The purpose of this experiment was to use gel-filtration chromatography to separate of mixture of blue dextran, cytochrome c and sodium chromate and determine the outer void (Vo), inner void (Vi), and elution volume (Ve) of the gel filtration column. To separate the mixture, first a 15 mL chromatographic column must be packed with a slurry of Sephadex G-75 (GE Healthcare, 17-0050-01) suspended in 20 mM, pH 7.0 sodium phosphate buffer. Then the sample mixture was eluted with the phosphate buffer into 1.5 mL portions. The portions were given a score (0-4) in terms of relative colour intensity and then plotted as an elution profile. Blue dextran was completely excluded from the column, sodium chromate was completely included in the column and cytochrome c was only partially included in the column. The outer void (VO) is equal to elution volume of blue dextran (VD), was 13.5 mL. The total volume (Vt) is equal to volume of elution of sodium chromate (VP) which is 29.25 mL. The inner void (Vi) was calculated to be 27.0 mL.
Collected portions of blue dextran, cytochrome c, and sodium chromate, from gel-filtration chromatography was given a score in terms of relative colour intensity. Colour intensity was measured on a scale of 0-4, where 0 described a fraction with a complete absence of colour, while 4 described a fraction with the most intense colour relative to the other fractions. The first colour observed was blue, then peach, and finally, yellow.
The Ve values obtained from the elution profile (Fig. 1.1 and Table 1.2) were 13.5 mL for blue dextran, 29.25 mL for cytochrome c, and 40.5 mL for sodium chromate. The Vo was 13.5 mL and the Vt was 40.5 mL (Table 1.2). The Vi was calculated to be 27.0 mL (Table 1.2). These values were obtained through the process of gel filtration. .
Gel filtration is a separation procedure that is based on each specific protein's Stoke's radius, which essentially describes the radius of the hydrated protein if it was a perfect sphere.