Journal of Chemistry

Review Article

Supercritical Algal Extracts: A Source of Biologically Active Compounds from Nature

Table 2

Comparison of SFE with conventional extraction methods, on the example of algae processing; the results of independent experiments are shown separately and marked with (a)–(d).

(a)


Extraction	Biomass pretreatment	Sample and solvent	Conditions	Extraction yield	Reference

Solvent extraction with -hexane at static and dynamic (Soxhlet) mode	Microalgal powder: Oven-drying at 85°C for 16 hours and grinding in a ring mill Microalgal paste (solid conc. 30%, by mass): centrifuging wet algae in benchtop centrifuge	Static mode: (i) Microalgal powder: 4 g (ii) Microalgal paste: 13.3 g (iii) Solvents for performance on powder: pure -hexane and mixture of -hexane and isopropanol (3 : 2), separately, both 300 mL (iv) Solvent for performance on paste: pure -hexane, 300 mL Dynamic mode (Soxhlet extraction): (i) Microalgal powder: 4 g (ii) Solvent: pure -hexane, 300 mL	Static mode: Ambient conditions, agitation: 800 rpm, : 7.5 h Dynamic mode: Rate of refluxes: 10 per hour, : 7.5 h Posttreatment: Removal of solid residues from extract by separation on a filtration paper	Results for microalgal powder: Lipid yield [g lipid extract/g d.m.]: (i) Static mode: 0.015 and 0.048, with and without cosolvent, respectively (ii) Dynamic mode: 0.057 Results for wet microalgal paste: Lipid yield after 80 min [g lipid extract/g d.m.]: Static mode: 0.010	[31]
SFE with CO₂		(i) Microalgal powder: 20 g (ii) Microalgal paste: 8 g Both powder and paste were formerly mixed with inert diatomaceous earth (d.e.) at ratios 2 : 1 w/w and 1 : 2 w/w, respectively Solvent f.r.: 400 mL/min	: 60 or 80°C, : 100–300 or 300–500 bar (lower- and higher-pressure experiments, resp.), : 80–120 min	Results for microalgal powder: Lipid yield after 80 min [g lipid extract/g d.m.]: (i) 60°C, 300–500 bar: 0.058 (ii) 80°C, 300–500 bar: 0.048 Effect of pressure: increase of the lipid yield almost twice in case of higher-pressure extractions, compared to lower-pressure experiments FAME content variations during 80 min of the process [g FAME/g d.m.]: (i) 60°C, 300–400 bar: 0.23–0.29 (ii) 80°C, 300–400 bar: 0.31–0.44 Effect of pressure: not significant Results for wet microalgal paste Lipid yield after 120 min [g lipid extract/g d.m.]: 60°C: 0.071	[31]

(b)


Extraction	Biomass pretreatment	Sample and solvent	Conditions	Extraction yield	Reference

Soxhlet extraction with mixture of -hexane and dichloromethane	() Drying by lyophilization (samples number 1 and ) or dehydration (sample number 3) () Pulverizing () Homogenization	(i) Samples: 1 g of each of 3 different matrixes collected from two locations in North-West Spain during two consecutive spring seasons) Each sample was mixed with 15 g of sea sand (ii) Solvent: -hexane : dichlo-romethane (50 : 50), 250 mL	: 7 h Extract posttreatment: Removal of solvent on a rotatory evaporator at 40°C, oven-drying obtained extract at 75°C for 90 min and then cooling to room temperature (20°C) Providing fraction of hydrocarbons: Redissolving the extract in -hexane (5 mL) and next passing through Sep-Pak silica solid-phase extraction (SPE) column, previously activated with -hexane (4 mL); eluating hydrocarbons from SPE cartridge with 10 mL of -hexane and then evaporating the eluent under a stream of air, redissolving the residue in -hexane (1 mL) and subjecting it to analysis (GC-FID)	Results for sample 1 (): Recoveries [%], RSD [%]: Pristane, sample at 5 mg/L: 74.1, 5.81; Pristane, sample at 40 mg/L: 90.5, 6.59; C₁₈, sample at 5 mg/L: 87.9, 5.36; C₁₈, sample at 40 mg/L: 97.8, 6.88; C₁₉, sample at 5 mg/L: 86.0, 1.52; C₁₉, sample at 40 mg/L: 96.7, 3.20; C₂₀, sample at 5 mg/L: 69.1, 4.90; C₂₀, sample at 40 mg/L: 96.5, 3.31; C₂₂, sample at 5 mg/L: 69.6, 4.98; C₂₂, sample at 40 mg/L: 99.4, 4.69; C₂₄, sample at 5 mg/L: 78.4, 4.42; C₂₄, sample at 40 mg/L: 99.8, 3.39; C₂₈, sample at 5 mg/L: 90.4, 7.58; C₂₈, sample at 40 mg/L: 101, 1.65; C₃₂, sample at 5 mg/L: 98.8, 1.05; C₃₂, sample at 40 mg/L: 99.7, 1.74; C₃₆, sample at 5 mg/L: 97.9, 0.58; C₃₆, sample at 40 mg/L: 97.4, 3.10 Aliphatic hydrocarbon yield [μg/g d.m.]: C₁₈: 4.32 ± 0.08, C₂₀: 5.35 ± 0.34, C₂₂: 3.99 ± 0.20, C₂₄: 2.65 ± 0.06, C₂₈: 1.06 ± 0.06, total: 14.9 ± 0.5 Results for sample 2 (): Aliphatic hydrocarbon yield [μg/g d.m.]: C₁₈: 5.14 ± 0.19, C₂₀: 5.88 ± 0.27, C₂₂: 5.03 ± 0.14, C₂₄: 2.50 ± 0.11, C₂₈: 0.64 ± 0.00, and total: 17.9 ± 0.3, Results for sample 3 (): Aliphatic hydrocarbon yield [μg/g d.m.]: C₁₈: 4.25 ± 0.25, C₂₀: 5.10 ± 0.30, C₂₂: 5.32 ± 0.44, C₂₄: 2.88 ± 0.25, C₂₈: 1.03 ± 0.15, and total: 17.0 ± 1.0,	[32]
SFE with CO₂ (+ methanol as modifier)		(i) Samples: 0.5 g of each of 3 matrixes mentioned above; each sample was preadsorbed onto 10% deactivated alumina (3 g) by admixing to obtain homogenous mixture, which was then complemented with alumina (1 g) (ii) Solvent: SC-CO₂ with methanol (200 mL)	Initial static equilibration period: : 100°C, : 229 bar, : 10 min Extraction: SC-CO₂ density: 0.55 g/mL, f.r.: 1 mL/min (dynamic or continuous flow mode), : 50 min, of nozzle: 45°C; of analyte-collecting trap: 40°C Providing fraction of hydrocarbons: eluating aliphatic hydrocarbons from analytes contained in the trap were with 5 portions of -hexane (1.5 mL per each fraction); concentrating obtained extract to 1 mL under a stream of air and then purifying (SPE column) and analyzing as it was described in case of Soxhlet extraction	Results for sample 1 (): Recoveries [%], RSD [%]: Pristane, sample at 5 mg/L: 36.8, 7.53; Pristane, sample at 40 mg/L: 52.3, 8.11; C₁₈, sample at 5 mg/L: 66.4, 9.28; C₁₈, sample at 40 mg/L: 76.0, 3.49; C₁₉, sample at 5 mg/L: 77.6, 4.69; C₁₉, sample at 40 mg/L: 89.1, 4.71; C₂₀, sample at 5 mg/L: 84.6, 4.01; C₂₀, sample at 40 mg/L: 94.3, 4.42; C₂₂, sample at 5 mg/L: 87.9, 3.04; C₂₂, sample at 40 mg/L: 98.4, 5.18; C₂₄, sample at 5 mg/L: 90.8, 2.46; C₂₄, sample at 40 mg/L: 100, 3.85; C₂₈, sample at 5 mg/L: 88.6, 5.75; C₂₈, sample at 40 mg/L: 100, 3.35; C₃₂, sample at 5 mg/L: 87.8, 8.40; C₃₂, sample at 40 mg/L: 98.6, 3.86; C₃₆, sample at 5 mg/L: 84.8, 5.76; C₃₆, sample at 40 mg/L: 98.1, 4.09 Aliphatic hydrocarbon yield [μg/g d.m.]: C₁₈: 6.28 ± 0.66, C₂₀: 6.66 ± 0.79, C₂₂: 4.21 ± 0.44, C₂₄: 2.56 ± 0.28, C₂₈: 0.71 ± 0.07, and total: 19.1 ± 2.0 Results for sample 2 (): Aliphatic hydrocarbon yield [μg/g d.m.]: C₁₈: 4.54 ± 0.23, C₂₀: 4.25 ± 0.10, C₂₂: 2.99 ± 0.04, C₂₄: 2.30 ± 0.18, C₂₈: 0.59 ± 0.05, and total: 13.6 ± 0.6, Results for sample 3 (): Aliphatic hydrocarbon yield [μg/g d.m.]: C₁₈: 7.90 ± 1.07, C₂₀: 6.73 ± 0.63, C₂₂: 4.49 ± 1.26, C₂₄: 2.99 ± 3.55, C₂₈: 1.15 ± 5.17, and total: 21.7 ± 0.2	[32]

(c)


Extraction	Biomass pretreatment	Sample and solvent	Conditions	Extraction yield	Reference

Solvent extraction with ethanol	Freeze-drying	(i) Sample: 1 g (ii) Solvent: 20 mL	: 30 min (soaking matrix in ethanol) Posttreatment: Separation of extract from solid residues by centrifuging (4000 ×g, 20°C, 10 min, extract = supernatant)	Results of AXA extraction: Yield ± δ [mg AXA/g d.m]: 1.16 ± 0.18 Recovery [% of yield obtained with BBM method]: 48 Results of chlorophyll extraction: Yield ± SD [mg chlorophyll/g d.m]: 16.1 ± 1.91 Recovery [% of yield obtained with BBM method]: 56	[33]
SFE with CO₂ (+ ethanol as modifier)	Freeze-drying	(i) Sample: 1 g (ii) Solvent: (a) Pure solvent: SC-CO₂ (b) Cosolvent: EtOH, 0.1, 0.2, 0.5, 1.0, 2.0, or 20 mL	: 60°C, : 200 bar, : 60 min Providing extract after SFE: soaking obtained biomass in 20 mL of ethanol for 30 min and then separation of supernatant by centrifugation (4000 ×g, 20°C, 10 min); in case of samples treated with 0.1–2.0 mL of cosolvent, proper volume of ethanol had to be added to complement it to 20 mL Extract posttreatment: Removal of chlorophyll to reduce the saturation of green color by treating extract with several acids, such as H₂SO₄, HCl, H₃PO₄, and CH₃COOH, at the concentration range of 0.002–0.1 N	Results of AXA extraction: Yield ± δ [mg AXA/g d.m]; recovery [% of yield obtained with BBM method]: Pure SC-CO₂: 2.02 ± 0.20, 83; SC-CO₂ + 0.1 mL of EtOH: 2.13 ± 0.36; 87; SC-CO₂ + 0.2 mL of EtOH: 2.33 ± 0.62, 96; SC-CO₂ + 0.5 mL of EtOH: 2.40 ± 0.37, 98; SC-CO₂ + 1.0 mL of EtOH: 2.32 ± 0.43, 95; SC-CO₂ + 2.0 mL of EtOH: 2.41 ± 0.49, 99; SC-CO₂ + 20 mL of EtOH: 2.46 ± 0.23, 101 Results for samples treated with pure SC-CO₂ and SC-CO₂ + cosolvent at the highest concentration showed statistically significant difference ( by Student’s -test) with AXA yield of both: conventional method and the rest of supercritical extracts Results of chlorophyll extraction: Yield ± SD [mg chlorophyll/g d.m], recovery [% of yield obtained with BBM method]: Pure SC-CO₂: 29.4 ± 1.70, 103; SC-CO₂ + 0.1 mL of EtOH: 28.8 ± 0.91, 104; SC-CO₂ + 0.2 mL of EtOH: 30.8 ± 2.31, 108; SC-CO₂ + 0.5 mL of EtOH: 28.7 ± 1.24, 104; SC-CO₂ + 1.0 mL of EtOH: 28.4 ± 0.91, 103; SC-CO₂ + 2.0 mL of EtOH: 29.1 ± 0.36, 102; SC-CO₂ + 20 mL of EtOH: 29.5 ± 1.04; 103 Results for all samples showed statistically significant differences (s.s.d., ) with chlorophyll yield obtained using conventional method and no s.s.d. between each other	[33]

(d)


Extraction	Biomass pretreatment	Sample and solvent	Conditions	Extraction yield	Reference

Solvent extraction with aqueous ethanol	() Grinding beforehand using a laboratory mill in equal short time intervals in order to avoid overheating () Passing the material through laboratory sieves (diameter 3 mm), periodically, and collecting the fine fraction after each sieving	(i) Sample: 30 g (ii) Solvent: aqueous ethanol at the concentration of 70% Solvent: sample ratio (w/w): 1 : 1	: 10 days Providing extract (fraction) of polysaccharides: Drying EtOH extract in air and then treating it twice with 0.1 M HCl, at ratio (w/w) 1 : 20, at 60°C for 120 min; neutralizing newly obtained extract and centrifuging to separate supernatant, from which WSPS fractions were isolated (concentrating supernatant in a rotary evaporator dialyzing against distilled H₂O lyophilization) HCl extract posttreatment: Separation of fucoidans from the polysaccharide fractions by anion-exchange chromatography (elution by a linear gradient of H₂O and 2 M NaCl solution), which resulted in providing one, two, or three fucoidan subfractions of different degree of sulfation (marked as , according to an increasing order of sulfated group content) fractions	Results for F. evanescens extracts: Yield of fucoidans [%, by d.m.]; content of total sugar [%, by mass]: 5.11; 48.0 Content of SO₃Na and polyphenols [%, by mass]: 34.6 and 0.5 Results for S. japonica extracts: Yield of fucoidans [%, by d.m.]; content of total sugar [%, by mass]: : 0.38; 46.4; : 1.28; 45.2 Content of SO₃Na and polyphenols [%, by mass]: : 14.0 and 0; : 26.3 and 0.1 Results for S. oligocystum extracts: Yield of fucoidans [%, by d.m.]; content of total sugar [%, by mass]: : 0.34; 46.0; : 0.65; 48.1; : 0.55; 44.0 Content of SO₃Na and polyphenols [%, by mass]: : 17.4 and 0.4; : 24.0 and 1.1; : 32.0 and 0.1	[34]
SFE with CO₂ (+ ethanol as modifier)		(i) Sample: 30 g (ii) Solvent: Treating F. evanescens: pure SC-CO₂ Treating S. japonica and S. oligocystum: both pure SC-CO₂ and SC-CO₂ + EtOH (5%) Tested fluid : sample ratios (w/w): from 10 : 1 to >30 : 1; fluid : sample ratio (w/w) chosen for experiments: 30 : 1	= 60°C, : 550 bar, : 60 min Providing fraction of polysaccharides and posttreatment of HCl extract were as described in case of solvent extraction	Results for F. evanescens extracts: Yield of fucoidans [%, by d.m.]; content of total sugar [%, by mass]: Pure SC-CO₂: 3.02; 49.2 Content of SO₃Na and polyphenols [%, by mass]: Pure SC-CO₂: 39.2 and 1.5 Results for S. japonica extracts: Yield of fucoidans [%, by d.m.]; content of total sugar [%, by mass]: Pure SC-CO₂, : 0.35; 48.5; pure SC-CO₂, : 1.26; 44.2; SC-CO₂ + EtOH (5%), : 1.35; 45.1 Content of SO₃Na and polyphenols [%, by mass]: Pure SC-CO₂, : 11.8 and 0.1; pure SC-CO₂, : 27.0 and 0.1; SC-CO₂ + EtOH (5%), : 27.3 and 0.1 Results for S. oligocystum extracts: Yield of fucoidans [%, by d.m.]; content of total sugar [%, by mass]: Pure SC-CO₂, : 0.38; 47.4; pure SC-CO₂, : 0.57; 45.2; pure SC-CO₂, : 0.27; 42.0; SC-CO₂ + EtOH (5%), : 0.55; 49.0; SC-CO₂ + EtOH (5%), : 0.32; 34.9 Content of SO₃Na and polyphenols [%, by mass]: pure SC-CO₂, : 16.4 and 2.1; pure SC-CO₂, : 23.4 and 1.7; pure SC-CO₂, : 34.0 and 0.1; SC-CO₂ + EtOH (5%), : 28.8 and 1.5; SC-CO₂ + EtOH (5%), : 32.0 and 0.5 Effect of different fluid : sample ratios: 10 : 1 ratio enabled to provide about 95%, by mass, of all extractable substances, while increasing ratio over 30 : 1 did not result in significant enhancement of SFE efficacy	[34]