Gemini nx c18 column

After acid hydrolysis and reaction of 1-phenyl-3-methyl-5-pyrazolone (PMP), the monosaccharide composition of EPS was detected with high-performance liquid chromatography (HPLC) as reported before.[22 (link)] Briefly, sample (5 mg) was hydrolyzed by 2 mL of trifluoroacetic acid (2 M) for 4 h at 120°C. Subsequently, the hydrolysate was dried under vacuum and redissolved by 1 mL of water. The resulting solution (100 μL) was mixed by 200 μL methanol solution of PMP (0.5 M) and 200 μL of NaOH solution (0.3 M) and reacted for 30 min at 70°C. The reaction was terminated through neutralization with 450 μL of HCl solution (0.3 M), and the product was then partitioned three times with chloroform. Afterward, the collected aqueous layer was filtered through a 0.45 μM filter membrane and subjected to HPLC that was conducted by a Phenomenex GEMINI-NX C₁₈ column (250 nm × 4.6 nm, 5 μm) on an Agilent 1200 instrument at 30°C. Potassium phosphate-buffered saline solutions (0.1 M, pH 6.7) containing 83% acetonitrile (solvent A) and 17% acetonitrile (solvent B) were used as mobile phases, and the wavelength of UV detection was set at 250 nm. The monosaccharide components were finally identified through comparing their retention times with those of standard saccharides.