I did a size-exclusion chromatography lab to seperate Serum Albumin (molecular w

Question

I did a size-exclusion chromatography lab to seperate Serum Albumin (molecular weight 66,437 Da) and Pyriodoxal 5'-Phosphate (247.14 Da). After the mixture was filtered through a column the fractions were collected in 12 cuvettes, and the absorbance for each cuvette was measured at 400 nm. Then a Dilute Reagent Dye was added and the absorbance was then measured at 600 nm. Below I have included my graphed data from the experiment. My question is: from this data, how do I determine if Serum Albumin or Pyriodoxal 5'-Phosphate eluted first from this data?

I was told that larger molecules will elute first, and smaller molecules will elute last. From this, I would guess serum albumin would elute first and Pyriodoxal 5 phosphate would elute last. However, I do not understand how to interpret my data below to approve or disprove my conclusion.

Absorbance vs. Eulate 0.800 0.700 0.600 0.500 0.400 0.300 2 0.200 0.100 0.000 0.100 0.200 Abs at 400 nm | Abs at 600 nm 1 2345 6 789 10 11 12 Elute Number

Explanation / Answer

In this question statement made by you is correct that in gel-filtration larger molecule comes first and smaller molecule comes at the last.

Hope you are also aware of the fact that small molecule travel inside the beads whereas larger molecules travels through the space between beads. All this conditions depend upon that what kind or pore size gel material has been chosen for the gel filtration chromatography.

Now the all interpretation is depend upon that what type of material is you are separating and what type of analytical procedures you are using. In this question one is albumin that is made up of amino acids and second substance is is chemical compound. So this question suggests two methods one is screening at 400 nm and other is adding a die that gives the color at 600 nm. That die is Bradford reagent that is made up of commassie dye and this dye interacts specifically with lysine and arginine residues of protein and gave the blue color that is detected at 595 or 600 nm. Here Pyriodoxal 5'-Phosphate is absorb at 400 nm that is absent in initial fraction and only very small amount is showing absorbance at 400 nm that might be due to some residue or albumin protein but the signal strength (absorbance at Y-axis is low) strong at 600 nm ( in 4th fraction), while adding dye, represent that protein albumin is coming out first. Similarly opposite fact is true for Pyriodoxal 5'-Phosphate that shows strong absorbance at 400 nm and low absorbance at 600 nm while adding dye (fraction no 6-10).

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