Biochemistry Lab

Abstract:

The experiment conducted was done for the purpose of practicing Gel-Filtration Chromatography by separation of Blue Dextran, Cytochrome and Potassium Chromate. A gel column was packed with a slurry of the column matrix and was slowly packed to condense the gel beads into a tight bed. The sample was then added as an equal ring around the top of the column. The valve was opened to begin flow and the sample began to elute into the gel column and separate. Throughout the filtration, clean buffer solution was carefully added to the top of the column to prevent the beads from drying. While the filtration proceeded, 1.5 mL fractions were collected and kept for visual observation following the filtration. Once the filtration had concluded, totaling 39 fractions being collected, the fractions were visually compared and were rated on a scale of 0-5, 0 being colourless, 1 being very pale colour and growing to 5 which was a dark colour. It was observed that the Blue Dextran eluted between the 10th and 12th fractions, Cytochrome between the 17th and 27th fractions, and Potassium Chromate between the 28th and 36th fractions. All other fractions were rated a 0 and were colourless. It can be concluded that, on the understanding that small molecules would elute quicker than larger molecules, Blue Dextran is the smallest molecule and Potassium Chromate is the largest.

Results

In Figure 1, the results are represented as a linear progression, beginning with the first fraction and continuing through the entire filtration. The first peak observed is the Blue Dextran elution, the next is the Cytochrome, and the last peak is the Potassium Chromate. It is seen that the transition from Cytochrome to Potassium Chromate was fairly close together and was not completely separated when filtering.

Question

Find Vo, Vi and Vt.

Vo 

Vi 

Vt