I have a BSA-FITC conjugate made in Sodium Bicarbonate pH 8.5 that I'd like to desalt (the excess dye) and buffer exchange (into PBS) via gravity. I equilibrated a G-25 column with PBS pH 7.4. I applied my conjugate (2.5 mL volume) and eluted it with 3.5 mL PBS so my fraction 2 (2.5 mL volume) is my conjugate in the correct buffer confirmed by pH and OD at 280 nm. What I don't understand is my next fraction F3 (3.5 mL volume) has a pH of 8.5, the bicarbonate buffer, but also has protein (maybe?). OD says there's no protein detected, but by agitating this fraction there seems to be protein in there. Also, by gravity the conjugate should be diluted, but my F2 has the same volume. It sounds to me I want to combine my F2 and F3 (hence why after desalting, sample comes out more diluted), but my F3 now has sodium bicarbonate buffer?
The length and cross-sectional area of the G25 column, as well as the applied sample volume, are relevant to how well the protein and salt will be separated.
Since the protein elutes in the void volume of the column, it probably will not be diluted by much as long as the column is not too large. This seems to be the case in your procedure, with most of the protein eluting in fraction 2, separated from most of the bicarbonate buffer. There will probably be some protein eluting in fraction 3 along with some of the bicarbonate. You can easily check fraction 3 for FITC-BSA by the absorbance or fluorescence of the fluorescein group, and compare it to fraction 2.
The pH of fraction 3 may be the same as the bicarbonate buffer of the sample, but the bicarbonate concentration may be lower. Depending on the relative buffering capacities and concentrations of the two buffers, a small proportion of the pH 8.5 buffer may overwhelm the buffering of the pH 7.4 buffer. (PBS usually contains only 10 mM phosphate buffer, whereas labeling reactions are usually done in 100 mM sodium bicarbonate.)
Since BSA and FITC are cheap reagents, I would be happy to have recovered most of the FITC-BSA in fraction 2 at the desired pH, and discard fraction 3.
How do you find out there is some protein in your third fraction?
The peaks on chromatography are usually wider and tail, so it's no surprise that you should have protein in your third fraction. Maybe you could try to collect smaller fractions (like 1 ml) to have higher resolution of your elution profile.
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