Anthocyanins formic acid solvent

Anthocyanins and anthocyanidins are readily separated on cellulose layers using developing solvents consisting hydrochloric acid-formic acid-water in differing proportions [24]. Glycosides and aglycones are readily separated on the same plate, provided that a sufficiently polar solvent is employed. 

The separation and identification of flavanol-anthocyanin adducts in wine and in model solutions were performed with RP-HPLC coupled to DAD or ESI-MS. The investigation was motivated by the assumption that the formation of flavanol-anthocyanin complexes may influence the organoleptic characteristics of wine during ageing. Measurements were carried out in an ODS column (250 X 2 mm i.d. particle size 5 pm) at 30°C. The flow rate was 0.25 ml/min. Solvent A was water-formic acid (95 5), solvent B consisted of ACN— solvent A (80 20, v/v). The Gradient elution began with 3 per cent B for 7min to 20 per... 

The optimum conditions for the MS detection of anthocyanin derivatives have also been intensively studied. Grape skin extract was prepared by macerating skins with methanol-formic acid (95 5) for three days changing the extracting agent each day. The combined extracts were evaporated and diluted with water. RP-HPLC measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 5 /an) at 30°C. The solvents were water-formic acid (90 10, v/v, A) and methanol-water-formic acid (45 45 10, v/v, B). The gradient was from 35 to 95 per cent B in 20 min to 100 per cent B in 5 min final hold... 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

Polyphenol Analyses. Skin and seed extracts were prepared as described elsewhere (4,15). Flavonol and anthocyanin composition of grape skin extracts and wines were determined by direct reversed-phase HPLC analysis with diode array detection. The chromatographic conditions were the same as described earlier (16) but the formic acid concentration in the elution solvents was raised to 5% to improve anthocyanin resolution. Quantitations were based on peak areas, using mdvidin-3-glucoside (at 530nm) and quercetin-3-glucoside (at 360 nm) response factors, respectively, for anthocyanins and flavonols. 

C. One gram of the sample is ground with mortar and pestle and extracted twice with 5 mL of a formic acid/methanol 1 9 (v/v) solution for 1 h under shaking at room temperature in the dark. The extracts are combined and centrifuged at 18.000 g for 10 min. In an alternative, 50% (v/v) acetic acid in water was proposed as solvent in the extraction of anthocyanins from skins (Yamane et al., 2006). 

Figure 3.15 LC-UV anthocyanin profile recorded at wavelength 520 nm of a hybrid grape crude extract (Clinton, Vitis labrusca x Vitis riparia). Analytical conditions column C18 (250 x 4.6mm, 5pm), binary solvent composed of A) H20/formic acid (90 10 v/v) and B methanol/H20/formic acid (50 40 10 v/v/v) gradient program from 15% to 45% of B in 15min, 45-70% B in 30min, 70-90% B in lOmin, 90-99% B in 5min, 99-15% B in 5min (flow rate 1 mL/min). (Reprinted from American Journal of Enology and Viticulture 51, Favretto and Flamini, Copyright 2000)...

Non-acidified methanol is the more suitable solvent to extract anthocyanins from grape reducing risks of hydrolysis of acetylated compounds 20 berry skins are extracted with 50 mL of methanol for 12h at room temperature (Revilla et al., 1998). Alternatively, the use of a methanol/water/formic acid 50 48.5 1.5 (v/v/v) mixture was reported as well (Gao et al., 1997). The volume of extract is reduced to about one-half under vacuum at 30 °C, adjusted to 100 mL with water, and 10 mL of this solution is diluted to 50 mL with water in order to further reduce the MeOH content. The resulting solution is purified by a SPE Ci8 cartridge (e.g., lg) previously activated by passage of... 

Analysis by the uv absorption of citrate buffered solutions enables the I anthocyanin to be determined from the maximum at 520nm. The distribution of component anthocyanins can be found by TLC on cellulose with various strongly acidic solvents (eg. cone HCl, formic acid, water, 19 19.5 61.5) of extracts of plant material obtained with 1% hydrochloric acid in methanol. The Rf values found for 3-monoglucosides of the anthocyaninidins of Vitis vinifera were, for the petunidin compound (0.13), for that of malvidin (0.22) and of peonidin (0.25) (ref. 11). ... 

Based on our own experience, solid phase extraction with 500 mg C18 SEP-Pak disposable cartridges works satisfactory in terms of extraction yield and repeatability of results. We use methanol/formic acid = 9/1 (v/v) for anthocyanin extraction, followed by evaporation to dryness and reconstitution in water/formic acid = 9/1 (v/v). This solution is brought onto cartridges equilibrated with water/formic acid = 9/1 (v/v). The samples are then washed with the same solvent and eluted with methanol/formic acid =9/1 (v/v) and again evaporated to dryness. After reconstitution in an appropriate solvent, the samples are ready for use. On average, the recovery of anthocyanins is better than 95 %. Repeated extraction (n=15) of a reference standard (en-3-O-gle) gave a scattering of less than 7 % (relative standard deviation). 

Separation of typical anthocyanins is carried out on cellulose layers in three solvent systems which all contain cone, hydrochloric acid, formic acid, and water in different proportions System 1 (19 19 62), system 2 (7 51 42), and system 3 (25 24 51). The mobile phase is run 18 cm (ca. 120 min.), and the 1 values of the individual compounds are given in Table 4. 

Standard-grade silica gel is recommended since the trace metals present aid the separation of peonidin and malvidin from cyanidin and delphinidin (Harbome, 1998). Preparative TLC of anthocyanins using 20 X 20 cm chromatoplates with 1-mm layers of a mixture of 2/3 silica gel (adsorbosil-2) and 1 /3 cellulose powder (MN-300, gypsum free) was described by Asen (1965). He employed the following solvent systems to purify milligram quantities of anthocyanins from plant tissues w-butanol/water (1 1), water/HCl/formic acid (8 4 1), 1 % HCl, and acetone/0.5 N HCl (1 3). Table 5.2 lists Rf values of some common anthocyanidins on microcrystalline cellulose (Strack and Wray, 1989). The simultaneous analysis of anthocyanidins and anthocyanins on cellulose layers using a solvent system of concentrated HCl/formic acid/water (24.9 23.7 51.4) has been demonstrated by Andersen and Francis (1985). 

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