Flavonoid chromatographic methods

The isolation of flavonoids from the methanol extract of G. uralensis was carried out under non-basic conditions, because some flavonoids isomerize under basic conditions, e.g. racemization of flavanones and isoflavanones, ring-open reaction of flavanones etc. Bioactive fractions were separated by some chromatographic methods and each step was monitored with anti-H. pylori activity with the paper disk method. Eighteen compounds were isolated from these bioactive fractions and... 

The low-molecular-weight water-soluble fraction of LCP flour was found by thin layer chromatographic methods to contain several flavonoid components. To establish the role of flavonoids in the production of yellow color in biscuits, these components were extracted from LCP and glandless cottonseed flours with 85Z aqueous isopropyl alcohol (which is a better solvent for flavonoids than water). Before removal of the flavonoids, the flours had been treated with petroleum ether to extract residual lipids that could interfere with flavonoid isolation. Extraction of the residual lipids did not significantly alter the color of biscuits prepared with the extracted flours (Figure 7). 

Chromatographic methods were developed to separate a few of the citrus flavonoids from the complex mixture of citrus flavonoids. The early paper chromatographic methods for flavanones (22, 23) were difficult to quantitate because of band broadening and uneven solvent development. Several thin layer chromatographic (TLC) methods were developed to separate the bitter from the nonbitter flavanone glycosides (24, 25, 26, 27). 

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. 

High-speed countercurrent chromatography (HSCCC) is a relatively new technique. It is a liquid-liquid partitioning chromatographic method in which the stationary phase is immobilized by a centrifugal force. HSCCC is the most advanced form of CCC in terms of partition efficiency and separation time. As solvent system, a mixture in different ratio is usually used, depending on the flavonoids nature ethyl acetate/... 

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. 

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 ... 

Sample extraction and hydrolysis details e.g., solvent extraction after freeze drying, with optimized acid or enzymatic hydrolysis Preparation of flavonoid standards and use of internal standards Chromatographic separation and detection method used, ideally RP-HPLC with UV or fluorescent detection Outline of quality assurance procedures employed... 

A rapid HPLC method for the simultaneous determination of the flavonoids hesperidin and naringin and their aglycones (hesperetin and naringenin) in soft drinks, marmalades, candies, yogurts, biscuits, and salad dressings was also developed (153). Samples were refluxed with 60% aqueous methanol at 90°C and chromatographed on a semimicro column and detected at 283 nm by UV. The detection limit for these phenolics was 0.5 /zg/g. 

There are several published information regarding the isolation and the identification of flavonoids in plant material using different methods, mainly chromatographic and spectroscopic. Today, high-performance liquid chromatography (HPLC) is established as the most convenient method which enables separation and identification of... 

High-performance liquid chromatography (HPLC) is the method for detection, identification and also quantification of flavonoids, phenolic acids and their derivatives. With this method, the sample is applied and eluted through a chromatographic column under specific conditions designed for optimum separation and resolution so that each compound or group of compound passes through the column with a... 

An example for the separation for flavonoids with HP-RPC is the screening method employed for the systematic identification of glycosylated flavonoids and other phenolic compounds in plant food materials by Lin et al20 These authors used an analytical 4.6 mm x 250 mm 5 pm C18 silica column at 25 °C with linear gradient elution (eluent A (0.1% FA in water and eluent B 0.1% FA in ACN) at 1.0 ml min-1. DAD was performed at 270, 310, 350, and 520 nm to monitor the UV/VIS absorption. The LC system was directly coupled to an ESI mass spectrometer without flow splitting and the mass spectra acquired in the positive and negative ionization mode. The same analytical scheme (aqueous MeOH extraction, reversed-phase liquid chromatographic separation, and diode array and mass spectrometric detection) can be applied to a wide variety of samples and standards and therefore allows the cross-comparison of newly detected compounds in samples with standards and plant materials previously identified in the published literature. 

Flavonoid constituents are present in a number of drugs, and therefore a consistent sample preparation method is suggested in Document 12 together with a uniform TLC system for the dry extracts. In the sample chromatograms included with the testing procedure, there are five dry extracts and the separation of a drug. This should confirm that the chromatographic system chosen could also be used for this apphcation. 

Procyanidins can usually not be completely separated from each other, from phenolic acids and from accompanying flavonoids by sample clean up and/or chromatographic procedures. The differentiation between these individual compounds can therefore only by accomplished by the selectivity of a suitable detection method. UV-DAD (ultra violet diode array detection), electrochemical and mass spectrometry detection have been used for the identification and quantitative determination of procyanidins. 

The chromatographic profile should feature the fundamental attributions of sameness and differences of the constituents of the investigated extract and define their quality in order to get reproducible biological data in terms of safety and efficacy. The analytical methods should be so precise that different batches of the extract in question will have the same efficacy. As described above, it has now become routine to define a ginkgo extract in terms of the content of the two groups, the flavonoid glycosides and... 

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