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?

Eulate A 400nm A 600 nm 0.000 0.001 0.000 0.012 0.010 0.057 0.068 0.022 0.675 0.007 -0.076 0.068 -0.057 -0.043 -0.056 0.066 0.056 Absorbance vs. Eulate 0.800 0.700 0.600 0.500 5 0.400 0.300 0.200 0.100 4 0.593 0.609 0.290 0.078 0.010 0.001 A 400nm 10 A 600 nm 12 0.000 -0.100 0.200 11 12 Eulate Number

Explanation / Answer

First of all, based on the principle of the chromatography method that you mention theoretically the smaller molecules move faster than the larger molecules so that first elutes the pyridoxal-5-phosphate and finally the albumin (Obviously depending on the solvent that has been used and the size that allows to pass the chromatographic matrix affect who comes first and who at the end).

The data you put is somewhat confusing, based on this principle of size exclusion chromatography the pyrodoxal-5-phosphate is the first to elute and finally albumin. You mention that a diluted reagent for dye was used and it is here the central point to think. Assuming that this reagent to dye is specific for pyridoxal-5-phosphate then you already have the answer, the pyridozal-5-phosphate elutes in the fourth cuvette (In addition to being a small molecule eluted all in a single fraction, on the other hand the albumin being a large molecule takes more fractions to completely elute).

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