Polyphenols separation

Figure 6.5 Mechanism of polyphenols separation using l-alkyl-3-methylimidazo-lium-based ionic liquids.

HPLC Separation of Polyphenolics Basic Protocol Reversed-Phase HPLC Analysis of Polyphenolics Separated 11.3.1... 

REVERSED-PHASE HPLC ANALYSIS OF POLYPHENOLICS SEPARATED INTO NONANTHOCYANIN AND ANTHOCYANIN FRACTIONS... 

The discussion in this chapter focused on the developments of analytical techniques in the field of polyphenols separation and fractionation and provided at the same time an overview on the methods developed to enable identification of these compounds. 

Figure 8 illustrates one of the processing schemes used for separating various components in a hydrocarbon-containing plant. Acetone extraction removes the polyphenols, glycerides, and sterols, and benzene extraction removes the hydrocarbons. If the biomass species in question contain low concentrations of the nonhydrocarbon components, exclusive of the carbohydrate and protein fractions, direct extraction of the hydrocarbons with benzene or a similar solvent might be preferred. 

DEGENHARDT A, ENGELHARDT U H, LAKENBRINK 0 and WINTERHALTER P (2000) Preparative separation of polyphenols from tea by high-speed countercurrent chromatography , J Agric Food Chem, 48, 3425-30. 

When appreciable amounts of pectin, proteins, lipids, unwanted polyphenols, or other compounds are suspected to be present in anthocyanin-containing extracts, some of them can be precipitated or the anthocyanins may be crystalhzed and separated from the others. Pectin and proteins can be removed by organic solvents such as methanol and acetone in order to reduce their solubility, then precipitated and separated by centrifugation. Gelatin was used to remove proanthocyanidin due to its high molecular weight. Anthocyanins were reported to be precipitated early by lead acetate to achieve isolation from other materials. ... 

Thousands of polyphenols from fruits (grapes, apples, etc.), vegetables (horse beans), and teas have been identified, many having good coloring properties, especially anthocyanins and some flavonoids. Well-documented reviews discuss the coloring capacities of some polyphenols including procyanidins. - Detailed presentations of anthocyanin and flavonoid properties and analysis are included in Sections 2.3, 4.3, and 6.3. The soluble proanthocyanidins of the colored horse bean Viciafaba L. seed coats were isolated and separated by solvent partition. 

Males et al. [103] used aqueous mobile phase with formic acid for the separation of flavonoids and phenolic acids in the extract of Sambuci flos. In a cited paper, authors listed ten mobile phases with addition of acids used by other investigators for chromatography of polyphenolic material. For micropreparative separation and isolation of antraquinone derivatives (aloine and aloeemodine) from the hardened sap of aloe (Liliaceae family), Wawrzynowicz et al. used 0.5-mm silica precoated plates and isopropanol-methanol-acetic acid as the mobile phase [104]. The addition of small amounts of acid to the mobile phase suppressed the dissociation of acidic groups (phenolic, carboxylic) and thus prevented band diffusions. 

Instant tea produced as described above will dissolve completely in hot water but not in cold water, as the caffeine-polyphenol complexes are insoluble under those conditions. Since virtually all instant tea manufacture in the U.S. is for iced tea preparation, process modification is required. This initial extract may be cooled to 5 to 10°C and the cold water insoluble material or cream be allowed to precipitate. Under these conditions, 20 to 35% of the extract solids may be separated by centrifugation. The supernatant solids will reconstitute in cold water after concentration and drying.105 It is also possible to process the cream to make a portion of it compatible with the product and thereby retain the caffeine and some polyphenolic components that are present in this fraction.106 Commercial use of the enzyme Tannase, which removes gallic acid from gallated tea polyphenols107 and reduces cream formation108 can be used to reduce cream losses and manufacture instant teas retaining more of the natural polyphenol content. 

A similar problem occurs with beer stabilization. A serious problem in the brewing industry is the tendency of some beers to develop hazes during long-term storage due to protein precipitation that is usually stimulated by small quantities of naturally occurring proanthocyanidin polyphenols. In the same way as observed for wine, the excess polyphenols are traditionally removed by treatment with insoluble PVPP, with the same resulting problems. To resolve the problems, several authors have proposed the use of laccase, which forms polyphenol complexes that may be removed by filtration or other separation means. 

DF and antioxidants are generally addressed separately as groups of food constituents in both chemical and nutritional studies. However, it is a little-known fact that a substantial proportion of the antioxidant polyphenols and carotenoids contained in fruit and vegetables are linked to DF (Saura-Calixto and others 2007), and some of the postulated benefits of fiber intake can be attributed to these associated compounds. These compounds are not bioaccessible in the human small intestine, but they... 

Because of the good separation capacity chemically modified stationary phases were proposed for the rapid and economical analysis of peppermint polyphenols [126],... 

RP-HPLC with gradient elution was employed for the study of the influence of theaflavins and thearubigins on the adsorption of black tea on calcium carbonate. Separation of tea constituents was performed in an ODS column (250 X 4.9mm i.d. particle size 5 im). Aqueous solvent was 1 per cent citric acid, pH adjusted to 2.8 with sodium hydroxide and the organic solvent was ACN. The gradient initiated at 8 per cent ACN, was increased to 31 per cent in 50min. Theaflavins and thearubigins were detected at 460 nm, while total polyphenolics were detected at 280 nm. The flow rate was 1.5 ml/min. The results demonstrated the involvement of theaflavins and thearubigins in the adsorption process [185],... 

RP-HPLC methods have been frequently applied for the investigation of various chemical, biochemical and biophysical processes in in vitro model systems. Thus, the separation of new compounds achieved by enzymatic oxidation of phloridzin was carried out by semi-preparative RP-HPLC. Phloridzin was incubated with a polyphenol oxidase prepared from apple pulp for 6h at 30°C under air agitation. After incubation the suspension was filtered, stabilized by NaF and injected into the RP-HPLC column using diluted acetic acid-ACN gradient. The new compounds were isolated and identified by NMR and MA techniques. The proposed mechanism of the formation of new phloridzin derivatives 3 and 4 is shown in Fig. 2.159. The results illustrate that RP-HPLC can be successfully used for the study of enzymatic processes in model systems [331],... 

Removing Interfering Polyphenols. The most troublesome problem encountered was high polyphenol content. If not removed, this dark-colored wood extractive decreased resolution during chromatographic separations, quickly render expensive chromatographic media (e.g., HPLC columns) nearly useless, and often precipitated enzymes during subsequent purification steps (14,15), We found it best to remove the bulk of this material prior to the first concentration step. 

Although effective, residual polyphenols in crude samples resulted in less separation than possible with this method. Such binding often resulted in peaks containing several different activities (9). And increased sample loading often broadened and reduced the number of peaks (9,74). Due to these interferences, two different scales of anion exchange chromatography were used. Analytical separations were used to gather information about the enzymes present and preparative separations were used to purify enzyme quantities sufficient for characterization. 

Plant susceptibility to ozone as determined by visible injury may be very closely related to quantities of o-diphenols associated with the chloroplasts and specific requirements for activation of polyphenol oxidase enzymes. There is a significant correlation between ozone injury and concentrations of total phenols expressed as percent caffeic acid equivalents in peanut cultivars. This concept is not intended to underestimate the importance of membranes that separate phenols and enzymes. Perhaps future research will demonstrate that membranes of resistant alfalfa, green bean and other species differ both qualitatively and quantitatively from those of susceptible plants of these species. 

The polar groups are, on the other hand, responsible for an induced polar selectivity. Analytes able to form hydrogen bonds like phenols are retarded more strongly with polar-embedded stationary phases than with the corresponding classical RP of an identical carbon content. This is demonstrated in Figure 2.5 for the separation of polyphenolic compounds present in red wine. The retention time of the polyphenolic compound kaempferol with the shielded phase is more than three times longer than with the corresponding RP column of an identical carbon content. The polar... 

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