Agilent 1260 infinity liquid chromatograph

¹H NMR spectra were recorded on a Bruker AC-400 (400.1 MHz) (Karlsruhe, Germany) relative to the tetramethylsilane signal in a solvent being either CDCl₃ (7.24 ppm) or D₂O (4.27 ppm (25 °C), 4.4 ppm (60 °C)). IR spectra of polymers were recorded on the IR Fourier spectrometer IRAffinity-1 of Shimadzu (Kyoto, Japan) in KBr tablets.
The determination of the molecular weights and dispersity (Đ) of homopolymers was carried out by size-exclusion chromatography using an Agilent-1260 Infinity liquid chromatograph (Santa Clara, CA, USA) equipped with refractometric, light-scattering, and viscometric detectors in combinations with two Agilent PLgel MIXED-C columns (7.5 × 300 mm, 5 µm). SEC analysis was performed in DMF containing 0.1 mol/L LiBr at 50 °C for PMAG, and in THF at 40 °C for PVSI.
Thermogravimetric analysis was carried out on TG209 F1 Libra (Netzsch, Germany) thermomicroscales with an automatic sample feeding system in a nitrogen atmosphere at a constant heating rate of 10 °C/min. The range of the studied temperatures varied from 40 to 600 °C. Preliminary sample preparation consisted of grinding and homogenization of copolymer samples.

The chlorogenic acid content in the I. britannica extract was quantified using an Agilent 1260 Infinity Liquid Chromatograph (Agilent Technologies, Santa Clara, CA, USA). This system was equipped with a quaternary pump delivery system (G1331B), an auto-sampler (G1329B), a column thermostat (G1316A), and a diode array and multiple wavelength detector (G1315D). A volume of 10 µL from each pre-treated sample was injected into an Eclipse XDB-C18 column (4.6 × 150 mm, 5 µm, Agilent Technologies Inc., Santa Clara, CA, USA) set at 35 °C, and detection was carried out at λ = 330 nm. The mobile phase was composed of water (J.T. Baker) and acetonitrile (J.T. Baker), both containing 0.1% (v/v) formic acid (Sigma-Aldrich, Saint Louis, MO, USA), which served as eluents A and B, respectively. The separation was executed at a flow rate of 0.8 mL/min using a gradient program. The gradient was programmed as follows:

0 to 5 min, 10 to 20% B;

5 to 10 min, maintaining 20% B;

10 to 15 min, 20 to 30% B;

15 to 25 min, 30 to 80% B;

25 to 28 min, 80 to 10% B;

28 to 30 min, holding at 10% B;

The analysis ran for a total of 30 min.

Isolated IAAs were analyzed by RP-HPLC using a method based on Jaskula et al. [23 (link)]. All analyses were performed on an Agilent 1260 Infinity liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a temperature controlled autosampler, binary pump, thermostatted column compartment, and a diode array detector. Instrument control and data analysis was performed in Agilent (Santa Clara, CA, USA) Chemstation (version D.04). Isocratic separations were performed on an Alltech (Columbia, MD, USA) Altima column (150 mm × 4.6 mm). Eluent A consisted of 18.2 MΩ deionized water adjusted to a pH of 2.80 with phosphoric acid. Eluent B was acetonitrile. The mobile phase consisted of 48% solvent A and 52% solvent B with a flowrate of 1.8 mL/min. Eluting peaks were monitored at 270 nm and 314 nm and were compared to the IAA standards from the American Society of Brewing Chemists for identification. The ratio of trans to cis IAAs was calculated for each chromatogram using integrated peak areas and Equation (1).
$\frac{T}{C}=\frac{\left(t-isocohumulone\right)+\left(t-isohumulone\right)}{\left(c-isocohumulone\right)+\left(c-isohumulone\right)}$