Bronisław K. Głód

Application of micro-TLC to the total antioxidant potential (TAP) measurement.

DPPH is commonly applied for estimation of antioxidant capacity of single and complex biological samples, and changes colour from purple to yellow during reduction to DPPH-H. Importantly, for some samples, for example coloured foods, such reaction cannot be used because of interference from pigments. Therefore, the number of reported quantitative protocols involving off- or on-line sample reaction with DPPH are based on chromatographic separation of target components. In typical planar chromatographic assay, developed plates are sprayed with DPPH solution for antioxidant screening. Such approach enables simple visualisation of separated spots exhibiting antioxidant activities, but unfortunately, such procedure may also give the misleading signal for coloured spots. In the present communication we examined a new approach for measuring antioxidant capacity using quantitative analysis of DPPH and DPPH-H molecules after reaction with the sample, and then separated from the interfering compounds by micro-thin-layer chromatography. Particularly, the antioxidant capacities of coloured food samples (such as herbs and meads) were determined and the results compared with those obtained using the classical photometric assay. The main advantages of the new micro-TLC assay are (i) low cost, (ii) multiple measurements, (iii) short analysis time, (iv) simplification of sample preparation and (v) effective separation of DPPH signal from interfering compounds.

A fast and simple method for the measurement of total antioxidant potential and a fingerprint of antioxidants.

The goal of this paper is to demonstrate a new approach to the chromatographic total antioxidant potential (TAP) assay and chromatographic fingerprints of honeys. The analyte is analyzed using reversed-phase high-performance liquid chromatography with electrochemical (amperometric) detection. The TAP measure was the total surface area of all recorded chromatographic peaks (on the amperometric detector in the oxidation potential range) on the chromatogram. The proposed assay is superior to already described assays because TAP can easily be related to the different potentials of working electrodes, as well as different groups of compounds separated on the column. The fingerprinting of antioxidants (antiradicals) has been applied for the quality assessment of honeys.

Application of UV detection in HPLC in the total antioxidant potential assay

AbstractHPLC has been already used for the TAP estimation. Phenylalanine, salicylic, p-hydroxybenzoic (pHBA) or terephthalic (TPA) acids have been used as sensor compounds. Products of their reaction with the hydroxyl radicals, generated in the Fenton-like reaction, were analyzed using electrochemical or fluorescence detection. This paper describes the TAP assay based on the hydroxyl radicals reaction with pHBA, reversed-phase-HPLC separation and UV photometric detection. The elaborated assay has been used to evaluate TAP values of some apiculture products.n

RP-HPLC, WITH FLUORESCENCE DETECTION, ASSAY FOR THE DETERMINATION OF TOTAL ANTIOXIDANT POTENTIAL (TAP)

Total Antioxidant Potential (TAP) measures the antioxidant properties of pure compounds as well as complex samples, such as food products. TAP is proportional to the product of concentrations of all antioxidants in the sample and their antioxidant powers (rate constants). TAP usually gives more information than analysis of individual antioxidants in the sample separately. In literature, hydroxyl radicals are analyzed indirectly as products of their reactions with p-hydroxybenzoic (pHBA) or terephthalic (TPA) acids. The 3,4-dihydroxybenzoic or hydroxyterephthalic acids are separated in the reversed phase HPLC and monitored using electrochemical or fluorescence detection, respectively. In our previous papers, we found that these methods can also be applied to the estimation of TAP. In this paper a TAP assay based on the hydroxyl radicals reaction with pHBA, RP-HPLC separation, and fluorometric (not electrochemical) detection is described. The assay will be compared with the previously investigated method, based on the reaction with TPA. The elaborated assay was used to evaluate TAP values of some alcoholic beverages.

Comparative Analysis of Antioxidative Activity of Flavonoids Using HPLC–ED and Photometric Assays

The present investigation reports on the application of a new antioxidant activity assay for the examination of flavonoids. It has been shown that the high-performance liquid chromatography with electrochemical detection (HPLC–ED) measurements allow to obtain additional information about the antioxidative properties of pure compounds by measuring their half-wave potential, the chromatographic peak height, and the product of the peak height and exponent of potential. In comparison to the classical electrochemical measurements, the HPLC–ED is characterized by a much smaller detection limit. The results were compared with the standard photometric measurement based on 1,1-diphenyl-2-picrylhydrazyl radicals. The possible antioxidant activity forecasting is also discussed.

Application of HPLC to Study the Reaction of Free Radicals with Antioxidants and/or Toxins

The application of HPLC in the antioxidative (antiradical) properties studies of various samples is discussed in this paper. The assay is based on the reaction of, characterized by strong oxidizing properties, hydroxyl radicals (generated in the Fenton-like reaction) with the sample and with the, so-called, sensor. The product of the sensor reaction with the radicals is analyzed using RP-HPLC with fluorescence detection. It is well known that antioxidants, which are healthy for living organisms, have a negative environmental effect. Therefore, the goal of the paper is to discuss if the same setup can be used to get rid of unwanted compounds (toxins) from the environment. It was found that the phenols and PAHs are degraded by hydroxyl radicals. The optimal (maximum degree of the degradation) conditions were obtained for and 0.1u2009mM Fe2

Anti-oxidative properties of bi-1,2,4-triazine bisulphides

The anti-oxidative properties of bitriazines (BTs) were evaluated using HPLC and cyclic voltammetry. In the first case, a RP-HPLC assay was made, using a fluorescence detector, hydroxyl radicals generated in Fenton reaction, and terephthalic acid as a spin trap. The measurements were performed using aqueous or methanolic solutions. It was found that when the BTs were dissolved in water they were antioxidants, while dissolved in methanol they were pro-oxidants. Their different physicochemical properties in both solvents were confirmed by voltammetric, chromatographic as well as spectrophotometric measurements.