Detection of Phenolic and Flavonoid. Compounds Using High ... chamber with respective mobile phase for phenolic compounds and flavonoids for chamber ...
Chapter 30
Detection of Phenolic and Flavonoid Compounds Using High Performance Thin Layer Chromatography (HPTLC)
Abstract High performance thin layer chromatography (HPTLC) is a sophisticated instrumental technique based on the full capabilities of thin layerchromatography. The advantages of automation, scanning, full optimization, selective detection principle, minimum sample preparation,hyphenation, etc., enable it to be a powerful analytical tool for chromatographic information of complex mixtures of inorganic, organic andbiomolecules which are characterized in this chapter. Introduction A wide variety of active phytochemicals,including flavonoids, terpenoids, lignans, sulfides, polyphenolics, carotenoids, coumarins, saponins, plant sterols, curcumins, phthalides, tannins, gallic acid, quercetin, phytosterols, alcohols, aldehydes have been identified from medicinal plants [3].These phtochemicals are estimated by a variety of techniques such as spectroscopy and chromatography. High performance thin layer chromatography (HPTLC) chromatographic fingerprints can be applied for this kind of certification. Aim To identify the phenolics and flavonoid contents in plant extract by HPTLC (Srivastava 2011). Principle The separation of the components of a mixture is due to their different affinities for a stationary phase such as a solid or a liquid and their differential solubility in a moving phase such as a liquid or gas. Materials Required 1. 2. 3. 4. 5.
Silica gel 60F254 TLC plate Hamilton syringe LINOMAT 5 instrument Photo-documentation chamber
© Springer International Publishing Switzerland 2016 P. Thangaraj, Pharmacological Assays of Plant-Based Natural Products, Progress in Drug Research 71, DOI 10.1007/978-3-319-26811-8_30
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6. Folin cio-calteu reagent 7. Ethanolic aluminium chloride (1 %) 8. Ethyl acetate and methanol Protocol Sample Preparation 1. Dissolve 50 mg of plant extract in 1 mL acetone and centrifuge. 2. Use the supernatant as test solution for HPTLC analysis. Sample Loading Load 3 µL of the above solution in the 5 × 10 silica gel 60F254 TLC plate (LINOMAT 5 instrument) using Hamilton syringe. Spot Development 1. Load the sample in a TLC plate and keep in TLC twin trough developing chamber with respective mobile phase for phenolic compounds and flavonoids for chamber saturation up to 20 min. 2. Develop the plate in the chamber using respective mobile phase up to 80 mm distance. Photo-Documentation 1. Develop the plate and dry in hot air to evaporate solvents from the plate. 2. Keep the plate in photo-documentation chamber and capture the images in a white light, UV 254 nm and UV 366 nm. Derivatization 1. Spray the respective spray reagents in a plate and dry at 120 °C in a hot air oven for 5 min. 2. Photo-document the plate in a white light for phenolic compounds and UV 366 nm for flavonoids using photo-documentation chamber. Scanning 1. Fix the plate in a scanner stage and scan the plate at 500 nm for phenolic compound and 366 nm for flavonoids. 2. Note the peak table and densitogram. 3. Mobile phase (for phenolic compounds and flavonoids). 4. Ethyl acetate–methanol–water (10:1.65:1.35) Spray Reagent For phenolic compounds 1. Apply the 20 % aqueous sodium carbonate solution over the plate followed with 25 % aqueous Folin cio-calteu reagent after brisk dry. 2. Dry the plate at 120 °C for 5 min.
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For flavonoids 1. Apply the 1 % ethanolic aluminium chloride solution over the plate and dry at 120 °C for 5 min. Retardation Factor 1. Retardation factor (Rf) is defined as the amount of separation due to the solvent migration through the sorbent layer as shown in the formula. 2. It depends on time of development and velocity coefficient or solvent front velocity. Rf ¼
Migration distance of the substance Migration distance of the solvent front from origin
References Egonstahl. (1990). A laboratory hand book of TLC (pp: 856–878). New York: Springer. Srivastava, M. M. (Ed.). (2011). HPTLC: High-performance thin-layer chromatography. ISBN 978-3-642-14024-2 e-ISBN 978-3-642-14025-9; doi:10.1007/978-3-642-14025-9 Heidelberg, Dordrecht, London, New York: Springer.
