O Estado da arte da cromatografia líquida bidimensional: conceitos fundamentais, instrumentação e aplicações

The constant evolution of science and the growing demand for new technologies have led to new techniques in instrumentation that can improve detection, separation, resolution, and peak capacity. Comprehensive two-dimensional liquid chromatography (LC×LC) is presented as a powerful tool in complex sample analyses. During an analysis, a sample is subjected to two independent separation mechanisms that are combined, resulting in increased resolving power. For appropriate application of LC×LC, understanding the influence of parameters that require optimization is necessary. The main purpose of optimization is to predict the combination of stationary phases, separation conditions, and instrumental requirements to obtain the best separation performance. This review discusses theoretical, intrumental, and chemometric aspects of LC×LC and focuses on its applications in foods. It aims to provide a clear understanding of the aspects that can be used as strategies in the optimization of this analytical method.

Separação e determinação quantitativa dos alcanos lineares e dos cíclicos/ramificados em petróleos brasileiros por aduto de ureia e cromatografia gasosa: um estudo de caso revisitado

A complete analysis of oils and their fractions allows correlations to be defined between their composition and derivatives or related geological materials. This work focused on optimization and implementation of a method for separation and quantification of n-alkanes in Brazilian oil samples by urea adduction and GC-FID techniques. Ten samples with different ºAPI were analyzed in triplicate to quantify individual n-alkanes and cyclic/branched alkane fraction. For individual quantification of n-alkanes, internal standardization with deuterated n-tetracosane was used. The use of urea adduction for the separation and quantification of n-alkanes was highly effective, with recovery values of above 80%.

DETECTION AND QUANTIFICATION OF PHYTOCHEMICAL MARKERS OF Ilex paraguariensis BY LIQUID CHROMATOGRAPHY

Ilex paraguariensis (yerba-mate) is used as a beverage, and its extract requires adequate quality control methods in order to guarantee quality and safe use. Strategies to develop and optimize a chromatographic method to quantify theobromine, caffeine, and chlorogenic acid in I. paraguariensis extracts were evaluated by applying a quality by design (QbD) model and ultra high-performance liquid chromatography (UHPLC). The presence of these three phytochemical markers in the extracts was evaluated using UHPLC-MS and was confirmed by the chromatographic bands in the total ion current traces (m/z of 181.1 [M+H]+, 195.0 [M+H]+, and 353.0 [M−H]−, respectively). The developed method was then transferred to a high-performance liquid chromatography (HPLC) platform, and the three phytochemical markers were used as external standards in the validation of a method for analyses of these compounds in extracts using a diode array detector (DAD). The validated method was applied to quantify the chlorogenic acid, caffeine, and theobromine in the samples. HPLC-DAD chromatographic fingerprinting was also used in a multivariate approach to process the entire data and to separate the I. paraguariensis extracts into two groups. The developed method is very useful for qualifying and quantifying I. paraguariensis extracts.

TREATMENT OF WASTE FROM ATOMIC EMISSION SPECTROMETRIC TECHNIQUES AND REUSE IN UNDERGRADUATE LAB CLASSES FOR QUALITATIVE ANALYSIS

Flame atomic absorption spectrometry (FAAS) and inductively coupled plasma optical emission spectrometry (ICP OES) are widely used in academic institutions and laboratories for quality control to analyze inorganic elements in samples. However, these techniques have been observed to underperform in sample nebulization processes. Most of the samples processed through nebulization system are discarded, producing large volumes of waste. This study reports the treatment and reuse of the waste produced from ICP OES technique in a laboratory of analytical research at the Universidade Federal do Ceará, Brazil. The treatment of the waste was performed by the precipitation of elements using (NH4)2CO3. Subsequently, the supernatant solution can be discarded in accordance with CONAMA 430/2011. The precipitate produced from the treatment of residues can be reused as a potential sample in undergraduate qualitative analytical chemistry lab classes, providing students the opportunity to test a real sample.

COMPARAÇÃO DE MÉTODOS POR CROMATOGRAFIA LÍQUIDA NA DETERMINAÇÃO DE MULTIRRESÍDUOS DE AGROTÓXICOS EM MORANGOS

Liquid chromatography is often used for the determination of pesticide multiresidues in foods. In Brazil, the strawberry crop is an example of a food with high levels of irregularities because of the application of pesticides. This is a major concern from the perspective of food safety, environmental protection, and certification for food export. The purpose of this study is to evaluate and compare chromatographic separation and detection methods in relation to a newly developed and validated method using ultra high performance liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) for the analytical determination of pesticides in strawberries. The comparisons were based on evaluations of the analysis time, consumption of the solvent in the mobile phase, injection volume, detectability, matrix effect, and recovery. The results showed that the LC–MS/MS and UHPLC–MS/MS techniques were both extremely efficient at analyzing pesticide residues with different physico-chemical parameters that were present at low concentrations in a complex matrix. The UHPLC separation method provided better chromatographic performance and productivity, which contributed favorably to routine analytical determinations. Detection by MS/MS had better detectability and selectivity compared with the diode array detector.

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Isolation of lignans glycosides from Alibertia sessilis (Vell.) K. Schum. (Rubiaceae) by preparative high-performance liquid chromatography

Enantiomeric aglycone lignans contained in a mixture were separated from a fraction of the extract of the stems of Alibertia sessilis (Vell.) K. Schum. (Rubiaceae) by preparative high-performance liquid chromatography. An efficient and fast separation can be achieved with methanol-water (30:70, v/v). Their structures were identified as (+)-lyoniresinol 3alpha-O-beta-glucopyranoside and (-)-lyoniresinol 3alpha-O-beta-glucopyranoside, being reported for the first time in Rubiaceae.

Flavonoids extraction from Alpinia zerumbet (Pers.) Burtt et Smith leaves using different procedures

The current study aims to verify the best method for a rapid and efficient extraction of flavonoids from Alpinia zerumbet. Dried leaves were extracted using distillated water and ethanol 70% by extraction methods of shaking maceration, ultrasonic, microwave and stirring. By the application of TLC and reversed-phase HPLC techniques the rutin and kaempferol-3-O-glucuronide were detected. Ethanol 70% was more efficient for flavonoids extraction than water. No significant yielding variation was verified for ultrasonic, microwave and stirring methods using ethanol 70% (11 to 14%). Relative concentration of rutin and kaempferol-3-O-glucuronide, respectively, was higher by ultrasonic (1.5 and 5.62 mg g-1 dried leaves) and by microwave (1.0 and 6.64 mg g-1 dried leaves) methods using 70% ethanol. Rapid and simplified extraction proceeding optimize phytochemical work and acquisition of secondary metabolites.

Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja) crops

Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja) was tested through dual culture in this plant with different plant pathogenic fungi and bacteria. Isolates that showed significantly higher antagonistic activity were fermented in liquid media and crude extracts from the supernatants had their biological activities assessed by optical density techniques. Inhibitory effecton tested pathogens was observed for concentrations between 0.5% and 1% of crude extracts. There was a correlation between the antimicrobial activity of extracts and the use of nutrient-rich media in bacteria fermentation. Using a bioguided method, a peptidic compound, active against Fusarium oxysporum, was obtained from the 7ANT04 strain (Pyrobaculum sp.). Analysis by nuclear magnetic resonance and liquid chromatography coupled to mass detector evidenced an 11-amino acid compound. Bioinformatic software using raw mass data confirmed the presence of a cyclic peptide conformed by 11 mostly non-standard amino acids.

ESTABLISHMENT TECHNIQUES FOR TROPICAL LEGUMES IN THE UNDERSTORY OF A EUCALYPTUS PLANTATION

This study evaluated establishment methods for a mixture of herbaceous forage legumes [Centrosema acutifolium, Clitoria ternatea, Pueraria phaseoloides, Stylosanthes Campo Grande (Stylosanthes capitata + S. macrocephala), Calopogonium mucunoides, Lablab purpureus, Arachis pintoi, and Aeschynomene villosa] under the shade of an Eucalyptus grandis plantation submitted to thinning (40%) 8 years after planting in Anhembi, São Paulo (22°40'S, 48°10'W, altitude of 455 m). The experiment started in December 2008 and consisted of the comparison of the following four types of seed incorporation by light disc harrowing: (1) broadcast sowing without seed incorporation; disc harrowing before (2) or after (3) planting, and (4) disc harrowing before and after planting. Ninety days after planting, the number of legume plants/m2 and the percentage of ground cover by the plants varied between the treatments tested; however, the treatments had no effect on the dry matter accumulation of forage legumes. Disc harrowing before planting yielded superior results compared to the treatments without disc harrowing and disc harrowing after planting. At the end of the experimental period, the plots contained Arachis, Centrosema, Stylosanthes, and Pueraria. The dry matter accumulated by Centrosema corresponded to 73% of total dry matter yield of the plots. The participation of Arachis, Centrosema and Stylosanthes in final dry matter composition of the plots varied according to establishment method. The advantages of the use of species mixtures rather than monocultures in the understory of forest plantations were discussed.

Data mining techniques for identification of spectrally homogeneous areas using NDVI temporal profiles of soybean crop

The aim of this study was to group temporal profiles of 10-day composites NDVI product by similarity, which was obtained by the SPOT Vegetation sensor, for municipalities with high soybean production in the state of Paraná, Brazil, in the 2005/2006 cropping season. Data mining is a valuable tool that allows extracting knowledge from a database, identifying valid, new, potentially useful and understandable patterns. Therefore, it was used the methods for clusters generation by means of the algorithms K-Means, MAXVER and DBSCAN, implemented in the WEKA software package. Clusters were created based on the average temporal profiles of NDVI of the 277 municipalities with high soybean production in the state and the best results were found with the K-Means algorithm, grouping the municipalities into six clusters, considering the period from the beginning of October until the end of March, which is equivalent to the crop vegetative cycle. Half of the generated clusters presented spectro-temporal pattern, a characteristic of soybeans and were mostly under the soybean belt in the state of Paraná, which shows good results that were obtained with the proposed methodology as for identification of homogeneous areas. These results will be useful for the creation of regional soybean "masks" to estimate the planted area for this crop.

Surgical techniques for maxillary bone grafting - literature review

For oral rehabilitation with implant-supported prostheses, there are required procedures to create the bone volume needed for installation of the implants. Thus, bone grafts from intraoral or extraoral donor sites represent a very favorable opportunity. This study aimed to review the literature on the subject, seeking to discuss parameters for the indications, advantages and complications of techniques for autogenous bone grafts.

Comparison of three diagnostic techniques for the detection of leptospires in the kidneys of wild house mice (Mus musculus)

Forty-one wild house mice (Mus musculus) were trapped in an urban area, near railways, in Santa Fe city, Argentina. Both kidneys from each mouse were removed for bacteriological and histological examination. One kidney was inoculated into Fletcher semi-solid medium and isolates were serologically typed. The other kidney was microscopically examined after hematoxylin-eosin, silver impregnation and immunohistochemical stains. Leptospires, all of them belonging to the Ballum serogroup, were isolated from 16 (39%) out of 41 samples. The presence of the agent was recorded in 18 (44%) and in 19 (46%) out of 41 silver impregnated and immunohistochemically stained samples respectively. Additionally, leptospires were detected in high number on the apical surface of epithelial cells and in the lumen of medullary tubules and they were less frequently seen on the apical surface of epithelial cells or in the lumen of the cortical tubules, which represents an unusual finding in carrier animals. Microscopic lesions consisting of focal mononuclear interstitial nephritis, glomerular shrinkage and desquamation of tubular epithelial cells were observed in 13 of 19 infected and in 10 of 22 non-infected mice; differences in presence of lesions between infected and non-infected animals were not statistically significant (P=0,14). The three techniques, culture, silver impregnation and immunohistochemistry, had a high agreement (k³0.85) and no significant differences between them were detected (P>0.05). In addition, an unusual location of leptospires in kidneys of carrier animals was reported, but a relationship between lesions and presence of leptospires could not be established.

Using passive techniques for vibration damping in mechanical systems

This paper examines two passive techniques for vibration reduction in mechanical systems: the first one is based on dynamic vibration absorbers (DVAs) and the second uses resonant circuit shunted (RCS) piezoceramics. Genetic algorithms are used to determine the optimal design parameters with respect to performance indexes, which are associated with the dynamical behavior of the system over selected frequency bands. The calculation of the frequency response functions (FRFs) of the composite structure (primary system + DVAs) is performed through a substructure coupling technique. A modal technique is used to determine the frequency response function of the structure containing shunted piezoceramics which are bonded to the primary structure. The use of both techniques simultaneously on the same structure is investigated. The methodology developed is illustrated by numerical applications in which the primary structure is represented by simple Euler-Bernoulli beams. However, the design aspects of vibration control devices presented in this paper can be extended to more complex structures.

Performance evaluation of 3D computer vision techniques

This work presents the implementation and comparison of three different techniques of three-dimensional computer vision as follows: • Stereo vision - correlation between two 2D images • Sensorial fusion - use of different sensors: camera 2D + ultrasound sensor (1D); • Structured light The computer vision techniques herein presented took into consideration the following characteristics: • Computational effort ( elapsed time for obtain the 3D information); • Influence of environmental conditions (noise due to a non uniform lighting, overlighting and shades); • The cost of the infrastructure for each technique; • Analysis of uncertainties, precision and accuracy. The option of using the Matlab software, version 5.1, for algorithm implementation of the three techniques was due to the simplicity of their commands, programming and debugging. Besides, this software is well known and used by the academic community, allowing the results of this work to be obtained and verified. Examples of three-dimensional vision applied to robotic assembling tasks ("pick-and-place") are presented.