Digitally-optimized HPTLC coupled with image analysis for pursuing polyphenolic and antioxidant profile during alfalfa sprouting

The utilization of Folin-Ciocalteu as a post-chromatographic derivatization reagent for direct quantitative estimation of polyphenolic compounds using HPTLC coupled with image analysis was developed, validated and implemented in this study. The phenolic compounds react with Mo+6; the hypothesized active center in molybdotungstophosphoric heteropolyanion causing its reduction into blue colored complex. In addition, a direct TLC-DPPH assay was developed and applied to determine the correlation between the polyphenolic content and their free radical scavenging ability during the sprouting of alfalfa seeds for eleven days. Moreover, different approaches for image to digital conversion routines were studied in this work. Digital camera and flatbed scanner as common in-house image acquisition tools were compared. In addition, three software packages (ImageJ®, JustTLC® and SorbfilTLC®) adopting completely different digitalization algorithms in quantitative image analysis were also competed. Different statistical data revealed that digital camera coupled with ImageJ® program was superior to other investigated approaches regarding linearity, precision, accuracy and detection of polyphenols as well as antioxidants at very low concentration levels.