44 February / March 2020
allowing the use of much higher mobile phase flow rates producing rapid preparative separations [17]. Given these attributes SFC chromatography is ideally suited to isolation and purification of cannabis extracts. In addition, super critical CO2
extraction
(SFE) of cannabis is routinely performed to produce a cannabis oil [18,19].
SCOPE
The preparative separation of cannabis mixtures to isolate specific components can be challenging. Traditional preparative liquid chromatography can be used to separate and isolate specific cannabis components. However, preparative liquid chromatography has several draw backs including the limits on flow rates and ultimately production throughput due to the relatively high viscosity of the mobile phase used. In addition, considerable amounts of ethanol and water are required for the liquid chromatographic separation of cannabis. In order to isolate the components, the ethanol/water mixture has to be removed or reduced in volume. This removal process is time consuming. The mixtures of CO2
low viscosity which can be used at very high flow rates to encourage higher production levels. In addition, CO2
is rapidly released
during component isolation and ethanol amounts are low and quickly removed.
One of the key factors for a successful SFC preparative separation and isolation of cannabinoids is stationary phase selection. There are a several of attributes that are necessary for the optimal stationary phase including:
1. The stationary phase should be designed to deliver the desired separation at the lowest level of organic modifier possible (in the case of Cannabis ethanol would be the organic modifier).
2. The stationary phase should be robust and easily scalable for preparative applications.
3. The stationary phase should not be expensive to manufacture.
Preliminary Investigations
Preliminary investigations for the SFC separations of cannabinoids employed modified polysaccharide phases coated phases for the SFC separations of natural products (NP) since they can be useful for the separation of structurally similar compounds. The GreenSep NP-I has been specifically optimised for the separation of 10 different cannabinoids. The chromatogram shown in Figure 1 is an example of the peak shape, performance and separation capacity obtainable with the GreenSep NP-I column with SFC for a high-resolution
Figure 4: Cannabinoid mixture chromatographed on GreenSep NP-III using 5% Ethanol modifier.
separation of a mixture of cannabinoids. Unfortunately, these polysaccharide phases whether coated or immobilised are expensive to manufacture, making these types of columns a major contributor to isolation costs.
Isolation of THCA and CBDA
Preparative SFC separations of cannabinoids have been performed using a column with 2-Ethyl pyridine bonded to silica as a stationary phase with ethanol used as co-solvent since it is less toxic compared to methanol or other organic solvents. This is of vital importance if the resulting isolate is for human consumption as no toxic solvent residues are present. A
chromatogram showing the separation of mixture of cannabinoids is shown in Figure 2. CBDA and THCA are both well separated from the other cannabinoids, however, to elute these two components in less than 10 minutes 20% ethanol co-solvent is required.
GreenSep NP- III permits both CBDA and THCA to elute in less 10 minutes with only 10% ethanol (chromatogram shown in Figure 3), half as much when compared to 2-ethyl pyridine.
GreenSep NP-III can be used at higher total flow rates requiring only 5% ethanol to elute both THCA and CBDA in less than 13 minutes, while still maintaining good chromatographic resolution (chromatogram shown in Figure 4).
Figure 2: Cannabinoid mixture chromatographed on GreenSep Ethyl Pyridine.
/ethanol mobile phase are very Figure 3: Cannabinoid mixture chromatographed on GreenSep NP-III using 10% Ethanol modifier.
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