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Efficient cannabidiol extraction with centrifugal partition chromatography

7th June 2019


Fig.1. The chemical structure of cannabidiol
Fig.2. the CPC 250 with PLC 2250 Purification System
Fig.3. How CPC works

Gregoire Audo details efficient cannabidiol extraction with centrifugal partition chromatography

Cannabidiol (CBD), the main non-psychotropic phytocannabinoid (pCBs) present in the Cannabis sativa L. (Cannabaceae) plant, is presented as a promising therapeutic tool in a wide range of non-psychiatric (impact on inflammation, oxidative stress) and psychiatric disorders such as anxiety, depression and psychosis.[1] It lacks the psychotomimetic and other psychotropic effects that the main plant compound Δ9-tetrahydrocannabinol (THC) antagonises. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still in progress.[2] Once the bioactivity of such compounds has been shown in a large variety of scientific studies, the availability of reliable isolation of CBD (Fig. 1) qualitatively and quantitatively, has proven to be crucial to perform.

All pCBs are uniquely found in cannabis, with the total number of identified pCBs currently reported as over 100 (together with over 500 non-cannabinoid constituents), which makes the isolation a tedious task. Classical chromatographic methods such as high-performance liquid chromatography (HPLC) and flash chromatography cannot always give the efficiency and rate needed to meet the demand for CBD research, and they are too expensive to do on a large scale. Because of the structural similarities of cannabis compounds and adsorption behaviour on solid stationary phases, the large-scale production of pure CBD remains difficult to achieve by conventional preparative separation techniques. The conventional techniques can require expensive consumables and frequent replacements, and can take days.

At the same time, centrifugal partition chromatography (CPC) attracts attention as an alternative for purification processes, especially for high added value active pharmaceutical compounds such as cannabis, by his high loading capacity, total recovery of the loaded sample, low solvent consumption and easy scale-up.

CPC (Fig. 2) operates via the same chromatographic principles as HPLC, but the two techniques use different chemistry and mechanics to perform separation. Unlike HPLC, CPC doesn’t use a cylindrical column to contain the stationary phase. Instead, the CPC column consists of CPC discs arranged on a rotor. The discs are a series of partition cells connected by narrow ducts. Each cell contains a liquid stationary phase that is held in place by centrifugal force, as the rotor (column) spins. Each cell contains a liquid stationary phase that is held in place by centrifugal force, as the rotor (column) spins. The mobile phase is pumped through the stationary phase and mobilises the compounds that will be eluted according to their partition behaviour (Fig. 3).

Another big advantage of the CPC is the huge range of solvents that can be used: water, alkanes, alcohol, or other commonly used liquids. The ratio of each solvent depends on the partition coefficient of the target compound into the biphasic solvent system. For CPC, the ideal partition coefficient is between 0.5 to 3. Within this range, the target compound will be efficiently elute into the CPC system. The choice to have an appropriate solvent system assures the separation of the target compounds, contamination-free from other compounds.

CPC has advantages over HPLC due to the elimination of silica. Having a liquid stationary phase can have a number of benefits. There is no non-specific adsorption to a solid support, and there is a much higher sample loading capacity as the volume normally taken up by the solid support is occupied by the liquid stationary phase. All of this increases the capacity for higher throughputs, less solvent usage and huge tolerance of extract compounds that can be achieved. Because of this, researchers can use three to five times less solvent with CPC than with traditional methods.[3] Additionally, once filtered, the solvent can be recycled for further lowering cost.

Preparative isolation of up to four different major cannabinoids could be achieved by using CPC as the single technique. The quality of the isolated cannabinoids (>95% pure by HPLC) is sufficient for many purposes, such as biological testing on a large scale. Additional GC-FID and TLC data supports the purity of the isolated compounds.

Gilson scientists injected 5g of crude cannabis oil and manage to extract 205mg with 99% (HPLC) of CBD.[4] A typical separation using classical LC would have required at least a two-step process over multiple days.[5,6] The huge advantage of using only liquids without any silica support leads to CPC technique recovery of more than 90% of the total weight of these compounds in the original sample, which is greater than the average recovery rate for HPLC. Finally, CPC is not limited to CBD extraction. The technique is also used in the purification of many others compounds from natural sources, such as [6]-gingerol from ginger (Zingiber officinale Roscoe[5] or liquiritin from Chinese licorice (Glycyrrhiza uralensis Fisch.).[7]

CPC is not only an effective method for extracting CBD from cannabis, but it can also be a great solution to purify other phytocannabinoid components.

As the CBD market grows, and demand for CBD-based products increases, researchers will need a more efficient way to purify CBD from cannabis for research. CPC uses less expensive reagents and produces a highly pure product more efficiently than HPLC and flash chromatography, which makes it ideal for the extraction step in the production of CBD or other cannabinoids.  

References

[1] Cannabidiol (CBD) and its analogs: a review of their effects on inflammation, S. Burstein, Bioorg. Med. Chem; Vol.23, 2015

[2] Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders, Campos et al.; Philos. Trans. R. Soc. B Biol. Sci.,
Vol. 367, 2012

[3] Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes; Oier Aizpuruia-Olaizola et al.; J Nat Prod. Vol. 79, No. 2, 2016

[4] CPC 250 PRO Purification of Cannabidiol from Cannabis sativa; Gilson Applications Laboratory.

[5] Caulerpenyne from Caulerpa taxifolia: A comparative study between CPC and classical chromatographic techniques; Estell Sfecci et al.; Phytochem Lett.
Vol. 20, 2017

[6] Effective Cannabinoid Purification by Flash Chromatography; John R. Bickler and Elizabeth Denton; Biotage. Presented at ACS 2016, Philadelphia, PA, 2016.

[ 7] Orthogonal Analysis Underscores the Relevance of Primary and Secondary Metabolites in Licorice; Simmler et al. J Nat Prod. Vol.77, 2014

Gregoire Audo works in the application laboratory department at Gilson Purification





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