Phytochemical analysis involves the systematic extraction, separation, and characterization of bioactive compounds from plant material. Plant secondary metabolites include alkaloids, flavonoids, terpenoids, phenolic acids, saponins, and tannins, many of which have pharmaceutical, nutritional, and industrial applications.
Extraction is the critical first step. Maceration involves soaking powdered plant material in a solvent at room temperature with occasional stirring. Percolation uses a continuous flow of solvent through the plant material. Soxhlet extraction cycles hot solvent through the sample for exhaustive extraction. Ultrasonic-assisted extraction uses cavitation to disrupt cell walls and enhance mass transfer. Supercritical fluid extraction using CO2 provides selective, solvent-free extraction of non-polar compounds.
Solvent selection follows like-dissolves-like principles. Hexane and dichloromethane extract non-polar compounds such as waxes and essential oils. Ethanol and methanol extract mid-polarity compounds including flavonoids and alkaloids. Water extracts polar compounds such as tannins and glycosides. Sequential extraction with solvents of increasing polarity fractionates the extract by compound class.
Phytochemical screening uses qualitative color reactions to detect compound classes. The Dragendorff test detects alkaloids. The Shinoda test detects flavonoids. The Liebermann-Burchard test detects terpenoids and sterols. The ferric chloride test detects phenolic compounds. These tests provide preliminary information about the chemical composition.
Quantitative analysis determines the concentration of specific phytochemicals. Total phenolic content is measured using the Folin-Ciocalteu method with gallic acid equivalents. Total flavonoid content uses the aluminum chloride colorimetric method. High-performance liquid chromatography (HPLC) with UV, diode array, or mass spectrometric detection quantifies individual compounds. Gas chromatography-mass spectrometry (GC-MS) is used for volatile compounds.
Structure elucidation of purified compounds uses nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, infrared spectroscopy, and X-ray crystallography. Dereplication using HPLC-MS and database searching identifies known compounds and focuses efforts on novel structures.
