Carol H. Collins

Validação em métodos cromatográficos e eletroforéticos

The validation of an analytical method is fundamental to implementing a quality control system in any analytical laboratory. As the separation techniques, GC, HPLC and CE, are often the principal tools used in such determinations, procedure validation is a necessity. The objective of this review is to describe the main aspects of validation in chromatographic and electrophoretic analysis, showing, in a general way, the similarities and differences between the guidelines established by the different Brazilian and international regulatory agencies.The validation of an analytical method is fundamental to implementing a quality control system in any analytical laboratory. As the separation techniques, GC, HPLC and CE, are often the principal tools used in such determinations, procedure validation is a necessity. The objective of this review is to describe the main aspects of validation in chromatographic and electrophoretic analysis, showing, in a general way, the similarities and differences between the guidelines established by the different Brazilian and international regulatory agencies.

Immobilization of horseradish peroxidase onto polyaniline polymers

Some chemically synthesized conductive polymers, based on polyaniline (PANI), were used as supports for Horseradish Peroxidase (HRP) immobilization. The polymers were first activated with glutaraldehyde (PANIG) and then HRP was successfully immobilized. Among the tested polymers, those with intermediate oxidation state (PANIG1, PANIG2 and PANIG3) were more suitable for HRP immobilization than that predominately reduced (PANIG2) or oxidized (PANIG5). PANIG1 presented the best performance for HRP immobilization, and the analysis of the protein versus active enzyme retention showed that immobilized HRP maintained 100% native activity. The effects of enzyme concentration, reaction time and pH on the immobilization efficiency were investigated.

Métodos de extração e/ou concentração de compostos encontrados em fluidos biológicos para posterior determinação cromatográfica

When organic compounds present in biological fluids are analysed by chromatographic methods, it is generally necessary to carry out a prior sample preparation due the high complexity of this type of sample, especially when the compounds to be determinated are found in very low concentrations. This article describes some of the principal methods for sample preparation in analyses of substances present in biological fluids. The methods include liquid-liquid extraction, solid phase extraction, supercritical fluid extraction and extraction using solid and liquid membranes. The advantages and disadvantages of these methods are discussed.

Speciation of aqueous chromium(VI) solutions with the aid of Q-mode factor analysis followed by oblique projection

The identities of the species of chromium(VI) that are present in aqueous solution, their spectra and their equilibria, continue to be a subject of discussion in the literature. In this paper, the composition of the Cr(VI) equilibria was estimated from the UV-vis spectra of dilute potassium dichromate solutions, without any prior knowledge of the quantities of pure components, with the aid of Imbrie Q-mode factor analysis (Q-mode FA) followed by Varimax rotation and Imbrie oblique projection. Combining these results with the k-matrix method, it was possible to obtain the spectra of the individual Cr(VI) species. Sets of 3.3x10(-4) and 3.3x10(-5) mol l(-1) Cr(VI) solutions were studied. In the pH range from 1 to 12, two factors were identified, which were related to the two species, chromate ion (CrO(4)(2-)) and bichromate ion (HCrO(4)(-)). When the analysis was extended to concentrated acid media, another factor appeared, which was related to chromic acid (H(2)CrO(4)). No evidence for the dichromate ion (Cr(2)O(7)(2-)) was seen at the Cr(VI) concentrations used. The spectra of the pure components were obtained and pK values for the first and second chromic acid dissociations were estimated as -0.54 and 5.8, respectively.

Nano-liquid chromatography in pharmaceutical and biomedical research.

Miniaturized separation techniques have emerged as environmentally friendly alternatives to available separation methods. Nano-liquid chromatography (nano-LC), microchip devices and nano-capillary electrophoresis are miniaturized methods that minimize reagent consumption and waste generation. Furthermore, the low levels of analytes, especially in biological samples, promote the search for more highly sensitive techniques; coupled to mass spectrometry, nano-LC has great potential to become an indispensable tool for routine analysis of biomolecules. This short review presents the fundamental aspects of nano-LC analytical instrumentation, discussing practical considerations and the primary differences between miniaturized and conventional instrumentation. Some theoretical aspects are discussed to better explain both the potential and the principal limitations of nano-LC. Recent pharmaceutical and biomedical applications of this separation technique are also presented to indicate the satisfactory performance for complex matrices, especially for proteomic analysis, that is obtained with nano-LC.

O estado da arte da cromatografia associada à espectrometria de massas acoplada à espectrometria de massas na análise de compostos tóxicos em alimentos

Chromatography combined with several different detection systems is one of the more used and better performing analytical tools. Chromatography with tandem mass spectrometric detection gives highly selective and sensitive analyses and permits obtaining structural information about the analites and about their molar masses. Due to these characteristics, this analytical technique is very efficient when used to detect substances at trace levels in complex matrices. In this paper we review instrumental and technical aspects of chromatography-tandem mass spectrometry and the state of the art of the technique as it is applied to analysis of toxic substances in food.

Reevaluation of ethanol as organic modifier for use in HPLC-RP mobile phases

Ethanol:water mixtures have been reevaluated for use as reversed phase mobile phases. Optimization of the chromatographic conditions for both C8 and C18 columns was carried out through a factorial design which evaluates temperature, ethanol concentration and mobile phase flow rate. With the C8 stationary phase, peak asymmetries were not significantly altered at the different points in the factorial design while the retention factors and resolutions were somewhat lower at higher temperatures, where the viscosity of EtOH is lower. Similar observations were obtained with the C18 phase. The efficiency of the ethanol:water mobile phase for the separation of mixtures containing neutral and basic compounds was compared with those obtained using methanol:water and acetonitrile:water mobile phases. Ethanol was shown to be a good organic modifier for RP-HPLC, with good chromatographic properties. This, considering the much lower toxicity of ethanol, the facility of its disposal, and its favorable cost, should make ethanol:H2O the mobile phase of choice for many RP-HPLC applications.

Stability of high-performance liquid chromatography columns packed with poly(methyloctylsiloxane) sorbed and radiation-immobilized onto porous silica and zirconized silica.

Reversed-phase packing materials were prepared from HPLC silica and from zirconized HPLC silica support particles having sorbed poly(methyloctylsiloxane) (PMOS) as the stationary phase. Portions of zirconized material were subjected to 80 kGy of ionizing radiation. Columns prepared from these packing materials were subjected to 5000 column volumes each of neutral and alkaline (pH 10) mobile phases, with periodic tests to evaluate chromatographic performance. It was shown that the PMOS stationary phase sorbed onto zirconized silica requires an immobilization treatment (such as gamma irradiation) for long term stability while prior surface zirconization of the silica support surface greatly improves the chromatographic stability of the stationary phase when using alkaline mobile phases.

Fases estacionárias para cromatografia líquida de alta eficiência em fase reversa (CLAE-FR) baseadas em superfícies de óxidos inorgânicos funcionalizados

Particles of porous silica or other solvent resistent inorganic oxides can be functionalized by aliphatic (e.g., C-8 or C-18) or other groups to give stationary phases for use in reversed phase HPLC. The functionalization can be done by bonding of individual groups to the surface of the support particles, by producing an organic polymeric film from pre-polymers, or by adsorbing/immobilizing pre-formed polymers on the surfaces. These three types of functionalization are reviewed.

Chromatographic evaluation of radiation-immobilized poly(methyloctylsiloxane) on titanium-grafted silica

Silica was chemically modified by reaction with titanium tetrachloride to give a titanium-grafted silica (Si–Ti). Poly(methyloctylsiloxane) (PMOS) was sorbed into the pores of this support, which was then γ-irradiated (80–120 kGy) and extracted with dichloromethane. Chromatographic performance of the stationary phase was evaluated in terms of the efficiency (plates/m), resolution (Rs), retention factor (k) and asymmetry (As) of a standard mixture of solutes of different polarities. γ-Irradiation increases the quantity of retained PMOS and the efficiency of the resulting columns. These stationary phases, initially loaded with 50% PMOS following titanium-grafting, retained 32±2% of the PMOS.