Armen CPC application note N°09

Purification of 4-methyl phenylcoumarins from Mesua elegans
by Multi Dual Mode Centrifugal Partition Chromatography
1
G.Audo, C. Le Quémener1, C. Gomathi2, K. Awang2, J.Boustie3
1

Armen instrument application laboratory, 56860 St Avé, France
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
Laboratoire de Pharmacognosie et de Mycologie, Faculté de Pharmacie, PNSCM Team, UMR 6226, Univ Rennes 1, 35043 Rennes, France23Introduction
Malaysian Clusiaceae includes some well-known important trees and is a family of the main canopy of the forest1. Mesua
species have been known to produce various interesting compound structures, which are used for its medicinal values such astreatment of fever, dyspepsia and renal diseases2.
Centrifugal Partition Chromatography (CPC) method was developed for the separation of 4-phenylcoumarins from Mesua
elegans hexanic extract. Comparison study between multiple CPC dual mode and isocratic mode were done for 50, 150, 300
and 500 mg injection.

MethodologySample (Figure 1) : Fraction F1 from hexane crude of Mesua elegans after silica gel column
chromatography (230-400 mesh; Hex:EtOAc 9.5:0.5)
Instrument : Armen SCPC-250 coupled with Armen Spot Prep II
Solvent : Hept/EtOAc/MeCN/H2O with 0.1% TFA (9:1:9:1)
Flow Rate : 10mL/min
Rotor Speed : 1600 rpm
Mode of elution : Isocratic or Multiple dual mode

Figure 1: HPLC analysis at 280nm of sample

Results and discussion
Two 4-phenylcoumarins compounds were isolated by using CPC (Figures 2 and 3) and the structures of these twocompounds were identified by 1H NMR, 13C NMR and LC-MS ; mammea A/BA 1 [(M+H)+ = 407.1855] and mesuagenin C 2[(M+H)+ = 461.2777].

Figure A: HPLC analysis at 280nm of fraction 4

Figure 2: CPC chromatogram of 300mg injection, isocratic mode

Figure B: HPLC analysis at 280nm of fraction 6

Figure C: HPLC analysis at 280nm of fraction 7

Figure 3: CPC chromatogram of 300mg injection, dual mode

Figure D: HPLC analysis at 280nm of fraction 10

Until 150mg injected, differences of HPLC purityfor both compounds between isocratic and multidual modes are not significant. But when
injected mass are higher, in isocratic mode, thepurity decrease below 90% whereas in multidual, compounds are up to 93% even when
500mg are injected (see Table 1, Figures A toD).

Mass
Injected
50 mg
150 mg
300 mg
500 mgIsocratic Mode: DSC
MammeaMesuagenin
A/BA1
C2
98.5%
98.1%
96.2%
94.6%
84.2%
91.1%
82.3%
79.4%

Multi-Dual Mode
MammeaMesuagenin
A/BA1
C2
99.66%
100%
97.6%
95.5%
93.0%
96.6%
93.2%
94.3%

Table 1: HPLC purity comparison at 293nm of compounds isolated in300mg injected by isocratic and multi-dual mode

Conclusion
AGP Software allows an automatic variation of ascending to descending mode (and vice versa) for multi-dual CPC
separation. By using this elution mode, two compounds had been successfully isolated with high purity.
In this study, optimization of multi-dual mode was done to increase quantity injected for a good ratio recovery/purity.
[1] Whitmore, T.C.(Ed.), (1973), Tree Flora of Malaya – Volume Two, p.162-236.
[2] Verotta, L., Lovaglio, E., Vidari, G., (2004), Phytochemistry, 65, 2867-2879.
Notes : This application note has been produced and edited using information that was available when the data was acquired for each article. This application note is subject to revision without
prior notice

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