Question

In Gel Filtration Chromatography, polyacrylamide beads and phosphate buffer were used. We wanted to separate Blue Dextran (2 mDa/blue), BSA (70 kDa, no color), myoglobin (17 kDa, reddish/brownish), and yellow food coloring (500 Da, yellow). For some reason, some fractions we collected earlier contained 3 substances (Blue Dextran, BSA, and myoglobin) together. A little later, fractions collected contained BSA and myoglobin, but the color of them was clear.

Why did fractions collected earlier contain Blue Dextran, BSA, and myoglobin though we wanted to make them separate? Why did the fractions collected a little later contain myoglobin and BSA but their color was not reddish/brownish? What could we do to separate all substances and get much better results?

I will appreciate your help very much!

Answer 1

An investigation shows that  the uptake and efflux of Blue Dextran in the isolated perfused rabbit lung. Blue Dextran is a high-molecular-weight glucose polymer (original mol wt 2 x 10(6) g/mol) containing covalently bonded Reactive Blue 2 dye (approximately mmol/g dextran). This blue dye is known for its high binding affinity to a wide variety of proteins, with a particularly high affinity for serum albumin. In isolated rabbit lungs perfused with a protein-free perfusate, both bolus injection and recirculation of Blue Dextran revealed a rapid saturable uptake. Once the lungs were loaded with Blue Dextran, efflux of the Blue Dextran accumulated in the lungs could be induced by addition of bovine serum albumin (BSA) to the recirculating perfusate. The amount of BSA-induced efflux of Blue Dextran from the lung was independent of perfusate flow. When the left pulmonary artery was ligated after the lungs had been loaded with Blue Dextran, the dye-induced BSA efflux was only about 50% of normal. Release of the ligature so that both lungs were perfused resulted in efflux of the remaining Blue Dextran. The combination of high airway pressure and low flow also reduced the dye efflux, and the effect was reversed by reducing the airway pressure. With the assumption that the high average molecular weight of Blue Dextran confines this molecule to interaction with proteins on the vascular surface, the results of this study suggest that Blue Dextran uptake and its BSA-induced efflux are proportional to the perfused vascular surface area in the lung.