Journal of Chromatography , 349 (1985) 69-75 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands CHROMSYMP. 674 SEPARATION OF NATURAL POLAR SUBSTANCES BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY, CENTRIFUGAL THIN-LAYER CHROMATOGRAPHY AND DROPLET COUNTER-CUR- RENT CHROMATOGRAPHY FULVIA ORSINI and LUISELLA VEROTTA* Dipartimento di Chimica Organica e Industriale, Via Venezian 21. 20133 Milan (Italy) SUMMARY Reversed-phase high-performance liquid chromatography, centrifugal thin- layer chromatography and droplet counter-current chromatography are complemen- tary, highly efficient techniques for isolating natural, very polar, substances from plant sources. The choice of method can be made directly by a preliminary thin-layer chromatographic investigation. An application to the isolation of some new triter- penic glycosides in PassiJEora quadrangularis L. is described. INTRODUCTION . Passzjlora extracts have been wisely used in the folk medicine of Europe and America because of their sedative and antihypertensive properties. Many PassiJora spp. have already been investigated, and some indole alkaloids of the harman type, C- and 0-glycosyl flavonoids, cyanogenic glycosides, and a cyclopropane triterpenic glycoside (passiflorine) have been identifiedlWs. In spite of the different classes of com- pounds found, the active principles of PassiJlora spp. have not yet been recognized, and pharmacological tests are currently under investigation. Prior to biological test- ing, we have studied the applicability of a number of isolation methods to the puri- fication of individual constituents of the aqueous soluble fraction of Pass$ora quad- rangularis L. EXPERIMENTAL Gel-Jiltration column chromatography (GFCC) Sephadex LH-20 (130 g; Pharmacia) was packed by a slurry technique into a glass column (950 x 30 mm I.D.) and eluted with methanol (300 ml) at a flow-rate of 12 ml/h. A total of 120 2-ml fractions were collected and monitored by thin-layer chromatography (TLC) on silica gel with solvent A. TLC Silica gel plates (5 x 10 cm), F254 TLC (Merck, Darmstadt, F.R.G.), were 0021-9673/85/%03.30 0 1985 Elsevier Science Publishers B.V.