The compositions of the pretreated and dioxane-extracted samples were measured using a modified version of the NREL standard method (NREL/TP-510-42618). Because only relatively small amounts of sample were available for compositional analysis, the analyses were performed on 100 mg samples instead of the standard of 300 mg. Sugars and sugar degradation products, i.e., furfural and hydroxymethyl furfural, were measured using high performance liquid chromatography (HPLC) equipped with refractive index and photodiode array detectors (Agilent 1100, Agilent Technologies, Palo Alto, CA). A Rezex RFQ Fast Acids column (100 × 7.8 mm, 8 μm particle size, Phenomenex, Torrance, CA) and Cation H+ guard column (BioRad Laboratories, Hercules, CA) operated at 85°C were used to separate sugar monomers, total oligomers, and degradation products present in the reaction solutions. The eluent was 0.01N H2SO4 at a flow rate of 1.0 mL min−1. Samples and standards were filtered through 0.45 μm nylon membrane syringe filters (Pall Corp., East Hills, NY) prior to injection (2.5 μL) onto the column. The HPLC was controlled and data was analyzed using Agilent ChemStation software (Rev.B.03.02).
Nylon membrane syringe filter
Manufactured by Pall Corporation
Sourced in United States
Nylon membrane syringe filters are laboratory equipment used for the filtration of samples prior to analysis. They are designed to remove particulates and clarify solutions, ensuring sample integrity and protecting analytical instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Rezex rfq fast acids column, by Phenomenex (2 mentions)
Cation h guard column, by Bio-Rad (2 mentions)
Agilent 1100, by Agilent Technologies (2 mentions)
6 n hcl, by Merck Group (2 mentions)
L norleucine, by Fujifilm (2 mentions)
Na s buffer, by Beckman Coulter (1 mentions)
L 8900 amino acid analyzer, by Hitachi (1 mentions)
4 protocols using nylon membrane syringe filter
1
Biomass Compositional Analysis Protocol
2
Quantitative PHB Content Determination
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The PHB content of the bacterial cells was determined by a quantitative method that used HPLC analysis to measure the crotonic acid formed by acid-catalyzed depolymerization of PHB (Karr et al., 1983 (link)). Cell mass samples were freeze-dried before analysis. PHB-containing dried bacterial cells (15–50 mg) were then digested in 96% H2SO4 (1 mL) at 90°C for 1 h. The reaction vials were then cooled on ice, after which, ice-cold 0.01N H2SO4 (4 mL) was added followed by rapid mixing. The samples were further diluted 20- to 150-fold with 0.01N H2SO4 before analysis by HPLC.
The concentration of crotonic acid was measured at 210 nm using an HPLC equipped with a photodiode array detector (Agilent 1100, Agilent Technologies, Palo Alto, CA). A Rezex RFQ Fast Acids column (100 × 7.8 mm, 8 μm particle size, Phenomenex, Torrance, CA) and Cation H+ guard column (BioRad Laboratories, CA) operated at 85°C were used to separate the crotonic acid present in the reaction solutions. The eluent was 0.01N H2SO4 at a flow rate of 1.0 mL min−1. Samples and crotonic acid standards were filtered through 0.45 μm pore size nylon membrane syringe filters (Pall Corp., NY) prior to injection onto the column. The HPLC was controlled and data were analyzed using Agilent ChemStation software (Rev.B.03.02).
The concentration of crotonic acid was measured at 210 nm using an HPLC equipped with a photodiode array detector (Agilent 1100, Agilent Technologies, Palo Alto, CA). A Rezex RFQ Fast Acids column (100 × 7.8 mm, 8 μm particle size, Phenomenex, Torrance, CA) and Cation H+ guard column (BioRad Laboratories, CA) operated at 85°C were used to separate the crotonic acid present in the reaction solutions. The eluent was 0.01N H2SO4 at a flow rate of 1.0 mL min−1. Samples and crotonic acid standards were filtered through 0.45 μm pore size nylon membrane syringe filters (Pall Corp., NY) prior to injection onto the column. The HPLC was controlled and data were analyzed using Agilent ChemStation software (Rev.B.03.02).
3
Quantitative Amino Acid Analysis
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The flag leaf blades at the heading stage and the mature seeds from transgenic plants, as well as WT, were harvested from the main tiller of each plant. For total amino acid analysis, 10mg of rice power of each sample was hydrolyzed with 1mL of 6N HCl (Sigma, United States) in a 2mL screw-cap tube before adding 10nmol L -(+)-norleucine (Wako Pure Chemicals, Japan). The samples were then heated at 110°C for 24h, followed by the treatment of 6h at 65°C in order to evaporate HCl completely. The residue was then dissolved in 1mL Na-STM buffer and centrifuged at 1,600 × g for 10min at room temperature. The supernatant was filtered with a 0.45μm nylon membrane syringe filter (Pall Life Sciences, United States) and transferred to an autosampler bottle for amino acid analysis. HPLC data were normalized with the level of L -(+)-norleucine per sample. Three biological replicates were designed for each sample. Seventeen amino acids were measured, including alanine (Ala), arginine (Arg), aspartic acid (Asp), cysteine (Cys), glutamic acid+glutamine (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine (Val) (Yang et al., 2018 (link)).
4
Free and Total Amino Acid Analysis in Rice
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For free amino acid (FAA) analysis, 50mg of milled and dried rice power was thoroughly mixed in 500 μL Na-S™ buffer (2% sodium citrate, 1% HCl, 0.1% benzoic acid; Beckman, USA) for 30min in a mixer and extracted for 10min via ultrasonication. The mixture was then centrifuged at 16 000rpm for 10min at room temperature. The supernatant was collected and filtered through a 0.45-μm nylon membrane syringe filter (Pall Life Sciences, USA) for injection and analysis using an L8900 Amino Acid Analyzer (Hitachi, Japan).
For total amino acid (TAA) analysis, 10mg of dry rice power was hydrolysed with 1mL of 6 N HCl (Sigma, USA) in a 2-mL screw-cap tube, followed by the addition of 10 nmol L(+)-norleucine (Wako Pure Chemicals, Japan). The samples were heated at 110°C for 24h, and the HCl was then evaporated for 6h at 65°C. Dried samples were dissolved in 1mL Na-S™ buffer and filtered for amino acid analysis as described above. Data obtained from HPLC were normalized with the level of L(+)-norleucine per sample. For each sample, two technical replicates were carried out.
For total amino acid (TAA) analysis, 10mg of dry rice power was hydrolysed with 1mL of 6 N HCl (Sigma, USA) in a 2-mL screw-cap tube, followed by the addition of 10 nmol L(+)-norleucine (Wako Pure Chemicals, Japan). The samples were heated at 110°C for 24h, and the HCl was then evaporated for 6h at 65°C. Dried samples were dissolved in 1mL Na-S™ buffer and filtered for amino acid analysis as described above. Data obtained from HPLC were normalized with the level of L(+)-norleucine per sample. For each sample, two technical replicates were carried out.
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