Flavones 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. 

Fig. 13 HPLC separation of flavanone glycosides and polymethoxylated flavones (PMFs) in orange juice spiked with didymin and PMFs.

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. 

FIGURE 1.8 High-performance hquid chromatography (HPLC) separation of urinary phytoestrogens (a) and standards (h) monitored at 260 and 280 nm (insert). Peak ideutificatioD DE, daidzein GE, genistein COM, coumestrol FOR, formononetin B-A, biochanin A flavone, internal standard. (Reprinted with pomission from Franke, A.A., Custer, L.J., Cema, C.M., and Narala, K., Proc. Soc. Exp. BioL Med., 208, 18-26, 1995.)... 

Spherical shape and narrow particle size distribution are the two key factors for a good HPLC stationary phase. Very recently, Zhang et al. present a chiral MOF with an average particle size of 5 pm for the HPLC separation of alcohol, ketone, flavone, phenol, base, and amide racemates. Ten racemates were well-separated on a 25-cm long MOF column with excellent selectivity. The stereoselectivity likely resulted from the interaction of the analytes with the inner pore space of MOF, which has the most appropriate size and steric fit. Besides, the dispersion, dipole-dipole, and hydrogen-bonding forces which come from the mobile phase may also play significant roles in chiral separation. These results show that chiral MOFs are practicable for HPLC enantioseparation. 

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. 

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. 

Traditionally, flavonoids have been separated and analyzed by HPLC and gas chromatography (GC). However, recent developments of SFC may permit a more accurate and complete analysis of plant phenolic compounds. Supercritical fluid chromatography brings together the advantages of both HPLC and GC techniques because it may be readily employed in the analysis of nonvolatile and thermolabile compounds and provides facile coupling to detector technologies such as mass spectrometry and Fourier transform infrared (FT-IR) spectroscopy. In recent years, SFC has been used to separate flavonoid compounds, most of which are polymethoxylated flavones and polyhydroxylflavonoids. 

Morin et al. successfully separated polymethoxylated flavones (PMFs) by packed-column SFC, illustrating that the SFC procedure is considerably faster than HPLC, with good resolution and adequate accuracy for the quantitative analysis of the PMFs. The chromatographic system... 

The first comprehensive 2D system was developed in the late 1970s by Erni and Frei, who applied IEX x RPC to the analysis of senna glycosides from plant extracts.61 In the subsequent decades, comprehensive MD-HPLC methods have been further developed, mainly for peptides and proteins,62 3 but also for separation of various natural products such as phenolic and flavone antioxidants64 and carotenoids.65 The theoretical aspects of MD-HPLC techniques have also been further developed.66-68... 

HPLC is the method of choice for the separation of complex mixtures containing non-volatile compounds such as various flavonoids in extracts prepared from different samples. A survey of literatures revealed that most researchers have used Cjg-reversed stationary phases, which proved to be superior to the normal phase technique. The reversed phases are suitable for separating flavonoids in a wide range of polarities, as Vande Casteele et al. have demonstrated the separation of 141 flavonoids from polar triglycosides to relatively non-polar polymetoxy-lated aglycones belonging to the classes of flavones, flavonols, flavanones, dihydroflavonols, chalcones, and dihydrochalcones. 

Morin et al. successfully separated polymethoxylated flavones (PMFs) by packed-column SFC, illustrating that the SFC procedure is considerably faster than HPLC, with good resolution and adequate accuracy for the quantitative analysis of the PMFs. The chromatographic system consisted of a bare silica column (250 X 4.6 mm I.D.) with a carbon dioxide—methanol mobile phase and UV detection (313 mn). The pressure was controlled by a manual backpressure regulator connected in series after the detector and... 

The first category comprise carotenoids, lipids and steroids, the second includes boswellic and fatty acids and vitamins and the last comprises flavones, alkaloids, cannabinoids, berberines and anthraquinones, to give just a few examples. Another method of classification is according to the separation mode, such as normal-phase, reversed-phase, ion-exchange, size-exclusion and affinity-based separations, likewise as in HPLC. In addition, it is worth mentioning that the instrumentation design incorporates pressurized CEC (PEC) and a microchip platform [7]. 

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