Chromatographic characterisation, under highly aqueous conditions, of a molecularly imprinted polymer binding the herbicide 2,4-dichlorophenoxyacetic acid

The affinity of a 2,4-dichlorophenoxyacetic acid (2,4-D) molecularly imprinted polymer (MIP), which was synthesised directly in an aqueous organic solvent, for its template (2,4-D) was studied and compared with the affinity exhibited by two other reference (control) polymers, NIPA and NIPB, for the same analyte. Zonal chromatography was performed to establish the optimal selectivity, expressed as imprinting factor (IF), under chromatographic conditions more aqueous than those described so far in the literature. Frontal analysis (FA) was performed on columns packed with these polymers, using an optimized mobile phase composed of methanol/phosphate buffer (50/50, v/v), to extract adsorption isotherm data and retrieve binding parameters from the best isotherm model. Surprisingly, the template had comparable and strong affinity for both MIP (K = 3.8 × 10⁴ M⁻¹) and NIPA (K = 1.9 × 10⁴ M⁻¹), although there was a marked difference in the saturation capacities of selective and non-selective sites, as one would expect for an imprinted polymer. NIPB acts as a true control polymer in the sense that it has relatively low affinity for the template (K = 8.0 × 10² M⁻¹). This work provides the first frontal chromatographic characterization of such a polymer in a water-rich environment over a wide concentration range. The significance of this work stems from the fact that the chromatographic approach used is generic and can be applied readily to other analytes, but also because there is an increasing demand for well-characterised imprinted materials that function effectively in aqueous media and are thus well-suited for analytical science applications involving, for example, biofluids and environmental water samples.     

Determination of competitive adsorption isotherms applying the nonlinear frequency response method: Part I. Theoretical analysis

In this work adsorption equilibria of binary mixtures are quantified analyzing the nonlinear frequency response of a chromatographic column. Local partial derivatives of an isotherm model can be estimated for certain steady-states from the low frequency asymptotes of the corresponding frequency response functions (FRFs). The required FRFs correspond to two different compounds and the type of the imposed inlet concentration changes, e.g. periodical inlet concentration changes of only one compound or of both of them. For an accurate determination of isotherm parameters, it is necessary to approach as close as possible the low frequency asymptotic behaviour of these functions. Based on principles valid for the FRFs corresponding to the adsorption of a single solute, frequencies needed to reach the low frequency asymptotes of the functions of interest for estimating competitive isotherms are defined in this paper. The relation between the accuracy of the isotherm parameters determined and numbers and types of periodical inlet concentration changes and steady-states analyzed is also evaluated.     

Binding behaviour of molecularly imprinted polymers prepared by a hierarchical approach in mesoporous silica beads of varying porosity

One of the most interesting methods for preparing molecularly imprinted polymers with controlled morphology consists in filling the pores of silica beads with an imprinting mixture, polymerizing it and dissolving the support, leaving porous imprinted beads that are the "negative image" of the silica beads. The main advantage of such an approach consists in the easy preparation of spherical imprinted polymeric particles with narrow diameter and pore size distribution, particularly indicated for chromatographic applications. In this approach it has been shown that the resulting morphology of polymeric beads depends essentially on the porosity and surface properties of the silica beads that act as microreactors for the thermopolymerization process. Anyway, it is not yet clear if the porosity of the silica beads influences the binding properties of the resulting imprinted beads. In this paper, we report the effect of different porosities of the starting mesoporous silica beads on the resulting binding properties of imprinted polymers with molecular recognition properties towards the fungicide carbendazim. The morphological properties of the imprinted beads prepared through this hierarchical approach were measured by nitrogen adsorption porosimetry and compared with a reference imprinted material prepared by bulk polymerization. The chromatographic behaviour of HPLC columns packed with the imprinted materials were examined by eluting increasing amounts of carbendazim and extracting the binding parameters through a peak profiling approach. The experimental results obtained show that the resulting binding properties of the imprinted beads are strongly affected by the polymerization approach used but not by the initial porosity of the silica beads, with the sole exception of the binding site density, which appears to be inversely proportional to them.     

Custom synthesis of molecular imprinted polymers for biotechnological application: Preparation of a polymer selective for tylosin

A molecularly imprinted polymer (MIP) selective for tylosin was designed and synthesised using a computational method (MIP “dialling”). In re-binding experiments the MIP demonstrated high affinity for tylosin in aqueous solutions and in organic solvents. The synthesised polymer was tested for re-binding with the template and related metabolites such as tylactone, narbomycin and picromycin. The HPLC analysis showed that the computationally designed polymer is specific and capable of separating the template from its structural analogues. The MIP was capable of recovering tylosin from broth samples. The polymer capacity for tylosin was estimated as 6.4 mg/g for MIP, which was suitable for practical application and tylosin recovery from broth samples. Among the advantages of this was the possibility to adsorb tylosin from a complex media with easy removal of oils and other impurities which are present in significant quantities, which can create problems for its chromatographic purification procedure. The MIP “dialling” procedure can have a general significance for the fast preparation of specific adsorbents for biotechnological applications.     

Determination of competitive adsorption isotherms applying the nonlinear frequency response method: Part II. Experimental demonstration

This paper demonstrates an experimental application of the nonlinear frequency response (FR) method extension to determine adsorption isotherms of binary mixtures. This method, based on the analysis of the response of a chromatographic column subjected to the sinusoidal inlet concentration changes, is shown to be an alternative for isotherm determination. The critical issue related to the successful application of the method is to reach experimentally the low frequency asymptotic behaviour of the corresponding frequency response functions (FRFs). Although, there are different possibilities to perform periodical inlet concentration changes, in this paper only simultaneous changes for both components were considered. The adsorption of phenol and 2-phenylethanol on octadecyl silica was analyzed experimentally using a mixture of methanol and water as a solvent. Parameters of competitive isotherms were also estimated for comparison using the classical perturbation method. Despite certain differences between competitive isotherms estimated with the two methods that were found, the obtained results show the potential of the nonlinear FR method for measuring competitive isotherms.     

Indirect competitive immunoassay for trichlorophenol determination: Rational evaluation of the competitor heterology effect

An improved competitive indirect immunoassay for the detection of 2,4,6-trichlorophenol (2,4,6-TCP) has been developed and optimized by preparing heterologous haptens that have been evaluated as coating antigens. The relation between the degree of heterology and immunoassay detectability has been investigated according to the geometric and electronic distribution similarities between the haptens and the analyte using molecular modeling tools. The assay has been characterized according to different physicochemical parameters such as the incubation time, the ionic strength, the effect of detergents and the pH. The resulting assay has an IC₅₀ of 1.44 μg l⁻¹ and a limit of detection (LOD) of 0.2 μg l⁻¹ and it shows a good accuracy and suitability to analyze trichlorophenol in drinking water.     

Environmental assessment of CFC alternatives: Rate constants for the reactions of OH radicals with fluorinated compounds

The rate constants for the reactions of OH radicals with CF₃OCHFCF₃, and CF₃CHFCF₃ have been measured over the temperature range 250-430 K. Kinetic measurements have been carried out using the flash photolysis, and laser photolysis methods combined, respectively, with the laser induced fluorescence technique. The influence of impurities in the samples has been investigated by using gas chromatography. No sizable effect of impurities was found on the measured rate constants of these fluorinated compounds, if the purified samples were used in the measurements. The following Arrhenius expressions were determined: k(CF₃OCHFCF₃) = (4.39 ± 1.38) × 10⁻¹³ exp[−(1780 ± 100)/T] cm³ molecule⁻¹ s⁻¹, and k(CF₃CHFCF₃) = (6.19 ± 2.07) × 10⁻¹³ exp[−(1830 ± 100)/T] cm³ molecule⁻¹ s⁻¹.     

An unsteady state method for the measurement of polymer thermal diffusivity: I. Development of a cell

In this work the development of a cell able to determine the thermal diffusivity of polymers in their fused or solid state, using an unsteady state technique, is presented. In this case a step down perturbation method was used because of its easy experimental setup. For each experimental run a temperature step down perturbation of 10 °C was applied, and the temperature evolution with time and position were registered and saved. Thermal diffusivities were then determined by regression using those data and a simple analytical model. The polymers used to test the cell were high density polyethylene and polypropylene (PP). For the first polymer, the thermal diffusivity was found to be 2.05×10⁻⁷ m²/s, which compare satisfactorily with a value of 1.97×10⁻⁷ m²/s calculated from reported values of density, heat capacity and heat conductivity. For the PP the value was found to be 7.08×10⁻⁷ m²/s with a positive deviation of 5.05% when compared with a computed value of 6.74×10⁻⁷ m²/s. Taking into account experimental errors, and the natural variations between different stocks of materials, the observed differences are acceptable.     

Dynamic control of protein conformation transition in chromatographic separation based on hydrophobic interactions: Molecular dynamics simulation

Conformational transitions of a protein in hydrophobic interaction based chromatography, including hydrophobic interaction chromatography (HIC) and reversed-phase liquid chromatography (RPLC), and their impact on the separation process and performance were probed by molecular dynamics simulation of a 46-bead β-barrel coarse-grained model protein in a confined pore, which represents the porous adsorbent. The transition of the adsorbed protein from the native conformation to an unfolded one occurred as a result of strong hydrophobic interactions with the pore surface, which reduced the formation of protein aggregates. The conformational transition was also displayed in the simulation once an elution buffer characterized by weaker hydrophobicity was introduced to strip protein from pore surface. The discharged proteins that underwent conformational transition were prone to aggregation; thus, an unsatisfactory yield of the native protein was obtained. An orthogonal experiment revealed that in addition to the strengths of the protein–protein and protein–adsorbent hydrophobic interactions, the elution time required to reduce the above-mentioned interactions also determined the yield of native protein by HIC and RPLC. Stepwise elution, characterized by sequential reduction of the hydrophobic interactions between the protein and adsorbent, was presented as a dynamic strategy for tuning conformational transitions to favor the native conformation and reduce the formation of protein aggregates during the elution process. The yield of the native protein obtained by this dynamic operation strategy was higher than that obtained by steady-state elution. The simulation study qualitatively reproduced the experimental observations and provided molecular insight that would be helpful for designing and optimizing HIC and RPLC separation of proteins.     

Fast high-throughput method for the determination of acidity constants by capillary electrophoresis: I. Monoprotic weak acids and bases

A new and fast method to determine acidity constants of monoprotic weak acids and bases by capillary zone electrophoresis based on the use of an internal standard (compound of similar nature and acidity constant as the analyte) has been developed. This method requires only two electrophoretic runs for the determination of an acidity constant: a first one at a pH where both analyte and internal standard are totally ionized, and a second one at another pH where both are partially ionized. Furthermore, the method is not pH dependent, so an accurate measure of the pH of the buffer solutions is not needed. The acidity constants of several phenols and amines have been measured using internal standards of known pK_a, obtaining a mean deviation of 0.05 pH units compared to the literature values.     

Synthesis of triarylphosphines having para -SH and -SMe groups: Preparation of their complexes and formation of a monolayer on a gold surface

The phosphines P(C₆H₄-4-SR)₃ (R = H, Me, 2-C₅H₉O) and (C₆H₄-4-SR)₂PCH₂CH₂P(C₆H₄-4-SR)₂ (R = H, Me) have been synthesized. The phosphines with -SMe groups can be prepared by reaction of 4-BrC₆H₄SMe with either BuLi or magnesium (to generate the corresponding Grignard compound) followed by reaction with PCl₃ or Cl₂PCH₂CH₂PCl₂, respectively. The methyl group can be eliminated by reaction with sodium in liquid NH₃. Other methods of protection/deprotection of the thiol group failed to afford the desired compounds. Reaction of 4-BrC₆H₄SH with dihydropyrane afforded the protected thiol 4-BrC₆H₄S-2-C₅H₉O from which the corresponding phosphine was successfully synthesized. However, attempts to remove the tetrahydropyranyl group by reaction with AgNO₃-HCl, gave an insoluble polymer as product. Reaction of P(C₆H₄SR)₃ (R = H, Me) with Ni(CO)₄ affords the corresponding mono phosphine complex quantitatively. The complex with the unprotected thiol group can be absorbed on a gold surface and the corresponding ν_CO bands were detected by grazing angle Fourier transform infrared reflection absorption spectroscopy (grazing angle FTIR-RAS). Reaction of Rh(acac)(CO)₂ with P(C₆H₄SR)₃ (R = Me) affords the complex Rh(acac)(CO)(P(C₆H₄SR)₃) (R = Me), but if R = H a polymer insoluble in any solvent was obtained. The same occurs in the case of PtCl₂(CO)(DMSO). Apparently, once P(C₆H₄SH)₃ is coordinated to a metal not in the zero oxidation state, oxidation of the thiol group to disulphide becomes very easy even in a dinitrogen atmosphere.     

1,4-Addition reaction of ethyl bromodifluoroacetate to Michael acceptors in the presence of copper powder: Improvement of the reaction using TMEDA as an additive

Reaction of ethyl bromodifluoroacetate with a variety of Michael acceptors was tremendously improved by the addition of TMEDA. Using this additive, 1,4-adducts were formed exclusively, and any 1,2-adducts or radical adducts were not obtained. THF or other low boiling solvents can be used as a solvent. This simplifies the work-up of the reaction effectively.     

In situ continuous monitoring of chloride, nitrate and ammonium in a temporary stream: Comparison with standard methods

A multi-parameter probe was used for in situ chloride, nitrate and ammonium measurements in a temporary stream (Ribeira da Pardiela, in the South of Portugal). Comparison with standard analytical methods was performed for all elements. For chloride, the results of the probe depicted the same behaviour as that obtained with the standard method, although it is clear that the matrix effects were present. For nitrate, the results obtained with the probe were in agreement with the other standard methods used, except for samples collected during drought, when the stream water became brownish and exhaled an offensive smell, due to the decomposition of organic matter. For ammonium, surprisingly the probe show to be the best option, the phenate method being affected by matrix effects. The results still suggest an interference of the bicarbonate ion on nitrate determination, but standard additions approach was shown to minimize most of the matrix effects. Recoveries were reasonable to good for all the three anions under scrutiny.     

Physico-chemical aspects of polyethylene processing in an open mixer: Part 27: Formal kinetics of aldehyde and carboxylic acid formation in the initial stages

There are many reactions susceptible to yield aldehydes and acids in polyethylene melts. It is β-scission of the alkoxy radicals formed on bimolecular hydroperoxide decomposition that is expected to be one of the main sources of the aldehydes that are formed at increasing rates in the early stages of polyethylene processing. Acid-catalyzed decomposition of allylic hydroperoxides is another source of substantial amounts of aldehydes. Formation and decomposition of α,γ- and α,β-di-hydroperoxides should yield acids. The activation energy estimated for these different processes is very large (about 57 kcal/mol) so that their contribution could be significant in the high temperature range only. This is different for the reaction of aldehydes with hydroperoxides to yield peroxy-hemiacetals. These intermediates can be expected mainly in the low temperature range where hydroperoxides are accumulating. Decomposition of the peroxy-hemiacetals gives acids as one of the main products. Free-radical induced oxidation of aldehydes is likely to yield peracids as far as oxygen addition is competitive with decarbonylation. The main problem is the transformation of the peracids into acids. The reaction with double bonds is expected to yield significantly more acids than thermal decomposition of peracids. If the last occurs, it will be followed mainly by decarboxylation. The overall activation energy for both processes of acid formation is negative (−18 to −20 kcal/mol). It is some combination of the various mechanisms examined that might account for the experimental activation energy for acid formation in the initial stages that is close to 18 kcal/mol.     

Selectivity patterns in micellar electrokinetic chromatography: Characterization of fluorinated and aliphatic alcohol modifiers by micellar selectivity triangle

The usefulness of the micellar selectivity triangle (MST) for prediction and interpretation of separation patterns in micellar electrokinetic chromatography (MEKC) separations is presented. In addition, we demonstrate the capability of controlling selectivity properties of micelles through addition of organic modifiers with known solvation properties as predicted by MST. The examples are modification of the hydrogen bond donor (HBD) micelle of lithium perfluorooctanesulfonate, the hydrogen bond acceptor (HBA) micelle of tetradecyltrimethylammonium bromide, and the sodium dodecyl sulfate micelles with intermediate hydrogen bonding properties with two hydrophobic organic modifiers. One is an aliphatic alcohol, n-pentanol that can act as both a HBA and a HBD; by contrast, the other organic modifier is a fluorinated alcohol, hexafluoroisopropanol that is a strong HBD modifier and would enhance the hydrogen bond donor strength of micelles. A test sample composed of 20 small organic solutes representing HBA, HBD, and non-hydrogen bond aromatic compounds was carefully selected. The trends in retention behavior of these compounds in different micelles are consistent with the selectivity patterns predicted by the MST scheme. To the best of our knowledge, this is the first report on the unique selectivity of fluorinated alcohols as modifiers in MEKC. These results demonstrate the usefulness of the MST scheme for identifying pseudo-phases with highly similar or different selectivities and can serve as a guide for judicious selection of modifiers to create pseudo-phases with desired selectivity behavior on a rational basis.     

Preparation and binding study of solid-phase microextraction fiber on the basis of ametryn-imprinted polymer: Application to the selective extraction of persistent triazine herbicides in tap water,rice, maize and onion

A monolithic ametryn molecular-imprinted polymer based on a simple polymerization method was fabricated for use as new solid-phase microextraction (SPME) fiber, which can be coupled with GC and GC/MS for selective extraction and analysis of triazine herbicides. Methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA) and ametryn bear role of functional monomer, cross-linker and template, respectively. In the optimized conditions the fabricated fiber showed better molecular recognition abilities for methylthiotriazine herbicides than chloro-triazine herbicides. By use of bi-Langmuir isotherm model the evaluated equilibrium constants for ametryn were 0.01 and 890.69 μM⁻¹, and the numbers of binding sites were 129.98 and 5.82 nmol g⁻¹, respectively. The high extraction efficiency was obtained for ametryn, prometryn, terbutryn, atrazine, simazine, propazine, and cyanazine, yielding the detection limits of 14, 28, 45, 56, 85, 95 and 74 ng mL⁻¹, respectively by GC with flame ionization detection. The reliability of the prepared fiber for extraction of ametryn and other analogues in real samples has been investigated and proved by using spiked samples such as tap water, rice, maize, and onion.     

Determination of amoxicillin in pharmaceuticals using sequential injection analysis (SIA): Evaluation of the presence of interferents using multivariate curve resolution

In this study, we report a method for determining amoxicillin in pharmaceuticals using sequential injection analysis (SIA) with a diode-array spectrophotometric detector. Before determining the amoxicillin, we use multivariate curve resolution with alternating least squares (MCR-ALS) to investigate whether any interferents are present. With a suitable analytical sequence, we can use SIA to generate a pH gradient and, for each sample, obtain a matrix of data that have been analysed by several chemometric techniques based on multivariate analysis: principal components analysis (PCA), simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) and MCR-ALS. In this way we obtain the concentration profiles and spectra of the species in the sample.We studied six pharmaceuticals containing amoxicillin. Two of these pharmaceuticals contained no interferents, one contained an interferent but amoxicillin had a selective spectral area with respect to it, and the other three contained interferents. For the first three samples, we set up a system of univariate calibration, which determines amoxicillin quickly (it can analyse 20 samples/h) using inexpensive instrumentation and reactives.     

Thermal decomposition of solid benzoyl peroxide in the presence of solid admixtures: Part I. Observations made under constant heating conditions

The effect of some solid admixtures on the decomposition of benzoyl peroxide in the solid state has been observed by means of differential enthalpic analysis. Admixtures which either might well participate in the radical chain process or only dilute the given reaction system have been chosen. These have been various kinds of carbon black differing from each other by free-spin concentration, chain structure and surface area, further activated charcoal and colloid silica, respectively.     

Extraction and preconcentration of polychlorinated dibenzo-p-dioxins using the cloud-point methodology: Application to their determination in water samples by high-performance liquid chromatography

A new analytical method based on the cloud-point extraction (CPE) methodology combined with high-performance liquid chromatography is used in this study for the extraction and quantification of polychlorinated dibenzo-p-dioxins. These compounds are extracted by using the non-ionic surfactant polyoxyethylene 10 lauryl ether (POLE) from different aqueous systems: fresh, brackish and sea waters. After the cloud-point extraction, which also allowed a preconcentration of the PCDDs being studied, the samples were analysed using a HPLC-UV system under the optimum chromatographic conditions. The proposed method showed very satisfactory recovery percentages for the PCDDs under study and standard deviation values lower than 2%.The results obtained were finally compared with those found using the traditional liquid-liquid extraction method.