Flavonols HPLC separation

It has been stated that the extraction, hydrolysis, and RP-HPLC separation method is specific and sensitive for the analysis of flavonols, flavones and flavanons. The data can be used for the estimation of the daily intake of these compounds [187]. 

Jungbluth, G. and Ternes, W., HPLC separation of flavonols, flavones and oxidized flavonols with LTV-, DAD-, electrochemical and ESI-ion trap MS detection, Fresenius J. Anal. Chem., 367, 661, 2000. 

Cheynier, V. and Rigaud, J., HPLC separation and characterization of flavonols in the skins of Vitis vinifera var. Cinsault. Am. J. EnoL Vitic. 37, 248, 1986. 

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,... 

The addition of micelles that migrate counter-current in the capillary can be used for the separation of apolar compounds, particularly when organic solvents such as methanol are added to the buffer solution. Using this method, flavone and flavonol aglycones present in honey were separated, although no specific advantage with HPLC separation using reversed-phase columns was observed. 

Pear Phenolics. The same HPLC analytical system gave good resolution of pear juice phenolics and enabled peak assignments and quantitation to be made in a similar manner to that described for apple phenolics (3,7). It was also necessary to isolate the procyanidins and catechins using the Sephadex clean-up procedure (4) in order to measure their concentration. Figure 6A shows the HPLC separation of cinnamics, flavonols, and arbutin in pear juice while Figure 6B is a HPLC chromatogram of the pear juice procyanidin fraction. 

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... 

The anthocyanin composition of flowers has also been frequently investigated by chromatographic methods. Acylated anthocyanins and flavonols have been extracted from the flowers of Dendrobium cv. Pompadour and separated by TLC and RP-HPLC techniques. Fresh flowers (approximately. 1 kg) were extracted with 101 of methanol-acetic acid-water (4 1 5, v/v), and the extract was concentrated and analysed by preparative and analytical RP-HPLC using ODS columns (150 X 19 mm i.d. and 250 X 4.6 mm i.d.) at 40°C. The gradient consisted of methanol-water-acetic acid in various volume ratios. Flow rates were 4 and 1 ml/min for preparative and analytical separations, respectively. Cyanidin 3- (6-malonylglucoside)-7,3 -di(6-synapilglucoside) and the demalonyl derivative were detected in the flowers [261],... 

The technique of CPC was also employed as a key step in the purification of 26 phenolic compounds from the needles of Norway spruce (Picea abies, Pinaceae). An aqueous extract of needles (5.45 g) was separated with the solvent system CHCl3-Me0H-i-Pr0H-H20 (5 6 1 4), initially with the lower phase as mobile phase and then subsequently switching to the upper phase as mobile phase. Final purification of the constituent flavonol glycosides, stilbenes, and catechins was by gel filtration and semipreparative HPLC. °... 

Various analytical methods exist for flavonoids. These range from TLC to CE. With the introduction of hyphenated HPLC techniques, the analytical potential has been dramatically extended. Gas chromatography (GC) is generally impractical, due to the low volatility of many flavonoid compounds and the necessity of preparing derivatives. However, Schmidt et al. ° have reported the separation of flavones, flavonols, flavanones, and chalcones (with frequent substitution by methyl groups) by GC. 

Among the numerous applications of SPE are separations of phenolic acids and flavonoids from wines and fruit juices. Sep-Pak Cig cartridges have been used for the fractionation of flavonol glycosides and phenolic compounds from cranberry juice into neutral and acidic parts before HPLC analysis. Antimutagenic flavonoids were identified in aqueous extracts of dry spinach after removal of lipophilic compounds by SPE. ... 

For fruits and their products, HPLC techniques for phenolics have been used to study the effect of processing, concentration, and storage on the phenolic composition of juices as well as a potential precursor for an off-flavor compound in juices. Phenolic analysis has been further applied to the detection of economic adulteration and especially to verify the authenticity of fruit juices. This is especially important when cheaper fruits can be added to more expensive ones in a fraudulent manner. In most fruits, the nonanthocyanin flavonoids consist mainly of flavonols and flavanols, with trace amounts of flavones. Glycosides are the predominant forms present. These most often are separated by reversed-phase HPLC on Cl8 columns with gradients consisting of acidified H20 and ACN, MeOH, or EtOH. 

For phenolic acids in bilberry juice, a reversed-phase HPLC method (16) using a linear-gradient elution of (a) 2% aqueous acetic acid and (b) acidified, aqueous acetonitrile on two Cl8 columns was able to separate the 12 phenolic acids and flavonoids (three flavonol glycosides and three flavonols) in ethyl acetate extract. Phenolics in blueberries were extracted, isolated, and... 

For the red wines (82-84), which were injected directly into the HPLC without sample preparation, a ternary-gradient system using aqueous acetic acid (1% and 5% or 6%), and acidified acetonitrile (acetonitrile-acetic acid-water, 30 5 6) was used for cinnamic acid derivatives, catechins, flavonols, flavonol glycosides, and proanthocyanidins. Due to the large number of peaks, the gradient was extended to 150 min for the resolution of many peaks of important phenolics. This direct injection method was able to separate phenolic acids and esters, catechins, proanthocyanidins, flavonols, flavonol glycosides, and other compounds (such as tyrosol, and rrans-resveratrol) in wine in a single analysis. However, use of acetic acid did not permit the detector (PDA) to be used to record the UV spectra of phenolics below 240 nm (84). 

Capillary electrophoresis (CE) has also been employed for the analysis of flavonols.17 In fact, CE methods coupled with electrochemical detection (ECD) are increasingly being employed for the analysis of flavonoids because of the minimal sample volume required, short analysis time, and high separation efficiency.17 The limit of detection for quercetin and kaempferol by CE/ECD methods are comparable to those obtained by LC/MS. The primary drawback of using CE is low repeatability of retention times as compared with HPLC. 

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