6.5. Supercritical Fluid

The supercritical fluid extraction (SFE) is a method which usually employs CO₂ as solvent. The use of supercritical fluids (SF) began in the mid-1980s for the extraction of components from plant matrix with great application in the pharmaceutical, cosmetic, and food industries. The SFE has been effective for the extraction of polar compounds such as polyphenols and nonpolar compounds such as lipids and carotenoids. A scheme of this equipment is presented in Figure 7.

Scheme of supercritical fluid equipment.

(1) Particle Size. In this process, the particle size is an important issue to consider. In general, the content of bioactive compounds in the final extract is inversely proportional to the particle size of the plant material. Commonly, it is preferred to work with small particle sizes and to sieve the solids [59, 115]. However, very small particle sizes might cause the agglomeration of the substrate in the compact channels of the equipment, which produces a poor solvent flow and a low recovery yield. On the other hand, the use of larger particles decreases the mass transfer because it hinders the solvent flow velocity. Therefore, in SFE operations, it is relevant to define the suitable mean particle diameter of the plant matrix [133].

(2) Flow Rate of CO₂ and the Role of Pressure and Temperature. In SFE, the flow rate of CO₂ is applied principally in function of the solute solubility. The flow rate must be sufficiently high to maximize the extraction rate. An optimum value of flow rate is located on the region where both solubility and mass transfer are significant factors [134]. Therefore, increasing flow rate of CO₂ will not have a strong effect in extraction, if diffusion from the inner cells of plant material is slow; temperature increase is more appropriated if diffusion from the inner cells needs to be faster [135]. An increase in temperature facilitates the diffusion of bioactive compounds to CO₂ and makes the solute vapor pressure to rise, whereas it reduces both the viscosity and surface tension of the water contained in the plant, allowing a greater penetration of the SF [136]. Temperature must be elevated carefully; its increase provokes the solvent density to decrease, diminishing the solubility of interest compounds [137, 138]. Thus, temperature is the main parameter that influences the selectivity and it is necessary to optimize it in order to increase yield [136, 137]. In recent experiments with beetroot leaves for the extraction of polyphenols, it was found that increasing the pressure increased the extraction kinetics. The researchers attributed this improvement in extraction thanks to the increase in fluid density [139]. However, temperature has a major effect in the extraction than flow rate and pressure.

(3) Interaction of CO₂ with Solvent and the Addition of Modifiers. For the extraction of phenolic compounds, CO₂ is not the best option, since its polarity is low compared to the polarity of phenolic compounds and their related compounds like flavonoids. This reduces the solubility of flavonoids in CO₂. Increasing pressure might enhance the solubility of flavonoids in CO₂ because of the augmentation of density [137]. Therefore, the extraction conditions for the supercritical CO₂ must be above its critical temperature and pressure (31°C and 74 bar, respectively). As mentioned before, temperature as the main parameter has more effect on solubility, and the increase of pressure does not always have a notable effect on it [140]. If temperature is not to be raised in order to preserve thermolabile compounds, the addition of modifiers to CO₂ improves the recovery of bioactive compound. The modifiers must fulfill the conditions to be considered as green solvents. Due to their low toxicity, the most used and recommended modifiers are water and ethanol (Table 4) although occasionally methanol and propanol are used. Ethanol addition to CO₂ has been found to improve the antioxidant properties, and this effect is attributed to the higher amount of phenolic compounds extracted [130, 138, 142]. Nevertheless, the employment of modifiers is not recommended if organic solvent-free extracts are required. In this case, it is preferable to manipulate the pressure and temperature conditions of the process to modify the density of CO₂ [144]. For this reason, CO₂ is the solvent par excellence in most of the SFE operations [145, 146].

Experimental conditions for supercritical fluid extraction of flavonoids.

Solv: solvent, Temp: temperature, TFC: total flavonoid content, EtOH: ethanol, and MeOH: methanol.

The SFE technique allows the extracts to be cleaner and easier to recover compared to other conventional and unconventional methods because it does not need to concentrate the extracts at the end of the process. It is noteworthy that the CO₂ used in some SFE processes is largely a by-product of industrial processes, for example, the beer production process, which reduces emissions to the environment [135, 145]. An additional advantage of SFE is the possibility of coupling with gas or liquid chromatographs at the end of the operation, which is normally used for the identification of highly volatile compounds. The SFE method is used for industrial scale, confirming the wide applicability for the process from milligrams in laboratory up to tons in the industry. However, flavonoid extraction by SFE has not been exploited and more research is necessary to understand how the parameters can be modulated in order to increase the yield. The main disadvantage of the method is that the initial cost of the equipment is very high and it requires more expertise to run SFE extraction procedures [135].