Empower pro

The HPLC method was used for detailed investigations of the chemical profile of the extracts from the infested and uninfested leaves. The analysis was performed using an Empower-Pro chromatograph (Waters), which consists of a quaternary pump (M2998 Waters) with a degasser and a UV-Vis diode array detection (DAD) system. Separation was performed on a column filled with modified silica gel RP-18 (Atlantis T3—Waters, 3 μm, 4.6 mm × 150 mm). The mobile phase consisted of A (1% acetic acid in water) and B (acetonitrile). The program of the gradient elution was set as follows: 20% B (0 to 10 min); 20–25% B (10 to 25 min); 25–45% B (25 to 40 min); the flow speed was 1 mL/min. The qualitative analysis of phenolic compounds was performed on the basis of retention times and diode array spectral characteristics in comparison with available standards of phenolic compounds. In addition, quantitative investigations were performed on the basis of the areas of the peaks of the tested compounds and calibration curves prepared separately for each standard compound. The DAD detection was conducted at 280 nm for phenolic acid derivatives and at 330 nm for flavonoid derivatives for the purpose of quantitative analysis. The content of phenolic derivatives was expressed as mg/g of dry matter.

Waters^® Empower™ Pro software was used for data acquisition. Metabolites were identified comparing their RT and m/z to the standard ones. The samples were quantified against standard curves of at least 6 points run in triplicate. Standard curves were run at the beginning and end of each chromatographic series. Quality control checks (blanks and standards) were run every 20 samples. Normalization was made using the total protein quantification obtained directly from the well using the bicinchoninic acid method, as previously described.

Sugars and amino acids were determined essentially as described by Ghaffari et al. [68 (link)]. Numerical analysis and quantification of individual compounds were carried out using the Empower Pro software (Waters, Milford, MA, USA) and authentic standards, respectively.

The dry extract of Galphimia glauca was analyzed in a modular HPLC system (Waters) constituted by a 2695 separation model (Alliance; Waters) and a 2996 photodiode detector (Waters). The equipment was controlled with a data capture computer software program (Empower pro; Waters). The chromatographic method was developed in a reverse-phase column (Alttima, RP- 18, 3 μm, 4.6 × 70 mm; Merck). The mobile phase comprised a 35:65 acetonitrile/water isocratic system eluted at a 1.7 mL/ min flow with a 21 min run time. The fingerprints were obtained at a 220 nm wavelength. For the calibration curve, four ascendant concentrations of G-B which were previously isolated from Galphimia glauca extract were injected in the same chromatographic method (Figure 3). This methodology allowed us to discover that the G. glauca extract contained 53 mg/g of G-B (Figure 1).

We determined the O₂ and CO₂ concentrations in the supplied and emitted gasses using a paramagnetic oxygen analyzer (Servomex 1100 A; Norwood, MA, United States) and an infrared CO₂ analyzer (Horiba PIR 2000; Japan) and measured the gas-flow rates with a bubble flow meter. We determined biomass dry weight as described (Herbert et al., 1971 ) and measured the substrate (Mtl or Ara) concentration in culture supernatants (from either the used HMY-Mtl or the HMY-Ara) by HPLC. To this end, we filtered the samples through 0.22-μm nitrocellulose membranes and manually injected 10 μl of each sample with a rheodyne 7725i injector into a Carbopac PA1 column maintained at 35 °C. The chromatography involved a mobile phase of 0.1 M NaOH and a flow rate of 1.0 ml.min^-1, with peak heights measured by a Waters 2465 electrochemical detector and the substrate concentrations quantified by means of the software Empower Pro, 2002 Waters.
We calculated the μ_max from the linear regression of the Napierian logarithmic values (ln) of biomass dry weight during the exponential growth phase and determined the qO_2max plus the CO₂ production by a mass-balance method (Cooney et al., 1977 (link)). We calculated the biomass yield and specific rates of substrate consumption as described (Roels, 1983 ).

Multi-wavelength HPLC runs were carried out by connecting on-line the SEC-HPLC system with a Waters 996 Photodiode Array Detector. Chromatograms at specific wavelengths (280 and 340 nm for proteins and EMB, respectively) and UV-vis spectra of peaks were extrapolated by means of Waters Empower Pro software. NS does not display any absorbance at 340 nm and EMB incubated in the absence of NS is eluted at much higher volume in SEC, as typical for small organic molecules (data not shown).