Extracted lipids were subjected to an acid-catalyzed transesterification process prior to analysis by gas chromatography with a flame ionization detector (GC-FID). Firstly, lipids were dissolved in 4 mL of n-hexane (99.0% Panreac AppliChem, Darmstadt, Germany). For the transesterification step, 1 mL of the diluted sample, 100 μL of heptadecanoic acid (internal standard, 98.0% Sigma Aldrich, Steinheim, Germany), and 1 mL of the mixture 10% MeOH:H2SO4 (10:1 (v/v)) were added to glass capped test tubes with a screw cap and heated at 100 °C for 2 h. The organic phase was collected and after residual water removal with Na2SO4, fatty acid methyl esters were analyzed by GC-FID (CP-3800 Varian, Agilent, Santa Clara, CA, USA) using a Teknokroma® TR WAX 30 m × 0.25 mm × 0.25 µm column. GC-FID operational conditions were as follows: carrier gas flow (He) 1 mL min−1; injector—250 °C; detector—280 °C; oven—40 °C with a ramp of 30 °C min−1 up to 150 °C followed by a ramp of 3 °C min−1 up to 250 °C. The volume of the injected sample was 1 µL and the split (1:10) injection technique was applied. For peak identification, a mixture containing saturated, monounsaturated, and polyunsaturated FAME ranging from C4:0 to C24:1 was used (SupelcoTM 37 Component FAME Mix, Sigma Aldrich, Steinheim, Germany). Results were expressed as percentages and as means of triplicates ± standard deviations.
Tr wax
Manufactured by Teknokroma
Sourced in Spain
TR-WAX is a stationary phase for liquid chromatography. It is designed for the separation and analysis of a wide range of compounds, including polar and non-polar substances. The core function of TR-WAX is to provide efficient and reliable chromatographic separations.
Automatically generated - may contain errors
Lab products found in correlation
Thermo trace gc ultra, by Thermo Fisher Scientific (2 mentions)
Heptadecanoic acid, by Merck Group (1 mentions)
N hexane, by ITW Reagents (1 mentions)
Supelco 37 component fame mix, by Merck Group (1 mentions)
Agilent 6850 gc, by Agilent Technologies (1 mentions)
Xs105, by Mettler Toledo (1 mentions)
5 protocols using tr wax
1
Transesterification and GC-FID Analysis of Lipids
2
Fatty acid profiling of yeast extracts
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Total lipids were extracted from the homogenized yeast samples with 8:4:3 (v/v/v) chloroform:methanol:saline solution (0.88% KCl) according to the Folch method (Folch et al., 1957) .
Fatty acid methyl esters (FAMEs) were prepared through acid-catalyzed transesterification and subsequently purified by thin-layer chromatography. FAMEs from the desaturase functional assays were analyzed using a Thermo Trace GC Ultra (Thermo Electron Corporation, Waltham, MA, USA) equipped with a fused silica 30 m × 0.25 mm open tubular column (Tracer, TR-WAX (film thickness 0.25 µm); Teknokroma, Spain), coupled to a flame ionization detector. Identification was carried out by comparing the retention times with those from commercial FAME standards. Further confirmation of peaks and analysis of FAMEs from the elongase assays was carried out using an Agilent 6850 GC equipped with a mass spectrometry detector (5975 Series) and a 30 m × 0.25 mm open tubular column (Tracer, DB5-MS (film thickness 0.25 µm); Teknokroma, Spain), and comparing the spectra against those from the NIST library. The conversion efficiency of all assayed P. littoralis enzymes toward the exogenously supplied PUFA substrates was calculated as: (all product areas/(all product areas + substrate area)) × 100 (Kabeya et al., 2021) (link).
Fatty acid methyl esters (FAMEs) were prepared through acid-catalyzed transesterification and subsequently purified by thin-layer chromatography. FAMEs from the desaturase functional assays were analyzed using a Thermo Trace GC Ultra (Thermo Electron Corporation, Waltham, MA, USA) equipped with a fused silica 30 m × 0.25 mm open tubular column (Tracer, TR-WAX (film thickness 0.25 µm); Teknokroma, Spain), coupled to a flame ionization detector. Identification was carried out by comparing the retention times with those from commercial FAME standards. Further confirmation of peaks and analysis of FAMEs from the elongase assays was carried out using an Agilent 6850 GC equipped with a mass spectrometry detector (5975 Series) and a 30 m × 0.25 mm open tubular column (Tracer, DB5-MS (film thickness 0.25 µm); Teknokroma, Spain), and comparing the spectra against those from the NIST library. The conversion efficiency of all assayed P. littoralis enzymes toward the exogenously supplied PUFA substrates was calculated as: (all product areas/(all product areas + substrate area)) × 100 (Kabeya et al., 2021) (link).
3
Gravimetric Quantification of Total Lipids
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Total lipids from lyophilized individuals were extracted according to Monroig et al. (2006) . After evaporation of the solvent mixture under nitrogen, the lipids were dried overnight in a vacuum desiccator and quantified gravimetrically (0.01 mg, Mettler Toledo XS 105, Barcelona, Spain).
Total lipids were stored at 10mg/ml in chloroform:methanol (2:1,v/v) which also contained 0.01% of butylated hydroxytoluene (BHT) as an antioxidant. Total lipid samples were stored at -30 °C, in sealed vials, under nitrogen. Fatty acid methyl esters (FAMEs) from total lipids were prepared by direct acid transmethylation and subsequently purified by thin layer chromatography (Silca gel G 60, Merck) using a mix of hexane:diethyl:ether:acetic acid (85:15:1.5, v/v/v) as the solvent phase.
FAMEs were analysed with a Thermo gas chromatograph (Thermo Trace GC Ultra, Thermo Electron Corporation, Waltham, MA, USA) fitted with an on-column injection system, a FID detector, and a silica capillary column (30 m × 0.25 mm × 0.25 µm film thickness, TR-WAX, Teknokroma, Spain), using helium as a carrier gas. The analytical temperature was programmed from 50 ºC to 220 °C. Chromatograms were integrated and analysed with Azur Datlys (St Martin d'Heres, France) software. FAMEs were identified by comparison of retention times of each peak with those of well characterised standards.
Total lipids were stored at 10mg/ml in chloroform:methanol (2:1,v/v) which also contained 0.01% of butylated hydroxytoluene (BHT) as an antioxidant. Total lipid samples were stored at -30 °C, in sealed vials, under nitrogen. Fatty acid methyl esters (FAMEs) from total lipids were prepared by direct acid transmethylation and subsequently purified by thin layer chromatography (Silca gel G 60, Merck) using a mix of hexane:diethyl:ether:acetic acid (85:15:1.5, v/v/v) as the solvent phase.
FAMEs were analysed with a Thermo gas chromatograph (Thermo Trace GC Ultra, Thermo Electron Corporation, Waltham, MA, USA) fitted with an on-column injection system, a FID detector, and a silica capillary column (30 m × 0.25 mm × 0.25 µm film thickness, TR-WAX, Teknokroma, Spain), using helium as a carrier gas. The analytical temperature was programmed from 50 ºC to 220 °C. Chromatograms were integrated and analysed with Azur Datlys (St Martin d'Heres, France) software. FAMEs were identified by comparison of retention times of each peak with those of well characterised standards.
4
Fatty Acid Composition Analysis of Cells
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The experimental procedure used for analysis of fatty acid composition of cells and TAG isolated from cells of R. opacus (and from cork granules) was done as described in Brandl et al., 1988 and Timm et al., 1990 . Briefly, samples were methylated using a mixture of methanol: sulphuric acid (85/15 v/v) during 3.5 h at 100 °C. Methyl esters were analyzed in a GC-FID (Varian 3800) equipped with a CP-Sil 52 CB 30 m × 0.32 mm × 0.25 μm capillary column (Teknokroma, TR-WAX) using splitless mode. The identification of fatty acids was performed comparing the respective retention factor values (Rf) to standard fatty acid methyl esters.
5
Quantification of Fatty Acid Methyl Esters
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Fatty acid methyl esters were quantified using gas chromatography (Varian 3800) equipped with a flame ionization detector (GC-FID). FAMEs were separated on Eq. CP-Sil 52 CB 30 m  0.32 mm  0.25 lm capillary column (Teknokroma, TR-WAX).
One microliter portion of the organic phase was analyzed using a split less mode. Helium was used as carrier gas at a flow rate of 1.0 ml/min. A temperature program was established for an efficient separation of the methyl esters. Initial oven temperature was set at 50 °C for 2 min with an increase of 10 °C min À1 to a final temperature of 225 °C. Injector and detector temperatures were set at 220 and 250 °C, respectively. The fatty acids were identified by comparison of the respective retention factor values (Rf) of standard fatty acid methyl esters.
One microliter portion of the organic phase was analyzed using a split less mode. Helium was used as carrier gas at a flow rate of 1.0 ml/min. A temperature program was established for an efficient separation of the methyl esters. Initial oven temperature was set at 50 °C for 2 min with an increase of 10 °C min À1 to a final temperature of 225 °C. Injector and detector temperatures were set at 220 and 250 °C, respectively. The fatty acids were identified by comparison of the respective retention factor values (Rf) of standard fatty acid methyl esters.
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