Phenolic compounds chromatographic conditions

RP-HPLC has been employed for the determination of flavonoids and other phenolic compounds in cranberry juice. The neutral and acidic analytes were preconcentrated octadecyl silica SPE cartridges conditioned with distilled water (neutral analytes) or with 0.01 M HC1 (acidic compounds). Hydrolysis of samples was carried out in aqueous methanol solution acidified with 6 M HC1 at 35°C for 16h. Chromatographic separation was performed in an ODS column (150 X 4.6mm i.d. particle size 5/.an). Solvents A and B were water-acetic acid (97 3, v/v) and methanol, respectively. The gradient started with 0 per cent B (flow rate, 0.9 ml/min), reached 10 per cent B in lQmin (flowrate, 1.0 ml/min) and increased to 70 per cent B in 40min (flowrate, 1.0 ml/min). Analytes were detected at 280 and 360 nm. Some typical chromatograms are presented in Fig. 2.71. The concentrations of flavonoids and phenolic acids are compiled in Table 2.69. It was stated that the SPE-HPLC procedure makes possible the simultaneous determination of phenolic compounds and flavonoids, therefore, it can be employed for the measurement of these classes of analytes in other fruit juices [188],... 

III. HPLC SYSTEMS FOR PHENOLIC COMPOUNDS A. Chromatographic Conditions... 

For phenolics in fruit purees and jams (54), an HPLC condition similar to that used for apple juice, but with acidified water (5% formic acid) and methanol, was utilized as a solvent system. In most cases, detection was achieved with diode array detection, at UV 280 nm and 320 nm. The different phenolic compounds were identified by their UV spectra and by chromatographic comparisons with authentic standards. Several classes of phenolic compounds (cinnamic acids, catechins, dihydrochalcones, and flavonol glycosides) could be detected along with arbutin in... 

Analysis of Phenolic Compounds. A Hewlett-Packard (Palo Alto, CA) Model 1090 HPLC System, was used to determine the levels of specific phenolic components. The HPLC system was equipped with a ternary solvent delivery system, a diode array UV-VIS detector, and HP ChemStation software for data collection and analysis. Full chromatographic traces were collected at 280, 520, 316, and 365 nm, and spectra were collected on peaks. The stationary phase was a Hewlett-Packard LiChrosphere C-18 coliram, 4mm X 250 mm, with 5 pM particle size packing. Operating conditions include an oven temperature of 40 C, injection volume of 25 pL, and flow rate of 0.5 mL/minute. The mefriod was based on a previously published method for phenolic components in wine (30) and used the modified solvent gradient shown in Table II. Solvent A was 50 mM dihydrogen ammonium phosphate, adjusted to pH 2.6 with orthophosphoric acid. Solvent... 

Figure 12.14 Chromatographic analysis of aniline (a) Precolumn chromatogram (the compound represented by the shaded peak is solvent flushed) (b) main column chromatogram without cryotrapping (c) main column chromatogram with ciyottapping. Conditions DCS, two columns and two ovens, with and without ciyottapping facilities columns OV-17 (25 m X 0.32 mm i.d., 1.0 p.m d.f.) and HP-1 (50 m X 0.32 mm, 1.05 p.m df). Peak identification is as follows 1, benzene 2, cyclohexane 3, cyclohexylamine 4, cyclohexanol 5, phenol 6, aniline 7, toluidine 8, nittobenzene 9, dicyclohexylamine. Reprinted with permission from Ref. (20).

This compound ionizes to form four ions by cleavage and rearrangement. The loss of a methyl radical leads to the ion at ml z 248 the ion at ml z 154 amu is the p-nitrothio-phenolate ion the ion at mlz 158 is the p-nitrophenolate ion and the ion at mlz 141 is due to loss of the p-nitrophenyl radical. The carrier gas was argon/methane, and the source temperature was 240°C. A PZI79 column was used in the gas chromatograph. This mass spectrum was obtained while the compound was in the source, as indicated in Figure 4. Under these conditions, variations in the field in the source did not alter... 

PB-HPLC-MS of Phenols and Conjugates. The SAX chromatographic separation of phenols, sulfates, and glucuronides is described in detail elsewhere (12). Briefly, a 25 cm x 4.6 mm SAX column (Supelco, Bellefonte, PA) is used with an acetonitrile-pH 4.5 ammonium formate buffer (2 3) mobile phase at a flow rate of 1.5 mL/min. Under these conditions, the nine model compounds were resolved to baseline in less than 20 minutes. 

High-performance liquid chromatography (HPLC) is the method for detection, identification and also quantification of flavonoids, phenolic acids and their derivatives. With this method, the sample is applied and eluted through a chromatographic column under specific conditions designed for optimum separation and resolution so that each compound or group of compound passes through the column with a... 

The extraction solvents are then evaporated in the rotary evaporator under vacuum condition to concentrate the extract, to which other solvents can be added for purification and separation. Solubihty of compounds in different solvents and pH are important criteria used for the separation and purification of polyphenolic compounds. Separation of phenolic compotmds from the mixtures of compounds can be done using solvent separation methods and various chromatographic methods (including solid-phase extraction). 

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