Alliance e2695 hplc system

The phenolic compounds present in methonolic extract were tentatively identified using the chromatographic separation method as previously reported [4 (link)].
Chromatographic separation was performed on an Alliance e2695 HPLC system (Waters, Bedford, MA, USA) equipped with a RP-xTerra MS column (150 × 4.6 mm i.d., 3.5 μm particle size), photodiode array detector (PDA) and interfaced with a triple quadruple mass spectrometer (MSD 3100, Waters) fitted with an ESI ion source. The sample (20 μL) was eluted through the column with a gradient mobile phase consisting of A (0.1% formic acid) and B (acetonitrile acidified with formic acid 0.1%) with a flow rate of 0.5 mL/min. The following multistep linear solvent gradient was used: 0–40 min: 14–26% B; 40–60 min: 15% B; 60–75: 0% B; 75–80 min: 14% B. The HPLC-PDA-ESI-MS chromatogram spectral data were stored and processed with Masslynx 4.1 data system. Each peak in the chromatogram was accomplished in a single chromatographic run in order to be identified [34 (link)].

Analyses of pigment compositions were performed using an Alliance e2695 HPLC system (Waters, Milford, CT, USA) equipped with a 2998 Photodiode Array (PDA) detector (Waters, Milford, CT, USA) and a Zorbax Eclipse Plus C18 column (250 × 4.6 mm, 5 μm, Agilent, Palo Alto, CA, USA). The temperature of the column oven was set at 30 °C. A mixture of H₃PO₄ solution (pH 2.5, phase A) and acetonitrile (phase B) were used as the mobile phase using the following gradient program: 0 min, 80% A, 20% B; 25 min, 20% A, 80% B; 35 min, 20% A, 80% B; 36 min, 80% A, 20% B; 41 min, 80% A, 20% B. The PDA was set at 200–600 nm, and the flow rate of the mobile phase was 0.8 mL/min [21 (link)].

Free polyamines in the diet were extracted according to the previous method with some modification [18 (link)]. Briefly, 0.5 g diet was weighed and homogenized in 3 mL of 5% ice-cold perchloric acid (v/v), followed by incubation for 30 min on ice. After centrifugation at 12,000×g for 30 min at 4 °C, 100 μL of the supernatant fluid was mixed with an equal volume of 1.5 mol/L HClO₄ and then neutralized by addition of 100 μL of 2 mol/L of K₂CO₃. The tubes were centrifuged at 13,000×g for 5 min, and the resulting supernatant fluid was used for HPLC determination of free polyamines. For determination of polyamines content in culture media and cells, IPEC-J2 cells were harvested by centrifugation at 1,000×g for 10 min. The supernatant fluid was obtained and deproteinized as described previously, whereas the cells were washed twice with ice-cold PBS (pH 7.4). Cells were lysed in 100 μL ice-cold PBS (pH 7.4) by sonication, and deproteinized as described previously. Polyamines in samples were determined by HPLC (Alliance e2695 HPLC system, Waters Corporation, MA, USA) using the OPA-NAC method [19 (link)].

The SEC–MALS experiments for samples purified by IMAC were performed using an Alliance e2695 HPLC system (Waters, Milford, MA, USA) equipped with a Superdex 200 Increase 10/300 GL column (Cytiva), which was connected in line with a DAWN HELEOS II multi-angle static light scattering detector (Wyatt Technology, Santa Barbara, CA, USA). The data were collected at 20 °C with 20 mM HEPES buffer (pH 7.5) containing 150 mM NaCl and analyzed using ASTRA 6 software (Wyatt Technology) [46 (link)]. Protein concentration was determined using a refractive index detector. A dn/dc value of 0.185 mL/g was generally used for the proteins.

The purity of the recombinant heterodimeric proteins after each stage of purification was assessed by both RP-HPLC and SDS-PAGE analysis. RP-HPLC was carried out using an Alliance e2695 HPLC system (Waters, Milford, MA, USA) with UV absorbance detection at 215 nm. The SCF-Lα-GCSF and GCSF-Lα-SCF proteins from IBs extracts, their folding intermediates, and fractions derived from the chromatography columns were analyzed on a C₁₈ reverse-phase column (Zorbax 300SB-C18, 4.6 × 250 mm; Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation of the proteins was performed in acetonitrile gradient (mobile phase A—0.1% trifluoroacetic acid (TFA) in water, mobile phase B—9.9% water, 90% acetonitrile, and 0.1% TFA) at a flow rate 1 mL/min, as follows: (1) initial equilibration at 10% B, (2) a 5-min gradient to 58% B, (3) a 74-min gradient to 81% B, (4) a 3-min gradient to 90% B, (5) a 4-min isocratic elution at 90% B, (6) a 3-min gradient to 10% B, and a final (7) 5-min isocratic elution at 10% B. The temperature of the column was maintained at 30 °C. SDS-PAGE was performed on a slab gel containing 15% polyacrylamide by the method of Laemmli (1970) (link).

Nineteen phenolic compounds and caffeine were quantified according to the following method. Powdered extracts and fractions were dissolved in 10% (v/v) DMSO in methanol using 20 min of sonication, and then passed through a 0.45-μm GHP syringe filter (Pall Corp.). The filtered samples were injected into an Alliance e2695 HPLC system (Waters Corp., Milford, MA, USA) equipped with an auto-sampler, a quaternary pump, and a Poroshell 120 SB ODS column (120 Å, 2.7 μm, 4.6 × 150 mm; Agilent Technologies, Santa Clara, CA, USA) with an injection volume of 5 μL. The eluent was passed through a Waters Isocratic Solvent Manger simultaneously into a single quadrupole mass detector (MS^S) and a UV detector, with a retention time gap (0.075 min on average) between the two. The elution program and the conditions for separation and mass detection are described in Supplementary Data I. For the UV detector, gallic acid, catechins, and theaflavins were detected at 275 nm, and flavonols and flavones were monitored at 365 nm. All data were collected and processed using Empower 3 software (Waters Corp.). The relative contents of flavonol glycosides (such as kaempferol, myricetin, and quercetin glycosides) were compared using the peak area of 365 nm.

HPLC analysis was performed using an Alliance e2695 HPLC system (Waters Corp., Milford, MA, USA) coupled to a 2475 fluorescence detector (Waters Corp., Milford, MA, USA).
The vials were maintained in a sample manager at 15 (±5) °C until a 100-μL aliquot was injected into an autosampler, after which it was injected onto an XBridge BEH C18 reversed-phase column (150 mm × 4.6 mm, length × internal diameter, 5 μm) thermostated at 30 °C. A binary mobile phase was used, at a flow of 1 mL/min. Mobile phase component A was 0.01 M sodium dihydrogen phosphate solution (phosphoric acid was used to adjust the pH to 4.8) containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, and component B was acetonitrile. Isocratic elution was carried out with mobile phases A and B at a ratio of 65:35 (V/V).
The excitation and emission wavelengths of channel A for fluorescence detection were set to 228.0 nm and 279.0 nm, respectively. Channel B was operated at excitation and emission wavelengths of 272.0 nm and 450.0 nm, respectively, for the detection of fluoroquinolones.