No single extraction procedure is ideally suited for all flavonoids due to the differences in their solubility characteristics. Also when the class of flavonoid present in the plant material is not known, a preliminary TLC examination of the hydrolyzed plant extract is essential in order to determine this. Chromatographic separation of the flavonoid rich fraction and its subsequent TLC analysis using known markers shall reveal the class of flavonoid contained. According to Harborne, a routine screening of plant tissues for the presence of flavonoids may be undertaken by two-dimensional paper chromatography of concentrated alcoholic extracts using butanol: acetic acid: water (4:1:5-top layer) and 5% acetic acid. Flavonoids having characteristic colours may be distinguished depending on their relative positions on the chromatogram. Some flavonoids are seen in visible light and majority may be visualized under UV light. Fuming the chromatogram with ammonia yields further information on the compounds from the colour changes observed. Use of standard markers such as rutin, quercetin, kaempferol, delphinidin, luteolin, and orientin will cover most classes of flavonoids.

Flavonoids of flower, fruit, leaves, bark, roots, rhizomes, and resinous exudates occur only as glycosides, while flavonoid aglycones are most typically found in wood tissues. Since most flavonoid glycosides are readily hydrolyzed by acid, care is to be taken where the fresh material is used. Hydrolyzed plant extracts on shaking with ethyl acetate segregate most flavonoids (except anthocyanins) into the organic layer.

Flavonoids may be extracted from fresh or dried plant material by extraction with methanol or ethanol. Preliminary extraction with petroleum ether removes waxy material.

1. Fresh plant material (flowers) is extracted with hot aqueous ethanol and filtered
2. The filtrate is concentrated in vacuo, and the residue washed with ether.
3. Dissolve it in dilute hydrochloric acid and extract with equal volume of ethyl acetate. Wash the ethyl acetate extract first with dilute hydrochloric acid and then with water.
4. Pass the ethyl acetate layer through anhydrous sodium sulphate, concentrate in vacuo to a syrupy mass, cover with toluene, and store in a refrigerator.
5. Collect the formed precipitate by centrifugation, dissolve in hot water, wash with ether, and set aside the aqueous layer.
6. Separate the crystallized flavonoid glycosides and purify by recrystallization from 50% aqueous ethanol.