Fatty acid methyl esters (FAMEs) were analyzed as previously described by Ackman [33 ]. In brief, FAME separation was carried out on a gas chromatography system equipped with a flame ionization detector, by using a fused silica capillary column. Both the detector and injector temperatures were fixed at 250 °C. Initially, the temperature of the fused silica capillary column was set at 120 °C for 3 min and increased later at a rate of 2 °C.min−1 to 180 °C, to be finally fixed at 220 °C for 25 min. Fatty acids were identified by comparison of the retention times with two complex qualitative standard mixtures PUFA No.1 and PUFA No.2 (Supelco, Sigma-Aldrich, Bellefonte, PA, USA). Measurements were carried out in triplicate.
Pufa no 1
Manufactured by Merck Group
Sourced in United States
PUFA No.1 is a laboratory equipment designed for the analysis of polyunsaturated fatty acids (PUFAs). It is a specialized instrument used to separate and quantify different PUFA compounds in various sample matrices.
Automatically generated - may contain errors
Lab products found in correlation
Pufa no 2, by Merck Group (4 mentions)
Pufa no 3, by Merck Group (4 mentions)
Bame mix, by Merck Group (2 mentions)
Supelco 37 component fame mix, by Merck Group (2 mentions)
Glc 110 mixture, by Matreya (2 mentions)
Finnigan trace gc ultra, by Thermo Fisher Scientific (1 mentions)
Fatty acid methyl ester mixes, by Merck Group (1 mentions)
Omegawax 250, by Merck Group (1 mentions)
Non adecanoic acid c19 0, by Matreya (1 mentions)
Sp 2560, by Merck Group (1 mentions)
Gc 2010 plus, by Shimadzu (1 mentions)
6 protocols using pufa no 1
1
GC-FID Analysis of Fatty Acid Methyl Esters
2
Fatty Acid Profiling in Human Milk
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A Perkin Elmer, Clarus 500 GC-FID gas chromatography (GC) equipped with Flame Ionization Detector (FID) and Thermal Conductivity Detector (TCD) was used for the analysis of fatty acids. Using a 30 m × 0.25 mm × 0.25 µm fused carbon-silica column with a temperature range from 40 °C to 260 °C (Stabilwax, Crossbond, Carbowax, Polyethylene glycol), the individual fatty acid methyl esters (FAMEs) were separated. The temperatures were set at 200 °C for the GC oven, 300 °C for FID, 150 °C for TCD, and 250 °C for the injector with a split ratio of 1:2. The flow rate for the carrier gas (nitrogen) was 0.76 mL/min and for the other gases was 450 mL/min for air and 45 mL/min for hydrogen. The injection volume of the sample was 5 µL and the sampling rate was 12.5 Hz. Eighty minutes was set as the total run time for each sample. PUFA No.1 (Marine source) and PUFA No.2 (Animal source) supplied by SUPELCO (USA) were used as an authentic FAMEs standard for identification according to the retention time. FAs to be detected were like the PUFAs used, which ranged from C14 to C24. Values of FAs were expressed as mg/100 mL of human milk and g/100 g of FAs (%).
3
Gas Chromatography Analysis of Argan Oil Triacylglycerols
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All reaction products (FFA, partial acylglycerols and TAG) were separated by silica gel thin-layer chromatography (TLC). The TAG band was recovered, methylated and analysed by gas chromatography (GC), as fatty acid methyl esters (FAME), according to the methodology described by [36 (link)]. A gas chromatograph Finnigan TRACE GC Ultra (Thermo Electron Corporation) with a capillary column (60 m × 0.25 mm ID × 0.25 µm film) Thermo TR-FAME, and an AS 3000 autosampler from Thermo Electron Corporation were used for FAME analysis. The flame ionization detector (FID) was set at 280 °C and supplied with air and hydrogen at 350 and 35 mL min−1, respectively, The injector (in splitless mode) was set at 250 °C. The temperature program of the chromatographic column was the following: 100 °C for 1 min, increase to 160 °C at 10 °C min−1, held for 10 min, increase to 235 °C at 4 °C min−1 and kept for 10 min. The carrier gas Helium was supplied at a flow rate of 1.2 mL min−1. Fatty acid methyl ester mixes (PUFA No1 and PUFA No 3, from Supelco) were used as external standard and methyl myristate (C14:0) as internal standard.
The incorporation degree (ID) of C8:0 or C10:0 in TAG of argan oil, was calculated as follows, Equation (1) [20 (link)]:
where MFA represents the C8:0 or C10:0 moles in the TAG and MT is the total amount of fatty acids (moles) in the TAG.
The incorporation degree (ID) of C8:0 or C10:0 in TAG of argan oil, was calculated as follows, Equation (1) [20 (link)]:
where MFA represents the C8:0 or C10:0 moles in the TAG and MT is the total amount of fatty acids (moles) in the TAG.
4
Fatty Acid Extraction and Quantification
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The methodologies for FA preparation and analysis were performed according to Silva et al. [19 (link)], with minor modifications. Briefly, for the initial saponification step, 150 µL of 2 M KOH (diluted in 67% ethanol; v/v) was added to 150 µL of homogenate. Samples were then kept at 80 °C for 1 h, cooled to room temperature, diluted 1:1 with water, acidified (HCl), and FAs isolated with hexane.
To the FA fractions isolated from each sample after saponification, 1.5 mL of acetyl chloride:methanol (1:20 v/v) solution was added for the derivatization step, and samples were kept at 80 °C for 1 h. After adding, 1 mL of Mili-Q water and 1 mL of hexane for phase separation, the organic layer was recovered to clean GC vials and solvent was evaporated in a vacuum concentrator (Speedvac™) for 10 min. Samples were then resuspended in 50 µL of hexane, and methylated nonadecanoic acid (50 µL; 10 mg mL−1) was added as an internal standard to each sample, prior to gas chromatography analysis. Fatty acid methyl ester mixes (PUFA No1 from marine source and PUFA No 3 from Menhaden oil) were used as external standards (Supelco, Bellefonte, PA, USA). Operating conditions were as described by Silva et al. [19 (link)]. Theoretical correction factor (FCT) for FID detectors was applied in FA quantification, according to Guo [36 ].
To the FA fractions isolated from each sample after saponification, 1.5 mL of acetyl chloride:methanol (1:20 v/v) solution was added for the derivatization step, and samples were kept at 80 °C for 1 h. After adding, 1 mL of Mili-Q water and 1 mL of hexane for phase separation, the organic layer was recovered to clean GC vials and solvent was evaporated in a vacuum concentrator (Speedvac™) for 10 min. Samples were then resuspended in 50 µL of hexane, and methylated nonadecanoic acid (50 µL; 10 mg mL−1) was added as an internal standard to each sample, prior to gas chromatography analysis. Fatty acid methyl ester mixes (PUFA No1 from marine source and PUFA No 3 from Menhaden oil) were used as external standards (Supelco, Bellefonte, PA, USA). Operating conditions were as described by Silva et al. [19 (link)]. Theoretical correction factor (FCT) for FID detectors was applied in FA quantification, according to Guo [36 ].
5
Quantification and Identification of Lipids
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The total lipids were quantified following the method of Folch et al. (60 (link)), modified by using dichloromethane:methanol (2:1) instead of trichloromethane:methanol (2:1). The fatty acids were transesterified to fatty acid methyl esters by acid-catalyzed methylation (61 (link)). Non-adecanoic acid (C19:0, Matreya LLC, State College, PA, USA) was added as an internal standard. Fatty acid methyl esters were analyzed by gas chromatography, using a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu Europe GmbH, Duisburg, Germany) equipped with a capillary column (Omegawax 250, 30 m × 0.25 mm × 0.25 μm; Supelco, Bellefonte, PA, USA) and a flame-ionization detector. The carrier gas was helium at 1.30 ml min−1, with a split ratio of 1:100, and the injection volume was 1.0 μl. The initial column temperature of 150°C was held for 7 min, increased at 3°C min−1 to 170°C and held for 25 min, and then increased at 3°C min−1 to 220°C and held for 30 min. The injector and detector temperatures were 250 and 260°C, respectively. Fatty acids were identified by comparing retention times with those of commercially available standards (Supelco 37 Component FAME Mix, BAME Mix, PUFA No.1, PUFA No.2, PUFA No.3, Sigma-Aldrich Co. LLC; GLC-110 Mixture, Matreya LLC) and quantified by using the internal standard (C19:0). Analyses were run in duplicate.
6
Fatty Acid Analysis in Red Blood Cells
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RBCs were separated from plasma by centrifugation at 3,000 rpm for 15 min at 4°C and fatty acids transesterified as previously described by Harris, Pottala (46 (link)). The resulting fatty acid methyl esters were analyzed by gas chromatography using a Shimadzu GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) equipped with a capillary column (SP-2560, 100 m × 0.25 mm × 0.20 μm; Supelco, Bellefonte, PA, United States of America) and a flame-ionization detector. The oven’s initial temperature of 50°C was held for 1 min, then increased at a rate of 50°C/min to 150°C and held for 20 min, then increased at 1°C/min to 190°C and held for 3 min, and further increased at 1°C/min to 220°C and held for 20 min. Injector and detector temperatures were set at 250 and 280°C, respectively. Hydrogen was the carrier gas at a linear velocity of 17.2 cm/s. Fatty acids were identified by comparing retention times to standards (Supelco 37 Component FAME Mix, BAME Mix, PUFA No.1, PUFA No.2, PUFA No.3, Sigma-Aldrich, St. Louis, MO, United States of America; GLC-110 Mixture, Matreya, Pleasant Gap, PA, United States of America). The omega-3 index was calculated as the sum of EPA and DHA expressed as a proportion of total identified fatty acids.
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