Two Dimensional Thin Layer Reaction Chromatography

Flavonoid glycosides as precursors of flavonoid aglyca can be detected using the two-dimensional thin-layer reaction chromatography [37, 67]. After the first development of the TLC plate (sorbens: Silica gel 60 F254), the sugars were hydrolyzed by hydrochloric acid applied in a microwave vapor-blast process. Turning the TLC plate by 90° a second development was carried out (solvent system: ethyl acetate/formic acid/water 8 + 1 + 1 for both developments). The spots were detected by NP/PEG-reagent. Tschirsch and Holzl adapted this method to acylated flavonoid glycosides, e.g., acylated apigenin-7-glucoside-derivatives from chamomile [146] (Figure 10.6). The acyl groups were removed by saponification, liberating the flavonoid glycoside. In this case concentrated ammonia was used after the first development (Figure 10.7). The duration and temperature of treatment varied from 1 min at room temperature to 1 hour at 70°C depending on the stability of the acyl group (sorbens: silica gel 60 F254; solvent system: ethylacetate/formic acid water 100 + 10 + 5 for both developments, detection by NP/PEG-reagent). The separation of apigenin-7-P-D-(6"-0-acetyl)- and (4"-0-acetyl)glucoside was possible [146]. The method is particularly useful when no authentic reference substances are available.

10.3.3 High-Pressure Liquid Chromatography

The first HPLC separation of quercetin, quercetin-7-glucoside, apigenin-7-glucoside, rutin, herni-arin, umbelliferone, and two unidentified phenyl carboxylic acids was done by Schilcher [117, 118]. All compounds were separated on an RP 8 column running a gradient of methanol-water (15-80%). The method was further developed and perfected for the qualitative and quantitative determination of chamomile flavonoids by Redaelli et al. [99, 100] in 1981 and by Dolle et al. in 1985. A comparison of three methods is shown in Table 10.6.

The method Dolle published allows reproducible determinations of the chamomile flavonoids [18]. Method no. 3 uses the diode-array technique for detection, giving reliable results. Several later publications used this detection method [14, 82, 124, 125, 126, 128, 146].

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