3.8. Purification of Flavonoids with Macroporous Resins

The resins were soaked in a solution of 95% (v/v) ethanol, equivalent to twice the volume of the resins, for 24 h. The ethanol was then filtered out and the resins were rinsed with deionized water to remove any traces of alcohol. Then, the washed resins were immersed with 5% HCl and 5% NaOH, respectively, for 4 h. Lastly, they were washed with deionized water until the pH of the filtrate reached neutrality and were soaked in deionized water at room temperature for standby.

Six types of macroporous resins (HPD100, HPD600, HPD826, D101, NKA-9, and ADS-17) were each weighed to be 1 g and then added separately into six 100 mL Erlenmeyer flasks. The resins in each flask were soaked in 20 mL of crude flavonoid-rich extract solutions with an initial content of 40 mg/mL. Then, the flasks were shaken using a shaking incubator with a speed of 120 rpm at 25 °C for 24 h. After absorption equilibrium was reached, the resins were filtered and the concentration of total flavonoids in the filtrate was determined. Subsequently, the remaining resin was washed with deionized water until the eluents were colorless. Then, 30 mL of 60% ethanol was added to the flasks for desorption. The flasks were shaken with a speed of 120 rpm at 25 °C for 24 h again. After desorption equilibrium was reached, the solutions were filtered and the total flavonoid contents of the supernatant were measured. The corresponding adsorption and desorption capacity and ratio of each resin were calculated using the equations below [44]:

where Q₁ and Q₂ are the adsorption and desorption capacity (amount) at equilibrium (mg/g), respectively; C₀ and C_e are the original and adsorption equilibrium concentrations of the total flavonoids in the solutions (mg/mL), respectively; V₀ is the initial sample volume added into the flask (mL); M is the dry weight of the resin (g); C_d is the equilibrium concentration of total flavonoids in the desorption solution (mg/mL); V₂ is the desorption solution volume (mL); and E₁ (%) represents the adsorption rate.

Static adsorption kinetics experiment: According to adsorption and desorption capacities, we selected the HPD100 resin for the adsorption kinetics study. We added 1 g of pretreated resin HPD100 in a 100 mL Erlenmeyer flask to 30 mL of crude flavonoid-rich extract solution (40 mg/mL) and then shook it at 25 °C and 120 rpm. We took out 500 μL of supernatants at specific time intervals (1 h) until equilibration and determined the concentrations of total flavonoids in the supernatants to plot the static adsorption kinetics curve.

Static desorption kinetic experiment: After completing the adsorption experiment, we collected the HPD100 resins and dried the surface water with filter paper. Subsequently, we immersed 0.5 g of resin saturated with adsorption in 30 mL of 70% ethanol (v/v) and incubated it at 25 °C and 120 rpm. We took out 500 μL of supernatants at specific time intervals (1 h) until equilibration and measured the concentrations of total flavonoids in the supernatants to plot the static desorption kinetics curve.

First, we added six aliquots (accurately weighed 1 g) of HPD100 macroporous resin separately into six 100 mL Erlenmeyer flasks. Next, we added 20 mL of sample solutions containing different concentrations of crude flavonoid-rich extracts (10, 20, 40, 60, 80, and 100 mg/mL). The flasks were continually shaken for 3 h at 120 rpm and 25 °C. We determined the original and adsorption equilibrium concentrations of total flavonoids in the solutions and then drew the adsorption concentration curve.

The dynamic experiments of adsorption and desorption were performed in a chromatographic column (1:10), wet-packed with a column volume that was around 25 mL of the selected HPD100 resin. The solution of crude flavonoid-rich extract from FRC was filtered through a 0.45 µm membrane and then applied to the resin at a suitable flow rate. The solutions of crude extracts with a concentration of 40 mg/mL and a volume of 250 mL were loaded at different flow rates of 1.5, 3, and 6 BV/h (bed volume per hour). The effluent liquid was collected every 25 mL and the total flavonoid concentration was detected. Then, the flow rate–volume curves were plotted.

To determine the effect of eluent concentration on desorption efficiency, ethanol/water mixtures with different ratios (10, 20, 40, 60, 80, and 95% v/v) were used as solvents. An amount of 0.5 g of HPD 100 resins loaded with crude flavonoid extract were placed in conical flasks, and 20 mL of each ethanol solution was added. The flasks were shaken at 25 °C and 120 rpm for 4 h to reach equilibrium. Then, the solutions were filtered and the total flavonoids contents of the filtrates were measured. The elution concentration curve was plotted based on the results.

A glass column was packed with 25 mL of HPD100 macroporous resin, which had a diameter-to-height ratio of 1:10. The crude flavonoid-rich extract was loaded onto the resin column until the adsorption equilibrium was reached. Then, the deionized water was first used for washing the resin column and then eluted with 80% ethanol solution at a flow rate of 3BV/h. The eluent was collected in 25 mL fractions, and the total flavonoid concentration in each fraction was measured with UV spectrophotometry. The dynamic elution curve was drawn based on the elution volume and flavonoid content.