Agilent 1100 hplc instrument

For chemostat cultivations, the glucose concentration in the feed medium was measured with a high-performance liquid chromatography (HPLC) setup (Shimadzu Corporation, Japan) equipped with a Rezex ROA-organic acid H⁺ column (300 mm by 7.8 mm; Phenomenex, USA). A refraction index detector (RID-10A; Shimadzu Corporation, Japan) was used for quantitation. The column was operated at 60°C, the flow rate was set at 1 ml min⁻¹, and 4 mM H₂SO₄ served as the mobile phase. Before injection, a 900-μl sample was mixed with 100 μl of 0.04 M H₂SO₄ and filtered through a 0.20-μm regenerated cellulose (RC) membrane filter (KC90.1; Roth, Germany). The injection volume was 10 μl.
Determinations of the glucose concentration in the mixing vessel, intracellular trehalose contents, and glucose equivalents of intracellular glycogen contents (see below) during retentostat cultivation and the preceding chemostat cultivation were done on an Agilent 1100 HPLC instrument (Agilent Technologies, USA) equipped with an Aminex HPX 87H ion exchange column (Bio-Rad, The Netherlands), operated at 60°C with 5 mM H₂SO₄ as the mobile phase at a flow rate of 0.6 ml min⁻¹. Detection was performed by means of a dual-wavelength absorbance detector (G1314A; Agilent, USA) and a refractive index detector (G1362A; Agilent, USA).

LC-ESI-MS/MS analysis was carried out based on the method modified from Simirgiotis et al. (2013) (link) as follows: a mass spectrometer of Esquire 4000 Ion Trap (Bruker Daltonics, Bremen, Germany) was connected to an Agilent 1100 HPLC instrument (Agilent Technologies, Waldbronn, Germany) with ESI interface. Data acquisition of full scan was performed from m/z 50 to m/z 2000 in a negative ion mode. Nitrogen was used as nebulizer gas with electrospray needle 4500 V. Using ultrahigh pure helium as the collision gas, induced dissociation spectra were obtained with fragmentation amplitude of 1.00 V (MS/MS). HPLC analysis was carried out using the condition as above of HPLC chromatograph.

Two-week-old rice plants grown on soil were treated with 30 mM acetic acid for 30 min as described above. Roots were harvested, ground to a fine powder, and subjected to extraction and quantification of jasmonates as described previously with slight modification^{49 (link)}. Briefly, the concentrated sample was subjected to LC–ESI–MS/MS, which was composed of a quadrupole tandem mass spectrometer (API-3000) with an electrospray ion source and an Agilent 1100 HPLC instrument (Agilent Technologies, Palo Alto, CA, USA) equipped with a CAPCELL CORE C18 column (length 50 mm, diameter 2.1 mm; OSAKA SODA CO., LTD Osaka, Japan). The solvents used for both columns were water (A) and acetonitrile containing 0.1% (v/v) acetic acid (B), respectively. A 15-min linear gradient (3–70% B) was applied just after sample injection (flow rate 0.2 ml min⁻¹). The multiple reaction-monitoring mode was used in ESI–MS/MS to monitor precursors and products. JA and JA-Ile were analyzed in the negative ion mode with nitrogen as the collision gas. JA, [²H₂]-JA, JA-Ile and [¹³C₆]-JA-Ile were determined with combinations of m/z 209/59, m/z 211/59, m/z 322/130, and m/z 328/136, respectively.

Identification of secondary metabolites present in the roots of E. planum was performed using the HPLC-MS system consisting of Agilent 1100 HPLC instrument with a photodiode-array detector PDAeλ (Palo Alto, CA, USA) and Esquire 3000 ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) with the XBridge C18 column (150 × 2.1 mm, 3.5 μm particle size) and the MSⁿ spectra were recorded in the negative and positive ion modes using a previously published approach [28 (link), 29 (link)]. The elution was conducted with water containing 0.1% formic acid (solvent A) and acetonitrile (solvent B). The gradient elution was started at 10% of B and linearly changed to 25% of B in 25 min and to 98% in 46 min of B over 10 min, followed by return to stationary conditions and reequilibration for 10 min. Moreover, the UHPLC-MS/MS was applied for the quantitative analysis of phenolic compounds (flavonoids and phenolic acids) and for the analysis of triterpenoid saponins. Methods and results were described previously [30 (link)].