Methods for Obtaining and Determination of Squalene from Natural Sources
Table 6
Summary of the analytic techniques used in quantification of squalene in the presence of acylglycerols, FASEs, free phytosterols, and tocopherols, from natural sources.
Technique
Conditions/observation
Reference
Densitometric estimation
Solvent: cyclohexane (Rf value SQ = 0.60 ± 0.02); SQ spots detected with iodine vapours; determination on a HPTLC unit at = 200 nm
Capillary column CP-Sil 8 CB (15 m, 0.1 mm, i.d. 0.25 mm, Chrompack, Middleburg, The Netherlands), oven temperature program = 60–140°C at 30°C/min and 340°C at 15°C (15 min hold); carrier gas = Helium at 41.3 kPa; FID temperature = 360°C
TLC + GC-FID (Shimadzu 17A, Japan); separation on a DB-5HT (5%-phenyl)-methylpolysiloxane nonpolar column (30 m, i.d. 0.32, Agilent Tech. Palo Alto, US); temperature program: injector and detector temperatures set at 370°C, column regime: 80–365°C at 15°C/min (8 min hold); split ratio = 1 : 50, using N2 as carrier gas, linear velocity = 30.6 cm/s at 80°C temperature = 200°C, range of / 30–600 at 1250 scan/s
3800-GC (Varian, USA) with FID, DB 225 column (30 m, i.d. 25 mm), carrier gas N2, linear velocity = 34.8 cm/s, split ratio: 75, temperature programme: 180°C (1 min hold) to 220°C, with 3°C/min (2 min hold); detector temperature set at 300°C, injector temperature 290°C
Visible/near-infrared scanning spectrophotometer (NIR Systems 6500, Perstorp Analytical Inc.); spectra recorded between 400 and 2,500 nm, at 2 nm intervals as (/R), R = reflected energy
ibid
GC
Hewlett-Packard 3500 GC with FID, CP-TAP column (25 m × 25 mm i.d.,Varian, USA), split ratio = 1 : 50, carrier gas He, 1 mL/min; temperature programme: oven initial temperature 80°C (3 min hold), rising to 150°C at 10°C/min, to 250°C at 5°C/min, to 340°C at 10°C/min (20 min hold)
439 Packard model GLC with FID connected to a Chrompac CR-3A integrator; 2 m × 2 mm i.d., glass column packed with 10% SE30 on Chromosorb W; temperature program: 200°C (3 min hold), raised to 270°C at 5°C, detector and injector temperatures set at 320°C
Hewlett-Packard model 1050 LC (Wilmington, DE) with manual injection valve (model 7125, Rheodyne, Cotati, CA) having a 20 µL loop, coupled with a Perkin-Elmer model 8500 GC (Norwalk, CT) with PTV injector (Perkin-Elmer) and FID. LC conditions: mobile phase = methanol/water, 50 × 4.6 mm i.d. column slurry packed with 10 µm silica, C4, Vydac 214 TPB, UV detection at 205 nm, LC column maintained at 45°C; mobile phase regime: methanol/water = 70 : 30 (hold 3 min) linear decrease to 22% water in 3 min, maintained 4 min, linear decrease in 2 min to 14% water, kept 3.5 min, to 0% in 3.5 min. GC conditions: 5% diphenyl/95% dimethyl polysiloxane fused silica column (30 m × 0.25 mm i.d., Sugelabor, Madrid, Spain), carrier gas = He; temperature programme: 130°C–230°C at 20°C/min, maintain 2 min, raise at 290°C at 3°C/min, maintain 30 min, detector temperature set at 320°C
Rapid method for quantitative determination of 10–150 µg squalene: (i) solution containing squalene is reduced to dryness under nitrogen, add H2SO4, maintain in water bath at 70°C, for 5 min → pale yellow colour develops, adding formaldehyde to intensify and stabilize the colour; (ii) optical density determination at nm
Elemental analyser coupled to isotope-ratio mass spectrometer for detection of SQ origin (vegetal/animal); Thermo Scientific Flash 1112 EA for IRMS coupled to a Thermo Scientific Delta V Series IRMS via a Thermo Scientific ConFlo IV interface; duration for squalene/squalane analysis is 400 s
