts were chromatographed on a C8 column using gradient elution with acetonitrile/water mixtures containing 0.1% acetic acid, with UV-vis followed by negative-ion mode MS detection. Using linear discriminant analysis of the HPLC-MS data (extracted ion chromatograms), oil samples belonging to seven botanical origins (hazelnut, sunflower, corn, extra virgin olive, soybean, peanut and grapeseed) were correctly classified with excellent resolution among all the categories.
Journal of Chromatography 12/2008; 1216(2):230-6. · 4.53 Impact Factor
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ABSTRACT: The esterification of fatty alcohol ethoxylates (FAEs) with phthalic anhydride in 1,4-dioxane was studied. At 110 degrees C and in the presence of urea, which increased the reaction rate, esterification was completed in 60 min. The reaction yield did not decrease when the sample contained up to 50% water. For the non-ethoxylated alcohols, the UV-vis response factors of the derivatives were not significantly different from each other (f=1 for C12E0 as reference). The response factors decreased when the number of ethylene oxide units, m, increased, reaching constant values of ca. 0.40 and 0.65 for the n=8 and 18 series, respectively, when m>or=3. Using a C8 column and gradient elution with acetonitrile/water plus 0.1% acetic acid, FAEs in industrial mixtures and cleaning products were characterized. At column temperatures of 25 and 35 degrees C, homologous series with even values of n from 8 up to 20, were resolved up to m=14 and 18, respectively, and at 25 degrees C, series with consecutive even and odd values of n were resolved up to m=7. Within the series, superior resolution of the ethoxymers with large values of m was achieved; however, the elution order was reversed for the m=1 and 0 ethoxymers, which produced overlapping of a few peaks within the series. A simple procedure for the accurate prediction of the concentrations of all the ethoxymers in industrial samples was described. After preconcentration with C18 solid-phase extraction cartridges, FAEs were also characterized and quantified in environmental samples (river and sea water). The LODs were ca. 2 microM (S/N=3) in the injected solutions.
Journal of Chromatography 08/2008; 1203(1):47-53. · 4.53 Impact Factor
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ABSTRACT: The esterification of fatty alcohol ethoxylates (FAEs) by melting with maleic anhydride has been studied in the presence of urea. The reaction rate and yield of non-ethoxylated and ethoxylated alcohols increase largely when grinded urea is suspended in the reaction medium, a 100% yield being achieved at 80 degrees C in 15 min. The procedure is tolerant to the presence of large amounts of water. The UV-vis response factors of the derivatives of individual FAE oligomers vary little with the number of carbon atoms in the alkyl chain, n, and the number of ethylene oxide units, m. Derivatised samples of industrial FAE mixtures, cleaning products and river and sea waters were chromatographed on a C8 column of the fused-core type using gradient elution with acetonitrile/water mixtures plus 0.1% acetic acid. The hydrocarbon series with even values of n from 8 up to 20, were well resolved using column temperatures in the 15-30 degrees C range. Superior resolution between the consecutive oligomers within the series was also obtained at large values of m; however, reversion of the elution order for the derivatives with m=1 and 0 hindered direct quantitation of a few oligomers at low m values. Full resolution of all the oligomers was achieved by deconvolution with a genetic algorithm assisted by the unconstrained local method of Powell. After preconcentration with C18 solid-phase extraction cartridges, both river and sea water samples also showed the signature of the FAE oligomers. The evaluation of total FAEs in these samples was demonstrated.
Journal of Chromatography 03/2008; 1180(1-2):32-41. · 4.53 Impact Factor
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ABSTRACT: A systematic derivation procedure that greatly facilitates the application of the Taylor method to the integration of kinetic models is developed. In addition, an algorithm that gives the integration step as a function of the required level of accuracy is proposed. Using the Taylor method, application of this algorithm is immediate and largely reduces the integration time. In addition, a new method of integration of kinetic models, whose most important feature is the self-adaptability to the stiffness of the system along the integration process, is developed. This “stiffness-adaptive” Taylor method (SAT method) makes use of several algorithms, combining them to meet the particular requirements of the integration of each species along the integration process. In comparison with the Runge–Kutta–Felhberg, Runge–Kutta–Calahan, Taylor, and Gear methods, the SAT method is the best to integrate non-stiff and stiff kinetic systems, giving the best accuracy and the smallest computing time. © 1992 by John Wiley & Sons, Inc.
Journal of Computational Chemistry 09/2004; 13(7):810 - 820. · 4.58 Impact Factor
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ABSTRACT: The program OPKINE is presented for the study of reaction mechanisms and multicomponent analysis in dynamic conditions. This program is written in FORTRAN-77 for IBM 30/90 and VAX 8300 computers, and permits the simultaneous evaluation of both rate constants and initial reagent concentrations or, alternatively, rate constants and sensitivities. Up to 20 kinetic curves, with up to 400 points each, can be treated to evaluate up to 40 parameters. Integration of the system of differential equations is performed by means of the Runge–Kutta–Fehlberg method. OPKINE is provided with the Simplex, and modified versions of the Davidon–Fletcher–Powell and Gauss–Newton–Marquardt optimization methods. A Monte Carlo procedure to search for the seeds is also available. Simulated experiments have been used to check the performances of the program. Complex kinetic mechanisms and mixtures of a large number of analytes can be managed with good results.
Journal of Computational Chemistry 09/2004; 12(3):283 - 291. · 4.58 Impact Factor
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ABSTRACT: The separation of linear alkylbenzene sulfonates (LAS) by nonaqueous capillary electrophoresis (NACE) using negative polarity, and a buffer containing acetic acid and an alkylamine in nonaqueous ethanol, has been investigated. Several primary, secondary, and tertiary alkylamines with alkyl chains of different length were compared. The solutes travelled against the electroosmotic flow (EOF), and at the same time were braked by association with the alkylamine molecules or with the alkylammonium ions. The best resolution between adjacent LAS homologues (R approximately 2.1), partial isomer resolution in two peaks, and at the same time an excellent repeatability, was obtained with a small dipentylamine excess over the acetic acid. When the buffer concentration increased, resolution between the homologues increased slightly (R approximately 2.4), and a different isomer group was partially separated. A background electrolyte (BGE) containing 10 mM acetic acid and 20 mM dipentylamine to separate and quantify the homologues within 25 min is recommended. The isomer peak profile with up to three peaks can be estimated using this buffer and another one with 80 mM acetic acid and 90 mM dipentylamine. The former BGE was used to determine LAS in liquid and powder laundry detergents. The detection limit for the determination of total LAS in these products was 2.5 microg mL(-1), and the peak area and migration time interday repeatabilities were below 4.3 and 2.8%, respectively.
Electrophoresis 07/2001; 22(10):2017-24. · 3.30 Impact Factor
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ABSTRACT: Octyl- and nonylphenol ethoxylates (OPEs and NPEs) with different numbers of ethoxy units (average values: n = 10 and N = 40 for OPEs, and n = 10 for NPEs) were separated by micellar electrokinetic chromatography under positive polarity using an 80 mM borate buffer of pH 8.5 containing sodium deoxycholate (SDC) or sodium cholate (SC). When sodium dodecyl sulfate (SDS) was added to the background electrolyte (BGE) in the absence of the bile salt, a single peak at a migration time longer than that of the EOF was obtained. Substituting the SDS by a bile salt, the homologues were resolved. At the same bile salt concentration, resolution between the homologues was higher with SDC than using SC. Optimum resolution between consecutive homologues was obtained with 50 mM SDC. In the presence of low or moderate amounts of acetonitrile or n-propanol, the background line improved significantly, whereas resolution may increase or decrease slightly. We propose a procedure for the determination of OPEs and NPEs with optimum resolution between the homologues as well as a modified procedure with improved selectivity for the single-run determination of other absorbing nonionic, cationic, and anionic (such as linear alkylbenzene sulfonates) surfactants in industrial and household cleaning products and its application to a variety of samples. The detection limit was ca. 28 microg x mL(-1) of total NPE (n = 10), and peak area repeatabilities at 50 microg x mL(-1) were 1.7% (intraday) and 5.6% (interday).
Electrophoresis 03/2001; 22(3):526-34. · 3.30 Impact Factor
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ABSTRACT: Mixtures of the cationic surfactants benzalkonium chloride (BKC) and cetylpyridinium chloride (CPC) were quickly resolved and reproducibly and reliably determined by using background electrolytes (BGEs) containing 80 mM borate, pH 8.5, bile salts and large concentrations of an organic solvent. When the bile salt is present, the separation mechanism changes from capillary zone electrophoresis (CZE) to a mixed micellar electrokinetic chromatography (MEKC)-CZE, with predominant MEKC interactions, which lead to an excellent resolution of all the solutes, including the C12-C18 homologues of BKC and CPC. A BGE containing 50 mM sodium deoxycholate and 30% ethanol for an extreme resolution, or 20% tetrahydrofuran for an adequate resolution within a much shorter analysis time, is recommended. The procedure was applied to the determination of the surfactants in industrial and household formulations, with excellent resolution between the homologues, detection limits of a few microg ml(-1) and reproducibilities below 2%.
Journal of Chromatography 11/2000; 895(1-2):227-35. · 4.53 Impact Factor
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ABSTRACT: An improved method for the determination of cow's milk in non-bovine cheese is reported: electrophoresis of whey proteins in acidic, isoelectric buffers. Two background electrolytes (BGEs) have been tested: (i) 50 mM iminodiacetic acid (pH=isoelectric point=2.30 at 25 degrees C), 0.5% hydroxyethylcellulose, 0.1% Tween 20 and 6 M urea (apparent pH 3.1), E=300 V/cm, for the separation of alpha-lactalbumins (alpha-LAs); (ii) a BGE with the same composition, but supplemented with 10% Tween 20, E=450 V/cm, for the fractionation of beta-lactoglobulins (beta-LGs). Surfactants have a discriminating effect on the retention behaviour of the bovine alpha-LA and beta-LG proteins, owing to the different strength of the protein-surfactant association complexes, and are needed for separating these two proteins from small peaks in the electropherograms generated by degradation of casein during cheese ripening. Novel equations are given for deriving the ratio of the area (or height) of bovine alpha-LA, or beta-LG, to the area (or height) of ovine or caprine alpha-LA or beta-LG (such ratios being typically used to determine the percentage of cow's milk in dairy products), since previous equations had marked drawbacks, such as non-linearity of the plots with increasing slopes at high cow's milk percentages, and too broad confidence limits at high cow's milk contents, where the peak area (or height) ratio tends asymptotically to infinite. With the novel procedures reported, contents of cow's milk as low as 1% can be quantified in goat's and ewe's cheeses. The present protocols give lower detection limits, are cheaper and more rapid than any other methodology reported in the literature, and can be easily applied to the routine quality control of binary and ternary cheeses.
Journal of Chromatography 06/2000; 878(2):261-71. · 4.53 Impact Factor
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ABSTRACT: A novel method is reported for analyzing adulteration of goat and ewe cheeses with cow's milk: capillary zone electrophoresis (CZE) in isoelectric, acidic buffers (50 mM imino diacetic acid, IDA, pH = pI 2.3). The cheese samples were extracted with a 20:80 v/v ethanol-water mixture in presence of 3 M urea and 1% beta-mercaptoethanol for 1 h. After centrifugation and lipid extraction, the samples were dissolved in 50 mM IDA, 6 M urea and 0.5% hydroxyethyl cellulose and analyzed by CZE at 700 V/cm. A total of 18 characteristic peaks were resolved among the three types of cheeses and 18 variables were defined as their respective areas. There was excellent similarity among the electrophoretic patterns obtained with cheeses of a given type of milk, while cheeses made with different types of milk were easily distinguishable. Most peaks were common to all cheeses, but the profile differed depending on the type of milk used. Principal component analysis, linear discriminant analysis, and partial least squares regression (PLS) were used for statistical analysis of the data obtained by CZE. In particular, by using PLS multivariate regression, the contents of cow's milk in presumably pure goat and ewe cheeses, as well as in binary and ternary mixtures, could be predicted with relative standard deviations of ca. 6-7%. In addition, the ripening time in goat and ewe cheeses could also be predicted.
Electrophoresis 03/2000; 21(3):633-40. · 3.30 Impact Factor
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ABSTRACT: The determination of the thyreostatics 2-thiouracil, its derivatives (4-methyl-2-thiouracil, 4-propyl-2-thiouracil and 4-phenyl-2-thiouracil) and methimazole in manufactured dried animal feed by micellar electrokinetic chromatography (MEKC) is described. A 99 +/- 5% extraction yield at the 20 micrograms g-1 level (n = 8) was achieved by shaking the milled fodder with methanol-1 M NaOH (80 + 20). Aliquots of the supernatant were injected in a 75 microns x 33.5 cm uncoated silica capillary using pressure; separation was performed at 23 degrees C with 15 kV (positive polarity) in a background electrolyte (BGE) containing 40 mM sodium dihydrogenphosphate, 50 mM sodium dodecyl sulfate and 15 mM Tween 20 at pH 9. When the surfactants were added to the BGE, all the thyreostatics were well resolved and the fodder extracts showed lower backgrounds. The peaks appeared within the 2.25-5.2 min range with efficiencies in the 2.5 x 10(4)-8 x 10(4) range; methimazole appeared in the vicinity of the electroosmotic migration time. Calibration curves were linear within the studied range (20-200 micrograms ml-1, r2 > 0.998). Limits of detection in the extracts of spiked fodder samples ranged from 0.25 to 0.4 microgram ml-1, which corresponded to 0.6-1.0 microgram of drug per gram of fodder. Peak area repeatabilities were about 4% at the 20 micrograms ml-1 level.
The Analyst 02/1999; 124(2):125-8. · 4.23 Impact Factor
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ABSTRACT: A capillary zone electrophoresis (CZE) procedure for the determination of l-ascorbic acid (AA) and total ascorbic acid (TAA, as the sum of AA and dehydroascorbic acid) in vascular plants, lichens, bryophytes, and liverworts is described. The samples were frozen in liquid nitrogen and extracted with 2% metaphosphoric acid. To determine AA, an aliquot was directly injected in a fused-silica capillary. The determination of TAA was performed upon its reduction with dl-homocysteine at pH 7. The background electrolyte contained 60 mM sodium chloride, 60 mM sodium dihydrogen phosphate, 1 x 10(-4)% hexadimetrine bromide, and NaOH up to pH 7. The procedure was rapid and highly reproducible; the limit of detection was 0.2 microg ml-1, which corresponded to 0.4 mg/100 g of sample. The method was validated by comparison with an enzymatic assay. While the enzymatic assay failed to quantify AA in some plants owing to strong background UV absorption, CZE was successful in all the extracts assayed.
Analytical Biochemistry 01/1999; 265(2):275-81. · 3.00 Impact Factor
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ABSTRACT: The spectrophotometric determination of the carbamate pesticides carbaryl, bendiocarb, carbofuran, methiocarb, promecarb and propoxur is proposed. The pesticides are hydrolyzed in alkaline medium to 1-naphthol or phenolates, which are coupled with diazotized trimethylaniline (TMA) in a sodium dodecyl sulfate micellar medium to form the azo dyes. The micellar medium increased the solubility of TMA and enhanced the sensitivity. The performance of TMA was compared with that of sulfanilic acid, which is the conventional reagent used in these determinations. Although the absorbance of the azo dyes of sulfanilic acid was also enhanced in the micellar solution, the sensitivity was still greater with TMA. Further, at the pH required for the coupling reaction (9.5), the absorbance of the blank was negligible with TMA but large with sulfanilic acid. Using TMA, the limits of detection were in the range 0.2-2 micrograms cm-3. The procedure was applied to the determination of carbaryl in spiked tap, river and pond water samples and to the evaluation of various carbamates in commercial pesticide formulations.
The Analyst 06/1997; 122(5):459-63. · 4.23 Impact Factor
