Flavonoids HPLC separation

Mabry. Identification of flavonoid SO103 compounds in HPLC separation of... 

HPLC separation, as described above, is restricted to rather simple compounds that represent only a small proportion of flavonoids. Indeed, proanthocyanidin analysis becomes increasingly difficult as their molecular weight increases, due to the larger number of possible structures, smaller amounts of each individual compound, and poorer resolution of the chromatographic profiles. This is especially true of grape proanthocyanidins, which, unlike those of apple or cacao consisting only of epicatechin units, are based on four major... 

In reversed-phase HPLC separation of polyphenolics on the basis of polarity, the elution order of polyphenolics may be predicted. The more-polar polyphenolics are generally eluted first under reversed-phase conditions. Glycosylation in flavonoids increases their polarity and therefore their mobility in the re-versed-phase system. The elution order of benzoic acids, hydroxycinnamic acids, and agly-cones of flavonoids can normally be determined on the basis of the number of polar hydroxyl groups and lipophilic methoxyl groups. For additional information about elution order for various classes of polyphenolics, see Background Information. 

An HPLC separation method with diode array detector and mass spectrometric (MS) detection equipped with atmospheric pressure ionization (API) was developed to determine flavone, flavonol, and flavanone in various vegetables, including green bean, broccoli, brussels sprouts, celery, kale, leek, onion, parsley, pepper (green, yellow, and red), and tomato (118). The flavonoids were analyzed as aglycones after acid hydrolysis. The extraction and acid hydrolysis conditions are based on previous work by Hertog et al. (119). Quercetin is the overall major flavonol, followed by kaempferol. The flavones, apigenin and luteolin, were found only in limited foods,... 

An HPLC-DAD method was developed for the separation and the determination of flavonoid and phenolic antioxidants in commercial and freshly prepared cranberry juice.Two sample preparation procedures were used with and without hydrolysis of the glycoside forms of flavonoids carried out by the addition of HCl in the step prior to solid-phase extraction (SPE). The flavonoid and phenolic compounds were then fractionated into neutral and acidic groups via a solid-phase extraction method (Sep-Pak Cig), followed by a RP HPLC separation with gradient elution with water-methanol-acetic acid and a detection at 280 and 360 nm. A comparison of the chromatograms obtained for extracts prepared with and without hydrolysis showed that flavonoids and phenolic acids exist predominantly in combined forms such as glycosides and esters. In a freshly squeezed cranberry juice, for instance, 400 mg of total flavonoids and phenolics per liter of sample was found, 56% of which were flavonoids. Quercetin was the main flavonoid in the hydrolyzed products, where it accounted for about 75% of the total flavonoids, while it was absent in the unhydrolyzed products. 

Jagota, N.K. Cheathan, S.F. HPLC separation of flavonoids and flavonoid glycosides using a polystyrene/ divinylhenzene column. J. Liq. Chromatogr. 1992, 15 (4), 603-615. 

Krauze-Baranowska, M. Baczek, T. Glod, D. Kaliszan, R. E.Wollenweber, HPLC separation of O-acylated flavonoids and biflavones from some species of Gymnospermae, Chromatographia, 2004, 60, 9-15. 

Coelution of procyanidins with cinnamics and flavonoids presented a major problem in quantitating the procyanidins. (Cinnamics and flavonoids could be measured in the presence of procyanidins by monitoring at 320 nm where procyanidins are nonabsorbing.) This was overcome with preliminary separation of compound classes with mini column gel filtration (4). Figure 4B shows an HPLC separation of apple juice procyanidins. Compounds absorbing at 320 nm which coeluted with catechin, epicatechin and the procyanidins have been eliminated by the cleanup procedure. The HPLC conditions (column, solvent gradient, etc.) are identical to those used for separation of cinnamics and flavonoids, the separation taking 60 min. 

For separation and determination of phenolic acids and flavonoids, HPLC is the established technique (Nave et al., 2007 Rodriguez-Delgado et al., 2001 Spranger et al., 2004 Vitrac et al., 2002). The chromatographic conditions include the use of, almost exclusively, a reversed phase C18 column UV-vis diode array detector, and a binary solvent system containing acidified water and a polar organic solvent (Tsao Deng, 2004). 

Banwart, W. L., P. M. Porter, T. C. Granato, and J. J. Hassett, HPLC separation and wavelength area ratios of more than 50 phenolic acids and flavonoids, J. Chem. Ecol., 77, 383-395 (1985). 

Chemical Structure of the Main Flavonoid Subclasses and Some of Their More Representative Components Together with Their Spectral Characteristics Recorded Online after Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Separation Using Methanol-Water Acidified with Sodium Phosphate (pH 3.3) ... 

FIGURE 3.3 Ultraviolet (UV)-visible spectra of the main classes of flavonoids recorded online after reversed-phase high-performance liquid chromatography (RP-HPLC) separation using an acetonitrile/4.5% formic acid (v/v) gradient. 

The leaf flavonoids of the cruciferous species such as Camelina sativa, Crambe abyssinica, Crambe hispanica, Thlaspi arvense, Brassica napus and Sinapis alba were separated and identified with the combination of HPLC, TLC and paper chromatography. Llavonoid aglycones were extracted by cutting fresh three-week-old leaves in tiny pieces and boiled in 50 ml of 2 M HC1 for 45 min. 

RP-HPLC found application in the separation and identification of the main flavonoids in weld (.Reseda luteola L.). The aerial parts of the weld were dried, ground and extracted with various solvents and solvent mixtures such as methanol, ethanol, water, methanol-water... 

Negative atmospheric pressure chemical ionization (APC) low-energy collision activation mss spectrometry has also been employed for the characterization of flavonoids in extracts of fresh herbs. Besides the separation, quantitative determination and identification of flavonoids, the objective of the study was the comparison of the efficacy of the various detection systems in the analysis of flavonoids in herb extracts. Freeze-dried herbs (0.5g of chives, cress, dill, lovage, mint, oregano, parsley, rosemary, tarragon and thyme) were ground and extracted with 20 ml of 62.5 per cent aqueous methanol. After sedimentation the suspension was filtered and used for HPLC analyses. Separations were carried out in an... 

Because of the advantageous dietary effects of flavonoids they have been vigorously investigated in food and food products. The objectives of these measurements were the separation and quantitation of well-known flavonoids in foods and the identification of new flavonoids. An HPLC-ESI MS method has been developed for the isolation and identification of new quercetin derivatives in the leaves of Eruca sativa (Mill). Fresh leaves (500g) were homogenized with 1 200 ml of methanol-water (7 3, v/v), the suspension was macerated for 24h at ambient temperature, then it was filtered, concentrated to 50 ml and diluted with water to 500 ml. The extract was applied to an Amberlite XAD-2 column (75 X 8cm i.d.) and was washed subsequently with 11 of water and 11 of diethyl ether. The glucoside fraction was eluted with 1.51 of methanol and the eluate was concentrated in vacuum and liophilized. 

Because of the importance of soybean and soybean products in both human and animal nutrition their flavonoid content has been investigated many times. Thus, HPLC-UV and HPLC-MS have been applied for the determination of flavonoids and other phytochemicals in soybean extracts and in onion with and without hydrolysis, lg of onion was homogenized and mixed with 8 ml of methanol-water (8 2, v/v). After 2h the suspension was centrifuged at 4°C and the supernatant injected. Powdered soybean (500 mg) was defatted by 2 X 10 ml of hexane and further treated as the onion sample. Flavonoids were hydrolyzed by mixing 2 ml of extract with 2 ml of 2 M HC1 and heated to 130°C for 2 h. The solution was neutralized with 4 M of NaOH. Separation was performed in an ODS column (125 X 4.6 mm... 

Another isocratic elution method was applied for the determination of flavonols in green and black tea leaves and green tea infusions by RP-HPLC. The chemical structures of the flavonols studied are shown in Fig. 2.66. Infusions of teas were prepared by mixing lg of tea leaves with 100 ml of boiling water for 5min, then they have filtered and used for HPLC analysis. The infusion step was repeated three times. Flavonoids were hydrolysed by mixing lg of tea leaves with 40 ml of 60 per cent aqueous ethanol and 5 ml of 6 M HC1. The suspension was heated at 95°C for 2 h, then filtered and the volume was adjusted to 50 ml with 60 per cent aqueous ethanol. Separation was performed in an ODS column (150 X 4.6mm i.d.) operated at 30°C. The isocratic mobile phase consisted of 30 per cent aqueous ACN in 0.025 M KH2P04, and the pH was adjusted to 2.5 with 6 M HC1. The... 

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