Flavonoids separation methods

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,... 

Another favorite substance for medicine and cosmetics is menthol. It is obtained from a number of heibs, peppermint (Mentha x piperita L.) being the most common source (Fig. 2.13). The peppermint plant contains about 1-3 % essential oil, which can be obtained by a chemical separation method called steam distilla-tiom About half of the oil is menthol and its derivatives, terpenes, and flavonoids. Menthol has a pleasant odor and eooling effect, and these two properties are why... 

The separation of complex flavonoid mixtures demands in many cases the use of several chromatographic techniques. Thus, a comparison of the HPTLC and high-performance liquid chromatographic (HPLC) behaviorof twenty-six flavonoids, anda method for establishing HPLC gradient elution conditions by using TLC data (23) is useful. 

Stalikas C. 2007. Extraction, separation, and detection methods for phenolic acids and flavonoids. J Sep Sci 30(18) 3268-3295. 

Different fruits and vegetables vary significantly in their structural constituents, macronutrients (proteins, oils, and carbohydrates), and micronutrients such as flavonoid profiles. It is almost impossible to develop one optimal method for extraction, separation, and analysis for each and every different fruit or vegetable. However, because of the relatively similar chemistry and biochemistry of flavonoids, some general statements can be abstracted from the existing literature. Flavonoids of fruits and vegetables... 

Quantification of individuals The foregoing quantitative methods provide rapid estimation of the total flavonoids or their subgroups however, they offer no information on specific flavonoids. To analyze the concentration of individual flavonoids, good separation procedures must first be developed, followed by quantification using various spectrophotometric or other types of detectors. Such a separation and... 

A new HP-TLC method has been applied for the quantitative analysis of flavonoids in Passiflora coerulea L. The objective of the experiments was the separation and identification of the compound(s) responsible for the anxiolytic effect of the plant. Samples were extracted with 60 per cent ethanol or refluxed three times with aqueous methanol, and the supernatants were employed for HPTLC analysis. Separation was performed on a silica layer prewashed with methanol and pretreated with 0.1 M K2HP04, the optimal mobile phase composition being ethyl acetate-formic acid-water (9 1 l,v/v). It was established that the best extraction efficacy can be achieved with 60 - 80 per cent aqueous methanol. The HPTLC technique separates 10 different flavonoids, which can be used for the authenticity test of this medicinal plant [121],... 

A simple TLC method has been developed for the separation and identification of flavons and flavon glycosides in the extract of Phillyrea latifolia L. The leaves (100 g) were defatted in 11 of chloroform for 24 h and then extracted with 2 X 11 of ethanol-water (80 20, v/v). The collected extracts were concentrated and extracted again with n-hexane to remove chlorophylls and other apolar constituents. Analytes were extracted with ethyl acetate. Both normal phase and RP-TLC have been used for the separation of flavonoids. The results are compiled in Table 2.36. It was concluded from the data that TLC can be successfully applied for the quality control of plant extracts containing various flavone derivatives [124],... 

Using numerical taxonomy it was found that the best separations were obtained by chloroform-methanol-(98-100 per cent) formic acid (44.1 3 2.35) and n-hexane-ethyl acetate-glacial acetic acid (31 14 5, v/v) as mobile phases. As the flavonoid profile of the propolis samples showed considerable differences, the method has been proposed for the authenticity test and traceability of various propolis products [141]. 

A new poly(7-oxobomene-5,6-dicarboxylic acid-Wod -norbomene)-coa(cd silica has been synthesized and applied for the separation of flavonoids in model systems and in the extracts of onion, elder flower blossom, lime blossom, St. John s Wort and red wine. Separation was performed in a (150 X 4 mm i.d. particle size 7 /rm) column at room temperature. Flavonoids (quercitrin, myricetin, quercetin, kaempferol and acacetin) were separated with gradient elution water-ACN (20 mmol TFA) from 78 22 to 70 30 v/v in 3min. The flow rate was 2 ml/min. The separation of the standard mixture is shown in Fig. 2.51. It has been stated that the method is rapid, accurate and the MS detection makes possible the reliable identification of flavonoids [153],... 

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. 

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... 

As the separation characteristics of liquid chromatographic and electrophoretic techniques markedly differ from each other, combined methods using the advantages of both procedures have been successfully used for the analysis of flavonoids. Thus, the use of CZE-UV, HPTLC-UV and GC-MS for the measurement of flavonoids in seeds and root exudates of Lotus pedunculatus has been reported. The rooting solution and seed exudate were passed through cellulose acetate filters to bind the flavonoids. After extraction,... 

M. Medic-Saric, G. Stanic and I. Bosnjak, The use of information theory and numerical taxonomy methods for evaluating the quality of thin-layer chromatographic separations of flavonoid constituents of matricariae flos. Pharmazie 56 (2001) 156-159. 

G. Kitanov, A method for separation of flavonoid and xanthone aglycones with monohydrox-yphenyl and o-dihydroxyphenyl moiety. Acta Pharm. Zagreb 50 (2000) 69-73. 

In earlier times, thin-layer chromatography (TLC), polyamide chromatography, and paper electrophoresis were the major separation techniques for phenolics. Of these methods, TLC is still the workhorse of flavonoid analysis. It is used as a rapid, simple, and versatile method for following polyphenolics in plant extracts and in fractionation work. However, the majority of published work now refers to qualitative and quantitative applications of high-performance liquid chromatography (HPLC) for analysis. Llavonoids can be separated. 

A number of techniques have been used for the preparative separation of flavonoids. These include HPLC, Diaion, Amberlite XAD-2 and XAD-7, and Fractogel TSK/Toyopearl HW-40 resins, gel filtration on Sephadex, and centrifugal partition chromatography (CPC). The choice of methods and strategies varies from research group to research group and depends often on the class of flavonoid studied. 

There is no single isolation strategy for the separation of flavonoids and one or many steps may be necessary for their isolation. The choice of method depends on the polarity of the compounds and the quantity of sample available. Most of the preparative methods available are described in a volume by Hostettmann et al. ... 

Various countercurrent chromatographic techniques have been successfully employed for the separation of flavonoids. Countercurrent chromatography is a separation technique that relies on the partition of a sample between two immiscible solvents, the relative proportions of solute passing into each of the two phases determined by the partition coefficients of the components of the solute. It is an all-liquid method that is characterized by the absence of a solid support, and thus has the following advantages over other chromatographic techniques ... 

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. 

---