Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography

A novel, rapid, and economical method to isolate and purify natural sulforaphane from broccoli seeds is described. The procedure involves solvent extraction of autolyzed seed meal, followed by separation by solid phase extraction (SPE) and purification by preparative high-performance liquid chromatography (HPLC). The SPE method provides higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction. High purity and recovery of sulforaphane product can be obtained by preparative HPLC with a C 18 column and 30% methanol in water as the mobile phase. The purified compound was characterized by MS and (1)H and (13)C NMR. The techniques described here are useful tools in the preparative-scale isolation of sulforaphane in a fast, cost-effective, and waste-conscious manner.     

Immobilized zinc affinity chromatography of pectin hydroxamic acids for purification of trypsin inhibitors from soybean and sweet potato

Commercial pectin (with a 94% degree of esterification, DE94) suspended in methanol was reacted with methanolic alkaline hydroxylamine at room temperature for 20 h to prepare pectin hydroxamic acids (PHAs). The prepared PHA was coupled to the epoxy-activated Sepharose 6B gel to get immobilized PHA resins. The immobilized PHA resin was then balanced in column with 2 mM ZnCl2 in 50 mM Tris-HCl buffer (pH 7.9) to test the immobilized Zn-PHA gel as solid phase for immobilized metal affinity chromatography for the purification of trypsin inhibitors (TIs) from soybean and sweet potato. Using TI activity staining, it was found that purified TIs from the commercial soybean and sweet potato after trypsin affinity column purification could be adsorbed onto an immobilized Zn-PHA affinity column and eluted by 100 mM EDTA in 10 mM Tris-HCl buffer (pH 7.9). The immobilized Zn-PHA affinity column was used for TI purifications from crude extracts of sweet potato. The recovery of TI activity for one step was 90%, with 19.74-fold purification increase.     

Centrifugal precipitation chromatography -- a novel chromatographic system for fractionation of polymeric pigments from black tea and red wine

A novel chromatographic system was developed and first applied to the fractionation of polymeric pigments from black tea and red wine. Centrifugal precipitation chromatography (CPC) generates solvent gradients through a long separation channel under a centrifugal force field. Tea and wine extracts are precipitated in a hexane- or methyl tert-butyl ether-rich environment and are exposed to a gradually increasing ethanol concentration. This causes a repetitive precipitation and dissolution of the biopolymers along the channel. Consequently, they are eluted in the order of their solubility in the organic solvent. It is shown by HPLC analysis of the separated fractions that monomers elute first, whereas fractionated polymers can be found at the end of the chromatographic run. This novel method allows gentle fractionation of polymeric tea and wine constituents and also has potential for use in preparative-scale separations.