Millex gv 0.22 μm filter unit

A 15 μM bovine Hb was incubated in the presence of 30 mM fructose in 50 mM phosphate buffer (pH 7.4). Each of BA, NBA, and ASA were added in pursuance of analyzing their protection against glycation process at the final concentration of 1.5 mM [16 (link)]. Due to the limitation of using sodium azide as a preservative agent in Hb incubation [25 (link)] sterilizing of all of the solutions was performed using low protein binding filter (Millex–GV 0.22 μm filter unit, Millipore). Finally, solutions were incubated at 37°C under sterile conditions. Control samples were prepared in a similar way in the absence of fructose. Sampling was done at appropriate time intervals up to 20 days of incubation, then samples were kept at ‒70°C until analyzing.

The HPLC-FD system consists of a Model Ternary Gradient Unit LG2080-02, a Model 3-Line Degasser DG-2080-53, and a Model FB-1520S fluorescence detector (excitation wavelength, 280 nm; emission wavelength, 340 nm) together with a Model Intelligent HPLC Pump PU-2080-Plus (JASCO, Tokyo, Japan). A Shodex Asahipak ES-502N 7C column (7.5 mm ID × 100 mm, Showa Denko Co., Tokyo, Japan) was used for the anion-exchange HPLC with an ethanol gradient on 0.05 M sodium acetate, 0.40 M Na₂SO₄ (acetate-sulfate buffer), pH 4.85, at the flow-rate of 1.00 mL/min^{15 (link),19 (link)}. The following ethanol concentrations were used for the analysis: 0–1 min, 0%; 1–50 min, linear increase from 0 to 10%; 50–55 min, linear decrease from 10 to 0%; 55–60 min, 0%. All solvents were filtered through a filter unit (0.22 μm, pore size, TPP) and the samples were filtered through filter units (Millex-GV 0.22 μm Filter Unit, Millipore, Bedford, MA, USA) before use.