Supercritical fluid extraction (SFE) represents an environmentally friendly alterna­tive to organic solvent extraction. Even though it was first introduced in 1879 by Hannay and Hogarth, the extraction method did not gain much scientific attention until around 1960 [18, 39]. In addition to avoiding the use of toxic solvent, SFE has many apparent advantages over organic solvent extraction. It produces extracts of higher purity, requires less processing steps and can be operated at moderate tem­peratures to minimize extract degradation [39, 42] . Supercritical fl uid generally has a high solvent power for non-polar components and a low affinity towards analytes with high molecular weights. The supercritical state is achieved when a substance is exposed to conditions exceeding its critical temperature (Tc) and pressure (Pc). In this state, the substance has a liquid-like density with a gas-like viscosity [6].

CO2 is the most commonly used fluid for SFE as it is cheap, non-flammable, inert, and readily available. Figure 5 shows the phase diagram for CO2 with its supercritical region. The critical temperature and pressure of CO2 are 304.1 K and 7.38 MPa respectively. Supercritical carbon dioxide (SCCO2) is often used in the extraction of thermolabile compounds as its low Tc enables complete extraction to occur without the application of excessive heating which may degrade analytes [29, 35]. SCCO2 is a highly effective extractant due to its high diffusivity and its easily manipulated solvent strength. The solvent power of SCCO2 towards a polar analyte can be improved by adding a polar modifier. The addition of methanol or water to SCCO2 allowed for successful extraction of polar compounds [35], while ethanol addition was found to increase the yield of lipids from Arthrospira maxima [36]. SCCO2 extraction has been applied in many fields including food industry, environ­mental science, and pharmaceuticals.

SFE can be classified as either an analytical or a preparative system. In the ana­lytical system, SFE apparatus is directly combined with a chromatographic device.

TEMPERATURE

Fig. 5 P-T phase diagram for carbon dioxide [35]

Even though this system enables rapid analytes examination, it cannot be used as a production system as any extracted analytes are immediately consumed during the chromatographic analysis. On the other hand, the preparative system has been used to produce pilot-scale quantities of various analytes from microorganisms, including chlorophyll from microalgae. Figure 6 shows a pilot-scale preparative SFE system. It consists of a solvent (in this case CO2) pump, a modifier pump, an extraction cell, valves, and two fractionation cells [17]. Detailed description of the SFE working

mechanism can be found elsewhere [17] . The microalgal biomass is placed in the extraction cell, while the supercritical fluid is depressurized in the fractionation cells equipped with temperature and pressure controllers. Upon depressurization, the supercritical fluid evaporates to the ambient as a gas, forcing analytes to precipitate in the fractionation cells. The dual-fractionation arrangement allows different ana­lytes to be precipitated in each cell based on their differential solubilities in the evaporating supercritical fluid.

The use SCCO. process to extract chlorophyll a from microalgal species has been reported [ 30 [ . Optimum extraction conditions were found to be 60°C and 400 bar for N. gaditana and 60°C and 500 bar for Synechococcus sp. Chlorophyll a yields for the two microalgae at these optimum conditions were, respectively, 2.24 and 0.72 mg chlorophyll/mg dry weight microalgae. Even though these yields were not comparable to those of traditional methanol extraction (18.5 mg chlorophyll/mg dry weight microalgae for N. gaditana and 4.10 mg chlorophyll/mg dry weight microalgae for Synechococcus sp.), SCCO2 extraction was found to be more selec­tive and the extracted chlorophyll a appeared to contain less impurities. An exten­sive evaluation comparing the two extraction systems for isolating microalgal chlorophyll is, unfortunately, not yet possible due to limited knowledge on the supercritical process. The commercial feasibility of using SCCO2 process to extract chlorophyll from microalgae is also a subject of further research.