Table 3.
Methods based on saponification for n-alkane determination in vegetable oils.
| Sample Amount—Internal Standard (I.S.) | Saponification and LLE for Unsaponifiable Extraction | Column Chromatography and/or Further Purification | Analysis (Injection Mode)—Column Type—Chromatographic Conditions | Ref. |
|---|---|---|---|---|
| 20 g oil sample + I.S. (n-C20) in 1 mL hexane | According to the Official European Community method—Annex XVII, for the determination of stigmastadienes in vegetable oils, 1995 *. | A glass column (1.5 cm i.d. × 50 cm) was filled with 15 g silica gel, slurried in hexane. After sample loading, the n-alkane fraction was eluted with hexane (50 mL) at 1 mL/min and concentrated to 1 mL. | GC-FID/ GC-MS Wide bore capillary column of borosilicate glass (30 m × 0.75 mm i.d.), coated with SPB-1, 1 µm film thickness. Oven temperature: 110 °C (6 min) to 300 °C at 5 °C/min. Injector: 300 °C; Detector: 320 °C; carrier gas: nitrogen at 14 mL/min. | [25] |
| 20 g oil sample + I.S. (n-C20) in 1 mL hexane | According to the Official European Community method—Annex XVII, for the determination of stigmastadienes in vegetable oils, 1995 *. | A glass column (2.0 cm i.d. × 50 cm) was filled with 15 g silica gel slurried in hexane. After sample loading, the n-alkane fraction was eluted with hexane (50 mL) and concentrated. | GC-FID (split/splitless injection; split ratio 1:20). Fused silica capillary column (30 m × 0.32 mm i.d.), coated with SPB 5; 1.0 µm film thickness. Oven temperature: 120 °C (4 min) to 280 °C (5 min) at 4 °C/min, then to 305 °C (10 min) at 4 °C/min; Injector and detector: 315 °C; carrier gas: helium (2 mL/min). | [27] |
| 20 g oil sample + I.S. (n-C11) | According to the Official European Community method—Annex XVII, for the determination of stigmastadienes in vegetable oils, 1995 *. | A glass column (1.5 cm i.d. × 50 cm) was filled with a 2 mm layer of anhydrous sodium sulphate and 15 g of silica gel and conditioned with hexane. After sample loading, the n-alkane fraction was eluted with n-hexane (120 mL) and concentrated to 1 mL. | GC-FID (on-column injection). Fused silica capillary column (25 m × 0.32 mm i.d.), coated with SE54, 0.25 µm film thickness. Oven temperature: 60 °C (1 min) to 290 °C (40 min) at 5 °C/min. Detector: 310 °C. | [30] |
| 20 g oil sample + I.S. (n-C20) in 1 mL hexane | According to the Official European Community method—Annex XVII, for the determination of stigmastadienes in vegetable oils, 1995 *. The solution was then passed through anhydrous sodium sulphate (50 g), washed with 20 mL of hexane and evaporated to dryness. | A glass column was filled with silica gel. After sample loading, the n-alkane fraction was eluted with hexane (30 mL). | GC-FID (split injection). Fused silica capillary column (30 m × 0.32 mm i.d.), coated with SE 54, 0.5 µm film thickness. Oven temperature: 100 °C (1 min) to 300 °C (10 min) at 4 °C/min. Injector: 280 °C; detector: 310 °C; carrier gas: helium (10 psi). | [1] |
| 20 g oil sample + I.S. (n-C20) in 1 mL hexane | Saponification: 30 min slight boiling with 75 mL of 10% ethanolic KOH. LLE: the saponified solution was transferred to a 500 mL separatory funnel, added to 100 mL of distilled water and extracted twice with 100 mL hexane. The combined extracts were concentrated to 1 mL. | A glass column (1.5 cm i.d. × 50 cm) was filled with 15 g silica gel, slurried in hexane. After loading the unsaponifiable fraction (together with washes), the n-alkane fraction was eluted with hexane (60 mL) at a rate of about 1 mL/min. | GC-FID/ GC-MS. Wide bore capillary column of borosilicate glass (30 m × 0.75 mm i.d.), coated with SPB-1, 1 µm film thickness. Oven temperature: 110 °C (60 min) to 300 °C at 5 °C/min. Injector: 300 °C; detector: 320 °C; carrier gas: nitrogen (14 mL/min). | [26] |
| 20 g of oil sample + I.S. (n-C20) in 1 mL hexane | Saponification 30 min slight boiling with 75 mL of 10% ethanolic KOH. LLE: The saponified solution was transferred to a 500 mL decanting funnel, 100 mL distilled water was added, and the mixture was extracted twice with 100 mL portions of hexane. The hexane solution was dried over anhydrous sodium sulfate, evaporated to dryness and dissolved in 1 mL of hexane | A glass column (1.5 cm i.d. × 40 cm) was filled with 15 g of silica gel. After sample loading, the n-alkane fraction was eluted with hexane (100 mL) and concentrated to about 0.5 mL. | GC-MS (on column injection). Fused silica capillary column (30 m × 0.25 mm i.d.), coated with DB-5, 0.25 µm film thickness. Oven temperature: 60 °C (1 min) to 120 °C at 3 °C/min, and finally to 300 °C at 7 °C/min. Detector: 300 °C; carrier gas: helium (1.3 mL/min). | [2] |
| 5 g of oil sample + I.S. (n-C16) | Saponification: ethanolic KOH 12% (w/v) at 60 °C for 1.5 h. After cooling, 50 mL of water was added and the unsaponifiable matter was extracted with 4 × 50 mL of petroleum ether. The combined extracts were washed with 50 mL of ethanol: water (1:1), dried over anhydrous sodium sulfate and evaporated to dryness. | The dry residue was dissolved in chloroform and deposited on a silica gel TLC plate. After developing the plate with hexane/diethyl ether (6:4, v/v), it was sprayed with 2,7-dichlorofluorescein and the band corresponding to hydrocarbons was scraped, extracted 3 times with chloroform/diethyl ether (1:1, v/v), filtered and dried in a rotary. evaporator. | GC-FID (split injection; 60:1 split ratio). Fused silica capillary column (30 m × 0.25 mm i.d.), coated with DB-5MS, 0.25 µm film thickness. Oven temperature: 150 °C to 300 °C (10 min) at 4 °C/min. Injector and detector: 250 °C.; carrier gas: helium (mL/min). The transfer line: 250 °C. Electron impact mass spectra were measured at an acceleration energy of 70 eV. | [23] |
| 5 g of oil sample + I.S. (n-C15) | Saponification: 19 mL of a 10% ethanolic KOH under reflux for 20 min. After cooling, 25 mL distilled water was added and the unsaponifiable fraction was extracted with 2 × 25 mL of n-hexane. The combined extracts were washed with 3 × 12.5 mL of ethanol:water mixture (1:1), dried on anhydrous sodium sulfate and evaporated to dryness. | A glass column (1.5 cm i.d. × 40 cm) was filled with 15 g of silica gel. After sample loading, the n-alkane fraction was eluted with n-hexane (40 mL) and concentrated to 0.5 mL. | GC-MS (splitless injection)—Fused silica capillary column (30 m × 0.25 mm i.d.), coated with TR-5MS, 0.25 µm film thickness. Oven temperature: 60 °C (3 min) to 300 °C (10 min) at 5 °C/min. Injector and transfer line at 300 °C. Ion source temperature: 225 °C; Carrier gas: helium (1 mL/min). | [3] |
* According to the official method of the European Community (13)—Annex XVII, designed for the determination of stigmastadienes in vegetable oils (ECC, 1995): Saponification: 30 min slight boiling with 75 mL of 10% etanolic KOH. LLE: the saponified solution was transferred to a 500 mL separatory funnel, added to 100 mL of distilled water and extracted twice with 100 mL hexane; the combined extracts were washed with a mixture of EtOH:H2O (1:1) until reaching neutral pH. The unsaponifiable fraction was dried over anhydrous sodium sulfate, evaporated to dryness and dissolved in 1 mL of hexane. LLE, liquid–liquid extraction.