The aim of this study was to develop a thin-layer chromatographic method of qualitative analysis, aiming to confirm the presence of the red beetroot pigments in a given sample. The TLC system developed for this purpose consists of the precoated RP-18 F254s TLC plates and the acetonitrile + methanol + water + glacial acetic acid, 2:7:1:0.1 (v/v/v/v) mobile phase. With the use of this system, a striking horizontal separation of betacyanin pigments is obtained for both the red beetroot juice and the commercial betanin sample (with the left-to-right resolution distance of the two bands equal to ca. 6 mm), and a unique pattern of the two skewed chromatographic bands is observed. This striking phenomenon has been given a thorough consideration, and its tentative physicochemical justification was provided, based on analogical cases reported and extensively discussed in our earlier studies. Characteristic fingerprint obtained both for the beetroot juice and the commercial sample of betanin (resembling two slant butterfly wings) can prove very helpful for qualitative confirmation of the presence (or otherwise) of the betanin pigment in the red color juices and beverages, as it was demonstrated upon an example of elderberry juice with a confirmed fortification with the betanin pigment.
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