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Extraction and separation of pinosylvins by CPC

Pinosylvins are stilbenoid toxins available in the heartwood and knots of Pinaceae, protecting them from microbiological attacks [1]. In case of oxidative stress, the pinosylvin is converted to methyl-ether.

1. picture: structure of pinosylvin and it’s monomethyl-ether

Extraction of pinosylvins can be easily realized from chopped wood and knots, a less expensive byproduct of wood production. Extraction with hexane yield mostly lipophilic compounds (low yield for PS), and alcohols can extract more hidrophilic compounds (low yield for PSMME). To ensure maximum yield acetone was used, since it extracts fast, end recycling is easy.

2. picture: CPC-UV chromatogram on a 250 ml Armen SCPC-250 instrument
Rotation speed: 2100 rpm (330 g)
Flow speed: 10 ml/min
3. picture: CPC-UV chromatogram on the RotaChrom Prototype CPC
Rotation speed: 630 rpm (240 g)
Flow speed: 200 ml/min

Although separation of PS and PSMME can be achieved by various methods, due to occuring of other stilbenoids and isomers a chromatographic separation is needed to provide pure PS and PSMME. The recently used flash chromatographic technics eliminate use of chlorinated solvents, however the cost and environmental load of silica gel and used solvent is still high for upscale.[2].

Centrifugal Partition Chromatography Chromatography (CPC) is a special chromatographic technique where both stationary and mobile phase are liquid, and the stationary phase is immobilized by a strong centrifugal force. The main advantage is the cost-effectiveness, since it does not need any expensive and bulky solid stationary phase, and both quantity and quality requirements for solvents are less decreased contrast to standard liquid chromatographic techniques.

4. picture: HPLC-UV chromatogram of the extract
5. picture: HPLC-UV chromatogram of the purified pinosylvin
6. picture: HPLC-UV chromatogram of the purified pinosylcinmonomethylether

We screened various combination of non-chlorinated solvents for a scalable and economic separation of pinosylvins by CPC. It was evident that a medium-polarity system was needed. Alkane – acetone – water systems gave good solubility and good selectivity not only for PS and PSMME, but also for the isomers. Ternary systems are easy to recycle and set with a density measurement. Quaternary mixtures from Arizona system provided no real advantage in selectivity, so they were neglected. Isopropanol instead of acetone gives similar selectivity, however due to higher viscosity and boiling point it is more difficult to work with.

Table of some solvent systems, partition coefficients and selectivity:

Sytem Ratio KPS KPSMME α
n-Pentane : Acetone : Water 1:1,5:1 0,25 0,70 2,8
n-Hexane : Acetone : Water 1:2,0:1 0,15 0,73 4,9
n-Heptane : Acetone : Water 1:2,0:1 0,08 0,79 9,8

For small-scale experiments, an Armen SCPC 250 ml column was used with an Armen SpotPrep chromatographic system. We injected 1 gram of concentrated extract in 10 ml of lower-phase. For scale-up, the RotaChrom Prototype CPC was used with 2l of column volume. 12 gram of extract was injected in 100 ml of lowerphase. Detection were carried out on 320 nm. In both cases, purities over 99% were easily achieved with >80% recovery for PS and >95% for PSMME proving that CPC is both high-efficiency, economic and scalable solution for a preparative separation.

The n-hexane : acetone : water system with upper phase density of 700 kg/m³ and lower phase density of 895 kg/m³ is clearly a best compromise for high selectivity, easy recycling, good yield and the use of ICH class-II solvent.

 

References

[1] Lee SK, Lee HJ, Min HY, Park EJ, Lee KM, Ahn YH, Cho YJ, Pyee JH. Antibacterial and antifungal activity of pinosylvin, a constituent of pine. Fitoterapia 2005; 76, 258-260
[2] Poljansek I, Oven P, Vek C, Willför S, Raitanen JE. Extraction and isolation of pinosylvins from pine wood residue. 21th International Symposium on Separation Science 2015, Ljubljana, P-11

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