Introduction

The Cannabis sativa L. (Cannabaceae) plant contains more than 500 potentially active compounds. Of particular interest are the terpenoid and cannabinoid compounds, which include cannabidiol (CBD) and tetrahydrocannabinol (THC), due to their wide-ranging therapeutic benefits. Specifically, CBD has been found to be effective for treating conditions, including pain, inflammation, and muscle spasticity in multiple sclerosis.1,2 The US Food and Drug Administration (FDA) recently approved Epidiolex®, the first cannabis-derived drug for treating children with rare forms of epilepsy. In addition to CBD, there is growing evidence to suggest that several other cannabis-derived compounds may offer potential medical benefits, and research in this area continues to expand at a rapid pace.

As the number of therapeutic applications of these compounds grows, a method that can be used to isolate cannabis compounds that are free of impurities consistently is essential for both researchers and manufacturers. Ideally, this method would also minimize the impact on the environment. Here, we describe Centrifugal Partition Chromatography (CPC), an efficient and easily scalable approach for the extraction of highly pure cannabis compounds. Also, CPC is cost-effective and dramatically reduces solvent use resulting in a more environmentally-friendly, green method.

Drawbacks of standard chromatography methods for cannabis compound purification

Traditional chromatography techniques such as high-performance liquid chromatography (HPLC) and flash chromatography are commonly used to isolate compounds from natural product extracts. However, these methods are time-consuming and cost-prohibitive for producing pure cannabis-derived compounds on a large scale. Both methods require the use of solid support silica resin, an expensive consumable that requires frequent replacement due to irreversible absorption of a variety of compounds during the separation process. In addition to the significant cost associated with these techniques, the negative impact on the environment can be substantial due to the excessive solvent and silica requirements for this procedure. Isolation of cannabis compounds by these methods also requires laborious pretreatment steps, making it a time-consuming process. Using a greener chromatography method, such as CPC, will enable laboratories to reduce their impact on the environment.

How CPC works

CPC is a liquid chromatography method that relies on two immiscible liquid phases. One serves as the mobile phase or the eluent, and the other as the stationary phase. The mixture of liquids such as water, ethanol, or heptane can be used as the stationary phase in place of the silica-based column in flash chromatography and HPLC. The stationary phase is retained in the column by a centrifugal field generated by the rotation of the CPC column. Once injected, the mobile phase flow elutes the target compounds in order of their partition coefficients, defined by their relative affinity for each of the CPC liquid phases (Figure 1). The reusable CPC column consists of several stacked stainless-steel disks, each engraved with numerous twin cells, linked by ducts. This configuration provides better retention of the stationary phase, allows for higher elution flow rates, and improves separation times.

Figure 1. Principle behind CPC

Efficient purification using CPC

CPC is an efficient method that requires short run times (e.g., 30 minutes) to purify compounds from a complex mixture with up to 98% analytical purity (Figure 2). Higher elution flow rates can be used enabling faster run times, thereby increasing productivity.3 The method can be easily scaled up for processing milligrams to kilograms of a product efficiently, unlike preparative HPLC and flash chromatography, which may require substantial stationary phase changes when moving to large-scale purification.

Because CPC columns rely on a liquid stationary phase, they do not need to be replaced like silica-based columns and cartridges used in standard chromatography methods. The CPC column can be loaded with different solvents to create the column required for a particular target compound, and purification parameters can be adjusted according to the desired purity level. Because there is no irreversible absorption of compounds as with silica-based columns, the CPC column can be reused after rinsing, resulting in significant cost savings.

In one small-scale study, 5 grams of crude hemp extract was injected into a Gilson CPC 250 PRO and PLC 2250 Purification System (Figure 2). Using this one-step method, 205 milligrams of up to 98% pure CBD was obtained, as determined by HPLC analysis, demonstrating the efficiency of CPC for cannabis compound purification.

Figure 2. CPC purification of CBD and THC from cannabis oil using a Gilson CPC 250 PRO and PLC 2250 Purification System

A green chromatography method

As demand for pure cannabis compounds increases, so too will solvent usage as manufacturers scale up purification to meet the demand.4 Traditional liquid chromatography techniques require high volumes of solvents such as methanol and acetonitrile, which are hazardous to humans and toxic to the environment. As more laboratories make the shift to environmentally sustainable methods, scientists are looking for ways to reduce their use of these solvents. However, efforts to replace them with less toxic solvents may result in reduced yields with lower purity, which in turn will require additional steps and more solvent use to achieve the desired results.

CPC uses some of the same solvents as traditional chromatography methods, but after the purification, distillation can be implemented to recover the solvents original quality, making them available for reuse in future runs. These advantages make CPC a more environmentally friendly, greener method.

Summary

CPC is an efficient technique for purifying valuable natural compounds. It offers numerous advantages to researchers and manufacturers to help them meet the demands of the rapidly growing cannabis market. It is an efficient method that can be used to produce large quantities of highly pure cannabis-derived compounds quickly and cost-effectively. Also, CPC uses considerably less solvent, making it a more environmentally sustainable, green method.

References

1. Russo, E.B. Cannabidiol claims and misconceptions.
doi: 10.1016/j.tips.2016.12.004., 2017

2. Zhornitsky, S., and Potvin, S. Cannabidiol in humans: the quest for therapeutic targets.
doi: 10.3390/ph5050529., 2012

3. Mandova T. and al. Assessment of two centrifugal partition chromatography devices. Application to the purification of Centaurium erythraea methanolic extract
doi:10.1016/j.phytol.2017.04.033, 2017.

4. http://industrialgreenchem.com

VERITY® Compact CPC System

The VERITY® Compact CPC System combines a PLC Purification System with a centrifugal partition chromatography (CPC) system for high-level target compound purification. This automated, cost-effective, liquid-liquid purification technique utilizes reusable, silica-free chromatographic columns to streamline your workflow and allow you to quickly isolate specific molecules with high yields and purity.

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