HPLC of phenolics

Other modes of detection, such as nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR), could be advantageous detection techniques, especially to validate the HPLC of phenolics in various foods, but the cost and complexity of the instrumentation limit the... 

As previously mentioned, the RP-HPLC of phenolic compounds is carried out with polar eluents. Simple HPLC systems use one pump that pumps a single solvent or, more frequently, a solvent mixture through the column. This is known as isocratic HPLC. More complex gradient systems use more than one pump governed by a computer and pump changing mobile phases that can involve several solvents. Water is usually one of the components in the mixmre the other components most frequently include methanol, acetonitrile, and, more rarely, tetrahydrofuran, etc. The pH of the eluent is always acidic, and this acidity can be attained by the addition of acids such as acetic, formic, phosphoric, trifluoroacetic, and others. The selection of the acid used is dependent on the specific separation problems that need to be solved. 

A conlometric array consisting of sixteen detectors was set up to generate voltammetric data for ELD after RP-HPLC of phenolics and flavonoids in juice beverages. Such detection conld be nsed for on-line resolntion of componnds with similar retention times. Within each class of componnds (phenolics and flavonoids), the oxidation potential changed with the snbstitntion pattern as depicted in eqnation 4. A mixture of twenty-seven reference componnds was resolved in a run of 45 min dnration. LOD was in the low (xgL range with a linear response range of at least three orders of magnitude . 

The analysis by HPLC of phenolic compounds in peach fruit in our laboratory is done as follows. AU chemicals are reagent-grade products. Three separate batches of 5 g each of mesocarp tissue are blended in a mortar with 10 ml of 80% (v/v) acetone, and the homogenate is filtered through a Buchner funnel with suction. The three extracts are collected, centrifuged to dryness at below 30 °C, lyophilized and stored until analysis. After dissolution of the extract, 1 ml of 50% methanol is added to each sample, and the solution is centrifuged for 10 min at 12 000 g. Portions (2[rl) of the supernatant are separated into components by HPLC. Authentic phenolic compounds are analyzed also. The unknown com-... 

HPLC DETERMINATION OF PHENOLS WITH PHOTOMETRIC AND AMPEROMETRIC DETECTORS... 

For selective estimation of phenols pollution of environment such chromatographic methods as gas chromatography with flame-ionization detector (ISO method 8165) and high performance liquid chromatography with UV-detector (EPA method 625) is recommended. For determination of phenol, cresols, chlorophenols in environmental samples application of HPLC with amperometric detector is perspective. Phenols and chlorophenols can be easy oxidized and determined with high sensitivity on carbon-glass electrode. 

The comparison of analytical characteristics HPLC methods of determination of phenols with application amperometric and photometric detectors was caiiy out in this work. Experiment was executed with use liquid chromatograph Zvet-Yauza and 100 mm-3mm 150mm-3mm column with Silasorb C18 (5 10 p.m). With amperometric detector phenols were detected in oxidizing regime on glass-cai bon electrodes. With photometric detector phenols were detected at 254 nm. 

HPLC method with amperometric detection was applied for detenuination of phenols in sea sediment and some dmg preparation. Peaks of phenol, guaiacol, cresols, hydroquinon and resorcinol were identified on chromatogram of birch tai. The HPLC method with electrochemical detectors was used for detenuination of some drug prepai ation of aminophenol derivate. So p-acetaminophenol (paracetamol) was determined in some drug. 

Phenols hold an important place among organic pollutants, which need to be constantly monitored in waters and in places of militai y activities. Sampling of phenol matrix is conducted with solid face extragents (SPE) with further HPLC or GC analysis. Application of the known SPE usually is ineffective as it doesn t give the possibility to provide full extraction of the analyt (microcontents) in the matrix media. Therefore SPE application needs further progress in their selectivity. 

More recently, the reaction advancement of resole syntheses (pH = 8 and 60°C) was monitored using high-performance liquid chromatography (HPLC), 13C NMR, and chemical assays.55,56 The disappearance of phenol and the appearances of various hydroxymethyl-substituted phenolic monomers and dimers have been measured. By assessing the residual monomer as a function of reaction time, this work also demonstrated the unusually high reactivity of 2,6-dihydroxymethyl-phenol. The rate constants for phenolic monomers toward formaldehyde substitution have been measured (Table 7.6). 

Figure 13, indicates that the first mole of phenol is released in <30 s, the same elapsed time for the chemiluminescence to reach a maximum intensity. In fact, the measured rate constant r, for the rise in the chemiluminescence emission, is identical to the rate of the first phenol s release from the oxalate ester. Furthermore, the slower rate of release of the second phenol ligand has a rate constant that is identical to the chemiluminescence decay rate f. Thus, the model allows a quantitative analysis of the reaction mechanism, heretofore not available to us. We intend to continue this avenue of investigation in order to optimize the chemiluminescence efficiencies under HPLC conditions and to delineate further the mechanism for peroxy-oxalate chemiluminescence. 

Knowledge of the identity of phenolic compounds in food facilitates the analysis and discussion of potential antioxidant effects. Thus studies of phenolic compounds as antioxidants in food should usually by accompanied by the identification and quantification of the phenols. Reversed-phase HPLC combined with UV-VIS or electrochemical detection is the most common method for quantification of individual flavonoids and phenolic acids in foods (Merken and Beecher, 2000 Mattila and Kumpulainen, 2002), whereas HPLC combined with mass spectrometry has been used for identification of phenolic compounds (Justesen et al, 1998). Normal-phase HPLC combined with mass spectrometry has been used to identify monomeric and dimeric proanthocyanidins (Lazarus et al, 1999). Flavonoids are usually quantified as aglycones by HPLC, and samples containing flavonoid glycosides are therefore hydrolysed before analysis (Nuutila et al, 2002). 

The use of HPLC for quantification of phenols is often limited to a single class of phenolics and then often only to low-molecular weight compounds that are available as standards. It is, therefore, often necessary to use colorimetric assays such as the Folin-Ciocalteau assay which rely on the reducing ability of phenols to quantify the amount of total phenolics in a sample (Waterman and Mole, 1994 Singleton et al, 1999 Schofield et al, 2001). The degree of condensation of polyphenols can be quantified by colorimetric assays such as the acid-butanol assay and the vanillin assay (Waterman and Mole, 1994 Schofield et al, 2001). 

Hong, V. and Wrolstad, R.E., Use of HPLC separation/photodiode array detection for characterization of anthocyanins, J. Agric. Food Chem., 38, 708, 1990. Osmianski, J. and Lee, C.Y., Isolation and HPLC determination of phenolic compounds in red grapes. Am. J. Enol. Vitic., 41, 204, 1990. 

Paganga, G. et al.. The polyphenolic content of fruit and vegetables and their antioxidant activities what does a serving constitute Free Radical Res., 30, 153, 1999. Maatta, K.R. et al.. High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection Rihes species, J. Agric. Food Chem., 51, 6736, 2003. 

HPLC has proved to be fast and sensitive for the analyses of phenolic plant constit-nents, and is especially useful for the analysis of anthocyanins. The first application of HPLC to anthocyanin analyses was in 1975 by Manley and Shubiak and it has now become the method of choice for the separation of mixtures of anthocyanins and anthocyanidins. HPLC is now used for anthocyanin qualitative, quantitative, and preparative work, offering improved resolution compared to chromatographic procedures previously employed. It also allows for simultaneous rapid monitoring of the eluting anthocyanins. ... 

Jorg, E. and Sontag, G. (1992). Determination of phenolic acids in honey by HPLC using coulometric dual electrode detection. Dtsch. Lebensm. Rundsh. 88,179-183. 

Sontag, G., Friedrich, O., Kainz, G., and Jorg, E. (1989). Determination of phenolic compounds by HPLC with electrochemical detection. Proc. EUR 5th Food Chem. Conference, Agric. Food Chem. Consum. 2, 703-707. 

Although the condensation of phenol with formaldehyde has been known for more than 100 years, it is only recently that the reaction could be studied in detail. Recent developments in analytical instrumentation like GC, GPC, HPLC, IR spectroscopy and NMR spectroscopy have made it possible for the intermediates involved in such reactions to be characterized and determined (1.-6). In addition, high speed computers can now be used to simulate the complicated multi-component, multi-path kinetic schemes involved in phenol-formaldehyde reactions (6-27) and optimization routines can be used in conjunction with computer-based models for phenol-formaldehyde reactions to estimate, from experimental data, reaction rates for the various processes involved. The combined use of precise analytical data and of computer-based techniques to analyze such data has been very fruitful. 

A wide variety of methodologies have been employed for the analysis of antioxidants in polymers and some standard methods are available. For high-density polyethylene ASTM method D5524 (ASTM International) — Determination of phenolic antioxidants in high-density polyethylene, describes a method whereby the sample is ground to a small particle size and then extracted by refluxing with cyclohexane. The cyclohexane extract is then examined by reverse-phase HPLC with UV detection. 

HPLC) for phenolic acids analysis. When procedure (ii) was applied, the ion-exchange resin was separated from the methanol phase and eluted with three 40 ml aliquots of 80% methanol. The resin bead eluates were evaporated to dryness and subjected to spectrophotometry (Shimadzu UV 160 spectrophotometer) for total phenolics and high-performance liquid chromatography (HPLC) for phenolic acids analysis. 

Experiment III. Determination of phenolic acids by HPLC analysis... 

Experiment III. Determination of phenolic acids by HPLC analysis Material required, Procedure, Statistical analysis and Precaution are the same as described in Section 3.3. 

De la Torre-Carbot K, Jauregui O, Gimeno E, Castellote AI, Lamuela-Raventos RM and Lopez-Sabater M. 2005. Characterization and quantification of phenolic compounds in olive oils by solid-phase extraction, HPLC-DAD, and HPLC-MS/MS. J Agric Food Chem 53(11 ) 4331 —4340. 

Ibern-Gomez M, Andres-Lacueva C, Lamuela-Raventos RM and Waterhouse AL. 2002. Rapid HPLC analysis of phenolic compounds in red wines. Am J Enol Vitic 53(3) 218—221. 

Tomas-Barberan FA, Gil MI, Cremin P, Waterhouse AL, Hess-Pierce B and Kader AA. 2001. HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines, peaches, and plums. J Agric Food Chem. 49(10) 4748 t760. 

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