The culture supernatant was collected by centrifugation for 5 min at 10000 × g and analyzed by an HPLC (Agilent Technologies, USA) equipped with a Cosmosil SC18-MS-II column (Nacalai Tesque, Japan) and UV detector (Agilent Technologies, USA) at 280 nm. The mobile phase contained solution A (methanol: acetic acid: water; 10:0.3:89.7 v/v/v) and 60% solution B (methanol: acetic acid: water; 90:0.3:9.7 v/v/v) at a flow rate of 0.3 mL/min as described by Nutaratat et al.24 (link). Isocratic elution was used instead of gradient elution. Authentic IAA (Sigma, USA) was used as a standard.
Cosmosil sc18 ms 2 column
Manufactured by Nacalai Tesque
Sourced in Japan
The Cosmosil SC18-MS-II column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a chemically bonded C18 stationary phase on a silica gel support. The column is suitable for use in both analytical and preparative HPLC applications.
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3 protocols using cosmosil sc18 ms 2 column
1
Quantification of Indoleacetic Acid
2
Trp-dependent IAA Biosynthesis in R. fluvialis
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To investigate whether R. fluvialis DMKU-CP293 employed Trp-dependent or Trp-independent IAA biosynthesis, culture media with and without tryptophan supplements were used. The inoculum was transferred into a 250 ml Erlenmeyer flask containing 50 ml mineral salt (MS) medium (gram per liter: KH2PO4, 1; K2HPO4, 1; NaCl, 1; MgSO4·7H2O, 4; (NH4)2 SO4, 4; Na3C6H5O7·7H2O, 0.5; Glucose, 5), supplemented with or without a 0.1% (w/v) L-tryptophan for investigation of Trp-dependent or Trp-independent IAA biosynthesis, respectively. The yeast inoculum cell density was estimated by measuring the optical density at 600 nm (OD600) using a spectrophotometer and adjusted to 0.2, prior to incubation on an orbital shaker at 200 rpm. The cultures were incubated at 30 °C, and samples of culture broth were taken at daily intervals for 5 days. The culture supernatant was analyzed for IAA using high-performance liquid chromatography (HPLC), equipped with a Cosmosil SC18-MS-II column (Nacalai Tesque, Kyoto, Japan) and UV detector (Agilent Technologies, Santa Clara, CA), methanol: acetic acid: water at the ratio 60: 40: 0.3 v/v/v were used as mobile phase with a flow rate of 0.5 ml/min as described by Nutaratat et al. [17 (link)].
3
Indole-3-Acetic Acid Production by Yeast
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Yeast was inoculated into 3 mL of yeast extract peptone dextrose medium (YPD) containing 1% yeast extract, 2% peptone, and 2% dextrose and incubated at 30 °C and 200 rpm for 16–18 h. Then, the yeast inoculum was transferred to a 125 mL Erlenmeyer flask containing 25 mL of YPD medium supplemented with 0.1% L-tryptophan. The initial OD600 was adjusted to 0.2 prior to incubation on a rotary shaker at 30 °C and 170 rpm for 3 days. Final OD600 values were spectrophotometrically determined. The samples were centrifuged at 14,000 × g for 5 min and the supernatants were collected for IAA analysis using high performance liquid chromatography (HPLC; Nexera LC-40 series, Shimadzu, Japan) with a Cosmosil SC18-MS-II column (Nacalai Tesque, Japan) and UV detector at 280 nm. A mixture of ethanol, acetic acid, and water (60:1.5:40 v/v/v) was used as a mobile phase with a flow rate of 0.3 mL/min, as described by Nutaratat et al. [41] (link). Authentic IAA (Sigma, USA) was used as a standard.
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