Purification of crownethers containing molecules by Centrifugal Partition Chromatography (CPC)
Isabelle Lachaise1, Sandrine Peyrat2, Valessa Barbier2
1 Plateforme Chromatographie Analytique et semi preparative, 2 Equipe Système Polymères Complexes

Institut de Chimie et des Matériaux(ICMPE), 2 rue Henri Dunant , 94320 Thiais, France

Introduction

At ICMPE (Thiais, France), researchers have developed new crownethers containing macromolecules
displaying membrane permeabilization properties. These molecules are currently of interest in cancer research. Theyare synthesised from the polymerisation of crownether containing monomers (see Figure 1). Monomers A and B areroutinely purified by extraction in hot pentane. 10 to 15 extraction cycles are required to obtain a suitable yield (4560%). In this application note we illustrate a new purification method for these monomers using Centrifuge Partition
Chromatography (CPC).
Samples
Synthesised samples of crownether containing monomers A and B have been chosen for purification by CPC(Figure 1). Crownether molecules are synthesized according a standard procedure as described in RSC Advances, 2012,
2, 8606–8609. Previous analysis of samples A and B by HPLC are shown in figure 1. For both samples, HPLC
chromatograms show the major compounds, (53 % for monomer A and 44% for monomer B), and 4 additionalmolecules.

Figure 1 : Analytical HPLC chromatograms of samples; 0.8ml/min, Injection volume : 10µl, 205 nm(left) Sample A : MeOH/H2O (70/30), Purosphere RP18 column (250mmx4.6mmx5µm); (right) Sample B : MeOH/H2O (85/15) betabasic-C18column (150mmx4.6mmx3µm). Insert : monomer chemical structure.

CPC experiments

CPC is a separation process that involves two immiscible solvent phases. One remains the stationary phase
while the other is the mobile phase. The aim is to find a suitable solvent system which provides both a good separation
and a high yield.
Several Arizona biphasic systems have been tested for the purification of both samples. Sample A has beensuccessfully purified using the L Arizona system Hept/EtOAc/MeOH/H2O (2/3/2/3) with a partition coefficient equal
to 1.13 in ascending mode.
CPC runs have been performed with 1.58 to 3.6 g of sample A. Subsequent HPLC analysis shows high purity fractions
were collected with a recovery of over 90%, (see table 2, and Figure 3).

Elution

Extrusion

Injectedmass

Run
time

Solvent
consumption

1,58 g2,01 g
3,6 g

45 mn

750 mlPurifiedproductmass
1,17 g
1,49 g2,78 gPurity92,36%
93,46%
94,62%

Table 2 : CPC of sample A ; CPC column volume:250 ml, 8ml/mn, 1600 rpm: Hept/EtOAc/MeOH/H2O (2 /3 /2 3), isocratic mode, UV
detection : 205, 245 nm

Due to its higher polarity, sample B could not be purified using an Arizona system Hept/EtOAc/MeOH/H2O as nosatisfying partition coefficient could be obtained between upper and lower phase. Nevertheless, purification wassuccessfully performed using a ternary polar system, BuOH/MeOH/H2O (45.8%/4.16%/50%). The partition coefficientwas 1.39 in descending isocratic mode and 1.07g of pure B (94,6% purity by HPLC) has been recovered (1.84 ginjected) (Figure 3).

Figure 3: HPLC analysis after purification by CPC (left) : sample A and (right): sample B (for conditions see figure1 sample B)

Conclusion

We have developed a new methodology by CPC for purification of crownethers containing molecules. CPC allows for thequick purification of several grams of sample with a good yield and a high purity of collected fractions. Further purifications
involving larger, and thus more polar, crownether macrocyles are currently under investigation in our laboratory.