We used sonication to lyse and homogenize fungal mycelia for the extraction of secondary compounds [34 (link)]. Around 500 mg of fungi (pure mycelial culture) were taken and dispersed in 10 mL of an 80% ethyl acetate solution. The contents were subjected for sonication in the ultrasonic bath sonicator (Elma Ultrasonic Cleaner S100H, Mumbai, India) at a 80 W cm2 intensity for 0.5 pulse cycles at a temperature of 40 °C. After the process of sonication, the contents were filtered using a Whatman No. 1 paper filter to eliminate the mycelia, and the remaining filter was centrifuged at 2500 rpm. The liquid containing the secondary metabolic compounds of the endophytic fungi was determined by GC-MS analysis. A Shimadzu Make QP-2010 on a non-polar 60 M RTX 5MS column was used for the GC-MS analysis, with helium as the carrier gas, containing 15 psi as constant pressure.
60 m rtx 5ms column
Manufactured by Shimadzu
The 60 M RTX 5MS column is a gas chromatography column designed for the separation and analysis of a wide range of compounds. It features a stationary phase composed of 5% diphenyl and 95% dimethylpolysiloxane, which provides excellent separation performance for a variety of sample types.
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3 protocols using 60 m rtx 5ms column
1
Fungal Metabolite Extraction via Sonication
2
GC-MS Analysis of Chemical Compounds
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GC-MS analysis was carried out using Shimadzu model QP-2010 with a nonpolar 60 M RTX 5MS column. Helium was used as the carrier gas and the temperature programming was set with initial oven temperature at 40 °C and held for 3 min and the final temperature of oven was 480 °C with rate at 10 °C. Two microliters (2 µl) sample was injected using a split less mode. Mass spectra was recorded over 35–650 amu range with electron impact ionization energy 70 eV. The total running time for a sample was 45 min. Relative quantitative determinations were made by relating respective peak areas to total ion chromatogram (TIC).
3
GC-MS analysis of plant extracts
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GCMS analysis of the samples was carried out using Shimadzu Make QP-2010 with a nonpolar 60 M RTX 5MS Column. Helium was used as the carrier gas and the temperature programming was set with the initial oven temperature at 400°C and held for 3 min and the final temperature of the oven was 4800°C with a rate at 100°C. A 2 μL sample was injected with split less mode. Mass spectra were recorded over 35-650 amu range with electron impact ionization energy 70 eV. The total running time for a sample is 45 min. The chemical components from the ethanolic extracts of plants were identified by comparing the retention times of chromatographic peaks using the Quadra pole detector with NIST Library to relative retention indices (Fig. 1 and2).
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