Adsorption and separation of flavonoid aglycones and flavonol aglycones by using Zr(IV) immobilized collagen fiber adsorbent as column packing material

Flavonoid aglycones and flavonol aglycones, are difficult to be separated due to their similar structures and limited hydroxyls. Herein, Zr(IV) immobilized collagen fiber (ZCF) was prepared by immobilizing Zr(IV) on glutaraldehyde cross-linked collagen fiber (GCF) to separate apigenin and kaempferol, the representative com-pounds of flavonoid aglycones and flavonol aglycones. TG, DSC, SEM and element mapping were applied to characterize ZCF. The static adsorption suggested that when the amount of immobilized Zr(IV) reached only 0.3 mmol/g, the adsorption difference between apigenin and kaempferol on ZCF was highly improved compared with GCF without Zr(IV). The adsorption rate of apigenin increased from 2.21% to 14.31%, while that of kaempferol significantly increased from 22.35% to 87.17%. In addition, the spectroscopy (UV-Vis, FT-IR) were applied to analyze the binding sites of flavonoids with Zr(IV), indicating that the strong adsorption of ZCF to kaempferol may be related to the 3-hydroxyl on the C-ring, while apigenin only can conduct a weak binding with ZCF through 5-hydroxyl on the A-ring. In ZCF column chromatography separation, apigenin and kaempferol could be separated effectively with purities of 94.02% and 89.23% by stepwise elution of 50% aqueous ethanol followed by 50% aqueous ethanol containing 0.005 mol/L citric acid. In separation on ZCF and GCF combined columns, the purities of apigenin and kaempferol reached 95.59% and 92.56%, respectively, and the trace amount of Zr(IV) shedding from ZCF was completely adsorbed by GCF avoiding contaminating the target components. In addition, the effective separation of apigenin and kaempferol in apium graveolens (celery) extract by ZCF column was also achieved. The above results indicated ZCF could selectively adsorb flavonol aglycones with 3-hydroxyl on the C-ring from the mixture with flavonoid aglycones. This investigation provides a novel column chromatography packing material for the fast separation of flavonoid aglycones and flavonol aglycones.

Automated summary

Flavonoid aglycones and flavonol aglycones, such as apigenin and kaempferol, were successfully separated using a Zr(IV) immobilized collagen fiber (ZCF). The adsorption capacity of ZCF for kaempferol was higher than apigenin, and efficient separation of flavonol aglycones from flavonoid aglycones was achieved through ZCF column chromatography.