The total lipids were extracted from the tissues (liver, brain cortex, and retina) using a 2:1 mixture of methanol/chloroform. As phospholipids are dominant in the brain and neurons, the phospholipid fraction was isolated further from the total lipid of the brain cortex in Exp. II using an SPE column (CHROMABOND NH2) (Macherey-Nagel GmbH & Co. KG, Düren, Germany). This was not feasible for retina lipids because there were only trace amounts of samples, even when pooled from four mice within a group. Extracts were taken to dryness and weighed to estimate the total lipid content. The total lipids or phospholipid fraction was subjected to transesterification by methanol/dichloromethane = 3/1, as described elsewhere [26 (link)], and the resulting fatty acid methyl esters were dissolved in n-hexane for fatty acid analysis in a Hewlett-Packard 5890 gas chromatograph with flame ionization detection on a SP-2380 fused silica capillary column (30 m × 0.25 mm× 0.2 µm; Supelco, Bellefonte, PA, USA) using nitrogen as the carrier gas (1.5 mL/min). The oven temperature program was set to 110℃ for 5 min, which was then increased at 10℃/min to 170℃, then at 3℃/min to 230℃, and held at 230℃ for 5 min. The fatty acid peaks were identified by a comparison of the retention times with authentic standards.
Sp 2380 fused silica capillary column
Manufactured by Merck Group
Sourced in United States
The SP-2380 fused silica capillary column is a laboratory instrument designed for chromatographic analysis. It is a highly specialized column used to separate and analyze a variety of chemical compounds. The column is made of fused silica, which provides stability and durability. Its core function is to facilitate the separation and identification of complex mixtures through the process of gas chromatography.
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6 protocols using sp 2380 fused silica capillary column
1
Lipid Profiling in Tissues
2
Yeast Fatty Acid Composition Analysis
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The yeast cells were harvested by centrifugation at 1500g for 5 min at 4 ºC and they were washed with distilled water before determining the total fatty acid composition using the one-step method proposed previously (Garcés and Mancha 1993) (link). A volume of 3.3 mL methanol/toluene/dimethoxypropane/sulphuric acid (39:20:5:2) and 1.7 mL heptane was added to the cell pellet, and the mixture was heated at 80 ºC for 1 h. After cooling, the upper phase containing the fatty acid methyl esters was transferred to a fresh tube, washed with 6.7% sodium sulphate and evaporated to dryness with nitrogen.
The methyl esters were dissolved in an appropriate volume of heptane and analysed by GLC (Hewlett-Packard 6890 gas chromatography apparatus; Palo Alto, CA, USA) using a Supelco SP-2380 fused-silica capillary column (30 m length, 0.25 mm i.d., 0.20 μm film thickness: Supelco, Bellefonte, PA, USA). Hydrogen was used as the carrier gas at 28 cm s -1 , the temperature of the flame ionization detector and injector was 200 ºC, the oven temperature was 170 ºC, and the split ratio was 1:50. Peaks were identified by comparing their retention times with those of the corresponding commercial standards.
The methyl esters were dissolved in an appropriate volume of heptane and analysed by GLC (Hewlett-Packard 6890 gas chromatography apparatus; Palo Alto, CA, USA) using a Supelco SP-2380 fused-silica capillary column (30 m length, 0.25 mm i.d., 0.20 μm film thickness: Supelco, Bellefonte, PA, USA). Hydrogen was used as the carrier gas at 28 cm s -1 , the temperature of the flame ionization detector and injector was 200 ºC, the oven temperature was 170 ºC, and the split ratio was 1:50. Peaks were identified by comparing their retention times with those of the corresponding commercial standards.
3
Glycosyl Linkage Analysis of Polysaccharides
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EPS matrix (2 mg) or CW preparation of S. indica or B. sorokiniana were ground with a stainless steel bead (5 mm) using a TissueLyser mill at 30 Hz for 1 min. The powdered material was subjected to glycosyl linkage analysis as described (Liu et al., 2015 (link)). Briefly, a methylation reaction was performed using NaOH/DMSO. The methylated compounds were hydrolyzed in 1 M trifluoroacetic acid, reduced using sodium borodeuteride (ACROS Organics, cat.no. 194950050) and per-o-acetylated. The resulting partially methylated alditol acetates were analyzed using an Agilent 5977A GC/MSD System equipped with a SP-2380 Fused Silica Capillary Column (Supelco). The glycosidic linkages were assigned based on retention time and mass spectrum fragmentation patterns compared to the CCRC spectral database (https://www.ccrc.uga.edu/specdb/ms/pmaa/pframe.html ).
4
Quantification of Tissue Fatty Acids
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Tissue FA (plasma, PFC, and RBC) were quantified by gas chromatography. Briefly, plasma was dried under nitrogen, PFC was lyophilized, homogenized and weighed, and RBC were vortexed and aliquoted directly. Internal standard (trinonadecanoic acid or pentadecanoic acid in toluene) was added to each sample, and 1.5 N methanolic hydrochloric acid was used for direct transesterification. Samples were heated for 2 h at 100°C. Following methylation, saturated sodium chloride was added, and lipids were extracted into toluene for direct injection. Calibration curves were generated using GLC-502B (Nu-Chek Prep, Elysian, MN) for FA reference standards. Samples were analyzed on an Agilent 6890 gas chromatographer (split injection) equipped with a name ionization detector. A 30 m × 0.32 mm × 0.2 μm SP-2380 fused silica capillary column (Supelco, Bellefonte, PA) was used with hydrogen as the carrier gas. Oven temperature was programmed from 140 to 190°C at 5°C/min, held for 1 min at 190°C, increased to 260°C at a rate of 17°C/min, then held for 3 min for a total run time of 18.12 min. The name ionization detector was set at 285°C. FA concentrations are expressed as weight percent of total FA.
5
Glycosyl Residue Linkage Analysis
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For glycosyl residue linkage analysis, the samples were permethylated, reduced, re-permethylated, depolymerized, reduced, and acetylated. The resulting partially methylated alditol acetates (PMAAs) were analyzed by gas chromatography-mass spectrometry (GC-MS) as previously described [66 (link)].
The fractionated wall samples from WT and PdGAUT12.1-KD lines were first suspended in 200 ul dimethyl sulfoxide and the samples stirred for 2 days at 25°C. The samples were permethylated using potassium dimsyl anion and iodomethane. The permethylated uronic acids were reduced using lithium borodeuteride and then permethylated again by the method described earlier [67 (link)] with sodium hydroxide and methyl iodide in dry DMSO. This additional permethylation was to insure complete methylation of the polymer. Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121°C), reduced with NaBD4, and acetylated using acetic anhydride/TFA. The resulting PMAAs were analyzed on an Agilent 7890A GC interfaced to a 5975C MSD (mass selective detector, electron impact ionization mode). Separation was performed on a Supelco SP-2380 fused silica capillary column (30 m × 0.25 mm ID).
The fractionated wall samples from WT and PdGAUT12.1-KD lines were first suspended in 200 ul dimethyl sulfoxide and the samples stirred for 2 days at 25°C. The samples were permethylated using potassium dimsyl anion and iodomethane. The permethylated uronic acids were reduced using lithium borodeuteride and then permethylated again by the method described earlier [67 (link)] with sodium hydroxide and methyl iodide in dry DMSO. This additional permethylation was to insure complete methylation of the polymer. Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121°C), reduced with NaBD4, and acetylated using acetic anhydride/TFA. The resulting PMAAs were analyzed on an Agilent 7890A GC interfaced to a 5975C MSD (mass selective detector, electron impact ionization mode). Separation was performed on a Supelco SP-2380 fused silica capillary column (30 m × 0.25 mm ID).
6
Fatty Acid Profiling of Oils
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For fatty acids determination, lipids were trans-esterified to their fatty acid methyl esters.
Methylation reaction was carried out for 1h after the addition of 2 mL of methanol/toluene/sulphuric acid (88/10/2; v/v/v) to 5 mg of oil. Methyl esters were then extracted with 2 mL heptane and analysed by GC. The chromatographic system consisted of an Agilent 6890 gas chromatograph (Palo Alto, CA) equipped with a Supelco SP-2380 fused silica capillary column (30 m length; 0.25 mm i.d.; 0.20 µm film thickness: Bellefonte, PA). The carrier gas (H2) flow was set at 28 cm/s. Injection and FID temperature was 200 ºC, whereas oven temperature was 170 ºC. Fatty acids were identified by comparing their retention times with those obtained from commercial standards. Their percentage was calculated according to the area obtained from each peak.
Methylation reaction was carried out for 1h after the addition of 2 mL of methanol/toluene/sulphuric acid (88/10/2; v/v/v) to 5 mg of oil. Methyl esters were then extracted with 2 mL heptane and analysed by GC. The chromatographic system consisted of an Agilent 6890 gas chromatograph (Palo Alto, CA) equipped with a Supelco SP-2380 fused silica capillary column (30 m length; 0.25 mm i.d.; 0.20 µm film thickness: Bellefonte, PA). The carrier gas (H2) flow was set at 28 cm/s. Injection and FID temperature was 200 ºC, whereas oven temperature was 170 ºC. Fatty acids were identified by comparing their retention times with those obtained from commercial standards. Their percentage was calculated according to the area obtained from each peak.
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