High molecular weight contaminants

Gel filtration will allow the high-molecular-weight contaminant to be removed. The remaining mixture of lower-molecular-weight proteins can be separated by ion-exchange chromatography, as described in Prob. 4.4. 

Activated carbon possesses extremely high surface area (38), often in excess of 1000 m /g. Much of that surface area is, however, associated with micropores—that is, pores <20 A (<2 nm) in diameter. The surface area associated with meso-pores—pores 20 to 500 A (2 to 50 nm) in diameter—is considerably lower (typically in the range 10—100 m /g). Most liquid-based applications (including fats and oils purification) involve the adsorption of high-molecular-weight contaminants whose molecular dimensions prevent penetration into micropores therefore, activated carbon containing significant mesoporosity is most desirable in these applications (39). 

Hum. Crude material isolated from the mycelium was relatively low in proteases and in high molecular weight impurities. Supernatant provided much more raw material initially (up to 300 yg/ml by HPLC) but this F-II was high in protease activity and was contaminated with huge amounts of proteinaceous material from the medium. The experiences learned in the development of process II, namely the ability of hydroxylapatite to remove very similar impurities and the success of durapore membranes to effectively remove large amounts of high molecular weight contaminants, spurred further work on a supernatant process. 

In the study, the relatively heavy concentrations of contaminants in the feed material for the hyperfiltration unit precluded effective use of other "tighter" types of membranes as the first pass barrier due to the potential for fouling. Optimally, a combination system employing the initial membrane used here to remove high molecular weight contaminants, followed by a "tighter" membrane to remove lower weight phenolics from the permeate of the first membrane would have been more likely to provide the full spectrum of contaminant removal desired. 

First, an ultrafiltration membrane with a cut off of 5 kDa was used to remove salts and small molecular weight inq)urities. In a second ultrafiltration step using a 30 kDa mend rane die eluate was collected. In this step high molecular weight contaminant and endotoxins were removed. As in the procedure described above, the aqueous solution was treated alternately with cation and anion exchange resins in order to remove charged by-products. Finally, 1 was isolated by freeze drying and obtained as a white fluffy powder. 

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