Carlos Ubide

Study of a fluorometric-enzymatic method for bilirubin based on chemically modified bilirubin-oxidase and multivariate calibration

The chemical derivatization of bilirubin oxidase (BOx) with a fluorescein derivative (FS) yields a chemically modified enzyme (BOx-FS), with excitation and emission maxima at 487 and 520 nm, respectively. During the oxygen oxidation reaction of bilirubins, in the presence of the modified enzyme, the change in the fluorescence of the modified enzyme depends on the concentration and type of bilirubin. This effect can be used for analytical purposes. Firstly, a theoretical-experimental study of the analytical system was carried out. The mechanism responsible for the fluorescence variation was clarified, a mathematical model developed and the variables affecting the fluorescence changes optimized. The concentration ranges in which the model can be applied (up to 12 mg bilirubin l(-1)), and the precision of the measurement (about 4%) were established for the three bilirubins. The application of the methodology to the simultaneous determination of direct and total bilirubins were studied by applying multivariate calibration methods to the whole kinetic profiles. A reduced calibration matrix (derived from a 5(3) base matrix) is proposed for calibration and different numerical methods were tested: Principal Components Regression (PCR), Partial Least Squares Regression (PLS) and Artificial Neural Networks (ANN). The simultaneous determination of direct and total bilirubin (average validation errors of about 9 and 10%, respectively) can be carried out from a single run. Furthermore, a semi-quantitative speciation of the three bilirubins (free, conjugated and albumin-bonded bilirubin) may be simultaneously obtained.

Simultaneous kinetic spectrophotometric determination of o-, m-and p-aminophenol using partial least squares calibration

The oxidative coupling reactions of p-aminophenol with m-and p-aminophenol in the presence of octacyanomolybdate(v) were used as the basis of a differential kinetic method for the simultaneous determination of o-, m-and p-aminophenol (o-, m-and p-AP) in binary and ternary mixtures. The influence of Mov concentration, acidity, ionic strength and temperature was studied in order to establish optimum conditions. Several oxidants, including Ce4+, CrVI, MnVII, H2O2 and metaperiodate and FeIII, MnIV, MoV and WV cyano complexes were tried. The MoV cyano complex was chosen because it has a suitable redox potential and gives rise to kinetically useful runs. The reaction must be implemented under sub-stoichiometric concentrations of oxidant and so the concentration of each aminophenol in the mixture influences the reaction of the others, originating in a system with a high degree of non-linearity. As a result, most methods of data treatment cannot be used and multivariate calibration (PLS) was applied to the data acquired by a diode-array spectrophotometer between 340 and 500 nm. PLS calibration with a number of factors ranging between six and nine allowed satisfactory results for all kinds of mixtures. Aminophenols can be determined over the concentration range 0.1–1.0 mmol l–1 for o-AP, 0.3–1.0 mmol l–1 for m-AP and 0.25–3.0 mmol l–1 for p-AP with errors of less than 10% in all instances. No systematic errors were detected.

Individual Kinetic Determinations Using Partial Least SquaresCalibration

The slow oxidation reactions of cysteine, tyrosine and tryptophan by hexacyanomanganate(IV) were used to investigate partial least squares calibration (PLS) as a method to be applied to the kinetic determination of individual species. All the three reactions are first order in hexacyanomanganate(IV) concentration and the reactions with cysteine and tyrosine are first order in amino acid concentration. The stoichiometry was studied in each case. The determination results were always compared with those obtained by applying the initial rate method to the same kinetic runs. When suitable kinetic curves are obtained and the initial rate is easily determined, both methods give similar results; however, when the initial rate is not easily determined (too fast reaction, slope that changes very quickly, etc.) the results obtained with PLS are better (better precision and wider dynamic range). Moreover, and in contrast to the initial rate method, PLS allowed tryptophan to be determined in the presence of some different feed matrices, provided the calibration set was of the same nature as the samples. This advantage is of general application and makes the analytical method more robust. PLS was tested on uncatalysed reactions but it could equally be applied to catalysed reactions.

Linear and non-linear multivariate calibration methods for multicomponent kinetic determinations in cases of severe non-linearity

Both p-aminophenol (p-AP) and p-phenylenediamine (p-PDA) undergo oxidative coupling reactions in the presence of octacyanomolibdate(V) as oxidant. The kinetics of these reactions can be followed spectrophotometrically. When they happen to be in the same reaction mixture, relevant characteristics are (1) related to reaction conditions; the reagent must be in deficit and (2) related to the reaction products; ‘cross-coupling’ reactions are also involved. All this gives rise to systems with severe non-linearities. This is the reason why they have been used here to test the predictive ability of several linear and non-linear algorithms in cases of special complexity. Principal component regression (PCR), partial least squares regression (PLS) and three different forms of applying artificial neural networks (ANNs), with and without reduction of variables, have been tested. All the models were applied to the same calibration and validation sets. Typical prediction errors ranged between 2 and 5%. Not very different results were found when independent accuracy and precision studies were accomplished, regardless of whether linear or non-linear calibration methods were used. This is probably due to differences in the spectroscopic as well as the kinetic behaviour of both analytes. These differences allowed the construction of ANN calibration models with only 30 original variables (kinetic profiles of 10 data points at three selected different wavelengths).

Efficient hydridoirida-β-diketone-catalyzed hydrolysis of ammonia- or amine-boranes for hydrogen generation in air

The dihydridoirida-β-diketone [IrH2{(PPh2(o-C6H4CO))2H}] (2) has been used as a homogeneous catalyst for the hydrolysis of ammonia- or amine-boranes to generate up to 3 equivalents of hydrogen in the presence of air. When using 0.5 mol% loading of 2, dimethylamine-borane is hydrolysed completely within 8 min at 30 °C and maintains its activity in consecutive runs. Ammonia-borane or tert-butylamine-borane is hydrolysed completely within 32 or 25 min respectively. Triethylamine-borane fails to be hydrolysed. Kinetic studies suggest a sequence of two consecutive first-order reactions, in which an intermediate builds up and finally falls, with the first step being the rate controlling step. ΔH1(‡) are in the range 65-85 kJ mol(-1) and negative values of ΔS1(‡) are obtained. A multinuclear NMR study of the catalyzed reaction shows the formation of a resting state (A) of the active catalyst proposed to be of the hydridodiacyl type [IrH(PPh2(o-C6H4CO))2(solvent)] with a hydride trans to the acyl group. In the absence of substrate a dormant species (B) is formed. By the reaction of hydridoirida-β-diketones with ammonia, the hydridoirida-β-ketoimine [IrHCl{(PPh2(o-C6H4CO))(PPh2(o-C6H4CNH))H}] (3) and the hydridobis(acylphosphane)aminoiridium(III) complex [IrH(PPh2(o-C6H4CO))2(NH3)] (4), with a hydride trans to phosphane, are formed. Aromatic amines such as aniline or anisidines afford cationic [IrH{(PPh2(o-C6H4CO))2H}(C6H4RNH2)]ClO4 (R = H (6); p-MeO (7); o-MeO (8)) hydridoirida-β-diketones with a coordinated amine group trans to the hydride. The dormant species B is proposed to be of the hydridobis(acylphosphine)aminoiridium(III) type with a hydride trans to the amine group.

New way of application of the bromate–bromide mixture in kinetic analysis☆

Abstract The bromine generated in situ by the bromate–bromide mixture has usually been used in kinetic analysis to determine either individual species that give reaction with bromine or the total concentration of mixtures of those analytes. In this paper the bromate–bromide mixture is proposed as a reagent for multicomponent kinetic determinations. Mixtures of acetylsalicylic acid (ASA) and paracetamol (AAP) have been used to test the discriminating ability of reactions with bromine. The reaction between bromine and AAP is fast and the reaction between bromine and ASA is negligible. However, ASA slowly hydrolyzes to yield salicylic acid (SA) and acetic acid, and the reaction between bromine and SA can be considered fast in the reaction conditions. This complex behaviour contains enough information to resolve mixtures of ASA and AAP after applying partial least squares regression (PLS) to the kinetic photometric data. After optimization, errors around 5–6% were found for the determination of ASA and AAP. Quimiometric considerations on the calibration models are made. The method has been successfully applied to the determination of ASA and AAP in pharmaceuticals.

Image analysis for maintenance of coating quality in nickel electroplating baths--real time control.

The aim of this paper is to show how it is possible to extract analytical information from images acquired with a flatbed scanner and make use of this information for real time control of a nickel plating process. Digital images of plated steel sheets in a nickel bath are used to follow the process under degradation of specific additives. Dedicated software has been developed for making the obtained results accessible to process operators. This includes obtaining the RGB image, to select the red channel data exclusively, to calculate the histogram of the red channel data and to calculate the mean colour value (MCV) and the standard deviation of the red channel data. MCV is then used by the software to determine the concentration of the additives Supreme Plus Brightner (SPB) and SA-1 (for confidentiality reasons, the chemical contents cannot be further detailed) present in the bath (these two additives degrade and their concentration changes during the process). Finally, the software informs the operator when the bath is generating unsuitable quality plating and suggests the amount of SPB and SA-1 to be added in order to recover the original plating quality.

Uncertainty due to the quantification step in analytical methods.

The quantification step is an important source of uncertainty in analytical methods, but it is frequently misunderstood and disregarded. In this paper, it is shown how this uncertainty is closely related to the linear response range of a method, and to the Pearson correlation coefficient of the calibration line. So, if there is a need for a pre-fixed quantification uncertainty, the linear response range will be affected. Some practical cases are given showing the quantification uncertainty significance. The theoretical equation giving the value of the quantification uncertainty is deduced from which new conclusions can be taken out. Because of that, the quantification uncertainty can easily be calculated and the parameters that really affect its value are shown along the paper. Some final considerations about detection limits and two-point calibration lines are also given. The paper can also be considered a reflection on uncertainty owed to calibration and on their consequences on the analytical methodology.

The hexacyanomanganate(IV)-hydrogen peroxide reaction. Kinetic determination of vanadium.

The reaction between hexacyanomanganate(IV) and hydrogen peroxide in acidic medium is strongly catalysed by vanadium. The stoichiometry has been proved to be Delta[Mn(IV)]/Delta[H(2)O(2)]=1. S-shaped absorbance-time curves are obtained for a wide range of conditions, which seems to be due to the transient formation of hydroperoxyl radicals, HO(2).; therefore, the reaction is autoinduced. The reaction kinetics is first order on the hexacyanomanganate(IV) concentration. Complex dependence of the initial rate on the H(2)O(2) as well as H(+) concentrations are observed both for the uncatalysed and for the catalysed reactions, but the rate laws are given and reaction mechanisms are proposed. The catalytic effect of vanadium has been used to determine traces of vanadium (the (IV) and (V) oxidation states are determined together). By applying the initial rate method a detection limit of 0.9 ng ml(-1) (18 nM), and a linear range up to 50 ng ml(-1) were found. The relative standard deviations for the 4 and 25.5 ng ml(-1) levels were 5.2 and 2.5% respectively. Positive kinetic interferences from Cr(VI), Hg(I) and Ag(I) have been observed; other interferences are less severe and Cr(IIl), Fe(II) and Fe(III), among many others, do not interfere. The Mn(IV) solution must be prepared daily and its conservation needs some care (0 degrees C) but the proposed method has been applied to the determination of vanadium in a phosphate rock (reference material, BCR No. 32) without previous separations. Recovery experiments have also been performed; excellent results were obtained in both cases.

Improved selectivity in multicomponent determinations through interference modelling by applying partial least squares regression to kinetic profiles

Binary and ternary mixtures of cysteine, tyrosine and tryptophan were used to show how kinetic methods together with partial least squares regression (PLSR) can be used to overcome interferences in multicomponent determinations. Two methods were compared: an equilibrium method which uses the characteristic UV spectra of amino acids and a kinetic method which follows the reaction kinetics of the oxidation of the amino acids by hexacyanomanganate(IV). In both cases, multi-wavelength detection with a diode-array spectrophotometer was used. The results obtained from the two methods are critically compared. Lower concentrations of tyrosine and tryptophan can be determined by the equilibrium method because of their high absorptivities. On the other hand, the kinetic method, in conjunction with PLSR, can be considered an example of how selectivity can be improved. This allowed the resolution of mixtures of tyrosine and tryptophan in the presence of several complex spiked matrices (aqueous extracts of feed samples that interfere to different extents) without any previous separation.