João Grosso Pacheco

Isolation of phenolic compounds from hop extracts using polyvinylpolypyrrolidone: characterization by high-performance liquid chromatography-diode array detection-electrospray tandem mass spectrometry.

The aim of the present work was the development of a suitable methodology for the separation and determination of phenolic compounds in the hop plant. The developed methodology was based on the sample purification by adsorption of phenolic compounds from the matrix to polyvinylpolypyrrolidone (PVPP) and subsequent desorption of the adsorbed polyphenols with acetone/water (70:30, v/v). At last, the extract was analyzed by HPLC-DAD and HPLC-ESI-MS/MS. The first phase of this work consisted of the study of the adsorption behavior of several classes of phenolic compounds (e.g. phenolic acids, flavonols, and flavanols) by PVPP in model solutions. It has been observed that the process of adsorption of the different phenolic compounds to PVPP (at low concentrations) is differentiated, depending on the structure of the compound (number of OH groups, aromatic rings, and stereochemistry hindrance). For example, within the phenolic acids class (benzoic, p-hydroxybenzoic, protocatechuic and gallic acids) the PVPP adsorption increases with the number of OH groups of the phenolic compound. On the other hand, the derivatization of OH groups (methylation and glycosylation) resulted in a greatly diminished binding. The use of PVPP revealed to be very efficient for adsorption of several phenolic compounds such as catechin, epicatechin, xanthohumol and quercetin, since high adsorption and recovery values were obtained. The methodology was further applied for the extraction and isolation of phenolic compounds from hops. With this methodology, it was possible to obtain high adsorption values (>or=80%) and recovery yield values (>or=70%) for the most important phenolic compounds from hops such as xanthohumol, catechin, epicatechin, quercetin and kaempferol glycosides, and in addition it allows the identification of about 30 phenolic compounds by HPLC-DAD and HPLC-ESI-MS/MS.

Analysis of aldehydes in beer by gas-diffusion microextraction: Characterization by high-performance liquid chromatography–diode-array detection–atmospheric pressure chemical ionization–mass spectrometry

In this work, a recently developed extraction technique for sample preparation aiming the analysis of volatile and semi-volatile compounds named gas-diffusion microextraction (GDME) is applied in the chromatographic analysis of aldehydes in beer. Aldehydes-namely acetaldehyde (AA), methylpropanal (MA) and furfural (FA)-were simultaneously extracted and derivatized with 2,4-dinitrophenylhydrazine (DNPH), then the derivatives were separated and analyzed by high-performance liquid chromatography with spectrophotometric detection (HPLC-UV). The identity of the eluted compounds was confirmed by high-performance liquid chromatography-atmospheric pressure chemical ionization-mass-spectrometry detection in the negative ion mode (HPLC-APCI-MS). The developed methodology showed good repeatability (ca. 5%) and linearity as well as good limits of detection (AA-12.3, FA-1.5 and MA 5.4microgL(-1)) and quantification (AA-41, FA-4.9 and MA 18microgL(-1)); it also appears to be competitive in terms of speed and cost of analysis.

Determination of Free and Total Sulfites in Wine using an Automatic Flow Injection Analysis System with Voltammetric Detection

An automated flow injection analysis (FIA) system, based on an initial analyte separation by gas-diffusion and subsequent determination by square-wave voltammetry (SWV) in a flow cell, was developed for the determination of total and free sulfur dioxide (SO2) in wine. The proposed method was compared with two iodometric methodologies (the Ripper method and a simplified method commonly used by the wine industry). The developed method displayed good repeatability (RSD lower than 6%) and linearity (between 10 and 250 mg l−1) as well as a suitable LOD (3 mg l−1) and LOQ (9 mg l−1). A major advantage of this system is that SO2 is directly detected by flow SWV.

Gas-diffusion microextraction.

Gas-diffusion microextraction (GDME) is an innovative technique that combines the advantages of membrane-aided gas-diffusion with microextraction concepts. GDME makes uses of a novel portable and low-cost device that comprises a small, commercially available, semi-permeable membrane. Furthermore, if derivatization is integrated into the GDME concept, considerable enrichment factors can be obtained. It may become a powerful tool for any analyst who intends to quantify volatile and semi-volatile analytes in various kinds of matrices. The analysis of vicinal diketones in beer was used as a case study to show GDME applicability and capabilities. Vicinal diketones were derivatized with o-phenylenediamine and then determined by HPLC-UV. Obtained results showed good repeatability and precision with extraction periods at the minute time span.

Development of a membraneless extraction module for the extraction of volatile compounds: application in the chromatographic analysis of vicinal diketones in beer.

A membraneless extraction module (MLEM) for the sample preparation of volatile compounds and its use for the chromatographic analysis of vicinal diketones in beer are reported. The extraction process is based on the same principles of gas diffusion (GD) and pervaporation (PV); however it does not use a membrane. This module has a lower chamber where the sample continuously flows, while volatile compounds evaporate to the headspace. Inside the module there is a suspended small reactor, where a small volume of a suitable acceptor solution is placed. This extraction module was tested in the determination of vicinal diketones (VDKs) in beer (CV=5%; LOD=4 microg L(-1)), showing applicability with real samples. Several parameters of the extraction process, such as temperature, sample flow and extraction time, were studied and optimized. This module proved to be a good tool for the sampling of volatile compounds, since the extraction is made without using a membrane avoiding all the robustness problems related with its use.