Due to the Sephadex (gel) and buffer mixture, the column was initially opaque, with a dark blue color solution containing a protein mixture of methylene blue and haemoglobin on top of the gel bed.

As the protein combination is introduced and travels through the gel bed, the dark blue solution will brighten in color.

The fraction was collected in a measuring cylinder and transferred to the designated test tubes, with the procedure repeated for the remaining 11 test tubes.

To keep the gel from drying out, the column is then topped up with buffer.

The brown colour began just below the light methylene blue solution, displaced before the blue solution, and accumulated in fractions 2 and 3.

The colour becomes a little less bright with fractions 3 and 4.

The methylene blue solution was split into fractions 7-12; the colour of the set is most brilliant in fractions 8-9, while it begins to fade in fraction 10.

This indicates that the separation is complete, with barely a trace of material remaining in the column.

Both the methylene dye and the protein were initially present in the protein combination.

Further separation of the protein (haemoglobin) causes the brown color seen at the bottom of the column, as protein elutes first due to size because it cannot penetrate the pores of the gel but can move around them, whereas smaller molecules can infiltrate the pores and take longer to elute, thus being last because their pores are smaller than the beads.