Organic chemistry

High-performance liquid chromatography

Reagent

High performance thin layer chromatography

Thin-layer chromatography

Chemistry

Derivatization

Formic acid

Silica gel

Methanol

Ethyl acetate

Chromatography

Journal of Liquid Chromatography &amp Related Technologies

HPTLC silica gel plates without and with fluorescence indicator F254 in combination with n-hexane–ethyl acetate–formic acid (20:19:1, v/v/v) as a developing solvent were explored for the HPTLC–densitometric and HPTLC–MS/(MSn) analyses of flavonoids. Pre-development of the plates with chloroform–methanol (1:1, v/v) was needed for reliable HPTLC–densitometric analyses of flavonoid aglycones in the whole RF range, while 2-step pre-development (1st methanol–formic acid (10:1, v/v), 2nd methanol), that decreased background signals of formic acid adducts, was required for HPTLC–MS analyses. Optimization with conditioning of the adsorbent layer with water before development and saturation of the twin trough chamber resulted in required decrease of the RF values of studied flavonoids (flavone, apigenin, luteolin, chrysin, quercetin dihydrate, myricetin, kaempferide, kaempferol, naringenin, pinocembrin).Detection was performed based on fluorescence quenching (on the plates with F254), natural fluorescence and after post-chromatographic derivatization with natural product reagent without or with further enhancement and stabilization of fluorescent zones with polyethylene glycol (PEG 400 or PEG 4000) or paraffin–n-hexane reagents. For all three reagents, drying temperature and time passed after drying influenced the intensity, which was increasing the first 20 min, and the stability (less than 2 h for PEGs and at least 24 h for paraffin–n-hexane) of the standards' zones.Optimal wavelengths for densitometric evaluation were selected based on in-situ absorption spectra scanned before and after derivatization and after stabilization. The developed method was tested via analyses of propolis, roasted coffee, rose hip, hibiscus, rosemary and sage crude extracts. To further increase the reliability of the obtained densitometric results HPTLC–MS/(MSn) analyses of all crude extracts were performed. Several phenolic and non-phenolic compounds were tentatively identified.Some possible interferences with phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) that are often present in the extracts together with flavonoids were also examined.

HPTLC–densitometric and HPTLC–MS methods for analysis of flavonoids