There are also some reports of CBD extraction using polar and non-polar mixed solvents for increased oil yield.However,mixed solvents sometimes result in more impurities and more complicated post processes that are needed.By principle,super-critical fluid is any substance at a temperature and pressure above its critical points but below the pressure required to compress it into solids.Under supercritical conditions,there is no distinction between liquid and gas phases,and the SCF exhibits both liquid and gas properties.Another characteristic of SCF is that a small change in pressure or temperature could result in large changes in the physical properties,making SCF an excellent ‘fine-tuned’ solvent.Super-critical fluid extraction is another commonly used extraction technology for high-value bioactive compounds,and the commonly used fluids are carbon-dioxide and water.Compared to ethanol and hydrocarbon extraction,SC-CO2 has the advantage of non-flammable,non-toxic,high selectivity,low solvent loss,easy solvent separation and recovery,and low solvent cost.Through adjusting the SFE conditions,full-spectrum CBD distillate with high terpene retention can be obtained.The upfront cost of SFE extraction equipment is much higher than the conventional solvent extractions,but the overall long-term operating cost is actually lower.SFE extracted CBD oil still needs winterization process to remove the lipids and wax,and the technology is not easy to be scaled up.Addition of 5% ethanol as co-solvent to SC-CO2 was proven to enhance the extraction of CBD.In recent years,several novel solvent extraction technologies have been studied.
Microwave-assisted extraction is an emerging green extraction technology and has shown some advantages over the conventional technologies,such as increased yield,indoor plant table reduced extraction time and solvent needs and lower energy consumption.Such superior characteristics should be attributed to the disruption effect of electromagnetic waves on the integrity and structure of cell wall,which enhances the mass transfer during the extraction.Ultrasound-assisted extraction is another efficient extraction technology that has been used for extracting bio-active compounds from plant materials.Agarwal et al.showed that UAE of CBD from hemp was more efficient than conventional extractions without ultrasound.Currently,studies on CBD extraction using different methods have been extensively studied but reports on the extraction of terpenes are scarce in both scientific research and industrial productions.This is partly because decarboxylation of CBD distillate at high temperatures is necessary in the industrial production,during which the terpenes are mostly vaporized and lost.Therefore,there is need to improve the extraction efficiency,yield and selectivity of CBD and collect the terpenes at the same time,and develop extraction equipment with higher through puts and lower costs.Attentions to food safety must be taken for the selection of solvents and extraction methods if the extracted CBD and terpenes are to be added in foods and beverages.Molecular distillation,also known as short-path distillation,is usually the first purification step for crude oil.As illustrated by Fig.7A,the molecular distillation involves an evaporation and a condensation process,and generally consists of a feeding system,a motor system and wiper blades,a couple of internal and external condenser sets for collection of different substances.The evaporation condensation chamber is vacuumed in favor for the evaporation of desired substances.The feed oil is spread into a thin layer onto a heated wall by wiper blades,on which the light molecules evaporate and escape the evaporator in direction to the condenser,and then condensate on the surface of the internal condenser.The distance between the evaporator and the condenser is comparable to the molecular mean free path of the vaporized light substance.Therefore,the light molecules can condensate and is collected,while the heavy substances flow into a separate collector.
After molecular distillation,the cannabinoids and heavier colorants,lipids and wax are separated,and terpenes can be collected separately due to their high volatility.However,the CBD and THC are usually collected in a mixture due to their similarities in the chemical composition and physical properties.Since THC is a psychoactive and controlled drug substance,it is strictly prohibited in any foods,and thus needs to be separated from the CBD.There are three typical chromatographical technologies in the hemp industry: high-pressure liquid chromatography ,centrifugal partition chromatography ,and flash chromatography.HPLC is the most commonly used chromatographic purification technologies; and flash chromatography is a rapid separation and purification technology that is commonly operated under lower pressures.CPC,on the other hand,a series-connected network of extraction cells,in which both the stationary phase and fluid phase are liquids.The stationary phase is immobilized by centrifugal forces,while the fluid phase containing CBD moves along the cascade and is purified.Fig.7B shows a schematic illustration of chromatographical separation of CBD and THC in the column and an example of commercial-scale HPLC system.As the driven by the high pressure,the mobile phase containing mixed cannabinoids flow through a column containing packing materials coated with specialized materials.Different eluents have different rates of adsorption and desorption on the coating materials,and thus move along the column at different rates.According to their retention times,different effluents can be collected separately.After purification,the oil turns into clear viscous liquid with golden color.To obtain a ‘CBD isolate’,which is typically a fine white powder ,a crystallization process is required by further refining the purified oil.
In the crystallization process,the purified oil is firstly dissolved in solvents in a large vessel,which is heated and mechanically stirred; then the temperature is gradually decreased and the agitation rate is slowed down to initiate the nucleation; once the nucleation starts,the agitation rate is increased sharply to break the thermodynamic balance and separate the crystals from the solution; finally the filtrated crystals are rinsed with pentane again to remove the remaining impurities.Commercial CBD isolates usually have at least 99.5% purity.Future research should focus on developing more cost-effective purification equipment and methods for hemp CBD and terpenes.Furthermore,research in isolation of different terpene species has not been well reported.Owing to the functionalities and health benefits,hemp CBD and terpenes are promising future functional food ingredients.Currently,the strict regulations and lack of consumer basis have significantly affected the hemp market and development of CBD and terpene products.Additionally,hemp research is still in its early stage and many technological issues remained to be resolved.Nonetheless,a bright future is expected.Research and success of using hemp CBD and terpenes as functional food ingredients need more efforts in the following aspects: Extensive scientific research needs to be conducted to provide evidence of the health benefits and safety of hemp CBD and terpenes for consumption as foods,which will help the policy makers to develop and standardize the national and global regulations for the development of hemp CBD and terpene foods.Research in improving the solubility and bio-compatibility with other major food components,and enhancing the stability,shelf life and bio-accessibility of the hemp CBD and terpene is needed and important in successful addition of them in various food product systems.More efforts are also critical in the education of consumers on the health benefits and safety of hemp CBD and terpenes to develop and expand the potential consumer basis.Efficient hemp drying technologies with high processing efficiency and throughput,but minimal loss of bio-active compounds is necessary to ensure the quality and safety of hemp plants after harvest,which can positively influence the downstream processing.
Efficient,safe,and cost-effective extraction and purification technologies need be developed and optimized to improve the yield and selectivity of CBD and terpenes and ensure the purity and safety of the extract for food applications.Although these challenges and research needs in the development of high value products from hemp remain,it is expected that hemp CBD and terpenes will become functional food ingredients to improve human health and get closer to the daily life of human beings in the future.The most employed method of extraction of cannabinoids from plant raw material is solid-liquid extraction using ethanol or acetone as extracting solvents due to their affinity and consequent high extracting efficiency for cannabinoids.High performance liquid chromatography and gas chromatography coupled to mass spectrometry are the analytical techniques of choice for the following qualitative and quantitative analysis.HPLC is usually employed when the acid and the neutral form of the investigated cannabinoids must be measured separately,while GC analyses enable the characterisation of the “total-cannabinoid content” as GC systems,by definition,work with high temperatures that lead unavoidably to the decarboxylation of the cannabinoid acids.The “total-cannabinoid content” is usually measured as it best represents the pharmacological activity of the material,unless differently stated by legislation.Thanks to their volatile nature,the isolation of terpenes,and in particular of mono and sesquiterpenes,hydroponic vertical farming from plant raw material is straightforward and their profiling can be performed by head space solid-phase microextraction online combined to GC-MS analysis.The recovery of cannabinoids from solid matrices by HS-SPME is also feasible but requires long sampling times in combination to high sampling temperatures due to their low volatility and low tendency to escape to the headspace.In 2004 Lachenmeier et al.optimised a successful HS-SPME method followed by on-coating derivatisation of the cannabinoids with N-methyl-N-trimethylsilyltrifluoroacetamide for the extraction of cannabinoids from hemp food products using 90°C as sampling temperature and 30 min of extraction time while in 2005 Ilias et al.showed that cannabinoids extraction should be performed at 150°C to maximise their recovery in short sampling times.However,in a very recent work,Czégény et al.investigated the effect of temperature on the composition of pyrolysis products of CBD in e-cigarettes.
They tested different operating temperatures and they proved that,depending on the temperature and atmosphere ,25–52% of CBD can be converted into other cannabinoids amongst which Δ9-THC,Δ8-THC,cannabinol and cannabichromene are the predominant pyrolysates.Even though when performing HS-SPME it is usually unlikely to reach such extreme temperatures,the results of Czégény et al.suggest that reduced sampling temperature should be preferred to obtain a truthful cannabinoid fingerprint profile in the plant raw material,CBD potential degradation should be investigated during the optimisation of the sampling temperature.As thoroughly described by Psillakis et al.,vacuum is a powerful experimental parameter to consider to increase the extraction kinetic of semi-volatile compounds during the HS-SPME process.This is because in the case of semivolatiles and under non-equilibrium conditions,a reduced pressure inside the sample container decreases the resistance to mass transfer in the gas zone at the solid-head space interface.As a consequence,higher extraction efficiencies for semi-volatile compounds can be achieved in shorter sampling time and potentially at milder extraction temperatures.This study investigates the advantages and disadvantages of using vacuum assisted HS-SPME over regular HS-SPME as sample preparation process to be exploited in analytical protocols aiming at comprehensively characterising both the terpene and cannabinoid profiles of Cannabis inflorescences in a single step employing a total analysis system.Due to their relative high molecular weights and boiling points cannabinoids have a low tendency to escape to head space and require high sampling temperatures to be recovered by HS-SPME.However,in view of a reliable and comprehensive HS-SPME method suitable for the simultaneous qualitative characterisation of both the terpenoid and the cannabinoid fractions of Cannabis inflorescences,high sampling temperatures are not advisable for two reasons.First,they may decrease the distribution coefficient between the fiber and the head space of the most volatile components reducing their recovery.In addition,high sampling temperature,especially when combined with relatively long extraction times,may induce decomposition of some compounds and/or creation of other components or artefacts.According to the theory,a reduced pressure inside the HS sample container increases the compounds’ molecular diffusion coefficient in air and favours their vapour flux at the solid surface.As a result,reducing the total head space pressure is an alternative and complementary strategy,compared to the adoption of high sampling temperatures,to speed up the extraction kinetic of semi-volatile compounds.In this study we compared the performances of Vac-HS-SPME to those of Reg-HS-SPME under different sampling temperatures and extraction times and we explored whether Vac-HS-SPME could be a more suitable sample preparation technique for the simultaneous characterisation of both the terpenoid and cannabinoid profiles of Cannabis inflorescences.For the following discussion CBD will be considered as representative of the plant cannabinoids,while -myrcene and trans–caryophyllene of the mono and sesquiterpene markers,respectively.Tables 1,2 and 3 report the average peak area and % RSD of all the investigated compounds when testing 10 mg of matrix under the different experimental conditions and the relative analyte abundance defined as the ratio between the average peak area obtained under vacuum to that measured at atmospheric pressure conditions at 150°C,90°C and 80°C,respectively.Fig.2 shows the extraction temperature profiles of -myrcene,trans–caryophyllene and CBD acquired when sampling 10 mg of the matrix for 5 min at the different investigated temperatures.Finally,Fig.3 provides the extraction times profiles for CBD for each sampling temperature.In all the cases,the results of both reduced and atmospheric pressure conditions are reported.