The separation of cytochrome c and albumin by gel filtration chromatography

In the lab today, we did the experiment in the title and i took 30 samples and measured the absorbance in a spectrophotometer at 280nm and 410nm. The experiment went a little like this: We dissolved cytochrome c and albumin in NaCl and watched it move through a column of Sephadex G100....? I didn't really understand it. The first question is - Why do the elution profiles show 2 peaks at 280nm but only 1 at 410nm? My graph does indeed show this but I don't know why, can anybody help? The second question is - In what order are the proteins eluted? Does the observed order conform to your expectations? Explain. PLEASE HELP ME, I AM SOOOOOO LOST!!

First, the Sephadex G100 inside the column is very tiny beads with very very tiny pores in them. Very large proteins cannot fit inside any of the pores and so as buffer is flowing through the column, very large proteins will flow through the column most rapidly. Intermediate-sized proteins can fit into some of the pores, but not all of them, and so will elute behind very large proteins. Very small proteins can fit inside more of the pores and so will flow most slowly through the column. So, the order of elution is from biggest proteins to smallest ones.

Now, ALL proteins absorb light at 280 nm because of the presence of aromatic amino acids in their structures. One of your proteins has an additional chemical group in its structure that causes the protein to have color--to absorb light in the visible range, in this case at 410 nm. So, absorbance at 280 nm will show two peaks while only one of those peaks will also absorb light at 410 nm.

The molar mass of albumin is probably somewhere around 68,000 D while cytochrome c is a much smaller protein, about 12,000 D. So, I would expect that the first protein is albumin and the second is cytochrome c.