Figure 1 The structure of ecdysone, the first ecdysteroid iso- lated from Bombyx mori pupae. Marshall PN and Lewis SM (1974) A rapid thin-layer chromatographic system for Romanowsky blood stains. Stain Technology 49: 235}240. Randerath K (1963) Thin-Layer Chromatography, pp. 211}214. London: Academic Press. Stahl E (ed) (1969) Thin-Layer Chromatography: A Laboratory Handbook. Berlin: Springer- Verlag. Wall PE (1988) Separation and quantiRcation of Fuchsin Basic using reversed-phase thin-layer chromatography. In: Dallas FAA, Read H, Ruane RJ and Wilson ID (eds) Recent Advances in Thin-Layer Chromatography, pp. 207}210. New York: Plenum Press. Wall PE (1989) HPTLC as a quantitative method for the determination of the purity of dyes of histological impor- tance. Journal of Planar Chromatography 2: 246}247. Wall PE (1991) Thin layer chromatographic separation of thiazins: problems and solutions. Journal of Planar Chromatography 4: 365}369. Wall PE (1993) The value of planar chromatography for the analysis of triphenylmethane dyes. Journal of Planar Chromatography 6: 394}403. ECDYSTEROIDS: CHROMATOGRAPHY R. Lafont and C. Blais, Ecole Normale Supe & rieure et Universite & Pierre et Marie Curie, Paris, France J. Harmatha, Academy of Sciences of the Czech Republic, Prague, Czech Republic I. D. Wilson, AstraZeneca Pharmaceuticals Ltd, Macclesfield, Cheshire, UK Copyright ^ 2000 Academic Press Introduction Ecdysteroids are present both in animals (mainly Arthropods) and plants and comprise about 300 different molecules related to ecdysone (Figure 1). Structural variation in the number of carbons on the side-chain and of substituents at various positions (Table 1) results in the presence of compounds displaying very different polarities. Most available chromatographic techniques have been applied to the isolation and analysis of ecdysteroids. Paper chromatography is now obsolete and no longer used. Gas chromatography (GC) is of limited use, as the derivatization procedures neces- sary to make volatile derivatives require careful control. Currently, the most widely used techni- ques are high performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), with the former providing the major analytical methods. Liquid^ Liquid Partitions The simplest separation method concerns partition- ing between two non-miscible solvents, and it is currently used for clean-up of biological samples. On this basis, several procedures have been designed, which allow the preparation of almost pure com- pounds in the gram scale. Solvent Partitioning Partition between n-butanol and water can be used to remove polar contaminants, whereas partition be- tween aqueous methanol and hexane removes non- polar materials. Lipids can also be removed from aqueous extracts with hexane}methanol (7 : 3, v/v), light petroleum or n-propanol}hexane (3 : 1, v/v). The nature of the contaminants to be removed and that of the ecdysteroids to be isolated govern the choice for a given partition system. The number of free -OH groups signiRcantly affects partition coefR- cients (Table 2). The combination of two successive partition steps allows the elimination of both polar and apolar con- taminants. It is thus possible to combine (1) chloro- form/water and (2) water/butanol. This results in a butanol-containing fraction that is signiRcantly enriched. It is possible to select a narrower range of polarity by replacing chloroform with a more polar organic solvent, e.g. isobutyl acetate, that neverthe- less allows ecdysteroids to remain in the water phase. III / ECDYSTEROIDS: CHROMATOGRAPHY 2631