515 pump

High-performance liquid chromatography (HPLC), a very popular and widely used method to separate, identify, and quantify compounds from a mixture, was used to identify the antimicrobial compounds in the bacterial extract. The compounds separated through TLC were curettaged and was subjected to reverse-phase HPLC (RP-HPLC) (Rao et al., 2017 (link)). The purity of the antimicrobial compounds was analyzed by analytical HPLC supported with Waters Spherisorb 5 μm ODS2 4.6 mm × 250 mm analytical cartridge (C-18 column) on a Waters 515 pump, isocratic reverse phase system with a 2,998 photodiode array detector at 210 nm, and the range given was 190–600 nm (Elshafie et al., 2017a (link), c (link)). The flowrate was 1.0 ml/min, and additional UV detector was measured at 254 nm using Empower 2 software. Methanol and water (2:1) were used as the mobile phase. The purified bacterial methanol extract was mixed with HPLC-grade methanol and filtered by using a 0.22-micromillipore membrane filter before injecting into the injection port. The samples were run for 15 min, and the retention time was noted; based on the percentage of the peak area, the purity of each compound was determined.

Adenine and guanine solutions standards and the supernatant of the adsorption-desorption experiments were analyzed via liquid chromatography. The analyses were performed using an HPLC system (515 pump, Waters Corp.) along with a single quadrupole mass detection system (SQ-2 Waters Corp.) and an electronic distance measurement instrument (EDM) in positive mode (ESI⁺). It is sometimes necessary to ionize the samples to obtain a better response; therefore, the supernatants’ pH value was adjusted with formic acid or ammonium hydroxide. Working conditions were adjusted for a 1.58 kV capillary for adenine and 2.47 for guanine, a 19 V cone for adenine and a 39 V cone for guanine, at a temperature of 350 °C, and a desolvation gas flow of 650 L h⁻¹ using a Symmetry C18 column (4.6 × 75 mm, size of 3.5 μm spherical particle, by Waters Corp.) under an isocratic elution with a mobile phase (80–20% methanol-water, HPLC-ESI-MS) and with a flow rate of 0.3 mL min⁻¹. A fixed sample volume (20 μL) was injected using a loop. We performed HPLC-ESI-MS analyses on all the samples to ensure the presence of the adenine (136 m/z) and guanine (152 m/z) ions after each adsorption–desorption process. The retention time for adenine was 2.09 min, and for guanine, it was 1.57 min.

3 mg copolymer were dissolved in 1 mL chromatography grade THF and was filtered by using a 0.45 μm nylon 66 filter membrane. Then using gel permeation chromatography (GPC) which was purchased from Waters Inc. (Milford, MA, USA) to define the molecular weight and distribution of the compounds, and THF was used as the eluent. The system is equipped with a column (7.8 × 300 mm, Waters Styragel, Waters Inc., Milford, MA, USA), a Waters 515 pump and a Waters 2414 refractive index detector. When the column temperature of GPC is 40 °C, the flow rate is 1 mL/min, and the baseline is smooth. The filtrate was pulled into an injection needle, and the sample was slowly and uniformly injected into the sampler when the air was removed. All data were obtained under the same standard curve.

All reagents were obtained commercially and used as received. Anhydrous CH₂Cl₂ was purified using standard methods. Branched LMW PEI 600 was obtained from Aladdin, Shanghai, China, while branched PEI 25 kDa was obtained from Sigma-Aldrich, St. Louis, MO, USA. Label IT^® Cy5^TM was provided by Mirus, Madison, WI, USA; pGL-3 was provided by Promega, Madison, WI, USA; pEGFP-N1 (coding for Enhanced Green Fluorescent Protein (EGFP) DNA) was provided by Clontech, Palo Alto, CA, USA. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen Corp (Carlsbad, CA, USA). The MicroBCA protein assay kit was obtained from Pierce, Rockford, IL, USA, while the luciferase assay kit was provided by Promega, Madison, WI, USA. The endotoxin-free plasmid purification kit was obtained from TIANGEN, Beijing, China.
¹H-NMR spectra were measured on a Bruker AV400 spectrometer. The molecular weight (Mw) was measured using gel permeation chromatography (GPC), with a Waters 515 pump, a Waters 2410 Refractive Index Detector (25 °C, incorporating Shodex columns OHPAK KB-803, Santa Barbara, CA, USA). The mobile phase involved 0.5 mol·L⁻¹ HAc/NaAc buffer at a flow rate of 0.5 mL·min⁻¹; poly(ethylene glycol) was used as the standard (average Mw: 900–80,000 Da).

The number and weight average molecular weight (M_n and M_w respectively) of PS samples were determined by GPC (Waters). The instrument was equipped with a Waters 515 pump, two PL mixed-D and mixed-E columns and a Waters 410 refractive index detector operating at 35 °C. Calibration was based on a series of six narrow MW linear polystyrene standards with molecular weights ranging from 580 to 578,500 g mol⁻¹. THF was used as the eluent at a flow rate of 1 ml min⁻¹.

Sugars, organic acids, HMF, and ethanol were measured using an HPLC system (Ultimate 3000, Dionex, USA) that had a refractive index detector and a UV detector. The temperature of the Aminex HPX-87H column was set at 65 °C and the flow rate of the mobile phase (0.01 N H₂SO₄) was 0.6 mL/min. The levels of galactose, xylose, and mannose were determined using an HPLC system (515 Pump, 717plus Autosampler, Waters, USA) that had an Asahipak NH2P-50 4E amine column and an evaporative light scattering detector (SEDEX 75, Sedere, France). The temperature of the column was kept at 30 °C, the mobile phase was a solution of acetonitrile and water (80:20), and the flow rate was 1.0 mL/min.